Major Allergen Measurements: Sources of Variability, Validation, Quality Assurance, and Utility for Laboratories, Manufacturers, and Clinics

The isolation and characterization of prominent allergenic proteins or glycoproteins is an important step in the development of allergenic extracts exhibiting improved definition, consistency, and clinical utility. Quantitative analyses specific for major allergenic components currently are being performed in numerous corporate and academic laboratories but have not been validated within or across laboratories in a systematic manner. In our laboratory, validation of double-bind (sandwich) ELISA assays for a diverse group of major allergens or extract components revealed a number of critical assay variables and reagent incubation conditions that directly influenced the precision, accuracy, specificity, and robustness of these tests. Data from ELISA methods for six allergens (Dermatophagoides farinae Der f 1, Alternaria Alt a 1, dog albumin, dog Can f 1, fire ant Sol i 3, and yellow jacket venom Ves 5) showed that up to twofold differences in results were observed when analysts or microplates were varied. Analyses of dog allergens using multiple reagents and concentrations indicated that twofold variations in results also can be produced by distinct combinations of materials or incubations from different assay steps. Data from Can f 1 and egg white analyses produced up to fivefold differences in antigen concentrations based on changes in the capture antibody source (mouse monoclonal versus rabbit polyclonal) or storage buffer. These results suggest that differences in major allergen concentrations reported by different testing laboratories may be related to assay differences as well as extract variations and raise questions as to the accuracy of major allergen concentrations and therapeutic dose recommendations reported at regional and national allergy meetings. Validated double-bind ELISA methods may be well suited for consistency monitoring and standardization of extracts provided that reference materials, reagent qualifications, and interlaboratory comparability are defined precisely.