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KMID : 0545120070170111885
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Journal of Microbiology and Biotechnology
2007 Volume.17 No. 11 p.1885 ~ p.1889
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Improvement of Antigen Blotting in a Tissue Blot Immunobinding Assay for the Detection of Two Chili Pepper Viruses
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Han Jung-Heon
Shin Jun-Sung
Kim Young-Ho
Kim Byung-Dong
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Abstract
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The tissue blot immunobinding assay (TBIA) is widely used for the detection and localization of plant viruses in various plant tissues. The basic experimental procedures of TBIA sampling and blotting were simplified using commercially available micropipette tips. This method was termed the ringblot immunobinding assay (R-BIA), as the blot on the membrane forms a ring shape. The detection efficacy of RBIA was tested for two chili pepper viruses, pepper mild mottle tobamovirus (PMMoV) and pepper mottle potyvirus (PepMoV), following the optimized serological procedures of TBIA (length of the incubation period and BSA concentration, and primary and secondary antibodies). Sensitivity of the RBIA was about 1 ng/ml of purified PMMoV in pepper leaf sap from a healthy pepper plant. R-BIA also showed high specificity in the detection of PMMoV and PepMoV. Moreover, the modified sampling and blotting procedures were simpler and more reliable than other TBIA methods (such as wholeleaf blotting and crushed-leaf blotting), suggesting that the RBIA may be used for medium- to large-scale detection of plant viruses in laboratories with minimal facilities
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KEYWORD
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Chili pepper, detection, pepper viruses, ring-blot, tissue blot immunobinding assay
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