Environmental and occupational respiratory disorders
Simultaneous detection of total and allergen-specific IgE by using purified allergens in a fluorescent multiplex array

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Background

Testing serum samples for total and allergen-specific IgE requires separate testing for each antibody and allergen specificity.

Objective

To apply fluorescent suspension array technology to allow simultaneous detection of total and allergen-specific IgE in serum in a single quantitative test.

Methods

A 7-plex suspension array for the simultaneous detection of total IgE and IgE specific to Der p 1, Der p 2, Fel d 1, Can f 1, Bet v 1, and Phl p 5 was developed, using mAb or purified allergens covalently coupled to fluorescent microspheres. The multiplex array was validated by comparing total and allergen-specific IgE levels in serum from patients with allergy with results obtained by enzyme immunoassays.

Results

There was a highly significant correlation between total IgE levels measured by multiplex array and fluorescent enzyme immunoassay (r = 0.97; P < .001; n = 63). Total and allergen-specific IgE levels also correlated with enzyme-linked and fluorescent enzyme immunoassay results (r = 0.44-0.94; n = 95 or 106). The multiplex array was reproducible (r = 0.86-0.99; mean coefficient of variance percentage, 12% to 25%). The sample volume required for a 7-plex assay was <20 μL per sample, compared with >400 μL in current immunoassays.

Conclusion

The multiplex array is a high-throughput system that allows simultaneous quantification of allergen-specific and total IgE.

Clinical implications

Our results suggest that fluorescent multiplex technology will facilitate large-scale epidemiologic studies of allergic sensitization. The reduced serum volume is an advantage for pediatric studies.

Section snippets

Human sera

Human sera originated from studies of allergic sensitization performed previously at the University of Virginia15, 16, 17 (n = 62) or were kindly provided by Dr Ronald van Ree from the European Union Development of certified reference materials for allergenic products and validation of methods for their quantification Allergen Standardization Project (n = 33)18 and by Dr Enrique Fernandez-Caldas of Laboratorios LETI, Spain19 (n = 63). Collection of sera was approved by the institutional review

Array for total IgE

To establish the best coupling concentration of IgE capture mAb in the array for detection of total IgE, activated microspheres were covalently coupled with capture mAb at concentrations of 50 to 800 μg per coupling batch (1.25 × 106 beads). A 2B12-IgE standard curve ranging from 0.05 to 1000 IU IgE/mL was prepared on each of the coupled bead sets. A coupling concentration of 400 μg was found optimal on the basis of signal intensity and dynamic range of the standard curve (Fig 1, A). After

Discussion

The study presented here used fluorescent multiplex suspension array technology to develop a method enabling the simultaneous detection of total and allergen-specific IgE. The benefits of multiplex technology over ELISA methods for various analytes have recently been reviewed.5, 25, 26 Speed, the ability to measure different antibody concentrations with minimum sample volume, and more desirable reaction kinetics and enhanced dynamic range are major benefits of multiplex technology versus ELISA.

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    Supported in part by Small Business Innovation Research contract ES55545 from the National Institute of Environmental Health Sciences.

    Disclosure of potential conflict of interest: E.-M. King, L. D. Vailes, and A. Tsay have received grant support from a National Institute of Environmental Health Sciences Small Business Innovation Research contract and are employed by Indoor Biotechnologies Inc. M. D. Chapman owns stock in and is employed by Indoor Biotechnologies Inc and has received grant support from the National Institute of Environmental Health Sciences. S. M. Santiover has declared that she has no conflict of interest.

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