Preparation Of Anti-ansamitocin Monoclonal Antibodies And Development Of ELISA Method

Antibody drug conjugates is an increasingly important new targeted drug used for cancertreatment. Developing this kind of drug requires their accurate characterization throughout allphases of the discovery, optimization and development life cycle. Assessment of biologicalefficacy and preclinical safety in vivo requires the deployment of a panel of bioanalyticalassays to quantitate total antibody (conjugated and unconjugated), conjugated antibody, freedrug, and immunogenicity in plasma samples from a variety of animals used in preclinicalstudies. Advantage of ELISA method is economical, fast and convenient compared withisotopic labeling, LC-MS detection method. Artificial antigen and antibody are the key fordeveloping an immunoassay method for rapid detecting small drug molecules.In this study, we synthesized two kinds of DM1(a ansamitocin derivative) antigen, whichare the KLH-MCC-DM1for Immunization and the Trastuzumab-2A-DM1for detection.Through determining the value of Absorption at a252nm wavelength before and after DM1coupling to carrier protein, we can know whether DM1was coupled to carrier protein, Theresults showed that two kinds of coupling methods are successful.The BALB/C mice were immunized with the prepared KLH-MCC-DM1, One cell clonestably secreting monoclonal antibody against ansamitocin was chosen after several rounds ofELISA screening, we used the clone to produce ascites and purified the monoclonal antibody.The antibody subtype is the IgG1, subtype of light chain is the lambda.ELISA method on DM1and trastuzumab detection was established by the monoclonalantibody obtained. The methodology validation results showed that existing of bFGF、EGF、VEGF or TNF-α had not interfered with trastuzumab detection;the average recovery of high,medium and low concentration samples between85~95%, the precision is less than10%;Thelower limit of quantitative limit is1.95ng/ml,accuracy is93.95%, the precision is5.5%; Theintraplate accuracy and interplate accuracy in80to100%range, precision is less than15%.Finally determination of Trastuzumab-2A-DM1metabolism in rat.