U.S. patent application number 10/739457 was filed with the patent office on 2004-07-08 for method for identifying a mycobacterium species.
This patent application is currently assigned to Kreatech Biotechnology B.V.. Invention is credited to Arias-Bouda, Lenka Pereira, Goerdayal, Soenita, Houthoff, Hendrik-Jan, Kolk, Arend, Kroon-Swart, Saskia, Kuyper, Sjoukje, Van Der Meulen, Remco.
Application Number | 20040132106 10/739457 |
Document ID | / |
Family ID | 8229027 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040132106 |
Kind Code |
A1 |
Houthoff, Hendrik-Jan ; et
al. |
July 8, 2004 |
Method for identifying a mycobacterium species
Abstract
The invention relates to a method for identifying a
Mycobacterium species comprising the steps of: a) contacting at
least one immuno-cross reactive antigen component of a
mycobacterial species with a sample of a body fluid of a human or
animal individual; b) contacting at least one antibody, which is
capable of reacting with a mycobacterial antigen, with said body
fluid sample; c) detecting the presence of antigen-antibody
complexes, and identifying the Mycobacterium species present in
said body fluid sample.
Inventors: |
Houthoff, Hendrik-Jan;
(Amsterdam, NL) ; Kroon-Swart, Saskia;
(Voorschoten, NL) ; Van Der Meulen, Remco;
(Amsterdam, NL) ; Goerdayal, Soenita; (Nieuwegein,
NL) ; Kolk, Arend; (Muiderberg, NL) ;
Arias-Bouda, Lenka Pereira; (Amstelveen, NL) ;
Kuyper, Sjoukje; (Amsterdam, NL) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Assignee: |
Kreatech Biotechnology B.V.
|
Family ID: |
8229027 |
Appl. No.: |
10/739457 |
Filed: |
December 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10739457 |
Dec 17, 2003 |
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09581013 |
Jul 7, 2000 |
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6733983 |
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09581013 |
Jul 7, 2000 |
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PCT/NL98/00701 |
Dec 8, 1998 |
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Current U.S.
Class: |
435/7.1 |
Current CPC
Class: |
G01N 33/5695
20130101 |
Class at
Publication: |
435/007.1 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 8, 1997 |
EP |
97 203 851.7 |
Claims
1. A method for identifying a Mycobacterium species comprising the
steps of: a) contacting at least one immuno-cross reactive antigen
component of a mycobacterial species with a sample of a body fluid
of a human or animal individual; b) contacting at least one
antibody, which is capable of reacting with a mycobacterial
antigen, with said body fluid sample; c) detecting the presence of
antigen-antibody complexes, and identifying the Mycobacterium
species present in said body fluid sample.
2. A method according to claim 1, wherein the sample of a body
fluid is chosen from the group consisting of serum, blood and
excretion fluids, such as sputum, saliva, CSF (cerebrospinal
fluid), or tear fluid, and solutions or preparations thereof.
3. A method according to any of the preceding claims, wherein the
at least one immuno-cross-reactive antigen component is bound to a
support.
4. A method according to any of the preceding claims, wherein the
at least one antibody for a mycobacterial species is bound to a
support.
5. A method according to any of the preceding claims, wherein steps
a) and b) are performed simultaneously.
6. A method according to claim 5, wherein the at least one
immuno-cross-reactive antigen component and the at least one
antibody for a mycobacterial species are bound to the same solid
support, and wherein said antibody does not react with the at least
one immuno-cross-reactive antigen component.
7. A method according to claim 6, wherein the support is chosen
from the group consisting of membranes, dip-sticks, filters,
spheres, granules and microtiter plates.
8. A method according to any of the preceding claims, wherein the
at least one antibody for a mycobacterial species is a monoclonal
antibody.
9. A method according to any of the preceding claims, wherein the
detecting the presence of antigen-antibody complexes is performed
by using an indirect or direct labeling method.
10. A method according to claim 9, wherein the detecting is
performed by using a label chosen from the group of biotin,
biocytin, iminobiotin, digoxigenin, avidin, streptavidin, colloidal
dye substances, fluorochromes, such as rhodamin, reducing
substances, such as eosin or erythrosin, (colored) latex sols,
carbon sols, metals, metal sols or other particulate sols, dansyl
lysin, Infra Red Dyes, coumarines, enzymes, and iodide labels.
11. A method according to any of the preceding claims, wherein the
Mycobacterium species is identified on the basis of one or more
reference patterns.
12. A method according to any of the preceding claims, wherein the
at least one immuno-cross reactive antigen component comprises the
total of a preparation of Mycobacterium species, or the total of
the culture medium of said species.
13. A method according to claims 1-11, wherein the at least one
immuno-cross reactive antigen component comprises a KP90, KS90,
antigen6, KP100 or SP100 fraction of a total preparation of a
Mycobacterium species, or a suitable fraction of a culture medium
of said species.
14. A method according to any of the preceding claims, wherein the
at least one antibody for a mycobacterial species comprises IgG,
IgA, IgM or any combination thereof.
15. A diagnostic kit comprising a support, on which at least one
immuno-cross reactive antigen component of a mycobacterial species
and at least one antibody, which is capable of reacting with a
mycobacterial antigen and which does not react with said at least
one immuno-cross reactive antigen component, are bound, and means
for detecting the presence of antigen-antibody complexes.
16. A diagnostic kit according to claim 15, wherein the support is
chosen from the group consisting of membranes, dip-sticks, filters,
spheres, granules and microtiter plates.
Description
[0001] The invention relates to a method for identifying a
Mycobacterium species responsible for a mycobacterial infection in
a human or animal, and to diagnostic kits for use in said
method.
[0002] The genus Mycobacterium contains about 50 species. It is
responsible for a number of diseases which are known collectively
as mycobacterioses. The best known and widest spread of these are
leprosy, caused by M. leprae, and tuberculosis caused by M.
tuberculosis. Both of these diseases affect more than ten million
people all over the world. Most other mycobacteria normally occur
only as environmental saprophytes. However, they can also cause
opportunist diseases, which happens often, but not exclusively, in
organisms suffering from problems with their immune systems, such
as AIDS patients or people undergoing immunosuppression. The
opportunist types comprise the slow-growing species M. avium, and
the closely related M. intracellulare and M. scrofulaceum (often
together referred to as the MAIS complex), M. kansai, M. marinum
and M. ulcerans, and the fast-growing species M. chelonae and M.
fortultum. Although quite rare in the Western world for several
decades, the occurrence of opportunist mycobacterial diseases and
tuberculosis has shown a significant increase with the incidence of
AIDS. Further, it has been reported that mycobacteria or antigens
of mycobacteria play a role in the etiology of a plurality of other
diseases, such as sarcoidosis and Crohn's disease, as well as
different auto-immune diseases, such as auto-immune dermatitis,
rheumatoid arthritis and diabetes. It has been suggested that this
role can be attributed to a structural mimicry between epitopes of
mycobacteria and those of the host organism.
[0003] The cell walls of mycobacteria are very complex and contain
many different lipids, some of which have structures unique to the
genus. These structures comprise mycolinic acids and esters,
peptido-glycolipide, arabino-galactane and lipo-arabinomanane. The
lipid-rich cell walls of a mycobacterial cell are responsible for
the notable coloring properites of the mycobacteria. They also
enable mycobacteria to counter an attack by the immune system of a
host organism. A number of species, after being taken up into
macrophages, surround themselves with a thick layer of secreted
lipids.
[0004] Many of the different components of the mycobacteria
interact with the immune system of a host organism. These
components comprise proteins and hydrocarbon antigens, which can
either be actively secreted by the mycobacteria or can form part of
the cell wall or cell membrane. In addition, they may be present in
the cytoplasm, for example in the cytoplasmic matrix, ribosomes and
enzymes. Mycobacteria further also possess immuno-modulating
components, such as immunosuppressing compounds and adjuvants. As
of consequence, a single mycobacterial species can induce a large
variety of immune responses in different forms having diverse
specificities. This makes it very difficult to derive protein
antigens which are suitable for the detection of species-specific
humoral responses as a basis for a highly sensitive and specific
diagnostic test for the above mentioned diseases, particularly
tuberculosis. Because mycobacteria have a frequent occurrence, both
human and animal body fluids contain nearly at all times
anti-mycobacterial antibodies.
[0005] In the past, researchers have attempted to develop a
sufficiently-sensitive diagnostic test for mycobacterioses. The
focus of these attempts has mostly been on finding species-specific
glycolipid antigens for the detection of specific humoral immune
responses, because of the problems with the specificity of protein
antigens.
[0006] In the international patent application 94/14069, it has
been disclosed to make use of the antibody response of an organism
to immuno-dominant mycobacterial cross-reactive antigen components
(further referred to as Im-CRAC) for developing a diagnostic test
for mycobacterial infections. The Im-CRAC is believed to provide
indirect information on the nature of the immune recognition of,
and response to, a specific mycobacterial pathogen.
[0007] The method proposed in WO-A-94/14069 is based on the
discovery that the clinical manifestation of mycobacterial diseases
is related to the varying capability of an individual host to
produce a humoral response to different mycobacterial
immuno-cross-reactive antigen components (Im-CRAC). Each
mycobacterial infection generates its own specific antibody
response to a number of specified antigens. Analysis of the
antibody-response, e.g. by immunoblotting, has demonstrated that
the immuno-dominant Im-CRAC vary in accordance with the
immunopathological manifestation of the mycobacterial diseases.
Said analysis results in different and distinguishing band patterns
of mycobacterial antigens for different individuals which are
infected with different Mycobacterium species. The band pattern,
which is obtained after an immunoblot, can be referred to as an
Antigen Bar Code. The antigen-antibody reactions which are shown in
the bar code are, when taken together, unique for a certain
Mycobacterium species.
[0008] The present invention aims at providing an improved method
for identifying a Mycobacterium species in a diagnostic test.
Although satisfactory in most aspects, it is still desirable to
have a diagnostic test which is even more sensitive than the method
described in WO-A-94/14069. Furthermore, there is a need for a
diagnostic test which can be used in the determination of previous
vaccination, and for monitoring therapy of organisms infected with
a Mycobacterium species. During therapy, there are situations where
low levels of certain antibodies occur, which may disturb the
accuracy and/or sensitivity of the test making use of the
antibody-antigen cross-reactions, as outlined hereinabove.
[0009] It has now been found that a highly sensitive diagnostic
test can be performed by contacting a sample of a body fluid with
both antibodies and antigens. It has been found that, besides
antibodies, several mycobacterial components are present in animal
and human body fluids, of which the presence can be determined by
using cross-reactions with a chosen set of antibodies in a reliable
manner.
[0010] The invention therefore provides a method for identifying a
Mycobacterium species comprising the steps of:
[0011] a) contacting at least one immuno-cross reactive antigen
component of a mycobacterial species with a sample of a body fluid
of a human or animal individual;
[0012] b) contacting at least one antibody, which is capable of
reacting with a mycobacterial antigen, with said body fluid
sample;
[0013] c) detecting the presence of antigen-antibody complexes, and
identifying the Mycobacterium species present in said body fluid
sample.
[0014] Surprisingly, a highly reliable test has been developed
based on the concept as outlined above. By using the method of the
invention it is possible to monitor the different stages of a
treatment of a mycobacterial disease.
[0015] Also, it is possible to determine whether an individual has
been vaccinated for a mycobacterial disease, and for which
mycobacterial disease. As not every organism infected with a
Mycobacterium species shows the same reaction to said infection, a
very reliable test is provided, in that a wide area of both
antigens and antibodies that may be present can be covered in the
test method.
[0016] The present method can be applied to any sample of body
fluid of any human or animal individual. Of course, the most
reliable results are obtained when a sample of a body fluid wherein
the presence of a Mycobacterium species is most often encountered.
Examples of suitable body fluids include serum, blood and excretion
fluids, such as sputum, saliva, CSF (cerebrospinal fluid), or tear
fluid. These body fluids can either be subjected to a method
according to the invention directly, or they can undergo some form
of pretreatment. Usually, the body fluids will be diluted by
dissolution in a buffer solution prior to the diagnostic test.
[0017] In a preferred embodiment, saliva or a solution or
preparation thereof is subjected to a diagnostic test for
identifying a Mycobacterium species. If necessary, the mucus-like
structure of the saliva can be removed by a treatment with for
instance xylomethasolin or by any other known method. The invention
also encompasses a diagnostic test method for identifying a
Mycobacterium species wherein a sample of saliva is contacted with
at least one immuno-cross-reactive antigen component of a
mycobacterial species, and/or at least one antibody which is
capable of reacting with a mycobacterial antigen, detecting the
presence of antigen-antibody complexes, and identifying the
Mycobacterium species present in the saliva sample. Surprisingly,
the high content of various enzymes in saliva does not disturb the
reliability of the diagnostic test to any substantial extent. In
the alternative, the effect of the enzymes on the outcome of the
test can suitably be annulled.
[0018] The performance of a test on saliva has surprisingly proven
to be very successful and reliable. Apparently, mycobacterial
infections are sufficiently manifested in saliva. The collection
and use of saliva has many advantages over the use of more
conventional body fluids, such as serum or blood. It is relatively
easy to collect, even under the difficult field conditions, that
are often encountered in Third World countries where both
tuberculosis and leprosy have high incidence. Furthermore, saliva
is non-invasive, which raises compliance of individuals to be
tested, requires minimal training to collect and has reduced
biohazard risk in collection, transport and testing, in particular
in areas where a high incidence of HIV-infections exists.
[0019] As has been set forth hereinabove, according to the present
invention a sample of a body fluid is contacted with both an
antigen or a preparation thereof and an antibody or a preparation
thereof. Very good results have also been obtained with a test
wherein a sample of a body fluid is contacted with only an
antibody, which is capable of reacting with a mycobacterial
antigen, or a preparation thereof. The inventions thus also
encompasses a method for identifying a Mycobacterium species
comprising the steps of contacting at least one antibody, which is
capable of reacting with a mycobacterial antigen, detecting the
presence of antibody-antigen complexes, which have been formed
between said antibody and one or more antigen components of the
Mycobacterium species, and identifying the Mycobacterium species
present in said body fluid sample.
[0020] In accordance with the invention, it is possible to contact
a body fluid sample first with the antigen and subsequently with
the antibody or vice versa. It is preferred, however, that the
sample of a body fluid is contacted with the antigen and the
antibody simultaneously. This can, for instance, be done by passing
the body fluid sample by two surfaces, to one of which the antigen
or the preparation thereof has been bound, and to the other of
which the antibody or the preparation thereof has been bound. On
the basis of this principle, the skilled person will be able to
develop various ways of performing the diagnostic test. It will be
apparent that when the method is chosen such that the antigen or
the preparation thereof and the antibody or the preparation thereof
are in contact with one another, the antigens and antibodies used
have to be chosen such that they do not react with each other.
[0021] The at least one immuno-cross-reactive antigen component may
comprise the total or a fraction of the components of Mycobacterium
or the total or a fraction of the culture medium of Mycobacterium.
In principle, any fraction of Mycobacterium or a culture medium
thereof can be used. Such a fraction can be obtained in a
conventional manner, and is advantageously separated by
electrophoresis prior to the method according to the invention as
has been described in WO-A-94/14069 to provide a banding pattern
after the test has been performed. Usually, the antigen component
will mainly comprise lipo-polysaccharides. However, often proteins
will also be present. According to the invention, both
lipo-polysaccharides and proteins, having antigen action, can be
used separately or combined.
[0022] Preferred antigen components are chosen from the
non-exhaustive group of KATG, MPT63 (=18 kD), MPT64 (=24 kD),
MPT51, MTC28, Ag85a (=30-31 kD), Ag85b (=Ag6), Ag85c, Ag5 (=CIE
Ag78 and =38 kD), DES, MPB70,80 (=22/23 kD), Lipoologosaccharide
(LOS), lipoarabinomannan (LAM), PMB67 (67 kD), Isocitrated
dehydrogenase, Malate dehydrogynase, 2,3-diacyl-trehalose (DAT),
Phenolicglycolipid (PGL), ESAT6 (=6 kD), hsp70=DnaK=Ag63 (=71 kD),
CIE Ag82=GroEL and homologues (=65 kD), GroES and homologues=BCGa
(=10 kD), Antigen60, as well as those having molecular weights of 6
kD, 10/12 kD, 16 kD, (often referred to as 14 kD), 18 kD, 19 kD, 21
kD, 22 kD, 23 kD, 24 kD, 28 kD, 29 kD, 30 kD, 30 kD, region 32 kD,
33 kD, 34 kD, 36 kD, 38 kD, 42 kD, 50-55 kD, 60 kD, 65 kD, 67 kD,
71 kD, 88 kD, and 95 kD.
[0023] Preferably, the at least one immuno-cross-reactive antigen
component comprises a KP90, KS90, antigen6, KP100, or SP100
fraction, or a fraction of a culture medium of a preparation of a
Mycobacterium species. It has been found that these antigen
preparation provide very reliable test results. Moreover, they are
sufficiently stable to be stored for a prolonged period of time
without affecting the reliability of the diagnostic test.
[0024] The at least one antibody for a mycobacterial species with
which the sample of a body fluid is contacted in accordance with
the invention, can be any antibody which is capable of reacting
with a certain mycobacterial antigen. Such an antibody or a
preparation thereof can be obtained in a manner known to the
skilled artisan. According to the invention, it is preferred to use
monoclonal antibodies, although the use of polyclonal antibodies
has also proven to be suitable. Preferably, IgG, IgA, IgM or a
combination thereof is used as the antibody. These antibodies have
proven to be very convenient to handle in a diagnostic test and
lead to very satisfying results.
[0025] In a preferred embodiment, the at least one
immuno-cross-reactive antigen component or the at least one
antibody for a mycobacterial species is immobilized on a support.
Both non-solid supports and solid supports can be used. Preferably,
a solid support is used.
[0026] Particularly preferred is the embodiment wherein both the
antigen component and the antibody are immobilized on a support.
When steps a) and b) are performed simultaneously, a particularly
useful test has proven to be based on an embodiment of the
invention wherein the antigen component and the antibody are
immobilized on the same support. As has been stated above, in that
case it is necessary that the antibody does not react with the at
least one immuno-cross-reactive antigen component. It is
advantageous to provide a layer of the at least one
immuno-cross-reactive antigen component on top of a layer of the at
least one antibody, or vice versa. Preferably, first a layer of the
at least one immuno-cross-reactive antigen component is applied to
the solid support, and then a layer of the at least one antibody is
applied thereon.
[0027] The at least one immuno-cross-reactive antigen component
will generally be used in a concentration from 0.1 to 20 .mu.g/ml,
and preferably from 1 to 10 .mu.g/ml. When the antigen component is
applied to a support, the amount of said component will depend on
the test and the support chosen. The at least one antibody will
generally be used in a concentration from 0.1 to 20 .mu.g/ml, and
preferably be from 1 to 10 .mu.g/ml. When the antibody component is
applied to a support, the amount of said component will depend on
the test and the support chosen. The ratio wherein the at least one
immuno-cross-reactive antigen component and the at least one
antibody are used, will usually be of from 1:10 to 10:1, preferably
from 1:2 to 2:1.
[0028] Preferred solid supports are chosen from the non-exhaustive
group consisting of membranes, such as nitrocellulose membranes,
dip-sticks, filters, spheres, granules and microtiter plates. The
antigen component and or the antibody may be immobilized on these
supports in a manner known in the art.
[0029] After the sample of the body fluid has been contacted with
the at least one immuno-cross reactive antigen component and the at
least one antibody, there may or may not have formed
antibody-antigen complexes. These complexes can be of two types;
either the antibody or the antigen component of these complexes
will be from the body fluid. Both or just one type of these
complexes may be present. The detecting of the two types of
complexes can be carried out separately or together. Preferably,
they are performed simultaneously. Suitable detecting methods are
immunoblotting, as has been disclosed in WO-A-94/14069, as well as
any of the usual direct and indirect labeling methods known in the
art. Suitable labels can be chosen from the non-exhaustive group of
biotin, biocytin, iminobiotin, digoxigenin, avidin, streptavidin,
colloidal dye substances, fluorochromes, such as rhodamin, reducing
substances, such as eosin or erythrosin, (colored) latex sols,
carbon sols, metals, metal sols or other particulate sols, dansyl
lysin, Infra Red Dyes, coumarines, enzymes, and iodide labels.
Particularly preferred is the use of gold labels, colloid labels,
latex sols, and enzymes. The use of these labels enable the
performance of a method according to the invention in a so-called
rapid test.
[0030] After the analysis, the Mycobacterium species can suitably
be identified. Although any conventional identification procedure
can be used, it is preferred that the Mycobacterium species is
identified on the basis of one or more reference patterns.
[0031] Of course, the invention also encompasses a diagnostic kit
for use in a method as described hereinabove. The kit comprises a
support, on which at least one immuno-cross reactive antigen
component of a mycobacterial species and at least one antibody,
which is capable of reacting with a mycobacterial antigen and which
does not react with said at least one immuno-cross reactive antigen
component, are bound, and means for detecting the presence of
antigen-antibody complexes. The support is preferably chosen from
the group of membranes, dip-sticks, filters, spheres, granules and
microtiter plates. The means for detecting may comprise the means
for detecting in a manner as described herein above.
[0032] The invention will now be elucidated by the following
non-restrictive examples.
EXAMPLES
[0033] Preparation of KP90/KS90
[0034] KP90 was prepared from starting material from crude
mycobacterial mass as described in WO-A-94/14069 (the contents of
which are incorporated herein by reference). Starting material made
from 1 gram lyophilized bacteria was centrifuged at 90,000.times.g
at 4.degree. C. for 2 hours. The pellet was washed two times with
PBS. Between the washing steps the sample was sonicated for
6.times.30 seconds on ice with interruptions of 10 seconds and
centrifuged at 90,000.times.g at 4.degree. C. for 2 hours. The
pellet was collected and resuspended in 10 ml 0.05 M Tris buffered
saline (TBS) pH 7.4. The supernatants, designated as KS90, can also
be used as antigen. The pellet was sonicated for 6.times.15 seconds
on ice with interruptions of 10 seconds. This preparation is
designated with the term KP90. After the protein concentration was
determined, quantities of 1.7 ml were frozen at concentrations of 1
mg/ml in 25 mM TBS/50% glycerol and stored at -20.degree. C. The
following components have been shown to be present in KP90: LAM
(++), 10 kD (+), 16 kD (+), 21 kD (+), 24 kD (?), 30 kD (+/-), 31
kD (-), 34 kD (+/-), 38 kD (-), 65 kD (++), and 95 kD (+).
[0035] Preparation of a Culture Fluid Fraction
[0036] After culturing Mycobacterium tuberculosis for 3 weeks, the
bacteria were removed by centrifugation and filtration. Components
from the culture fluid were precipitated by 0-45% ammonium sulphate
precipitation. After centrifugation, the precipitate was dialyzed
and further purified by ion exchange chromatography. This fraction
is called antigens. Further purification can be performed by
hydrophobic interaction chromatography.
[0037] Preparation of Monoclonal Antibodies (MoAb)
[0038] IgG monoclonal antibodies were prepared according to the
literature (Clin. Exp. Immunol. (1984) 58:511-521)
[0039] Preparation of Saliva
[0040] Saliva was collected using the Omni-Sal.TM. saliva
collection device (Saliva Diagnostic Systems) and stored at
-20.degree. C. The tube contains 1 ml of a preservative solution.
The pad is designed to hold 1 ml of fluid when saturated, resulting
in a 1:2 dilution of saliva. Before use the saliva sample was
pretreated with protease inhibitor and triton X and nonidet P40
(0.01%)
[0041] Preparation of Serum
[0042] Blood was obtained by venipuncture and processed to serum
using standard methods.
[0043] EIA
[0044] Coat
[0045] Before coating KP90 was sonicated for 5.times.10 seconds on
ice with 10 seconds interruptions.
[0046] Microtiter plates were coated with KP90 in several dilutions
in PBS pH8.0 for 22 hrs at 37.degree. C., or with one or more MoAbs
(IgG or IgM) against components of M. tuberculosis in several
dilutions in PBS pH 8.0 overnight at 4.degree. C. MoAbs that were
tested were a.o.: a MoAb against a 38 kD protein of M.
tuberculosis, and a MoAb against LAM. Combination coatings of MoAbs
and KP90 were made by coating first with KP90 and then with one or
more MoAbs, or by first coating with KP90 on one half of the
microtiter plate well, while the plate was placed under an angle
followed by coating with one or more MoAbs after the plate was
turned 180.degree..
[0047] After coating the plates were blocked with 3% BSA for 1 hour
at room temperature dried at 37.degree. C. and stored at 4.degree.
C.
[0048] Test
[0049] Sera were tested in a 1:200 and 1:400 dilution. Saliva
samples were tested in several dilutions ranging from 1:1 to
1:100.
[0050] 100 .mu.l was pipetted into the coated wells of a microtiter
plate and incubated for 1 hour at 37.degree. C. Non-binding serum
components are washed away in a washing step with PBS. A second
incubation with a conjugate, either anti-Hu-IgA, or anti-Hu-IgM,
labeled with peroxidase, or another MoAb against the same component
of M. tuberculosis which is monitored labeled with peroxidase, or a
combination of these two is performed for 1 hour at 37.degree. C.
Excess conjugate is then washed away.
[0051] Indication of the presence of human antibodies of the
sub-type IgA or IgM binding specifically to KP90 and/or the
presence of M. tuberculosis components in the sample takes place by
adding TMB (tetramethylbenzidine) to the wells.
[0052] Binding enzyme results in the occurrence of a blue color
which, after addition of a coloring stop solution, changes to
yellow. This yellow color has an absorption maximum of 450 nm.
[0053] Interpretation of the test results takes place on the basis
of the so-called cut-off sample. A test sample can be considered
positive when the result found in the test scores higher than the
cut-off sample. The cut-off sample is based on results with a large
panel of positive and negative subjects.
[0054] Results
[0055] After performing the procedures described above under `Test`
and `Coat`, the results as shown in Table I were obtained regarding
the presence of antibodies in saliva. For determining
anti-KP90-IgA, a saliva dilution of 1:100 was find to be suitable.
For determining anti-KP90-IgG, a saliva dilution of 1:20 appeared
to be suitable. In Table I, samples 38, 39, 40, 44, 4,18, 25, 31,
and 33 were TB-positive, whereas the other samples were
TB-negative.
[0056] A combination of an anti-Mycobacterium IgA and IgG antibody
detection is saliva, wherein different cut-offs are employed, has
additional value in diagnostics. Also, testing saliva in
combination with serum has been found to have additional value in
Mycobacterium diagnostics.
1TABLE I detection of anti-Mycobacterium IgA and IgG in saliva
samples IgA (saliva IgG (saliva 1:100) +/- bij OD 1:20) +/- bij OD
Saliva OD450 CO = 0, 7 OD 450 CO = 0, 22 1 0.689 - 0.074 - 2 0.345
- 0.064 - 3 0.302 - 0.050 - 5 0.321 - 0.065 - 6 0.462 - 0.063 - 7
0.141 - 0.071 - 9 0.509 - 0.075 - 10 0.609 - 0.066 - 12 0.446 -
0.162 - 15 0.173 - 0.054 - 16 0.335 - 0.108 - 22 0.561 - 0.100 - 23
0.389 - 0.096 - 28 0.177 - 0.064 - 32 0.305 - 0.178 - 34 0.683 -
0.173 - 35 0.55 - 0.213 - 36 0.567 - 0.070 - 38 0.742 + 0.234 + 39
0.351 - 0.364 + 40 0.555 - 0.301 + 44 0.801 + 0.235 + 4 1.074 +
1.152 + 18 0.833 + 0.106 - 25 1.064 + 0.678 + 31 0.059 - 2.204 + 33
1.945 + 0.077 -
[0057] In order to determine the presence of LAM in saliva, saliva
was first prepared as described above. Additionally, 1 ml samples
were dialyzed in order to remove triton and salt, whereafter they
were freeze-dried and taken up in 200 .mu.l PBS. Of the thus
obtained samples, 100 .mu.l was pipetted in each well of a
microtiter plate provided with a coat as described above. Detection
took place as described above using rabbit anti-M. tuberculosis
antibodies, which were in their turn detected using
anti-rabbit-IgG-peroxidase conjugate. The results are shown in
Table II.
2TABLE II detection of LAM in saliva Saliva OD1 OD2 mean 1
TB-positive 0.924 0.97 0.947 2 TB-positive 0.737 0.79 0.764 3
TB-positive 0.472 0.453 0.463 4 TB-positive 0.722 0.734 0.728 5
TB-positive 0.439 0.431 0.435 6 TB-positive 0.599 0.603 0.601 7
TB-positive 0.72 0.693 0.707 8 TB-positive 0.69 0.565 0.628 9
TB-positive 0.643 0.669 0.656 mean 0.659 10 TB-negative 0.289 0.285
0.287 11 TB-negative 0.353 0.338 0.346 12 TB-negative 0.294 0.293
0.294 13 TB-negative 0.296 0.294 0.295 14 TB-negative 0.298 0.297
0.298 15 TB-negative 0.315 0.309 0.312 16 TB-negative 0.35 0.34
0.345 17 TB-negative 0.353 0.344 0.349 18 TB-negative 0.253 0.261
0.257 mean 0.309
[0058] Further, IgG, IgA, and LAM were detected in sputum samples
analogous to what has been described above for saliva. Anti-M.
tuberculosis antibodies were determined in sputum using KP90.
[0059] To detect antibodies in sputum, samples of sputum were
diluted 1:20 or 1:25 (for IgG and IgM) or 1:100 (for IgA) in serum
dilution buffer from the KREATECH IgA EIA kit. The samples were
vigorously shaken for at least one hour. Subsequently, the samples
were tested for the presence of anti-tuberculosis antibodies by
ELISA as described above.
[0060] KP90 was coated on a microtiter plate. The cut off ratio was
8 for IgG and IgM and 0.6 for IgA with serum 8-59 as a reference to
calculate the ratio. The results are shown in Table III.
3TABLE III detection of antibodies in sputum Sputum IgG IgA IgM 1
TB-negative negative positive negative 2 TB-negative negative
positive negative 3 TB-negative negative negative negative 4
TB-negative negative negative negative 5 TB-negative negative
positive negative 6 TB-negative negative negative negative 7
TB-negative negative negative negative 8 TB-negative negative
negative negative 9 TB-positive positive positive positive 10
TB-positive positive negative positive 11 TB-positive positive
negative negative 12 TB-positive negative positive positive
[0061] Western Blot
[0062] Gelelectrophoresis and preparation of membrane for assays
was performed as described in WO-A-94/14069, page 13 under point
2.
[0063] Strips were incubated with 1:100 or 1:200 diluted human
serum or saliva collected with an Omni-Sal device dilutions ranging
from 1:1 to 1:100.
[0064] For the detection of antibodies in serum and saliva
immunodetection was performed as described in WO-A-94/14069.
[0065] Dot Blot Assay
[0066] Antigen (KP90) 1-10 .mu.g/ml and MoAb 0.5-5 .mu.g/ml were
spotted separately onto nitrocellulose membrane. The membrane
(blocked or unblocked with BSA) was incubated with serum (1:200
dilution) or saliva sample (1:1-1:100 dilution) in PBS/tween/BSA
for 1 hour at room temperature. After washing the membrane with
PBS/tween the membrane was incubated incubation with conjugate:
[0067] 1. Indirect Label:
[0068] a combination of anti-Hu-IgA or anti-Hu-IgG labeled with
peroxidase and a MoAb (IgG or IgM) against the same component of M.
tuberculosis which is monitored labeled with peroxidase. Detection
was performed using AEC substrate (0.8% 3-amino-9-ethylcarbazole in
dimethylformamide) 1:10 diluted in AEC buffer (50 mM Acetate
Buffer, pH5.0, 0.1% ureumperoxide), DAB (3,3'-diamino-benzidine
tetrahydrochloride) using standard methods, or the ECL detection
system of Amersham.
[0069] 2. Direct Label:
[0070] a combination of anti-human-IgA or anti-human-IgG labeled
with gold and a MoAb (IgG) against the same component of M.
tuberculosis, which is monitored, labeled with gold. The IgA-gold
conjugate was obtained commercially or made as described in e.g.
WO-A-96/35696. The MoAb-gold conjugate was made as follows: The
gold particles were obtained commercially. The conjugate was made
according to standard procedures or in the case of platinum-based
linker as described in e.g. WO-A-96/35696.
[0071] Rapid Strip Test
[0072] Antigen (KP90) 1-10 .mu.g/ml and one or more MoAbs (IgG)
0.5-5 .mu.g/ml are immobilized on different lanes on nitrocellulose
membrane. A control lane depending on the Ab which is detected
(anti-IgA or anti-IgG/proteinA) is also applied above the other
lines. The nitrocellulose is used blocked with 0.1% BSA or
unblocked. On a attached absorbent pad anti-IgA-gold conjugate and
MoAb-gold conjugate (Moab against the same antigen as the
immobilized MoAb but recognizing another determinant) are dried.
The IgA-gold conjugate was obtained commercially or made as
described in WO-A-96/35696. The MoAb-gold conjugate was made as
described above. The sample (serum or saliva) is diluted in 1 ml
PBS (pH 7.4) in an glass tube. The test strip is then dropped into
the tube with the absorbent pad downwards. After 15 min to 2 hours,
when the control line was colored, the results were determined.
[0073] Second procedure: Antigen (KP90) 1-10 .mu.g/ml and one or
more MoAbs (IgG) 0.5-5 .mu.g/ml are immobilized on different lanes
on nitrocellulose membrane (see FIG. 1). A control lane depending
on the Ab which is detected (anti-IgA or anti-IgG/proteinA) is also
applied above the other lines. An absorbent pad was attached. The
whole sample was labeled with gold conjugate through a platinum
based linker (see e.g. WO-A-96/35696). Then the strip was dropped
into the sample with the absorbent pad downwards. After 15 min to 2
hours, when the control line was colored, the results were
determined.
[0074] Agglutination Test
[0075] Coating
[0076] The latex particles are washed several times with Borate (pH
8.5) or PBS, by filtration, or by use of ion-exchange resins
depending on the quantity needed. Antigen (KP90 or antigens) or
antibody are coated on latex particles by incubating, for 1-16
hours at 37.degree. C., 20.degree. C. or 4.degree. C., 1 ml of 1%
suspension of the appropriate particles in Borate (pH 8.5) or PBS
containing 0.01% tween-20 and a final protein concentration up to 5
mg/ml. Subsequently the latex is centrifuged and the supernatant
containing any unabsorbed ligand is discarded. After several
washing steps (see above) the coated latex is re-suspended in MES
buffer containing 0.1% BSA and can be used in a latex agglutination
test or stored at +4.degree. C.
[0077] Agglutination
[0078] The coated latex particles are mixed in several dilutions
with serum or saliva sample in a drop on a glass slide.
Agglutination is assayed after 1 at room temperature to 16 hours at
+4.degree. C. incubation time. The agglutination can be scored by
eye (floc appearance) or after filtration of the mixture. In the
latter case a pore size of the membrane is chosen which allows non
agglutinated latex particles to pass and retains agglutinated
particles. By using colored particles the membrane is colored in
case of a positive reaction.
[0079] By using different colored latex particle for antigen (KP90
or antigen6) and antibody both reactions can be monitored
separately.
[0080] Results
[0081] Serum was tested in accordance with the above procedure
using KP90 and polystyrene particles. The incubation time of the
test was from 5 minutes at room temperature. The results are shown
in FIG. 1. FIG. 1A shows a positive reaction, whereas FIG. 1B shows
a negative reaction.
* * * * *