TY - BOOK AU - Ravindra Dattatraya Padalkar AU - Jayaprakash V (Guide) TI - Production and Application of Monoclonal Antibodies Against Duck Plague Virus U1 - 636.089 6 PY - 1999/// CY - Mannuthy PB - Department of Microbiology, College of Veterinary and Animal Sciences N2 - 2. Monoclonal fltl~ibodies (Mabs) were raised against the vaccine strain r of OPY and three strains of OPY Viz, Vaccine (oPY - Y), IYRl (OPY -I) and AlIeppy strain (OPY -A) were used to raise polyclonal serum in the present investigation. DPY - Y was revived in 11 day old chicken embryo and the embryo death was recorded fout to five days PI with congestion all over the body . and spleen and necrotic foci in liver. The cytopathy in CEF cell culture observed was rounding ami clumping of the cells, syncytium formation and bridge fortnation with extensive vacuolation in the Cytoplasm. The detachment of the cells was observed at 120 h PI. DPY -I a virulent strain was inoculated in the ducklings, death was tecorded in all the inoculated birds with extensive hemorrhages on Serous membranes, muscles and visceral organs. Necrotic foci on liver,' enlargement and congestiol1 of liver and spleen, and white hecrotic foci in the gizzard were evident. The virus was further passaged ill DOE and .' '" '. " . . .. ," . ~ ". ," I' .' , , ", . cultivated in bulk in DEF cell culture. ,'.' < • .. ~ . • . . The DPY - Y and DPY -J\ were titrated in CEF cell culture and the TCID 50 was 4.7 X 10 ~ per ml of the inoculum for DPY-Y and 3.2 X 104 for DPV -J\. DPV -I cultivated in DEF cell culture had a TCIDso of 1 XI 0 5 'per ml of the inoculum All the strains were partially purified at 100000 g for 4.5 hat 4" C in Beckman ultra centrifuge and the protein concentration of the virus was estimated by biuret method and was found to be 11 mg, 8 mg and 7 mg for DPV-Y, J\ and I respectively. All the three strains of DPV were inoculated in rmce to raise polyclonal serum. Four mice out of five inoculated with DPV-Y showed ' ELISJ\ titres more than l : 12800, one mouse showed a titre of I :6400. The mice inoculated with DPY -A showed a titre of more than 1: 12800 and those inoculated with DPV-I, 1 :~/WO ELISA 'Was' used to test the sera samples of the mice inoculated with DPV strains. The test was found to be highly sensitive, easy to perform and less time consuming. The test therefore can be recommended for routine diagnosis of DPY '. ~:"'---' " ••• I I' ..•.. r: Polyclonal serum was used to study the cross reactivity of the three strains of DPY with ELlSA. DPY -V polyclonal serum reacted with the homologous virus with a titre of 1: 12800. It also showed similar titer with other two heterologous strains. Polyclonal serum raised against DPY -A had a titre of I: 12800 with homologous and heterologous strains of DPV: DPV-l reacted with homologous strain at a titre of 1 ;1,)400. Similar titres were observed with hcrtologous strains. Immuno peroxidase test was used for the detection of the tissue antigens in DPV infected CEF monolayers and in liver and spleen sections. Polyclonnl scrum raised against DPV - V detected homologous virus in the CEF monolayers and hetrologous virus in the liver and spleen sections. The staining reaction was observed us dark brown deposits at the VIruS localization. However background tissue was also stained brown to faint yellow in the stained preparation . ..•.. The virus neutralization test was used to study the cross neutralization by employing polyclonal serum raised against the three. strains ofOPV. Polyclonal ~rUm raised against DPV-V showed a YNT 01'64 with a VNI of 1.8 with homologous virus and a VNT 45 with VNI 1.65 with other two strains of DPV. Polyclonal serum raised against DPY -A neutralized the homologous virus at a titre of 32 with a VNI of 1.5. It also neutralized the vaccine strain of IJPV with same VNT and VNI, However it neutralized DPV-I \ ith VNT 24 and VNI 1.35. DPV-I polyclonal scrum neutralized the homologous virus at a titre of 45 with a VNI of I.G5 . The same neutralized DPV-V and DPV-A with a VNT 32 and VNI 1.5. Monocloual Antibody Production The plates were seeded with the feeder cells by collecting the spleen cells from a normal healthy mouse. The same plates were used for seeding the fused cells. Fusion of the myeloma cells and the splenocytes from the donor mice primed with the vaccine strain of DrV was under aken in presence of poly ethylene glyco] ,1)~U 4000). Small colonies of the hybrids were observed in the wells and the growth of these was monitored. As soon as the hybrids attained n satisfactory growth i.e. ~n :-,e! ,_,:1 t coverage of the well the hybrid supornatant was tested for antibody production with the help of indirect ELlSA and the positive hybrids were selected. Cloning of the positive hybrids was done by limiting dilution method to ascertain 1110110- clonality. A tnln! er seven clones producing desired antibodies against UPV vaccine strains were grown and Mabs were used for testing the cross reactivity of the three strains of DPV. Out of 7 Mabs tested Mab I reacted with homologous virus with a titre of 32. It also reacted with other two heterologous strains with the similar titre. Mab 3, 4 and 6 had a titre of 16 with homologous as well as heterologous virus strains of Dry and Mab 2, 5 and 7 showed a titre of 8 with all the strains ofDPY. No differences were recorded in the- EUSJ\ titres of the 7 Mabs with homologous as well as heterologous strains of DPV. This suggested that there may be a close antigenic and serologic relationship within the three strains ofDPV under study. ? r Immuno peroxidase test was used to detect the viral antigens in the DPY infected CEF and in the liver and spleen tissue sections from the birds infected with virulent Alleppy and IYRI viruses. All the seven Mabs detected homologous as well as heterologous virus in the infected CEF an~ in the liver and spleen sections. The reaction was very clear and background staining was almost negligible. The viral antigen.deposits could be detected as dark red brown deposits in the cytoplasm and nucleus of the cells. All the seven Mabs were tested for cross neutralization and the results indicated that, Mab 1 neutralized the homologous virus and had a VNT of 8 with YNI 0.9. It also neutralized the heterologous strains with similar YNT • ..• and VNI. Mab 2, 4 and 6 had a VNT of 6 with homologous virus and a VNI 0.79. These neutralized the other two virus strains with a VNT 4 and VNI 0.75. Mab 3 had a VNT Of 2.4 with VNI 0.39 with homologous virus and a VNT of 2 with VNI of 0.35 with the heterologous strains. However, Mab 5 and 7 did not neutralize the homologous as well as hetrologous viruses and may be a non neutralizing. The survivabiIity and longevity trial was conducted to ascertain the appropriate time of collection of supernatant along with the usual parameters like change in the pI-I and 80 per cent coverage of the well. The criterion designated for the collection of the hybridoma supernatant like change of pH and 80 per cent coverage of the well seems to ,be appropriate for deciding the medium change and collection of the supernatant. The conclusions from the present study are, in comparison with the 'polyclonal serum Mabs are highly specific in detection of the viral antigens in the tissue sections infected with the virus and the background staining reaction was completely reduced, thereby refining the test and making the diagnosis very easy. The test can be recommended for the diagnosis ofDPV. " . -t As regards the ELISA test it has bee: I observed that the test is highly sensitive , easy to perform and less time consunung and should be recommended as routine diaFlloslic test for DPV.Y :.; YNT is highly specific with polyelonal sera us well as with Mab. The specificity of the test is increased fairly by using Mabs. As regards the possible strain variation of the DPY it can be concluded that there may not be any strain variation in the strains tested. The probable reason for the break down of immunity and vaccination failure maybe because of low titered vaccine used in the field for vaccination of the birds or thc virus may be acquiring virulence by sub clinical passage in the ducks in the field which might result in the breakdown of immunity and failure of the vaccine. A potent tissue culture adapted vaccine may be useful in solving the problem. UR - http://krishikosh.egranth.ac.in/handle/1/5810144168 ER -