[Show abstract][Hide abstract] ABSTRACT: 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 the standardization of allergen measurements.
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.
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 greater than twofold were observed for Fel d 1, Can f 1, and Der f 1, which were confirmed by the 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.
This study shows that a single standard of eight purified allergens can be used to compare allergen measurements by immunoassay. This approach will improve the continuity of environmental exposure assessments and provide improved standardization of allergy diagnostics and vaccines used for immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.
[Show abstract][Hide abstract] ABSTRACT: Testing serum samples for total and allergen-specific IgE requires separate testing for each antibody and allergen specificity.
To apply fluorescent suspension array technology to allow simultaneous detection of total and allergen-specific IgE in serum in a single quantitative test.
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.
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 microL per sample, compared with >400 microL in current immunoassays.
The multiplex array is a high-throughput system that allows simultaneous quantification of allergen-specific and total IgE.
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.
Full-text · Article · Nov 2007 · The Journal of allergy and clinical immunology
[Show abstract][Hide abstract] ABSTRACT: 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 5 0.97; P < .001; n 5 63). Total and allergen-specific IgE levels also correlated with enzyme-linked and fluorescent enzyme immunoassay results (r 5 0.44-0.94; n 5 95 or 106). The multiplex array was reproducible (r 5 0.86-0.99; mean coefficient of variance percentage, 12% to 25%). The sample volume required for a 7-plex assay was <20 mL per sample, compared with >400 mL 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. (J Allergy Clin Immunol 2007;120:1126-31.) Key words: Multiplex array, total IgE, allergen-specific IgE, enzyme immunoassay, allergic sensitization, allergy diagnostics, asthma Reliable methods for assessing IgE mediated sensitiza-tion to specific allergens are crucial for the investigation and diagnosis of allergic diseases. Allergy diagnosis is routinely performed by skin prick testing or using in vitro tests, such as RAST, ELISA, or fluorescent enzyme immu-noassay (FEIA). Although skin testing is sensitive and convenient, in vitro testing has some advantages includ-ing precise quantitation, safety, lack of drug interference, and the possibility of long-term storage of specimens. 1 For some allergens such as foods, in vitro IgE testing strengthens the probability that the clinical diagnosis is correct. 2 Sampson 3 established 95% predictive decision points for food allergen-specific IgE, as measured using FEIA. All current methods for in vitro IgE analysis such as RAST, ELISA, and FEIA have a central limitation: tests for each antibody or allergen specificity have to be per-formed separately, which is time-consuming and asso-ciated with higher costs and an increased possibility of technical errors. The limitations of current in vitro proce-dures are an impediment for large studies of sensitization and for prospective studies to be performed over a period of several years. The use of significant amounts of serum is a further limitation, especially for pediatric studies. In recent years, bead-based suspension arrays have been developed that enable the simultaneous detection of multiple analytes. The technology uses polystyrene beads that are internally labeled with a distinguishable fluoro-phore, allowing them to be identified by a laser-based scanner. Antigens, antibodies, or other molecules are co-valently coupled to the surface of the beads. Combining different bead types with the respective different capture antigens allows the simultaneous detection of multiple an-alytes. Quantification of multiple cytokines in serum and culture supernatants is a common application of fluores-cent multiplex array. 4,5 Other applications include moni-toring environments for chemical and biological warfare agents 6 and detecting IgG Abs to toxins, bacterial se-rogroups, or capsular polysaccharides. 7-10 There is good evidence that measuring IgE Abs to 2 to 4 major allergens from a given source is effective for diagnostic purposes.
[Show abstract][Hide abstract] ABSTRACT: Current enzyme immunoassay methods for detection of common indoor allergens in environmental dust samples are labor-intensive and time consuming.
To develop and validate a fluorescent multiplex array to measure 6 (Der p 1, Der f 1, Der p 2, Der f 2, Fel d 1, and Can f 1) indoor allergen levels simultaneously.
A multiplex array for 6 allergens, using mAbs covalently coupled to fluorescent microspheres, was developed using a single universal standard composed of purified natural allergens. The multiplex array was validated by comparing the measured dust mite, cat, and dog allergen levels in household dust samples to those obtained by standard ELISA methods.
Linear regression analysis showed a highly significant quantitative correlation between the multiplex array and ELISA for dust mite, cat, and dog allergens: R(2) values ranging from 0.90 to 0.99 (P < .001). In addition, the sensitivity, limit of detection (<0.1 ng/mL), reproducibility, intra-assay coefficient of variance (<5%), and interassay coefficient of variance (<25%) of the fluorescent multiplex array were shown to be equal to or better than the ELISA method.
A multiplex array has been developed to measure simultaneously 6 indoor allergens from a single sample. The array will facilitate epidemiologic studies and indoor air quality assessments and can, in principle, be expanded to include other allergens and biologics.
The multiplex array lends itself to clinical studies, population-based environmental surveys, and allergen avoidance studies comparing allergen exposure in large populations over several time points.
Full-text · Article · Mar 2007 · Journal of Allergy and Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: Respiratory symptoms related to both endotoxins and animal allergens continue to be an important cause of occupational disease for animal technicians and scientists working with rodents. Better sampling methods for airborne allergens and endotoxin are needed to help standardize compliance with federal occupational health regulations. Using an ion-charging device, we sampled 20 mouse rooms and four rat rooms at the University of Virginia, along with 43 domestic living rooms in houses in the Charlottesville area with at least one cat or dog. The use of filter tops on cages corresponds to a 50-fold reduction in mean levels of both airborne allergens (P < 0.001) and endotoxin (P < 0.001). The use of vented cages with filtered exhaust ports was associated with additional reductions. However, the mean airborne endotoxin level in all rooms using filter tops without a filtered exhaust port on the cages was significantly lower (P = 0.003) than the level in domestic living rooms. Our results for maximum airborne allergens or endotoxin are comparable with previous reports. However, the sensitivity of the technique allows an accurate assessment of low-level exposure, which makes it possible to evaluate the effect of cage designs. In addition, this approach allows direct comparison with results for airborne allergen and endotoxin in domestic homes. The results could allow a more consistent approach to the application of occupational health guidelines.
Full-text · Article · Apr 2005 · Contemporary topics in laboratory animal science / American Association for Laboratory Animal Science
[Show abstract][Hide abstract] ABSTRACT: Mouse urinary allergens are an important cause of occupational asthma in animal facilities. Domestic exposure to mouse allergens is a risk factor for asthma among inner-city residents.
We sought to develop a sensitive and specific assay for assessing environmental mouse allergen exposure.
An ELISA for recombinant (r)Mus m 1 was developed by using rabbit polyclonal antibodies to rMus m 1 that were affinity purified against the natural allergen. Assay specificity was established by means of immunoblotting and ELISA. Mus m 1 levels in mouse, other mammalian allergenic products, and house dust samples from inner-city homes were compared.
Polyclonal antibodies to Mus m 1 showed a single 20-kd band on immunoblots against rMus m 1 and male mouse urine. Parallel dose-response curves were obtained by using mouse urine extract and natural Mus m 1 or rMus m 1. Mus m 1 was detected in mouse allergenic products (0.10-10.0 microg/mL) and in gerbil allergenic products (0.1 microg/mL) but was less than the limit of detection in epithelial extracts from 10 other animal species. Environmental measurements showed an excellent correlation between Mus m 1 levels in house dust extracts from inner-city asthma studies by using 2 different Mus m 1 standards (n=22; r=0.99; P <.001).
A highly sensitive ELISA has been developed with rMus m 1. This assay is suitable for monitoring domestic and environmental exposure to mouse urinary allergens.
Full-text · Article · Aug 2004 · Journal of Allergy and Clinical Immunology
[Show abstract][Hide abstract] ABSTRACT: RationaleA consistent method for measuring airborne allergens and endotoxin in animal houses is essential for evaluating the risk of sensitization and the impact of measures taken to reduce exposure.
[Show abstract][Hide abstract] ABSTRACT: RationaleTo develop purified Group 1 and Group 2 mite allergens with verifiable allergen and protein content, as part of the EU CREATE program, which will serve as international standards for in vitro assays and for research use.
[Show abstract][Hide abstract] ABSTRACT: Peanut allergy is an important health problem in the United States, affecting approximately 0.6% of children. Inadvertent exposure to peanut is a risk factor for life-threatening food-induced anaphylaxis.
The purpose of this investigation was to develop an immunoassay for a major peanut allergen, Ara h 1, to detect peanut allergen in foods so that the risk of inadvertent exposure can be reduced.
A specific 2-site monoclonal antibody-based ELISA was developed to measure Ara h 1 in foods. The sensitivity of the assay was 30 ng/mL. Ara h 1 was measured in foods (n = 83) with or without peanut and in experiments to optimize allergen yield and to determine peanut contamination in spiked foods.
Ara h 1 levels in food products ranged from less than 0.1 microg/g to 500 microg/g. Ara h 1 measured in ng/mL was transformed to microg/g for food products. Peanut butter contained the highest amounts of Ara h 1. Peanut extracts contained from 0.5 to 15 mg Ara h 1/g of peanut depending on the extraction conditions. Optimal extraction of Ara h 1 was obtained by using phosphate buffer with 1 mol/L NaCl and Tween at 60 degrees C. Ara h 1 was not always detected in presence of chocolate under the extraction conditions tested. Spiking experiments showed that the assay could detect approximately 0.1% Ara h 1 contamination of food with ground peanut. There was an excellent correlation between Ara h 1 levels and peanut content measured by using a commercial polyclonal antibody-based ELISA (r = 93, n = 31, P <.001).
A new sensitive and specific monoclonal antibody-based ELISA was used to monitor Ara h 1 content in food products. This assay should be useful for monitoring peanut contamination in the food manufacturing and processing industry and in developing thresholds for sensitization or allergic reaction in persons with peanut allergy.
Full-text · Article · Mar 2003 · Journal of Allergy and Clinical Immunology