Optimizing a protein-specific ELISA for the detection of protein-marked insects

A series of tests were conducted to determine if the sensitivity and efficiency of an established rabbit immunoglobulin (IgG)-specific enzyme-linked immunosorbent assay (ELISA) could be improved for detecting a protein mark on insects. Five variations of ELISA were examined for their ability to detect a rabbit IgG mark on the convergent lady beetle, Hippodamia convergens Guérin-Méneville. The conventional sandwich ELISA was the most sensitive immunoassay based on the strength of the ELISA reaction and the proportion of individuals scoring positive for rabbit IgG. ELISAs were also conducted on rabbit IgG-marked beetles that were either homogenized or soaked in sample buffer prior to the assay. Results showed that homogenized beetles yielded higher ELISA values in the conventional ELISA than soaked beetles, but the qualitative response (i.e., percentage scoring positive for the mark) was about the same for up to 18 days after marking. Therefore, in some instances, simply soaking an individual beetle might be a viable alternative to the labour-intensive homogenization of the sample. Sandwich ELISAs with immunoreagent incubation intervals held constant at 5, 10, 20, or 60 min were also examined for their ability to detect rabbit-IgG-marked beetles. Results showed that the ELISA with immunoreagent incubations of 60 min yielded a significantly higher ELISA reading than the shorter intervals. However, all the marked beetles examined scored positive for the presence of rabbit IgG, regardless of the incubation interval. Finally, a test was conducted to determine if the conventional ELISA could also detect the presence of relatively inexpensive normal rabbit serum on marked beetles. Beetles marked with rabbit serum diluted one part rabbit serum to one, four, or eight parts water yielded statistically similar ELISA values to those beetles marked with the conventional rabbit IgG mark.