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 AbstractThe 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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