The immunoassay continues to be an important bioanalytical technique for fundamental biological studies and clinical diagnosis. This proposal suggests a new approach to the heterogeneous assay involving the use of a microimmunoreactor column coupled to a flow injection analysis (FIA) system. The focus will be on the rapid analysis of small volume (10uL) samples with analyte concentrations in the sub-nanogram/mL range. The success of this approach will depend upon the availability of well- characterized immunochemical reagents of high activity. A series of synthetic procedures are described for coupling antibody or antibody fragments to a surface in a preferred orientation. This will encourage rapid reaction with soluble components and minimal non-specific interactions with the immunosorbent surface. The enzyme immunoassay forms the basis for detection, which is accomplished electrochemically or fluorometrically. The optimization of system performance requires a detailed understanding of the reactions occurring at the immunosorbent surface. The immunoreactor, which is in effect a flow reactor, can be used to study the kinetics of immunological reactions and to exploit differences in rates for analytical purposes. Monoclonal antibodies or mixtures of monoclonal antibodies will be used to obtain enhancement in selectivity. Heterobifunctional antibodies will be prepared which will permit linkage of two proteins without loss of biological activity. The techniques developed will be applied particularly to analysis of haptens in the sub-nanogram/mL range where the radioimmunoassay is normally employed. Two methods are proposed for non-competitive binding assays which should provide greater dynamic range and would be simpler to employ. Immunoassays will be developed for human cytokeratins. The distribution of these proteins in urine may be indicative of bladder cancer either as a cancerous or precancerous state.
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