Characterization of a 12kDa Protein Recognized by MAb Bb1H9 and Its Applications in Detection of Added Bovine Blood Cell Protein in Dietary Products

• Other Authors: Ofori, Jack Appiah (authoraut)
• Format: Others
• Language: English; English
• Published: Florida State University
• Subjects: Nutrition; Food; Exercise
• Online Access: http://purl.flvc.org/fsu/fd/FSU_migr_etd-5873

Although the use of hemoglobin-containing food additives is increasing, there is virtually no method available for monitoring their presence in food and dietary products. We have previously developed a competitive ELISA (cELISA) for the detection of bovine blood in food using a monoclonal antibody (MAb), Bb1H9, which recognizes a 12kDa antigenic protein. Because 12kDa approximates that of a monomer of hemoglobin (14.4kDa) and further studies have shown the 12kDa protein to be present in the red cells of bovine blood, we hypothesized that this protein is a subunit of hemoglobin. This study sought to verify this hypothesis and hence investigate the usefulness of assays based on MAb Bb1H9 in monitoring the presence of bovine hemoglobin-containing food additives. Extracts obtained from raw and heat-treated bovine blood fractions and products comprising of bovine hemoglobin-containing ingredients were analyzed with indirect ELISA (iELISA), cELISA and western blot. Target proteins resolved by two-dimensional electrophoresis were subjected to N-terminal sequencing. Results indicated that the 12kDa protein is a monomer of the tetrameric hemoglobin molecule (64.5kDa) and that MAb Bb1H9 was able to bind both in the presence and in the absence of heme. MAb Bb1H9 was also found to react with a linear (ERMFLSF) epitope on the alpha chain and possibly, a conformational epitope on the beta subunit of hemoglobin. MAb Bb1H9 in cELISA format proved useful against all products tested except for the bovine hemoglobin hydrolysate (RIF) and sodium erythorbate (DAU) containing products. MAb Bb1H9 in western blot format, however, proved useful for the detection of the product, DAU. MAb Bb1H9 also proved useful in the detection of bovine hemoglobin hydrolysates that have been hydrolyzed with pepsin for up to 6h. MAb Bb1H9 is expected to become a valuable regulatory tool for labeling law enforcement to protect the interest of individuals (e.g. Jews, Muslims, and vegans) that avoid consuming blood-derived products for religious or ethical reasons; and to deter rogue manufacturers who may be tempted to use such blood-derived proteins fraudulently to increase the apparent meat content. === A Dissertation submitted to the Department of Nutrition, Food and Exercise Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy. === Fall Semester, 2011. === October 7, 2011. === Bovine, competitive ELISA, Hemoglobin, Monoclonal antibody === Includes bibliographical references. === Yun-Hwa Peggy Hsieh, Professor Directing Dissertation; Kenneth H. Roux, University Representative; Shridhar K. Sathe, Committee Member; Jeong-Su Kim, Committee Member.