Immunodetection of Porcine Blood in Foods

Justification: Different porcine blood proteins have been widely used as emulsifier, binder and/or colorant in processed foods. However, misusage of porcine blood ingredients, such as mislabeling and substitution, can cause religious objections, law violation, food safety and/or food quality decrease. These issues highlight the need of detecting unfavorable porcine blood in foods to fight food fraud. Porcine whole blood, plasma and blood cells can be applied individually or in combination as food additives. Therefore, the study was divided into two parts. The objectives of the part 1 is (1) to develop and characterize a monoclonal antibody (mAb) that is specific for porcine hemoglobin; (2) to develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) that can detect porcine blood adulteration in foods. The objective of part Ⅱ is to characterize two mAbs which have the target protein in porcine plasma. Methods: In part Ⅰ, mAbs were developed using hybridoma technique and purified using immunoaffinity. Western blot was applied to verify the target protein; to study the mAb selectivity; and to study the effect of pH on storage stability (29 days at 4 °C) and thermostability (50 ºC, 100 °C and 121 ºC for 15 min) of target protein. Indirect non-competitive ELISA (inELISA) was performed to study antibody affinity and storage stability of target protein, and to choose the optimized condition for icELISA. Eventually, an optimized icELISA and extraction buffer was developed. The assay was validated by FDA Guidance for Industry. In part Ⅱ, immunoaffinity column was applied to isolate the target protein. The isolated proteins were sequenced. The immunoreactivity of target protein was verified using four commercial antibodies (anti-transferrin, anti-haptoglobin, anti-plasminogen and anti-C7). To further investigate the isoelectric point (pI) and disulfide information of target protein, two-dimensional gel electrophoresis and non-reducing SDS-PAGE were performed, respectively. Results: In part Ⅰ, mAb13F7 was chosen after screening test because it has the best selectivity to porcine blood. The target protein of the mAb was porcine hemoglobin (PHb) subunit (14 kDa). Although this mAb could cross-react with hemoglobin from bovine, horse and sheep, their hemoglobin band color intensity was much less than that of PHb according to Western blot. From inELISA and icELISA, this mAb showed a high immunoaffinity to PHb compared with bovine hemoglobin. The affinity constant of this mAb is in a nanomolar range, which can be considered as high-affinity antibody. As for thermostability, PHb can maintain the best molecular integrity and immunoreactivity at alkaline pH compared to acidic pH and neutral pH. During storage at 4ºC up to one month, PHb remained intact without any degraded peptides observed and the immunoreactivity did not change significantly (P > 0.05). Finally, a sample extraction buffer (12.5 mM NaHCO3 and 25 Mm NaCl) and an anti-PHb cELISA were developed. After assay validation, the optimized cELISA was PHb-specific and had a working range from 0.5 ppm to 1000 ppm. This assay was sensitive (limit of detection: 0.5 ppm) and reproducible with low inter- and intra- coefficient of variances (CVs < 15%). In part Ⅱ, the target protein was successfully isolated and its immunoreactivity has been confirmed. Target protein sequence was obtained. In total, four commercial antibodies were tested, neither of them showed similar band pattern as mAb19C5 and mAb16F9. The target analyte is still under investigation. Significance: The established icELISA assay in part Ⅰ can be used to detect trace amount of porcine blood in foods to fight food fraud. It also has the potential to be used in identifying diseased pork through determining residual hemoglobin concentration in pork. It is suitable for (1) government to enhance food regulation; (2) food industry to surveillance product quality; and (3) third party authority to certify halal/kosher foods or evaluate food authenticity.