Studies on the smear technique for detecting virus antigens in an insect vector by use of fluorescent antibodies

Abstract

The direct and indirect methods with fluorescein-labeled antibodies were compared for the detection of wound-tumor virus (WTV) antigens in smears of leafhoppers Agallia constricta Van Duzee exposed to the virus. In order to obtain specific staining, commercial fluorescein-labeled sheep serum prepared against rabbit gamma globulin for use in the indirect method was passed through a DEAE cellulose column, a procedure known to remove fractions staining nonspecifically. The material that stained specifically by both methods was in the form of particles with brilliant yellow-green fluorescence. WTV-antiserum before and after conjugation had titers of 1 2560 and 1 320 , respectively, showing that some antibodies were either lost or inactivated during the conjugation procedure. For detection of WTV antigens in smears of exposed leafhoppers by the direct method, conjugated WTV antiserum could be diluted only to 1 2 , whereas by the indirect method unconjugated WTV antiserum was satisfactory at a dilution of 1 80 . Also there was much less non-specific fluorescence in smears of leafhoppers stained by the indirect method than by the direct method. Before discovering the superiority of the indirect method of staining the smears, 379 exposed leafhoppers were examined by the direct method. Of these 204 were positive and 134 negative both by smear test (direct method) and by transmission. Eighteen leafhoppers were positive by smear test but did not transmit WTV, and 23 leafhoppers did transmit but were negative by the smear test. Of 319 healthy leafhoppers smeared along with exposed leafhoppers, all, with one exception, which was scored a doubtful positive, were scored negative by the smear test.