Type of Document	Thesis
Author	Monaghan, Erin Kelly
URN	etd-08282003-181925
Title	Enzyme Linked Immunosorbent Assay (ELISA) For Detection Of Sulfur-Rich Protein (SRP)In Soybeans(Glycine Max L.) And Certain Other Edible Plant Seeds
Degree	Master of Science
Department	Nutrition, Food, and Exercise Science, Department of
Advisory Committee	Advisor Name Title Shridhar K. Sathe Committee Chair Cathy W. Levenson Committee Member Kenneth H. Roux Committee Member
Keywords	(SRP) In Soybeans
Date of Defense	2003-06-01
Availability	unrestricted
Abstract The sulfur-rich protein (SRP) in soybeans is a 7S basic globulin that accounts for up to 5% of the total extractable seed proteins. Sulfur-containing amino acids are the limiting amino acids in most food legumes and therefore there is a continued interest in increasing the expression of sulfur containing proteins in legumes to improve protein quality. The objective of our investigation was to develop a sensitive method for detection of SRP and use the method to detect the presence of SRP or SRP-like proteins in various edible plant seeds. Rabbit polyclonal antibodies (pAbs) raised against SRP were used in inhibition ELISA, Western blotting and Dot blotting to develop a sensitive assay and to assess cross-reactivity of pAbs to non soybean proteins. Typically, primary and secondary antibody dilutions of 1:10,000 (v/v) and 1:5,000 (v/v) in 0.1% BSA in borate saline buffer (BSB, 0.1M, pH 8.45), respectively, were used. All protein extractions were done in either BSB (pH 8.45), 0.1 M NaOH (pH 12.22) or 70% (v/v) aqueous ethanol and cross reactivity was assessed by comparing sample IC50 (50% inhibitory concentration) to IC50 value for SRP. Inhibition ELISA assays could detect SRP at concentrations as low as 400 ng/ml in aqueous extracts, Western blotting and Dot blotting could detect SRP at concentrations as low as 50 ng and 1 ng, respectively. The assays indicated the presence of cross-reactive proteins in several of the BSB (pH 8.45) extracted samples, including soybean, winged bean, black gram, mung bean, navy bean, tepary bean, pinto bean, chickpea, blackeye pea, horse bean, black bean, Great Northern bean, lima bean, small red bean, wheat berries and moth bean. The assays also detected cross-reactive proteins in several of the 0.1 M NaOH (pH 12.22) extracted samples, including soybean, winged bean, black gram, mung bean, navy bean, horse bean, moth bean, black bean, Great Northern bean, tepary bean, pinto bean, chickpea, lima bean, blackeye pea, small red bean. In addition, cross-reactive proteins were also detected in samples extracted with 70% (v/v) aqueous ethanol, including soybean, winged bean, wheat bran and black gram.
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