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 the 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 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.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 the continuity of environmental exposure assessments and provide improved standardization of allergy diagnostics and vaccines used for immunotherapy.

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.

Assessments of allergen exposure by ELISA rely on standards of known allergen content. However, there are only two WHO/IUIS International Reference Preparations, which are extracts on known allergen content (not purified allergens). Our aim was to develop a single "Universal Standard" containing purified natural allergens with verifiable allergen content to serve as a standard for ELISA and multiplex assays. Six purified allergens (Der p 1, Der f 1, mite Group 2, Fel d 1, Can f 1, Bla g 2) were formulated into a Universal Standard at concentrations of 1300-5000 ng/ml. Protein content was compared by amino acid analysis, advanced protein assay and A280 nm. ELISA was used to compare purified allergen standards with current extract-based commercial standards. There was good agreement between all three protein assays for purified natural and recombinant mite Group 1 and Group 2 allergens. Overlapping curves were obtained for Der p 1, Fel d 1, and Can f 1 in the Universal Standard and commercial ELISA standard. There was a 1.7 fold difference for mite Group 2 allergens, and ∼8-fold difference for Der f 1 and a ∼5-fold difference for Bla g 2. This suggests that Der f 1 and Bla g 2 are significantly over-estimated using current extract-based ELISA standards, as compared to the purified proteins. A single Universal Standard of purified natural allergens can be used for allergen exposure assessment by ELISA or multiplex array technology. Allergen standards should be established based on protein determinations and International Reference Standards should be developed for calibration purposes.

RATIONALE: The detection of indoor allergens is labor intensive and time consuming when multiple allergens are analyzed by separate ELISA. A multiplex array for indoor allergens (JACI, 2006; 117:S28) has been developed to measure eight important household allergens (Der p 1, Der p 2, Der f 1, Der f 2, Fel d 1, Can f 1, Rat n 1, and Mus m 1) in a single quantitative test. METHODS: A suspension array for eight allergens was developed using mAb covalently coupled to fluorescent microspheres. The assays in the multiplex array were validated by comparing allergen levels in ∼50 dust samples by ELISA and by multiplex array using a single universal allergen standard containing purified natural allergens. RESULTS: Linear regression analysis showed a highly significant quantitative correlation between the multiplex array and ELISA for each of the allergens: R2 values >0.90, p<0.001 for all allergens. The limits of detection of the multiplex array are an order of magnitude greater than ELISA, <0.1 ng/mL, and interassay CVs are less than 20%. CONCLUSIONS: The simultaneous quantification of multiple allergens in a high throughput multiplex array has been developed. The array will be applicable to large population studies where the assessment of multiple allergens is required. The array will allow for greater efficiency, reproducibility and standardization in the measurement of indoor allergens.

RATIONALE: Sensitization and exposure to indoor allergens is a major risk factor for asthma. Allergen avoidance is recommended as a primary treatment strategy. Allergen levels in homes can vary by over 100 fold and exposure assessment typically uses laboratory based ELISA techniques. Our objective was to develop a rapid lateral flow screening test to detect multiple indoor allergens in homes, workplaces, public and commercial buildings. METHODS: Lateral flow tests for mite Group 1 and Group 2 allergens, Fel d 1 and Can f 1 were used to measure allergen levels in ~170 dust and air samples on a three point visual scoring scale. Allergen levels were quantified by mAb ELISA and the results were compared. RESULTS: There was a highly significant correlation between lateral flow tests and ELISA for all four allergens over a 1-100ug/g range, with r values from 0.55 - 0.82 (Pearson's correlation, p<0.001). Results for Fel d 1 showed a weaker correlation, especially at high Fel d 1 values (>20ug/g), suggesting a prozone effect for this allergen. Overall, there was 85% agreement between lateral flow and ELISA results: A prototype credit card size test which simultaneously measured all four allergens was produced. CONCLUSIONS: The multiplex rapid test for indoor allergens will enable patients with allergic asthma to test their home environment for allergens and to take suitable control procedures to reduce exposure. The allergen screening test can be used by allied health and indoor air quality professionals, as well as consumers, to assess environmental allergen exposure.

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.

Background: 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. Objective: We sought to develop a sensitive and specific assay for assessing environmental mouse allergen exposure. Methods: 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. Results: 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 mg/mL) and in gerbil allergenic products (0.1 mg/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). Conclusions: 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. (J Allergy Clin Immunol 2004;114:341-6.)

Rationale A 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. Methods Levels of airborne mouse and rat allergens in animal rooms were measured using a recently described method for collecting airborne allergens employing an ion charging device with a flow rate of 1.7m 3 /min for 24 hours (Clin Exp All 33:986). The allergens were measured using a two-site monoclonal assay for rat and a monoclonal primary/polyclonal secondary assay for mouse (Indoor Biotechnologies). Endotoxin was assayed in parallel using the Limulus Amoebocyte Lysate test QCL 1000 (Bio-Whittaker). Results Airborne mouse and rat was present in relevant animal rooms, however there was a very wide range of results for both allergens ( 3 to >6ng/m 3 ) and endotoxin ( 3 to >1000pg/m 3 ). The primary factor influencing airborne allergen and endotoxin was the presence of filter tops on individual animal cages for mouse (allergen p=.003, endotoxin p=.01) and rat (allergen p =.013, endotoxin p =.001). Filter tops reduced the airborne allergen and endotoxin levels by >98%. Conclusions Though previous studies have attributed reduced allergen levels to filter tops, this study shows the same effect on endotoxin levels. The reduced levels are similar to levels of airborne allergens found in domestic studies and thus filter tops may provide sufficient protection from exposure for animal technicians.

Rationale To 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. Methods Natural allergens, or recombinant allergens from Ecoli or Ppastoris were purified by affinity chromatography or HPLC and analyzed by SDS-PAGE, protein assay, amino acid analysis, mass spectrometry and by ELISA. Purified allergens were compared by ELISA with three existing allergen standards and with six commercial Dpt or Df extracts. Results Amino acid analysis showed good agreement between the protein content, with recombinant:natural protein ratios of 2.4-2.7 and 0.51-1.4 for Group 1 and Group 2. Q-TOF MS showed major peaks at MW 14027-14096 for purified Group 2 allergens. Multiple isoforms were detected in natural allergens. ELISA showed parallel dose response curves and consistent quantitative relationships between all five allergen standards with detection limits ranging from 4-13ng/ml (Group 1) and 0.5-1ng/ml (Group 2). Inter-assay ELISA CV's were 12-23% Group 1 (IBI), 37-45% Group 1 (ALK) and 26-36% Group 2. Allergen levels in Dpt or Df extracts ranged from 8-1000μg/vial (Group 1) and 3-170μg/vial (Group 2). Conclusions Purified mite allergen standards have been developed and the quantitative relationship between current mite references and CREATE reference materials has been established. Immunoassays for both major mite allergens have been validated. The purified CREATE references are suitable for international comparisons of allergen levels by immunoassay and should have applications in standardizing allergenic products and environmental exposure measurements.

BACKGROUND 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. OBJECTIVE 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. METHODS 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. RESULTS 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). CONCLUSION 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.

SummaryBackground International guidelines recommend allergen avoidance for asthma management, but do not include making assessments of allergen exposure. Mite allergen exposure cannot be assumed, especially in geographical regions where climatic conditions vary.Objective To develop a rapid test that would enable consumers to detect mite allergen in the home.Methods A lateral flow test using gold labelled antibody for mite group 2 allergen was developed as part of a detection kit incorporating the MITEST dust sampling device. Dust samples were assayed by ELISA for group 1 and group 2 allergens and by using the rapid test. The tests were compared as indices of mite allergen exposure.Results There was a good correlation between group 1 and group 2 levels by ELISA (n = 349, r = 0.60, P < 0.001). In a multi‐centre study of 65 homes (263 dust samples) in five countries, there was a strong correlation between ELISA and the rapid test. Most samples with high scores in the test (43/48, 90%) contained > 1 µg/m2 group 2 allergen, whereas most low samples contained < 1 µg/m2 (50/64, 78%). Differences between mean group 2 levels of samples that scored low (0.28 µg/m2), medium (1.68 µg/m2) or high (3.18 µg/m2) on the test were highly significant (P 0.007 to < 0.001).Conclusions A simple rapid test has been developed that detects mite allergen in the home within 10 min. The mite screening test should educate consumers about allergen exposure and encourage compliance with allergen‐avoidance procedures. This technology has applications for the detection of other common environmental allergens.

Immunoassays for indoor allergens were first developed in the late 1980s, and the last decade has seen an explosion of research and clinical studies on the role of household allergen exposure in causing allergic disease, especially asthma. Monoclonal antibody-based enzyme immunoassays (ELISA) have become a cornerstone of laboratory assessment of exposure to mite, cat, dog, and cockroach allergens (1, 2). The panel of assays available for allergen measurements has grown steadily and includes assays for Blomia tropicalis, Lepidoglyphus destructor, and bovine and equine allergens (3-19) (Table 1). Over 300 publications using allergen immunoassays are now on MEDLINE databases, covering a wide range of scientific studies. These include the following: • Sites and distribution of allergen accumulation within the home; seasonal variation in allergen levels. • Case control studies and population studies designed to establish risk levels for allergen exposure leading to sensitization and to asthma symptoms. • Analysis of the aerodynamic properties of indoor allergens. • Standardization of allergenic products used for diagnosis and treatment. • Design and monitoring of allergen avoidance studies and assessment of the efficacy of allergen control products, devices, chemicals and procedures (e.g., mattress encasings, air filters, and vacuum cleaners). This allows manufacturers to design products that are of proven efficacy by using objective methods to demonstrate their effects on allergens. As a result of these studies, the evidence that exposure to indoor allergens is an important risk factor for asthma has become stronger, and for some allergens, particularly dust mite, there is convincing evidence for a dose-response relationship between sensitization and exposure (2, 20). Expert panel reports and position statements from the European Union (EU), the US National Heart, Lung, and Blood Institute (NHLBI), and the American Academy of Allergy, Asthma, and Immunology (AAAAI) have recommended allergen avoidance as an integral part of asthma management (2, 21-23). However, until recently, measuring allergen exposure in homes has largely been a procedure carried out by research laboratories. Over the past 2 years, commercial reference laboratories in Europe and in the USA have been provid-ing allergen analysis services, and patient-based allergen-detection tests and personal monitoring systems are being introduced. This review focuses on recent advances in developing these systems and how measurements of allergen exposure can be used to improve clinical practice and provide benefits for allergic patients. Monoclonal antibody (mAb)-based ELISA techniques have become established as the reference standard for indoor allergen analysis. The advantages of ELISA are high throughput, accurate quantitation (ng/g or μg/g allergen protein), and defined specificity, by using mAbs to allergens of known molecular structure and allergenic importance (Table 1). Successful strategies for developing these assays, including selection of mAbs and assessment of assay variability and performance, have recently been reviewed (24). A number of assays are available for assessing exposure to the most common dust-mite species, as well as animal allergens and cockroach allergens. The gaps in the repertoire are for assays for American cockroach allergen and fungal allergens. Although an assay for Per a 3 of American cockroach has been described, the assay has not yet been validated for environmental measurements (15). Recently, two assays for the Alternaria allergen Alt a 1 have been described and used for comparing Alt a 1 levels in allergen extracts (18, 19, 25). The problem with these and other assays for fungal allergens (e.g., Asp f 1) has been in validating the assays for environmental exposure measurements. Asp f 1 is not expressed in spores, which are the most common form of Aspergillus in house dust (16, 17). Similarly, it has proved difficult to measure Alt a 1 in house-dust samples, and this may reflect the fact that extensive fungal contamination is required to produce allergen that can be detected in the assays. Alt a 1 can be eluted from Alternaria spores; however, the spore count has to be very high (>100 000 spores/ml) for allergen to be detectable, and it seems unlikely that such high spore concentrations will routinely be found in domestic environments (18). Producing and maintaining allergen standards is critical to the reproducibility and accuracy of allergen assays. In the past, standards have been established by comparing the allergen content of a reference preparation (e.g., mite or cat extract) relative to a purified sample of a given allergen (Der p 1 or Fel d 1). It is important to realize that until recently all allergen standards were simply extracts that had been calibrated to a known concentration: they were not purified allergens. Standards have been established by researchers, regulatory agencies, or manufacturers and biotechnology companies for “in house” or commercial use (reviewed in Refs. 24, 26-28). However, currently, there are no international standards for purified allergens. Such standards would be of great value in comparing allergen measurements in epidemiologic studies and for standardization of allergenic products used for diagnosis and immunotherapy. A new initiative to develop recombinant allergen standards is being organized by the WHO/IUIS Allergen Standardization Committee. The aim is to develop recombinant allergen standards for mite (groups 1 and 2), birch (Bet v 1), grass pollen (Phl p 1 and Phl p 5), and olive pollen (Ole e 1) allergens. The program will involve comparison of both natural and recombinant allergens, and criteria will be established for protein measurements and for assessing allergenic activity. Currently, bacterially expressed rBlo t 5 and rAlt a 1 are being used as standards in ELISA, and most of the allergens listed in Table 1 have been expressed as recombinants with immunoreactivity comparable to the natural allergen (29, 30). The WHO/IUIS Allergen Standardization program is being funded by the EU and will involve collaborations between academic researchers, allergen manufacturers, and biotechnology companies. The aim is to produce the purified allergens that will serve as primary standards for immunoassays. For example, mite-allergen Der p 2 has been expressed at high level in E. coli. Recombinant Der p 2 is highly immunoreactive and has been used in NMR and x-ray crystallographic studies to determine the tertiary structure of the allergen and the location of mAb-binding epitopes (31-33). Thus, it will become possible to establish a primary standard using rDer p 2 that can be stored in WHO-approved repositories, and this would be the international standard for Der p 2 measurements (analogous to current IgE standards). The limitations of ELISA are that dust extraction, sample preparation, and the assays themselves are time-consuming, and their use is confined to well-equipped research or clinical laboratories. The level of public awareness of the role of indoor allergens in asthma has grown dramatically over the past 10 years and has spawned the development of simple qualitative or semiquantitative tests that enable patients themselves to assess mite-allergen levels in their homes. The first of these tests was the chemical test for guanine (the Acarex test), a surrogate test for mites (the major producers of guanine in houses dust) (34). This test correlates reasonably well with levels of mite allergen and, although it has not been widely used, is still marketed in Europe. The production of allergen-specific mAbs made it possible to develop innovative tests for specific mite (and other) allergens, including DUSTSCREEN, the ACLOTEST, and, most recently, rapid lateral flow tests which enable allergen to be detected in 5–10 min (35-37). These tests use specific mAbs or polyclonal antibodies bound to membranes in simple dipstick or cassette formats. DUSTSCREEN is a nitrocellulose-based strip test which detects multiple allergens in a single test with one dust sample. The nitrocellulose strip has six capture mAb “spots” for different allergens, which are detected by a cocktail of enzyme-labeled mAb. This test can be quantified with a strip reader and shows good correlation with ELISA (35). However, DUSTSCREEN takes several hours to perform, and it is best suited for pharmacy, clinic, or office use where allergen levels on dust samples can be obtained the same day. ACLOTEST, a dipstick test for mite, uses polyclonal rabbit antibodies conjugated to colloidal dye (Samaron Pink) for detection and takes 30–60 min to develop (36). Results are graded according to the intensity of the pink color that develops and correlated with ELISA. The most recent advance is the development of true rapid tests for mite allergen which use lateral flow technology and gold-labeled mAb for detection (Fig. 1). The lateral flow test has been developed for mite group 2 allergen and has the advantages that it detects both Dermatophagoides pteronyssinus and D. farinae, is quite sensitive (detection limit ∼100 pg), and gives results in 10 min (37). The rapid test uses technology similar to that of pregnancy tests and is designed for home use as part of a kit which includes a simple, disposable, plastic dust sampling and extraction device (the MITEST dust collector) (38). The MITEST collector samples per unit area (0.25 m2) and allows dust to be collected and extracted within 2 min. Prototype rapid tests have also been developed for mite group 1 allergen and Fel d 1. In principle, lateral flow technology can be applied to other allergens, such as those of cockroach, rodent urine, foods, and latex, thus providing simple tests of exposure for a variety of environmental allergens. Rapid lateral flow tests for allergen detection by gold-labeled mAb. Capture anti-Der p 2 mAb is “striped” onto the membrane, and gold-labeled secondary mAb is impregnated in a pad with an absorbent wick at one end of the membrane. When dust extract is applied to the wick, allergen diffuses into the pad and binds to the gold-labeled mAb. This complex flows further along the membrane until it meets the line of capture mAb, where the complex binds and forms a visible red line. The intensity of color development is proportional to the allergen concentration in the sample. Dust analyses provide a useful, but indirect measure of personal allergen exposure, and the shortcomings of dust analysis have prompted attempts to monitor airborne allergen levels. Airborne measurements of animal allergens can often be made by conventional ELISA under undisturbed conditions (11, 39-42). The sensitivity of such measurements has recently been increased by 5–10-fold by commercial ELISA amplification systems, which use either catalyzed reporter deposition or polymeric streptavidin to amplify the signal, and which have detection limits in the low pg range (12, 43-45). However, even with amplification systems, detection of mite and cockroach allergens in the air has proved difficult (2, 39, 46). Tovey and colleagues have developed a novel personal sampling device that enables inhaled particulate allergen to be directly visualized and measured. The HALOGEN system uses silicone intranasal samplers which can be worn by the patient for 4–8 h during normal domestic activities. Particles that are inhaled during respiration are deposited onto small plastic slides coated with allergen-specific mAb and can be immunostained to form an allergen “halo”, or directly assayed for allergen by amplified ELISA (reviewed in Ref. 47). The HALOGEN system has been successfully used to detect inhaled particles bearing Der p 1, Fel d 1, pollen allergens, and fungal spores (47-50). Allergen avoidance has been recognized as an integral part of the management of patients with asthma, together with antiasthma drugs and immunotherapy. This recognition is based on the extensive epidemiologic studies on sensitization and exposure to indoor allergens that have been carried out worldwide, in which immunoassays for measuring exposure have played a major role. Currently, several prospective studies are underway to investigate the effects of early childhood exposure on asthma prevalence, including controlled trials of avoidance in infancy to determine whether effective reductions of allergen exposure at home decrease the prevalence of the disease (20, 51-53). The outcomes of these studies could have an enormous impact on the significance of indoor allergens from a public health perspective. The AAAAI position statement on environmental allergens emphasizes the fact that patient adherence to allergen avoidance recommendations is often low, at 20–50% in clinical trials and probably lower in clinical practice (23). This underscores the need for better patient education and care to ensure that avoidance procedures are properly implemented. Monitoring allergen exposure can play a role in demonstrating whether patients are exposed to significant concentrations of indoor allergens and in encouraging compliance with avoidance procedures. Curiously, the NHLBI expert panel report suggested that patients’ exposure could be assumed on evidence of allergen sensitization (skin tests or in vitro IgE tests) and clinical history (22). However, patient history may not be a good guide to mite or cockroach sensitization, or to exposure to animal allergens if the patient does not keep furred pets. Even if sensitization can be demonstrated, patients may not be, or may not believe themselves to be, significantly exposed at home (54). Climatic and housing conditions vary widely in Europe and across the USA, making it unwise to make assumptions about allergen exposure. Studies in the USA have shown that allergen exposure in Boston (MA) varies according to housing type: centrally heated apartments which have low relative humidity (<45% relative humidity [RH]) for 6 months had extremely low levels of mite allergen (<0.5 µg/g dust), whereas single family homes with >50% RH year-round had 10–100-fold higher allergen levels (Fig. 2) (55). Other studies show that mite allergens can occur in apparently dry arid regions in homes that use evaporative cooling systems (56). Animal allergens are found in significant quantities in homes that do not contain pets, in schools, and in other public places, and cockroach allergens are found in about 20% of homes in the USA that have no visible evidence of cockroach infestation (40, 54, 57-59). Year-round comparison of temperature and relative humidity (RH) (A) and mite-allergen levels (B) in houses and apartments in Boston, MA. The apartments had a common heating system which maintains high temperature and low RH from November to June. In contrast, single-family homes had lower temperatures, RH >50% year-round, and ∼100-fold higher allergen levels. Reproduced courtesy of Dr Ginger Chew (Columbia University) and with the permission of the publishers (Allergy 1999;54:1058–1066). These observations suggest that assessments of allergen exposure should not be assumed. Over the past 2 years more than a dozen commercial reference laboratories in the USA and in Europe have begun to provide allergen-analysis services. Interestingly, in the USA, with the exception of contract research studies, these services are most often used by indoor air quality (IAQ) specialists and environmental companies, not allergists! Although making house visits to collect samples for allergen analysis is time-consuming, and not reimbursed by health authorities, a new study carried out by DeBlay et al. in Strasbourg (France) shows that use of an “indoor environmental technician” (IET) to visit homes resulted in greater compliance with avoidance measures and significantly greater reductions in allergen levels (60). This multicenter study involved around 380 rhinitis/asthma patients divided into two groups, those who received avoidance advice from their allergist alone, and those who received it from their allergist and from the IET during a home visit. The problem with allergen avoidance has always been that even if avoidance reduces allergen exposure by 80–90%, reservoir levels may remain higher than proposed threshold levels for sensitization, e.g., 2 µg/g mite allergen, in homes with very high allergen levels (>10 µg/g). This is a potential problem for patient-based tests, such as lateral flow tests, which may give positive reactions even when substantive allergen reduction is achieved. At present, such tests should be used as screening tests to demonstrate exposure, while ELISA is the preferred technique for following avoidance procedures. Allergists should consider indoor allergen exposure when making recommendations about immunotherapy and about allergen avoidance. Precedents from grass pollen and ragweed hay fever studies suggest that immunotherapy is most effective when given out of season, when allergen exposure in negligible. An earlier report suggested that monitoring allergen exposure is beneficial in evaluating mite immunotherapy among asthma patients (61). Demonstrating that the patient is sensitized and exposed to relevant allergen could help to reinforce advice about immunotherapy or about the selection of appropriate avoidance procedures. Several allergen control devices and procedures have been systematically tested for their effects on allergens. These include mattress encasings, vacuum cleaners, acaricides, protein denaturants, air cleaners, air ducts, and procedures that reduce ambient humidity (62, 63). Allergists can provide patients with clear information about the efficacy of these procedures and make appropriate practical recommendations. The greatest benefits are to improve patient knowledge of the relationship between allergen exposure and allergic symptoms, so that patients can make informed choices about controlling allergen exposure in the home (63). Patient-based tests and personal sampling systems should encourage compliance with avoidance procedures. The use of environmental technicians, allied health personnel, or IAQ specialists may help with implementation and encourage changes in lifestyles to reduce allergen exposure. The net result is that both allergists and patients benefit from objective information about allergen exposure. It has been estimated that a twofold reduction in allergen exposure could reduce the risk of developing asthma and asthma severity (64-66). While this effect may be optimistic for a relatively modest reduction in allergen exposure, it suggests that reducing allergen levels of key reservoirs (in bedrooms, living rooms, and basements) by more than 50% may reduce asthma morbidity. Over the next decade, we can look forward to further applications and advances in immunoassay technology that should provide better understanding of the role of allergen exposure in asthma and lead to the development of effective intervention strategies. We thank Wanda Harvey for secretarial support. This work was supported by National Institutes of Health grants AI 32557 and AI 34607. Dr Chapman is founder of the INDOOR biotechnologies companies (Charlottesville, VA, and Manchester, UK), which are involved in the production of allergen detection systems and provide reference laboratory services.

by an ELISA that uses a chimeric mouse/human antibody to Der p 2 to construct the IgE binding standard curve.A good correlation was found between binding of IgE antibodies from IO sera of Bla g I allergic patients to rBla g I and IgE binding to the natural allergen (r = 0.8; p < 0.01).In summary.a eucaryotic expression system was used to express rBla p 1 that behaves like natural Bla g I.This recombinant allergen will be used to study diagnosis of cockroach allergy by skin testing cockroach sensitized individuals, and to perform crystallographic studies of the three dimentional structure of Bla g I.