Abstract
The reliability of the standard double antibody sandwich enzymelinked immunosorbent assay (DAS-ELISA) was compared with a shorter, two-step DAS procedure in which sample and conjugate were mixed and incubated together in one step. The two assays were compared using beet western yellows virus and potato leafroll, M, S, X, and Y viruses. The two-step procedure was more sensitive,i.e., it detected small quantities of virus with greater statistical reliability than the standard procedure. At high virus concentrations, the standard produced stronger ELISA reactions than the two-step assay, but both assays were reliable. Since all of the viruses tested withstood high incubation temperatures, the incubation period for the two-step procedure could be reduced to 1 hr at 30 or 37 C. Therefore, assays could be completed within 2 hr using the two-step procedure compared with 2 days for the standard procedure. Reliable results were achieved with samples prepared by grinding tissues in buffer or, more simply, by adding pure, pressure extracted juice directly to conjugate in assay wells. Coating plates with gamma globulins or with F(ab′)2 fragments of gamma globulins gave equally reliable results with all viruses except potato leafroll, where coating with gamma globulins was superior.
Compendio
Se comparó la confiabilidad del método estándar de doble anticuerpo en la técnica de detección por conjugados enzimáticos (DAS-ELISA) con un procedimiento más corto de dos pasos, en el cual la muestra y el conjugado se mezclaron e incubaron al mismo tiempo en un paso. Se compararon las dos pruebas usando el virus occidental del amarillamiento de la remolacha y los virus del enrollamiento de la papa, M, S, X e Y. El procedimiento de dos pasos mostró mayor sensibilidad, o sea que detectó pequeñas cantidades de virus con una mayor confiabilidad estadística que el procedimiento estándar. A altas concentraciones del virus, el procedimiento estándar produjo reacciones más marcadas que el procedimiento de dos pasos, pero ambas pruebas fueron confiables. Considerando que todos los virus probados soportaron temperaturas altas, el período de incubación con el procedimiento de dos pasos podría ser reducido a 1 hr a 30 ó 37 C. Por lo tanto usando este procedimiento las pruebas pueden completarse en 2 hr, en lugar de 2 días que requiere el procedimiento estándar. Se consiguieron resultados confiables con muestras preparadas triturando el tejido en bufer o simplemente agregando directamente en los hoyos de prueba jugo puro extraído a presión. El revestimiento de las placas con gama globulinas o con fragmentos de gama globulinas F(ab′)2 dió igualmente resultados confiables con todos los virus, con excepción del virus del enrollamiento, donde el revestimiento con gama globulinas fué superior.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Literature Cited
Barbara, D.J. and M.F. Clark. 1981. A simple indirect ELISA using F(ab′)2 fragments of immunoglobulin. J Gen Virol 58:315–322.
Bar-Joseph, M., S.M. Garnsey, D. Gonsalves, M. Mosconitz, D.E. Purcifull, M.F. Clark and G. Loebenstein. 1979. The use of enzyme-linked immunosorbent assay for the detection of citrus tristeza virus. Phytopathology 69:190–194.
Clark, M.F. 1981. Immunosorbent assays in plant pathology. Annu Rev Phytopathol 19:83–106.
Clark, M.F. and A.N. Adams. 1977. Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483.
Engval, E. and B.R. Clark. 1980. Enzyme-linked immunosorbent assay (ELISA): Theoretical and practical aspects.In: Enzyme-immunology (E.T. Maggio, Ed.). CRC Press, Inc., Boca Raton, FL, pp 167–179.
Flegg, C.L. and M.F. Clark. 1979. The detection of apple chlorotic leafspot virus by a modified procedure of enzyme-linked immunosorbent assay. Ann Appl Biol91: 61–65.
Kaniewski, W.K. and P.E. Thomas. 1986. Two-step ELISA methods for quick and reliable detection of potato viruses. Phytopathology 76:884.
Stobbs, L.W. and D. Barker. 1985. Rapid sample analysis with a simplified ELISA. Phytopathology 75:492–495.
Van Retenmortel, M.H.V. 1982. Serology and immunochemistry of plant viruses. Academic Press, New York. 302 pp.
Voller, A., D.E. Bidwell and A. Bartlett. 1979. The Enzyme-Linked Immunosorbent Assay (ELISA). Dynatech, Europe, Guernsey, 125 pp.
Vuurde, J.W.L. van and D.Z. Maat. 1985. Enzyme-linked immunosorbent assay (ELISA) and disperse dye immunoassay (DIA): comparison of simultaneous and separate incubation of sample and conjugate for the routine detection of lettuce mosaic virus and pea early browning virus in seeds. Neth J Pl Pathol 91:3–13.
Author information
Authors and Affiliations
Additional information
Scientific Paper No. 7759.
Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the project by the USDA and does not imply its approval to the exclusion of other products that may also be suitable. This article reports the results of research only.
Rights and permissions
About this article
Cite this article
Kaniewski, W.K., Thomas, P.E. A two-step ELISA for rapid, reliable detection of potato viruses. American Potato Journal 65, 561–571 (1988). https://doi.org/10.1007/BF02908337
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02908337