A KINETIC ENZYME-IMMUNOASSAY FOR THE QUANTITATION OF ANTIBODIES TO A HUMANIZED MONOCLONAL-ANTIBODY IN HUMAN SERUM

A kinetic enzyme immunoassay was developed and validated to quantitate human antibodies to the humanized monoclonal antibody CAMPATH(1)-1H ( C1H) in human serum. The assay was configured using C1H-coated 96-well plates which were blocked with bovine serum albumin, and incubated wi th dilutions of human serum containing anti-C1H antibody. Antibody was detected using biotinylated C1H followed by streptavidin-conjugated a lkaline phosphatase and p-nitrophenyl phosphate. Absorbance data were collected for 10 min, and mOD min(-1) data were exported to MultiCalc data analysis software. A 4-parameter logistic-log algorithm was shown to model the data through the range of the standard curve within 15% of nominal values. The overall assay performance coefficient of variat ion by ANOVA was 9.2%. The lower limit of detection was defined at 160 Units ml(-1). The anti-idiotype antibody standard stock solution is s table at 4 degrees C and at -80 degrees C for at least 11 months in bu ffer. The anti-idiotype antibody controls are stable for at least seve n freeze-thaw cycles and at least 6 months in human serum stored at -2 0 degrees C. A strategy was devised by which to establish the specific antibody potency for any given batch of anti-C1H antibody standard re lative to the Reference Standard. This EIA has been used to quantify a nd characterize anti-C1H antibody in human serum in support of clinica l safety and efficacy studies. (C) 1998 Elsevier Science B.V. All righ ts reserved.