Development of an indirect enzyme-linked immunosorbent assay for detecting serum antibodies of soybean antigen protein

The study is aimed to develop an indirect enzyme-linked immunosorbent assay(ELISA) for detection of serum specific antibodies of soybean antigen protein.Soybean antigen proteins were purified by Agarose gel chromatography and used to inject Kunming mice with different doses.An indirect ELISA was developed with the purified antigen protein at different concentrations,and the optimal antigen concentration and serum dilution were determined by phalanx titration.The other working conditions were also optimized.The serum antibody levels of immuned mice were detected by the established ELISA.The optimal coating concentration of 11S protein and serum dilution were 5.0 μg·mL-1 and 1∶800,respectively.The optimal coating concentration of 7S protein and serum dilution were 2.5 μg·mL-1 and 1∶1600,respectively.Both intra-and inter-assay repeatability of ELISA were less than 10%.The optimal immune times and doses of 11S and 7S protein were 2 and 1000 μg·kg-1,respectively.The developed indirect ELISA had good specificity,sensitivity and reproducibility.This study provides a reliable method for the clinical detection of allergic reactions induced by soybean antigen protein.