This kit is a Enzyme-Linked Immunosorbent Assay (ELISA). Add samples to the pre-coated plate. Then add biotinylated antigen. The antigens in the samples compete with the biotinylated antigen to bind to the capture antibody and incubate. Unbound antigen is washed away during a washing step. An avidin-HRP is then added and then incubate. Unbound avidin-HRP is washed away during a washing step. TMB Substrate is then added and color develops. The reaction is stopped by addition of acidic stop solution and color changes into yellow that can be measured at 450 nm. The intensity of the color developed is inversely proportional to the concentration of 5-MTHF in the sample. The concentration of 5-MTHF in the sample is then determined by comparing the O.D. of the samples to the standard curve.