ARS | Publication request: Optimizing a Protein-Specific Elisa to Detect Protein-Marked insects

Submitted to: International Journal Of Pest Management
Publication Acceptance Date: October 28, 2003
Publication Date: September 1, 2004
Citation: Hagler, J.R. 2004. Optimizing A Protein-Specific Elisa To Detect Protein-Marked Insects. International Journal Of Pest Management. P. 209-214

Interpretive Summary: 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 rabbit IgG marked lady beetles. Results showed that there is great variation in the ability of the different ELISAs to detect rabbit IgG-marks on beetles. The conventional protein marking ELISA described over a decade ago remains superior to the more recent ELISA protocols tested. The labor required to conduct the ELISA can be reduced while maintaining efficacy by soaking the insect instead of homogenizing it in sample buffer. Furthermore, incubation intervals for each step of the ELISA should be at least 60 minutes due to the decreased sensitivity observed for ELISAs with shorter incubation intervals. Finally, inexpensive rabbit serum may be a suitable marker alternative to expensive rabbit IgG for many MRR studies.

Technical Abstract: 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. Five variations of ELISA were examined for their ability to detect a rabbit IgG mark on the convergent lady beetle, Hippodamia convergens Guérin-Meneville. 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 simply soaking an individual beetle is a viable alternative to the labor intensive homogenization of the sample. Sandwich ELISAs with immunoreagent incubation intervals held constant at 5, 10, 20, or 60 minutes were also examined for their ability to detect rabbit-IgG-marked beetles. Results showed that the ELISA with 60 minute immunoreagent incubations 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 1, 4, or 8 parts water yielded statistically similar ELISA values to those beetles marked with the conventional rabbit IgG mark.