A SENSITIVE ENZYME-IMMUNOASSAY FOR THE QUANTITATION OF HUMAN CTLA4IG FUSION PROTEIN IN MOUSE SERUM - PHARMACOKINETIC APPLICATION TO OPTIMIZING CELL-LINE SELECTION

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the qu antitation of human CTLA4Ig in mouse serum was validated. The EIA meth od employed a technique in which a monoclonal anti-CTLA4 antibody was adsorbed onto 96-well polystyrene microtiter plates and used to captur e the CTLA4Ig in mouse serum samples. The captured CTLA4Ig was then de tected using a goal anti-human IgG(Fc) antiserum conjugated to the enz yme horseradish peroxidase. The validation included assessments of met hod accuracy and precision, range of reliable response, lower limit of quantitation (LLQ), inter-analyst robustness, storage stability in mo use serum and assay specificity. The results indicate that this valida ted assay is precise, accurate, and reproducible. This EIA has a range of reliable response in 10% mouse serum of 0.14-4.58 ng ml(-1) result ing in a 100% serum equivalent curve of 1.4-45.8 ng ml(-1) Assessment of individual standard curve variations indicated a reproducible respo nse with R-2 values of greater than or equal to 0.995. The LLQ was est ablished at 1.4 ng ml(-1). The accuracy and precision estimates, based on the quality control values, were within 3.8% and 5.2% respectively , for CTLA4Ig. Stability of CTLA4Ig was established in mouse serum for 5 days at both 4 degrees C and room temperature, for 2 months at -70 degrees C and through five freeze-thaw cycles. This validated assay wa s successfully employed in the assessment of pharmacokinetic character istics of CTLA4Ig in mice and to aid in the selection of an optimal CT LA4Ig-producing cell line.