Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses

Sudharshana, M R and Reddy, D V R (1989) Penicillinase-based enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of Virological Methods, 26 (1). pp. 45-52. ISSN 0166-0934

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A penicillinase (PNC)-based, enzyme-linked immunosorbent assay (ELISA) was standardized to detect maize mosaic virus (MMV) in sorghum leaf extracts, peanut mottle virus (PMV) in pea leaf extracts, and tomato spotted wilt virus (TSWV) in peanut leaf extracts. Rabbit Fc-specific antibodies were conjugated with PNC by a single step glutaraldehyde bridge. Among several indicators tested, bromothymol blue (BTB) was found suitable for measuring PNC activity under simulated conditions. Two reagents, starch-iodine complex (SIC) and a mixed pH indicator, containing bromocresol purple and BTB (2:1) used earlier for the PNC-based ELISA, were compared with BTB for utilization in the PNC-based ELISA. SIC gave a slightly higher virus titre than BTB or the mixed pH indicator, but it often gave nonspecific reactions. Sodium or potassium salts of penicillin-G at 0.5 − 1.0 mg/ml and BTB at 0.2 mg/ml were found to be suitable as substrate-indicator mixture for PNC-based ELISA. The sensitivity of the PNC system was comparable to those of the alkaline phosphatase (ALP) and horseradish peroxidase (HRP) systems in detecting MMV, PMV, and TSWV. The PNC conjugate could be used at a greater dilution than those of the ALP and HRP conjugates and the BTB substrate mixture was stable for at least 3 weeks at 4°C. Penicillin is readily available in developing countries, and at a substantially lower cost than p-nitrophenyl phosphate, the commonly used substrate for ALP in the plate ELISA. Thus the PNC-based ELISA provides a less expensive means for assaying plant viruses by ELISA.

Item Type: Article
Uncontrolled Keywords: ELISA; Bromothymol blue; Penicillinase; Maize mosaic virus; Peanut mottle virus; Tomato spotted wilt virus
Subjects: Mandate crops > Groundnut
Mandate crops > Sorghum
Others > Plant Virology
Others > Maize
Depositing User: Mr Siva Shankar
Date Deposited: 30 Nov 2012 03:46
Last Modified: 30 Nov 2012 08:30
Official URL:
Acknowledgement: We are grateful to Dr. A.J. Gibbs, Research School for Biological Sciences, Australian National University, and Dr. D. McDonald, Legumes Pathology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), for their helpful comments on the manuscript.
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