We describe an immunoassay that is based on the interchain interaction of separated VL and VH chains from a single chain antibody variable region. In the presence of antigen, the chains reassociate. VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 were immobilized on microtiter plates. Samples were coincubated with an M13-displayed VH chain, and assayed with peroxidase-labeled anti-M13 antibody. Signal was detected in direct proportion to the amount of HEL in the sample. Wide dynamic range with < 15 ng/ml sensitivity was attained.