Application of monoclonal antibodies in the avian leukosis virus GS‐antigen ELISA

Abstract

Five hybridoma cell lines which secrete antibodies to avian leukosis/ sarcoma (ALV) group-specific (gs) antigens (gag gene products) have been established. The hybrid cells resulted from fusion of P3/X63-Ag8.653 myeloma cells with splenocytes of BALB/c mice which had been immunised with purified avian myeloblastosis virus (AMV). Screening of supernatant fluids was performed by an indirect double antibody sandwich enzyme-linked immunosorbent assay (IDAS-ELISA) and the immunoelectroblotting technique. Three hybrid clones secrete monoclonal antibodies (MCA) to 27,000 dalton polypeptides (p27) and two hybridomas produce monoclonal antibody directed to 19,000 dalton phosphoprotein (pp19). All five monoclonal antibodies belong to mouse immunoglobulin isotype IgG(1). MCAs directed to different ALV-p27 epitopes can replace polyclonal rabbit or hamster anti-gs sera in the DAS-ELISA in use for the detection of congenitally ALV-shedding hens. In albumen samples a 16- to 32-fold increase of sensitivity of ALV gs-antigen detection was obtained as compared to the DAS-ELISA employing polyclonal sera. Gs-antigens of a purified AMV preparation were detectable up to minimal concentrations of 13 pg/ml.

Application of monoclonal antibodies in the dutch avian leukosis control programme

“…Since neither the CFT nor the DAS-ELISA as applied here discriminated between gsa of exogenous and endogenous origin the increased sensitivity of the latter test system also increased the chance of detecting endogenous gsa (De Boer and Osterhaus, 1985). Circumstantial evidence for the shedding of gsa of endogenous origin has been described but this usually resulted in relatively low levels of endogenous gsa (Fadly et al, 1981;De Boer et al, 1983).…”

“…Various other extremely sensitive MCA combinations yielded in the DAS-ELISA too many 'false positive' reactions due to the detection of gsa of endogenous origin (De Boer and Osterhaus, 1985). Two modifications of the test procedure were introduced: in order to avoid non-specific binding 4% foetal calf serum was added to the diluents of MCA HRP conjugates and the cut-off level between ELISA-negative and ELISA-positive wells was set at twice the value of the mean absorbance of a series of eight control wells.…”

“…The hybrid cells resulted from fusion of P3/X63-Ag8.653 mouse myeloma cells with splenocytes of BALB/c mice which had been immunised with purified avian myeloblastosis virus (De Boer and Osterhaus, 1985). Three hybrid clones secrete MCAs directed to two topologically distinct epitopes of the 27,000 dalton polypeptide (p27), the major component of ALV-gsa.…”

Expression of avian leukaemia virus env-gp85 in Spodoptera frugiperda cells by use of a baculovirus expression vector

“…Monoclonal antibodies (MAb) directed against ALV gp85 were prepared as described for ALV p27 (De Boer & Osterhaus, 1985). MAb CVI-ALV-61.6 stained an 85K protein in immunoblots using density gradient-purified ALV of subgroup A (RAV-1) (G. F. De Boer et al, unpublished results).…”

“…The chickens received booster injections of 4 x 107 Sf9 cells expressing recombinant gp85 in incomplete Freund's adjuvant 4 weeks later. Blood samples were collected at 2 week intervals after the immunizations and tested by an indirect ELISA (IDAS-ELISA) using microtitre plates coated with affinity chromatography-purified ALV gp85, according to the procedures described by De Boer & Osterhaus (1985), and by immunoblots of purified ALV subgroup A (RAV-1) virus. The plaque reduction assay was performed as described by Spencer (1987).…”