A multi‐allergen standard for the calibration of immunoassays: CREATE principles applied to eight purified allergens

Abstract

Background Allergen measurements are widely used for environmental exposure assessments and for determining the potency of allergen vaccines, yet few purified allergen standards have been developed. The aim of the study was to develop a single standard containing multiple purified allergens that could be used in enzyme immunoassays and in multiplex arrays for standardization of allergen measurements. Methods Eight purified allergens were formulated into a single multi-allergen, or “universal”, standard based on amino acid analysis. Dose response curves were compared with previous individual ELISA standards and allergen measurements of house dust extracts to obtain correction factors. Measured allergen concentrations were also modeled using linear regression and the predictive accuracy was determined. Results Parallel dose response curves were obtained between the universal allergen standard and the individual ELISA standards, with close agreement between curves for 5/8 allergens. Quantitative differences of >2-fold were observed for Fel d 1, Can f 1 and Der f 1, which were confirmed by analysis of house dust extracts. Correction factors were developed that allowed ELISA data to be expressed in terms of the universal standard. Linear regression data confirmed the predictive accuracy of the universal standard. Conclusion This study shows that a single standard of eight purified allergens can be used to compare allergen measurements by immunoassay. This approach will improve continuity of environmental exposure assessments and provide improved standardization of allergy diagnostics and vaccines used for immunotherapy.

Comparison of buffers for extraction of mite allergen der p 1 from dust

“…Harmonising protocols for indoor allergen measurement can make results more comparable and lower inter-laboratory variability. Recently, Filep et al (22) have developed a single standard for eight common indoor allergens. Adding a such control sample with declared allergen levels can improve performance and reduce variability between laboratories.…”

Monitoring of occupational and environmental aeroallergens – EAACI Position Paper

“…Recently, monoclonal antibodies have been used in fluorescent multiplex arrays that allow detection of multiple allergens in a single test. This principle has been successfully applied in a test system for up to eight allergens, which can be measured simultaneously under the same assay conditions (Multiplex Array for Indoor Allergens; MARIA TM system, Indoor Biotechnologies Inc, Charlottesville, VA, USA), thus improving standardization and reproducibility, and increasing the efficiency of measurements (46)(47)(48).…”

“…The factor used for the conversion from potency relative to the reference material into mass units has a substantial (<2-fold) uncertainty in the current literature. It is very important to recognize that measurement data produced by different immunoassays can only be compared if assay calibration standards are validated and tested against known national or international standards, where these are available (CREATE; 47,54). The aims of the CREATE project were to identify candidate reference materials and ELISA reagents that would subsequently be prepared by regulatory authorities.…”

Cat and House Dust Mite Allergen Content Is Stable in Frozen Dust over Time

“…However, the standard used in the present study was a multi-allergen standard, termed the universal allergen standard, developed more recently (1000 ng/mL), which consists of eight purified allergens. Conversion factors have been defined in order to facilitate transition between the universal allergen standard and previous standards, as described by Filep et al [11]. Microtiter plates (Maxisorp, Nunc, Roskilde, Denmark) were coated over night at 4-8C with mAb 6F9, at 2 g/mL in 50 mM carbonate/bicarbonate buffer (pH 9.6).…”