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

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Title: Developement of monoclonal antibodies for a multiple antigen ELISA to verify safe cooking end-point temperature in beef and pork
Author: Hafley, Brian Scott
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 .
URI: http : / /hdl .handle .net /1969 .1 /4802
Date: 2007-04-25

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Developement of monoclonal antibodies for a multiple antigen ELISA to verify safe cooking end-point temperature in beef and pork. Available electronically from http : / /hdl .handle .net /1969 .1 /4802 .

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