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As the No.1 food plant protein in North America, soybean proteins have been comprehensively used as the major food ingredient in the human diet and animal feeding. Bioactive soybean lectin, also known as soybean agglutinin (SBA), is generally recognized as an antinutritional protein in uncooked or undercooked soybean products. It has been reported that bioactive SBA could be a potential cause of poor digestion, nutrient deficiencies, obesity, and chronic inflammation in human diets. Although SBA is not generally considered toxic for animals, it may be responsible for growth-depressing effects due to its interference with the digestive system. The majority of SBA can be destroyed and lose its antinutritional activity during the proper cooking process. However, there is no specific regulation on the pre-treatment of soybean meal in animal feeding. Though SBA is recognized as an antinutrient in the human diet and animal feeding, it has a promising future in clinical treatment. It can be used as a carrier for drug delivery and has been verified to have antitumor and anticarcinogenic activities. SBA inhibits tumor cells' growth, enhances the antitumor cytotoxicity, and acts as a biomarker for cancer cell characterization. Based on the current research, SBA maintains its bioactivity (hemagglutinating activity and anticancer activity) under its native condition. It means tetramer SBA owns its bioactivity. After heat treatment, the hemagglutinating activity and anticancer activity all decreased. There is a lack of studies focusing on the thermal properties of SBA, especially its molecular conformation changes and immunoreactivity. Therefore, this study aimed to investigate the effect of temperature on SBA molecular conformation and immunoreactivity.Four monoclonal antibodies (mAbs) specific to SBA (mAb1A10, 2H1, 5A5, 5G12) was purified from the supernatant using immunoaffinity chromatography. The mAb1A10, 5A5, and 5G12 were characterized using immunoblot. Two-dimensional (2D) gel electrophoresis was performed to verify SBA purity and isoelectric point (pI). Native and non-native gel electrophoresis were used to study the temperature (60 °C, 70 °C, 80 °C for 10 min) effect on SBA molecular conformation. Western blot (WB) was performed to investigate the temperature effect on immunoreactivity of SBA. Hemagglutination assay was used to study temperature effect on SBA hemagglutinating activity. SBA was a tetramer (about 120 kDa) under native condition and was dissociated to monomer (about 30 kDa) under non-native condition. A tetrameric SBA was observed from three native gel electrophoresis in both unheated and heated (60 ⁰C and 70 °C) samples, while heat-induced SBA aggregates were observed at 70 °C and 80 ⁰C. No SBA monomer was observed in both unheated and heated samples. The immunoreactivity of two anti-SBA monoclonal antibodies (mAb1A10 and 5A5) was not affected by different heat treatment. As for another anti-SBA mAb5G12, its immunoreactivity decreased as a function of temperature. SBA aggregates were not immunodetectable by mAb5G12 after heating at 80 ⁰C, indicating that mAb5G12's conformational epitope was heat-sensitive. From non-native gel electrophoresis, a monomer SBA was observed in both unheated and heated samples, while heat-induced SBA aggregates were shown at 80 ⁰C. mAb1A10 and 5A5 detected all these protein bands observed in the non-native gel. SDS affected mAb5G12 immunoreactivity with SBA.