THE PATENTS ACT, 1970
COMPLETE SPECIFICATION
Section 10
"Method for Quantification of Antigen'
Serum Institute of India Ltd. a Corporation organized and existing under the laws oj India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pane 411 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

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METHOD FOR QUANTIFICATION OF ANTIGEN
FIELD OP INVENTION
This invention relates to a method of quantification of glycoprotein antigen of active, inactive or fragmented virus, without or with adjuvant.
BACKGROUND OF THE INVENTION
Before inactivated tissue culture rabies vaccines released for use in human or animals, such vaccines are assayed by an in vivo .test for immunogenicity. The rabies vaccine in general ' is tested using NIH mouse protection test, which was first developed by Saligman. The test in use is a further standardized procedure by WHO involving two immunizations at 7-days interval, while European pharmacopoeia practices one immunization with different dilutions of the test and reference vaccines followed by intracerebral challenge with a standard rabies virus strain (CVS) ' 14 days later (Saligman Jr EB, 1973). This makes the NIH test expensive and cumbersome that involves the use of live rabies virus and requires long duration. The later test has several disadvantages such as 28 days needed for the completion of the test, the need for about 100
laboratory animals per test, and the use of infectious
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rabies virus. Moreover, the potency results show very high variation in the test results (Lafon M. et al, 1985; Barth R et al., 1988).
Hence, in vitro assays for quantification of the GP antigen are recommended as these assays do not take much time and enable in-process antigen estimation in the production process. These methods are also biohazard-safe and do not violate animal welfare. In this regard, antibody binding test was developed by Arko et al. 1973, which was modified by Barth et al. 1982, who showed that for few years this method was a reliable potency test. However, the need for tissue culture, infectious virus and immunofluorescence complicate this method further. The single radial diffusion test (SRD), which was developed by Ferguson and Schild for the potency assay of rabies vaccines is based on the fact that the content of the rabies virus glycoprotein (GLP) is correlated with the potency of the rabies vaccines (Ferguson and Schild, 1982). This assay was applied after a collaborative study (Ferguson et al. 1984) on human rabies vaccines in several institutions such as Food and Drug Administration in the USA (Fitzgerald and Needy, 1986). However, the low sensitivity of 1 IU/ml and the
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impossibility of testing adjuvanated vaccines restrict the use of SRD test.
Direct ELISA based methods have been described, which use specific antibodies raised against the rabies viral glycoprotein (GP) for quantitation. ELISA with polyclonal antibody against rabies virus GP has been tried for rabies vaccine potency test (Atanasiu et al 1973, Vander Marell and Wezel 1981). The ELISA, however, was found very sensitive method for antigen and antibody determination in rabies research (WHO, 1982) . ELISA has also been recommended for rabies vaccine potency test by various experts (Bogel K. 1984; WHO expert committee, 1984). Later, use of polyclonal antibodies for the direct ELISA was replaced with monoclonal antibodies as monoclonal antibodies recognize a correctly folded glycoprotein (Lafon et al., 1985). Thereafter, an immunocapture ELISA was studied in comparison with direct ELISA and- revealed that the MAb immunocapture ELISA was more sensitive and specific
(Nagarajan et al. 2006). Presently existing ELISA methods
have limitation for analyzing GP content in the adjuvanated vaccines, virus and viral debris that demands a simple, quick, precise and sensitive method (Pierre et al 1990; Rooijakkers et. al 1996).
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SUMMARY OF THE INVENTION
The present invention provides a method for quantification of glycoprotein (GP) of active, inactive or fragmented virus, without or with adjuvant. The instant method comprises immobilization of antibody specific to glycoprotein of interest on to a solid surface, capturing the GP on the antibody affording an antibody-GP immuno-complex, treating the complex with polyclonal anti-GP antibodies followed by treatment with an enzyme labeled secondary antibody and enzyme-substrate chromogen reaction.
The method of instant invention allows direct
quantification of the GP from non-adjuvanated vaccine,
adjuvanated vaccines, live or inactivated virus,
disintegrated viral components, GP in soluble or embedded
form.
The method of the present invention provides a convenient, simple, sensitive and reproducible method to assay a test sample for the presence of total GP in the vaccine samples. Thus, the method may be employed for the quantification of GP at any step during manufacturing and diagnosis to
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determine the extent of rabic infection in the rabic patients.
DETAILED DESCRIPTION OF THE INVENTION
The following description and definitions are provided in order to provide clarity as to the intent or scope of their usage in the specification and claims.
The immunoassay of the present invention provides a convenient, simple, sensitive and reproducible method to assay a test sample for the presence of total GP in the vaccine samples. The assay also enables accurate and quantitative measurement of GP in adjuvanated vaccines (e.g. aluminium phosphate gel adsorbed vaccines). The method exemplifies quantification of Rabies glycoprotein. Rabies GP is considered to be the major protective viral antigen (Wiktor et al). In order to optimize rabies vaccine potency test it is essential to have an assay system, which permits a rapid and accurate estimation of the rabies virus glycoprotein (GP) content of the vaccines. Several tests have been described for quantitation of the glycoprotein. They use tissue culture techniques, enzyme immunoassays techniques, or the single radial diffusion assay (Arko RJ
et al., 1973; Barth R et al., 1981; Atanasiu P et al.,
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1980; Van der Marel P and Van Wezel AL, 1981; Ferguson M et al., 1984).
Unfortunately, no efforts have been made in developing an effective assay, which can determine the potency of adjuvanated vaccines. The method of ELISA reported earlier rely upon direct antigen coating and has been described in the art (patent EP0589348). These techniques are less sensitive method as compared to immunocapture (sandwich) method as the immobilization of antigen can never be 100% and there will be very high competition of different proteins present in virus or virus harvest for binding onto solid phase (Pierre Perrin et al., 1990).
The instant invention overcomes drawbacks of the prior art and provides a better method for quantification of glycoprotein antigen.
The antigen of the instant invention can be in soluble or embedded form. The embedded form of antigen includes antigen in viral coat as well as adjuvanated antigen. The adjuvant selected herein can be any suitable adjuvant used in vaccine preparation as known to a person skilled in the art. The preferred adjuvant is aluminium salts or liposomes
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or nano-particles. The liposomes or nano-particles can be prepared from material comprising of natural, semisyntyhetic or synthetic lipid, polypeptide, carbohydrate, other suitable polymers or their derivatives.
The antigen of the instant invention may be present inside the viral coat of active or inactive virus. The antigen can also be in fragmented form bearing the antigenic epitope.
As per the method of instant invention, one can adsorb the anti rabies GP monoclonal antibodies to the solid support. In the adsorption technique, monoclonal antibody is allowed to adsorb onto the polystyrene surface for a suitable period of time, preferably 1 hour at 37 °C or overnight at 4°C. The art of developing monoclonal antibodies have been described in a number of publications (Kohler G and Milstein C 1975). The monoclonal antibody specific for
exposed native epitope of viral glycoprotein is indigenously developed that reacts with the exposed epitope of the embedded glycoprotein.
The plate can be then treated with a blocking agent. The blocking agent can be an inert protein. The blocking agent is used to prevent adsorption, in subsequent steps, of
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reagents, reactant or proteinaceous material to the solid phase. Preferably, commonly available and inexpensive proteins like albumin and casein are used but virtually any protein inert to the antigen, antibody or other reagents can be used.
The sample to be tested can comprise glycoprotein which may be aluminium phosphate gel adsorbed vaccine, active or inactive virus or viral components, soluble or embedded in viral coat. The test sample can be obtained from an infected patient called *Rabic patient'. The rabic patient sample can be serum or infected tissue.
The test samples can be treated with suitable detergents for releasing glycoprotein from the viral coat. A number of detergents can be used bearing in mind that the used detergent should not interfere with the immunoreaction. One such commercially available detergent is mulgofen, which has been reported for recovering membrane proteins (David AW et al. 1977) .
After incubation, the solid matrix can be washed with water or suitable buffer to remove unbound components. The
instant employs a phosphate buffered saline containing 0.1%
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of a commercially available detergent, Tween 20 surfactant (PBST). It is important to note that the buffer used should
not be one that would alter the conformation of the
antigenic epitopes of the GP molecule.
After washing, the solid matrix with monoclonal antibodies GP complex (if present in the test sample) can be exposed to a preparation of polyclonal antibodies specific for GP under such conditions as to allow the antibodies to bind to other antigenic epitopes present on GP. A number of publicly available antibodies suitable for this purpose may also be used. For example, anti-rabies sheep serum specific for GP may be obtained from U-SFDA, USA or NIBSC, London. The solid matrix can be washed to remove unbound primary antibodies using suitable washing buffer.
A secondary reagent that specifically binds the primary
antibody can then be added to the matrix. Most commonly,
the secondary reagent is an antibody that has specificity
for antigenic epitopes present on the primary antibody. The
secondary antibody is labeled with an enzyme, such as
horseradish peroxidase. For example, anti sheep antibodies
labeled with horseradish peroxidase may be used. Techniques
for enzyme labeling of antibodies and techniques suitable
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for quantification of the amount of chromophore produced are well known to those in the art of enzyme linked immunoassay and need not be described further here, The enzyme labeled antibodies may also be obtained commercially.
After incubation under conditions suitable for the labeled antibodies to bind to their respective epitopes, the amount of antibody bound to the matrix is determined. The relative concentration of GP present in the test sample can then be quantified on the basis of the amount of antibody bound
that is detected by using enzyme substrate chromogen
reagent followed by spectrometric measurements.
The specificity of the assay was established using rabic
glycoprotein (100% reactivity), blind vaccine (0%

reactivity), adjuvant (0% reactivity, rabic virus culture
media (0% reactivity) and host cells / tissues (0% reactivity) using 5th international standard of rabies vaccine (NIBSC). Sensitivity of the assay was found >0.05 IU/ml.
The linearity of the assay was checked at four different concentrations (0.500, 0.250, 0.125, 0.0625 and 0.0312
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IU/ml) of the standard sample. The correlation coefficient (r2) of the standard curve was found > 0.99 which shows the assay to be linear and in range.
The accuracy of the assay was found 100% with <5% variation at 95% CL. The coefficient of variation (CV) of the assay
was found < 2.5%.
These and other aspects of the invention will become more, readily apparent upon review of the examples below. Although the example is intended to illustrate one method of performing the invention, the method of the invention should not be limited by the particulars described below unless specified in the claims.
EXAMPLE 1
Quantification of rabies glycoprotein in virus culture,
culture supernatant and purified virus
The capture antibody (10ng/well), IgG2b MAb, was adsorbed onto the surface of ELISA wells in microtiter plates (F8 Nunc immunomodules, Nunc, Denmark) for either 1 hour at 37°C, or overnight at 4°C. The wells were blocked with blocking buffer 1% BSA (w/V) in 10mM phosphate buffer (pH
7.4) for 1 hour at 37°C. After completion of incubation,
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ELISA strips were washed thrice with washing buffer (10mM phosphate buffer with 100 mM NaCl and 0.1 % Tween-20 pH
7.4) using microtiter plate washer (Biotek Instruments, USA). Dilutions of NIBSC standard (0.500, 0.250, 0.125, 0.0625 and 0.0312 IU/mL) and test samples (active virus, inactive virus, viral fragments, virus in tissue and cell culture media, concentrated virus and its fragments,
soluble and embedded glycoprotein; dilutions: 1:2 - 1:1024) were prepared in the dilution buffer containing 1% (v/V) mulgofen detergent. 100 ml different dilutions of standard as well as test samples were added in the prelabelled microwells of ELISA strips (each dilution in triplicate) . For blank wells, dilution buffer was added in triplicate. The ELISA strips were incubated in a shaker incubator maintained at 37+1°C and 80-90 rpm for 30 min. After completion of incubation, the strips were washed thrice with washing buffer using plate washer. 1:1000 dilution of anti rabies sheep antibodies was prepared in the dilution buffer and 100 ml of diluted anti rabies sheep antibodies was added in each micro well of ELISA strip. The ELISA strips were incubated in an incubator at 37+1°C for 30 min. After completion of incubation, the strips were washed thrice with washing buffer using plate washer. 1:5000
dilution of HRP conjugated anti sheep antibodies was
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prepared in the dilution buffer and 100 m1 of diluted antibodies was added in each micro well of ELISA strips. The ELISA strips were incubated in an incubator at 37 + 1°C for 30 min. After completion of the incubation the strips were washed again with washing buffer three times using plate washer and 100 ml of freshly prepared TMB-H2O2 substrate-chromogen reagent was dispensed in each micro well of ELISA strips. The ELISA strips were kept at 37 + 1°C for 15 min in the dark for color development for 15 min. The reaction was quenched by adding 50ml/well of stop solution (2.5 N H2S04) and absorbance was read within 15 min. 450 nm using ELISA plate reader (Biotek Instruments, USA) and the GP content in the samples was calculated with the standard curve.
EXAMPLE 2
Quantification of rabies glycoprotein in Adjuvanated
Vaccine
The capture antibody (10ng/well), IgG2b MAb, was adsorbed onto the surface of ELISA wells in microtiter plates (F8 Nunc immunomodules, Nunc, Denmark) for either 1 hour at 37°C, or overnight at 4°C. The wells were blocked with blocking buffer 1% BSA (w/V) in 10mM phosphate buffer (pH
7.4) for 1 hour at 37°C. After completion of incubation,.
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ELISA strips were washed thrice with washing buffer (10mM phosphate buffer with 100 mM NaCl and 0.1 % Tween-20 pH 7.4) using microtiter plate washer (Biotek Instruments, USA). Dilutions of NIBSC standard (0.500, 0.250, 0.125, 0.0625 and 0.0312 IU/mL) and adjuvanated vaccine sample (Alum, liposome and nano-particles; dilutions: 1:2 1:1024) were prepared in the dilution buffer containing 1% (v/V) mulgofen detergent. 100 ml different dilutions of standard as well as test samples were added in the prelabelled microwells of ELISA strips (each dilution in triplicate) . For blank wells, dilution buffer was added in triplicate. The ELISA strips were incubated in a shaker incubator maintained at 31+1°C and 80-90 rpm for 30 min. After completion of incubation, the strips were washed thrice with washing buffer using plate washer. 1:1000 dilution of anti rabies sheep antibodies was prepared in the dilution buffer and 100 ml of diluted anti rabies sheep antibodies was added in each micro well of ELISA strip. The ELISA strips were incubated in an incubator at 37+1°C for 30 min. After completion of incubation, the strips were washed thrice with washing buffer using plate washer. 1:5000 dilution of HRP conjugated anti sheep antibodies was prepared in the dilution buffer and 100 ml of diluted antibodies was added in each micro well of ELISA strips.
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The ELISA strips were incubated in an incubator at 31+1°C for 30 min. After completion of the incubation the strips
were washed again with washing buffer three times using plate washer and 100 ml of freshly prepared TMB-H202 substrate-chromogen reagent was dispensed in each micro well of ELISA strips. The ELISA strips were kept at 37 + 1°C for 15 min in the dark for color development for 15 min.
The reaction was quenched by adding 50ml/well of stop solution (2.5 N H2SO4) and absorbance was read within 15 min. 450 nm using ELISA plate reader (Biotek Instruments, USA) and the GP content in the samples was calculated with the standard curve.
EXAMPLE 3
Quantification of rabies glycoprotein in rabic patients
The capture antibody (10ng/well), IgG2b MAb, was adsorbed onto the surface of ELISA wells in microtiter plates (F8 Nunc immunomodules, Nunc, Denmark) for either 1 hour at 37°C, or overnight at 4°C. The wells were blocked with blocking buffer 1% BSA (w/V) in 10mM phosphate buffer (pH
7.4) for 1 hour at 37°C. After completion of incubation, ELISA strips were washed thrice with washing buffer (10mM phosphate buffer with 100 mM NaCl and 0.1 % Tween-20 pH
7.4) using microtiter plate washer (Biotek Instruments,
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USA). Dilutions of NIBSC standard (0.500, 0.250, 0.125, 0.0625 and 0.0312 IU/mL) and test samples (patient serum and tissue from infected site; dilutions: 1:2 - 1:1024) were prepared in the dilution buffer containing 1% (v/V) mulgofen detergent. 100 ul different dilutions of standard as well as test samples were added in the pre-labelled microwells of ELISA strips (each dilution in triplicate). For blank wells, dilution buffer was added in triplicate. The ELISA strips were incubated in a shaker incubator maintained at 37 + l°C and 80-90 rpm for 30 min. After completion of incubation, the strips were washed thrice with washing buffer using plate washer. 1:1000 dilution of anti rabies sheep antibodies was prepared in the dilution buffer and 100 ml of diluted anti rabies sheep antibodies was added in each micro well of ELISA strip. The ELISA
strips were incubated in an incubator at 37+1°C for 30 min. After completion of incubation, the strips were washed thrice with washing buffer using plate washer. 1:5000 dilution of HRP conjugated anti sheep antibodies was prepared in the dilution buffer and 100 ml of diluted antibodies was added in each micro well of ELISA strips. The ELISA strips were incubated in an incubator at 37+l°C for 30 min. After completion of the incubation the strips
were washed again with washing buffer three times using
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plate washer and 100 ml of freshly prepared TMB-H202 substrate-chromogen reagent was dispensed in each micro
well of ELISA strips. The ELISA strips were kept at 31+1°C for 15 min in the dark for color development for 15 min. The reaction was quenched by adding 50ml/well of stop solution (2.5 N H2SO4) and absorbance was read within 15 min. 450 nm using ELISA plate reader (Biotek Instruments, USA) and the GP content in the samples was calculated with the standard curve.
Validation of the method for quantification of glycoprotein antigen
The specificity of the assay was established using rabic glycoprotein (100% reactivity), blind vaccine (0% reactivity), adjuvant (0% reactivity, rabic virus culture media (0% reactivity) and host cells / tissues (0% reactivity) using 5th international standard of rabies vaccine (NIBSC). Sensitivity of the assay was found >0.05 IU/ml.
The linearity of the assay was checked at four different concentrations (0.500, 0.250, 0.125, 0.0625 and 0.0312 IU/ml) of the standard sample. The correlation coefficient
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(r2) of the standard curve was found > 0.99 which shows the assay to be linear and in range.
The accuracy of the assay was found 100% with <5% variation at 95% CL. The coefficient of variation (CV) of the assay was found < 2.5%.

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REFERENCES
1. Ferguson M, Schild GC (1982). A single radial
immunodiffusion technique for the assay of rabies glycoprotein antigen: application for potency tests of vaccines against rabies. J Gen Virol, 59: 197-201.
2. Nagarajan T, Reddy GS, Subramanian M, Rajalakshmi S,
Thiagarajan D, Tordo N, Jallet C and Srinivasan VA (2006).
A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture. Biologicals, 34: 21-27.
3. Seligman Jr EB. (1973) The NIH test for potency. In:
Kaplan MM, Koprowski H, editors. Laboratory techniques in
rabies. 4th ed. Geneva: WHO; 1973, p. 279-286.
4. Ferguson M, Seagroatt V and Schild GC (1984). A
Collaborative study on the use of single radial
immunodiffusion for the assay of rabies virus glycoprotein.
J Biol Stand, 12: 283-294.
5. Fitzgerald EA and Needy CF. (1985) Use of the single
radial-imunodif fusion test as a replacement for the NIH
mouse potency test for rabies vaccine. IABS symposium on
reduction of animal usage in the development and control of
biological products London, U K, 24-26 April, Dev Biol
Stand 1986; 64: 73-97. Basel: S. Karger.
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6. Atanasiu P. (1973) The immunoperoxidase reaction for demonstration of rabies virus In: Kaplan MM, Koprowski H, eds. Laboratory techniques in rabies. Geneva: WHO, 1913: 358-360.
7. Van der Marel P, Van Wezel AL. (1981) Quantitative determination of rabies antigen by ELISA. Dev Biol Stand, 50: 267-275.

8. World Health Organization. Report of consultation on the application of monoclonal antibody in the characterization and differentiation of laboratory and field strains of rabies virus. WHO/Rab Res/82, 15/1982: 1-3.
9. Bogel K. Proposals for human rabies vaccines and their application. In: Vodopija I, Nicholson KG, Smerdel S, Bijok U, eds. Improvements in rabies post exposure treatment. Proceedings Dubrovnik Meeting, Zagreb, Yugoslavia, 30 May-1 June 1984, 1985; 203-208.

10. WHO expert committee on rabies. Seventh Report. WHO Tech Rep Ser 1984; 709.
11. Lafon MP, Perrin P, Versmisse P, Sureau P (1984) . Use of a monoclonal antibody for quantitation of rabies vaccine glycoprotein by enzyme immunoassay. J Biol Stand, 13: 295-301.
12. Perrin P, Morgeaux S and Sureau P (1990). In vitro rabies vaccine potency appraisal by ELISA: advantages of —
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the immunocapture method with a neutralizing anti glycoprotein monoclonal antibody. Biologicals, 18: 321-330.
13. Arko RJ, Wiktor TJ, Sikes RK (1973) The antibody binding test for vaccine potency. In Kaplan MM, Koprowski H, eds. Laboratory techniques in Rabies. 3rd edition. Geneva: WHO: 292-296.
14. Barth R, Gross Albenhausen E, Jaeger O, Mileke L
(1981) . The antibody binding test, a useful method for quantitative determination of inactivated rabies virus antigen. J Biol Stand, 9: 81-89.
15. Atanasiu P, Perrin P, Delagneau JF (1980) . Use of an
enzyme immunoassay with protein A for rabies antigen and
antibody determinations. Dev Biol Stand, 46: 207-215.
16. Wiktor TJ, Gyorgy E, Schlumberger HD, Sokol F,
Koprowski H (1973). Antigenic properties of rabies virus
components. J Immunol, 110: 269-276.
17. Kohler G, Milstein C (1975). Continuous cultures of
fused cells secreting antibody of predefined specificity.
Nature, 256: 495-497.
18. Pierre P, Sylvie M and Pierre S (1990). In vitro rabies
vaccine potency appraisal by ELISA: Advantages of the
immunocapture method with a neutralizing anti-glycoprotein
monoclonal antibody. Biologicals, 18: 321-330.
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19. Rooijakkers EJM, Uittenbogaard JP, Groen J, Osterhaus
ADME (1996). Rabies vaccine potency control: comparision of
ELISA systems for antigenicity testing. J. Virology
Methods, 58: 111-119.

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CLAIMS
1. A method for quantification of glycoprotein antigen using sandwich ELISA technique.
2. A method of claim 1, wherein the glycoprotein is in soluble or embedded form.
3. A method of claim 1, wherein the antigen is present in active virus, inactive virus or fragmented virus.
4. A method of .claim 1, wherein the antigen is adjuvanated or non-adjuvanated.
5. A method of claim 1, wherein the antigen is sampled from
rabic patient.
6. A method of claim 5, wherein the sample is rabic patient
serum or infected tissue.
7. A method of claim 1-4, comprising:
(a) immobilizing an anti-glycoprotein antibody onto a solid surface
(b) treating the solid surface with blocking agent to block the free surface
(c) contacting a test sample containing the antigen to the said anti GP antibody immobilized surface to capture the GP by said anti GP antibody
(d) rinsing away the non GP and unbound material
(e) contacting said captured GP-antibody complex to an anti GP immunoglobulin
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(f) rinsing away the unbound material
(g) contacting anti-immunoglobulin antibody bearing a
label;
(h) quantifying the amount of said label bound.
8. A method of claim 7, wherein said antigen is rabies glycoprotein.
9. A method of claim 7, wherein said test sample is treated with a suitable detergent to release embedded GP.

10. A method of claim 7, wherein said anti-GP antibody is a monoclonal antibody.
11. A method of claim 10, wherein said monoclonal antibody is specific to any strain of rabies virus.
12. A method of claim 10, wherein the viral strain is selected from PM, ERA or suitable strain.
13. A method of claim 11, wherein said monoclonal antibody is an IgG antibody.
14. A method of claim 13, wherein the monoclonal antibody is IgG2b isotype.
15. A method of claim 7, wherein said blocking agent is any neutral protein to the reactants or reagent used.
16. A method of claim 15, wherein the neutral protein is selected from bovine serum albumin or casein.
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7. The method of claim 7, wherein said anti GP
immunoglobulin is anti rabies glycoprotein
antibodies/antiserum.
18. A method of claim 7, wherein said anti immunoglobulin
..antibody/antisera comprise anti GP serum or polyclonal,.
antibodies raised in experimental animals.
19. A method of claim 18, wherein the experimental animal is selected from sheep, goat, rabbit, guinea pigs, mice or-rat.
20. A method of claim 7, wherein said antibody is detected with a secondary reagent capable of specifically binding to said antibody and said secondary reagent is labeled.
21. A method of claim 20, wherein said antibodies is labeled with an enzyme.
22. A method of claim 21, wherein the enzyme is horseradish peroxidase or alkaline phosphatase.
23. A method of claim 1 or 7, wherein the GP is in an aqueous solution.
24. A method of claim 23, wherein the aqueous solution is a buffer of pH ranging from 4.5 to 9.5.
25. A method of claim 23, wherein the aqueous solution comprised a surfactant.
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26. A method of claim 25, wherein the surfactant is
selected from Tween-20, Tween-80, Triton X-100, Mulgofen, etc.
27. A process of claim 26, wherein the surfactant concentration is in the range of 0.1% to 1.0%
28. A method of claim 23, wherein the aqueous solution further comprises a salt.
29. A method of claim 29, wherein the salt is any chloride salt.
30. A method of claim 29, wherein the salt concentration is in the range of from 10 mM to 1M.

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ABSTRACT
A monoclonal antibody based immunocapture ELISA was developed for the quantitation. The assay includes immobilization of the monoclonal antibody to rabies glycoprotein on the microtiter wells and blocking the free polystyrene surface with suitable blocking reagent. The monoclonal antibody is developed indigenously to specifically react with the exposed epitope of glycoprotein embedded in the viral coat. The immobilized antibody captures the glycoprotein when incubated with rabies virus sample (active and inactivated viral harvests or adjuvanated vaccine samples). The monoclonal antibody captured rabies glycoprotein is then incubated with sheep anti rabies immunoglobulin for detecting other epitopes of virus glycoprotein that leads to antibody-antigen-antibody immune complex. Finally, the immune complex was signaled with an enzyme labeled antibodies that was spectrophotometrically measured by reacting with the enzyme substrate that generates free radicals which oxidize a reducing agent to a colored product.
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