Detection of Three Plant Viruses by Dot-Immunobinding Assay

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Abstract
An indirect dot-immunobinding assay (DIA) and a double antibody sandwich (DAS)-DIA were evaluated and compared with DAS-ELISA for detection of tobacco mosaic virus (TMV), tobacco ringspot virus (TbRSV), and tomato ringspot virus (TmRSV). Indirect DIA successfully detected 30 pg (30 ng/ml in a 1-.mu.L sample) of purified TMV or TbRSV and 100 pg (100 ng/ml in a 1-.mu.L sample) of TmRSV. DAS-DIA and ELISA detected 50 ng (10 ng ml in a 5-ml sample) and 2.5 ng (10 ng/ml in a 250-.mu.l sample), respectively, of all three purified viruses. Detection levels similar to those given above for indirect DIA, DAS-DIA, and ELISA were achieved when purified virus was diluted in healthy tobacco or Chenopodium quinoa sap. With indirect DIA, absorption of the first antibody with healthy plant sap and incubation of the membrane, on which the sample had been adsorbed, in 2% Triton X-100 was necessary to eliminate background color. Absorption of antisera was not necessary with DAS-DIA or ELISA. Both DIA procedures compared favorably with ELISA in discriminating between healthy and TmRSV-infected C. quinoa, geranium, dandelion, tobacco, and plantain plants.