Development of a monoclonal antibody-based sandwich ELISA for detection of the latex allergen Hev b 1

Background: Natural rubber latex (NRL) products are complex mixtures consisting of different allergenic components. Among them, Hev b 1 belongs to the important and well-characterized ones. To quantify the relevant allergen Hev b 1 in NRL products, a two-site monoclonal antibody (mAb)-based assay was developed. Methods: Two Hev b 1-specific mAbs with different epitope recognition and ability to bind simultaneously to an Hev b 1 molecule were used in the study. Both mAbs (114F9 and 114G9) were enriched by in vitro production in a modular minifermenter and affinity purified. Wells of micro-ELISA plates coated with captured mAb 114G9 were incubated with samples containing Hev b 1. Bound Hev b 1 was detected by a combination of biotinylated mAb 114F9 as detection antibody and peroxidase-labeled avidin. Results: The optimized sandwich ELISA was highly reproducible in the linear range of the standard curve and Hev b 1 concentrations ranging from 12.5 to 400 ng/100 muI could be detected. The assay was suitable for the detection of Hev b 1 concentrations in latex sap and latex products, e.g. gloves, with a detection limit of 1.25 mug of Hev b 1/g of rubber. In a preliminary study with five different brands of latex gloves, Hev b 1 concentrations were found to be in the range of 18-40 mug per gram of rubber material, corresponding to 2-4% of the total extractable protein content in latex glove extracts. Conclusions: A sensitive sandwich assay was developed to quantify the latex allergen Hev b 1. This assay can be used to standardize latex extracts with regard to the content of the major allergen Hev b 1. Copyright (C) 2000 S. Karger AG, Basel.