Title of Invention	A TEST STRIP FOR QUALITATIVE DETECTION OF ANTIBODIES TO PESTE DES PETITS RUMINANT VIRUS
Abstract	A test strip for the qualitative detection of antibodies to peste des etits ruminants virus having three different surfaces, namely the absorbent pad, nitrocellulose (NC) membrane mounted on a laminate and sample pad, wherein the antigen is coated on the membrane along with the antigoat antibody prepared in rabbits, 5 mm apart using a line printer at medium speed. Dated this 7th day of September 2006

Title of Invention

A TEST STRIP FOR QUALITATIVE DETECTION OF ANTIBODIES TO PESTE DES PETITS RUMINANT VIRUS

Abstract

A test strip for the qualitative detection of antibodies to peste des etits ruminants virus having three different surfaces, namely the absorbent pad, nitrocellulose (NC) membrane mounted on a laminate and sample pad, wherein the antigen is coated on the membrane along with the antigoat antibody prepared in rabbits, 5 mm apart using a line printer at medium speed. Dated this 7th day of September 2006

Full Text	A strip for peste des etits ruminants virus Field of Invention: This invention relates to a lateral flow strip to test for the qualitative presence of antibodies to peste des etits ruminants virus. Background of the Invention: Testing of body fluid samples for disease detection is normally done either direct detection of organisms or demonstration of antibodies. Most of the tests performed rely on several sophisticated equipments and technical skill and laboratory area for performing such tests. The most common protein-based method for detection of PPR is the immuno-capture enzyme linked immuno-sorbent assay (ELISA) where in conventionally, Rinderpest virus (RPV crossreactive monoclonal antibodies mAbs) or polyclonal sera is used as coating antibody and PPRV specific mAb is used as a detection antibody. The antigen detection method targets the nucleocapsid (N) protein of PPRV since it is the most abundant protein present in virus-infected cells. Genome based methods for detection PPRV include techniques such as nucleic acid hybridization and reverse transcription polymerase chain reaction (RTPCR) RTPCR targeting either the fusion (F) gene or (N) gene has been successfully used in PPR detection. In India, plate ELISA kits have been prepared. However this test takes at least 5-6 hours to perform and requires sophisticated equipments for its performance. Moreover the results are not permanent and this test requires ELISA reader for measurement of absorbance. It is done on ELISA plates and uses a cell culture adapted virus antigen. The test also requires separation of bound and unbound analytes by washing between each step. Detection of PPR is mainly based on conventional antigen detection techniques such as agar gel immuno diffusion, counter immuno electrophoresis, haemagglutination test, immuno capture ELISA or virus isolation. These techniques are largely replaced by genome-based detection techniques such as reverse transcription polymerase chain reaction (RTPCR) and nucleic acid hybridization by virtue of its high sensitivity and specificity. However, genome-based methods are cumbersome and cannot be applicable to large numbers of samples. The nucleocapsid protein (N) of PPRV is the most abundant and immunogenic viral protein making it an ideal candidate for antigen-based diagnosis of PPR. The N gene of PPRV is about 1700 nucleotide long coding for 525 amino acids. It can be divided into three regions based on the rate of homology: the 5'end of the m RNA (1-500 nucleotide position) with moderate homology, the middle which is very well conserved and finally the 3'end with low homology. The most variable 3'end of the gene has been deleted and hence the expressed protein used in the present kit as coating antigen is capable of reacting with antibodies from all animals infected with different strains of PPRV circulating in India or elsewhere. Strip tests are known and have been used for detection of various analytes. However, the inventive step in the present strip test is that a truncated recombinant N protein is used as a coating antigen and the protein A gold conjugate is used in liquid phase enabling the test to be performed in a tube in vertical position and not in a lateral flow based method. This would reduce the cost of the test in avoiding plastic housing. However, there is a strip test for antigen detection using monoclonal antibodies to the PPR virus coupled to latex microspheres to both rinderpest virus and PPRV. The inventive step is the use of a truncated nucleocapsid protein that is expressed in bacteria as a coating antigen. Protein A gold conjugate is used in the liquid phase (instead of impregnating it in the conjugate matrix). Objectives of the Invention; The objective of this invention is to develop a user-friendly field based test for detection of antibodies to PPR virus. Yet another objective is to develop a specific and sensitive assay for detection using a truncated recombinant nucleocapsid protein of the virus as coating antigen. Further objective of this invention is to use a Protein A gold conjugate in the liquid phase instead of impregnating it in conjugate matrix. Other objective is to delete the most variable 3'end of the gene so that the expressed protein used as a coating antigen is capable of reacting with antibodies from all animals infected with different strains of PPRV. Yet another objective is to reduce the cost of the individual assay and to cater to "herd testing" using protein A coupled colloidal gold in the liquid phase. Further objective is to make this test useful to test antibodies in the serum of sheep, goats, bovine and buffaloes using the same conjugate. Detailed description of the Invention: The present invention provides a test strip having three different surfaces, namely the absorbent pad, nitrocellulose (NC) membrane mounted on a laminate and sample pad. The antigen is coated on the membrane along with the anti-goat antibody prepared in rabbits (commercial) 5mm apart using a line printer at medium speed. The antigen is a truncated nucleocapsid protein of PPRV whose gene has been expressed in a prokaryotic vector system. The recombinant plasmid harboring the deleted nucleocapsid gene (the 3'end of the gene is deleted) is transformed into host E coli cells and induced to produce the recombinant protein using the chemical, 1PTG. Then after 4 hours of induction, the cell extract having the expressed protein is expressed using ultra sonication of cells and the recombinant protein purified by ultra centrifugation. This purified recombinant protein is used as a coating antigen for detection of antibodies specific to it in the sera of sheep, goat, bovine etc. The recombinant antigen and antigoat antibody coated Nc strips are allowed to dry under vacuum overnight at 37°C. Then the absorbent pad and sample pad are then affixed on the NC laminate overlapping the NC membrane on the top and bottom respectively. Then strips of 4-5 mm width are cut and packaged in to aluminum pouches containing silica gel. At the time of use, these strips are taken out and kept in a small tube containing two drops of buffer provided, two drops of serum to be tested and one drop of the gold conjugate provided. The mixture migrates upwards due to capillary action and if antibodies are present in the serum sample being tested, two lines would appear. If no antibodies were present only one line would appear. If no lines appear the test is invalid. The invention has the following steps: (i) Expression of the protein using a truncated nucleocapsidprotein gene cloned in a plasmid vector by transformation of the plasmid in E. coli BL21 cells. The transformed colony is grown in Luria Bertani broth and after optical density reaches 0.4 the broth culture in induced using 0.4 mM of IPTG for a period of four hours. After 4 hours the bacterial cells are pelleted and sonicated in a buffer and the supernatant containing the recombinant protein is purified by ultracentrifugation. The purified protein has been characterized in SDA-PAGE and Western blotting. The purified protein is used for coating the nitrocellulose (NC) laminate. (ii) Rabbit anti-goat lgG (from Bangalore Genei) is used for coating another line 4 mm above the antigen coated line on the NC laminate to ascertain whether the system is working well (positive control line). The imprinting of the antigen and the anti-goat lgG is done using a line printer (Advanced Microdevices Pvt. Ltd., Ambala) (Figure 1). (iii) The line imprinted NC laminate is dried at 37°C overnight in vaccum. (iv) Then it is cut into strips of 4-5 mm width and sealed in a aluminum pouch containing a desiccant and stored at refrigerator temperature (around 4°C). (v) When the test is to performed, the strips are removed and placed in a tube containing 2 drops of buffer containing Tris HCI, bovine serum albumin and sodium azide; 2 drops of the serum to be tested and one drop of the commercial protein A colloidal gold conjugate (Advanced Microdevices Pvt. Ltd., Ambala). (vi) Allow the mixture in the tube to ascend the strip and read the results after 5-10 minutes. (vii) If two pink lines are visible the serum is positive for antibodies against the coated necleoprotein (Figure 2 and 3). (viii) If one line is present the serum being tested does not have any antibodies to the nucleoprotein of PPR virus. (ix) If no lines are evident the test is invalid and needs to be repeated. Figure 1: NC laminate imprinted with the recombinant antigen and anti-goat IgG prepared in rabbit along with the affixed absorbent and sample pads along with a separate tube containing serum to be tested, protein A gold conjugate and buffer. 1-Absorbent pad, 2-Control line, 3-Test line, 4-Sample pad, 5-Serum + colloidal gold + buffer. Figure 2: Appearance of lines after flow of the mixture from the tube in to the NC Laminate. 1-Absorbent pad, 2-Control line, 3-Test line, 4-Sample pad, 5-Serum + colloidal gold + buffer. Figure 3: The tests that have been performed and read. Some of the sera samples are positive and some negative. However all the strips show the positive control line. The present invention uses a recombinant protein (N) as coating antigen and is based on the principle of lateral flow. This test requires no gadgets and can be performed in the field. Another highlight of this testing system is that no separation of bound and unbound analytes is needed for the reading of the results. Moreover, contrary to earlier testing methods such as ELISA, these strips can be preserved forever for any future requirement. This test measures antibodies to the more abundantly present N protein of the PPR virus. We claim: 1. A test strip for the qualitative detection of antibodies to peste des etits ruminants virus having three different surfaces, namely the absorbent pad, nitrocellulose (NC) membrane mounted on a laminate and sample pad, wherein the antigen is coated on the membrane along with the antigoat antibody prepared in rabbits, 5 mm apart using a line printer at medium speed. 2. The antigen as claimed in claim 1 is a truncated nucleocapsid protein of PPRV whose gene has been expressed in a prokaryotic vector system. 3. The nucleocapsid protein as claimed in claim 2 is produced by recombinant plasmid harboring the deleted nucleocapsid gene through transformation into the host E. colicells using 1PTG chemical. 4. The recombinant plasmid harbouring the nucleocapsid gene as claimed in claim 3 wherein most variable 3'end of the gene has been deleted. 5. The nucleocapsid protein as claimed in claim 2 wherein the coating antigen is capable of reacting with antibodies from all animals infected with different strains of PPRV for the removal of 3'end. 6. The antigen as claimed in claim 1, wherein is being used in tubes using gold conjugate in liquid phase. 7. The strip as claimed in claim 1, wherein its use provides qualitative results in 5-10 minutes.

Full Text

A strip for peste des etits ruminants virus
Field of Invention: This invention relates to a lateral flow strip to test for the qualitative presence of antibodies to peste des etits ruminants virus.
Background of the Invention:
Testing of body fluid samples for disease detection is normally done either direct detection of organisms or demonstration of antibodies. Most of the tests performed rely on several sophisticated equipments and technical skill and laboratory area for performing such tests.
The most common protein-based method for detection of PPR is the immuno-capture enzyme linked immuno-sorbent assay (ELISA) where in conventionally, Rinderpest virus (RPV crossreactive monoclonal antibodies mAbs) or polyclonal sera is used as coating antibody and PPRV specific mAb is used as a detection antibody. The antigen detection method targets the nucleocapsid (N) protein of PPRV since it is the most abundant protein present in virus-infected cells.
Genome based methods for detection PPRV include techniques such as nucleic acid hybridization and reverse transcription polymerase chain reaction (RTPCR) RTPCR targeting either the fusion (F) gene or (N) gene has been successfully used in PPR detection.
In India, plate ELISA kits have been prepared. However this test takes at least 5-6 hours to perform and requires sophisticated equipments for its

performance. Moreover the results are not permanent and this test requires ELISA reader for measurement of absorbance. It is done on ELISA plates and uses a cell culture adapted virus antigen. The test also requires separation of bound and unbound analytes by washing between each step. Detection of PPR is mainly based on conventional antigen detection techniques such as agar gel immuno diffusion, counter immuno electrophoresis, haemagglutination test, immuno capture ELISA or virus isolation. These techniques are largely replaced by genome-based detection techniques such as reverse transcription polymerase chain reaction (RTPCR) and nucleic acid hybridization by virtue of its high sensitivity and specificity. However, genome-based methods are cumbersome and cannot be applicable to large numbers of samples.
The nucleocapsid protein (N) of PPRV is the most abundant and immunogenic viral protein making it an ideal candidate for antigen-based diagnosis of PPR. The N gene of PPRV is about 1700 nucleotide long coding for 525 amino acids. It can be divided into three regions based on the rate of homology: the 5'end of the m RNA (1-500 nucleotide position) with moderate homology, the middle which is very well conserved and finally the 3'end with low homology. The most variable 3'end of the gene has been deleted and hence the expressed protein used in the present kit as coating antigen is capable of reacting with antibodies from all animals infected with different strains of PPRV circulating in India or elsewhere.

Strip tests are known and have been used for detection of various analytes. However, the inventive step in the present strip test is that a truncated recombinant N protein is used as a coating antigen and the protein A gold conjugate is used in liquid phase enabling the test to be performed in a tube in vertical position and not in a lateral flow based method. This would reduce the cost of the test in avoiding plastic housing.
However, there is a strip test for antigen detection using monoclonal antibodies to the PPR virus coupled to latex microspheres to both rinderpest virus and PPRV. The inventive step is the use of a truncated nucleocapsid protein that is expressed in bacteria as a coating antigen. Protein A gold conjugate is used in the liquid phase (instead of impregnating it in the conjugate matrix).
Objectives of the Invention;
The objective of this invention is to develop a user-friendly field based test for detection of antibodies to PPR virus.
Yet another objective is to develop a specific and sensitive assay for detection using a truncated recombinant nucleocapsid protein of the virus as coating antigen.
Further objective of this invention is to use a Protein A gold conjugate in the liquid phase instead of impregnating it in conjugate matrix.

Other objective is to delete the most variable 3'end of the gene so that the expressed protein used as a coating antigen is capable of reacting with antibodies from all animals infected with different strains of PPRV.
Yet another objective is to reduce the cost of the individual assay and to cater to "herd testing" using protein A coupled colloidal gold in the liquid phase.
Further objective is to make this test useful to test antibodies in the serum of sheep, goats, bovine and buffaloes using the same conjugate.
Detailed description of the Invention:
The present invention provides a test strip having three different surfaces, namely the absorbent pad, nitrocellulose (NC) membrane mounted on a laminate and sample pad. The antigen is coated on the membrane along with the anti-goat antibody prepared in rabbits (commercial) 5mm apart using a line printer at medium speed.
The antigen is a truncated nucleocapsid protein of PPRV whose gene has been expressed in a prokaryotic vector system. The recombinant plasmid harboring the deleted nucleocapsid gene (the 3'end of the gene is deleted) is transformed into host E coli cells and induced to produce the recombinant protein using the chemical, 1PTG. Then after 4 hours of induction, the cell extract having the expressed protein is expressed using ultra sonication of cells and the recombinant protein purified by ultra centrifugation. This purified recombinant protein is used as a

coating antigen for detection of antibodies specific to it in the sera of sheep, goat, bovine etc.
The recombinant antigen and antigoat antibody coated Nc strips are allowed to dry under vacuum overnight at 37°C. Then the absorbent pad and sample pad are then affixed on the NC laminate overlapping the NC membrane on the top and bottom respectively. Then strips of 4-5 mm width are cut and packaged in to aluminum pouches containing silica gel.
At the time of use, these strips are taken out and kept in a small tube containing two drops of buffer provided, two drops of serum to be tested and one drop of the gold conjugate provided. The mixture migrates upwards due to capillary action and if antibodies are present in the serum sample being tested, two lines would appear. If no antibodies were present only one line would appear. If no lines appear the test is invalid.
The invention has the following steps:
(i) Expression of the protein using a truncated nucleocapsidprotein gene cloned in a plasmid vector by transformation of the plasmid in E. coli BL21 cells. The transformed colony is grown in Luria Bertani broth and after optical density reaches 0.4 the broth culture in induced using 0.4 mM of IPTG for a period of four hours. After 4 hours the bacterial cells are pelleted and sonicated in a buffer and the supernatant containing the recombinant protein is purified by

ultracentrifugation. The purified protein has been characterized in SDA-PAGE and Western blotting. The purified protein is used for coating the nitrocellulose (NC) laminate.
(ii) Rabbit anti-goat lgG (from Bangalore Genei) is used for coating another line 4 mm above the antigen coated line on the NC laminate to ascertain whether the system is working well (positive control line). The imprinting of the antigen and the anti-goat lgG is done using a line printer (Advanced Microdevices Pvt. Ltd., Ambala) (Figure 1).
(iii) The line imprinted NC laminate is dried at 37°C overnight in vaccum.
(iv) Then it is cut into strips of 4-5 mm width and sealed in a aluminum pouch containing a desiccant and stored at refrigerator temperature (around 4°C).
(v) When the test is to performed, the strips are removed and placed in a tube containing 2 drops of buffer containing Tris HCI, bovine serum albumin and sodium azide; 2 drops of the serum to be tested and one drop of the commercial protein A colloidal gold conjugate (Advanced Microdevices Pvt. Ltd., Ambala).

(vi) Allow the mixture in the tube to ascend the strip and read the results after 5-10 minutes.
(vii) If two pink lines are visible the serum is positive for antibodies against the coated necleoprotein (Figure 2 and 3).
(viii) If one line is present the serum being tested does not have any antibodies to the nucleoprotein of PPR virus.
(ix) If no lines are evident the test is invalid and needs to be repeated.
Figure 1: NC laminate imprinted with the recombinant antigen and anti-goat IgG prepared in rabbit along with the affixed absorbent and sample pads along with a separate tube containing serum to be tested, protein A gold conjugate and buffer.
1-Absorbent pad, 2-Control line, 3-Test line, 4-Sample pad, 5-Serum + colloidal gold + buffer.
Figure 2: Appearance of lines after flow of the mixture from the tube in to the NC Laminate.
1-Absorbent pad, 2-Control line, 3-Test line, 4-Sample pad, 5-Serum + colloidal gold + buffer.

Figure 3: The tests that have been performed and read. Some of the sera samples are positive and some negative. However all the strips show the positive control line.
The present invention uses a recombinant protein (N) as coating antigen and is based on the principle of lateral flow. This test requires no gadgets and can be performed in the field. Another highlight of this testing system is that no separation of bound and unbound analytes is needed for the reading of the results. Moreover, contrary to earlier testing methods such as ELISA, these strips can be preserved forever for any future requirement. This test measures antibodies to the more abundantly present N protein of the PPR virus.

We claim:
1. A test strip for the qualitative detection of antibodies to peste
des etits ruminants virus having three different surfaces,
namely the absorbent pad, nitrocellulose (NC) membrane
mounted on a laminate and sample pad, wherein the antigen is
coated on the membrane along with the antigoat antibody
prepared in rabbits, 5 mm apart using a line printer at medium
speed.
2. The antigen as claimed in claim 1 is a truncated nucleocapsid
protein of PPRV whose gene has been expressed in a
prokaryotic vector system.
3. The nucleocapsid protein as claimed in claim 2 is produced by
recombinant plasmid harboring the deleted nucleocapsid gene
through transformation into the host E. colicells using 1PTG
chemical.
4. The recombinant plasmid harbouring the nucleocapsid gene as
claimed in claim 3 wherein most variable 3'end of the gene has
been deleted.

5. The nucleocapsid protein as claimed in claim 2 wherein the
coating antigen is capable of reacting with antibodies from all
animals infected with different strains of PPRV for the removal
of 3'end.
6. The antigen as claimed in claim 1, wherein is being used in
tubes using gold conjugate in liquid phase.
7. The strip as claimed in claim 1, wherein its use provides
qualitative results in 5-10 minutes.

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Patent Number

243290

Indian Patent Application Number

1633/CHE/2006

PG Journal Number

41/2010

Publication Date

08-Oct-2010

Grant Date

04-Oct-2010

Date of Filing

08-Sep-2006

Name of Patentee

DEPARTMENT OF BIOTECHNOLOGY

Applicant Address

BLOCK-2 7TH FLOOR CGO COMPLEX, LODHI ROAD, NEW DELHI-110 003.

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. VENKATACHALAM	ANIMAL SCIENCES UNIVERSITY, CHENNAI-51,
2	DR. M. S. SHAILA,	MICROBIOLOGY, INDIAN INSTITUTE OF SCIENCE, BANGALORE-560 012
3	DR. GOPAL DHINAKAR RAJ	ANIMAL BIOTECHNOLOGY, MADRAS VETERINARY VOLLEGE, CHENNAI-600 007

PCT International Classification Number

A61B17/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA