Optimizing immunomarking systems and development of a new marking system based on wheat

Immunomarking systems used to track large-scale movement patterns of insects are highly dependent on the efficiency of the enzyme linked imunosorbent assay (ELISA) reaction and logistical factors (e.g. concentration of marker applied, ability of the marker to wet the insect cuticle, and trapping methods). This paper examines ways to increase ELISA efficiency and mediate logistical factors, and provides information on a new immunomarking protein based on wheat gluten. The present studies on improving efficiency of the ELISA reactions showed that specially treated microplate surfaces were needed for soymilk and gluten assays, but not for egg albumin and casein assays. Sample dilution was investigated and was found to improve the signal/noise (S/N) ratio for the albumin and casein assays, but S/N ratios for the gluten and soymilk assays were less sensitive. However, for all assays, marked specimens were still detectable even with dilutions down to 6% of the original sample, which would allow more tests to be run on the same initial sample volume. For the logistical factors, these studies showed that marking of an insect by having it walk across a dried residue could be virtually eliminated for the casein and soymilk assays when the concentration applied was reduced to < 4%, but residues of 0.125% egg that had been aged in the field seven days still marked 37.5% of test insects placed on the residues. Also, the adjuvant Sylgard(®) 309 used at 80 ppm enhanced wetting of the insect cuticle and had little or no effect on the ELISA reaction, but the wetting agents R-11 and Silwet(®) L-77 were much more likely to negatively affect ELISA performance. Five different trapping adhesives were also evaluated and found to reduce ELISA efficiency 38-45% for the casein assay and 61-78% for the soymilk assay, while the albumin and gluten assays were unaffected. The information provided in this paper can be used to help correct for inherent differences in marking efficiency of the different proteins by manipulation of sample preparation, adjuvants, and concentrations applied.