U.S. patent application number 11/480482 was filed with the patent office on 2006-11-02 for dot-elisa for the detection of animal viruses.
This patent application is currently assigned to The Penn State Research Foundation. Invention is credited to Huanguang Lu.
Application Number | 20060246429 11/480482 |
Document ID | / |
Family ID | 29712217 |
Filed Date | 2006-11-02 |
United States Patent
Application |
20060246429 |
Kind Code |
A1 |
Lu; Huanguang |
November 2, 2006 |
Dot-elisa for the detection of animal viruses
Abstract
A monoclonal antibody-based Dot-ELISA assay for the rapid
detection of animal viruses such as avian influenza virus. The
assay includes applying a specimen suspected of containing an
animal virus on a porous membrane and treating the specimen with a
solution of citric acid or lactic acid and a solution containing a
mucolytic agent and a detergent. The treated specimen is then
contacted with a primary monoclonal antibody for detecting the
virus. If present, the primary moncolonal antibody bind with an
antigen of the animal virus specimen. The specimen is contacted
with an anti-monoclonal antibody conjugate (secondary antibody) and
incubated to facilitate binding of the antigen-bound monoclonal
antibody to the conjugate. The bound conjugate and antigen-bound
monoclonal antibody is contacted with a coloring reagent to allow
visual detection of the presence of the animal virus in the
specimen.
Inventors: |
Lu; Huanguang; (State
College, PA) |
Correspondence
Address: |
MCDERMOTT WILL & EMERY LLP
600 13TH STREET, N.W.
WASHINGTON
DC
20005-3096
US
|
Assignee: |
The Penn State Research
Foundation
University Park
PA
|
Family ID: |
29712217 |
Appl. No.: |
11/480482 |
Filed: |
July 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10448333 |
May 30, 2003 |
7083912 |
|
|
11480482 |
Jul 5, 2006 |
|
|
|
60385841 |
May 31, 2002 |
|
|
|
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
G01N 33/56983 20130101;
G01N 2469/10 20130101; Y10S 435/962 20130101; G01N 2333/11
20130101 |
Class at
Publication: |
435/005 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Claims
1. A method for the detection and identification of an animal virus
in a specimen, comprising the steps of: (a) providing a substrate
for performing a monoclonal antibody-based assay; (b) applying to
the substrate a specimen suspected of containing an animal virus;
(c) applying to the substrate a solution containing an organic
acid; (d) applying to the substrate a solution containing a
mucolytic agent and a detergent; (e) contacting the substrate with
a primary monoclonal antibody and for a time sufficient to allow
the monoclonal antibody and an antigen of said animal virus
specimen to bind together to form an antigen-bound primary
monoclonal antibody; (f) contacting the antigen-bound primary
monoclonal antibody with an anti-monoclonal antibody conjugate for
a time sufficient to facilitate binding of the antigen-bound
monoclonal antibody to the conjugate; and (g) applying a color
reagent to the substrate, the color reagent capable of binding to
the conjugate and developing a colored marking to allow visual
detection of the presence of animal virus in the specimen.
2. The method of claim 1, wherein the organic acid is citric acid
or lactic acid.
3. The method of claim 2, wherein the substrate is a porous
membrane.
4. The method of claim 3, wherein the porous membrane is a
nitrocellulose membrane.
5. The method of claim 2, wherein the color reagent is an alkaline
phosphatase substrate.
6. The method of claim 1 wherein the solution of step (d)
comprises: a mucolytic agent selected from the group consisting of
dithiothreitol, N-acetyl-L-cysteine, and bromhexine hydrochloride,
and a non-ionic detergent selected from the group consisting of an
octoxynol and an octylglucoside.
7. The method of claim 6, wherein the solution of step (d)
comprises dithiothreitol and an octoxynol.
8. The method of claim 7, wherein the solution of step (d) further
includes sodium azide.
9. The method of claim 1, wherein the animal virus has a monoclonal
antibody specific to said virus.
10. The method of claim 1, wherein the animal virus comprises avian
influenza virus. avian infectious bronchitis virus, infectious
bursal disease virus, infectious larygotrachitis virus, fowl
adenovirus, fowl pox virus, avian reovirus, avian rotavirus, swine
influenza virus or equine influenza virus.
11. A monoclonal body assay kit for the detection and
identification of an animal virus comprising: a substrate for
performing a monoclonal antibody-based assay, a solution of an
organic acid, a solution of a detergent and a mucolytic agent, a
primary monoclonal antibody for detecting the virus, an
anti-monoclonal antibody conjugate, and a coloring reagent.
12. The monoclonal body assay kit of claim 11, wherein the solution
of the detergent and a mucolytic agent comprises: a mucolytic agent
selected from the group consisting of dithiothreitol,
N-acetyl-L-cysteine, and bromhexine hydrochloride, and a non-ionic
detergent selected from the group consisting of an octoxynol and an
octylglucoside.
13. The monoclonal body assay kit of claim 12, wherein the solution
of the detergent and a mucolytic agent comprises dithiothreitol and
an octoxynol.
14. The monoclonal body assay kit of claim 13, wherein the solution
of a detergent and a mucolytic agent further includes sodium
azide.
15. The monoclonal body assay kit of claim 11, wherein the organic
acid is citric acid or lactic acid.
16. The monoclonal body assay kit of claim 15, wherein the color
reagent is an alkaline phosphatase substrate.
17. The monoclonal body assay kit of claim 16, wherein the porous
membrane is nitrocellulose.
18. A solution for use in a monoclonal body assay comprising: a
mucolytic agent selected from the group consisting of
dithiothreitol, N-acetyl-L-cysteine, and bromhexine hydrochloride,
and a non-ionic detergent selected from the group consisting of an
octoxynol and an octylglucoside.
19. The solution claim 18, wherein the solution comprises
dithiothreitol and an octoxynol.
20. The solution of claim 19, wherein the solution further includes
sodium azide.
Description
CROSS REFERENCE TO A RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/385,841, filed May 31, 2002.
FIELD OF THE INVENTION
[0002] The present invention generally relates to methods for
detecting and identifying animal viruses present in a clinical and
virus cultural specimen with available monoclonal antibodies to the
specific virus.
BACKGROUND OF THE INVENTION
[0003] Avian influenza is a rapidly spread aerosolized bird disease
and rapidly infects flocks or birds when outbreak occurs. Isolation
of avian influenza virus (AIV) using embryonating chicken eggs
(ECE) has been the standard test, although it is a labor intensive
and time consuming procedure. All 15 subtypes of AIV replicate
successfully in ECE as measured by hemagglutination (HA) test. The
hemagglutination-inhibition (HI) and neuraminidase-inhibition (NI)
tests are commonly used to identify each specific subtype of AIV.
During the past decade, new methodologies using monoclonal
antibodies-based assays and molecular techniques have been evolved
and studied in the demonstration of antigens of AIV directly from
clinical and field specimens. Although these advanced methods are
not yet applicable for routine diagnostic or surveillance purposes,
they have been employed in certain circumstances for the
identification of AIV in conjunction with virus isolation in ECE.
To enhance the rapid detection of AIV in flocks enables the growers
and public authorities to better control the rapid spread of
disease.
[0004] The present invention provides a rapid test for such animal
viruses that is accurate and far less costly than conventional
testing methods. Isolation of AIV using ECE has been the gold
standard to date, but it is a time consuming procedure. The
procedure requires at least a minimum of 2 to 3 days to determine a
test result. Avian influenza is a vexing problem for poultry
producers. Avian influenza infections can range from subclinical
with no symptoms, to mild with some production losses, to severe
with high rates of illness and death. The present invention is
directed to a monoclonal antibody -based dot enzyme-linked
immunosorbent assay (MAb-based Dot-ELISA) test which is cheaper and
a far more rapid test for detecting such viruses than the
procedures and tests described above.
SUMMARY OF THE INVENTION
[0005] The object of this invention is to provide a rapid and less
costly method for detecting animal viruses. To accomplish the
object described above according to the present invention, there is
provided a method using a MAb-based Dot-ELISA which comprises:
[0006] (a) providing a substrate for performing a monoclonal
antibody-based assay;
[0007] (b) applying to the substrate a specimen suspected of
containing an animal virus;
[0008] (c) applying to the substrate a solution containing an
organic acid;
[0009] (d) applying to the substrate a solution containing a
mucolytic agent and a detergent;
[0010] (e) contacting the substrate with a primary monoclonal
antibody and for a time sufficient to allow the monoclonal antibody
and an antigen of said animal virus specimen to bind together to
form an antigen-bound primary monoclonal antibody;
[0011] (f) contacting the antigen-bound primary monoclonal antibody
with an anti-monoclonal antibody conjugate for a time sufficient to
facilitate binding of the antigen-bound monoclonal antibody to the
conjugate; and
[0012] (g) applying a color reagent to the substrate, the color
reagent capable of binding to the conjugate and developing a
colored marking to allow visual detection of the presence of animal
virus in the specimen.
[0013] A further object of the invention is to provide for a
monoclonal body assay kit for the detection and identification of
an animal virus comprising a substrate for performing a monoclonal
antibody-based assay, a solution of an organic acid, a solution of
a detergent and a mucolytic agent, a primary monoclonal antibody
for detecting the virus, an anti-monoclonal antibody conjugate, and
a coloring reagent.
[0014] A still further object of the invention is to provide a
solution of an organic acid for removal of non-viral substances
containing in a test sample that may cause false positive and a
solution of a detergent and a mucolytic agent for virus protein
extraction.
[0015] It is still a further object of the invention that the
method and monoclonal assay kit can be used to detect and identify
animal viruses such as AIV, avian infectious bronchitis virus,
infectious bursal disease virus, infectious larygotrachitis virus,
fowl adenovirus, fowl pox virus, avian reovirus, avian rotavirus,
swine influenza virus or equine influenza virus.
[0016] Other objects and characteristics of the present invention
will become apparent from the further disclosure of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention relates to a MAb-based Dot-ELISA test
that specifically detects the proteins associated with an avian
virus or other animal virus by using specific monoclonal antibodies
to that virus. The procedure and reagents used in the MAb-based
Dot-ELISA test for detection of different avian and other animal
viruses are identical except the primary monoclonal antibodies,
which require homologous specific to a specific virus, are
different.
[0018] The MAb-based Dot-ELISA test of the present invention
detects the AIV directly from clinical and field specimens. The
novel MAb-based Dot-ELISA test detects antigens of all AIV subtypes
when using group-specific monoclonal antibody (MAb) to AIV nucleic
proteins and specifically detects H7N2 subtype when using H7N2
subtype-specific MAb. The MAb-based Dot-ELISA test was specific for
AIV as no cross-reactions were obtained with other avian
viruses.
[0019] The invention is directed to a method for the detection and
identification of an animal virus in a specimen using a MAb-based
Dot-ELISA test. The method comprises the steps of:
[0020] (a) providing a substrate for performing a monoclonal
antibody-based assay;
[0021] (b) applying to the substrate a specimen suspected of
containing an animal virus;
[0022] (c) applying to the substrate a solution containing an
organic acid;
[0023] (d) applying to the substrate a solution containing a
mucolytic agent and a detergent;
[0024] (e) contacting the substrate with a primary monoclonal
antibody and for a time sufficient to allow the monoclonal antibody
and an antigen of said animal virus specimen to bind together to
form an antigen-bound primary monoclonal antibody;
[0025] (f) contacting the antigen-bound primary monoclonal antibody
with an anti-monoclonal antibody conjugate for a time sufficient to
facilitate binding of the antigen-bound monoclonal antibody to the
conjugate; and
[0026] (g) applying a color reagent to the substrate, the color
reagent capable of binding to the conjugate and developing a
colored marking to allow visual detection of the presence of animal
virus in the specimen.
[0027] The animal viruses to be tested include AIV, avian
infectious bronchitis virus (IBV), infectious bursal disease virus
(IBDV), infectious larygotrachitis virus (ILTV), fowl adenovirus
(FAV), fowl pox virus, avian reovirus, avian rotavirus, swine
influenza virus or equine influenza virus. It is not intended that
the MAb-based Dot-ELISA test should be limited to these particular
animal viruses or their accompanying antigens. Rather, it is
contemplated that the MAb-based Dot-ELISA test method of the
present invention may also be used for the detection of other avian
and animal viruses by using available monoclonal antibodies
specific to those viruses.
[0028] The novel features in MAb-based Dot-ELISA test method
involve (i) the treatment solutions A and B, (ii) the selection and
use of a substrate, a porous membrane such as nitrocellulose, and
(iii) the production and application of specific MAb to specific
animal viruses (e.g., AIV H7N2, IBV). The procedure is based on
antigen-antibody reaction. Other reagents in the procedure, such as
detector block solution, washing solution, enzyme labeled goat
anti-mouse IgG conjugate (secondary antibody), and alkaline
phosphatase substrate, are commercially available. These reagents,
while available for use in the MAb-based Dot-ELISA test procedure,
are not specific for the detection of viruses.
[0029] The MAb-based Dot-ELISA test specifically detects the
proteins associated with AIV and IBV. The novel MAb-based Dot-ELISA
test method of the invention is a rapid and effective diagnostic
test. One individual can test up to 100 samples in one run within
two hours.
[0030] The MAb-based Dot-ELISA test method of the present invention
detects live virus antigens, as well as inactivated antigens of
AIV, directly from clinical and field specimens. The sensitivity of
the MAb-based Dot-ELISA test for AIV detection ranges from 25-70%
while the specificity ranges from 80-100% in testing various
clinical and field specimens in comparison with virus isolation
using ECE. Sensitivity is a test value that measures the proportion
of animals that are truly infected or the proportion of specimens
that are truly positive. Specificity is a test value that measures
the proportion of animals that are truly not infected or the
proportion of specimens that are truly negative. Sensitivity and
Specificity are calculated based on a 2.times.2 contiguous table
which classifies test results of a diagnostic test (e.g., MAb-based
Dot-ELISA test) by a standard test (e.g., virus isolation in ECE)
as follows: TABLE-US-00001 Animal Status Infected Non-infected
Total Test + a b a + b Sensitivity = a/(a + c) Positive Test - c d
c + d Specificity = d/(b + d) Negative Total a + c b + d N
[0031] Hemagglutination (HA) and hemagglutination-inhibition (HI)
are standard tests used for identification and serotyping of AIV
after the virus is isolated in ECE or tissue cultures. The
MAb-based Dot-ELISA test method agrees with HA and HI tests in 100%
sensitivity and 100% specificity in testing AIV from ECE or tissue
culture materials.
[0032] The MAb-based Dot-ELISA test method detects AIV antigens in
allantoic fluid samples (virus culture in ECE) that contain a
concentration as low as 0.4 HA unit in a 5 .mu.l sample. The
MAb-based Dot-ELISA test method is specific for AIV and no
cross-reactions are obtained with other avian viruses. The
MAb-based Dot-ELISA test method is highly sensitive and comparable
to a commercial Directigen.RTM. test in the detection of AIV. The
Directigen.RTM. test is designed to detect influenza type A viruses
in humans and it also can be used to detect AIV since AIV belongs
to influenza type A viruses. The Directigen.RTM. test has been used
for AIV detection during AIV outbreaks in recent years, but it is a
very costly test, about $18 per test. Because of the cost, it is
not feasible for mass testing during outbreaks or routine
surveillance as one device for one test sample. Furthermore, the
Directigen.RTM. test only detects common antigens associated with
AIV and does not apply for subtyping of the 15 H subtypes of
AIV.
[0033] The MAb-based Dot-ELISA test method of the present invention
using MAb to IBV has also been successfully applied for the
detection and identification of IBV. It achieves 100% sensitivity
and 100% specificity in comparison with an indirect fluorescent
antibody test (IFA). The IFA is used as a standard diagnostic test
for the detection and identification of IBV from a
specimen-inoculated ECE. IBV containing in a diagnostic specimen
generally requires 3 to 4 serial passages in ECE to be detectable
by the IFA. The MAb-based Dot-ELISA test of the present invention
detects IBV 1 or 2 passages, far earlier than the IFA.
[0034] The testing method of the present invention includes a
nitrocellulose membrane and Dot-ELISA standard reagents such as a
detector block solution, a washing solution, an enzyme labeled goat
anti-mouse IgG (secondary antibody), and an alkaline phosphatase
substrate of 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue
tetrazolium (BCIP/NBT). The MAb-based Dot-ELISA test method
includes two additional treatment solutions: solution A comprising
an organic acid for removal of non-viral substances containing in a
test sample that may cause false positive and solution B comprising
a detergent and a mucolytic agent for virus protein extraction.
[0035] The organic acid of solution A can be citric acid or lactic
acid. A citric acid solution is preferred and can be prepared by
mixing 150 mM citric acid in distilled water.
[0036] Solution B is prepared by mixing 1.5% (v/v) mucolytic agent,
6% (v/v) detergent and 0.2% (w/v) sodium azide. The mucolytic agent
can be dithiothreitol, N-acetyl-L-cysteine or bromhexine
hydrochloride. The detergent can be an octoxynol (such as
Triton.TM. X-100, Triton.TM. X-114 or Igepal.TM. CA-630), or an
octylglucoside (such as octyl-.beta.-D-glucopyranoside), or other
similar non-ionic detergent.
[0037] The following are representative examples of using the novel
MAb-based Dot-ELISA test method of the present invention for the
detection of animal viruses. These examples are not to be
considered as limiting the scope of the invention in any
manner.
EXAMPLE 1
[0038] AIV subtype-specific MAb to H7N2 were produced at the
Monoclonal Laboratory at Penn State University. The general or
group MAb to all subtypes of AIV were provided by Dr. Michael L.
Perdue of ARS, USDA, Beltsville, Md.
[0039] Reference strains of AIV subtypes H3N2, H4N8, H5N3, H6N8,
H7N2 and H9N1 stored in The Pennsylvania State University's Animal
Diagnostic Laboratory were used for sensitive and specific testing
of the MAb-based Dot-ELISA test in the detection of AIV. All 15
subtypes H1 through H15 of AIV were tested at the National
Veterinary Services Laboratories in Ames, Iowa. Other avian
viruses, including IBV, ILTV, PMV, FAV, fowl pox virus, avian
reovirus and avian rotavirus, were used for specific testing of the
MAb-based Dot-ELISA test for AIV.
[0040] The novel MAb-based Dot-ELISA test procedure of the present
invention comprises the following steps:
[0041] (1) Preparing test strips (50-60 mm.times.10 mm) of
nitrocellulose membrane, placing the test strips on chromatography
paper (20.times.20 cm), and then applying test samples to the
strips (5 .mu.l per sample, 5-6 samples per strip), and allowing
the test strips to air dry.
[0042] (2) Treating the test strips with solution A comprising 150
mM citric acid prepared in distilled water for approximately 5
minutes.
[0043] (3) Washing the test strips with a wash solution two times,
for about 30-60 seconds each time.
[0044] (4) Treating the strips with solution B comprising 1.5%
(v/v) dithiothreitol, 6% (v/v) Triton.TM. X-100 and 0.2% sodium
azide for approximately 5 minutes and washing twice as described in
step (3).
[0045] (5) Applying a block solution to the strips, incubating for
approximately 10 to 30 minutes and then allowing the strips to air
dry.
[0046] (6) Applying appropriate monoclonal antibodies for detecting
a specific virus to the strips and incubating for about 15-30
minutes.
[0047] (7) Washing the test strips with washing solution three
times, approximately 2 minutes each time.
[0048] (8) Adding secondary antibody of goat anti-mouse IgG
conjugate to the strips and incubating for approximately 15-30
minutes.
[0049] (9) Washing the strips as described in step (7).
[0050] (10) Adding BCIP/NBT coloring reagent and incubating for
approximately 5-10 minutes in the dark for color development. If
the antigen is present, a purple color will indicate the presence
of the virus. The intensity of the purple color will indicate the
relative concentration of the antigen in the specimen. The reaction
of the coloring reagent can be stopped by adding distilled H.sub.2O
or tap water when a clear purple dot develops on a positive control
sample. If the antigen is absent, no color develops.
[0051] All reactions are conducted at ambient temperature in clean
Pitch dishes. The test strips are placed on chromatography paper to
air dry before and after each reaction.
EXAMPLE 2
[0052] A combination of MAb-based Dot-ELISA test and virus
isolation in ECE was successfully used for the rapid laboratory
diagnosis of AIV within 24 hours during the 2001/02 outbreak in
Pennsylvania. This study was conducted at The Pennsylvania State
University's Animal Diagnostic Laboratory. Clinical and field
specimens including tracheal swabs, cloacal swabs, environmental
swabs and watery manure samples were collected from AIV affected
and suspicious flocks and processed with viral transfer medium.
These specimens were screened first for the presence of antigens of
AIV by the MAb-based Dot-ELISA test, and then were inoculated into
ECE for virus isolation following standard procedures with the
modification of daily withdrawing of allantoic fluid samples. After
20-24 hours, and again 40-48 hours, 0.2-0.5 ml of allantoic fluid
was drawn from the ECE that had been inoculated with a specimen
which was positive for AIV by MAb-based Dot-ELISA test or from a
case clinically suspicious for AIV infection. After allantoic fluid
samples were drawn, the ECE were resealed and placed beck to egg
incubator for continuous incubation up to 72 to 96 hours. The early
incubation allantoic fluid samples were tested for AIV by the
MAb-based Dot-ELISA test and hemagglutination (HA) test.
[0053] Among 7 flocks affected by AIV during the 2001/02 outbreak
in Pennsylvania, the MAb-based Dot-ELISA test detected AIV directly
from clinical specimens before virus isolation from 2 broiler
breeder flocks, and 4 of 5 broiler flocks. The presence of AIV was
confirmed by virus isolation in ECE within 24 hours by means of the
modified procedure.
[0054] Findings in this study showed that the H7N2 virus present in
a clinical specimen grew rapidly in ECE and yielded sufficient HA
titers for AIV identification within 24 hours post inoculation if
the specimen was positive or suspicious for AIV by the MAb-based
Dot-ELISA test. The combination of AIV screening test by MAb-based
Dot-ELISA test and virus isolation in ECE provides a rapid and
effective laboratory diagnosis of AIV during an outbreak.
[0055] The MAb-based Dot-ELISA test is a rapid same day test and
comparable to the commercial Directigen.RTM. test in the detection
of AIV antigens from clinical and field specimens when group
specific MAb to AIV nucleic proteins are utilized. The MAb-based
Dot-ELISA test also specifically detects H7N2 subtype of AIV when
using H7N2 subtype-specific MAb.
EXAMPLE 3
[0056] The MAb-based Dot-ELISA test was also evaluated for its
ability to detect and antigenically characterize IBV and was
compared to that of established ECE and indirect IFA procedures.
IBV isolated in ECE from tracheal and rectal swabs collected from
commercial layer flocks (and/or SPF sentinels placed in flocks)
experiencing egg production and/or shell quality problems in
Pennsylvania were used for this procedural evaluation and
comparison.
[0057] Of 81 field samples tested, 73, 75 and 78 samples were found
to contain IBV antigen by IFA, by characteristic chicken embryo
lesions assay and by the MAb-based Dot-ELISA test, respectively.
Seventy one of 81 samples assayed were positive by all three
laboratory procedures. Statistical comparisons of these results
strongly suggest that the procedures are equally sensitive in
detecting IBV in field samples. A number of false positive (3
Dot-ELISA; 2 IFA) and negative (4 IFA) reactions were identified.
Additionally, statistical comparisons of the three different
procedures to demonstrated that MAb-based Dot-ELISA test was
significantly more sensitive than IFA (P<0.01) and chicken
embryo lesion (P<0.001) assays.
COMPARATIVE RESULTS
[0058] The method of the present invention provides a low cost and
more rapid determination of viruses than available commercial kit.
By way of example, a comparison to a commercial Directigen.RTM. is
described below.
[0059] A commercial Directigen.RTM. kit for the direct detection of
human influenza A antigen is manufactured by Becton Dickinson
Microbiology Systems in Cockeysville. Md. and distributed by VWR
Scientific Products Corporation. The test specimens used for both
MAb-based Dot-ELISA test and Directigen.RTM. test were tracheal
swabs, cloacal swabs, and manure samples. Other environmental
samples were collected from experimentally H7N2 infected SPF
chickens, H7N2 positive flocks of field outbreak, and routine AIV
surveillance submissions. All the clinical and field samples were
processed with viral transfer medium not containing serum and
filtered through 0.45 .mu.m filters. These specimens were first
tested for the presence of antigens of AIV by the MAb-based
Dot-ELISA test using AIV group- or subtype-specific MAb and then
were inoculated into embryonating chicken eggs for virus isolation.
The virus isolation results were determined by the HA and HI
tests.
[0060] The MAb-based Dot-ELISA test was found to be specific for
all subtypes of AIV by using group-specific MAb to AIV nucleic
protein. The H7N2 MAb was specific for H7N2 subtype and had no
reaction to other subtypes. Reactions of the MAb-based Dot-ELISA
test using AIV group and subtype-specific MAb did not occur with
other viruses tested, namely, IBV, ILTV, PMV, FAV, fowl pox virus,
avian reovirus and avian rotavirus.
[0061] The MAb-based Dot-ELISA test was highly sensitive and
comparable to the commercial Directigen.RTM. test in the detection
of AIV. Both assays were evaluated using two-fold serial dilutions
of HA unites of H5N3 reference strain and field isolates of H7N2
virus obtained in Pennsylvania poultry in 1997 and 2001. The
MAb-based Dot-ELISA test detected AIV antigens in allantoic fluid
specimens as low as 0.4 HA unit using a 5 .mu.l sample per test.
The reactions increased in intensity as the HA units were
increased. Reactions were not clearly observed on the test
specimens which contained less than 0.4 HA unit. By comparison, the
Directigen.RTM. test detected the same concentration of 0.4 HA unit
of allantoic fluid specimens. The results are summarized in Table 1
below. TABLE-US-00002 TABLE 1 Comparison of the sensitivities of
the MAb-based Dot-ELISA test and Directigen .RTM. test in detection
of AIV in allantoic fluid samples measured by HA units MAb-based
Dot-ELISA Two-fold dilutions of AIV.sup.a test using AIV HA units
in AF sample group-specific MAb Directigen .RTM. test 6.4 +++ +++
3.2 +++ +++ 1.6 ++ ++ 0.8 + + 0.4 + + 0.2 - - 0.1 - - 0.05 - -
.sup.aThe AIV subtypes used for this test including 2 field
isolates of H7N2 (PA/H7N2/chicken/3779-2/97 and
PA/H7N2/broiler/8015/01) and 2 reference strains of H5N3 and
H3N2.
Sensitivity and Specificity of MAb-Based Dot-ELISA Test
[0062] Sensitivity (Se) and specificity (Sp) of the MAb-based
Dot-ELISA test in the detection of AIV from clinical and field
specimens are set forth in Table 2 below. The Table is divided into
results for four trials. Three trials were directed to SPF chickens
infected with H7N2 virus. The fourth trial is directed to H7N2
outbreak in broiler chickens in Pennsylvania in 2001/02.
[0063] Three clinical trials of SPF chickens infected with H7N2
virus were conducted as models for Se and Sp evaluation of the
MAb-based Dot-ELISA test for screening a large number of samples to
detect antigens of AIV. Se and Sp of the MAb-based Dot-ELISA test
in the detection of AIV were calculated using a 2.times.2
contiguous table set forth above, in which the virus isolation
results in ECE were used as a gold standard test. The MAb-based
Dot-ELISA test detected virus antigens directly from tracheal,
cloacal and environmental swabs, in which the Se ranged from 25% to
70% and the specificity ranged from 80% to 100% in testing various
clinical specimens. An average of 45-57% Se and 85-90% Sp was
obtained in calculation of total test specimens together per trial.
The results are summarized in Trials 1-3 of Table 2.
[0064] In testing field specimens collected from affected broiler
flocks of the 200 1/02 Al outbreak in Pennsylvania, the MAb-based
Dot-ELISA test obtained over 40-88% or an average of 68% of Se, and
80-100% or an average of 90% of Sp in the detection of AIV directly
from tracheal swabs and cloacal swabs of live birds, lung, and body
exudative fluids of dead birds, manure and environmental samples in
affected chicken houses. The results are summarized in Trial 4 of
Table 2 below. The Dot-ELISA yielded 100% Se and 100% Sp in testing
allantoic fluid samples after the clinical and field specimens were
passaged through embryonating chicken eggs for virus isolation. The
MAb-based Dot-ELISA test was highly sensitive for the detection of
AIV in allantoic fluids and agreed with the HA/HI test for AIV
identification. TABLE-US-00003 TABLE 2 Sensitivity (Se) and
specificity (Sp) of the MAb-based Dot-ELISA test in the detection
of AIV from tracheal, cloacal and environmental swabs of
experimentally infected SPF chickens and a field Al outbreak in
broiler chickens. AIV clinical Trials and type of Number of
specimens specimens Se % (a/(a + c)) Sp % (d/(b + d)) 2.1. Trial-1:
40 five-week-old SPF chickens inoculated with AIV
(PA/H7N2/chicken/3779-2/97) Tracheal swabs of group A.sup.a and
B.sup.b 37 25.00 (5/25) 100 (17/17) combined Cloacal swabs of group
A and B combined 93 70.97 (22/31) 82.26 (51/62) Environmental swabs
of group A and B 45 68.42 (13/19) 84.62 (22/26) combined Total
swabs tested.sup.c 175 57.14 (40/70) 85.71 (90/105) Allantoic fluid
samples.sup.d 175 100 (70/70) 100 (105/105) 2.2. Trial-2: 30
ten-week-old SPF chickens inoculated with AIV
(PA/H7N2/chicken/3779-2/97) Tracheal swabs of group A.sup.a 154
41.67 (10/24) 90.77 (118/130) Tracheal swabs of group B.sup.b 151
44.44 (12/27) 86.29 (107/124) Cage swabs of group A and B 29 40.00
(4/10) 94.74 (18/19) Filter dust swabs of group A and B 23 62.50
(5/8) 93.33 (14/15) Total swabs tested.sup.c 357 44.93 (31/69)
89.24 (257/288) Allantoic fluid samples.sup.d 357 100 (169/169) 100
(257/257) 2.3. Trial-3: 26 fourteen-week-old SPF chickens
inoculated with AIV (PA/H7N2/chicken/3779-2/97) Tracheal swabs of
group A.sup.a 115 47.83 (11/23) 91.30 (84/92) Tracheal swabs of
group B.sup.b 117 37.04 (10/27) 86.67 (78/90) Cage swabs of group A
and B 29 50.00 (5/10) 89.47 (17/19) Filter dust swabs of group A
and B 20 70.00 (7/10) 100 (10/10) Total swabs tested.sup.c 281
47.14 (33/70) 89.57 (189/211) Allantoic fluid samples.sup.d 281 100
(70/70) 100 (211/211) 2.4. The 2001/02 AIV (H7N2) outbreak in
broiler chickens in Pennsylvania Tracheal swabs 40 80.77 (21/26)
100 (14/14) Cloacal swabs 34 40.00 (10/25) 88.89 (8/9)
Environmental swabs 39 88.89 (16/18) 80.95 (17/21) Total swabs
tested.sup.c 113 68.12 (47/69) 90.91 (40/44) Allantoic fluid
samples.sup.d 113 100 (69/69) 100 (44/44) .sup.aGroup A birds in
each of the three trials received a volume of 0.5 ml of 50,000
chicken embryo lethal dose 50% (ELD.sub.50) per bird via a
combination of oral, eye and nasal droplets. .sup.bGroup B birds in
each of the three trials received a volume of 0.5 ml of 500,000
chicken embryo lethal dose 50% (ELD.sub.50) per bird via a
combination of oral, eye and nasal droplets. .sup.cTotal swab
samples tested by the MAb-based Dot-ELISA test before inoculation
to ECE. .sup.dAllantoic fluid samples tested by the MAb-based
Dot-ELISA test after one passage of the swab samples in ECE.
Comparison of Sensitivity and Specificity Data for the MAb-Based
Dot-ELISA Test and Directigen.RTM. Tests
[0065] No significant difference was seen between the MAb-based
Dot-ELISA test and Directigen.RTM. test for their Se and Sp in the
detection of AIV. Both assays detected AIV directly from clinical
samples of tracheal, cloacal and environmental swabs from
experimentally infected SPF chickens and 200 1/02 H7N2 outbreaks in
broiler flocks in Pennsylvania. The results are summarized in
Tables 3 and 4 below. TABLE-US-00004 TABLE 3 Comparison of the
MAb-based Dot-ELISA test and Directigen .RTM. test in the detection
of AIV directly from clinical specimens of tracheal, cloacal and
environmental swabs collected from experimentally infected SPF
chickens (Al trial-I). 3.1. Sensitivity (Se) and (specificity) Sp
of MAb-based Dot-ELISA and Directigen .RTM. in testing a sample
group of samples. Virus Isolation in ECE + - Total Dot-ELISA + 9 1
10 Se = 9/21 = 42.86% - 12 7 19 Sp = 7/8 = 87.50% Total 21 8 29
Directigen .RTM. + 8 1 9 Se = 8/21 = 38.10% - 13 7 20 Sp = 7/8 =
87.50% Total 21 8 29 3.2. Comparison of the MAb-based Dot-ELISA
test and Directigen .RTM. test by the McNemar's chi-square test.
Directigen .RTM. test + - Total Dot-ELISA + 9 1 10 - 0 19 19 Total
9 20 29 X.sup.2 = (|0 - 1|- 1).sup.2/(0 + 1) = 0 Since X.sup.2 = 0
< X.sup.2.sub.1,0.95 = 3.84, thus p < 0.05 Therefore, the two
assays are not significantly different.
[0066] TABLE-US-00005 TABLE 4 Comparison of the MAb-based Dot-ELISA
test and Directigen .RTM. test in the detection of AJY from
clinical specimens of tracheal, cloacal and environmental swabs
collected from broiler flocks affected with H7N2 virus during the
2001/02 outbreaks in Pennsylvania. 4.1. Sensitivity (Se) and
specificity (Sp) of MAb-based Dot-ELISA and Directigen .RTM. in
testing a sample group of samples. Virus Isolation in ECE + - Total
Dot-ELISA + 43 4 47 Se = 43/69 = 62.32% - 26 40 66 Sp = 40/44 =
90.91% Total 69 44 113 Directigen .RTM. + 40 2 42 Se = 40/69 =
57.97% - 29 42 71 Sp = 42/44 = 95.45% Total 69 44 113 4.2.
Comparison of the MAb-based Dot-ELISA test and Directigen .RTM.
test by the McNemar's chi-square test Directigen .RTM. Utest + -
Total Dot-ELISA + 41 6 47 - 1 65 66 Total 42 71 113 X.sup.2 = (|1 -
6|- 1).sup.2/(1 + 6) = 2.28 Since X.sup.2 = 2.28 <
X.sup.2.sub.1,0.95 = 3.84, thus p < 0.05 Therefore, the two
assays are not significantly different.
[0067] The results of comparison studies between the MAb-based
Dot-ELISA test and Directigen.RTM. test indicate that both assays
possessed equivalent Se and Sp and thus both assays are equally
sensitive and specific in the detection of AIV. However, both
assays in the AIV detection were less sensitive than virus
isolation in ECE (Tables 3.1 and 4.1). This is because ECE can
amplify AIV to a detectable titer during a period of 2-to-3 days of
incubation although an inoculum contains a very low concentration
of the virus, whereas the MAb-based Dot-ELISA test and
Directigen.RTM. test do not amplify AIV. Thus, they require a
minimum concentration of virus particles (e.g., 0.4 HA unit)
presenting in a test specimen to produce a visualized positive
reaction.
[0068] Nonetheless, specimens collected from AIV-infected birds
during an outbreak shall have adequate virus particles to be
detectable by the MAb-based Dot-ELISA test or Directigen.RTM. test.
The MAb-based Dot-ELISA test results in testing clinical and field
specimens from AIV-infected birds in this study indicated that
between 45% and 68% in average of such specimens containing AIV
(H7N2) were successfully identified as AIV positive by the
MAb-based Dot-ELISA test (Table 2). Specifically in trial 1 (Table
2.1), the MAb-based Dot-ELISA test detected over 70% of AIV
positive cloacal swabs, but only 25% of AIV positive tracheal
swabs. The low 25% Se in testing tracheal swabs was because the
tracheal swabs were collected after two weeks post inoculation,
when the virus concentration in tracheas became very low; whereas
cloacal swabs were collected during the period that birds actively
released virus through intestines and thus the high 70% Se was
achieved. Therefore, a sufficient number of samples and early
sampling of various sources during an outbreak are essential for
the MAb-based Dot-ELISA test to successfully detect AIV and achieve
a high Se in the detection.
Specificity of the MAb-Based Dot-ELISA Test in Testing of AIV
Surveillance Samples
[0069] AIV surveillance samples submitted to the laboratory of the
present inventor are routinely processed for virus isolation using
ECE and are also tested for the presence of AIV antigens by the
MAb-based Dot-ELISA test. From April to December 2001, a total
number of 1589 AIV surveillance samples in 32 submissions have been
tested by both methods. The 1589 samples were all negative for
virus isolation in ECE, 13 out of the 1589 samples in 7 submissions
had positive reactions by the MAb-based Dot-ELISA test. The
specificity of the MAb-based Dot-ELISA test for identification of
AIV negative samples among these 32 submissions ranged from 92% to
100%, or 99.18% of mean Sp, or 0.82% (13/1589) false positive in
comparison with virus isolation in ECE. The results are summarized
in Table 5 below. TABLE-US-00006 TABLE 5 Specificity (Sp) of the
MAb-based Dot-ELISA test on AIV surveillance samples in the
identification of AIV negative specimens in comparison to virus
isolation in ECE No. of No. of Dot-ELISA Serial No. of Number of
positives by positives by Specificity submissions samples Dot-ELISA
virus isolation (%) 1 51 0 0 100 2 70 0 0 100 3 42 0 0 100 4 70 2 0
97.14 5 58 0 0 100 6 55 0 0 100 7 45 0 0 100 8 37 0 0 100 9 47 0 0
100 10 61 0 0 100 11 47 0 0 100 12 70 1 0 98.57 13 45 1 0 97.77 14
55 0 0 100 15 35 0 0 100 16 35 0 0 100 17 77 0 0 100 18 31 0 0 100
19 30 0 0 100 20 36 1 0 97.22 21 54 0 0 100 22 30 0 0 100 23 39 0 0
100 24 40 0 0 100 25 40 3 0 92.51 26 43 1 0 97.67 27 52 0 0 100 28
22 0 0 100 29 10 0 0 100 30 58 0 0 100 31 24 0 0 100 32 120 4 0
96.67 Total 1589 13 0 99.18
[0070] Findings indicate that the MAb-based Dot-ELISA test is
highly sensitive in detecting antigens of AIV. It detected AIV in
allantoic fluids that contained a concentration as low as 0.4 HA
units. The MAb-based Dot-ELISA test using group-specific monoclonal
antibody to AIV nucleic protein detected AIV hemagglutinin subtypes
H3 through H9 tested. A subtype-specific MAb to H7N2 virus
specifically reacted with its homologous H7N2 subtype and had no
cross-reaction to other subtypes of AIV. The availability of
monoclonal antibody to AIV subtype H7 allows the MAb-based
Dot-ELISA test to single out a virulent subtype rapidly from other
virulent AIV strains.
[0071] The MAb-based Dot-ELISA test agreed with HA and HI tests for
AIV identification using allantoic fluids after clinical specimens
were passed through ECE. Allantoic fluids harvested from ECE were
highly sensitive by the MAb-based Dot-ELISA test and its results
were the same to the HA and HI tests for AIV identification.
Therefore, some substances from fecal materials created
non-specific reactions on the MAb-based Dot-ELISA test strips would
be verified as false positives after they were passed through ECE.
On the other hand, a positive reaction by the MAb-based Dot-ELISA
test, but negative for virus isolation in ECE, may be a true
positive for AIV since the MAb-based Dot-ELISA test detects AIV
antigens of both live and dead viruses.
[0072] Furthermore, the novel MAb-based Dot-ELISA test can be
effectively used as a rapid screening and diagnostic test to
identify AIV in various sources of surveillance samples and
clinical diagnostic specimens. Although the MAb-based Dot-ELISA
test can yield false positives under certain circumstances when
test samples are composed of dirty substances or chemicals such as
dark diarrhea-like manure samples, very bloody tracheal swabs,
cloacal swabs containing heavy diarrhea-like manure or
environmental swabs containing chemical disinfectants, false
positive reactions from these kinds of samples can be ruled out by
using clean and quality samples. The Directigen.RTM. test also
yielded false positive results in this study (Table 3.1 and 4.1).
Although the causative substances are not clearly understood, it
appears that specimens containing biological or bacterial
components or certain chemical substances likely cause false
positive reactions in both MAb-based Dot-ELISA test and
Directigen.RTM. test. The optimal samples for the MAb-based
Dot-ELISA test are tracheal and cloacal swabs from suspicious or
affected birds, fresh watery wet droppings and chicken house dust.
The MAb-based Dot-ELISA test is a same-day test and feasible for
mass testing, an individual can screen up to 100 clinical samples
in one run within few hours. The MAbs-based Dot-ELISA test
significantly improved the ability to rapidly detect antigens of
AIV during a field outbreak thus enhancing the ability to control
spread, and it has great economic potential in long-term prevention
and control of AIV outbreaks.
[0073] The novel monoclonal antibody-based MAb-based Dot-ELISA test
can be effectively used as a rapid screening and diagnostic test to
identify animal viruses in different kinds of surveillance samples
and clinical diagnostic specimens. This is a same-day test that can
be conducted on site. An individual can screen up to 100 clinical
samples in one run within two hours. Although the MAb-based
Dot-ELISA test can yield false positives to certain kinds of field
specimens composed of dirty substances or chemicals under certain
circumstances, true positive reactions can be determined with clean
and quantity samples. The optimal samples for the MAb-based
Dot-ELISA test are tracheal and cloacal swabs from suspicious or
affected birds and other animals, fresh watery wet droppings and
chicken house dust.
[0074] The MAb-based Dot-ELISA test significantly improves the
ability to rapidly detect antigens of animal viruses. It can be
applied in mass testing during a field outbreak, or it can be used
for routine surveillance. The MAb-based Dot-ELISA test method of
the present invention costs less than $.50 per test. Its
sensitivity and specificity in the detection of AIV, for instance,
are comparable to the commercial Directigen.RTM. kit, which costs
over $18.00 per test. The MAb-based Dot-ELISA kit of the present
invention has great economic potential in the prevention and
control of outbreaks involving animal viruses.
[0075] While the invention has been particularly shown and
described with reference to preferred embodiments thereof, it will
be understood by those skilled in the art that various alterations
in form and detail may be made therein without departing from the
spirit and scope of the invention.
* * * * *