Development of a Sandwich ELISA for Quantification of Gly m 4, a Soybean Allergen.

Abstract

Gly m 4 is a key soybean allergen that causes allergic symptoms in the skin, gastrointestinal tract, or respiratory tract of sensitive individuals. To understand naturally variable levels of Gly m 4 among conventional soybean varieties, a sandwich ELISA was developed and validated using a mouse anti-Gly m 4 monoclonal antibody and a goat anti-Gly m 4 polyclonal antibody as capture and detection antibodies, respectively. The ELISA shows high specificity to Gly m 4 without any cross-reactivity to other soybean proteins and has a quantification range of 7.8-250 ng/mL using an Escherichia coli-produced recombinant Gly m 4, with 2.1 ng/mL being the limit of detection. Within the quantification range, the coefficients of variation of the intra-assay and interassay precision are less than 5 and 12%, respectively. Moreover, extraction efficiency and dilutional parallelism experiments were completed to demonstrate the assay is accurate. The validated assay was used to quantify Gly m 4 levels in 128 soybean samples from 24 conventional soybean varieties grown at 8 distinct geographical locations. There was a 13-fold difference between the least and greatest amounts of Gly m 4 concentrations among the samples, and the results demonstrate that the most significant sources of variability in Gly m 4 levels in the conventional varieties were related to location and variety.