An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin

Immunoassay technology is routinely used to measure concentrations of prote ins and polypeptides in biological matrices. increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharm acokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report descr ibes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY35510 1 and LY396623, one and two amino acid changes relative to native human lep tin, respectively). Rabbits were immunized with either LY355101 or LY396623 . Antisera were screened to determine if any showed increased specificity f or the analog relative to native human leptin. Antisera showing increased s pecificity for the leptin analog were then treated by immunoabsorption agai nst native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays, which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 ph armacokinetics that were not confounded by the high levels of endogenous hu man leptin found in obese subjects. This technique has the potential for br oad application in the development of assays capable of specifically measur ing protein analogs without cross-reactivity to an endogenous substance. (C ) 2000 Elsevier Science B.V. All rights reserved.