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 CAMPATH1-1H (C1H) in human serum. The assay was configured using C1H-coated 96-well plates which were blocked with bovine serum albumin, and incubated with dilutions of human serum containing anti-C1H antibody. Antibody was detected using biotinylated C1H followed by streptavidin-conjugated alkaline 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 variation 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 stable at 4 degrees C and at -80 degrees C for at least 11 months in buffer. The anti-idiotype antibody controls are stable for at least seven freeze-thaw cycles and at least 6 months in human serum stored at -20 degrees C. A strategy was devised by which to establish the specific antibody potency for any given batch of anti-C1H antibody standard relative to the Reference Standard. This EIA has been used to quantify and characterize anti-C1H antibody in human serum in support of clinical safety and efficacy studies.
Division of Bioanalysis and Drug Metabolism, Glaxo Wellcome Inc., Research Triangle Park, NC 27709, USA.
L M
LM
Thurmond
M J
MJ
Reese
R J
RJ
Donaldson
B S
BS
Orban
D000074323:Alemtuzumab; D000465:Algorithms; D000704:Analysis of Variance; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000097:blood*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm / Q000276:immunology*; D005069:Evaluation Studies as Topic; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007700:Kinetics; D051379:Mice; D051381:Rats; D012015:Reference Standards
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000912:Antibodies, Neoplasm; D000074323:Alemtuzumab