Monoclonal antibody based two‐site enzyme immunoassays for wheat gluten proteins. 1. Kinetic characteristics and comparison with other Elisa formats

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Abstract
Solid‐phase two‐site enzyme immunoassays were developed using a number of monoclonal antibodies with differing specificities for wheat grain gluten proteins. While polystyrene microwells performed well in the assay, very high levels of non‐specific binding occured with PVC (Polyvinylchloride) microwells in the absence of ‘capture’ (solid‐phase bound) antibody. This was due to binding of complexes of antigen and labelled antibody, and could only be reduced by the use of rather stringent blocking conditions. Assays involving either the simultaneous or sequential addition of antigen and labelled antibody were developed, the latter providing superior results. A number of successful two‐site assays were constructed using single monoclonal antibodies, suggesting that repeating epitopes, known to occur in gluten proteins, were commonly recognized. Pairs of monoclonal antibodies with dissimilar antigenic specificites did not function in the two‐site assay. A comparison of monoclonal antibody based two‐site and antigencompetition immunoassays for gliadin was made. Several antibodies performed considerably better in one format than the other; thus, both assay formats will be of use in quantification of specific gluten protein subclasses.

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