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Soybeans (Glycine max. L), as one of the most widely grown crops, account for 90% of U.S. oilseed production. Despite their economic, nutritional, and consumer value, soybeans are among the top eight types of foods responsible for eliciting allergies in sensitive individuals. Soybean lectin is a tetrameric 7S protein with the binding specificity for GalNAc and Gal residues. It is one of the soybean allergens as well as an anti-nutrient that can cause reduced nutritional value of soybean and soybean derived food products. It is also a known toxicant able to agglutinate human red blood cells. Detection of soybean lectin in human and animal food supply is therefore important. The current available methodology for detection and quantification of soybean lectin rely on a variety of techniques that often lack specificity and sensitivity. The objective of this study was to obtain a validated method that is precise and accurate in the measurement of soybean lectin. A mouse monoclonal antibody based indirect ELISA was constructed for soybean lectin detection. Using low pressure column chromatography, soybean (certified seeds Hutcheson) lectin was purified. Using the purified soybean seed lectin, murine mAb 5A5 was produced and purified using Protein-G affinity column chromatography. Borate saline buffer, 0.1 M, pH 8.45 was used to solubilize proteins from petroleum ether defatted seed flour. Dot-blot and Western-blot immunoassays were used to screen mAb 5A5 for soybean lectin detection, specificity and sensitivity. Under the test conditions, the mAb 5A5 was specific for soybean lectin detection. The mAb 5A5, at concentration of 1520 ng/ml and 61 ng/ml, was able to detect 5 and 50 ng of the purified lectin, respectively, using dot- and Western- blot immunoassays. The mAb 5A5 detected the lectin in soluble soy proteins of 58 tested soybean seeds and 11 commercial soybean products using 1 and 10 microgram soy protein respectively for dot- and western blot immunoassays. No cross-reactive protein was found in 21 selected dry beans with mAb 5A5. Using purified murine anti-soybean lectin mAb 5A5 as detection Ab, a mouse monoclonal antibody based indirect ELISA demonstrated sensitivity, specificity, and reliability at trace levels. The indirect ELISA was validated to have LOD of 30 ng/ml (0.03 ppm), protein concentration at 50% máximum signal of 786.3ng/ml. This ELISA assay is reproducible and accurate with CVs <10% in intra- and inter-assay, and the average recoveries within 15% of the actual value. In conclusion, the indirect ELISA is sensitive, specific, and robust for the detection soybean lectin under the test conditions.