Japanese Journal of Phytopathology
Online ISSN : 1882-0484
Print ISSN : 0031-9473
ISSN-L : 0031-9473
On the Screening Procedures of ELISA for Monoclonal Antibodies against Three Luteoviruses
Kazusato OHSHIMAEishiro SHIKATA
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1990 Volume 56 Issue 2 Pages 219-228

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Abstract

Total of one hundred and thirty-five antibody-secreting hybridomas were fused from luteovirus-immunized spleen cells. Hybridomas secreting monoclonal antibodies (MoAbs) for potato leafroll virus (PLRV), beet western yellows virus (BWYV) and tobacco necrotic dwarf virus (TNDV), were screened by four different procedures of enzyme-linked immunosorbent assay (ELISA). There are procedure 1; antigen adsorption ELISA (AAI-ELISA), in which purified virus in phosphate buffered saline (PBS) at pH 7.4 was adsorbed onto the microplate wells, procedure 2; AAI-ELISA in which purified virus in sodium carbonate-bicarbonate buffer (SCB) at pH 9.6 was adsorbed onto the microplate wells, procedure 3; indirect double antibody sandwich ELISA (IDAS-ELISA) in which polyclonal antibody was used as trapping antibody and purified preparations diluted in PBS-T (containing Tween-20) as antigens were used, procedure 4; IDAS-ELISA in which polyclonal antibody was used for trapping antibody and crude saps of virus infected plants in PBS-T as antigens were used. Based on the results, all of the antibody-secreting hybridomas can be screened by AAI-ELISA of procedure 1. On the other hand IDAS-ELISA is indispensable for screening virus-specific MoAbs. We recommend that combination of two ELISA procedures, such as AAI-ELISA of procedure 1 and IDAS-ELISA of procedure 4, will be used for the screening of luteovirus antibody-secreting hybridomas.

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