Loss of ELISA specificity due to biotinylation of monoclonal antibodies

A significant degree of nonspecificity was found in ELISA determinations of soluble urokinase receptor (suPAR) in human blood plasma when biotinylated monoclonal antibodies (Mabs) were used for the detection layer. Surface pl asmon resonance studies using both nonbiotinylated and biotinylated antibod ies demonstrated that biotinylation reduced specific binding of the antibod ies to their target antigen, suPAR. Furthermore, biotinylation produced a n ew interaction with unknown human plasma protein(s), unrelated to suPAR. No nspecific interaction with plasma protein(s) was also observed after biotin ylation of a Mab having no specific target antigen in human plasma and, in both cases, the level of nonspecific interaction was directly related to th e degree of antibody biotinylation. These results reinforce earlier observa tions that biotinylation of antibodies can reduce the affinity of antibodie s, but also indicate that, in addition, biotinylation can reduce the specif icity of immunoassays for plasma proteins. (C) 2000 Elsevier Science B.V. A ll rights reserved.