Developement of monoclonal antibodies for a multiple antigen ELISA to verify safe cooking end-point temperature in beef and pork

Abstract

Four proteins exhibiting different rates of denaturation or precipitation with increasing cooking temperature from 63 to 73°C for beef and 67 to 79°C for pork were selected for developing a ratio model and incorporating the results into a mathematical expression. Monoclonal antibodies (Mabs) against lactate dehydrogenase isozyme 5 (LDH-5), bovine serum albumin (BSA), porcine enolase, and bovine myoglobin were developed for use in a sandwich enzyme-linked immunosorbent assay (ELISA) to simultaneously investigate changes in protein concentration with incremental increases in temperature. Four groups of mice were immunized separately with commercially available or purified protein (LDH-5, BSA, enolase, or myoglobin). After reporting ample blood serum titers, spleen cells were harvested and fused with SP2 myeloma tumor cells using an electro fusion cell manipulator. Hybridoma containing wells were screened against their respective protein to isolate hybridomas secreting protein specific Mabs. Tissue culture flask produced Mabs were used initially in sandwich ELISA assay testing. Mabs were tested against ground beef and pork cooked to instantaneous endpoint temperatures (EPTs). A 6 g section removed from the geometric center of each sample was homogenized in phosphate buffer, centrifuged, and a 1 ml aliquot collected for analysis. Microtiter plates were coated with goat anti-mouse IgG antibody (2 mg/ml) to act as a capture antibody for the protein specific monoclonal antibody concentrated from cell culture supernatant. Serial diluted muscle (beef or pork) extract (10 ml) from each EPT was applied to a microtiter plate. A protein A/G purified polyclonal antibody (Pab) was applied, followed by a goat anti-rabbit IgG peroxidase conjugated antibody. Concentration was determined by comparison to a standard curve. After multiple cell fusions, 24, 29, 66, and 12 cell lines secreting protein specific Mabs against LDH-5, BSA, enolase, and myoglobin, respectively, were produced. Six Mabs against LDH-5 reported R2 values > 0.9 indicating high specificity and affinity for LDH-5. Sandwich ELISA assays development with Mabs against BSA, enolase, and myoglobin was not as successful. Mouse ascites produced Mabs against BSA, enolase, and myoglobin were also unsuccessful when used in a sandwich ELISA. However, preliminary data suggested a multiple antigen ratio model still remained a viable option.