FIELD OF INVENTION
This invention relates to a process for simultaneous detection and serotyping of foot-and-mouth disease virus (FMDV) in a sample.
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 Antigenic differentiation can be done at present only in the laboratory that possess a bio-containment facility for handling and processing of Foot-and-mouth Disease suspected clinical samples collected from diseased animals. The vast geographical distribution of the different serotypes makes it still more difficult to identify and differentiate due to failure of the suspected clinical materials to reach such laboratories in time. Delay in identification of serotypes leads to loss of valuable epidemiological data about the disease. Various efforts are being made to hasten the process of foot and mouth disease virus identification. Most of the techniques are based on molecular techniques. However, simple immunological techniques that can be applied at penside are also being thought of These techniques concentrate only on detection on FMD virus but not differentiating the antigenic serotype of the virus.

SUMMARY OF THE INVENTION
The present invention provides a process for simultaneous detection and serotyping of foot-and-mouth disease virus (FMDV) in a sample. The present invention further provides a device and kit for detection and serotyping of foot-and-mouth disease virus (FMDV) in a sample.
One aspect of the present invention relates to a process for simultaneous detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample, the process comprising applying the sample to a loading region of a device comprising a sample pad, a glass fiber membrane comprising immobilized control phase comprising labeled binding partner in the form of a microsphere or colloidal metal conjugated monoclonal antibody mixture, a laminated nitrocellulose membrane having at least more than one immobilized phase containing antibody of specific serotypes on the predetermined region of the membrane and an absorbent pad; wherein antibodies are derived from FMDV or obtainable from FMDV; observing changes in appearance of said immobilized phases; and detecting presence of Foot and Mouth Disease Virus (FMDV) and determining the serotype of FMDV in the sample
Another aspect of the present invention relates to a device for detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample, the device comprising a sample pad, a glass fiber membrane containing labeled binding partner in the form of a microsphere or colloidal metal conjugated monoclonal antibody mix, a laminated nitrocellulose membrane containing monoclonal antibodies and an absorbent pad;
Yet another aspect of the present invention relates to a kit for detection and serotyping of Foot and Mouth disease Virus (FMDV) in a sample, the kit comprising the device as disclosed in the present invention and reagents.

Further aspect of the present invention relates to a kit for detection and serotyping of Foot and Mouth disease Virus (FMDV) in a sample, the kit comprising a strip having a reactivity zone containing at least more than one immobilized phase comprising antibodies derived against foot and mouth disease virus serotypes selected from a group consisting of O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3 and a control for confirming normal operation of the kit, with or without a housing.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1 shows the assembly of the device proposed for the identification and serotype differentiation of foot-and-mouth disease virus Figure 2
a) shows detection of Foot and Mouth Disease Virus from clinical samples
b) shows detection of O serotype of Foot and Mouth Disease Virus from clinical
c) shows detection of Asial serotype of Foot and Mouth Disease Virus from clinical samples with identification of serotype
d) shows detection of a serotype serotypes other than 0 and Asial of Foot and
Mouth Disease Virus from clinical samples
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for simuUaneous detection and serotyping of foot-and-mouth disease virus (FMDV) in a sample. The present invention further provides a device and a kit for detection and serotyping of foot-and-mouth disease virus (FMDV) in a sample.
The present invention particularly provides a novel process for simultaneous detection and serotyping of foot-and-mouth disease virus (FMDV) in a sample by using lateral flow assay technique and suitable reagents that can be used not only to

identify the disease in suspected animals but also antigenicaly differentiate the serotypes.
In accordance with the present invention, one embodiment provides a process for simultaneous detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample, the process comprising applying the sample to a loading region of a device comprising a sample pad, a glass fiber membrane comprising immobilized control phase comprising labeled binding partner in the fonn of a microsphere or colloidal metal conjugated monoclonal antibody mixture, a laminated nitrocellulose membrane having at least more than one immobilized phase containing antibody of specific serotypes on the predetermined region of the membrane and an absorbent pad; wherein antibodies are derived from FMDV or obtainable from FMDV; observing changes in appearance of the immobilized phases; and detecting presence of Foot and Mouth Disease Virus (FMDV) and determining the serotype of FMDV in the sample
In another embodiment, the present invention provides the process for simultaneous detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample, wherein the sample is selected from a group consisting of vesicular fluid, tongue epithelium feet epithelium and clinical specimen.
In another embodiment, the present invention provides a colored detection reagent as the labeled binding partner for the process for simultaneous detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample.
In yet another embodiment, the present invention provides the monoclonal antibody mixture comprises antibodies against FMDV serotype specific or a combination of antibodies against different serotypes of FMDV selected from a group consisting of O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3 serotypes of FMDV.

In still yet another embodiment, the present invention provides a device for detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample, said device comprising a sample pad, a glass fiber membrane containing labeled binding partner in the form of a microsphere or colloidal metal conjugated monoclonal antibody mix, a laminated nitrocellulose membrane containing monoclonal antibodies and an absorbent pad.
In further embodiment, the present invention provides the device for detection and serotyping of Foot and Mouth Disease Virus (FMDV) in a sample, wherein the monoclonal antibody mixture comprises antibodies against FMDV serotype specific or a combination of antibodies against different serotypes of FMDV selected from a group consisfing of O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3 serotypes of FMDV.
In another embodiment, the present invention provides a kit for detection and serotyping of Foot and Mouth disease Virus (FMDV) in a sample, wherein the kit comprising the device as disclosed in the present invention and reagents.
In yet another embodiment, the present invention further provides a kit for detection and serotyping of Foot and Mouth disease Virus (FMDV) in a sample, the kit comprising a strip having a reactivity zone containing at least more than one immobilized phase comprising antibodies derived against foot and mouth disease virus serotypes selected from a group consisting of O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3 and a control for confirming normal operation of the kit, with or without a housing.
The present invention further provides a kit for foot-and-mouth disease virus detection and serotyping comprising a strip including a reactivity zone containing at least more than one immobilized phase selected from antibodies thereon, derived from "FMDV or obtainable from FMDV through an immunological reaction, and a

control for confirming normal operation of the kit, provided on a predetermined region of wicking membrane; without or with a housing protecting the strip from any possible contaminants,
It includes a test sample application port and indicator windows for observing results of reaction in the reactivity zone and control zone on the strip.
The device
The assembly of the device disclosed in the present invention is depicted in Figure 1. A suspension of suspected material (vesicular fluid / tongue epithelium / feet epithelium) is overlaid on to the sample pad (2) of a plastic cassette (1) and allowed to react with a mixture of FMDV serotype specific monoclonal antibodies conjugated with colloidal gold particles (4), coated and dried on a glass fiber membrane (3). Due to capillary action, the contents in the sample pad move on to nitrocellulose membrane (5). A detecting antibody (6) precoated and dried on the NC membrane traps the antigen-anfibody complex in case of a positive sample. Aggregation of the complex produces a defined color (color of the colloidal gold). The mixture continues to move on the NC membrane and binds with one of the two test antibodies (7 or 8) depending on the virus serotype to form aggregation and produces a defined color (color of the colloidal gold). The excess and unbound monoclonal antibodies (MAbs) conjugated with colloidal gold particles continue to move on the NC membrane. Precoated and dried goat anti mouse IgG antibody (9) on the membrane traps the unbound and excess MAbs conjugated with colloidal gold giving another defined color (color of the colloidal gold). Excess sample is absorbed by the absorbent pad (10). Thus in case of a positive reaction the control band and the test bands are formed and incase of a negative reaction only the control band fonns.

In an embodiment, the disclosure provides a method for foot-and-mouth disease virus antigen serotyping from clinical and biological samples, comprising the steps of applying a partially processed test sample to a loading region of a strip; coupling a colored detection reagent to an analyte of interest in the test sample to form a complex there between; developing the specific complex onto a wicking membrane; and observing changes in appearance of a reactivity zone having at least more than one immobilized phase for specific serotypes selected from antibody on the predetermined region of the wicking membrane, derived from FMDV or obtainable from FMDV through an immunological reaction to determine the serotype of foot-and-mouth disease viruses causing the disease.
In another embodiment, the disclosure provides a kit for foot-and-mouth disease virus identification and serotyping comprising a strip including a reactivity zone containing at least more than one immobilized phase selected from antibodies thereon, derived from "FMDV or obtainable from FMDV through an immunological reaction, and a control for confirming normal operation of the kit, provided on a predetermined region of wicking membrane; without or with a housing protecting the strip from any possible contaminants,
In another embodiment, the disclosure provides a kit for foot-and-mouth disease virus identification and serotyping comprising a test sample application port and indicator windows for observing results of reaction in the reactivity zone and control zone on the strip.
In an embodiment, the disclosure provides a kit for foot-and-mouth disease virus identification and serotyping comprising a sample pad, a glass fiber membrane coated with mixture of FMDV serotype specific monoclonal antibodies conjugated with colloidal gold particles, nitrocellulose membrane (NC) facilitating the movement of the contents of the sample pad and the detection antibody, two test

antibodies for specific serotypes and goat anti mouse IgG antibody precoated and dried on the NC membrane.
In an embodiment, the disclosure provides a method for differentiating the antigenic serotype in a test sample wherein the presence of the test line 'U' and control line 'C indicates that the suspected samples has Foot-and-mouth disease virus.
In another embodiment, the disclosure provides a method for differentiating the antigenic serotype in a test sample wherein the presence of two test lines namely, 'U' and 'O' and control line 'C indicates that the serotype of sample is type O.
In a preferred embodiment, the disclosure provides a method for differentiating the antigenic serotype in a test sample wherein the presence of two test lines 'U' and 'As' and control line 'C indicates that the serotype of sample is type Asial.
In another embodiment, the disclosure provides a method for differentiating the antigenic serotype in a test sample wherein the presence of only test line 'U' and control line 'C indicates that the sample contains FMD virus which is not 'O' and 'Asial' but belonging to some other serotype may be 'A' or SAT viruses.
In still another embodiment, the disclosure provides a method for differentiating the antigenic serotype in a test sample wherein presence of only the control line 'C indicates that the sample does not contain any FMD virus.
In yet another embodiment, the disclosure provides a method for differentiating the antigenic serotype in a test sample wherein absence of control line 'C, even after development of any other line indicates that the test has to be repeated.
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 includes 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 disclosure 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
Detection and differentiation of FMDV serotypes
The process of detection and serotyping of FMDV is carried out by using the device as described in the Figure 1. A suspension of suspected material (vesicular fluid / tongue epithelium / feet epithelium) is overlaid on to the sample pad (2) and allowed to react with a mixture of FMDV serotype specific monoclonal antibodies conjugated with colloidal gold particles (4), coated and dried on a glass fiber membrane (3). Due to capillary action, the contents in the sample pad move on to nitrocellulose membrane (5). A detecting antibody (6) precoated and dried on the NC membrane traps the antigen-antibody complex in case of a positive sample. Aggregation of the complex produces a defined color (color of the colloidal gold). The mixture continues to move on the NC membrane and binds with one of the two test antibodies (7 or 8) depending on the virus serotype to form aggregation and produces a defined color (color of the colloidal gold). The excess and unbound monoclonal antibodies (MAbs) conjugated with colloidal gold particles continue to

move on the NC membrane. Precoated and dried goat anti mouse IgG antibody (9) on the membrane traps the unbound and excess MAbs conjugated with colloidal gold giving another defined color (color of the colloidal gold). Excess sample is absorbed by the absorbent pad (10). Thus in case of a positive reaction the control band and the test bands are formed and incase of a negative reaction only the control band forms.
Example 2
Foot-and-mouth disease virus identification in the field
A small portion of the tongue epithelium from the affected cattle was gently harvested into the buffer vial. The sample was mixed vigorously until it formed a uniform suspension and was allowed to settle. About 100-200 yd (two to three drops) of the suspected clinical sample suspension was applied on the sample pad of the device (Figure 1). The sample was incubated for about 5-10 minutes for the assay to complete. After drying the membrane was dried the color developed was analyzed. Presence of the test line 'U' and control line "C indicated that the suspected samples has Foot-and-mouth disease virus.
Example 3
Foot-and-mouth disease virus identification in buffalo
A small portion of the dental pad lesion or buccal lesion from the affected buffalo was gently harvested into the buffer vial. The sample was mixed vigorously until it formed a uniform suspension and was allowed to settle. About 100-200 ^il (two to three drops) of the suspected clinical sample suspension was applied on the sample pad of the device (Figure 1). The sample was incubated for about 5-10 minutes for

the assay to complete. After drying the membrane was dried the color developed was analyzed. Presence of the test line 'U' and control line 'C indicated that the suspected samples has Foot-and-mouth disease virus.
Example 4
Foot-and-mouth disease virus identification in swine
A small portion of the dental pad lesion or buccal lesion from the snout or limb of the affected swine was gently harvested into the buffer vial. The sample was mixed vigorously until it formed a uniform suspension and was allowed to settle. About 100-200 \x\ (two to three drops) of the suspected clinical sample suspension was applied on the sample pad of the device (Figure 1). The sample was incubated for about 5-10 minutes for the assay to complete. After drying the membrane was dried the color developed was analyzed. Presence of the test line 'U' and control line 'C indicated that the suspected samples has Foot-and-mouth disease virus.
Example 5
Foot-and-moutii disease virus identification and serotyping in the field
A small portion of the dental pad lesion or buccal lesion from the snout or limb of the affected swine was gently harvested into the buffer vial. The sample was mixed vigorously until it formed a uniform suspension and was allowed to settle. About 100-200 |.d (two to three drops) of the suspected clinical sample suspension was applied on the sample pad of the device (Figure 1). The sample was incubated for about 5-10 minutes for the assay to complete. After drying the membrane was dried the color developed was analyzed. Presence of the test line 'U' and control line 'C indicated that the suspected samples has Foot-and-mouth disease virus (Figure 2a).

Presence of two lines test lines 'U' and 'O' and control line 'C indicated that the serotype of sample is type 0 (Figure 2b). Presence of two lines test lines 'U' and 'As' and control line 'C indicates that the serotype of sample is type Asial (Figure 2c). Presence of only test line 'U' and control line C indicates that the sample contains FMD virus which is not 'O' and 'Asial' but belonging to some other serotype may be 'A' or SAT viruses (Figure 2d). Presence of only the control line 'C indicates that the sample does not contain any FMD virus. Absence of control line 'C, even after development of any other line indicates that the test has to be repeated (Figure 2 a, b, c, d).
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.

lWe Claim:
1. A process for simultaneous detection and serotyping of Foot and Mouth Disease
Virus (FMDV) in a sample, said process comprising:
a. applying the sample to a loading region of a device comprising a sample
pad, a glass fiber membrane comprising immobilized control phase
comprising labeled binding partner in the form of a microsphere or
colloidal metal conjugated monoclonal antibody mixture, a laminated
nitrocellulose membrane having at least more than one immobilized phase
containing antibody of specific serotypes on the predetermined region of
the membrane and an absorbent pad; wherein antibodies are derived from
FMDV or obtainable from FMDV:
b. observing changes in appearance of said immobilized phases; and
c. detecting presence of Foot and Mouth Disease Virus (FMDV) and
determining the serotype of FMDV in said sample
2. The process as claimed in claim 1, wherein the sample is selected from a group consisting of vesicular fluid, tongue epithelium feet epithelium and clinical specimen.
3. The process as claimed in claim 1, wherein the labeled binding partner is colored detection reagent.
4. The process as claimed in claim 1, wherein the monoclonal antibody mixture comprises antibodies against FMDV serotype specific or a combination of antibodies against different serotypes of FMDV selected from a group consisting of O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3 serotypes of FMDV.

5. A device for detection and serotyping of Foot and Mouth Disease Virus (FMDV)
in a sample, said device comprising a sample pad, a glass fiber membrane
containing labeled binding partner in the form of a microsphere or colloidal
metal conjugated monoclonal antibody mix, a laminated nitrocellulose membrane
containing monoclonal antibodies and an absorbent pad;
6. The device as claimed in claim 5, wherein the monoclonal antibody mixture
comprises antibodies against FMDV serotype specific or a combination of
antibodies against different serotypes of FMDV selected from a group consisting
of O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3 serotypes of FMDV.
7. A kit for detection and serotyping of Foot and Mouth disease Virus (FMDV) in a
sample, said kit comprising the device as claimed in claim 2 and reagents.
8. A kit for detection and serotyping of Foot and Mouth disease Virus (FMDV) in a
sample, said kit comprising a strip having a reactivity zone containing at least
more than one immobilized phase comprising antibodies derived against foot and
mouth disease virus serotypes selected from a group consisting of O, A, C, Asia
1, SAT 1, SAT 2 and SAT 3 and a control for confirming normal operation of the
kit, with or without a housing.
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