ISHS Acta Horticulturae 129: III International Symposium on Small Fruit Virus Diseases
USE OF THE BARBARA-CLARK F(AB')2 INDIRECT ELISA TEST FOR DETECTION OF VIRUSES IN SMALL FRUITS |
| Authors: | R. H. Converse, Robert R. Martin |
| Abstract:
An indirect ELISA test described in 1982 by D. J. Barbara and M. F. Clark (J. Gen. Virol. 58:315–322) utilizes enzyme-conjugated Staphylococcus aureus Protein A that binds specifically with the Fc portion of immunoglobulin molecules. This is done in polystyrene wells in which viral antigen is first trapped by homologous F(ab')2 fragments prepared by pepsin digestion and subsequent dialysis. In our tests we measured A405 values of single Rubus and Fragaria isolates of tobacco streak virus (TSV) using homologous (TSV-Rubus) rabbit antiserum in double antibody sandwich (DAS) vs. Barbara-Clark (B-C) tests. Preparation of both the immunoglobulin-alkaline phosphatase conjugate (1:1000) used for the DAS tests and the commercial (Zymed Laboratories, Burlingame, California, USA) protein-A-alkaline phosphatase (1:1000) used for the B-C tests was accomplished by means of the standard, one-stage Avrameas glutaraldehyde method. Immunoglobulin was used at 1 ug/ml for both tests, and F(ab')2 was used at 2.5 ug/ml for the B-C tests. The threshold level (A405 healthy sap mean plus 2 standard deviations) for the B-C tests was 0.049 (0.029 ± 0.01) compared to 0.085 (0.059 ± 0.013) for the DAS tests. DAS A405 mean values for TSV in Rubus and Fragaria were 0.721 and 0.846, respectively. The comparable B-C values 0.558 and 0.673, respectively, were about 21% less. However, in studies of more distantly related antigens, when we used the TSV-Rubus antiserum, DAS mean values for the bean red node strain of TSV were lower than comparable B-C values (0.221 and 0.335, respectively). We concluded that the somewhat lower sensitivity of the B-C test compared to the DAS test for homologous virus samples was offset by its lower threshold level. The increased sensitivity for detecting heterologous strains of a virus and a universal, commercially available enzymatic marker make the B-C test a valuable, rapid technique for detecting and for studying relationships in several small fruit viruses that we have investigated. | |
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