Rapid detection of specific polyclonal and monoclonal antibodies against bovine leukemia virus

ELISA and Western blot have been used for detecting specific antibodies or antigens for routine diagnostic laboratory tests and experimental protocols , as well as for screening hybridomas secreting antibodies. Although these techniques are sensitive, some slow growing hybridomas are identified as po sitive only when they are grown slowly long time. We standardized the dot-E LISA, a more sensitive technique, for the detection of antibodies against B LV. The main advantages of the dot-ELISA described in this study are (a) it s sensitivity, detecting hybridomas which would otherwise be considered neg ative and discarded from the results of indirect ELISA and/or Western blot; and (b) the possibility of economizing reagents using as little as 1 mu l of the antigen and 0.5 mu l of antibody and conjugate. Different BLV-antige n preparations were bound to nitrocellulose membranes (NC), including cells lysed chemically (LYS) or by sonication (SOC), semi-purified virus (PV), a nd supernatant from infected cultures, either without treatment (SUP) or so nicated (SOS). The antigen preparations most adequate for detecting monoclo nal antibodies against BLV and polyclonal antibodies in cattle sera were un diluted cell lysates (LYS) and semi-purified BLV (PV). When testing bovine sera, the supernatant (SUP) and sonicated supernatant (SOS) antigens gave a high background due to the presence of FCS which reacted with the anti-bov ine labeled antibodies. In this study, 59 BLV specific antibody secreting h ybridomas were identified using the dot-ELISA, compared to only 20 detected using iELISA, and doubtful reactions due to nonspecific binding to fetal c alf serum (FCS) and cellular components were measured. The results of the i mproved dot-ELISA described may be stored at room temperature for future re ference. Results were consistently reproducible in coated nitrocellulose me mbranes kept at different storage temperatures (- 20 degrees C, 4 degrees C , and 25-30 degrees C) 48 h, 1 week and 5 months. (C) 1999 Elsevier Science B.V. All rights reserved.