Calbiotech Vitamin B12 ELISA

PRINCIPLE OF THE TEST 

The B12 test kit is a solid phase enzyme-linked immunoassay (ELISA), based on the principal of delayed competitive binding. Streptavidin coated wells are incubated with extracted Vitamin B12 standards, controls, samples, and Intrinsic Factor-Biotin conjugate at room temperature for 45 minutes. During the incubation, the biotin-labeled intrinsic factor will bind to the Vitamin B12 in the sample, standard, or quality control serum. After the 45 minute incubation, Vitamin B12-Enzyme conjugate is added which competes with the Vitamin B12 of the sample, standard, or quality control serum for remaining sites on the Intrinsic Factor for an additional 30 minutes. All unbound conjugates are then removed and the wells are washed. Next, a solution of TMB Reagent is added and incubated at room temperature for 15 minutes, resulting in the development of blue color. The color development is stopped with the addition of stop solution, and the absorbance is measured spectrophotometrically at 450 nm. A standard curve is obtained by plotting the concentration of the standard versus the absorbance. The color intensity is inversely proportional to the amount of Vitamin B12 in the sample. The total assay procedure run time is 1.5 hours.