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 t...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.展开更多
文摘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.
