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

摘要

Four proteins exhibiting different rates of denaturation or precipitation withincreasing cooking temperature from 63 to 73????C for beef and 67 to 79????C for pork wereselected for developing a ratio model and incorporating the results into a mathematicalexpression. Monoclonal antibodies (Mabs) against lactate dehydrogenase isozyme 5(LDH-5), bovine serum albumin (BSA), porcine enolase, and bovine myoglobin weredeveloped for use in a sandwich enzyme-linked immunosorbent assay (ELISA) tosimultaneously investigate changes in protein concentration with incremental increasesin temperature.Four groups of mice were immunized separately with commercially available orpurified protein (LDH-5, BSA, enolase, or myoglobin). After reporting ample bloodserum titers, spleen cells were harvested and fused with SP2 myeloma tumor cells usingan electro fusion cell manipulator. Hybridoma containing wells were screened againsttheir respective protein to isolate hybridomas secreting protein specific Mabs. Tissue culture flask produced Mabs were used initially in sandwich ELISA assaytesting. Mabs were tested against ground beef and pork cooked to instantaneous endpointtemperatures (EPTs). A 6 g section removed from the geometric center of eachsample was homogenized in phosphate buffer, centrifuged, and a 1 ml aliquot collectedfor analysis.Microtiter plates were coated with goat anti-mouse IgG antibody (2 mg/ml) to actas a capture antibody for the protein specific monoclonal antibody concentrated from cellculture supernatant. Serial diluted muscle (beef or pork) extract (10 ml) from each EPTwas applied to a microtiter plate. A protein A/G purified polyclonal antibody (Pab) wasapplied, 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 specificMabs against LDH-5, BSA, enolase, and myoglobin, respectively, were produced. SixMabs against LDH-5 reported R2 values > 0.9 indicating high specificity and affinity forLDH-5. Sandwich ELISA assays development with Mabs against BSA, enolase, andmyoglobin 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.