Measurement of igg-blocking antibodies by elisa using monoclonal antibody and fluorogenic substrate

Lee Ha-Baik, Fred Rommel, N. Franklin Adkinson

Research output: Contribution to journalArticlepeer-review

Abstract

An enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antihuman γ-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 < 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 < 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 (> 98%). This application of ELISA for ragweed IgG antibody measurement has performance specifications equal or superior to previously developed radioimmunoassay and ELISA systems.

Original languageEnglish (US)
Pages (from-to)261-267
Number of pages7
JournalInternational archives of allergy and immunology
Volume85
Issue number3
DOIs
StatePublished - 1988

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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