Covalent binding of proteins to grafted plastic surfaces suitable for immunoassays. II. Picograms of IgE detected in BAL fluid in sarcoidosis.
An ELISA is described for the quantitation of minute amounts of human IgE using microtiter strips which were surface modified according to a procedure previously described. This ELISA used an IgG fraction of a rabbit polyclonal anti-IgE antibody coupled to the carboxylated surface, which prior to coupling had been preactivated with a water soluble carbodiimide. A biotinylated affinity purified rabbit anti-IgE antibody was used together with streptavidin conjugated alkaline phosphatase and biotinylated enzyme for the detection of bound analyte. Sarcoidosis is a systemic disorder of as yet unknown cause with epithelioid granuloma formation in the lung as a prominent feature. With the bronchoalveolar lavage technique, epithelial lining fluid was obtained and analyzed for IgE content since differences of immunoglobulin concentration in bronchoalveolar lavage fluid (BALF) may reflect disease activity. Our modified ELISA system was used for measuring IgE in BALF from healthy controls and sarcoid patients. The assay using modified strips demonstrated a considerably improved sensitivity compared to the use of physical adsorption of first antibody to untreated strips. The detection limit of the improved assay was approximately 3 pg IgE/sample (100 microl). Quantitative recovery of IgE was demonstrated within the measuring range (51.2-12500 pg IgE/ml) and more than 40% (23/54) of the sarcoid patients showed values above the lowest standard concentration (51.2 pg/ml). In contrast, none of the healthy controls (0/22) had detectable IgE as defined by the detection limit of the assay.
Larsson
P H
PH
Department of Clinical Immunology and Transfusion Medicine, Karolinska Hospital, Stockholm, Sweden. per.larsson@niwl.se
Eklund
A
A
Johansson
S G
SG
Larsson
K
K
D000328:Adult; D000818:Animals; D001992:Bronchoalveolar Lavage Fluid / Q000276:immunology; D016022:Case-Control Studies; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis*; D008171:Lung Diseases / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D010969:Plastics; D011485:Protein Binding; D011817:Rabbits; D012507:Sarcoidosis / Q000276:immunology*; D012680:Sensitivity and Specificity; D013499:Surface Properties
D010969:Plastics; D007073:Immunoglobulin E