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Extension of the enzyme linked immuno sorbent assay method to the measurement of the specific radioactivity of viruses in crude cellular extracts


, : Extension of the enzyme linked immuno sorbent assay method to the measurement of the specific radioactivity of viruses in crude cellular extracts. Journal of Virological Methods 6(6): 347-356

The double-antibody sandwich method of enzyme-linked immunosorbent assay, which allows accurate quantitative determination of plant viruses, was extended to a radiochemical procedure which permits direct measurement of the specific radioactivity of virus labeled in vivo and present in very crude plant homogenates. Evidence is presented showing that 20-50% of the virus introduced in the polystyrene wells during the antigen incubation step could be trapped in the sandwich. The percentage of the virus bound increased with the concentration of the coating antibody and was almost proportional to the concentration of the antigen and to the incubation time of the antigens. Complete dissociation of the double-antibody sandwich was achieved by incubation with 0.2 M KOH or NaOH (pH 13.3), and the label carried by the virus was measured by scintillation counting of the solubilization fluid. The ratio infected/healthy was much higher for the radiochemical procedure than for the immunosorbent assay itself since binding of the virus to the coating antibody was not accompanied by any nonspecific trapping of radioactive contaminants in the double-antibody sandwich. The procedure was highly sensitive since the background corresponded to the scintillation counting background. The detection of label carried by tobacco mosaic virus was possible when the tobacco samples contained at least 5 ng of virus carrying a label as low as 40 dpm 3H or 20 dpm 14C.

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