Monoclonal antibody-based ELISA for the quantification of porcine hemoglobin in meat products

•A mAb, 13F7 specific to mammalian hemoglobin, was developed.•PHb had the best solubility, molecular integrity & immunoreactivity at alkaline pH.•The degree of PHb aggregation increased as a function of temperature.•PHb had no changes in molecular integrity & immunoreactivity during storage.•A mAb-based icELISA was established to determine PHb in spiked pork and chicken. Misusage of porcine blood proteins, such as misbranding and substitution, can cause religious objections, law violation, and food quality concerns. These issues highlight the need for detecting unlabeled or overuse of porcine blood in foods. Compared with acidic and neutral pHs, porcine hemoglobin (PHb) at alkaline pH retained the best solubility, molecular integrity, and immunoreactivity after heat treatment. PHb at acidic and alkaline pHs remained stable during storage at 4 °C for 29 days. A monoclonal antibody (mAb) specific to mammalian hemoglobin, 13F7, was developed. A mAb13F7-based indirect competitive ELISA (icELISA) was optimized for the quantification of PHb in meat products. This assay had a wide working range from 0.5 ppm to 1000 ppm. It was sensitive (limit of detection: 0.5 ppm), precise and reproducible with low inter- and intra-coefficient of variances (