Development of Recombinant Antigen based Elisa for the Diagnosis of Peste Des Petits Ruminants
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Date
2014-05-10
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Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar
Abstract
Peste des petits ruminants (PPR) is a highly contagious economically important
viral disease of small ruminants. Currently, in India Monoclonal antibody based
competitive ELISA employing whole virus is used for serosurveillance / seromonitoring
of PPR. Recombinant antigen-based diagnostic assay would be of immense value during
DIVA strategy and in post-eradication phase to replace the existing whole virus based
diagnostic kits. The present study envisages cloning and expression of immunodominant
ectodomain of PPRV F protein in prokaryotic system and to assess its potential
diagnostic value by ELISA. Ectodomain region gene sequences corresponding to 222
amino acids, were amplified from PPR vaccine virus, cloned into pET33b vector and
expressed in E. coli at an optimal temperature of 37 °C with 1 mM IPTG for 5 hours. The
expressed immunodominant truncated ectodomain of the PPRV F protein (31 kDa) was
characterized by SDS-PAGE and Western blot using anti-his-tagged-conjugate, antiserum
raised against recombinant PPRV F protein, hyper immune serum against whole
PPR virus and convalescent sera from sheep and goats. Further, expressed protein
showed good immunoreactivity by ELISA, with antibodies raised against both PPRV and
recombinant PPRV F. The antibody response mounted against the recombinat PPRV F
protein in immunised rabbits was detected by whole virus antigen based indirect ELISA
indicating the native confirmation of the expressed protein in E. coli. The purified
recombinant PPRV F protein based ELISA was standardized with 4 μg/well of antigen,
1/10 dilution of serum and 1:3000 dilution of anti-goat / sheep conjugate. A cut off value
(PP) of 30 per cent was fixed upon comparison with competitive ELISA by two sided
contingency table. The assay showed 80.95 per cent sensitivity and 91.89 per cent
specificity with good agreement of kappa value (0.694) and excellent area under cuve
(0.9548). The study revealed that recombinant truncated ectodomain of PPRV F protein
can be used as diagnostic antigen in both seromonitoring and serosurveillance of PPR in
sheep and goats.
Description
Ph.D. Thesis
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