Immunological Examinations of Species Variability in Predator Gut Content Assays: Effect of Predator:Prey Protein Ratio on Immunoassay Sensitivity

Abstract

In qualitative predator gut content immunoassays, sensitivity of an immunoassay is important for determining whether a predator contains a targeted prey antigen. If the immunoassay employed is insensitive, the probability of obtaining a false-negative reaction is high. The sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) developed to detect pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), egg(s) in whole body homogenized predators varied in efficacy between species. Specifically, the indirect ELISA was more effective at detecting egg antigen in small predators than in large predators. In this study, we examined the effect that the predator:prey protein ratio has on the sensitivity of an indirect ELISA. Our results suggest that when assaying whole body homogenized predators, care must be taken not to overload an ELISA microplate with nontarget(predator)proteins.Predatorsamplesshould be diluted to less than 125 mg of total protein per sample. Any protein concentration above 125 mg per ELISA microplate well will likely result in an ELISA false-negative reaction. In another experiment, we compared the efficacy of an indirect ELISA with a dot blot immunoassay. Adults of Hippodamia convergens Guerin-Meneville (Coleoptera: Coccineliidae) that had eaten one pink bollworm egg were homogenized in variable dilutions of phosphate-buffered saline (PBS) and each sample was analyzed for pink bollworm egg antigen using both immunoassays. The dot blot immunoassay was more reliable than the indirect ELISA for detecting minute traces of egg antigen in the samples. Generally, the volume of PBS that the H. convergens were homogenized in had little effect on the qualitative outcome of the dot blot. However, the indirect ELISA was more effective when H. convergens was homogenized in a larger volume of PBS. This suggests that the efficacy of an indirect ELISA can be improved for large, protein-rich predators by grinding them in a larger volume of PBS, thus minimizing the total protein in a given sample. r 1997 Academic Press