Enzootic bovine Leukosis: development of an indirect enzyme linked immunosorbent assay (I-Elisa) in seroepidemiological studies

Abstract

Bovine Leukemia Virus (BLV) is the etiologic agent of Enzootic Bovine Leukosis, a retrovirus exogenous to the bovine species. Once infected, there is no detectable viraemia but instead there is a strong and persistent immunological response to BLV structural proteins, essentially the gp51 envelope glycoprotein and the mayor core protein p24. We describe the test procedure of an indirect ELISA (I-ELISA) using polyclonal reagents for the detection of antibodies to BLV. For comparison, the sera were simultaneously tested by agar gel immunodiffussion (AGID) test, which is currently used as diagnostic standard for BLV infection. The antigen applied does not require a high degree of purification and the data from the analysis of the negative sera showed that the establishment of a cut-off level corresponding to 3 times the standard deviation (SD) above the mean for the negative control set of sera provided acceptable specificity, reducing the risk of false positives results. A comparison of the results obtained by AGID test and I-ELISA showed that considering a total of 465 serum samples, all of the 234 samples (50%) that were positive by AGID were positive to the I-ELISA. Of 225 serum samples which yielded negative results in the AGID test, 69 (15%) were found to be positive by the I-ELISA and 156 (33%) were negative by both techniques. Few sera (2%) that were non-specific by AGID were defined as negative or positive by I-ELISA.

Adaptation of an indirect enzyme-linked immunosorbent assay for seroepidemiological studies of enzootic bovine leukosis

“…This provided a high specificity and reduced the risk of false positive results. This cutoff value is in accord with Gonzalez who reported that mean value + 2SD (0.11) and mean + plus 3SD (0.18) represent good specificity and sensitivity of the test, respectively (Ridge and Vizard 1993;Gonzalez et al 1999).…”

“…Using a crude antigen in an indirect ELISA can cause a high background (Gonzalez et al 1999); in this regard, we set up a cutoff value on the basis of the high numbers of negative sera (confirmed by CI-ELISA) from different herds and various ages. To establish a reliable cutoff that covers 99% of negative control set of sera, threefold standard deviation (SD) value above the mean of negative control absorbances was considered.…”

Testes de diagnóstico para o vírus da leucemia bovina

“…Impurities are responsible for high background values and for non specific reactions. Usually, high purification of antigens increases productions costs (González et al, 1999a). The polymerase chain reaction (PCR) is a useful and sensitive tool for fast and early detection of BLV proviral DNA in blood, organs and tumoral samples (González et al, 1999b).…”

Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

“…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].…”

“…In experimentally infected animals anti-gp51 antibodies seem to be the first to appear with titres consistently higher than anti-p24 antibodies [7] [8] [9]. Other authors reports that high sensitivity tests are able to detect anti-p24 antibodies before the appearance of anti-gp51 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].…”