Development of Recombinant Antigen based Elisa for Diagnosis of Brucellosis
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Date
2013-05-28
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Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar
Abstract
Brucellosis is a major zoonotic disease affecting both animals and humans.
Highly sensitive and specific diagnostic tests are needed for the detection of brucellosis.
BLS gene of 474 bp from strains of B. suis 1330, B. melitensis BVM 25b and B. abortus
S99 was amplified by PCR using designed primers, cloned into pTZ57R/T vector and
confirmed by RE digestion of plasmid with EcoRI and NotI, nucleotide sequencing and
by colony PCR. BLS gene was sub-cloned into pET32a prokaryotic expression system.
The recombinant plasmid pETBLS was then transformed in to E. coli BL21 cells. 18 kDa
rBLS protein was expressed and purified to homogeneity using Ni-NTA agarose metal
affinity column. The concentration of recombinant protein was found to be 0.847 mg/ml.
The specificity of purified rBLS protein was confirmed by immunoblot analysis with
swine Brucella positive serum and further immunoblotting studies were carried out with
various dilutions of sera and conjugate. A dilution of 1:1,000 for sera and 1:6,000 for anti
species conjugate was optimum. Further, rBLS protein was found immuno-reactive when
probed with bovine, sheep and goat and human Brucella positive and negative sera. The
purified rBLS protein based ELISA was standardized with 200 ng of protein with a serum
dilution of 1:25, one hundred each of swine, bovine, sheep and goat and human sera
sample were used. Results revealed good reactivity of rBLS antigen in comparison with
S-LPS antigen against same set of sera samples of different host species. Correlation was
found between the OD values of S-LPS ELISA and rBLS ELISA with "Correlation
coefficient (r)" ranging between 0.907 and 0.971 (P<0.0001); and the variation was found
to be non significant (P < 0.05). Further, to rule out the cross reactivity, Y. enterocolitica
O:9 and E. coli O:157 positive sera were subjected to rBLS ELISA, the result showed
low OD values of 0.246 and 0.128 respectively. In conclusion, rBLS ELISA could be
used for screening of animal and human brucellosis with common conjugate Protein G.
However, screening large scale field sera sample and validation has to be carried out for
its wider application.
Description
Ph.D. Thesis