Development Of Enzyme-Linked Immunosorbent Assay For Detection Of The Trypsin Inhibitor In Soybean Meal

Among the many factors that have been implicated as having an adverse effect on the nutritional value of soybean meal is one of the important protease inhibitors, that has the ability to inhibit the proteolytic activity of proteases of diverse origin. These trypsin inhibitors induce pancreatic hypertrophy/hyperplasia. Concomitant with this increase in the size of the pancreas is an increase in the secretion of digestive enzymes, including trypsin, chymotrypsin, and elastase. This led to the hypothesis that the growth depression caused by the trypsin inhibitors is the consequence of an endogenous loss of amino acids in the form of enzymes being secreted by hyperactive pancreases. As ELISA is selective, sensitive, objective and accurate and practicable, it can cover any gaps of the classic chemical analysis or instrumental analysis, and so that it is being used with increasing frequency in feed safety testing and quality evaluation.A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean meal is described. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. And the specific trypsin inhibitor activity of the first elution peak is 2317 TIU/mg with a purification fold of 115. But there is no band of it through SDS-PAGE analysis. These observations suggest that it may belong to BBI family. The second elution peak with a specific trypsin inhibitor activity of 5657 TIU/mg was found to consist of a single polypeptide band around 20 KDa through SDS-PAGE analysis. So it belongs to KTI family.Based on anti-trypsin-inhibitor polyclonal antibody, an ELISA method was developed to detect the trypsin inhibitor in soybean meal. The formula was y= 20.841x+ 50.961, R2=0.9941. When the spiked concentrations of TI were 0.8μg/ml,1.6μg/ml, 2.4μg/ml in blank feed, the recovery was 93.7%～104.9% with coefficients of variation of 2.8%～5.1%. The enzymic chemical assay and ELISA showed good agreement on testing the soybean meal and KTI standard of different heat treatmentConclusion can be drawn that the immunoassay presented here tends to be more accurate and specific than the enzymatic chemical analysis and can be safely used as a screening assay to monitor the soybean meal.