Studies on the detrimental effects of bivalent binding in a microtitration plate ELISA and possible remedies

During the development of a microtitration plate enzyme-linked immunosorbent assay (ELISA) for 3-acetyldeoxynivalenol, using an immobilised hapten:protein conjugate, problems were encountered in obtaining an inhibition curve with hapten. Investigations into this phenomenon were conducted. Univalent Fab fragments were prepared and their binding compared with untreated antiserum on plates coated with different immobilised coating concentrations and with conjugate of a lower hapten:protein ratio. The studies showed that standard curves obtained with Fab fragments using the coating conjugate of high hapten ratio were more sensitive and showed a greater degree of inhibition. However with the conjugate of lower hapten ratio the untreated antiserum gave the more sensitive curve although inhibition was incomplete. From these results it is concluded that the antiserum probably contains two populations of antibodies, one of which binds bivalently to the plates at all concentrations of immobilised hapten.