Date of Award
Doctor of Philosophy
Molecular, Cellular, and Developmental Biology
Hybridomas secreting monoclonal antibodies (M-Abs) against three isolates of barley yellow dwarf virus (BYDV) were established. Two M-Abs were generated against the MAV isolate, one against RPV, and six against P-PAV. None of the M-Abs reacted with healthy host components. Reactions of M-Abs, or unlabelled polyclonal antisera, in an indirect enzyme-linked immunosorbent assay (ELISA) indicate the presence of a common epitope on all three virus isolates. BYDV dissociated when incubated in carbonate-bicarbonate coating buffer at pH 9.6; stabilization was achieved by predialysis with either 2% formaldehyde or 2% glutaraldehyde. In indirect ELISA, unlabelled polyclonal antisera bound to both stabilized and dissociated homologous and heterologous BYDV isolates. However, conjugated polyclonal antisera were incapable of binding to any dissociated isolates or to stabilized heterologous isolates;M-Abs, independently produced against each of the BYDV isolates, were able to efficiently detect all BYDV isolates in serologically specific electron microscopy (SSEM). SSEM would not, however, detect unrelated viruses. The procedure was highly sensitive, detecting 7.5 pg of virus, and specific; SSEM performed on a mixture of BYDV and soybean mosaic virus (SMV) could detect only BYDV particles;Two M-Abs, specific for different epitopes on SMV, were used in a double-antibody sandwich ELISA (M-Ab-ELISA). When compared witha polyclonal antibody solid phase radioimmunoassay (SPRIA), M-Ab-ELISA was more sensitive than SPRIA and detected less than 10 ng of SMV/ml as compared to 25 ng SMV/ml, respectively. When seeds from 33 field plots, in which 0%, 30%, or 50% of the soybean plants had been inoculated with SMV, were assayed by both systems, results of the two tests correlated for 31 of 33 (94%) seed samples;Immunoaffinity columns, prepared by covalently binding M-Abs to an agarose support matrix, were used to purify SMV. Affinity purified SMV was substantially identical in infectivity, u.v. absorbance profile, 280/260 nm absorbance ratio, sedimentation coefficient, electrophoretic pattern of coat protein, morphology, and antigenicity, to virus purified by standard procedures.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Diaco, Robert, "Production and use of monoclonal antibodies for molecular characterization, affinity purification, and serological detection of economically significant plant viruses " (1985). Retrospective Theses and Dissertations. 7839.