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 (II4F9 and II4G9) were enriched by in vitro production in a modular minifermenter and affinity purified. Wells of micro-ELISA plates coated with captured mAb II4G9 were incubated with samples containing Hev b 1. Bound Hev b 1 was detected by a combination of biotinylated mAb II4F9 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 microl 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 microg 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 microg 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.
Raulf-Heimsoth
M
M
Research Institute for Occupational Medicine (BGFA), Ruhr University Bochum, Bochum, Germany. raulf@bgfa.ruhr-uni-bochum.de
Sander
I
I
Chen
Z
Z
Borowitzki
G
G
Diewald
K
K
van Kampen
V
V
Baur
X
X
D000485:Allergens / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D052179:Antigens, Plant; D004784:Environmental Monitoring; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007840:Latex / Q000592:standards; D020315:Latex Hypersensitivity / Q000276:immunology; D009784:Occupational Diseases / Q000276:immunology; D010940:Plant Proteins / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C098306:Hev b 1 protein, Hevea brasiliensis; D007840:Latex; D010940:Plant Proteins