This ELISA is designed, developed and produced for the quantitative measurement of Vitamin B12 in human plasma or serum samples. Assay calibrators and patient samples are added directly to wells of a microplate that is coated with an anti-Vitamin B12 monoclonal antibody. After the first incubation period, the antibody on the wall of the microtiter well captures sample Vitamin B12 and HRPconjugated Vitamin B12 in a competitive nature. Excessive amounts of unbound proteins and HRP-conjugated Vitamin B12 in each microtiter well are washed away. For the detection of the captured sample Vitamin B12, the well is incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the complex bound to the Vitamin B12 on the wall of the microtiter well is directly proportional to the amount of Vitamin B12 in the sample. A calibrator standard curve is generated by plotting the absorbance versus the Vitamin B12 concentration for each calibrator on point-to-point or cubical scales or 4 parameter curve fits. The concentration of Vitamin B12 in test samples is determined directly from this standard curve.