Further evaluation of factors affecting a rapid ELISA procedure for detection of IgG antibodies to Candida albicans

Abstract

A rapid enzyme-linked immunosorbent assay (ELISA) was developed to detect IgG antibodies against a cytoplasmic antigen of Candida albicans. The optimum conditions and time required for each step were investigated. Pre-equilibration at 37 ° C and constant agitation of immunoreactants resulted in a total test time of 1 h for examination of single serum dilutions. Initial binding of the antigen at 37 ° C to the solid phase occurred within 4–6 min and was dependent on concentration. A serum dilution of 100-fold resulted in adequate discrimination between precipitin test-positive and -negative sera. Interaction of antibody and the bound antigen was maximal after 6 min incubation at 37 ° C and was dependent upon the precipitin titre of the serum. Ten minutes was selected as the optimal incubation time for each of the stages, by which time maximal binding had occurred, irrespective of antibody affinity for antigen. The ELISA was completed by incubation at 37 ° C for 10 min with alkaline phosphatase-conjugated anti-human IgG. Discrimination between positive and negative test sera was achieved by incorporating in each test run precipitin test-positive and -negative reference sera from groups of persons with or without C. albicans colonization or infection. Closest agreement between sera positive for C. albicans precipitins by counter-immunoelectrophoresis and the rapid ELISA was seen when reference negative sera were selected from non-colonized individuals.