Browsing by Author "KAVITHA, KANDIMALLA"
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ThesisItem Open Access STANDARDIZATION OF INDIRECT ENZYME LINKED IMMUNOSORBENT ASSAY FOR ANTIBODY TITRATION TO PESTE DES PETITS RUMINANTS IN VACCINATED SHEEP(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2007-10) KAVITHA, KANDIMALLA; DHANALAKSHMI, K(MAJOR); NARASIMHA REDDY, Y; ANJANEYULU, YABSTRACT : The present study was taken up with the objective of standardization of indirect Enzyme Linked Immunosorbent Assay (I-ELISA) using polyclonal serum for antibody titration to PPRV in vaccinated sheep sera. The test was compared with SNT and FAT. Hyper-immune sera against PPRV, for use in indirect ELISA, were raised in four sheep by injecting each with 20 doses of PPR vaccine at weekly intervals. Sera after last injection were checked for antibody by cELISA and used as positive controls in the indirect ELISA test. For standardization of Indirect ELISA, partially purified PPRV antigen was prepared from PPR virus (vaccine strain) grown in vero cells by precipitation with polyethylene glycol (PEG 6000) at 8 %. Optimization of antigen concentration and serum dilutions were done by checker board titration. It was found that 2.5 μg of antigen per well and 1:100 dilution of serum samples were optimum and eliminated false positives. Indirect ELISA was employed for monitoring of antibody to PPRV in 20 sheep vaccinated against PPR. Titres of 1:100 and above were considered as positive for PPR antibody. Indirect ELISA detected antibodies in 16 animals on day 14 post vaccination (PV) while maximum of 18 animals were positive on day 21 and 28 PV. Two animals never responded to the vaccine as detected by indirect ELISA. Serum neutralization test (SNT) was also carried out for the sera collected from the vaccinates with a view to compare the tests. All the animals except one, had neutralizing antibody titres from day 14 PV and neutralizing antibody titres persisted till the end of the experiment. Fluorescent antibody technique (FAT) was applied for antibody detection to PPRV in the sheep sera. A maximum of seventeen animals were positive by FAT on day 21 and 28 PV. However three animals did not show any antibody response by FAT. On comparison it was found that SNT was the most sensitive test to detect antibody to PPRV. On comparison with SNT, ELISA had sensitivity of 84 % and specificity of 100 %. FAT had sensitivity of 82.35 % and specificity of 100 %. In conclusion it was found that, in view of limitations of SNT for application on large scale indirect ELISA and FAT were reliable, less expensive and effective to monitor the vaccine responses/vaccine efficacy in PPR vaccinated sheep.