Interest of epitopic dissection in immunoanalysis of proteins and peptides: review of theoretical and practical aspects.

The literature abounds with reports showing discrepancies in immunoassays of proteins and peptides. Whereas the isomorphism and polymorphism of proteins remains largely hidden in immunoassays making use of polyclonal antibodies, the use of monoclonal antibodies uncovered the difficulty of accurately assaying microheterogeneous analytes. Indeed, most proteic hormones are not entities with unique structures but rather mixtures of molecular forms with slight differences in structure which may reflect large variations in biological and immunological activities; the monoclonal antibodies appeared clearly less suited than the polyclonal for testing a mixture of isoforms. Protein microheterogeneity also has an impact on assay standardisation, since reference preparations may contain several isoforms of the analyte. Using recombinant glycoprotein does not solve the problem. Regarding the problem of discrepancy in immunoanalysis of proteins and peptides, we could establish, in a previous work, that discrepancy among lutropin assay kits may be related to various causes: i) differences in standard preparation and calibration curves; ii) microheterogeneity of lutropin molecules leading to missing some isoforms due to the restricted epitopic specificity of the monoclonal antibodies used in the kits. The epitopic dissection we engaged in appeared thus instrumental in explaining these discrepancies. It allowed us to enumerate epitopes on the surface of lutropin molecules, to elucidate the immunological structure and, finally, to characterize monoclonal antibodies used in commercially available lutropin assay kits with regard to their epitopic specificity. This work allowed us to interpret the discrepancy in serum lutropin concentration which was related to the use of monoclonal antibody with given specificity. Epitopic dissection may thus be instrumental in explaining discrepancy among immunoassays of proteins and peptides and in improving the accuracy of kits.