Enzyme-linked immunosorbent assay incubation time

Enzyme-linked immunosorbent assay (ELISA) is comparable to the immuno-radiometric assay except that an enzyme tag is attached to the antibody instead of a radioactive label. ELISAs have the advantage of nonradioactive materials and produce an end product that can be assessed with a spectrophotometer. The molecule of interest is bound to the enzyme-labeled antibody, and the excess antibody is removed for immunoradiometric assays. After excess antibody has been removed or the second antibody containing the enzyme has been added (two-site assay), the substrate and cofactors necessary are added in order to visualize and record enzyme activity. The level of molecule of interest present is directly related to the level of enzymatic activity. The sensitivity of the ELISAs can be enhanced by increasing the incubation time for producing substrate. 

Elements in an immunoassay that could impact its robustness include incubation temperatures, light exposure (enzyme-linked immunosorbent assay, ELISA), and different lots of matrix (plasma, serum CSF). The ruggedness of the analytical method can be tested by implementing changes to the analysts, different instruments, batch size, and the day, time, or other environment factors otherwise should not greatly impact the consistency of the assay. 

Experiment I. In a time-course experiment, mucosal PGE production and phospholipid fatty acid profile were assessed at d 0,4,8,12, and 16 of dietary treatment in formula-fed and naturally reared piglets (n = 5 piglets per time per dietary treatment). Mucosal cells were scraped from proximal ends of the small intestine and frozen at -80°C for later lipid analysis. Lipids were extracted by a modified Folch procedure (15). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated by thin-layer chromatography (16), and fatty acids in each phospholipid fraction were analyzed by gas chromatography. For eicosanoid measures, fresh mucosal tissue was incubated in Kreb s Ringer bicarbonate buffer as described previously (17). PGE2 was extracted from the incubation media with ethyl acetate and quantified using a competitive enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). 

An enzyme-linked immunosorbent assay (ELISA) was performed as described previously. Briefly, each well of a 96-well microtiter plate was coated with 100 pi of the indicated concentration of sample in PBS, and incubated for 2 hr. The wells were washed three times with PBS containing 0.05% Tween 20 (washing buffer), then blocked with 0.5% gelatin in PBS for 1 hr. After washing 3 times, the wells were incubated for 1 hr with 100 pi of the primary antibody including monoclonal anti-CML antibody (2G11 1 pg/ml), monoclonal anti-CEL antibody (CEL-SP 5 pg/ml) and monoclonal anti-GA-pyridine... 

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