Abstract
Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51-ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.
Highlights
With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual Enzyme-linked immunosorbent assay (ELISA) test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively
The bovine leukemia virus (BLV) is the causative agent of Enzootic Bovine Leukosis (EBL), a lymphoproliferative type disease that affects B cells, and the provirus is integrated into the genome, transforming infected animals in Lifetime carriers [1]
The sera samples were tested for the presence of BLV specific antibodies by agar gel immunodiffusion (AGID) and a commercial indirect ELISA test
Summary
The bovine leukemia virus (BLV) is the causative agent of Enzootic Bovine Leukosis (EBL), a lymphoproliferative type disease that affects B cells, and the provirus is integrated into the genome, transforming infected animals in Lifetime carriers [1]. Given the multiple transmission routes, both natural and iatrogenic and the minimum volumes of peripheral blood required to transmit the virus, this disease has a high potential for disease transmission [2] [3]. The Bovine leukemia virus (BLV) is an exogenous B-lymphotropic retrovirus that naturally provokes B-lymphoproliferative disorders in cattle and can be used to induce a related B-cell-associated pathology in experimentally infected sheep [6]. Other authors reports that high sensitivity tests are able to detect anti-p24 antibodies before the appearance of anti-gp antibodies; and the anti-p24 antibodies could be an equal or greater degree than anti-gp51 [3] [10]. The immunoglobulin isotype in the immune response varies according to the stages of LEB disease, in asymptomatic animals IgM and IgG1 anti p24 and gp51were detected, whereas the detection of IgA occurs in animals with lymphoma [11]
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