Characterization of a 12 kDa Thermal‐Stable Antigenic Protein in Bovine Blood

Abstract

MAb Bb1H9 represents the first antibody with the capacity to recognize bovine hemoglobin both in the absence and presence of the heme group, regardless of the heat treatment. MAb Bb1H9 can therefore be utilized in immunoassays by manufacturers and regulators to detect any ingredients containing hemoglobin or globin (hemoglobin without the heme group) in both raw and processed food and feed materials for product quality control and labeling law enforcement.

Immunodetection of Porcine Red Blood Cell Containing Food Ingredients Using a Porcine-Hemoglobin-Specific Monoclonal Antibody

“…Several such poorly separated spots were also visible at a molecular weight of around 26 kDa. A similar observation was reported previously where TDGE of bovine hemoglobin produced several spots with molecular weight around 12 kDa [ 6 ]. Other researchers have also reported the presence of multiple spots upon subjecting hemoglobin from other species to TDGE [ 10 ], an observation that has been ascribed to the presence of impurities, dimerization, or variable phosphorylation [ 11 ].…”

“…Two-dimensional electrophoresis (TDGE) was carried out on purified porcine hemoglobin (25 μg per 125 μL of rehydrating buffer) with isoelectric focusing (using the PROTEAN IEF Cell, Bio-Rad) as the first dimension and SDS-PAGE as the second dimension as previously described [ 6 ]. The gel was subsequently subjected to western blot as described above to determine the isoelectric point of the 12 kDa protein that is recognized by mAb 24C12-E7.…”

“…Two of these porcine blood antibodies that bind cooperatively to a 90 kDa antigenic protein in the plasma fraction of porcine blood, when used in sELISA, have been found effective in detecting porcine plasma-derived ingredients in foods and dietary supplements [ 5 ] Among the same panel of developed mAbs that bind to porcine blood, mAb 24C12-E7 was found to be porcine blood-specific and to react with a 12 kDa antigenic protein in the RBC fraction of porcine blood. In a previous study where we raised bovine-blood-specific antibodies for the purpose of probing the presence of bovine blood material in both feed and food using immunoassay [ 1 ], further studies revealed that the 12 kDa antigenic protein recognized by one of the antibodies, mAb Bb1H9, was a monomer of the tetrameric bovine hemoglobin molecule [ 6 ]. Based on this hind sight information, we anticipated this 12 kDa protein recognized by the porcine-blood-specific mAb 24C12-E7 likely to be a peptide of porcine hemoglobin.…”

Monoclonal Antibodies as Probes for the Detection of Porcine Blood-Derived Food Ingredients

“…To overcome the shortcomings of the aforementioned methods, in our laboratory we have developed sandwich and competitive enzyme-linked immunosorbent assays (ELISA) for detecting the presence of bovine blood in feed and meat products. The competitive ELISA (cELISA) employs monoclonal antibody (mAb) Bb1H9, which recognizes a 12 kDa protein in ruminant blood that has been identified to be a monomer of the tetrameric hemoglobin molecule . The sandwich ELISA (sELISA), which utilizes two mAbs (Bb6G12 and Bb3D6) that recognize a 60 kDa antigenic protein in bovine plasma, has been shown to be effective in detecting diversely processed plasma-derived proteins in both laboratory-adulterated ground meats and commercially available dietary supplements .…”