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Description:
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Four proteins exhibiting different rates of denaturation or precipitation with
increasing cooking temperature from 63 to 73à °C for beef and 67 to 79à °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 . |