U.S. patent application number 17/224544 was filed with the patent office on 2021-10-21 for methods, devices, kits and compositions for detecting tapeworm.
The applicant listed for this patent is IDEXX Laboratories, Inc.. Invention is credited to David Allen Elsemore, Jinming Geng.
Application Number | 20210324055 17/224544 |
Document ID | / |
Family ID | 1000005692703 |
Filed Date | 2021-10-21 |
United States Patent
Application |
20210324055 |
Kind Code |
A1 |
Geng; Jinming ; et
al. |
October 21, 2021 |
Methods, Devices, Kits and Compositions for Detecting Tapeworm
Abstract
Methods, devices, kits and compositions for detecting the
presence or absence of one or more tapeworm coproantigens in a
sample are disclosed herein. The methods, devices, kits and
compositions of the present invention may be used to confirm the
presence or absence of tapeworm in a fecal sample from a mammal and
may also be able to distinguish between different tapeworm species
and in the presence of one or more infections with helminths (such
as roundworm, hookworm, whipworm and heartworm), Giardia and
parvovirus. Confirmation of the presence or absence of tapeworm in
the mammal may be made, for example, for the purpose of selecting
an optimal course of treating the mammal and/or for the purpose of
determining whether the mammal has been rid of the infection after
treatment has been initiated.
Inventors: |
Geng; Jinming; (Scarborough,
ME) ; Elsemore; David Allen; (South Portland,
ME) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IDEXX Laboratories, Inc. |
Westbrook |
ME |
US |
|
|
Family ID: |
1000005692703 |
Appl. No.: |
17/224544 |
Filed: |
April 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16591329 |
Oct 2, 2019 |
11001626 |
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17224544 |
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62746805 |
Oct 17, 2018 |
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62741849 |
Oct 5, 2018 |
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62740100 |
Oct 2, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/33 20130101; G01N 33/5308 20130101; G01N 33/563
20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; G01N 33/563 20060101 G01N033/563; G01N 33/53 20060101
G01N033/53 |
Claims
1-93. (canceled)
94. A device for detecting the presence or absence of one or more
tapeworm coproantigens from a fecal sample, the device comprising a
solid support, wherein the solid support has immobilized thereon
one or more antibodies selected from the group consisting of: (a) a
first antibody capable of specifically binding a coproantigen from
a first tapeworm taenia pisiformis, but not a coproantigen from a
second tapeworm Taenia taeniaeformis or a coproantigen from a third
tapeworm Dipylidium caninum; (b) a second antibody capable of
specifically binding the coproantigen from the second tapeworm, but
not the coproantigen from the first tapeworm or the coproantigen
from the third tapeworm; and (c) a third antibody capable of
specifically binding the coproantigen from the third tapeworm, but
not the coproantigen from the first tapeworm or the coproantigen
from the second tapeworm.
95. The device according to claim 94, wherein the solid support
further comprises one or more lectins immobilized thereon.
96. The device of claim 95, wherein the solid support has
immobilized thereon two or more antibodies.
97. The device of claim 95, wherein the one or more lectins is
immobilized onto the solid support.
98. The device of claim 94, further comprising (d) one or more
types of Giardia coproantigen, roundworm coproantigen, whipworm
coproantigen, and/or hookworm coproantigen, wherein the one or more
types of roundworm coproantigen, whipworm coproantigen, and
hookworm coproantigen are specifically bound to the antibodies.
99. The device of claim 94, wherein the solid support has
immobilized thereon two or more antibodies.
100. The device of claim 94, further comprising one or more species
of tapeworm antigen, wherein the one or more species of tapeworm
antigen are specifically bound to the antibodies.
101. The device of claim 94, wherein: (a) the first antibody was
raised against a whole extract of the first tapeworm, E/S material
of the first tapeworm. Worm Wash material of the first tapeworm, or
TCA soluble material of the first tapeworm; (b) the second antibody
was raised against a whole extract of the second tapeworm, E/S
material of the second tapeworm, Worm Wash material of the first
tapeworm, or TCA soluble material of the second tapeworm; or (c)
the third antibody was raised against a whole extract of the third
tapeworm, E/S material of the third tapeworm, Worm Wash material of
the first tapeworm, or TCA soluble material of the third
tapeworm.
102. (canceled)
103. The device of claim 94, wherein the first antibody does not
specifically cross-react with one or more coproantigens selected
from the group consisting of: tapeworm Taenia taeniaeformis
coproantigen, tapeworm Dipylidium coproantigen, hookworm
coproantigen, roundworm coproantigen, whipworm coproantigen,
Giardia coproantigen and. parvovirus coproantigen.
104. The device of claim 103, wherein the hookworm is Ancylostoma,
the roundworm is Toxocara, and the whipworm is Trichuris.
105. The device of claim 103, wherein the tapeworm Dipylidium is
Dipylidium caninum; the hookworm is Ancylostoma caninum or
Ancylostoma iubaeforme; the roundworm is Toxocara canis or Toxocara
cati; the whipworm is Trichuris vulpis or Trichuris felis; the
Giardia is Giardia lamblia; and the parvovirus is feline parvovirus
or canine parvovirus.
106. (canceled)
107. The device of claim 94, wherein the second antibody does not
specifically cross-react with one or more coproantigens selected
from the group consisting of: tapeworm Taenia pisiformis
coproantigen, tapeworm Dipylidium coproantigen, hookworm
coproantigen, roundworm coproantigen, whipworm coproantigen,
Giardia coproantigen and parvovirus coproantigen.
108. The device of claim 107, wherein the hookworm is Ancylostoma,
the roundworm is Toxocara, and the whipworm is Trichuris.
109. The device of claim 107, wherein the tapeworm Dipylidium is
Dipylidium canimum; the hookworm is Ancylostoma caninum or
Ancylostoma tubaeforme, the roundworm is Toxocara canis or Toxocara
cati; the whipworm is Trichuris vulpis or Trichuris felis; the
Giardia is Giardia lamblia; and the parvovirus is feline parvovirus
or canine parvovirus.
110. (canceled)
111. The device of claim 94, wherein the third antibody does not
specifically cross-react with one or more coproantigens selected
from the group consisting of: tapeworm Taenia coproantigen,
hookworm coproantigen, roundworm coproantigen, whipworm
coproantigen, Giardia coproantigen and parvovirus coproantigen.
112. The device of claim 111, wherein the hookworm is Ancylostoma,
the roundworm is Toxocara, and the whipworm is Trichuris.
113. The device of claim 111, wherein the tapeworm Taenia is Taenia
pisiformis or Taenia taeniaeformis; the hookworm is Ancylostoma
caninum or Ancylostoma tubaeforme; the roundworm is Toxocara canis
or Toxocara cati; the whipworm is Trichuris vulpis or Trichuris
felis; the Giardia is Giardia lamblia; and the parvovirus is feline
parvovirus or canine parvovirus.
114. (canceled)
115. (canceled)
116. (canceled)
117. (canceled)
118. The device of claim 94, wherein the first, second and third
antibodies do not specifically bind any coproantigen derived from
at least one member selected from the group consisting of:
roundworm coproantigen, whipworm coproantigen, hookworm
coproantigen, Giardia coproantigen, and parvovirus
coproantigen.
119. The device of claim 94, wherein the device further includes
one or more reagents for the detection of one or more of the group
consisting of: one or more helminthic worm parasites; non-worm
parasites, heartworm, one or more viruses, one or more fungi, one
or more protozoa and one or more bacteria.
120. The device according to claim 94, wherein the solid support
further has immobilized thereon one or more antibodies selected
from the group consisting of: (i) an antibody capable of
specifically binding a roundworm coproantigen, but not a
coproantigen derived from the group consisting of whipworm,
hookworm, tapeworm, Giardia and parvovirus; (ii) an antibody
capable of specifically binding a whipworm coproantigen, but not a
coproantigen derived from the group consisting of roundworm,
hookworm, tapeworm, Giardia and parvovirus; and (iii) an antibody
capable of specifically binding a hookworm coproantigen, but not a
coproantigen derived from the group consisting of whipworm,
roundworm, tapeworm, Giardia and parvovirus; (iv) an antibody
capable of specifically binding Giardia coproantigen, but not
coproantigen selected from the group consisting of roundworm
coproantigen, whipworm coproantigen coproantigen, hookworm
coproantigen, tapeworm Taenia coproantigen, tapeworm Dipylidium
coproantigen and parvovirus coproantigen; and (v) an antibody
capable of specifically binding parvovirus coproantigen, but not
coproantigen selected from the group consisting of roundworm
coproantigen, whipworm coproantigen, hookworm coproantigen,
tapeworm Taenia coproantigen, tapeworm Dipylidium coproantigen, and
Giardia coproantigen.
121. The device according to claim 120, wherein the solid support
has two or more antibodies, three or more antibodies, four or more
antibodies, five or more antibodies, six or more antibodies, seven
or more antibodies or eight or more antibodies.
122. The device of claim 120, wherein the hookworm is Anyclostoma,
the roundworm is Toxocara and the whipworm is Trichuris.
123. The device of claim 122, wherein the hookworm is Ancylostoma
caninum or Ancylostoma tubaeforme; the roundworm is Toxocara cati;
or Toxocara cati; the whipworm is Trichuris vulpis or Trichuris
felis; the Giardia is Giardia lamblia; and the parvovirus is feline
parvovirus or canine parvovirus.
124. The device of claim 94, wherein the device is an enzyme-linked
immunosorbent assay device.
125. The device of claim 124, wherein the enzyme-linked
immunosorbent assay device is a lateral flow immunoassay
device.
126. The device of claim 94, wherein the sample is from a canine or
a feline.
127. The device of claim 94, wherein at least one of the
coproantigens is glycosylated.
128. A kit for detection of one or more platyhelminthic
coproantigens in a mammalian sample, the kit comprising the device
of claim 94, and one or more reagents sufficient for the detection
of the one or more coproantigens.
129. The kit of claim 128, wherein the one or more reagents are
selected from the group consisting of one or more indicator
reagents, one or more antibody labeling compounds, one or more
antibodies, one or more antigen capture reagents, one or more
inhibitors, and one or more wash reagents.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of priority from U.S.
Provisional Application Ser. Nos. 62/740,100, filed Oct. 2, 2018;
62/741,849, filed Oct. 5, 2018; and 62/746,805, filed Oct. 17,
2018, all which are incorporated by reference in their
entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0002] The present invention relates to compositions, devices, kits
and methods for the detection of and distinguishing between
tapeworm species in mammals. More particularly, the present
invention relates to antibodies and antibody compositions, devices,
kits, and methods for detecting the presence or absence of tapeworm
species in a sample from a mammal and for distinguishing between
tapeworm antigens.
2. Description of the Prior Art
[0003] Parasitic worm infections are common in animals and, if not
diagnosed and treated, can cause serious disease or death. Current
methods for diagnosis of parasitic worm infections primarily
involve microscopic examination of fecal samples, either directly
in fecal smears or following concentration of ova and parasites by
flotation in density media or by sedimentation. Despite this
procedure's high adoption, the method has significant shortcomings.
These microscopic methods are time consuming and require
specialized equipment. In addition, the accuracy of results of
these methods is highly dependent upon the skill and expertise of
the operator. For example, the presence of tapeworm is determined
by looking for eggs or proglottids, but these are excreted
intermittently and in small numbers. Not surprisingly, tapeworm
infection is often not diagnosed on routine fecal examination.
[0004] Stool handling is disagreeable and hazardous. Sanitary and
inoffensive procedures for processing stool are awkward and often
complex. Such procedures may include weighing, centrifuging and
storing, and are difficult except in a clinical laboratory equipped
with a suitable apparatus, protective equipment, and a skilled
technician. Therefore, any reduction in the number of steps
required to perform a fecal test and any reduction in contact
between test operator and the test material is desirable. Clinical
laboratories have been using the immunoassay methods for the
detection of various viruses, bacteria and non-helminth parasites
and organisms in feces. However, there remains a need for a simple
immunoassay method for the detection of a parasitic worm infection
in feces, whole blood or in serum.
SUMMARY OF THE INVENTION
[0005] In one aspect, the invention is directed to an isolated
antibody that can specifically bind to a tapeworm coproantigen such
as Taenia coproantigen, e.g., Taenia pisiformis coproantigen or
Taenia taeniaeformis coproantigen, or a Dipyhdium caninum
coproantigen. The antibody can be detectably labeled or attached to
a solid support.
[0006] In one embodiment, the isolated antibody specifically binds
to Taenia pisiformis coproantigen. The antibody does not
specifically cross-react with one or more coproantigens selected
from the group of: tapeworm Taenia taeniaeformis, tapeworm
Dipyhdium, hookworm (e.g., Ancylostoma), roundworm (Toxocara),
whipworm (e.g., Trichuris), Giardia and parvovirus; the tapeworm
Dipyhdium is Dipyhdium caninum; the hookworm is Ancylostoma caninum
or Ancylostoma tubaeforme; the roundworm is Toxocara canis or
Toxocara cati; the whipworm is Trichuris vulpis or Trichuris felis;
the Giardia is Giardia lamblia, and the parvovirus is feline
parvovirus or canine parvovirus.
[0007] In another embodiment, the isolated antibody specifically
binds to Taenia taeniaeformis coproantigen. The antibody does not
specifically cross-react with one or more coproantigens selected
from the group of: tapeworm Taenia pisiformis, tapeworm Dipyhdium,
hookworm (e.g., Ancylostoma), roundworm (Toxocara), whipworm (e.g.,
Trichuris), Giardia and parvovirus; the tapeworm Dipyhdium is
Dipyhdium caninum; the hookworm is Ancylostoma caninum or
Ancylostoma tubaeforme; the roundworm is Toxocara canis or Toxocara
cati; the whipworm is Trichuris vulpis or Trichuris felis; the
Giardia is Giardia lamblia, and the parvovirus is feline parvovirus
or canine parvovirus
[0008] In other embodiments, the isolated antibody binds to
Dipyhdium caninum coproantigen. The antibody does not specifically
cross-react with one or more coproantigens selected from the group
of: tapeworm Taenia, tapeworm Dipyhdium, hookworm (e.g.,
Ancylostoma), roundworm (Toxocara), whipworm (e.g., Trichuris),
Giardia and parvovirus where the tapeworm Taenia is Taenia
pisiformis or Taenia taeniaeformis; the hookworm is Ancylostoma
caninum or Ancylostoma tubaeforme; the roundworm is Toxocara canis
or Toxocara cati; the whipworm is Trichuris vulpis or Trichuris
fells; the Giardia is Giardia lamblia, and the parvovirus is feline
parvovirus or canine parvovirus.
[0009] In another aspect, the invention is directed to an
immunocomplex comprising a tapeworm coproantigen and one or more
antibodies specifically bound to the tapeworm coproantigen. In one
embodiment, the tapeworm coproantigen can be the coproantigen from
Taenia pisiformis, Taenia taeniaeformis, Taenia crassiceps or
Dipyhdium caninum. In another embodiment, the antibody is obtained
by immunization with a whole extract of the tapeworm, E/S material
of the tapeworm, Worm Wash of the tapeworm, or TCA soluble material
of the tapeworm. In another embodiment, the antibody is
specifically bound to a tapeworm coproantigen from a sample such as
a fecal sample obtained from a tapeworm-infected mammal. In some
embodiments, the mammal is further infected with one or more of
roundworm, whipworm and hookworm and the antibody does not
specifically bind to any antigen from the one or more of roundworm,
whipworm, or hookworm that may be present in the sample. In some
embodiments, the antibody can be detectably labeled or bound to a
solid support.
[0010] In another aspect, the invention is directed immunocomplex
comprising a tapeworm coproantigen, an antibody specifically bound
to the tapeworm coproantigen, and a lectin bound to the tapeworm
coproantigen. The lectin can bind specifically to one specific type
of carbohydrate or can bind to two or more carbohydrates on the
tapeworm coproantigen.
[0011] In another aspect, the invention provides a device for
specifically binding and isolating helminthic antigens from a
sample, for example coproantigens from a fecal sample, the device
comprising a solid support, wherein the solid support has
immobilized thereon one or more antibodies selected from the group
consisting of (a) a first antibody capable of specifically binding
coproantigen from a first tapeworm, but not coproantigen from a
second tapeworm or coproantigen from a third tapeworm; (b) a second
antibody capable of specifically binding the coproantigen from the
second tapeworm, but not the coproantigen from the first tapeworm
or the coproantigen from the third tapeworm; and (c) a third
antibody capable of specifically binding the coproantigen from the
third tapeworm, but not the coproantigen from the first tapeworm or
the coproantigen from the second tapeworm. The device, may be, but
is not limited to being, for example, an ELISA device, such as a
lateral flow immunoassay device or microtiterplate device. Samples
that may be tested for tapeworm by the device include, but are not
limited to being, feces, digestive tract mucous, urine, whole
blood, serum, mammary milk and whole tissue, such as tissue from
mammary gland, instine, liver, heart, lung, esophagus, brain,
muscle, and eye, for example. The device further may include, but
need not include, one or more reagents for the detection of one or
more of the group consisting of: one or more helminthic worm
parasites, non-worm parasites, one or more viruses, one or more
fungi, one or more protozoa, and one or more bacteria. In some
embodiments, the solid support further has immobilized thereon one
or more antibodies selected from: (i) an antibody capable of
specifically binding a roundworm coproantigen, but not a
coproantigen derived from the group consisting of whipworm,
hookworm, tapeworm, Giardia and parvovirus; (ii) an antibody
capable of specifically binding a whipworm coproantigen, but not a
coproantigen derived from the group consisting of roundworm,
hookworm, tapeworm, Giardia and parvovirus; (iii) an antibody
capable of specifically binding a hookworm coproantigen, but not a
coproantigen derived from the group consisting of whipworm,
roundworm, tapeworm, Giardia and parvovirus; (iv) an antibody
capable of specifically binding Giardia coproantigen, but not
coproantigen selected from the group consisting of roundworm
coproantigen, whipworm coproantigen, hookworm coproantigen,
tapeworm Taenia coproantigen, tapeworm Dipyhdium coproantigen, and
parvovirus coproantigen; and (v) an antibody capable of
specifically binding parvovirus coproantigen, but not coproantigen
selected from the group consisting of roundworm coproantigen,
whipworm coproantigen, hookworm coproantigen, tapeworm Taenia
coproantigen, tapeworm Dipyhdium coproantigen, and Giardia
coproantigen. In embodiments, the hookworm is Anyclostoma, the
roundworm is Toxocara and the whipworm is Trichuris. In other
embodiments, the hookworm is Ancylostoma caninum or Ancylostoma
tubaeforme; the roundworm is Toxocara canis or Toxocara cati; the
whipworm is Trichuris vulpis or Trichuris fells; the Giardia is
Giardia lamblia, and the parvovirus is feline parvovirus or canine
parvovirus.
[0012] In some embodiments, the solid support has immobilized
thereon two or more antibodies, three or more antibodies, four or
more antibodies, five or more antibodies, six or more antibodies,
seven or more antibodies or or eight or more antibodies to allow
for multiplexing.
[0013] In another aspect, the invention provides a device for
detecting the presence or absence of one or more tapeworm
coproantigens from a fecal sample; the device comprising a solid
support, a lectin and one or more antibodies selected from the
group consisting of: (a) a first antibody capable of specifically
binding a coproantigen from a first tapeworm species, but not a
coproantigen from a second tapeworm species or a coproantigen from
a third tapeworm species; (b)a second antibody capable of
specifically binding the coproantigen from the second tapeworm
species, but not the coproantigen from the first tapeworm or the
coproantigen from the third tapeworm species; and (c) a third
antibody capable of specifically binding the coproantigen from the
third tapeworm species, but not the coproantigen from the first
tapeworm species or the coproantigen from the second tapeworm
species, wherein the solid support has immobilized thereon one or
more lectins or one or more antibodies. In some embodiments, the
lectin is immobilized on the solid support. In other embodiments,
the first, second and third antibodies are immobilized on the solid
support. In other embodiments, the device further comprising one or
more species of tapeworm antigen, wherein the one or more species
of tapeworm antigen are specifically bound to the antibodies. In
some embodiments, the first tapeworm species is Taenia pisiformis,
the second tapeworm species is Taenia taeniaeformis, and/or the
third tapeworm species is Dipylidium caninum.
[0014] In yet another aspect, the invention provides a method of
detecting the presence or absence of one or more helminthic
antigens in a sample, for example coproantigens from a fecal
sample, the method comprising: (a) contacting a sample from a
mammal with one or more antibodies selected from the group
consisting of: (i) a first antibody capable of specifically binding
coproantigen from a first tapeworm, but not coproantigen from a
second tapeworm or coproantigen from a third tapeworm; (ii) a
second antibody capable of specifically binding the coproantigen
from the second tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the third tapeworm; and (iii) a
third antibody capable of specifically binding the coproantigen
from the third tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the second tapeworm; (b) forming
antibody-coproantigen complexes in the presence of the
coproantigens, if any, in the sample; and (c) detecting the
presence or absence of the antibody-coproantigen complexes, if any.
The coproantigens of tapeworm can include coproantigens of Taenia
species, e.g., Taenia pisiformis and Taenia taeniaeformis; and
Dipylidium species, e.g., Dipylidium caninum. In one embodiment,
the step of detecting the presence or absence of the complexes
further includes the step of providing a lectin that binds to at
least one of the complexes. In some embodiments, the lectin can be
detectably labeled or immobilized onto a solid support. In other
embodiments, the first, second and third antibodies are detectably
labeled or immobilized on a solid support. In some embodiments, the
first, second and third antibodies can be immobilized on a solid
support and the lectin can be detectably labeled. In other
embodiments, first, second and third antibodies can be detectably
labeled and the lectin can be immobilized on a solid support.
[0015] In another embodiment, wherein prior to the step of
contacting the fecal sample from a mammal with at least one
antibody, the method further comprising the step of contacting the
fecal sample with one or more lectins. The one or more lectins can
be detectably labeled or immobilized onto a solid support.
Alternatively, the first, second and third antibodies can be
detectably labeled or immobilized on a solid support. In one
embodiment the first, second and third antibodies can be
immobilized on a solid support and the one or more lectins can be
detectably labeled. In another embodiment, the first, second and
third antibodies can be detectably labeled and the one or more
lectins can be immobilized on a solid support.
[0016] In one aspect, the method is carried out to test a fecal
mammalian sample for tapeworm coproantigen. The method, however, is
not limited to being carried out to test a fecal sample. In
addition to feces, the sample therefore may be, but is not limited
to being whole blood, serum, mammary milk and whole tissue, such as
tissue from mammary gland, intestine, liver, heart, lung,
esophagus, brain, muscle, and eye, for example.
[0017] In yet another aspect, the invention provides a method of
diagnosing whether a mammal is infected with one or more parasitic
worms, the method comprising the steps of: (a) contacting a sample
from a mammal with one or more antibody selected from the group
consisting of: (i(i) a first antibody capable of specifically
binding coproantigen from a first tapeworm, but not coproantigen
from a second tapeworm or coproantigen from a third tapeworm; (ii)
a second antibody capable of specifically binding the coproantigen
from the second tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the third tapeworm; and (iii) a
third antibody capable of specifically binding the coproantigen
from the third tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the second tapeworm; (b) forming
antibody-coproantigen complexes in the presence of the
coproantigens, if any, in the sample; (c) detecting the presence or
absence of the antibody-coproantigen complexes, if any; and (d)
diagnosing the mammal as having: (i) a first tapeworm infection if
a first tapeworm antibody-coproantigen complex is present; (ii) a
second tapeworm infection if a second tapeworm
antibody-coproantigen complex is present; and (iii) a third
tapeworm infection if a third tapeworm antibody-coproantigen
complex is present. In one embodiment, two or more antibodies,
three or more antibodies, four or more antibodies, five or more
antibodies, six or more antibodies, seven or more antibodies or
eight or more antibodies are selected. In some embodiments, the
step of detecting the presence or absence of the complexes further
includes the step of providing one or more lectins that binds to at
least one of the complexes. In some embodiments, the lectin can be
detectably labeled or immobilized onto a solid support. In other
embodiments, the first, second and third antibodies are detectably
labeled or immobilized on a solid support. In some embodiments, the
first, second and third antibodies can be immobilized on a solid
support and the lectin can be detectably labeled. In other
embodiments, first, second and third antibodies can be detectably
labeled and the lectin can be immobilized on a solid support.
[0018] In another embodiment, wherein prior to the step of
contacting the fecal sample from a mammal with at least one
antibody, the method further comprising the step of contacting the
fecal sample with one or more lectins. The lectin can be detectably
labeled or immobilized onto a solid support. Alternatively, the
first, second and third antibodies can be detectably labeled or
immobilized on a solid support. In one embodiment the first, second
and third antibodies can be immobilized on a solid support and the
lectin can be detectably labeled. In another embodiment, the first,
second and third antibodies can be detectably labeled and the
lectin can be immobilized on a solid support.
[0019] In a further aspect, the invention provides a method of
diagnosing and treating a mammal infected with one or more
parasitic worms, the method comprising the steps of: (a) contacting
a sample from a mammal with at least one antibody selected from the
group consisting of: (i(i) a first antibody capable of specifically
binding coproantigen from a first tapeworm, but not coproantigen
from a second tapeworm or coproantigen from a third tapeworm; (ii)
a second antibody capable of specifically binding the coproantigen
from the second tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the third tapeworm; and (iii) a
third antibody capable of specifically binding the coproantigen
from the third tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the second tapeworm; (b) forming
antibody-coproantigen complexes in the presence of the
coproantigens, if any, in the sample; (c) detecting the presence or
absence of the antibody-coproantigen complexes, if any; and (d)
diagnosing the mammal as having: (i) a first tapeworm infection if
a first tapeworm antibody-coproantigen complex is present; (ii) a
second tapeworm infection if a second tapeworm
antibody-coproantigen complex is present; and (iii) a third
tapeworm infection if a third tapeworm antibody-coproantigen
complex is present; and (e) administering an effective amount of
one or more therapeutic agents to treat the mammal having the
first, second, or their tapeworm infection or combination thereof.
In one embodiment, two or more antibodies are selected. In other
embodiments, step (e) further includes one or more additional
therapeutic agents to treat infection by one or more helminthic
worm parasites, one or more non-worm parasites, one or more
viruses, one or more fungi, one or more protozoa, or one or more
bacteria. In other embodiments, step (e) further includes one or
more therapeutic agents to control, repel or kill an intermediate
host of a platyhelminthic worm parasite, helminthic worm parasite,
non-worm parasite, virus, fungus, or bacterium. Representative
examples of intermediate hosts include fleas and canine chewing
lice which can carry tapeworm eggs.
[0020] In further embodiments of the above methods, step (a) group
further consists of:: (i) an antibody capable of specifically
binding a roundworm coproantigen, but not a coproantigen derived
from the group consisting of whipworm, hookworm, tapeworm, Giardia
and parvovirus; (ii) an antibody capable of specifically binding a
whipworm coproantigen, but not a coproantigen derived from the
group consisting of roundworm, hookworm, tapeworm, Giardia and
parvovirus; (iii) an antibody capable of specifically binding a
hookworm coproantigen, but not a coproantigen derived from the
group consisting of whipworm, roundworm, tapeworm, Giardia and
parvovirus; (iv) an antibody capable of specifically binding
Giardia coproantigen, but not coproantigen selected from the group
consisting of roundworm coproantigen, whipworm coproantigen,
hookworm coproantigen, tapeworm Taenia coproantigen, tapeworm
Dipylidium coproantigen, and parvovirus coproantigen; and (v) an
antibody capable of specifically binding parvovirus coproantigen,
but not coproantigen selected from the group consisting of
roundworm coproantigen, whipworm coproantigen, hookworm
coproantigen, tapeworm Taenia coproantigen, tapeworm Dipylidium
coproantigen, and Giardia coproantigen. In some embodiments, the
hookworm is Anyclostoma, the roundworm is Toxocara and the whipworm
is Trichuris. In other embodiments, the hookworm is Ancylostoma
caninum or Ancylostoma tubaeforme; the roundworm is Toxocara canis
or Toxocara cati; the whipworm is Trichuris vulpis or Trichuris
felis; the Giardia is Giardia lamblia, and the parvovirus is feline
parvovirus or canine parvovirus. Thus, the fecal sample is
contacted with at two or more antibodies, three or more antibodies,
four or more antibodies, five or more antibodies, six or more
antibodies, seven or more antibodies or eight or more antibodies to
allow for multiplexing.
[0021] In further embodiments of the above methods, step (d)
diagnosing further comprises: a roundworm infection if a roundworm
antibody-coproantigen complex is present; a whipworm infection if a
whipworm antibody-coproantigen complex is present; a hookworm
infection if a hookworm antibody-coproantigen complex is present; a
Giardia infection if a Giardia antibody-coproantigen complex is
present; and a parvovirus infection if a parvovirus
antibody-coproantigen complex is present.
[0022] In some embodiments, the step of detecting the presence or
absence of the complexes further includes the step of providing one
or more lectins that binds to at least one of the complexes. In
some embodiments, the lectin can be detectably labeled or
immobilized onto a solid support. In other embodiments, the first,
second and third antibodies are detectably labeled or immobilized
on a solid support. In some embodiments, the first, second and
third antibodies can be immobilized on a solid support and the
lectin can be detectably labeled. In other embodiments, first,
second and third antibodies can be detectably labeled and the
lectin can be immobilized on a solid support.
[0023] The method may also be used to test for and distinguish
between environmental contamination with tapeworm. Environmental
samples that may be tested for tapeworms by the device include, but
are not limited to soil, decomposing material, or fecal matter from
residential settings including yards, gardens, sand boxes,
playgrounds. Testing locations may also include parks, beaches,
forests, farms, or other locations exposed to fecal material from
dogs, cats, or other mammalian hosts of tapeworms. Feces from
indoor and outdoor litter boxes may also be tested.
[0024] In yet another aspect, the present invention includes a kit
for carrying out one or more steps of the method of the invention.
The kit may optionally include, for example, the device and one or
more of the compositions of the present invention and instructions
for carrying out the method of the present invention. The kit may
further optionally include, for example, one or more indicator
reagents, one or more antibody labeling compounds, one or more
antibodies, one or more antigen capture reagents, one or more
inhibitors, and one or more wash reagents to be used as part of the
device and/or to be used in carrying out the method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the OD determinations of antibody ADX131
against worm extract and fecal extract from canines that are
negative or positive for tapeworm infection by following the method
of the present invention as shown in Example 1A. Positive=Fecal
extract (FEX) from T. pisiformis infected dog; Negative=FEX from
dog not infected with T. pisiformis; Worm Extract=T. pisiformis
worm extract (WE).
[0026] FIG. 2 shows the OD determinations of antibody ADX132
against worm extract and fecal extract from canines that are
negative or positive for tapeworm infection by following the method
of the present invention as shown in Example 1A. Positive=Fecal
extract (FEX) from T. pisiformis infected dog; Negative=FEX from
dog not infected with T. pisiformis; Worm Extract =T. pisiformis
worm extract (WE).
[0027] FIG. 3 shows a microtiter plate in which an ELISA assay was
carried out using fecal extracts of canines infected with tapeworm
by following the method of the present invention as shown in
Example 1B. H11 is a negative control. A5, B6, B8, E6 and E10 are
fecal extracts from five T. pisiformis positive dogs. Each of the
other wells has a fecal extract from one of the hookworm
Ancylostoma caninum infected dogs, or the roundworm Toxocara canis
infected dogs, or the whipworm Trichuris vulpis infected dogs, or
the D. caninum infected dogs.
[0028] FIG. 4 shows the specificity of a sandwich coproantigen
ELISA assay using antibody ADX131 coated onto a microtiter plate,
and ADX132-HRP conjugate applied after addition of the patient
sample. The assay was run on D. caninum WE, T. pisiformis WE, T.
crassiceps WE, T. taeniaeformis WE, T. taeniaeformis E/S, as well
as the FEX from a D. caninum positive dog, a D. caninum negative
dog, a D. caninum positive cat, a D. caninum negative cat, a T.
pisiformis positive dog, a T. pisiformis negative dog, a pool of
three T. taeniaeformis positive felines, a pool of three T.
taeniaeformis negative felines, hookworm Ancylostoma caninum WE,
roundworm Toxocara canis WE, and whipworm Trichuris vulpis WE as
discussed in Example 1B.
[0029] FIG. 5 shows the sensitivity of a sandwich coproantigen
ELISA assay using antibody ADX185 coated onto a microtiter plate,
followed by addition of the patient sample, then followed by
addition of T. taeniaeformis WE rabbit pAb-HRP conjugate. The assay
was run on FEX from 7 T. taeniaeformis positive felines, and 3 T.
taeniaeformis negative felines as discussed in Example 2B (part
B).
[0030] FIG. 6 shows the specificity of a sandwich coproantigen
ELISA assay using antibody ADX184 coated onto a microtiter plate,
and ADX193-HRP conjugate applied after addition of the patient
sample. ADX193 is a T. taeniaeformis E/S mouse mAb, described
below. The assay was run on D. caninum WE, T. pisiformis WE, T.
crassiceps WE, T. taeniaeformis WE, T. taeniaeformis E/S, as well
as the fecal extracts from a D. caninum positive dog, a D. caninum
negative dog, a D. caninum positive cat, a D. caninum negative cat,
a T. pisiformis positive dog, a Taenia pisiformis negative dog, a
pool of three T. taeniaeformis positive felines, and a pool of
three T. taeniaeformis negative felines as discussed in Example 2B
(part B).
[0031] FIG. 7 shows the results of an ADX184/ADX193-HRP ELISA assay
run on fecal extracts from six T. taeniaeformis positive cats,
fecal extracts from four T. taeniaeformis negative cats, and one T.
taeniaeformis E/S protein sample as discussed in Example 2C (part
A).
[0032] FIG. 8 shows the results of a coproantigen ELISA assay using
antibody ADX191 coated onto microtiter plates and ADX194 conjugated
with HRP. Both mouse mAbs were used at a concentration of 3 ug/ml.
This ELISA was run on fecal extracts from six T. taeniaeformis
positive cats, fecal extracts from four T. taeniaeformis negative
cats, and one T. taeniaeformis E/S protein sample as discussed in
Example 2D.
[0033] FIG. 9 shows the results of an ELISA assay using D. caninum
WE rabbit pAb coated onto plates, contacted with FEX at 5 .mu.g/ml,
then contacted with D. caninum WE rabbit pAb-HRP conjugate at 3
ug/ml, then contacted with a color substrate. The D. caninum WE
rabbit pAb ELISA assay was run on worm extracts from several
helminth species: D. caninum WE, Taenia pisiformis WE, T.
crassiceps WE, hookworm Ancylostoma caninum WE, roundworm Toxocara
canis WE, whipworm Trichuris vulpis WE as discussed in Example 3B
(part A).
[0034] FIG. 10 shows the results of a coproantigen ELISA assay
where D. caninum WE mouse pAb was coated onto plates, contacted
with FEX, then contacted with D. caninum WE mouse pAb-HRP
conjugate, then contacted with a color substrate as discussed in
Example 3E (part A). The assay was run on fecal extracts from 4 D.
caninum positive dogs and 3 D. caninum positive cats; one D.
caninum negative cat infected with Giardia and T. taeniaeformis,
one D. caninum negative cat infected with T. taeniaeformis, and one
D. caninum negative dog that was infected with Toxocara canis and
Taenia pisiformis as discussed in Example 3E (part A).
[0035] FIG. 11 shows the results of a first coproantigen ELISA
assay which used mouse mAb ADX226 coated onto plates, contacted
with FEX, then contacted with mouse mAb ADX251-HRP conjugate
followed by color substrate. The assay was run on fecal extracts
from 3 D. caninum positive dogs, 5 D. caninum negative dogs; 3 D.
caninum positive cats, and 1D. caninum negative cat as discussed in
Example 3F (part A).
[0036] FIG. 12 shows the results of a second coproantigen ELISA
assay which used mouse mAb ADX251 was coated onto plates, contacted
with FEX, then contacted with mouse mAb ADX227-HRP conjugate,
followed by color substrate. The assay was run on fecal extracts
from 3 D. caninum positive dogs, 5 D. caninum negative dogs; 3 D.
caninum positive cats, and 1 D. caninum negative cat as discussed
in Example 3F (part A).
[0037] FIG. 13 shows the results of a third coproantigen ELISA
assay which used mouse mAb ADX251 coated onto plates, contacted
with FEX, then contacted with D. caninum_WE rabbit pAb-HRP
conjugate, followed by color substrate. The assay was run on fecal
extracts from 3 D. caninum_positive dogs, 5 D. caninum_negative
dogs; 3 D. caninum_positive cats, and 1 D. caninum_negative cat as
discussed in Example 3F (part A).
[0038] FIG. 14 shows the results of a first set of coproantigen
ELISA assay configurations where D. caninum WE mouse mAbs ADX224,
ADX225, ADX226 and ADX227 were coated individually onto plates,
contacted with FEX, then contacted with D. caninum WE rabbit
pAb-HRP conjugate followed by color substrate as discussed in
Example 3G (part A).
[0039] FIG. 15 shows the results of a second set of coproantigen
ELISA assay configurations, where D. caninum WE mouse mAbs ADX226
and ADX227 were coated individually onto plates, contacted with
FEX, then contacted with D. caninum WE mouse mAb-HRP conjugate
ADX227-HRP followed by color substrate as discussed in Example 3G
(part A).
[0040] FIG. 16 shows the results of determining whether the
coproantigen bound by D. caninum WE mouse mAb ADX226 is
glycosylated by testing the ability of 21 different lectins to bind
the coproantigen using a commercial kit (Biotinylated lectin kits
I, II and III from Vector Laboratories, Burlingame, Calif.) as
discussed in Example 3H (part 1).
[0041] FIG. 17 shows the results of determining whether the
coproantigen bound by the antibody D. caninum WE rabbit pAb is
glycosylated by testing 21 different lectins for their ability to
bind the coproantigen using a commercial kit (Biotinylated lectin
kits I, II and III from Vector Laboratories, Burlingame, Calif.) as
discussed in Example 3H (part 2).
[0042] FIG. 18 shows the results of determining whether the
coproantigen(s) bound by the D. caninum WE rabbit pAb and ADX187 (a
D. caninum WE mouse mAb) are glycosylated by testing 21 different
lectins for their ability to bind the coproantigen using a
commercial kit (Biotinylated lectin kits I, II and III from Vector
Laboratories, Burlingame, Calif.) as discussed Example 3H (part
3).
[0043] FIG. 19 shows microtiter plates in which a series of T.p.,
T. taeniaeformis, D.c., hookworm, roundworm, whipworm and Giardia
ELISA assays were carried out using fecal extracts of a variety of
infected canines and felines as discussed in Example 4. Fecal
extracts from the following sources were tested in the ELISA
assays: T. pisiformis positive dog (FIG. 19, column 1), T.
taeniaeformis infected cat (FIG. 19, column 2), D. caninum infected
dog (FIG. 19, column 3), D. caninum infected cat (FIG. 19, column
4), hookworm A. caninum infected dog (FIG. 19, column 5), hookworm
A. tubaeforme infected cat (FIG. 19, column 6), roundworm T. canis
infected dog (FIG. 19, column 7), roundworm T. cati infected cat
(FIG. 19, column 8), whipworm T. vulpis infected dog (FIG. 19,
column 9), whipworm T. felis infected cat (FIG. 19, column 10),
Giardia infected dog (FIG. 19, column 11), Giardia infected cat
(FIG. 19, column 12), and two parvovirus infected cats (FIG. 19,
columns 13 and 14). In FIG. 19, columns 1 through 12 are an image
of a single microtiter plate, and columns 13 and 14 are an image of
another, separate microtiter plate.
[0044] FIG. 20 shows a microtiter plate in which a series of ELISA
assays capable of detecting two or three tapeworm species from the
genus Taenia, for example T. pisiformis, T. taeniaeformis, and the
genus Dipylidium, for example D. caninum were carried out using
fecal extracts from a variety of infected canines and felines as
discussed in Example 5. Fecal extracts from the following sources
were tested in the ELISA assays: Four T. pisiformis positive dogs
(FIG. 20, columns 1, 4, 7 and 10), four T. taeniaeformis infected
cats (FIG. 20, column 2, 5, 8 and 11), two D. caninum infected dogs
(FIG. 20, column 3 and 6), two D. caninum infected cats (FIG. 20,
column 9 and 12). In FIG. 20, columns 1 through 12 are an image of
a single microtiter plate.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
I. Introduction
[0045] The present invention is generally directed to methods,
devices, and kits for detecting and distinguishing between tapeworm
species in a fecal sample obtained from a mammal. The present
invention relates to tapeworm coproantigens from a variety of
tapeworm species including Taenia pisiformis, Taenia taeniaeformis,
and Dipylidium caninum. In particular, the present invention
relates methods, devices and kits for detecting tapeworm infection
and distinguishing between two or more tapeworm species.
[0046] The present invention provides a superior alternative to the
existing microscopic inspection techniques. This is true because
the present invention provides devices, kits and methods for
detecting the presence or absence of tapeworm in a sample from a
mammal that: (1) are both easy to use and yield consistently
reliable results; (2) allow for the absence or presence of a
specific tapeworm species in a mammal to be confirmed regardless of
whether that mammal is infected with one or more tapeworm species
and/or other helminthic worm parasites such as hookworm, roundworm,
whipworm and/or heartworm; (3) can detect tapeworm prior to the
time that the ova and protoglottids first appear in the infected
host's feces; and (4) can distinguish between different tapeworm
species as well as helminthic worm parasites such as roundworm,
whipworm and hookworm infections.
[0047] The present invention is based in part on the discovery of
unexpected properties of compositions specific to tapeworm
infections. Specifically, it was determined that antibodies raised
against tapeworm specific polypeptides (or raised against an
extract of whole tapeworm, E/S material of the tapeworm, Worm Wash
of the tapeworm, or TCA soluble material of the tapeworm) can be
used to capture, detect, and distinguish between tapeworm antigens
from different species in a mammal. The specificity for each type
of tapeworm species is surprising because tapeworms are related
cesotodes, and an antibody raised against a protein isolated from
any one of these tapeworm species would be expected to crossreact
with one or more of the other tapeworms species, host antigens, or
other host components.
[0048] It was further determined that this antibody can be used to
capture and detect tapeworm antigens in a mammal before eggs and
protglottids are visible in feces. This ability to detect tapeworm
so soon after infection, and before the appearance of any ova in
the feces of the infected mammal, is surprising because ova and
protglottides generally do not appear in the feces of an infective
host until weeks after the host becomes infected.
[0049] The present invention therefore includes methods, devices,
and kits that use antibodies and/or fragments thereof to
specifically capture and detect and distinguish between antigen of
different tapeworm species in a mammal. The ability of the present
invention to detect and diagnose a specific tapeworm species even
when one or more other tapeworm species or different worm types are
also present allows the mammal's caregiver the opportunity to
optimally select a treatment for ridding the tapeworm as well as
other worms such as roundworm, whipworm and/or hookworm from the
mammal. Further, the ability of the present invention to, in some
cases, detect tapeworm before eggs and protglottids appear in feces
provides the possibility that the caregiver may begin such
treatment before the mammal becomes severely sickened. An
intervention prior to appearance of ova and protglottids in the
feces would also greatly reduce or eliminate the possibility that
the infestation is spread to other animals or humans.
II. Definitions and Uses of Term
[0050] The term "compositions of the invention" refers to all of
the nucleic acids, polypeptides, glycoproteins, carbohydrates,
glycolipids, antibodies, and mixtures that include one or more of
those nucleic acids, polypeptides, glycoproteins, carbohydrates,
glycolipids, and antibodies and one or more other compounds, that
can be used to detect the presence or absence of tapeworm in a
sample obtained from a mammal by carrying out the method of the
present invention that are explicitly described, implicitly
encompassed or otherwise disclosed herein.
[0051] "A sample from a mammal" in which tapeworm can be detected
by the present invention includes all bodily components and
extracts thereof, such as any fluid, solid, cell or tissue, that
are capable of tapeworm antigen. Exemplary samples therefore
include, but are not limited to being, feces, milk, whole blood and
portions thereof, including serum, and further include tissue
extracts, including tissue from mammary gland, intestine, liver,
heart, lung, esophagus, brain, muscle, and eye, for example. The
sample may be taken directly from the mammal or the sample may be
taken from anything that has contacted the mammal. For example, the
sample may be fresh or decaying fecal droppings from the mammal. As
another example, the sample may include soil, dirt, sand, plant
material, or any other material that may be mixed with bodily
components that may be left behind by a mammal, such as feces, for
example. As such, the sample may be taken from an environmental
source, including soil, decomposing material, or fecal matter from
forests, farms, or residential settings, including litter boxes,
yards, gardens, sand boxes, playgrounds, parks, and beaches. No
matter the origin or the content of the sample, this sample
sometimes is referred to herein as the "sample", the "mammalian
sample", the "test sample" or the "sample under test".
[0052] As used herein, "nucleic acid" is synonymous with, and
therefore is used interchangeably with, "gene", "DNA", "cDNA",
"EST", "polynucleotide", "oligonucleotide", "polynucleic acid",
"RNA" and "mRNA". A nucleic acid may be in double-stranded form or
it may be in single-stranded form. Further, a nucleic acid is
either naturally isolated, such as from a whole tapeworm or a
portion thereof, for example, or it is artificially synthesized,
either in a recombinant host organism or by any other artificial
means known to the skilled artisan, such as by employing a
PCR-based technique, by creating a transgenic organism that
synthesizes the nucleic acid, by using a DNA synthesizing machine,
or by any another molecular-based technique, for example.
[0053] "Polypeptide", "peptide" and "protein" are synonymous terms
that are used interchangeably herein to refer to a polymer of amino
acid residues. A polypeptide, peptide and protein of the present
invention may be either naturally isolated, such as from a whole
tapeworm or from a portion of tapeworm for example, or artificially
synthesized, either in a recombinant host organism or by any other
artificial means known to the skilled artisan. A polypeptide,
peptide and protein of the present invention may be
glycosylated.
[0054] The term "antibody" or "antibody of the present invention"
refers to any antibody that is able to specifically bind to one or
more antigens for the particular worm without binding to antigens
from the other worms. For example antibodies to the one tapeworm
species are able to specifically bind to the antigens of the
tapeworm species, but not to any antigens from different tapeworm
species. The antibodies of the present invention may be raised
against one or more immunogenic polypeptides, glycoproteins,
carbohydrates, or glycolipids of the present invention. Unless
otherwise stated, it is to be understood that the antibody of the
present invention may include a mixture of two or more different
types of antibody. For example, the antibody may be a mixture of
two types of antibodies, wherein one of the two types specifically
binds to one particular antigen and the other of the two types
specifically binds to some other antigen.
[0055] The term "first antibody" as used herein means one or more
antibodies capable of specifically binding a coproantigen from a
first tapeworm species, but not coproantigen from a second tapeworm
species or coproantigen from a third tapeworm species.
[0056] The term "second antibody" as used herein means one or more
antibodies capable of specifically binding the coproantigen from
the second tapeworm species, but not the coproantigen from the
first tapeworm species or the coproantigen from the third tapeworm
species.
[0057] The term "third antibody" as used herein means one or more
antibodies capable of specifically binding the coproantigen from
the third tapeworm species, but not coproantigen from the first
tapeworm species or the coproantigen from the third tapeworm
species.
[0058] The "immunogenic polypeptide of the present invention" and,
more simply, "the polypeptide of the present invention", is an
immunogen against which the antibodies of the present invention may
be raised. All "polypeptides of the present invention" are
immunogenic and therefore may be used to elicit an immune response
in a host animal to produce the antibodies of the present
invention. Unless otherwise stated, it is to be understood that the
polypeptide of the present invention may be one component of a
mixed composition of a plurality of components.
[0059] An "immunogen" is any agent, such as the immunogenic
extract, polypeptide, glycoprotein, carbohydrate, or glycolipid of
the present invention, for example, that is capable of eliciting an
immune response in an animal that is exposed to that agent.
[0060] The term "tapeworm", as used herein, refers to
platyhelminths worm parasites such as intestinal tapeworms which
includes the genera Taenia and Dipylidium. Thus, the term
"tapeworm", as used herein, does not refer to the entirety of the
class cestoda, but rather to the subclass eucestoda, including the
order pseudophyllidea. Representative examples of tapeworm include
Taenia pisiformis, Taenia taeniaeformis, Taenia crassiceps,
Dipylidium caninum, Diphyllobothrium mansonoide, Diphyllobothrium
latum, and Spirometra erinaceieuropaei.
[0061] A "tapeworm coproantigen" or a "coproantigen from tapeworm"
is any tapeworm product that is present in the feces of a mammal
having a tapeworm infection and that may be specifically bound by
one or more of the antibodies of the invention. For example, a
tapeworm coproantigen may be, but is not limited to being, one or
more of the polypeptides of the invention.
[0062] The term "roundworm", as used herein, refers to helminths
such as intestinal roundworms of the order Ascaridida, which
includes the genera Toxocara, Toxascaris, Baylisascaris, Ascaridia,
Parascaris, Ascaris, Anisakis, and Pseudoterranova. Exemplary
roundworms therefore include Toxocara canis, Toxocara cati and
Toxascaris leonina. Thus, the term "roundworm", as used herein,
does not refer to the entirety of the phylum Nematoda. Therefore,
"roundworm" does not include any member of the genera Ancylostoma,
Uncinaria, Necator, Trichuris, Wuchereria, Brugia or
Dirofilaria.
[0063] A "roundworm coproantigen" or a "coproantigen from
roundworm" is any roundworm product that is present in the feces of
a mammal having a roundworm infection and that may be specifically
bound by one or more of the antibodies of the invention. For
example, a roundworm coproantigen may be, but is not limited to
being, a novel C-terminal 7 kD isoform of DIV6728, which is a
excretory/secretory protein of T. canis, is present in feces of T.
canis-infected canines as early as 38 days after the canines first
became infected with the T. canis. Therefore, a "roundworm
coproantigen" may be this novel C-terminal 7 kD isoform of DIV6728
(which is referred to herein as "Copro6728") that has been observed
in canine feces as discussed in U.S. Pat. No. 7,951,547.
[0064] The term "whipworm", as used herein, refers to helminths
such as intestinal whipworms of the genera Trichuris and
Trichocephalus. Exemplary whipworms therefore include Trichuris
vulpis, Trichuris campanula, Trichuris serrata, Trichuris fells,
Trichuris suis, Trichuris trichiura, Trichuris discolor and
Trichocephalus trichiuris. Further, the term "whipworm", as used
herein, does not refer to the entirety of the phylum Nematoda. For
example, "whipworm" does not include any member of the genera
Ancylostoma, Uncinaria, Necator, Toxocara, Toxascaris, Ascaris,
Wuchereria, Brugia or Dirofilaria.
[0065] A "whipworm coproantigen" or a "coproantigen from whipworm"
is any whipworm product that is present in the feces of a mammal
having a whipworm infection and that may be specifically bound by
one or more of the antibodies of the invention. For example, a
whipworm coproantigen may be, but is not limited to being,
"DIV6901" or "DIV6902, hereinafter, this particular antibody is
referred to as "anti-DIV6901" or "anti-DIV6902" as discussed in
U.S. Pat. No. 7,951,547.
[0066] The term "hookworm," as used herein, refers to helminthes
such as intestinal hookworm of the genera Ancylostoma, Necator and
Uncinaria. Exemplary hookworms therefore include Ancylostoma
caninum, Ancylostoma braziliense, Ancylostoma duodenal, Ancylostoma
ceylanicum, Ancylostoma tubaeforme and Ancylostoma pluridentatum,
Necator americanus, and Uncinaria stenocephala. Further, the term
"hookworm," as used herein, does not refer to the entirety of the
phylum Nematoda. For example, "hookworm" does not include any
member of the genera Trichuris, Trichocephalus Toxocara,
Toxascaris, Ascaris, Wuchereria, Brugia or Dirofilaria.
[0067] A "hookworm coproantigen" or a "coproantigen from hookworm"
is any hookworm product that is present in the feces of a mammal
having a hookworm infection and that may be specifically bound by
one or more of the antibodies of the invention. For example, a
hookworm coproantigen may be, but is not limited to being, a novel
N-terminal 28 kDa isoform of ASP5, which is a excretory/secretory
protein of Ancylostoma, present in feces of Ancylostoma-infected
canines as early as 9 days after the canines first became infected
with the Ancylostoma. Therefore, a "hookworm coproantigen" may be
this novel N-terminal 28 kDa isoform of ASP5 (which is referred to
herein as "CoproASP5") that has been observed in canine feces as
discussed in U.S. Pat. No. 7,951,547.
[0068] The term "Giardia", as referred herein, refers to protozoans
of the genus Giardia. Exemplary Giardia species therefore include
Giardia lamblia, also known asGiardia intestinalis.
[0069] A "Giardia coproantigen" or "coproantigen of Giardia" is any
Giardia product that is present in the feces of a mammal having a
Giardia infection and that may be specifically bound by one or more
of the antibodies of the invention.
[0070] "Lectins" are proteins that recognize and bind specific
monosaccharide or oligosaccharide structures (carbohydrates). A
lectin usually contains two or more binding sites for carbohydrate
units. The carbohydrate-binding specificity of a certain lectin is
determined by the amino acid residues that bind the carbohydrate.
The binding strength of lectins to carbohydrates can increase with
the number of molecular interactions. The dissociation constant for
binding of lectins to carbohydrates is about K.sub.d of 10.sup.-5
to 10.sup.-7. Lectins can be labeled with any type of label known
in the art, including, for example, fluorescent, chemiluminescent,
radioactive, enzyme, colloidal metal, radioisotope and
bioluminescent labels.
[0071] In embodiments of the invention, lectins used are those that
specifically bind tapeworm coproantigen. In embodiments of the
invention lectins that specifically bind O-glycosylated proteins
are useful in the invention. Such lectins include, for example, ECL
lectin (Erythina cristagalli), GSL I (Griffonia Simplicifolia
Lectin I), GSL II (Griffonia Simplicifolia Lectin II), jacalin, LCA
lectin (Lens cuhnaris), RCA 123 (Ricinus Communis), and PSA lectin
(Phaseolus vulgaris leucoagglutinin), WGA (wheat germ agglutinin)
and sWGA (succinylated wheat germ agglutinin). Lectins are
commercially available from, e.g., Vector Laboratories, Burlingame,
Calif., USA.
[0072] Lectins can be used that specifically bind to carbohydrates
on human, canine, feline, equine, bovine, ovine, or simian tapeworm
antigens.
[0073] "Specific for", "specifically binds", and "stably binds"
means that a particular composition of the invention, such as an
antibody, polypeptide, or oligonucleotide of the present invention,
for example, recognizes and binds to one or more other agents with
greater affinity than to at least one other agent. As one example,
an antibody of the present invention is said to be "specific for",
to "specifically bind", and to "stably bind" tapeworm antigens
whenever that antibody is able to recognize and bind to those
roundworm antigens with greater affinity than to any other antigens
from a non-tapeworm parasitic worm. Such binding specificity can be
tested using methodology well known in the art, for example, ELISA
or a radioimmunoassay (MA). Based on information observed regarding
the binding specificity of a particular composition of the
invention, the method of the present invention can be carried out
under conditions that allow that composition to bind to (and
therefore to allow the detection of such binding to) a particular
agent or agents, but not to significantly bind other agents, while
those conditions are maintained. As one example, the method of the
present invention can be carried out under conditions that allow an
antibody of the present invention to bind to (and therefore to
allow the detection of such binding to) one or more antigens of a
species of tapeworm antigens present in a particular sample, but
not significantly to any antigen from other tapeworm species or
other helminthic worm species may be present in that sample,
thereby allowing for the distinction between species of tapeworms
and roundworm, whipworm and hookworm.
[0074] "Detecting tapeworm" means detecting one or more
tapeworm-specific products, including one or more of the
polypeptides, antibodies and nucleic acids of the present
invention, or one or more tapeworm antigens, for example. The
presence of one or more such tapeworm products in a sample from a
mammal is indicative that the mammal has a tapeworm infection,
regardless of whether any whole tapeworm organism or ovum thereof
is also present in that sample. Conversely, the absence of one or
more such tapeworm products a sample from a mammal is indicative
that the mammal does not have a tapeworm infection.
[0075] "Treatment" means the administration of a therapeutic agent
to a patient by any suitable administration route to reduce or
eliminate parasitic worms (helminths) or other internal parasites
from the body either by controlling, stunning or killing them
and/or that reduces or eliminates infestation by intermediate hosts
such as insects, e.g.fleas, which can transmit infection with
parasitic worms or other internal parasites, without causing
significant damage to the patient.
III. Antibodies of the Invention
[0076] The present invention further includes antibodies and
antigen-binding fragments thereof that are raised against and that
specifically bind all or part of one or more polypeptides of the
present invention, and also includes compositions that include said
antibodies and antigen-binding fragments thereof. When contacted to
a sample obtained from a mammal, these antibodies and
antigen-binding fragments are able to specifically bind to a
particular helminthic worm antigen. For example the tapeworm
antibodies and antigen-binding fragments are able to specifically
bind tapeworm antigens present in the sample, but are not able to
specifically bind any antigen from other worms such as roundworm,
hookworm or whipworm that may be present in the sample. The
antibodies of the present invention are suitable for being used
only to capture one or more tapeworm antigens, only to detect one
or more tapeworm antigens, or more preferably, to both capture and
detect one or more tapeworm antigens.
[0077] The antibodies of the present invention may belong to any
antibody class, including for example, IgG, IgM, IgA, IgD and IgE,
and may be prepared by any of a variety of techniques known to the
skilled artisan. (See, e.g., Dean, Methods Mol. Biol. 80:23-37
(1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg, Methods
Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol. 32:389-99
(1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993);
Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al. Crit.
Rev. Immunol. 12:125-68 (1992); Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory (1988); and Making
and Using Antibodies: A Practical Handbook, Howard and Kaser, eds.,
CRC Press (2006), each one of which is incorporated herein by
reference in its entirety.)
[0078] In one technique, the polypeptide of the invention is
introduced into a host animal, such as into rabbit, mouse, rat,
guinea pig, goat, pig, cow, sheep, donkey, dog, cat, chicken, or
horse, for example. An enhanced immune response may be elicited in
the host animal by associating the polypeptide with a carrier
and/or by exposing the host to an adjuvant, but it is to be
understood that the present invention does not require that the
polypeptide be associated with a carrier or that the host be
exposed to the adjuvant. An exemplary carrier that may be used for
this purpose is bovine serum albumin, bovine thyroglobulin, and
soybean trypsin inhibitor. Exemplary adjuvants include Freund's
complete or incomplete adjuvant and MDL-TDM adjuvant. Regardless of
whether the polypeptide is associated with such a carrier or
whether the host is exposed to an adjuvant, booster immunizations
optionally may be made with the host animal being bled one or more
times thereafter. Polyclonal antibodies (pAbs) that specifically
bind the polypeptide may then be purified from antisera obtained
from the bleed or bleeds. Such purification may be achieved, for
example, by employing affinity chromatography techniques that
involve associating the polypeptide to a solid support. Such
affinity chromatography techniques are well known by the skilled
artisan.
[0079] In several embodiments, the tapeworm antibody of the present
invention is an antibody that is raised in rabbit by immunizing
that host animal with extract of whole tapeworm, E/S material of
the tapeworm, Worm Wash of the tapeworm, or TCA soluble material of
the tapeworm, as described below.
[0080] It is also to be understood that the antibodies of the
invention optionally may be polyclonal or monoclonal antibodies
(mAbs), single chain antibodies (scFv), chimeric antibodies, and
fragments thereof. Monoclonal antibodies that are specific for the
polypeptide of interest may be obtained and purified, for example,
by preparing cell lines that generate antibodies having the desired
specificity to the polypeptide of interest. Cell lines of this kind
may be derived from cells of a particular type (e.g., spleen cells)
that are isolated from a host animal that had previously been
immunized with the polypeptide as described before. In such a case,
these cells could then be immortalized, for example, by fusing them
with myeloma cells by carrying out any one of a variety of fusion
techniques known to the skilled artisan. In one exemplary
technique, the cells from the immunized host animal are
co-incubated with their fusion partner, e.g., the myeloma cells, in
the presence of a detergent for a short period of time before being
plated on a medium that supports the growth of hybrid cells (but
not the myeloma fusion partner). Such selection may be achieved,
for example, by using hypoxanthine, aminopterin, and thymidine
(HAT). When hybrid cells emerge during selection, in perhaps one or
two weeks after commencing the selection process, single hybrid
colonies (and their supernatants) are tested for their ability to
bind the polypeptide or polypeptides against which the host animal
was immunized. Hybrid colonies having the most optimal binding
specificity would represent the best candidates from which
monoclonal antibodies may be isolated. These monoclonal antibodies,
for example, may be isolated directly from the supernatant (i.e.,
medium) in which these colonies are grown by employing any one of a
variety techniques known to the skilled artisan.
[0081] The antibodies of the invention also may be a single chain
antibody (scFv), or an antigen binding fragment of an antibody.
Antigen-binding fragments of antibodies are a portion of an intact
antibody comprising the antigen binding site or variable region of
an intact antibody, wherein the portion is free of the constant
heavy chain domains of the Fc region of the intact antibody.
Examples of antibody fragments include Fab, Fab', Fab'-SH,
F(ab').sub.2 and F.sub.v fragments. In addition to production and
purification from animals or mammalian cells, antibodies, antibody
fragments, or non-antibody scaffolds can be selected based upon
various in vitro technologies, including phage display, ribosomal
display, or bacterial display.
[0082] Antibodies, including secondary antibodies, may be labeled
with any type of label known in the art, including, for example,
fluorescent, chemiluminescent, radioactive, enzymes, colloidal
particles, radioisotopes and bioluminescent labels. In various
embodiments of the invention, the one or more of the antibodies of
the invention are labeled with an enzyme, a colloidal particle, a
radionuclide or a fluorophor. The particulate label can be, for
example, a colored latex particle, dye sol, or gold sol conjugated
to an antibody.
IV. Methods, Devices and Kits of the Invention
A. Devices and Kits of the Invention
[0083] The present invention, in one aspect, is a detecting the
presence or absence of one or more tapeworm antigens from a sample,
the device comprising a solid support, wherein the solid support
has immobilized thereon one or more antibodies selected from the
group consisting of (a) a first antibody capable of specifically
binding coproantigen from a first tapeworm, but not coproantigen
from a second tapeworm or coproantigen from a third tapeworm; (b) a
second antibody capable of specifically binding the coproantigen
from the second tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the third tapeworm; and (c) a
third antibody capable of specifically binding the coproantigen
from the third tapeworm, but not the coproantigen from the first
tapeworm or the coproantigen from the second tapeworm; and
optionally, (d) one or more types of Giardia coproantigen,
roundworm coproantigen, whipworm coproantigen, and/or hookworm
coproantigen, wherein the one or more types of roundworm
coproantigen, whipworm coproantigen, and hookworm coproantigen are
specifically bound to the antibodies. See U.S. Pat. No. 7,951,547,
which is incorporated by reference in its entirety. The device is
arranged to aid specifically binding and isolating tapeworm
coproantigens from any Giardia, roundworm, whipworm and hookworm
antigen in a sample from a mammal.
[0084] In one aspect, the device includes a solid support, wherein
one or more antibodies of the invention are immobilized on the
solid support. The solid support may be, but is not limited to
being, the inner, bottom surface of a well of a microtiter plate, a
microparticle, the channels of a microfluidic device, a cartridge,
a membrane, or a substrate that is included as part of a lateral
flow device, for example. An exemplary microtiter plate is an
Immulon 1B 96-well plate (which is commercially available from
Thermo Scientific of Milford, Mass.), but it is to be understood
that the skilled artisan will recognize that a large variety of
other microtiter plates that are not the Immulon 1B 96-well plate
allow for the immobilization of antibodies thereon, and therefore
would be suitable for providing the solid support of the present
invention.
[0085] An exemplary lateral flow device is the lateral flow device
that is described in U.S. Pat. No. 5,726,010, which is incorporated
herein by reference in its entirety. The device for performing a
lateral flow assay may be a SNAP.RTM. device, which is commercially
available from IDEXX Laboratories, Inc. of Westbrook, Me. However,
it is to be understood that the skilled artisan will recognize that
a large variety of other lateral flow devices that are not
SNAP.RTM. devices or described by U.S. Pat. No. 5,726,010 allow for
the immobilization of an antibody thereon, and therefore would be
suitable for being used as the device of the present invention.
These devices can include, for example, lateral flow devices that
use colloidal gold technology.
[0086] Antibodies used in the device of the invention may be
immobilized on the solid support by any methodology known in the
art, including, for example, covalently or non-covalently, directly
or indirectly, attaching the antibodies to the solid support.
Therefore, while these antibodies may be attached to the solid
support by physical adsorption (i.e., without the use of chemical
linkers), it is also true that these antibodies may be immobilized
to the solid support by any chemical binding (i.e., with the use of
chemical linkers) method readily known to one of skill in the
art.
[0087] In some embodiments, the first antibody can be raised
against a whole extract of the first tapeworm species, E/S material
of the first tapeworm species, Worm Wash material of the first
tapeworm species, or TCA soluble material of the first tapeworm
species; (b) the second antibody can be raised against a whole
extract of the second tapeworm species, E/S material of the second
tapeworm species, Worm Wash material of the first tapeworm species,
or TCA soluble material of the second tapeworm species; or (c) the
third antibody can be raised against a whole extract of the third
tapeworm species, E/S material of the third tapeworm species, Worm
Wash material of the first tapeworm species, or TCA soluble
material of the third tapeworm species.
[0088] In some embodiments, the solid support further has
immobilized thereon one or more antibodies selected from: an
antibody capable of specifically binding a roundworm coproantigen,
but not a whipworm or hookworm coproantigen; an antibody capable of
specifically binding a whipworm coproantigen, but not a roundworm
or hookworm coproantigen; an antibody capable of specifically
binding a hookworm coproantigen, but not a whipworm or roundworm
coproantigen;an antibody capable of specifically binding Giardia
coproantigen, but not coproantigen selected from the group
consisting of roundworm coproantigen, whipworm coproantigen,
hookworm coproantigen, tapeworm Taenia coproantigen, tapeworm
Dipyhdium coproantigen, and parvovirus coproantigen; and an
antibody capable of specifically binding parvovirus, but not
coproantigen selected from the group consisting of roundworm
coproantigen, whipworm coproantigen, hookworm coproantigen,
tapeworm Taenia coproantigen, tapeworm Dipyhdium coproantigen, and
Giardia coproantigen. In embodiments, the hookworm is Anyclostoma,
the roundworm is Toxocara and the whipworm is Trichuris. In other
embodiments, the hookworm is Ancylostoma caninum or Ancylostoma
tubaeforme; the roundworm is Toxocara canis or Toxocara cati; the
whipworm is Trichuris vulpis or Trichuris felis; the Giardia is
Giardia lamblia, and the parvovirus is feline parvovirus or canine
parvovirus.
[0089] It is also to be understood that the solid support may be
any suitable material for the immobilization of the antibodies of
the invention. For example, the solid support may be beads,
particles, tubes, wells, probes, dipsticks, pipette tips, slides,
fibers, membranes, papers, natural and modified celluloses,
polyacrylamides, agaroses, glass, polypropylene, polyethylene,
polystyrene, dextran, nylon, amylases, plastics, magnetite or any
other suitable material readily known to one of skill in the art.
In some embodiments, the solid support may comprise a plurality of
particles, microparticles, chips or beads. The plurality of
particles, microparticles, chips or beads may be attached to the
device, or be loosely associated with the device. The plurality of
particles, microparticles, chips or beads may be situated on the
surface of the device or within the device. The plurality of
particles, microparticles or beads may be stationary on or within
the device, or they may be able to move within or through the
device.
[0090] The device optionally may include one or more labeled
antigen capture reagents that may be mixed with a sample from a
mammal prior to application to a device of the invention. When the
labeled capture antigen reagent is included, the labeled antigen
capture reagent may or may not be deposited or dried on a solid
surface of the device. "Antigen capture reagent" refers to any
compound that is specific for the antigen or antigens of interest.
The labeled antigen capture reagent, whether added to the mammalian
sample or pre-deposited on the device, may be, for example, a
labeled antibody specific for a roundworm antigen, including, but
not limited to, the antibodies of the present invention.
[0091] The device also may optionally include a liquid reagent that
transports (such as when the device is a SNAP.RTM. device, for
example), or otherwise facilitates removal of (such as when the
device includes a microtiter plate, for example), unbound material
(e.g., unreacted portions of the mammalian sample, such as, for
example, unreacted portions of fecal extract, and unbound antigen
capture reagent) away from the reaction zone (solid phase). The
liquid reagent may be a wash reagent and serve only to remove
unbound material from the reaction zone, or it may include a
detector reagent and serve to both remove unbound material and
facilitate antigen detection. For example, in the case of an
antigen capture reagent conjugated to an enzyme, the detector
reagent includes a substrate that produces a detectable signal upon
reaction with the enzyme-antibody conjugate at the reaction zone
(solid phase). Alternatively, in the case of a labeled antigen
capture reagent conjugated to a radioactive, fluorescent, or
light-absorbing molecule, the liquid reagent acts merely as a wash
solution facilitating detection of complex formation at the
reactive zone by washing away unbound labeled reagent.
[0092] The liquid reagent may further include a limited quantity of
an "inhibitor", i.e., a substance that blocks the development of
the detectable end product. A limited quantity is defined as being
an amount of inhibitor sufficient to block end product development
until most or all excess, unbound material is transported away from
the second region, at which time detectable end product is
produced.
[0093] The device of the present invention may also include various
binding reagents immobilized at locations distinct from the antigen
capture reagent or reagents. For example, an immunoreagent (an
antibody, antigen or polypeptide) that recognizes a
species-specific (e.g., roundworm-specific) antibody portion of a
labeled antibody or antigen capture reagent, or an enzyme portion
of an enzyme-labeled reagent, can be included as a positive control
to assess the viability of the reagents within the device. For
example, a positive control may be an anti-horseradish peroxidase
antibody that has been raised in, for example, goat or mouse.
Additionally, a reagent, e.g., an antibody, isolated from a
non-immune member of the species from which the antibody portion of
the antigen-antibody complex was derived can be included as a
negative control to assess the specificity of immunocomplex (i.e.,
antigen-antibody complex) formation.
[0094] In addition to being designed to specifically binding and
isolating tapeworm coproantigens in a mammalian sample, the device
of the invention optionally may be designed to allow one or more
other diagnostic tests to be performed. For example, the solid
support may also include reagents for the detection of one or more
helminthic worms such as roundworm, whipworm, heartworm and
hookworm, one or more non-worm parasites, one or more viruses (such
as parvovirus), one or more fungi, one or more protozoa (such as
Giardia), or one or more bacteria. The reagents for the detection
of one or more non-worm parasites, one or more viruses, one or more
fungi, one or more protozoa, or one or more bacteria may be, for
example, one or more antibodies or one or more antigens recognized
by antibodies specific for one or more non-worm parasites, one or
more viruses, one or more fungi, one or more protozoa, or one or
more bacteria.
[0095] In one embodiment, the device of the present invention is a
microtiter plate that includes a plurality of wells, wherein each
well includes a solid support having one or more antibodies of the
invention immobilized thereupon.
[0096] The microtiter plate may be used in conjunction with a
method of the present invention to detecting the presence or
absence of one or more helminthic coproantigens in a sample. For
example, a tapeworm infection may be diagnosed in a mammal by
detecting one or more tapeworm antigens with an antibody that is
immobilized on the solid support. In one embodiment, the antigens
that are detected are coproantigens. "Coproantigens" are any
product or products of gastrointestinal parasites such as tapeworms
that are present in a fecal sample from the host species (e.g., a
dog or cat) and that can specifically bind to antibodies.
Coproantigens therefore may be whole worm, worm eggs, worm
fragments, or products secreted, excreted or shed from worm or a
combination thereof.
[0097] In addition to the microtiter plate, there are a large
number of other alternative approaches for performing multiple
immunoassays in parallel (i.e., multiplex immunoassays) which are
known in the art. Typically, multiplex immunoassays can be
performed in a single vessel, in a composition or device where the
components of the multiple immunoassays are in fluid communication.
These technologies may be based on arrays, microarrays, and/or
microbeads or microparticles. In arrays or microarrays, the capture
reagents are typically spotted or otherwise deposited in discreet
areas of a single device, such as a membrane. In bead-based
approaches, capture reagents for each assay are attached to beads,
where the beads of each assay are distinguishable from the beads of
the other assays. The distinguishing may occur by color of light,
physical position, barcode, or the like. See, for instance, Tighe,
P. J., Ryder, R. R., Todd, I. and Fairclough, L. C. (2015), ELISA
in the multiplex era: Potentials and pitfalls. Prot. Clin. Appl.,
9: 406-422. doi:10.1002/prca.201400130).
[0098] A multitude of such multiplexing platforms are commercially
available. Examples include Luminex.RTM. (see, e.g., U.S. Pat. No.
7,523,637); .pi.Code.TM. MicroDiscs (Plexbio, see, e.g., US Pat.
Appl. No. US2014/0274778); Bracoded Magntic Beads and methods and
instruments for running assays therewith (e.g., Applied BioCode,
Inc., Santa Fe Springs, Calif., USA; magnetic barcoded chips (e.g.,
Applied BioCode, Inc.; e.g., U.S. Pat. No. 8,232,092); microfluidic
tests strips (e.g., LumiraDx.RTM., U.S. Pat. No. 9,919,313); and
planar light deck technologies such as LightDeck.RTM. (mBio.RTM.,
U.S. Pat. No. 9,739,714). Additional multiplex immunoassay
technologies include the MULTI-ARRAY (Meso Scale Diagnostics,
Rockville, Md., USA), the Bio-Plex.RTM. Multiplex System (Bio-Rad,
Hercules, Calif., USA), the Access 2 (Beckman Coulter, Atlanta,
Ga.; and the ProcartaPlex.RTM. and ProQuantum.RTM. technologies
(ThermoFisher Scientific, Waltham, Mass., USA) (see Fu Q, Zhu J,
Van Eyk J E. Comparison of multiplex immunoassay platforms. Clin
Chem. 2010 February;56(2):314-8.
doi:10.1373/clinchem.2009.135087).
[0099] In another aspect of the invention, the device for detecting
the presence or absence of one or more tapeworm coproantigens from
a fecal sample comprises a solid support, one or more lectins and
one or more antibodies selected from the group consisting of: (a) a
first antibody capable of specifically binding a coproantigen from
a first tapeworm species, but not a coproantigen from a second
tapeworm species or a coproantigen from a third tapeworm species;
(b) a second antibody capable of specifically binding the
coproantigen from the second tapeworm species, but not the
coproantigen from the first tapeworm or the coproantigen from the
third tapeworm species; and (c) a third antibody capable of
specifically binding the coproantigen from the third tapeworm
species, but not the coproantigen from the first tapeworm species
or the coproantigen from the second tapeworm species. In one
embodiment, the solid support has immobilized thereon one or more
antibodies, two or more antibodies, three or more antibodies, four
or more antibodies, five or more antibodies, six or more
antibodies, seven or more antibodies, or eight or more antibodies.
The labeled lectin can bind to the carbohydrates on the tapeworm
coproantigen captured by the immobilized antibodies for detection
purposes. In another embodiment, the lectin can be immobilized on
the solid support. The immobilized lectin can capture tapeworm
coproantigen, if present, and the resulting complex can be detected
by one or more labeled antibodies. In another embodiment, the
device further comprises one or more species of tapeworm antigen,
wherein the one or more species of tapeworm antigen are
specifically bound to the antibodies.
[0100] The invention further includes assay kits (e.g., articles of
manufacture) for detecting and distinguishing between different
species of tapeworm in a mammalian sample. In some embodiments, the
assay kits can further detect and distinguish co-infection with
helminthic worms such roundworm, whipworm and/or hookworm in the
mammalian sample. A kit therefore may include one or more devices
and/or compositions of the present invention. For example, the kit
may include anti-tapeworm antibodies and means for determining
binding of the antibodies to tapeworm antigens and means for
determining binding of the antibodies to tapeworm antigens. In one
particular example, such a kit includes the device having an
immobilized anti-tapeworm antibody to one or more different species
of tapeworm, one or more antigen capture reagents (e.g., a
non-immobilized labeled antigen capture reagent and an immobilized
antigen capture reagent) and wash reagent, as well as detector
reagent and positive and negative control reagents, if desired or
appropriate. Other components such as buffers, controls, and the
like, known to those of ordinary skill in art, may be included in
such test kits. The relative amounts of the various reagents can be
varied, to provide for concentrations in solution of the reagents
that substantially optimize the sensitivity of the assay.
Particularly, the reagents can be provided as dry powders, usually
lyophilized, which on dissolution will provide for a reagent
solution having the appropriate concentrations for combining with a
sample. The present kit may further include instructions for
carrying out one or more methods of the present invention,
including instructions for using any device and/or composition of
the present invention that is included with the kit.
B. Methods of the Invention
[0101] The present invention further includes methods for using one
or more of the devices, kits and/or compositions of the present
invention to detect the presence or absence of one or more tapeworm
antigens in a sample. The methods therefore may be carried out to
detect the presence or absence of one or more species of tapeworm
in a sample, such as, for example, a fecal sample, that is obtained
from a mammal, including, but not limited to, a canine, feline,
porcine, bovine or human. Further, the methods may be carried out
to detect one or more of helminthic worms such as roundworm,
whipworm, hookworm and heartworm, non-worm parasites such as
Giardia, and viruses such as parvovirus in the sample.
[0102] In the methods of the present invention, detection of one or
more species of tapeworm, may be accomplished by detecting the
presence or absence of one or more tapeworm antigens. When the
sample under test for tapeworm coproantigens is feces, the soluble
portion of the feces may be collected by any protocol known in art.
For example, in addition to the specific protocol described in the
Example section herein, the soluble portions of the sample
generally may be collected by using filtration, extraction,
centrifugation, or simple mixing followed by gravimetric settling.
The skilled artisan will recognize that there are a variety of ways
of extracting and preparing non-fecal samples from a mammal as
well. For example, the sample may be a bodily fluid that is
naturally excreted or otherwise released by the mammal or that is
artificially obtained from the mammal. Such artificial extraction
may be carried out by milking the mammal or by injecting a syringe
into the mammal and drawing the fluid into the syringe. Once
obtained, the fluid optionally may be fractionated (for example,
serum may be fractionated from whole blood as then used as the
sample). As another example, the sample may be obtained by swabbing
the mammal, such as the oral cavity of the mammal, for example. As
yet another example, tissue sections may be obtained by biopsy.
[0103] The methods include contacting the mammalian sample with one
or more antibodies specific for tapeworm coproantigens under
conditions that allow an antigen/antibody complex, i.e., an
immunocomplex, to form. That is, an antibody specifically binds to
a coproantigen present in the sample. The skilled artisan is
familiar with assays and conditions that may be used to detect such
antigen/antibody complex binding. For example, the antigen/antibody
complex may be detected using a secondary antibody that binds to
the antigen/antibody complex. The formation of a complex between
antigen and antibodies in the sample may be detected using any
suitable method known in the art.
[0104] Further, the relative amount of antibody-antigen complexes
that are formed in one particular reaction may be measured with
respect to those formed in any other reaction by any methodology
known in the art for achieving that goal. When it is determined
that a sample under test has a specific tapeworm antibody-antigen
complexes, it can be concluded, based upon the specific complexes
formed, that a specific tapeworm is present in the host mammal and
which tapeworm is present (tapeworm species Taenia pisiformis and
Dipylidium caninum for instance). When this is true, it may be
concluded that the mammal from which the test sample was obtained
harbors an intestinal tapeworm infection. The conclusions that the
mammal being tested harbors an intestinal tapeworm infection may be
made by a clinician at a diagnostic service provider or by a
caregiver of the mammal, such as the mammal's veterinarian, for
example. When a caregiver of a mammal determines (or is otherwise
informed that) a mammal harbors a tapeworm infection and which
tapeworm is present, the caregiver may then subject the mammal to a
course of treatment that is optimally designed to rid the mammal of
the tapeworm specifically, rather than of a parasitic worm
infection generally. Further, the present invention can be used to
confirm that any animal that has received treatment for the
specific tapeworm infection has been rid of that infection. A
caregiver who learns that a sample includes both tapeworm and
roundworm, but not hookworm, for example, could use that knowledge
to treat the mammal from which the sample was taken specifically
for tapeworm by administering to that mammal a drug optimally
effective against tapeworm and a second drug optimally effective
against roundworm. Absent such knowledge, the caregiver may, for
example, otherwise treat the mammal with a drug that is optimally
effective against only tapeworm, only roundworm, or neither
tapeworm nor roundworm (in such cases, the mammal would be at risk
of receiving suboptimal treatment). In addition, humans who may
come in contact with the infested animal or its excretions may be
advised to take precautions against acquiring the parasite or
parasites. In this context, it is important to determine the worm
species with high specificity worms can cause significant disease
(e.g., larval migrans) in humans.
[0105] A patient suffering from infections with intestinal
parasites may be treated with certain therapeutics known to
eliminate the parasites from the patient. Thus, a patient whose
feces is found to contain coproantigen from an intestinal parasite
may be treated with an appropriate therapeutic with the goal of
reducing or eliminating the intestinal parasite.
[0106] Patients suffering from infections with intestinal parasitic
worms such as tapeworms, hookworms, whipworms and/or roundworms can
be treated with de-worming drugs, also known as anthelmintics (or
anthelminthics). Such anthelmintics are widely known to those
skilled in the art. Anthelmintics for the treatment of tapeworms
include, without limitation praziquantel, nitazoxani.de,
albendazole, epsiprantel, fenbendazole, or combinations thereof.
Anthelmintics may be administered by a variety of suitable routes,
including orally, parenterally, such as subcutaneously,
intravenously, intramuscularly or interperitoneally, or topically
(cutaneously), such as directly on to exposed skin surface, to a
patient in the treatment and/or prevention of intestinal
parasites.
[0107] Giardia is a microscopic parasite that causes the diarrheal
illness known as Giardiasis. Giardia, including Giardia
intestinalis, Giardia lamblia and Giardia duodenalis, is found on
surfaces or in soil, food, or water that has been contaminated with
feces from infected humans or animals. Giardia coproantigen can be
detected with a number of commercially available tests, including
VetScan.RTM. Canine Giardia Rapid Test (Abaxis, Union City, USA),
Anigen.RTM. Rapid CPV-CCV-Giardia Antigen Test (BioNote, Seoul,
Korea), SNAP.RTM. Giardia Test (IDEXX, Westbrook, Me., USA) and
Giardia Antigen by ELISA test (IDEXX), ProSpecT.RTM.
Giardia/Cryptosporidium Microplate Assay (Thermo Fisher Scientific,
Waltham, Mass., USA) and Witness.RTM. Giardia Test (Zoetic,
Parsippany, N.J., USA). (Barbecho J M, Bowman D D, Liotta. J L.
Comparative performance of reference laboratory tests and in-clinic
tests for Giardia in canine feces. Parasil Vectors. 2018 Aug.
1;11(1):444. doi: 10.1186/s13071-018-2990-6. PMID: 30068364; PMCID:
PMC6090814.)
[0108] Therapeutics for the treatment of Giardia infections are
widely known to those skilled in the art. Giardia infections may be
treated with a one or more of several drugs including fenbendazole,
albendazole, metronidazole, tinidazole, nitazoxanide, paromomycin,
quinacrine, and furazolidone, febantel, pyrantel pamoate,
praziquantel or combinations thereof. These drugs may be
administered by a variety of suitable routes, including orally,
parenterally, such as subcutaneously, intravenously,
intramuscularly or interperitoneally, or topically (cutaneously),
such as directly on to exposed skin surface, to a patient in the
treatment and/or prevention of Giardia.
[0109] Intermediate hosts may be involved in transmitting one or
more worm or non-worm parasites, fungi, viruses and bacteria to the
patient and thus a successful therapeutic intervention includes
strategies to control or prevent reinfection. For instance, as
tapeworm infections can be transmitted by intermediate hosts such
as fleas and canine chewing lice, a successful therapeutic
intervention in the case of tapeworm infection includes strategies
to control any intermediate host (e.g., flea) infestation that may
be present on the patient. Thus, controlling intermediate hosts
such as fleas aids in preventing reinfection of the patient with
tapeworm. Therapeutics for the treatment or control of flea
infestation are well known to those skill in the art and include
selamectin, fipronil, imidacloprid, indoxacarb, pyrethrin,
permethrin, flumethrin, spinosad, nitenpyram, afoxolaner,
fluralaner, saralane, nitenpyram, methoprene, pyriproxyfen, and
lufenuron, or combinations thereof. Flea control agents may be
administered by a variety of suitable routes including orally,
parenterally, such as subcutaneously, intravenously,
intramuscularly or interperitoneally, or topically (cutaneously),
such as directly on to exposed skin surface, to a patient.
Representative suitable flea control forms includes sprays,
powders, collar, oral compositions, or topical treatments.
[0110] The steps of the method of the present invention may include
applying a mammalian sample to a device of the invention, which
includes a (a) first antibody capable of specifically binding
coproantigen from a first tapeworm, but not coproantigen from a
second tapeworm or coproantigen from a third tapeworm; (b) a second
antibody capable of specifically binding the coproantigen from the
second tapeworm, but not the coproantigen from the first tapeworm
or the coproantigen from the third tapeworm; and (c) a third
antibody capable of specifically binding the coproantigen from the
third tapeworm, but not the coproantigen from the first tapeworm or
the coproantigen from the second tapeworm to form
antibody-coproantigen complexes in the presence of the
coproantigens, if any, in the sample; and detecting the presence or
absence of the antibody-coproantigen complexes, if any.
[0111] In some embodiments, the device can further include one or
more antibodies selected from: an antibody capable of specifically
binding a roundworm coproantigen, but not a whipworm or hookworm
coproantigen; an antibody capable of specifically binding a
whipworm coproantigen, but not a roundworm or hookworm
coproantigen; an antibody capable of specifically binding a
hookworm coproantigen, but not a whipworm or roundworm
coproantigen;an antibody capable of specifically binding Giardia
coproantigen, but not coproantigen selected from the group
consisting of roundworm coproantigen, whipworm coproantigen,
hookworm coproantigen, tapeworm Taenia coproantigen, tapeworm
Dipyhdium coproantigen, and parvovirus coproantigen; and an
antibody capable of specifically binding parvovirus, but not
coproantigen selected from the group consisting of roundworm
coproantigen, whipworm coproantigen, hookworm coproantigen,
tapeworm Taenia coproantigen, tapeworm Dipyhdium coproantigen, and
Giardia coproantigen. In embodiments, the hookworm is Anyclostoma,
the roundworm is Toxocara and the whipworm is Trichuris. In other
embodiments, the hookworm is Ancylostoma caninum or Ancylostoma
tubaeforme; the roundworm is Toxocara canis or Toxocara cati; the
whipworm is Trichuris vulpis or Trichuris felis; the Giardia is
Giardia lamblia, and the parvovirus is feline parvovirus or canine
parvovirus.
[0112] In one embodiment, the step of detecting the presence or
absence of the antibody-coproantigen complexes, if any, further
includes the step of providing one or more lectins that binds to at
least one of the complexes. The lectin can be detectably labeled or
immobilized onto a solid support. Alternatively, the first, second
and third antibodies can be detectably labeled or immobilized on a
solid support. In one embodiment the first, second and third
antibodies can be immobilized on a solid support and the lectin can
be detectably labeled. In another embodiment, the first, second and
third antibodies can be detectably labeled and the lectin can be
immobilized on a solid support.
[0113] In another embodiment, the step of contacting the fecal
sample from a mammal with one ore more antibodies further includes
the step of contacting the fecal sample with one or more lectins.
The lectin can be detectably labeled or immobilized onto a solid
support. Alternatively, the first, second and third antibodies can
be detectably labeled or immobilized on a solid support. In one
embodiment, the first, second and third antibodies can be
immobilized on a solid support and the lectin can be detectably
labeled. In another embodiment, the first, second and third
antibodies can be detectably labeled and the lectin can be
immobilized on a solid support.
[0114] The antibodies and lectins may be directly or indirectly
attached to a solid support or a substrate such as a microtiter
well, antibody-immobilizing portion of a SNAP.RTM. device, magnetic
bead, non-magnetic bead, column, matrix, membrane, fibrous mat
composed of synthetic or natural fibers (e.g., glass or
cellulose-based materials or thermoplastic polymers, such as,
polyethylene, polypropylene, or polyester), sintered structure
composed of particulate materials (e.g., glass or various
thermoplastic polymers), or cast membrane film composed of
nitrocellulose, nylon, polysulfone or the like (generally synthetic
in nature). All of these substrate materials may be used in
suitable shapes, such as films, sheets, or plates, or they may be
coated onto or bonded or laminated to appropriate inert carriers,
such as paper, glass, plastic films, or fabrics. Suitable methods
for immobilizing antibodies, peptides and lectins on solid phases
include ionic, hydrophobic, covalent interactions and the like.
[0115] The methods of the present invention do not require the use
of solid phases or substrates, however. The skilled artisan will
recognize that there are a number of ways that the present method
may be carried out to detect the presence or absence of roundworm
without involving the use of solid phases or substrates. In just
one example, immunoprecipitation methods that do not require the
use of solid phases or substrates may be carried out.
[0116] In some embodiments of the invention, the antigen/antibody
complex is detected when an indicator reagent, such as an enzyme
conjugate, which is bound to the antibody, catalyzes a detectable
reaction. Optionally, an indicator reagent including a signal
generating compound may be applied to the antigen/antibody complex
under conditions that allow formation of a detectable
antigen/antibody/indicator complex. Optionally, the antibody may be
labeled with an indicator reagent prior to the formation of an
antigen/antibody complex.
[0117] The formation of an antigen/antibody complex or an
antigen/antibody/indicator complex in some of the methods of the
present invention specifically may be detected by, e.g.,
radiometric, enzymatic, chemiluminescent, colorimetric,
turbidimetric, fluorometric, photometric, size-separation, surface
plasmon resonance, or precipitation methods. Detection of an
antigen/antibody complex also may be accomplished by the addition
of a secondary antibody that is coupled to an indicator reagent
including a signal generating compound. Indicator reagents
including signal generating compounds (labels) associated with a
polypeptide/antibody complex may be detected using the methods
described above and may include chromogenic agents, catalysts such
as enzyme conjugates, fluorescent compounds such as fluorescein and
rhodamine, chemiluminescent compounds such as dioxetanes,
acridiniums, phenanthridiniums, ruthenium, and luminol, radioactive
elements, direct visual labels, as well as cofactors, inhibitors,
magnetic particles, and the like. Examples of enzyme conjugates
include alkaline phosphatase, horseradish peroxidase,
beta-galactosidase, and the like. The selection of a particular
label is not critical, but it will be capable of producing a signal
either by itself or in conjunction with one or more additional
substances.
[0118] Methods of the invention include, but are not limited to
those based on competition, direct reaction or sandwich-type
assays, including, but not limited to ELISA, RIA,
immuno-fluorescent assays (IFA), hemagglutination (HA),
fluorescence polarization immunoassay (FPIA), and microtiter plate
assays (i.e., any assay done in one or more wells of a microtiter
plate). One assay of the invention includes a reversible flow
chromatographic binding assay, which may be performed, for example,
by using a SNAP.RTM. device. See U.S. Pat. No. 5,726,010.
[0119] In some embodiments, the method of the invention facilitates
sandwich or competition-type specific binding assays. In a sandwich
assay, antigen capture reagents are immobilized in a reactive zone.
These antigen capture reagents may specifically bind to antigens in
the sample being tested for tapeworm. Following binding of the
antigen from the sample, the antigen capture reagent/antigen
complex is detected by any suitable method. For example, the
complex may be reacted with labeled specific binding reagents
(e.g., an enzyme-antibody conjugate) and antigen detected (e.g.,
upon reaction with substrate).
[0120] In other embodiments of the method of the present invention,
a competition assay is performed. In a competition assay, antigen
capture reagents are immobilized at the reactive zone and are
contacted simultaneously with antigen from a sample and labeled
antigen (e.g., an antigen-enzyme conjugate). The amount of label
detected at the reactive zone is inversely proportional to the
amount of antigen in the sample.
[0121] In some embodiments of the method, antibodies specific for
tapeworm coproantigens are attached to a solid phase or substrate.
A sample potentially including an antigen from tapeworm is added to
the substrate. Antibodies that specifically bind tapeworm antigens
are added. The antibodies may be the same antibodies used on the
solid phase or they may be from a different source or species.
Further, these antibodies may be linked to an indicator reagent,
such as an enzyme conjugate. Wash steps may be performed prior to
each addition. A chromophore or enzyme substrate may be added and
color may be allowed to develop. The color reaction may be stopped
and the color may be quantified using, for example, a
spectrophotometer, and/or the color may be subjectively assessed by
the human eye.
[0122] In other embodiments of the method, antibodies specific for
tapeworm coproantigens are attached to a solid phase or substrate.
A sample potentially including a tapeworm antigen is added to the
substrate. Second anti-species antibodies that specifically bind
the coproantigens are added. These second antibodies are from a
different species than are the solid phase antibodies. Third
anti-species antibodies that specifically bind the second
antibodies and that do not specifically bind the solid phase
antibodies are added. The third antibodies may include an indicator
reagent, such as an enzyme conjugate. Wash steps may be performed
prior to each addition. A chromophore or enzyme substrate may be
added and color may be allowed to develop. The color reaction may
be stopped and the color may be quantified using, for example, a
spectrophotometer, and/or the color may be subjectively assessed by
the human eye.
[0123] In a specific example, the method of the present invention
is performed in conjunction with a device that is a lateral flow
assay device by adding a prepared mammalian sample to a flow matrix
of the device at a first region (a sample application zone). The
prepared sample is carried in a fluid flow path by capillary action
to a second region of the flow matrix where a particulate label
capable of binding and forming a first complex with an antigen in
the sample exists. The particulate label can be, e.g., a colored
latex particle, dye sol, or gold sol conjugated to an antibody
specific for a roundworm antigen. The first complex is carried to a
third region of the flow matrix where an antibody that specifically
binds a roundworm antigen is immobilized at a distinct location. A
second complex is formed between the immobilized antibody and the
first complex. The particulate label that is part of the second
complex can be directly visualized by the human eye.
[0124] Each specific worm antibody may be an immobilized antigen
capture reagent in a reaction zone (solid phase). A second antigen
capture reagent, i.e., a second specific tapeworm antibody that has
been conjugated to a label, either may be added to the sample
before the sample is added to the device, or the second antigen
capture reagent can be incorporated into the device. For example,
the labeled antigen capture reagent may be deposited and dried on a
fluid flow path that provides fluid communication between a sample
application zone and the solid phase. Contact of the labeled
antigen capture reagent with the test sample can result in
dissolution of the labeled antigen capture reagent.
[0125] In one embodiment of the method of the present invention,
specific worm coproantigen is detected by ELISA. Specific examples
of the ELISA method of the present invention is described in the
Example section included herein. Although the present invention is
described with respect to those specific ELISA methods, however, it
is to be understood that those of ordinary skill in the art will
recognize that alternative, additional or substitute ELISA steps
may be used without deviating from the basic goal achieved through
this method of the invention.
[0126] In another embodiment of the present invention, tapeworm
coproantigen is detected by using a lateral flow device, such as a
SNAP.RTM. device, for example.
[0127] Further, the methods of the invention for detection of
tapeworm infection can be combined with other diagnostic assays to
detect the presence of other organisms or conditions. For example,
assays of the invention can be combined with reagents that detect
one or more helminthic worms, non-worm fecal parasites, one or more
viruses, one or more fungi, one or more protozoa, one or more
bacteria, one or more blood-borne parasites or occult blood or a
combination thereof. By providing two or more unique binding sites
in a single assay device (such as, for example, two unique spots on
a SNAP.RTM. assay device), the present invention allows for
detection of two or more organisms from a single sample. In one
embodiment, there are three unique spots for detection of past or
present infection or infestation from three organisms (the spots
being either antigen or antibody binding reagents) from a single
sample (i.e., the same individual sample is exposed to the three
capture reagents on a single device). In yet another embodiment,
there are four unique spots for detection of past or present
infection or infestation from four organisms (the spots being
either antigen or antibody binding reagents) from a single sample
(i.e., the same individual sample is exposed to the four capture
reagents on a single device. It is to be understood, however, that
the same device may include more than four unique spots and/or
allow for the detection of more than four organisms.
[0128] The reagents for the detection of one or more helminthic
worms, non-worm parasites, one or more viruses, one or more fungi,
one or more protozoa, or one or more bacteria may be, for example,
one or more antibodies or one or more antigens recognized by
antibodies specific for one or more helminthic worms, non-worm
parasites, one or more viruses, one of more fungi, or one or more
bacteria. In some embodiments, the reagents can include one or more
antibodies or one or more antigens recognized by antibodies
specific for one or more helminthic worm parasites (e.g.,
roundworm, whipworm, hookworm, and heartworm), non-worm parasites,
one or more viruses (e.g., parvovirus such as canine parvovirus or
feline parvovirus), one or more fungi, one or more protozoa (e.g.,
Giardia such as Giardia lamblia) or one or more bacteria.
[0129] The method further may optionally include using one or more
nucleic acids from tapeworm, including, but not limited to, the
nucleic acids of the present invention, to determine the presence
or absence of one or more tapeworm species in a mammalian sample.
Such use of these nucleic acids for determining the presence of the
helminth may be carried out before, after or concomitantly with the
carrying out of any other aspects of the method, including the
detection of one or more tapeworm species by antibody. Therefore,
in one aspect, after one or more tapeworm species is detected or
not detected in a particular sample and the mammal from which the
sample was obtained is diagnosed as either having or not having a
tapeworm infection, the sample (or a later-obtained sample from the
diagnosed mammal) may be tested for the presence or absence of any
one or more of the nucleic acids, including any one or more nucleic
acids of the invention. Anyone failing to detect a specific
helminth in a particular mammal by using one or more nucleic acids
(after the helminth had been detected by using one or more
antibodies) would need to take into consideration the possibility
that the antibodies had detected helminthic corpoantigen prior to
the appearance of detectable helminthic nucleic acid in the sample.
In such an instance, the mammal's caregiver may elect to ignore the
observation that the nucleic acid had failed to detect the helminth
and proceed with treating the mammal specifically for helminth
infection based on the observation that the antibodies had in fact
detected helminth. In another aspect, the nucleic acids are used to
determine the presence or absence of helminths in a particular
mammal, and then the presence or absence of helminths is further
evaluated by using the antibodies of the present invention.
Detection of one or more helminthic nucleic acids may be carried
out by using any nucleic acid detection techniques known to the
skilled artisan. For example, such detection may be carried out by
performing a PCR-based technique, such as, but limited to, for
example, a real-time PCR-based technique. Exemplary PCR-based
techniques are described in, e.g., PCR Protocols (Methods in
Molecular Biology), 2.sup.nd ed., Bartlett and Stirling, eds.,
Humana Press (2003); and Sambrook and Russell, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press (2001); each
one of which is incorporated herein by reference in its
entirety.
[0130] The present invention is specifically described with
reference to the following Examples; however, it is not to be
construed as being limited thereto.
EXAMPLE A
[0131] Unless otherwise indicated, the following materials and
techniques were used to generate data described in one or more of
Examples 1-5 as described below.
[0132] Preparation of worm extracts of Taenia pisiformis and
Dipylidium caninum. Taenia pisiformis and Dipylidium caninum worm
extracts were purchased from Antibody systems, Inc. Hurst, Tex.,
U.S.A. The worm extracts were centrifuged at 10,000 g for 30
minutes at 4.degree. C. Supernatant were collected, dialyzed into
PBS, pH 7.0 (membrane molecular weight cutoff 12-14 kD, Part
132678, Spectrum, Repligen, Waltham Mass., USA) and protein
concentration were determined with Bradford assay.
[0133] Preparation of worm extracts of Taenia taeniaeformis. Taenia
taeniaeformis were obtained from Ross University School of
Veterinary Medicine, Saint Kitts. The whole worms were washed
several times with cold PBS, pH 7.0, to remove any fecal materials
and mucus from the hosts in room temperature and homogenized at
4.degree. C. with a tissue grinder until no obvious tissue chunks
were visible to the naked eye. The homogenized materials were
transferred to a 50 ml Falcon tube and the grinder was rinsed 2-3
times with cold PBS, pH 7.0. The homogenized tapeworm, together
with the rinses of the grinder, were centrifuged at 10,000 g for 30
min at 4.degree. C. and the supernatants were collected before
dialyzed into PBS, pH 7.0 (membrane molecular weight cutoff 12-14
kD, Part 132678, Spectrum, Repligen, Waltham Mass., USA). Protein
concentration was determined with Bradford assay.
[0134] Preparation of Dipylidium caninum TCA soluble fraction. This
antigen was prepared by disrupting worms in an aqueous buffer
solution (phosphate buffer, pH, 7.2), centrifuging to remove
insoluble and particulate components, adding of 30% trichloroacetic
acid (TCA) dropwise to a final concentration of 15%, stirring for
another 15 min at 18-27.degree. C. after all the TCA added, sitting
on the bench for another 45 minutes undisturbed at 18-27.degree.
C., centrifuging to remove insoluble components, dialyzing against
an aqueous buffer solution (0.01 M phosphate buffer, pH 7.2) with
Spectrapor I dialysis tube, MWCO: 6-8 K, and lyophilizing with a
lyophilizer.
[0135] Preparation of Worm Wash. Frozen raw worm specimen were
rinsed 4 times in PBS buffer at pH7.2. The buffer solutions from
the first 3 rinse steps were discarded. The buffer solution from
the fourth rinse step was centrifuged at 10,000 g for 20 minutes,
and the supernatant was collected. The supernatant was concentrated
using an iCON Concentrator (MWCO: 20 mL/9K; Thermo Scientific). The
resulting concentrate was termed "Worm Wash" (WW).
[0136] Preparation of E/S material. The Excreted/Secreted (E/S)
materials were collected by keeping the tapeworms alive in a T-150
flask with tissue culture medium (EMEM with D-glucose, Gentamicin
and Fungizone, pH 7.2-7.3) in a 37.degree. C. incubator with 5% CO2
for two weeks. Briefly, living tapeworms were washed several times
with warmed medium to remove any fecal residues, placed in a T-150
Tissue culture flask with 100 ml warmed medium (EMEM). The
viability of the tapeworms was verified daily and the medium was
changed three times a day. The used media were pooled and
concentrated with an Icon Concentrator (MWCO: 9 K; Thermo
Scientific), and the resulting concentrated E/S material was used
for antibody production.
[0137] Polyclonal antibody (pAb) preparation. Polyclonal antibodies
were raised with Specific Pathogen Free (SPF) rabbits at SDIX, LLC
(Windham, Me., U.S.A.). The immunogens were whole worm extracts
(Dipylidium caninum and Taenia pisiformis from Antibody Systems
Inc., Hurst, Tex.; T taeniaeformis from IDEXX Laboratories, Inc.)
or E/S material (IDEXX Laboratories, Westbrook, Me.). Briefly,
rabbits were challenged subcutaneously with the same immunogen in
different adjuvants four times over a period of 50 days. Serum was
collected at the end of the immunization procedure.
[0138] Monoclonal antibody (mAb) preparation. Murine monoclonal
antibodies were produced according to standard procedures unless
otherwise noted. Briefly, 3-5 Balb/c mice were immunized with the
immunogen, and spleen cells harvested after completion of the
immunization schedule. Spleen cells were fused with myeloma cells.
Through several rounds of screening, and selecting with HAT medium,
isotyping and subcloning, specific hybridoma cell lines secreting
the desired mAb secreted were obtained.
[0139] Antibody purification and isolation. Both rabbit polyclonal
antibody and murine monoclonal antibody were purified with Protein
G Sepharose 4 Fast Flow (Thermo Fisher Scientific) affinity
chromatography using AKTA purification system. Briefly, rabbit
serum or TCF (terminal culture fluid) were diluted with the washing
buffer, loaded on to the Protein G column. The column was washed
with washing buffer thoroughly before the antibody being eluted
from the column. The eluted antibody was neutralized with 1 M Tris
buffer, pH 8.0, then dialyzed into 10 mM PBS, pH 7.2, and stored in
-20.degree. C. for future usage.
[0140] Fecal extract preparation. Samples from fresh, unpreserved
canine or feline fecal samples (1 gram) were suspended in 4 ml of
diluent solution ("diluent solution" is 0.05 M Tris base; 1 mM
EDTA; 0.45% Kathon; 16 mg/ml gentamicin sulfate; 0.05% Tween-20;
40% fetal bovine serum; 10% rabbit serum; and 5% mouse serum). The
suspension was centrifuged at 4000 rpm for 20 minutes to produce a
first supernatant. The first supernatant was centrifuged at 10000 g
for 10 minutes to produce a second supernatant, which is referred
to herein as "fecal extract."
[0141] ELISA assays. Purified polyclonal Ab or monoclonal Ab (100
.mu.l/well and 3 .mu.g/ml) was immobilized by physical adsorption
on Immulon 1B 96-well plates overnight at 4.degree. C. The plates
were then blocked with 1% BSA in 0.1M Tris pH 7.0 at 4.degree. C.
overnight, followed by drying at room temperature. Approximately
100 .mu.l of fecal extract was added to each well and allowed to
incubate at room temperature for one hour. The wells were then
washed five times with a PBS-Tween-20 solution according to
standard methods known to those of ordinary skill in the art. In a
separate reaction vessel, free rabbit pAb (a different antibody, in
case of mAb, against the same target was used) was labeled with
horseradish peroxidase (HRP) by using the crosslinker succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to create a
conjugate, and 3 .mu.g/ml of this conjugate was added to each well
having immobilized pAb or mAb. Following a 30-minute incubation
period at room temperature, unbound conjugate was washed from the
wells by using PBS-Tween-20 solution according to standard methods
known to those of ordinary skill in the art. 50 .mu.l of TMBLUE.TM.
peroxidase substrate (IDEXX Laboratories, Westbrook, Me.) was then
added to each well and the plates were incubated for 10 minutes at
room temperature. After stopping each enzymatic reaction with 0.1%
sodium dodecyl sulfate (SDS) following the 10-minute incubation
period, the optical density (OD) value of each well of the 96-well
plate was measured at A650 by standard spectrophotometric
techniques by using an ELISA plate reader to generate an "OD650
value" (or, more simply, an "OD value") for each well. In this
arrangement, the OD value obtained for any particular well of the
96-well plate was directly proportional to the amount of
specifically bound antigen present in the well. OD values of 0.1 or
below were regarded as negative results, and OD values were
regarded as positive results unless otherwise noted in the
Examples.
[0142] Carbohydrate/glycosylation characterization using
Carbohydrate-proving ELISA with Jacalin-biotin. Murine IgM mAb
ADX226 coated immulon 1B plate was used for the
carbohydrate-proving ELISA. Approximately 100 .mu.l of fecal
extract was added to each well and allowed to incubate at room
temperature for one hour. The wells were then washed five times
with a PBS-Tween-20 solution according to standard methods known to
those of ordinary skill in the art. Jacalin-Biotin (0.25 ug/ml) was
added to each well, followed by one-hour incubation at room
temperature. Unbound Jacalin-biotin (Vector Laboratories, 30 Ingold
Road, Burlingame, Calif.) was washed from the wells by using
PBS-Tween-20 solution according to standard methods known to those
of ordinary skill in the art. Streptavidin-HRP conjugate (Vector
Laboratories, 30 Ingold Road, Burlingame, Calif.) was added to each
well and incubate at room temperature for 30 minutes. Unbound
Streptavidin-HRP conjugate was washed away again by using PBS-Tween
20 solution. 50 .mu.l of TMBLUE.TM. peroxidase substrate (IDEXX
Laboratories Inc., Westbrook, Me.) was then added to each well
following the 30 min with Streptavidin-HRP conjugate and the plates
were incubated for 1 minute at room temperature. After stopping
each enzymatic reaction with 0.1% sodium dodecyl sulfate (SDS)
following the 1-minute incubation period, the optical density (OD)
value of each well of the 96-well plate was measured at A650 as
illustrated above in fecal assay ELISA section.
EXAMPLE 1A
[0143] Raising and screening of Murine Monoclonal Antibodies
against Taenia pisiformis. Murine mAbs (monoclonal antibodies) were
raised by immunization of mice with worm extract (WE) of T.
pisiformis (hereinafter, T. pisiformis extract or extract of T.
pisiformis), and hybridomas were raised. Candidate hybridomas were
screened for the ability of secreted mAbs to bind the immunogen
(i.e., extract of T. pisiformis) coated onto microtiter plates in a
ELISA capture assay. From this screen, one hundred (100) hybridomas
(i.e., 100 mAbs) that were positive in this screen (i.e. able to
bind the T. pisiformis extract) were chosen for further analysis.
The 100 candidate mAbs were further screened for their ability to
function in a coproantigen ELISA assay, by a capture assay screen.
Microtiter plates were coated with a rabbit polyclonal antibody
that had been raised by standard immunization of rabbits with T.
pisiformis extract. FEX (fecal extract) made from canine fecal
samples was added. The fecal samples were from dogs known to be T.
pisiformis infected and received from IDEXX Reference Laboratories.
One of the 100 candidate mAbs supernatant was added to each well,
followed by goat a-mouse IgG conjugated to a label. Five mAbs
(02D09, 03H02, 07C02, ADX131 and ADX13) performed particularly well
in this assay and were chosen for further analysis.
[0144] The five mAbs were further screened for their ability to
function when paired with each other in a sandwich assay. For this
purpose, each of the five mAbs was coated onto the wells of
microtiter plates and incubated with FEX (Taenia pisiformis
positive samples: n=10; T. pisiformis negative samples: n=10),
followed by one of the five mAbs conjugated to a label, or a Taenia
pisiformis WE mouse pAb. Five mAbs performed well in combination
with at least one of the other mAbs, and were therefore chosen for
further study. From the five mAbs that performed well, two (ADX131
and ADX132) were chosen for further testing because they resulted
the in low background and high signal when paired with each other
in the coproantigen ELISA.
[0145] ADX131 and ADX132 were each paired with HRP conjugates of
02D09, 03H02, 07C02, ADX131 and ADX132 in another ELISA essay. An
additional pairing with T. pisiformis WE rabbit pAb was used as a
positive control. ADX131 and ADX132 were individually coated onto
the wells of microtiter plates; incubated with FEX from a T.
pisiformis infected dog, a T. pisiformis uninfected dog, or T.
pisiformis worm extract; followed by the conjugates and the HRP
substrate. In each of the five antibody pairings, ADX131 resulted
in strong signal with the FEX from infected dog and with the worm
extract. In four out of the five antibody pairings, ADX131 resulted
in a negative result (i.e., signal below threshold) with the FEX
from uninfected dog, except that the pairing with 03H02 resulted in
a signal that was slightly above the chosen threshold of 0.1 OD
(FIG. 1). In all five antibody pairings, ADX132 resulted in strong
signal with the FEX from infected dog and with the worm extract. In
all five antibody pairings, ADX132 resulted in low background
signal with the FEX from uninfected dog (FIG. 2).
EXAMPLE 1B
[0146] Taenia pisiformis coproantigen ELISA specificity and
sensitivity evaluation. To assess the specificity of the assay, the
T. pisiformis coproantigen ELISA assay (using ADX131 coated on
plate and ADX132-HRP conjugate) was tested against FEX from canines
infected with Hookworm Ancylostoma caninum (n=44), roundworm
Toxocara canis (n=9), whipworm Trichuris vulpis (n=36), D.
caninum_(n=44), or Taenia pisiformis (n=5). The result (see FIG. 3)
showed that all 5 T. pisiformis samples were positive in the assay
and all of the others were negative. The T. pisiformis coproantigen
ELISA did not cross-react with hookworm Ancylostoma caninum,
roundworm Toxocara canis, whipworm Trichuris vulpis, or D. caninum.
Thus, the T. pisiformis coproantigen ELISA assay was 100% sensitive
and specific in this experiment. To assess the specificity of the
assay, a sandwich coproantigen ELISA was built with ADX131 coated
onto a microtiter plate, and ADX132-HRP conjugate applied after
addition of the patient sample. The assay was run on D. caninum WE,
T. pisiformis WE, T. crassiceps WE, T. taeniaeformis WE, T.
taeniaeformis E/S, as well as the FEX from a D. caninum positive
dog, a D. caninum negative dog, a D. caninum positive cat, a D.
caninum negative cat, a T. pisiformis positive dog, a T. pisiformis
negative dog, a pool of three T. taeniaeformis positive felines,
and a pool of three T. taeniaeformis negative felines. The
beforementioned WEs were used at a concentration of 1 .mu.g/ml
protein. The assay was additionally run on several WE samples at 2
.mu.g/ml protein and/or 10 .mu.g/ml protein. The WE samples run at
these higher concentrations were D. caninum WE, T. pisiformis WE,
T. taeniaeformis WE, T. crassiceps WE, hookworm Ancylostoma caninum
WE, roundworm Toxocara canis WE, and whipworm Trichuris vulpis WE.
Among these samples, the assay was positive only on T. pisiformis
WE and the T. pisiformis positive dog (FIG. 4). Thus, the
ADX131/ADX132-HRP ELISA was highly specific in this experiment.
[0147] To assess the specificity of the T. pisiformis coproantigen
ELISA assay (using ADX131 coated on plate and ADX132-HRP
conjugate), the assay was run on FEX from 822 canine samples. Of
these samples, 1 sample had been confirmed as T. pisiformis
positive by microscopy (i.e., proglottids were observed by
microscopy), and 821 had been confirmed as T. pisiformis negative
by microscopy. The results showed that of the 821
microscopy-negative samples, 818 were negative in the T. pisiformis
coproantigen ELISA assay, and 3 were positive. The
microscopy-positive sample was positive in the T. pisiformis
coproantigen ELISA assay. Thus, the T. pisiformis coproantigen
ELISA assay was 99.7% specific in this experiment.
EXAMPLE 1C
[0148] Antigen characterization: Glycosylation of the antigens
bound byanti-T. pisiformis mAb ADX131. In order to determine
whether the coproantigen bound by mAb ADX131 is glycosylated, the
ability of 21 different lectins were tested for their ability to
bind the coproantigen using a commercial kit (Biotinylated lectin
kits I, II and III from Vector Laboratories, Burlingame, Calif.).
The assays were carried out according to the manufacturer's
instructions. Briefly, FEX from T. pisiformis positive canines was
added to plates coated with ADX131. After washing, the
lectin::biotin conjugates were added, followed by streptavidin-HRP
and the color substrate. In this test, the following lectins
resulted in high signal and low background, indicating that they
bound to T. pisiformis coproantigen: WGA, succinylated WGA, PSA,
GSLII and LCA.
[0149] Antigen characterization: Glycosylation of the antigens
bound by anti-T. pisiformis mAb ADX132.In order to determine
whether the coproantigen bound by mAb ADX132 is glycosylated, the
ability of ADX132 to bind FEX substances bound by 21 different
lectins was tested using a commercial kit (Biotinylated lectin kits
I, II and III from Vector Laboratories, Burlingame, Calif.). The
assays were carried out according to the manufacturer's
instructions. Briefly, wells microtiter plates were coated with one
of lectins, and FEX from T. pisiformis positive canines was added,
followed by ADX132-HRP conjugate and the color substrate. In this
test, the following lectins bound to coproantigen: WGA, UEA and
GSLII.
[0150] The lectin binding data indicates that the T. pisiformis
coproantigen bound by the ADX131/ADX132 assay is glycosylated. The
lectin binding data further indicates that the ADX131/132
coproantigen contains the following moieties: non-reduced GlcNac;
reduced GlcNac; fucose; GSL II; and mannose.
EXAMPLE 2A
[0151] Preparation of rabbit polyclonal antibodies against T.
taeniaeformis. Worm extracts (WE), E/S material and worm wash (WW)
of T. taeniaeformis were prepared as described above. Rabbits were
immunized with T. taeniaeformis WE, T. taeniaeformis E/S, or T.
taeniaeformis WW as described above to raise the polyclonal
antibodies T. taeniaeformis WE rabbit pAb, T. taeniaeformis E/S
rabbit pAb and T. taeniaeformis WW rabbit pAb.
A. Initial Assessment of T. taeniaeformis WE Rabbit pAb-Rabbit pAb
ELISA Assay, and T. taeniaeformis E/S Rabbit pAb-Rabbit pAb ELISA
Assay.
[0152] For an initial assessment of the performance of coproantigen
ELISA assays with T. taeniaeformis WE rabbit pAb, and T.
taeniaeformis E/S rabbit pAb, feline fecal extracts from seven T.
taeniaeformis positive and six T. taeniaeformis negative cats were
tested.
[0153] T. taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay: In
this ELISA assay, T. taeniaeformis WE rabbit pAb was coated onto
plates, contacted with FEX, then contacted with T. taeniaeformis WE
rabbit pAb-HRP conjugate, then contacted with a color substrate. In
this assay, all seven T. taeniaeformis positive samples were
positive, while all six T. taeniaeformis negative samples tested
negative. Therefore, sensitivity and specificity for the T.
taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay were 100% in
this experiment.
[0154] T. taeniaeformis E/S rabbit pAb-rabbit pAb ELISA assay: In
this ELISA assay, T. taeniaeformis E/S rabbit pAb was coated onto
plates, contacted with FEX, then contacted with T. taeniaeformis
E/S rabbit pAb-conjugate, then contacted with a color substrate. In
this assay, all seven T. taeniaeformis positive samples were
positive, while all six T. taeniaeformis negative samples tested
negative. Therefore, sensitivity and specificity for the T.
taeniaeformis E/S rabbit pAb-rabbit pAb ELISA assay were 100% in
this experiment.
B. Performance of T. taeniaeformis WE Rabbit pAb-Rabbit pAb ELISA
Assay on Canine and Feline Fecal Samples.
[0155] The performance of coproantigen ELISA assay with T.
taeniaeformis WE rabbit pAb (as described under Section A) was
further assessed on canine and feline fecal extracts. The canine
sample set included fecal extracts from 31 T. taeniaeformis
positive canines and 74 T. taeniaeformis negative canines. The
feline sample set included fecal extracts from 13 T. taeniaeformis
positive felines and 39 T. taeniaeformis negative felines.
[0156] For canines, among the 31 positive samples, the T.
taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay yielded positive
signal in 8 samples. Among the 74 negative canine samples, the T.
taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay yielded positive
signal in 6 samples. Therefore, the sensitivity was 25.8% and the
specificity was 91.9% in this experiment.
[0157] For felines, among the 13 positive samples, the T.
taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay yielded positive
signal in 12 samples. Among the 39 negative samples, the T.
taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay yielded positive
signal in 4 samples. Therefore, the sensitivity was 92.3% and the
specificity was 89.7% in this experiment.
C. Performance of T. taeniaeformis E/S Rabbit pAb-Rabbit pAb ELISA
Assay on Canine and Feline Fecal Extracts.
[0158] The performance of coproantigen ELISA assay with T.
taeniaeformis E/S rabbit pAb (as described under Section A) was
further assessed on canine and feline fecal extracts. The canine
sample set included fecal extracts from 31 T. taeniaeformis
positive canines and 74 T. taeniaeformis negative canines. The
feline sample set included fecal extracts from 13 T. taeniaeformis
positive felines and 39 T. taeniaeformis negative felines.
[0159] For canines, among the 31 positive samples, the T.
taeniaeformis E/S rabbit pAb-rabbit pAb ELISA assay yielded
positive signal in 4 samples. Among the 74 negative canine samples,
the T. taeniaeformis E/S rabbit pAb-rabbit pAb ELISA assay yielded
positive signal in 1 sample. Therefore, the sensitivity was 12.9%
and the specificity was 98.6% in this experiment.
[0160] For felines, among the 13 positive samples, the T.
taeniaeformis E/S rabbit pAb-rabbit pAb ELISA assay yielded
positive signal in 12 samples. Among the 39 negative samples, the
T. taeniaeformis E/S rabbit pAb-rabbit pAb ELISA assay yielded
positive signal in 3 samples. Therefore, the sensitivity was 92.3%
and the specificity was 92.3% in this experiment.
D. Performance of T. taeniaeformis WW Rabbit pAb-Rabbit pAb ELISA
Assay on Canine and Feline Fecal Samples.
[0161] The performance of coproantigen ELISA assay with T.
taeniaeformis WW rabbit pAb was assessed on canine and feline fecal
extracts. The canine sample set included fecal extracts from 31 T.
taeniaeformis positive canines and 74 T. taeniaeformis negative
canines. The feline sample set included fecal extracts from 13 T.
taeniaeformis positive felines and 39 T. taeniaeformis negative
felines.
[0162] For canines, among the 31 positive samples, the T.
taeniaeformis WW rabbit pAb-rabbit pAb ELISA assay yielded a
positive signal in 16 samples. Among the 74 negative canine
samples, the T. taeniaeformis WE rabbit pAb-rabbit pAb ELISA assay
yielded positive signal in 3 samples. Therefore, the sensitivity
was 51.6% and the specificity was 98.6% in this experiment.
[0163] For felines, among the 13 positive samples, the T.
taeniaeformis WW rabbit pAb-rabbit pAb ELISA assay yielded a
positive signal in 12 samples. Among the 39 negative samples, the
T. taeniaeformis WW rabbit pAb-rabbit pAb ELISA assay yielded
positive signal in 3 samples. Therefore, the sensitivity was 92.3%
and the specificity was 95.9% in this experiment.
EXAMPLE 2B
[0164] Preparation of mouse polyclonal and monoclonal antibodies
against T. taeniaeformis WE and T. taeniaeformis E/S. Worm extract
(WE) of T. taeniaeformis and E/S material of T. taeniaeformis was
prepared as described above. Mice were immunized with T.
taeniaeformis WE or T. taeniaeformis E/S as described above to
raise the mouse polyclonal antibodies T. taeniaeformis WE mouse pAb
and T. taeniaeformis E/S mouse pAb. These polyclonal antibodies
specifically their respective immunogens when the immunogens were
coated onto microtiter plates.
A. Preparation of T. taeniaeformis WE Mouse mAb
[0165] Hybridomas were derived from the mice immunized with T.
taeniaeformis WE as described above. From the resulting mouse
monoclonal antibody (mouse mAb) secreting hybridomas, two T.
taeniaeformis WE mouse mAbs were selected because they resulted in
high signal and low background in ELISA assays: ADX184 (IgG) and
ADX185 (IgM).
B. Performance of Coproantigen ELISA with T. taeniaeformis WE Mouse
mAbs
[0166] To assess sensitivity, a sandwich coproantigen ELISA assay
was built with ADX185 coated onto a microtiter plate, followed by
addition of the patient sample, then followed by addition of T.
taeniaejrmis WE rabbit pAb-HRP conjugate. The assay was run on FEX
from 7 T. taeniaeformis positive felines, and 3 T. taeniaeformis
negative felines. As shown in FIG. 5, the assay detected
coproantigen in 7 out of the 7 positive samples, and in zero out of
the 3 negative samples. Thus, the sensitivity of the ADX185/T.
taeniaeformis WE rabbit pAb-HRP ELISA was 100% in this
experiment.
[0167] To assess specificity, a sandwich coproantigen ELISA was
built with ADX184 coated onto a microtiter plate, and ADX193-HRP
conjugate applied after addition of the patient sample. ADX193 is a
T. taeniaeformis E/S mouse mAb, described below. The assay was run
on D. caninum WE, T. pisiformis WE, T. crassiceps WE, T.
taeniaeformis WE, T. taeniaeformis E/S, as well as the fecal
extracts from a D. caninum positive dog, a D. caninum negative dog,
a D. caninum positive cat, a D. caninum negative cat, a T.
pisiformis positive dog, a Taenia pisiformis negative dog, a pool
of three T. taeniaeformis positive felines, and a pool of three T.
taeniaeformis negative felines. The beforementioned WEs were used
at a concentration of 1 .mu.g/ml protein. The assay was
additionally run on several WE samples at 10 .mu.g/ml protein and 2
.mu.g/ml protein. The WE samples run at these higher concentrations
were D. caninum WE, T. pisiformis WE, T. taeniaeformis WE, T.
crassiceps WE, hookworm Ancylostoma caninum WE, roundworm Toxocara
canis WE, and whipworm Trichuris vulpis WE. Among these samples and
as shown in FIG. 6, the assay was positive only on T. taeniaeformis
WE (at 10 .mu.g/ml) and the pool of T. taeniaeformis positive
felines. Thus, the ADX184/ADX193-HRP ELISA was highly specific in
this experiment.
C. Preparation and Screening of T. taeniaeformis E/S Mouse mAb
[0168] Hybridomas were derived from the mice immunized with T.
taeniaeformis E/S as described above. From the resulting mouse
monoclonal antibody (mouse mAb) secreting hybridomas, five T.
taeniaeformis E/S mouse mAbs were selected: ADX190, ADX191, ADX192,
ADX193, ADX194. These five mouse mAbs were further assessed for
their performance in an ELISA assay when each was paired with the
anti-E/S mouse pAb -HRP conjugate. The of the five assays was run
on T. taeniaeformis E/S, fecal extracts from seven T. taeniaeformis
positive cats, and fecal extracts from three T. taeniaeformis
negative cats. Each of the five assays detected all 7 positive
feline samples. Each was also negative on the samples from the
three negatives felines, although ADX193 and ADX194 had a higher
background on one out the three negative feline samples.
EXAMPLE 2C
[0169] Performance of an ELISA using ADX184 with ADX193-HRP
[0170] An ELISA was built with ADX184 coated onto microtiter plates
and ADX193 conjugated with HRP. Both mouse mAbs were used at a
concentration of 5 ug/ml.
[0171] The ADX184/ADX193-HRP ELISA was run on fecal extracts from
six T. taeniaeformis positive cats, fecal extracts from four T.
taeniaeformis negative cats, and one T. taeniaeformis E/S protein
sample. As shown in FIG. 7, the assay detected coproantigen in all
six positive fecal extracts and the E/S sample, but did not detect
coproantigen in any of the four negative fecal extracts.
[0172] The ADX184/ADX193-HRP ELISA was run on fecal extracts from
68 T. taeniaeformis positive cats, and from 108 T. taeniaeformis
negative cats that were D. caninum_positive. The assay resulted in
a positive signal in 55 out of the 68 T. taeniaeformis positive
samples (80.9% sensitivity), and resulted in a positive signal in
one out of the 108 T. taeniaeformis negative samples (99.1%
specificity; 0.9% cross-reactivity with D. caninum).
EXAMPLE 2D
[0173] Performance of an ELISA using ADX191 with ADX194-HRP
[0174] A coproantigen ELISA was built with ADX191 coated onto
microtiter plates and ADX194 conjugated with HRP. Both mouse mAbs
were used at a concentration of 3 ug/ml. This ELISA was run on
fecal extracts from six T. taeniaeformis positive cats, fecal
extracts from four T. taeniaeformis negative cats, and one T.
taeniaeformis E/S protein sample. As shown in FIG. 8, the assay
detected all six positive fecal extracts and the E/S sample, but
did not detect coproantigen in any of the four negative fecal
extracts.
EXAMPLE 3A
[0175] A. Preparation of Rabbit pAb against Dipylidium caninum TCA
Soluble Fraction (D. caninum TCA Rabbit pAb)
[0176] A TCA fraction of D. caninum worms was prepared as described
above and used to immunize two rabbits. Antiserum from one of these
rabbits was chosen for further analysis.
B. Assessment of a D. caninum TCA Rabbit pAb ELISA Assay with
Canine and Feline Samples.
[0177] In this coproantigen ELISA assay, the D. caninum TCA rabbit
pAb was coated onto plates, contacted with FEX, then contacted with
D. caninum TCA rabbit pAb-HRP conjugate, then contacted with a
color substrate as described above.
[0178] Canine: The D. caninum TCA rabbit pAb ELISA assay was run on
fecal extracts from 58 D. caninum positive dogs; the ELISA yielded
a positive signal in 57 out of the 58 samples. The assay was also
run on fecal extracts from 27 D. caninum negative dogs; the ELISA
yielded a positive signal in 8 out of the 27 samples. Therefore,
the assay was 98.3% sensitive and 70.4% specific in this
experiment.
[0179] Feline: The D. caninum TCA rabbit pAb ELISA assay was run on
fecal extracts from 31 D. caninum positive cats; the ELISA yielded
a positive signal in 28 out of the 31 samples. The assay was also
run on fecal extracts from 13 D. caninum negative cats; the ELISA
yielded a positive signal in 5 out of the 13 samples. Therefore,
the assay was 90.3% sensitive and 61.5% specific in this
experiment.
EXAMPLE 3B
[0180] A. Preparation of Rabbit pAb against D. caninum WE Fraction
(D. caninum WE Rabbit pAb).
[0181] WE was prepared from D. caninum worms (Antibody Systems) as
described above and used to immunize two rabbits. Antiserum from
one of these rabbits was chosen for further analysis.
B. Assessment of a D. caninum WE Rabbit pAb ELISA Assay.
[0182] In this ELISA assay, the D. caninum WE rabbit pAb was coated
onto plates, contacted with FEX at 5 ug/ml, then contacted with D.
caninum WE rabbit pAb-HRP conjugate at 3 ug/ml, then contacted with
a color substrate as described above. This D. caninum WE rabbit pAb
ELISA assay was run on worm extracts from several helminth species:
D. caninum WE, Taenia pisiformis WE, T. crassiceps WE, hookworm
Ancylostoma caninum WE, roundworm Toxocara canis WE, whipworm
Trichuris vulpis WE. As shown in FIG. 9, only the D. caninum WE
yielded a positive signal in this ELISA, demonstrating specificity
of the assay in this experiment.
[0183] The performance of the D. caninum WE rabbit pAb ELISA assay
was assessed in an additional experiment as follows. The assay was
run on fecal extracts from 44 D. caninum positive dogs and 48 D.
caninum negative dogs. The assay resulted in a positive signal in
16 out of the 44 positive samples and zero negative samples.
Therefore, the assay was 36.4% sensitive and 100% specific in this
experiment.
EXAMPLE 3C
[0184] A. Preparation of Rabbit pAb against D. caninum E/S (D.
caninum E/S Rabbit pAb)
[0185] E/S was prepared from a D. caninum worm from a dog as
described above and used to immunize two rabbits. Antiserum from
one of these rabbits was chosen for further analysis.
B. Assessment of a D. caninum E/S Rabbit pAb ELISA Assay
[0186] In this coproantigen ELISA assay, the D. caninum E/S rabbit
pAb was coated onto plates, contacted with FEX, then contacted with
D. caninum WE rabbit pAb-HRP conjugate, then contacted with a color
substrate as described above.
[0187] The assay was run on fecal extracts from 7 D. caninum
positive dogs. The assay resulted in a positive signal in 4 out of
the 7 positive samples. The assay resulted in a positive signal in
one out of the four D. caninum negative dogs. Thus, the assay was
57.1% sensitive and 75% specific in this experiment.
EXAMPLE 3D
[0188] A. Preparation of Rabbit mAb against D. caninum WE (D.
caninum WE Rabbit mAb)
[0189] WE was prepared from whole D. caninum worms from dogs as
described above. Rabbits were immunized with the Dipylidium caninum
WE according to a standard immunization procedure (SDIX, Windham,
Me., USA). Following a boost at least 3 weeks after the initial
immunization, a blood sample from the rabbit was used for
monoclonal antibody development (ImmunoPrecise Antibodies, Ltd.,
(IPA), Victoria, British Columbia, Canada). B cells from the rabbit
were collected and the B cell culture supernatants were evaluated
on D. caninum WE coated 96-well plates and probed with secondary
anti-rabbit IgG antibody. Approximately 50 .mu.l of B cell
supernatants from positive wells were tested for specific binding
in a second screen (IDEXX) with various fecal extracts. The B cells
of the best 32 candidates from this second screen were preserved in
lysis buffer for cloning. RNA was isolated from these 32 candidates
and rabbit antibody heavy and light (kappa) chain variable regions
were cloned into separate mammalian expression vectors (IPA).
Tissue culture supernatants collected from the mammalian cells
transfected with the antibody heavy and light chain vectors were
evaluated again with various fecal extracts. The top five clones of
recombinant rabbit mAb DNA constructs were sequenced (IPA).Two out
of these five rabbit mAb clones were chosen for further analysis:
RDX13 and RDX12.
B. Assessment of a ELISA Assay with D. caninum WE Rabbit mAb on
Ccanine and Feline Samples.
[0190] In this series of coproantigen ELISA assays, the mouse mAb
ADX226 was coated onto plates, contacted with FEX, then contacted
with RDX13-HRP and/or RDX12-HRP conjugates, then contacted with a
color substrate as described above.
[0191] In a first experiment, the RDX13-HRP and RDX12-HRP
conjugates were mixed, and the assay was run on fecal extracts from
38 D. caninum positive dogs, 28 D. caninum negative dogs, 20 D.
caninum positive cats and 7 D. caninum negative cats. The assay
yielded a positive signal in 28 out of the 38 positive dogs, 2 out
of the 28 negative dogs, 18 out of the 20 positive cats, and zero
out of the 7 negative cats. Thus, the assay resulted in 73.7%
sensitivity and 92.9% specificity for dog samples, and 90.0%
sensitivity and 100% specificity in cat samples, in this
experiment.
[0192] In a second, third and fourth experiment, the RDX13-HRP and
RDX12-HRP conjugates were used both individually and mixed, and the
assay was run on fecal extracts from 37 D. caninum positive dogs,
28 D. caninum negative dogs, 21 D. caninum positive cats and 7 D.
caninum negative cats.
[0193] In the second experiment, RDX13-HRP was used alone, the
assay yielded a positive signal in 20 out of the 37 positive dogs,
one out of the 28 negative dogs, 15 out of the 21 positive cats,
and zero out of the 7 negative cats. Thus, the assay resulted in
54.1% sensitivity and 96.4% specificity for dog samples, and 71.4%
sensitivity and 100% specificity for cat samples, in this
experiment.
[0194] In the third experiment, RDX12-HRP was used alone, the assay
yielded a positive signal in 13 out of the 37 positive dogs, one
out of the 28 negative dogs, 16 out of the 21 positive cats, and
zero out of the 7 negative cats. Thus, the assay resulted in 35.1%
sensitivity and 96.4% specificity for dog samples, and 76.2%
sensitivity and 100% specificity for cat samples, in this
experiment.
[0195] In the fourth experiment, the RDX13-HRP and RDX12-HRP
conjugates were mixed, the assay yielded a positive signal in 22
out of the 37 positive dogs, one out of the 28 negative dogs, 19
out of the 21 positive cats, and zero out of the 7 negative cats.
Thus, the assay resulted in 59.5% sensitivity and 96.4% specificity
for dog samples, and 90.5% sensitivity and 100% specificity for cat
samples, in this experiment.
EXAMPLE 3E
[0196] A. Preparation of Mouse pAb against D. caninum WE (D.
caninum WE Mouse pAb)
[0197] WE was prepared from D. caninum worms from dogs as described
above and used to immunize mice. The resulting antiserum, D.
caninum WE mouse pAb, was used in ELISA experiment described
below.
B. Assessment of a ELISA Assay with D. caninum WE Mouse pAb on
Canine and Feline Samples.
[0198] In this coproantigen ELISA assay, the D. caninum WE mouse
pAb was coated onto plates, contacted with FEX, then contacted with
D. caninum WE mouse pAb-HRP conjugate, then contacted with a color
substrate as described above.
[0199] The assay was run on fecal extracts from 4 D. caninum
positive dogs and 3 D. caninum positive cats; one D. caninum
negative cat infected with Giardia and T. taeniaeformis, one D.
caninum negative cat infected with T. taeniaeformis, and one dog
infected with Toxocara and Taenia pisiformis. As shown in FIG. 10,
the assay yielded a positive signal in 4 out of the 4 positive
dogs, in zero out of the one negative dog, in 3 out of the 3
positive cats, and in zero out of the 2 negative cats. Therefore,
the assay resulted in a high degree of specificity and sensitivity
in this experiment.
EXAMPLE 3F
[0200] A. Preparation of Mouse mAb against D. caninum TCA Soluble
Fraction (D. caninum TCA Mouse mAb)
[0201] TCA soluble fraction was prepared from D. caninum worms from
dogs as described above and used to immunize mice (MBS). The spleen
of an immunized mouse was used to generate a mouse mAb, ADX251,
which was used in the following ELISA experiments.
B. Assessment of ELISA assays with D. caninum TCA mouse mAb on
Canine and Feline Samples.
[0202] In a first coproantigen ELISA configuration, mouse mAb
ADX226 was coated onto plates, contacted with FEX, then contacted
with mouse mAb ADX251-HRP conjugate followed by color
substrate.
[0203] The assay was run on fecal extracts from 3 D. caninum
positive dogs, 5 D. caninum negative dogs; 3 D. caninum positive
cats, and 1 D. caninum negative cat. As shown in FIG. 11, the assay
yielded a positive signal in 3 out of the 3 positive dogs, in zero
out of the five negative dogs, in 3 out of the 3 positive cats, and
in zero out of the 1 negative cat. Therefore, the assay resulted in
a high degree of specificity and sensitivity in this
experiment.
[0204] In a second coproantigen ELISA configuration, mouse mAb
ADX251 was coated onto plates, contacted with FEX, then contacted
with mouse mAb ADX227-HRP conjugate, followed by color substrate.
The assay was run on fecal extracts from 3 D. caninum positive
dogs, 5 D. caninum negative dogs; 3 D. caninum positive cats, and 1
D. caninum negative cat. As shown in FIG. 12, the assay yielded a
positive signal in 3 out of the 3 positive dogs, in zero out of the
five negative dogs, in 3 out of the 3 positive cats, and in zero
out of the 1 negative cat. Therefore, the assay resulted in a high
degree of specificity and sensitivity in this experiment.
[0205] In a third coproantigen ELISA configuration, mouse mAb
ADX251 was coated onto plates, contacted with FEX, then contacted
with D. caninum_WE rabbit pAb-HRP conjugate, followed by color
substrate. The assay was run on fecal extracts from 3 D. caninum
positive dogs, 5 D. caninum_negative dogs; 3 D. caninum positive
cats, and 1 D. caninum negative cat. As shown in FIG. 13, the assay
yielded a positive signal in 3 out of the 3 positive dogs, in zero
out of the five negative dogs, in 3 out of the 3 positive cats, and
in zero out of the 1 negative cat. Therefore, the assay resulted in
a high degree of specificity and sensitivity in this
experiment.
EXAMPLE 3G
[0206] A. Preparation of Mouse mAb against D. caninum WE (D.
caninum WE Mouse mAb)
[0207] WE was prepared from D. caninum_worms from dogs as described
above and used to immunize mice. From these mice, four monoclonal
antibodies (ADX224, ADX225, ADX226 and ADX227) were generated as
described above and chosen for further analysis.
B. Assessment of ELISA Assays with D. caninum WE Mouse mAb on
Canine and Feline Samples.
[0208] The following ELISA configurations were run on fecal
extracts from 3 D. caninum positive dogs, 4 D. caninum negative
dogs; 3 D. caninum positive cats, and 1 D. caninum negative
cat.
[0209] In a first set of coproantigen ELISA configurations, the D.
caninum WE mouse mAbs were coated individually onto plates,
contacted with FEX, then contacted with D. caninum WE rabbit
pAb-HRP conjugate followed by color substrate. The results are
shown in FIG. 14. Thus, an assay with ADX224 on the plate detected
coproantigen in 5 out of six positive samples and in zero negative
samples. An assay with ADX225 on the plate detected coproantigen in
six out of six positive samples and in zero negative samples. An
assay with ADX226 on the plate detected coproantigen in six out of
six positive samples and in zero negative samples. The assay with
ADX227 on the plate detected coproantigen in six out of six
positive samples and in zero negative samples.
[0210] In a second set of coproantigen ELISA configurations, each
of the four D. caninum WE mouse mAbs were coated individually onto
plates, contacted with FEX, then contacted with each of the four D.
caninum WE mouse mAb-HRP conjugates followed by color substrate.
The results are shown in FIG. 15. Among the tested combinations,
two pairings were chosen. An assay with ADX226 on the plate and
ADX227-HRP detected coproantigen in six out of six positive samples
and in zero negative samples; and an assay with ADX227 on the plate
and ADX227-HRP detected coproantigen in six out of six positive
samples and in zero negative samples. This successful pairing of
ADX227 with itself suggests that the coproantigen detected by
ADX227 contains a repetitive epitope.
EXAMPLE 3H
[0211] A. Antigen Characterization: Glycosylation of the Antigens
Bound by the anti-D. caninum Antibody ADX226
[0212] In order to determine whether the coproantigen bound by D.
caninum WE mouse mAb ADX226 is glycosylated, the ability of 21
different lectins were tested for their ability to bind the
coproantigen using a commercial kit (Biotinylated lectin kits I, II
and III from Vector Laboratories, Burlingame, Calif. The assays
were carried out according to the manufacturer's instructions.
Briefly, ADX226 was coated into Immulon lb plates. FEX from D.
caninum positive or negative canines was added to plates. After
washing, the lectin::biotin conjugates were added, followed by
streptavidin-HRP and the color substrate. The results are shown in
FIG. 16. In this test, the following lectins bound specifically and
strongly to ADX226 coproantigen: PSA, LCA, Jacalin, ECL, GSL1,
RCA123, and WGA. This lectin binding data indicates that the D.
caninum_coproantigen bound by the ADX226 is glycosylated.
B. Antigen Characterization: Glycosylation of the Antigens Bound by
the anti-D. caninum WE Rabbit pAb
[0213] In order to determine whether the coproantigen bound by the
antibody D. caninum WE rabbit pAb is glycosylated, 21 different
lectins were tested for their ability to bind the coproantigen
using a commercial kit (Biotinylated lectin kits I, II and III from
Vector Laboratories, Burlingame, Calif.). The assays were carried
out according to the manufacturer's instructions. Briefly, D.
caninum WE rabbit pAb was coated into Immulon 1b plates. FEX from
D. caninum positive or negative canines and felines was added to
plates. After washing, the lectin::biotin conjugates were added,
followed by streptavidin-HRP and the color substrate. In this test,
the following lectins bound to D. caninum WE rabbit pAb
coproantigen: Jacalin, WGA, succinylated WGA, GSL II. The results
are shown in FIG. 17. This lectin binding data indicates that the
D. caninum coproantigen bound by D. caninum WE rabbit pAb is
glycosylated. This data also shows that an antibody-lectin sandwich
assay can be used to detect D. caninum coproantigen.
C. Antigen Characterization: Glycosylation of the Antigens Bound by
the anti-D. caninum Antibodies D. caninum WE Rabbit pAb and
ADX187
[0214] A similar assay configuration was used to determine whether
the coproantigen(s) bound by the D. caninum WE rabbit pAb and
ADX187 (a D. caninum WE mouse mAb) are glycosylated, 21 different
lectins were tested for their ability to bind the coproantigen
using a commercial kit (Biotinylated lectin kits I, II and III from
Vector Laboratories, Burlingame, Calif.). In this configuration,
the 21 different biotinylated lectins were coated into Immulon 1b
plates. FEX from D. caninum positive or negative canines was added
to plates. After washing, the D. caninum WE rabbit pAb-HRP or
theADX187-HRP conjugates were added, followed by streptavidin-HRP
and the color substrate. The results are shown in FIG. 18. In this
test, the following lectins bound to D. caninum WE rabbit pAb
coproantigen: Jacalin, WGA, and succinylated WGA. The same set of
lectins bound the ADX187 coproantigen: Jacalin, WGA, and
succinylated WGA. This lectin binding data confirms that the D.
caninum coproantigen(s) bound by D. caninum WE rabbit pAb and
ADX187 is glycosylated. This data also shows that an
antibody-lectin sandwich assay can be used to detect D. caninum
coproantigen.
[0215] Further testing of O-glycosylation of the coproantigen
recognized by ADX226In this experiment, ADX226 was coated onto
microtiter plates, followed by FEX, then jacalin-biotin, then
streptavidin-HRP, and finally the HRP substrate. The FEX were
prepared from fecal extracts of 20 D. caninum positive dogs, 24 D.
caninum negative dogs, 12 D. caninum positive cats, and 3 D.
caninum negative cats. The assay resulted in a positive signal in
18 out of the 20 positive dogs (95% sensitivity), zero out of 24
negative dogs (100% specificity), 11 out of 12 positive cats (91.7%
sensitivity), and zero out of 3 negative cats (100% specificity).
This data demonstrates that the ADX226 coproantigen is
O-glycosylated and that a sandwich immunoassay using an antibody
and a lectin can be used to detect tapeworm coproantigen.
EXAMPLE 4
[0216] A series of fecal ELISA sandwich assays was performed in
microtiter plates essentially as described in section "EXAMPLE A"
above to test the specificity of ELISA assays for the detection of
tapeworm T. pisiformis, tapeworm T. taeniaeformis, tapeworm D.
caninum, hookworm Ancylostoma caninum (A. caninum), hookworm
Ancylostoma tubaeforme (A. tubaeforme), roundworm Toxocara canis
(T. canis), roundworm Toxocara cati (T. cati), whipworm Trichuris
vulpis (T. vulpis), whipworm Trichuris felis (T. felis) and
protozoon Giardia lamblia (Giardia).
[0217] Fecal extracts from the following sources were tested in the
ELISA assays: T. pisiformis positive dog (FIG. 19, column 1), T.
taeniaeformis infected cat (FIG. 19, column 2), D. caninum infected
dog (FIG. 19, column 3), D. caninum infected cat (FIG. 19, column
4), hookworm A. caninum infected dog (FIG. 19, column 5), hookworm
A. tubaeforme infected cat (FIG. 19, column 6), roundworm T. canis
infected dog (FIG. 19, column 7), roundworm T. cati infected cat
(FIG. 19, column 8), whipworm T. vulpis infected dog (FIG. 19,
column 9), whipworm T. felis infected cat (FIG. 19, column 10),
Giardia infected dog (FIG. 19, column 11), Giardia infected cat
(FIG. 19, column 12), and two parvovirus infected cats (FIG. 19,
columns 13 and 14). In FIG. 19, columns 1 through 12 are an image
of a single microtiter plate, and columns 13 and 14 are an image of
another, separate microtiter plate.
[0218] Tapeworm T. pisiformis coproantigen ELISA (FIG. 19, Row A).
A sandwich ELISA assay for the detection of T. pisiformis
coproantigen was built. Briefly, mAb ADX131 was coated onto
microtiter plates, incubated with fecal extract, then incubated
with mAb ADX132-HRP conjugate, followed by detection with a
peroxidase substrate. ADX131 was coated at a concentration of 3
ug/ml. ADX132-HRP was used at a concentration of 3 ug/ml. The
results show that this T. pisiformis coproantigen ELISA yielded a
positive signal with FEX from T. pisiformis infected dog. However,
this T. pisiformis ELISA did not detect coproantigen in FEX from
any of the following: Tapeworm T. taeniaeformis infected cat, D.
caninum infected dog, D. caninum infected cat, hookworm A. caninum
infected dog, hookworm A. tubaeforme infected cat, roundworm T.
canis infected dog, roundworm T. cati infected cat, whipworm T.
vulpis infected dog, whipworm T. felis infected cat, Giardia
infected dog, Giardia infected cat, and Parvovirus infected cat
(FIG. 19, Row A). Therefore, this T. pisiformis ELISA specifically
detected T. pisiformis coproantigen and did not crossreact with
coproantigen from any of the following: Tapeworm T. taeniaeformis,
tapeworm D. caninum, hookworm A. caninum, roundworm T. canis,
roundworm T. cati, whipworm T. vulpis, whipworm T. felis, protozoon
Giardia lamblia, and feline parvovirus. Thus, this T. pisiformis
ELISA was highly species specific. This T. pisiformis ELISA is
useful for the detection or diagnosis of infection or infestation
with T. pisiformis. This T. pisiformis ELISA can be used to
distinguish infection with T. pisiformis from infection with
tapeworm T. taeniaeformis, tapeworm D. caninum, hookworm A.
caninum, hookworm A. tubaeforme, roundworm T. canis, roundworm T.
cati, whipworm T vulpis, whipworm T. felis, protozoon Giardia
lamblia, canine parvovirus and feline parvovirus.
[0219] Tapeworm T. taeniaeformis coproantigen ELISA (FIG. 19, Row
B). A sandwich ELISA assay for the detection of Taenia
taeniaeformis coproantigen was built. Briefly, mAb ADX184 was
coated onto microtiter plates, incubated with fecal extract, then
incubated with mouse mAb ADX193-HRP conjugate, followed by
detection with a peroxidase substrate. ADX184 was coated at a
concentration of 5 ug/ml. ADX193-HRP was used at a concentration of
3 ug/ml. The results show that this T. taeniaeformis coproantigen
ELISA yielded a positive signal with FEX from T. taeniaeformis
infected dog. However, this T. taeniaeformis ELISA did not detect
coproantigen in FEX from any of the following: Tapeworm T.
pisiformis infected dog, D. caninum infected dog, D. caninum
infected cat, hookworm A. caninum infected dog, hookworm A.
tubaeforme infected cat, roundworm T. canis infected dog, roundworm
T. cati infected cat, whipworm T. vulpis infected dog, whipworm T.
felis infected cat, Giardia infected dog, Giardia infected cat, and
Parvovirus infected cat (FIG. 19, Row B). Therefore, this T.
taeniaeformis ELISA specifically detected T. taeniaeformis
coproantigen and did not crossreact with coproantigen from any of
the following: Tapeworm T. pisiformis, tapeworm D. caninum,
hookworm A. caninum, hookworm A. tubaeforme, roundworm T. canis,
roundworm T. cati, whipworm T. vulpis, whipworm T. felis, protozoon
Giardia lamblia, and feline parvovirus. Thus, this T. taeniaeformis
ELISA was highly species specific. This T. taeniaeformis ELISA is
useful for the detection or diagnosis of infection or infestation
with T. taeniaeformis. This T. taeniaeformis ELISA can be used to
distinguish infection with tapeworm T. taeniaeformis from infection
with tapeworm T. pisiformis, tapeworm D. caninum, hookworm A.
caninum, hookworm A. tubaeforme, roundworm T. canis, roundworm T.
cati, whipworm T. vulpis, whipworm T. felis, protozoon Giardia
lamblia, canine parvovirus and feline parvovirus.
[0220] Tapeworm D. caninum coproantigen ELISA (FIG. 19, Row C). A
sandwich ELISA assay for the detection of D. caninum coproantigen
was built. Briefly, mAb ADX226 was coated onto microtiter plates,
incubated with fecal extract, then incubated with mAb RDXS-HRP
conjugate, followed by detection with a peroxidase substrate.
ADX226 was coated at a concentration of 3 ug/ml. RDXS-HRP was used
at a concentration of 3 ug/ml. The results show that this D.
caninum coproantigen ELISA yielded a positive signal with FEX from
D. caninum infected dog and D. caninum infected cat. However, this
D. caninum ELISA did not detect coproantigen in FEX from any of the
following: Tapeworm T. pisiformis infected dog, T. taeniaeformis
infected cat, hookworm A. caninum infected dog, hookworm A.
tubaeforme infected cat, roundworm T. canis infected dog, roundworm
T. cati infected cat, whipworm T. vulpis infected dog, whipworm T.
felis infected cat, Giardia infected dog, Giardia infected cat, and
Parvovirus infected cat (FIG. 19, Row C). Therefore, this D.
caninum ELISA specifically detected D. caninum coproantigen and did
not crossreact with coproantigen from any of the following:
Tapeworm T. taeniaeformis, tapeworm T. pisiformis, hookworm A.
caninum, hookworm A. tubaeforme, roundworm T. canis, roundworm T.
cati, whipworm T. vulpis, whipworm T. felis, protozoon Giardia
lamblia, and feline parvovirus. Thus, this D. caninum ELISA was
highly species specific and is useful for the detection or
diagnosis of infection or infestation with D. caninum. This D.
caninum ELISA can be used to distinguish infection with D. caninum
from infection with tapeworm T. pisiformis, tapeworm T.
taeniaeformis, hookworm A. caninum, hookworm A. tubaeforme,
roundworm T. canis, roundworm T. cati, whipworm T. vulpis, whipworm
T. felis, protozoon Giardia lamblia, canine parvovirus and feline
parvovirus.
[0221] Hookworm Ancylostoma coproantigen ELISA (FIG. 19, Row D). A
sandwich ELISA assay for the detection of A. caninum coproantigen
was built. Briefly, a polyclonal rabbit anti-ASP5-1 antibody was
coated onto microtiter plates, incubated with fecal extract, then
incubated with mouse anti-ASP5-1 mAb-HRP conjugate, followed by
detection with a peroxidase substrate as discussed in U.S. Pat. No.
7,951,547, which is incorporated by reference in its entirety. The
rabbit polyclonal antibody was coated at a concentration of 5
ug/ml. The mouse mAb was used at a concentration of 4 ug/ml. The
results show that this Ancylostoma coproantigen ELISA yielded a
positive signal with FEX from A. caninum infected dog and A.
tubaeforme infected cat. However, this Ancylostoma ELISA did not
detect coproantigen in FEX from any of the following: Tapeworm T.
pisiformis infected dog, tapeworm T. taeniaeformis infected cat,
tapeworm D. caninum infected dog, tapeworm D. caninum infected cat,
roundworm T. canis infected dog, roundworm T. cati infected cat,
whipworm T. vulpis infected dog, whipworm T. felis infected cat,
Giardia infected dog, Giardia infected cat, and Parvovirus infected
cat (FIG. 19, Row D). Therefore, this Ancylostoma ELISA
specifically detected A. caninum and A. tubaeforme coproantigen and
did not crossreact with coproantigen from any of the following:
Tapeworm T. taeniaeformis, tapeworm T. pisiformis, tapeworm D.
caninum, roundworm T. canis, roundworm T. cati, whipworm T. vulpis,
whipworm T. felis, protozoon Giardia lamblia, and feline
parvovirus. Thus, this Ancylostoma ELISA was highly specific and
useful for the detection or diagnosis of infection or infestation
with hookworm A. caninum and A. tubaeforme. Furthermore, this
Ancylostoma ELISA can be used to distinguish infection with
hookworm A. caninum or A. tubaeforme from infection with tapeworm
T. pisiformis, tapeworm T. taeniaeformis, tapeworm D. caninum,
roundworm T. canis, roundworm T. cati, whipworm T. vulpis, whipworm
T. felis, protozoon Giardia lamblia, canine parvovirus and feline
parvovirus.
[0222] Roundworm Toxocara coproantigen ELISA (FIG. 19, Row E). A
sandwich ELISA assay for the detection of T. canis and T. cati
coproantigen was built. Briefly, a mouse mAb raised against T.
canis protein DIV6728C was coated onto microtiter plates, incubated
with fecal extract, then incubated with an HRP conjugate of another
mouse mAb raised against T. canis protein DIV6728C, followed by
detection with a peroxidase substrate as discussed in U.S. Pat. No.
7,951,547, which is incorporated by reference in its entirety. ADX5
was coated at a concentration of 6 ug/ml. ADX10-HRP was used at a
concentration of 5 ug/ml. The results show that this Toxocara
coproantigen ELISA yielded a positive signal with FEX from T. canis
infected dog and T. cati infected cat. However, this Toxocara ELISA
did not detect coproantigen in FEX from any of the following:
Tapeworm T. pisiformis infected dog, T. taeniaeformis infected cat,
tapeworm D. caninum infected dog, tapeworm D. caninum infected cat,
hookworm A. caninum infected dog, hookworm A. tubaeforme infected
cat, whipworm T. vulpis infected dog, whipworm T. felis infected
cat, Giardia infected dog, Giardia infected cat, and Parvovirus
infected cat (FIG. 19, Row E). Therefore, this Toxocara ELISA
specifically detected Toxocara coproantigen and did not crossreact
with coproantigen from any of the following: Tapeworm T.
taeniaeformis, tapeworm T. pisiformis, tapeworm D. caninum,
hookworm A. caninum, hookworm A. tubaeforme, whipworm T. vulpis,
whipworm T. felis, protozoon Giardia lamblia, and feline
parvovirus. Thus, this Toxocara ELISA was highly specific and
useful for the detection or diagnosis of infection or infestation
with T. canis and T cati. This roundworm Toxocara ELISA can be used
to distinguish infection with roundworm T. canis or T. cati from
infection with tapeworm T. pisiformis, tapeworm T. taeniaeformis,
tapeworm D. caninum, hookworm A. caninum, hookworm A. tubaeforme,
whipworm T. vulpis, whipworm T. felis, protozoon Giardia lamblia,
canine parvovirus and feline parvovirus.
[0223] Whipworm Trichuris Coproantigen ELISA (FIG. 19, Row F).
[0224] A sandwich coproantigen ELISA assay for the detection of T.
vulpis coproantigen was built. Briefly, a mouse mAb raised against
T. vulpis protein DIV6901 (ADX6) was coated onto microtiter plates,
incubated with fecal extract, then incubated with an HRP conjugate
of another mouse mAb raised against T. vulpis protein DIV6901
(ADX14), followed by detection with a peroxidase substrate as
discussed in U.S. Pat. No. 7,951,547, which is incorporated by
reference in its entirety. ADX6 was coated at a concentration of 3
ug/ml. ADX10-HRP was used at a concentration of 3 ug/ml. The
results show that this Trichuris coproantigen ELISA yielded a
positive signal with FEX from T. vulpis infected dog and T. felis
infected cat. However, this Trichuris ELISA did not detect
coproantigen in FEX from any of the following: Tapeworm T.
pisiformis infected dog, T. taeniaeformis infected cat, tapeworm D.
caninum infected dog, tapeworm D. caninum infected cat, hookworm A.
caninum infected dog, hookworm A. tubaeforme infected cat,
roundworm T. canis infected dog, roundworm T. cati infected cat,
Giardia infected dog, Giardia infected cat, and Parvovirus infected
cat (FIG. 19, Row F). Therefore, this Trichuris ELISA specifically
detected T. vulpis and T. felis coproantigen and did not crossreact
with coproantigen from any of the following: Tapeworm T.
taeniaeformis, tapeworm T. pisiformis, tapeworm D. caninum,
hookworm A. caninum, hookworm A. tubaeforme, roundworm T. canis,
roundworm T. cati, protozoon Giardia lamblia, and feline
parvovirus. Thus, this Trichuris ELISA was highly specific and
useful for the detection or diagnosis of infection or infestation
with T. vulpis and T. felis. This whipworm Trichuris ELISA can be
used to distinguish infection with whipworm T. vulpis or T. felis
from infection with tapeworm T. pisiformis, tapeworm T.
taeniaeformis, tapeworm D. caninum, hookworm A. caninum, hookworm
A. tubaeforme, roundworm T. canis, roundworm T. cati, protozoon
Giardia lamblia, canine parvovirus and feline parvovirus.
[0225] Giardia coproantigen ELISA (FIG. 19, Row G). A sandwich
coproantigen ELISA assay for the detection of Giardia lamblia
coproantigen was built with an antibody pair that specifically
detects soluble antigen (SNAP.RTM. Giardia Test, IDEXX
Laboratories, Inc. Westbrook, Me. USA) (Olson Me., Leonard N.J.,
Strout J. Prevalence and diagnosis of Giardia infection in dogs and
cats using a fecal antigen test and fecal smear. Can Vet J. 2010
June;51(6):640-2. PMID: 20808578). Briefly, a rabbit anti-Giardia
polyclonal antibody was coated onto microtiter plates, incubated
with fecal extract, then incubated with an HRP conjugate of a mouse
anti-Giardia monoclonal antibody, followed by detection with a
peroxidase substrate. The polyclonal antibody was coated at a
concentration of 3 ug/ml. The mAb-HRP was used at a concentration
of 3 ug/ml. The results show that this Giardia coproantigen ELISA
yielded a positive signal with FEX from Giardia infected dog and
Giardia infected cat. However, this Giardia ELISA did not detect
coproantigen in FEX from any of the following: Tapeworm T.
pisiformis infected dog, T. taeniaeformis infected cat, tapeworm D.
caninum infected dog, tapeworm D. caninum infected cat, hookworm A.
caninum infected dog, hookworm A. tubaeforme infected cat,
roundworm T. canis infected dog, roundworm T. cati infected cat,
whipworm T. vulpis infected dog, whipworm T. felis infected cat and
Parvovirus infected cat (FIG. 19, Row G). Therefore, this Giardia
ELISA specifically detected Giardia lamblia coproantigen and did
not crossreact with coproantigen from any of the following:
Tapeworm T. taeniaeformis, tapeworm T. pisiformis, tapeworm D.
caninum, hookworm A. caninum, hookworm A. tubaeforme, roundworm T.
canis, roundworm T. cati, whipworm T. vulpis, whipworm T. vulpis
and feline parvovirus. Thus, this Giardia ELISA was highly species
specific and useful for the detection or diagnosis of infection
with Giardia lamblia. This Giardia ELISA can be used to distinguish
infection with Giardia lamblia from infection with tapeworm T.
pisiformis, tapeworm T. taeniaeformis, tapeworm D. caninum,
hookworm A. caninum, hookworm A. tubaeforme, roundworm T. canis,
roundworm T. cati, whipworm T. vulpis, whipworm T. felis, canine
parvovirus and feline parvovirus.
[0226] The positive infection status of the parvovirus infected
cats (FIG. 19, columns 13 and 14) was confirmed with the SNAP.RTM.
Parvo Test (M. Abd-Eldaim, M. Beall and M. A. Kennedy. "Detection
of feline panleukopenia virus using a commercial ELISA for canine
parvovirus" Vet Ther. 2009 Winter; 10(4): E1-6) according to the
manufacturer's instructions (IDEXX Laboratories Inc., Westbrook,
Me., USA). This multiplex fecal ELISA assay is capable of the
simultaneous, specific detection of tapeworm T. pisiformis,
tapeworm T. taeniaeformis, tapeworm D. caninum, hookworm A.
caninum, hookworm A. tubaeforme, roundworm T. canis, roundworm T.
cati, whipworm T. vulpis, whipworm T. felis, and protozoon Giardia
lamblia.
[0227] This data demonstrates that the methods of the invention can
be used to readily detect and distinguish between infections with
least eight different intestinal parasites in a species
specific-manner.
EXAMPLE 5
[0228] A series of fecal ELISA assays capable of detecting two or
three tapeworm species from the genus Taenia, for example T.
pisiformis, T. taeniaeformis, and the genus Dipylidium, for example
D. caninum were developed. The ELISAs were performed in microtiter
plates essentially as described in section "EXAMPLE A" above.
[0229] Fecal extracts from the following sources were tested in the
ELISA assays: Four T. pisiformis positive dogs (FIG. 20, columns 1,
4, 7 and 10), four T. taeniaeformis infected cats (FIG. 20, column
2, 5, 8 and 11), two D. caninum infected dogs (FIG. 20, column 3
and 6), two D. caninum infected cats (FIG. 20, column 9 and 12). In
FIG. 20, columns 1 through 12 are an image of a single microtiter
plate.
[0230] Coproantigen ELISA for the detection of tapeworms of the
genus Taenia (FIG. 20, Row A). A sandwich ELISA assay for the
detection of coproantigen from animals infected with Taenia
_tapeworms was built. Briefly, mAb ADX131 and mAb ADX184 were
coated at a concentration of 3 ug/ml each onto microtiter plates,
incubated with fecal extract, then incubated with mAb ADX132-HRP
and ADX193-HRP conjugates at a concentration of 3 ug/ml each,
followed by detection with a peroxidase substrate. The results show
that this Taenia coproantigen ELISA yielded a positive signal with
FEX from T. pisiformis infected dogs (FIG. 20, Row A, Columns 1, 4,
7 and 10) and T. taeniaeformis infected cats (FIG. 20, Row A,
Columns 2, 5, 8 and 11). However, this Taenia ELISA did not detect
coproantigen in FEX from tapeworm D. caninum infected dogs (FIG.
20, Row A, Columns 3 and 6) or D. caninum infected cats (FIG. 20,
Row A, Columns 9 and 12). Therefore, this Taenia ELISA specifically
detected T. pisiformis and T. taeniaeformis coproantigen and did
not crossreact with coproantigen from D. caninum. This Taenia ELISA
is useful for the detection or diagnosis of infection or
infestation with one or more Taenia tapeworms, including T.
pisiformis and T. taeniaeformis. This Taenia ELISA can be used to
distinguish infection with one or more Taenia tapeworms, including
T. pisiformis and T. taeniaeformis, from infection with Dipylidium
tapeworm, including D. caninum.
[0231] Coproantigen ELISA for the detection of tapeworms T.
pisiformis and D. caninum (FIG. 20, Row B). A sandwich ELISA assay
for the detection of coproantigen from animals infected with
tapeworms T. pisiformis and D. caninum was built. Briefly, mAb
ADX131 and mAb ADX226 were coated at a concentration of 3 ug/ml
each onto microtiter plates, incubated with fecal extract, then
incubated with mAb ADX132-HRP and mAb RDX5-HRP conjugates at a
concentration of 3 ug/ml each, followed by detection with a
peroxidase substrate. The results show that this ELISA yielded a
positive signal with FEX from T. pisiformis infected dogs (FIG. 20,
Row B, Columns 1, 4, 7 and 10), tapeworm D. caninum infected dogs
(FIG. 20, Row B, Columns 3 and 6) and D. caninum infected cats
(FIG. 20, Row B, Columns 9 and 12). However, this ELISA did not
detect coproantigen in FEX from T. taeniaeformis infected cats
(FIG. 20, Row B, Columns 2, 5, 8 and 11). Therefore, this ELISA
specifically detected T. pisiformis and D. caninum coproantigen and
did not crossreact with T. taeniaeformis coproantigen. This ELISA
is useful for the detection or diagnosis of infection or
infestation with tapeworms T. pisiformis and/or D. caninum and can
be used to distinguish infection with tapeworms T. pisiformis
and/or D. caninum from infection with tapeworm T.
taeniaeformis.
[0232] Coproantigen ELISA for the detection of tapeworms T.
taeniaeformis and D. caninum (FIG. 20, Row C). A sandwich ELISA
assay for the detection of coproantigen from animals infected with
tapeworms T. taeniaeformis and D. caninum_was built. Briefly, mAb
ADX184 and mAb ADX226 were coated at a concentration of 3 ug/ml
each onto microtiter plates, incubated with fecal extract, then
incubated with mAb ADX193-HRP and RDXS-HRP conjugates at a
concentration of 3 ug/ml each, followed by detection with a
peroxidase substrate. The results show that this ELISA yielded a
positive signal with FEX from T. taeniaeformis infected cats (FIG.
20, Row C, Columns 2, 5, 8 and 11), tapeworm D. caninum infected
dogs (FIG. 20, Row C, Columns 3 and 6) and D. caninum infected cats
(FIG. 20, Row C, Columns 9 and 12). However, this ELISA did not
detect coproantigen in FEX from T. pisiformis infected dogs (FIG.
20, Row C, Columns 1, 4, 7 and 10). Therefore, this ELISA
specifically detected T. taeniaeformis and D. caninum coproantigen
and did not crossreact with T. pisiformis coproantigen. This ELISA
is useful for the detection or diagnosis of infection or
infestation with tapeworms T. taeniaeformis and/or D. caninum and
can be used to distinguish infection with tapeworms T.
taeniaeformis and/or D. caninum from infection with tapeworm T.
pisiformis.
[0233] Coproantigen ELISA for the detection of tapeworms T.
pisiformis, T. taeniaeformis and D. caninum (FIG. 20, Row D). A
sandwich ELISA assay for the detection of coproantigen from animals
infected with_tapeworms T. pisiformis, T. taeniaeformis and D.
caninum was built. Briefly, mAb ADX131, mAb ADX184 and mAb ADX226
were coated at a concentration of 3 ug/ml each onto microtiter
plates, incubated with fecal extract, then incubated with mAb
ADX132-HRP, mAb ADX193-HRP and RDX5-HRP conjugates at a
concentration of 3 ug/ml each, followed by detection with a
peroxidase substrate. The results show that this ELISA yielded a
positive signal with FEX from T. pisiformis infected dogs (FIG. 20,
Row D, Columns 1, 4, 7 and 10), T. taeniaeformis infected cats
(FIG. 20, Row D, Columns 2, 5, 8 and 11), tapeworm D. caninum
infected dogs (FIG. 20, Row D, Columns 3 and 6) and D. caninum
infected cats (FIG. 20, Row D, Columns 9 and 12). Therefore, this
ELISA detected T. pisiformis, T. taeniaeformis and D. caninum
coproantigen. This ELISA is useful for the detection or diagnosis
of infection or infestation with tapeworms T. pisiformis, T.
taeniaeformis and/or D. caninum . These data demonstrate this ELISA
assay can be used to readily detect infection with Taenia and/or
Dipylidium tapeworms in dogs and cats.
* * * * *