After the alkaline denaturation (to produce folate from the endogenous joining protein) and you will stabilization (to end oxidization), an aliquot of handled attempt was transferred towards good streptavidin-decorated well. Following an aggressive binding response (folate throughout the solution decide to try competes with HRP-branded folate to own a small level of joining websites into an effective biotinylated FBP), the brand new FBP complexes is actually seized by streptavidin on wells. Unbound conjugates were eliminated from the washing.
The new likely HRP conjugates for twenty-five-OH nutritional D, B12 and you can folate was basically measured because of the an effective luminescent reaction (18). Reagent that has luminogenic substrates (luminol by-product and peracid salt) and you may an electron transfer representative (replaced acetanilide) is put into the latest wells. Oxidization of luminol derivative is actually catalyzed of the HRP inside this new bound conjugates, for this reason promoting white.