FIELD OF INVENTION
The present invention relates to detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) using combination of monoclonal antibodies and chicken IgY antibodies.
BACKGROUND OF THE INVENTION
Foot and mouth disease is a major disease of economic importance. It is caused by a virus that is the prototype of the Apthousvirus genus of Picornaviridae family. It affects all cloven hoofed animals including wild animals. The virus has seven antigenicaly distinct serotypes O, A, C, Asial and South African Territory viruses SAT-1, SAT-2 and SAT-3. Diagnosis of the disease is basically made by observing the clinical signs like appearance of vesicles in the tongue, buccal mucosa, dental pad and in the interdigital space of the hoof. Identification and serotyping of the causative agent is done in a containment facility using an antigen capture sandwich ELISA format that involves raising antiserum using laboratory animals such as rabbits and guinea pigs. This requires a small animal house and maintenance of laboratory animals under ideal conditions. Moreover the preparation of antiserum involves collection of blood in these animals which is an invasive procedure and causes lot of stress to these animals. Further, it is not possible to maintain these animals for a long period for production of more antiserum due to shorter life span and damages caused due to repeated bleeding for antiserum. With the increasing awareness of the animal rights movement globally, the use of laboratory animals for the production of immune serum is becoming more and more difficult. Alternative strategies have to be devised to continue with the ELISA procedure.
SUMMARY OF THE INVENTION
The present invention relates to detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) using sandwich ELISA method. The present invention particularly discloses a process of detection and serotyping of FMDV using the monoclonal antibodies and chiken IgY antibodies.
One aspect of the present invention relates to a process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample, the process comprising:
• providing a surface which is coated with monoclonal antibodies specific to FMDV serotype;
• contacting said surface with a labeled chicken IgY antibodies specific to FMDV serotype, wherein the contact of the surface and the labeled chicken IgY antibody is carried out in the presence or absence of the sample; and
• comparing the quantity of the labeled chicken IgY antibodies bound to the surface in the presence of the sample with the quantity of the labeled chicken IgY bound to the surface in the absence of the sample,
Another aspect of the present invention relates to a kit for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV); the kit comprising the monoclonal antibodies and chicken IgY specific to FMDV serotypes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) using sandwich ELISA method. The present invention particularly discloses a process of detection and serotyping of FMDV using the monoclonal antibodies and chicken IgY antibodies.
The present invention further relates to use of purified chicken IgY that is specific for FMDV serotypes for serological assays such as virus detection and antibody estimation.
The present invention provides a process for development of antibodies against the FMD viruses by using hybridoma technology and preparation of immunized chicken IgY that is specific for FMDV serotypes against which it is raised. The layer birds were immunized with the antigen and the eggs were collected for up to 72 weeks. The IgY fraction was purified from the egg yolk and was used in the ELISA application.
The process disclosed in the present invention is non invasive to the birds and the birds can be maintained for fairly long periods. The antibodies produced are consistent and one to two birds are sufficient to produce huge amounts of purified IgY thereby removing the batch to batch variation seen in the antiserum raised in the laboratory animals.
One embodiment of the present invention provides the monoclonal antibodies specific to FMDV serotypes.
Another embodiment of the present invention provides the chicken IgY antibodies specific to FMDV serotypes.
Another embodiment of the present invention provides a combination of chicken IgY antibodies and monoclonal antibodies for detection and serotyping of FMDV in a sample.
Yet another embodiment of the present invention provides a combination of chicken IgY antibodies to FMDV serotypes and monoclonal antibodies specific to FMDV serotypes to develop sensitive and specific serological assays for antigen and antibody detection.
In one embodiment, the present invention provides a process of production of antibodies against Foot-and-Mouth Disease Virus in chicken and purifying the IgY from the egg yolk.
Further embodiment of the present invention provides a process for production of monoclonal antibodies against the FMD viruses using hybridoma technology.
The chicken IgY antibodies were raised in the chicken and purified from the egg yolk. Detailed procedure is provided in Example 1. The procedure is non invasive to the birds and the birds can be maintained for fairly long periods. The antibodies produced are consistent and one to two birds will be sufficient to produce huge amounts of purified IgY thereby removing the batch to batch variation seen in the antiserum raised in the laboratory animals.
In accordance with the present invention, one embodiment provides a process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample, the process comprising providing a surface which is coated with monoclonal antibodies specific to FMDV serotype; contacting the surface with a labeled chicken IgY antibodies specific to FMDV serotype, wherein the contact of the surface and the labeled chicken IgY antibody is carried out in the presence or absence of the sample; and comparing the quantity of the labeled chicken IgY antibodies bound to the surface in the presence of the sample with the quantity of the labeled chicken IgY bound to the surface in the absence of the sample,
Another embodiment of the present invention provides the monoclonal antibodies specific to 0, A, C, Asia 1, SAT-1, SAT-2 and SAT-3 serotypes of FMDV useful for the process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample.
Yet another embodiment of the present invention provides the chicken IgY antibodies specific to 0, A, C, Asia 1, SAT-1, SAT-2 and SAT-3 serotypes of FMDV useful for the process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample.
Still another embodiment of the present invention provides the chicken IgY antibodies, wherein the chicken IgY antibodies are labeled with an enzyme conjugate selected from a group consisting of Horse Radish Peroxidase, Alkaline Phosphatase, chemiluminiscent and fluorescent dyes.
Still yet another embodiment of the present invention provides the monoclonal antibodies prepared by hybridoma technology.
In further embodiment, the present invention provides the chicken IgY antibodies produced by hyper immunization of layer birds with specific FMDV antigens of serotypes 0, A, C, Asia 1, SAT-1, SAT-2 and SAT-3
One embodiment of the present invention provides the process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample, wherein the contact of the surface and labeled chicken IgY antibodies in the presence of the sample is performed by contacting the surface with the sample, followed by washing the surface to remove unbound chicken IgY antibodies.
Another embodiment of the present invention provides the process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample, wherein washing is performed using Phosphate Buffer with about 0.05% Tween 20.
Another embodiment of the present invention provides the process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample, wherein the detecting is performed using an enzyme-linked immunosorbent assay (ELISA) or a solid phase binding assay or electro-immunoblot transfer assay.
In another embodiment of the present invention provides a kit for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV); wherein the kit comprising the monoclonal antibodies and chicken IgY specific to FMDV serotypes.
Detection of Foot-and-Mouth disease virus from suspected samples
Detection of FMDV in a suspected sample was performed using the monoclonal antibodies specific to FMDV serotypes and the chicken IgY specific to FMDV serotypes using the sandwich ELISA method. The details are provided in Example 2.
Detection of Foot-and-Mouth disease virus from tissue culture supernatants
Detection of FMDV in a tissue culture sample was performed using the monoclonal antibodies specific to FMDV serotypes and the chicken IgY specific to FMDV serotypes using the sandwich ELISA method. The details are provided in Example 3.
Detection of Foot-and-Mouth disease virus from tissue culture obtained samples as an in process control test
Detection of FMDV in a sample as an in process control test was performed using the monoclonal antibodies specific to FMDV serotypes and the chicken IgY specific to FMDV serotypes using the sandwich ELISA method. The details are provided in Example 4.
It will be appreciated by persons skilled in the art that the present invention is not limited by what has been particularly shown and described hereinabove. Rather the scope of the present invention inc ludes both combinations and sub combinations of the features described hereinabove as well as modifications and variations thereof
which would occur to a person of skill in the art upon reading the foregoing description and which are not in the prior art.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete invention and the description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all and only experiments performed.
Example 1
Production of chicken IgY antibodies specific to FMDV serotypes
The layer birds were immunized with the antigen and the eggs were collected for up to 72 weeks. The IgY fraction was purified from the egg yolk and was used for detection of FMDV serotypes using sandwich ELISA method. The procedure is non invasive to the birds and the birds can be maintained for fairly long periods. The antibodies produced are consistent and one to two birds will be sufficient to produce huge amounts of purified IgY thereby removing the batch to batch variation seen in the antiserum raised in the laboratory animals.
Example 2
Detection of Foot-and-Mouth disease virus from suspected samples
Monoclonal antibodies were coated at appropriate dilutions on to an ELISA plate. After overnight incubation in a moist chamber the plates were washed with Phosphate buffered saline with Tween 20 (0.05%). The plates were again washed with Phosphate buffered saline with Tween 20 (0.05%); the suspected samples
were added as 20% suspensions in 0.04 M Phosphate buffer and incubated at 37°C for an hour. The plates were again washed with Phosphate buffered saline with Tween 20 (0.05%). The horse raddish peroxidase conjugated serotype specific purified chicken JgY in Phosphate buffered saline with Tween 20 (0.05%) containing 10% bovine serum was added to the corresponding wells and incubated at 37°C for an hour. The plates were washed with Phosphate buffered saline with Tween 20 (0.05%) and a mixture of Tetra methyl benzidene (TMB) and Hydrogen peroxide was added and incubated at room temperature away from light. The plates were read at 450 nm. The serotype was declared based on the specific color development. The results are provided in Table 1.
Table 1

Example 3
Detection of Foot-and-Mouth disease virus from tissue culture supernatants
Monoclonal antibodies were coated at appropriate dilutions on to an ELISA plate. After overnight incubation in a moist chamber the plates were washed with Phosphate buffered saline with Tween 20 (0.05%). After washing the plates with
Phosphate buffered saline with Tween 20 (0.05%), the tissue culture supernatant samples were added directly and incubated at 37°C for an hour. The plates were again washed with Phosphate buffered saline with Tween 20 (0.05%). The horse raddish peroxidase conjugated serotype specific purified chicken IgY in Phosphate buffered saline with Tween 20 (0.05%) containing 10% bovine serum was added to the corresponding wells and incubated at 37°C for an hour. The plates were washed with Phosphate buffered saline with Tween 20 (0.05%) and a mixture of Tetra methyl benzidene (TMB) and Hydrogen was added and incubated at room temperature away from light. The plates were read at 450 nm. The serotype was declared based on the specific color development. The results are provided in Table 2.

Example 4
Detection of Foot-and-Mouth disease virus from tissue culture obtained samples as an in process control test
Monoclonal antibodies were coated at appropriate dilutions on to an ELISA plate. After overnight incubation in a moist chamber the plates were washed with Phosphate buffered saline with Tween 20 (0.05%). After washing the plates with
Phosphate buffered saline with Tween 20 (0.05%), the in process samples were added directly and incubated at 37DC for an hour. The plates were again washed with Phosphate buffered saline with Tween 20 (0.05%). The horse raddish peroxidase conjugated serotype specific purified chicken IgY in Phosphate buffered saline with Tween 20 (0.05%) containing 10% bovine serum was added to the corresponding wells and incubated at 37°C for an hour. The plates were washed with Phosphate buffered saline with Tween 20 (0.05%) and a mixture of Tetra methyl benzidene (TMB) and Hydrogen was added and incubated at room temperature away from light. The plates were read at 450 nm. The serotype was declared based on the specific color development. Thus this ELISA can be put to use for regular identification and serotyping of FMD viruses for in process samples as a quality control test.

The foregoing descriptions of specific embodiments of the present invention have been presented for the purpose of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variation are possible in light of the above teaching. It is intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

I/We Claim:
1. A process for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV) in a sample, said process comprising:
a. providing a surface which is coated with monoclonal antibodies specific to FMDV serotype;
b. contacting said surface with a labeled chicken TgY antibodies specific to FMDV serotype, wherein the contact of the surface and the labeled chicken IgY antibody is carried out in the presence or absence of the sample;
c. comparing the quantity of the labeled chicken IgY antibodies bound to the surface in the presence of the sample with the quantity of the labeled chicken IgY bound to the surface in the absence of the sample,
2. The process as claimed in claim 1, wherein the monoclonal antibodies are specific to O, A, C, Asia 1, SAT-1, SAT-2 and SAT-3.
3. The process as claimed in claim 1, wherein the chicken IgY antibodies are specific to O, A, C, Asia 1, SAT-1, SAT-2 and SAT-3.
4. The process as claimed in claim 1, wherein the chicken IgY antibodies are labeled with an enzyme conjugate selected from a group consisting of Horse Radish Peroxidase, Alkaline Phosphatase, chemiluminiscent and fluorescent dyes.
5. The process as claimed in claim 1, wherein the monoclonal antibodies are prepared by hybridoma technology.
6. The process as claimed in claim 1, wherein the chicken IgY antibodies are produced by hyper immunization of layer birds with specific FMDV antigens of serotypes O, A, C, Asia 1, SAT-1, SAT-2 and SAT-3
7. The process as claimed in claim 1, wherein die contact of the surface and labeled chicken IgY antibodies in the presence of the sample is performed by contacting the surface with the sample, followed by washing the surface to remove unbound chicken IgY antibodies.
8. The process as claimed in claim 1, wherein the washing is performed using Phosphate Buffer with about 0.05% Tween 20.
9. The process as claimed in claim 1, wherein the detecting is performed using an enzyme-linked immunosorbent assay (ELISA) or a solid phase binding assay or electro-immunob lot transfer assay.
10. A kit for detection and serotyping of Foot-and-Mouth Disease Virus (FMDV); said kit comprising the monoclonal antibodies and chicken IgY specific to FMDV serotypes.