Measurement of IgG-blocking antibodies by ELISA using monoclonal antibody and fluorogenic substrate.
An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman gamma-chain antibody and a fluorogenic substrate has been developed for quantitation of IgG-blocking antibodies in human serum. Generation of fluorescent product was linear with time to 60 min. Using optimal conditions the ELISA was sensitive to less than 1 ng/ml of specific IgG to short ragweed pollen. The assay demonstrated consistently parallel dilution curves with 51 sera (mean interdilutional coefficient of variation = 8.8%). Reproducibility was determined by constructing precision profiles for intra and interassay variation for the entire working range of the assay. Intraassay CVs ranged from a mean of 13% at threshold to less than 5% at higher antibody concentration. Interassay reproducibility similarly ranged from 18 to 10%. In this assay the effect of serum dilution on nonspecific binding was minimal and specific binding of 4-10 ng IgG antibody to the antigen-adsorbed wells was largely complete (75.8 +/- 4.8%) and highly specific (greater than 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.
Lee
H B
HB
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md.
Rommel
F
F
Adkinson
N F
NF
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis*; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D007074:Immunoglobulin G / Q000032:analysis / Q000276:immunology*; D051379:Mice; D011058:Pollen / Q000276:immunology; D011237:Predictive Value of Tests; D014468:Umbelliferones
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D014468:Umbelliferones; C048844:anti-IgG; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone