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

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.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

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.

Cockroach allergens: function, structure and allergenicity.

Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

Evaluation of German cockroach (Orthoptera:Blattellidae) allergen and seasonal variation in low-income housing.

Six apartments in a low-income housing project were evaluated for German cockroach. Blattella germanica (L.), infestation and concentration of an allergen derived from these cockroaches (Bla g II). Kitchen and living room samples were collected monthly for 1 yr. In addition, airborne sampling was carried out in 5 kitchens. The kitchen had the highest allergen concentration in 65% of visits and the highest number of cockroaches trapped in 69% of visits. In the kitchen, the highest cockroach levels were seen in June, whereas the values for Bla g II peaked in August. In keeping with this, the closest correlation was between Bla g II (microgram/g dust) and the number of cockroaches found 2 mo earlier. Airborne samples were assayed for 2 separate allergens. Bla g II and Bla g I. No allergen was detectable in the absence of disturbance. By contrast, during disturbance with a vacuum cleaner both Bla g II and Bla g I were detectable in the air of each apartment. Results suggest that immunochemical assay of a major allergen in dust samples from the kitchen floor may be used to monitor exposure to German cockroaches, also that cockroach levels may be used as an indicator or predictor of allergen in dust.

Influence of footing surface on mounting and other sexual behaviors of estrual Holstein cows.

Seven ovariectomized Holstein cows, treated sequentially with progesterone and estradiol benzoate to induce estrus, were used to determine whether cows showed a preference for mounting and displaying other sexual behaviors toward estrual or nonestrual cows tied on dirt or concrete surfaces. Preference tests were conducted in a test area that consisted of equal-sized concrete and dirt surfaces; two cows, designated A and B, were tied on the two surfaces, one on either side. Cows A and B were treated so that on each of four test days both were estrual, only A was estrual, only B was estrual or neither A nor B were estrual. On each test day, five estrual test cows were introduced individually into the test area for two 30-min test periods. During the first test period, tied cow A was on concrete and tied cow B was on dirt, but during the second test period, their positions were switched. Test cows were able to move freely from surface to surface and to interact with tied cow A or B. Estrual test cows spent 21.6 +/- 1.4 min of each 30-min test period on dirt, regardless of the estrual status of the cow tied on concrete or dirt. Mounting activity was 3- to 15-fold greater on dirt than on concrete when there was an estrual cow tied on dirt, regardless of the estrual status of the cow on concrete. Mounting activity was fourfold greater on concrete than on dirt when there was a tied estrual cow on concrete and a tied nonestrual cow on dirt.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of various steroid milieus or physiological states on sexual behavior of Holstein cows.

Two experiments were conducted to determine how steroid milieus and pregnancy affect sexual behavior. Experiment 1 was arranged as a Latin square with five ovariectomized cows and five steroid milieus: no steroid (N); progesterone (P4); estradiol benzoate (EB); P4 + EB; and P4 followed by EB (P4:EB). Progesterone was administered via pessary (2 g of P4) for 5 d and EB was injected (1 mg i.m.) on the day before a test day. On a test day, cows were exposed for four 30-min periods, twice each with a tied or a loose estrual test cow (prepared using P4:EB). Sexual behaviors recorded were attempted mounts, successful mounts, front mounts, stands, head butts, chin rests, and vulvar sniffs. Cows exhibited more (P less than .05) sexual behavior during periods with the loose estrual test cow than with the tied estrual test cow. Cows receiving P4 alone ranked lowest among treatments for each behavior, whereas cows receiving EB or P4:EB ranked highest or second-highest. Progesterone prevented stands in cows given P4 + EB, but these cows displayed mounting behaviors similar to those of cows given EB and P4:EB. Cows given P4 + EB were similar to those given N for most behaviors. In Exp. 2, 118 intact, lactating cows were observed in groups of four or five for mounting of estrual test cows during 24, 30-min observation periods on 8 d over 2 yr. The design was an incomplete block with physiological state, parity, estradiol, progesterone, and a calculated estrogen:progesterone ratio included in the model. Each block included one or two cows at 23 +/- .8 d after insemination, divided retrospectively into one pregnant and two non-pregnant groups (low [less than 1 ng/mL] vs high progesterone), and other cows at 89 +/- 1.0, 152 +/- 1.2, and 234 +/- 1.7 d of gestation (six physiological groups). Most cows were observed once, but 27 cows were included twice during 2 yr. Only 60% of the 118 cows made attempted or successful mounts even though estrual test cows were always receptive. Physiological state was not associated with amount of mounting because very active (greater than or equal to five attempts) and inactive cows were represented in all physiological groups. The estrogen:progesterone ratio on test day accounted for small, but significant, variation in mounting behavior. For cows observed on two different days, correlations between successive observations were .46 for attempted mounts, .78 for successful mounts, and .71 for total mounts.(ABSTRACT TRUNCATED AT 400 WORDS)

Tissue localization and regulation by juvenile hormone of human allergen Bla g 4 from the German cockroach, Blattella germanica (L.).

The German cockroach, Blattella germanica (L.), produces several potent protein aeroallergens, including Bla g 4, a approximately 20 kDa lipocalin. RT-PCR, Northern analyses and in situ hybridization showed that Bla g 4 is expressed only in the adult male reproductive system. Western blotting and ELISA with rBla g 4 antiserum detected immunoreactivity in the utricles and the conglobate gland, but not in other tissues of the male reproductive system. The Bla g 4 protein content of males increased from adult emergence to day 14, but during copulation Bla g 4 was depleted in the male and transferred to the female within the spermatophore. Topical application of juvenile hormone III stimulated Bla g 4 production by both conglobate gland and utricles.

Recombinant mite allergens. New technologies for the management of patients with asthma.

The introduction of molecular cloning techniques has led to advances in allergen identification and sequencing, production of recombinant allergens, identification of B-cell and T-cell epitopes, and tertiary structural analysis of allergen molecules. Over 10 groups of mite allergens have been cloned from Dermatophagoides spp., as well as several homologous allergens from Euroglyphus maynei, Lepidoglyphus destructor, and Blomia tropicalis. The availability of these allergens has made it feasible to consider their use for both diagnostic and therapeutic purposes. Several recombinant Dermatophagoides allergens show comparable reactivity on skin testing and in serologic assays to natural allergens, and cocktails of the recombinant proteins could be used as diagnostic reagents. New technologies have been developed for detection of allergen-specific IgE and for environmental allergen detection using rapid diagnostic tests. Novel approaches to immunotherapy are also being investigated, including T-cell-peptide based vaccines, allergen variants which lack IgE reactivity, and naked DNA vaccines. The application of allergen biotechnology should lead to improvements in the management of mite-allergic patients with asthma and represents a logical step toward reducing asthma mortality and morbidity.

Defined epitopes: in vivo and in vitro studies using recombinant allergens.

BACKGROUND: The aim of the studies was to investigate the biologic activity of recombinant cockroach and mite allergens and their potential for use in diagnosis and treatment of allergic disease. METHODS: Cockroach allergens Bla g 2, Bla g 4 and Bla g 5 and mite group 5 allergens were produced in bacterial expression vectors and used for immediate skin and serum IgE antibody tests. RESULTS: The cockroach allergens showed very good skin test reactivity in allergic patients, giving positive reactions at 10(-2)-10(-5) microg/ml; controls were negative at 10(0) microg/ml. These reactions correlated with serum IgE antibody results. The prevalence of reactivity to group 5 mite allergens varied with exposure. There was a high prevalence (70%) of sensitization to Blomia tropicalis allergen, Blo t 5, among patients from Brazil and Singapore, whereas < 20% of patients from Charlottesville, US and Manchester, UK gave positive skin tests to Blo t 5 (p < 0.001). CONCLUSIONS: The results show that recombinant allergens retain biologic activity and suggest that cocktails of two to four recombinant allergens could be used for diagnostic or therapeutic purposes. The phased introduction of recombinant allergens should improve the management of allergic disease.

High-level expression of cockroach allergen, Bla g 4, in Pichia pastoris.

Exposure to cockroach allergens is a risk factor for allergic disease and has been linked to an increase in asthma morbidity among cockroach-sensitive inner-city children. Bla g 4 is a ligand-binding protein (or calycin) that causes IgE antibody responses in 40% to 60% of patients allergic to cockroaches. Recombinant Bla g 4 was expressed in Escherichia coli as an 18 kd protein but provided poor yields (only 0.25 mg/L culture). To improve yields, Bla g 4 was expressed in the Pichia pastoris yeast system as a 23 kd secreted protein at concentrations of 50 mg allergen/L. By cross-inhibition radioimmunoassay, Bla g 4 expressed in E. coli or P. pastoris provided overlapping inhibition curves. Both allergen preparations bound comparable levels of serum IgE antibody and showed similar skin test reactivity in individuals allergic to cockroaches (10[-1] to 10[-3] microg/ml). Deglycosylation of Pichia-expressed Bla g 4 with endoglycosidase F resulted in an 18 to 20 kd doublet, and liquid chromatography-mass spectrometry results suggested that the 20 kd band contained residual sugar residues. Both glycosylated and deglycosylated Pichia Bla g 4 showed comparable inhibition of IgE antibody binding in radioimmunoassay. Pichia-produced Bla g 4 had the same antigenic reactivity as that produced in E. coli, and glycosylation had no effect on IgE antibody binding. The high yield of Bla g 4 obtained in the Pichia system will facilitate studies on the structure and function of calycin allergens and on the immune response of asthma patients to cockroach allergens.

Cockroach allergens: environmental distribution and relationship to disease.

Cockroach allergy has been recognized as an important cause of asthma. Exposure to high levels of cockroach allergens in the home is a major risk factor for symptoms in sensitized individuals. Previously identified allergens from Blatella germanica and Periplaneta americana include Bla g 2 (inactive aspartic proteinase), Bla g 4 (calycin), Bla g 5 (glutathione-S-transferase), Bla g 6 (troponin), the Group 1 cross-reactive allergens Bla g 1 and Per a 1, Per a 3 (arylphorin), and Per a 7 (tropomyosin). The primary site of cockroach allergen accumulation is the kitchen. However, lower levels of allergen can be found in bedding, on the bedroom floor, and in sofa dust. Strategies for decreasing exposure to cockroach have been investigated. The results suggest that a sustained decrease in cockroach allergen levels is difficult to accomplish, even after successful extermination of cockroach populations. The use of recombinant cockroach allergens may lead to the development of new approaches to asthma treatment in the future.

Molecular cloning of Per a 1 and definition of the cross-reactive Group 1 cockroach allergens.

BACKGROUND: Sensitization to allergens produced by German and American cockroaches is strongly associated with the cause of asthma. Most of the cockroach allergens identified to date have been species specific. OBJECTIVE: The aim of this study was to identify and sequence cross-reactive cockroach allergens. METHODS: A Periplaneta americana cDNA library was screened with IgE antibody from patients in the United States who were allergic to cockroach and who were sensitized to Blattella germanica. RESULTS: A cDNA clone was isolated that contained an 870-bp sequence with a 695-bp open reading frame, encoding a 231 amino acid protein, molecular weight 26.2 kd. Plaque immunoassays using anti-Bla g 1 and anti-Per a 1 mAbs and a panel of human IgE antibodies showed that the protein expressed by these clones was Per a 1. Sequence homology searches showed that Per a 1 was homologous to 5 previously reported, but unidentified, sequences from B germanica and P americana. These sequences encoded proteins with multiple molecular sizes containing approximately 100 amino acid repeats. The Per a 1 sequence also showed 31% identity to a mosquito precursor protein, ANG12, which may be involved in digestion. The Per a 1 cDNA was expressed in Pichia pastoris to produce purified recombinant allergen (yield, 14 mg/L). CONCLUSION: The results define the molecular structure and antigenic relationships between a new family of cross-reactive "Group 1" allergens produced by both P americana and B germanica. These recombinant allergens and specific mAbs will provide tools to improve the diagnosis and treatment of allergic diseases caused by cockroaches.

Molecular cloning of German cockroach (Blattella germanica) allergens.

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.

Identification of a novel cat allergen--cystatin.

BACKGROUND: Cat allergen is an important cause of sensitization among children with asthma in Japan. Although there is good evidence that cats produce other allergens, only one major allergen, Fel d 1, has been studied in detail. AIMS: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using IgE antibodies to cat dander and selected clones were sequenced and expressed. RESULTS: One cDNA clone contained an open reading frame encoding a 98-amino acid residue protein. Sequence homology searches revealed a high degree of identity with bovine and human cystatin A, 79 and 75%, respectively. This cat cystatin clone contained the conserved cysteine protease motif and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE Ab to cat cystatin. This cysteine protease inhibitor motif was partially conserved in dog allergens, Can f 1 and Can f 2, which are lipocalins. Recombinant cystatin was produced in Escherichia coli cells and purified as an 11-kD protein, corresponding to the predicted MW of cystatin. The structure of cat cystatin was modeled on human cystatin B using the SWISS-MODEL. CONCLUSION: A newly identified allergen, cystatin, has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

Molecular cloning, expression and modelling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor.

BACKGROUND: Cats are an important source of indoor allergens. However, only two cat allergens, Fel d 1 and albumin, have been cloned and sequenced. IgE antibodies to Fel d 1 and albumin do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens. OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding cystatin, was expressed in E. coli. The three dimensional structure of cat cystatin was modelled using the SWISS-MODEL computer program. RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human cystatin A, respectively. The cat cystatin sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed cystatin clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, Can f 1 and Can f 2, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat cystatin. The three-dimensional structure of cat cystatin was modelled on human cystatin structures. CONCLUSION: A newly identified allergen, cystatin (Fel d 3), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

Monoclonal antibodies to group II Dermatophagoides spp. allergens: murine immune response, epitope analysis, and development of a two-site ELISA.

BACKGROUND: Group II allergens are a major cause of sensitization in patients allergic to mites. To facilitate the antigenic analysis of group II allergens and to develop improved methods of allergen detection, we compared IgG anti-group II antibody responses in inbred mouse strains and raised a panel of monoclonal antibodies (mAbs). METHODS: IgE antibody responses were compared by antigen-binding radioimmunoassay. Epitope specificity of the mAbs was analyzed by two-site binding assays and by cross-inhibition radioimmunoassays. RESULTS: Comparison of polyclonal IgG antibody responses in five BALB congenic strains showed that H-2d mice had poor responses, whereas H-2b and H-2k mice had strong, cross-reactive, IgG anti-group II responses. The specificities of nine anti-Der p II IgE mAbs raised in A/J mice were compared with specificities of seven mAbs produced previously. Most mAbs (11 of 16) recognized common epitopes on Der p II and Der f II: three were specific to Der p II, and two showed high binding to Der f II. Epitope analysis showed that the mAbs defined four cross-reactive, nonoverlapping sites on the group II allergens. Binding of several combinations of mAbs was compared, and a two-site ELISA for group II antigens was developed. Linear regression analysis showed an excellent correlation between results of this assay and group II radioimmunoassay of house dust samples (n = 40, r = 0.85, p < 0.001). CONCLUSIONS: There are multiple cross-reactive B-cell epitopes on group II allergens. The group II ELISA has several important applications, including assessment of environmental allergen exposure, monitoring of the efficacy of avoidance procedures, and standardization of commercial mite allergen extracts.

Quantitation of the major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products.

BACKGROUND: Alternaria is one of the most important fungi associated with allergic disease, whereas Aspergillus fumigatus is involved in a broad spectrum of pulmonary diseases. Currently, fungal extracts used for diagnosis in the United States are unstandardized, and their allergenic content cannot be compared directly. OBJECTIVE: The goal of this study was to compare the variability of major allergen levels among US allergenic products derived from fungi: specifically, Alt a 1 levels in Alternaria alternata extracts, and Asp f 1 levels in A fumigatus extracts. METHODS: A novel 2-site monoclonal antibody ELISA was used for measuring Alt a 1 using recombinant Alt a 1 as a standard. Asp f 1 was also measured by ELISA. Allergenic products produced by 8 US manufacturers over a 2-year period were compared, as were multiple lots produced by a single company. RESULTS: Alt a 1 levels in Alternaria extracts from 8 companies produced in 1998 and 1999 ranged from less than 0.01 to 6.09 microg/mL (mean 1.4 +/- 1.6 microg/mL, n = 15). In general, Alt a 1 levels were consistent within and between companies (1.4 +/- 1.1 microg/mL, n = 27), with 21 of 32 (66%) of all extracts tested containing 0.7 to 2 microg/mL Alt a 1. Aspergillus extracts showed much greater variability in Asp f 1 levels, with extracts from 8 companies containing from less than 0.1 to 64 microg/mL Asp f 1 (mean 16.3 +/- 23.9 microg/mL, n = 15). Overall variability was greater for Aspergillus products within and between manufacturers (22 +/- 22 microg/mL Asp f 1, n = 20). CONCLUSIONS: ELISA-based assays for specific allergens showed greater consistency among allergenic products derived from Alternaria than from Aspergillus. These assays should facilitate improved quality control and standardization of fungal allergen extracts and lead to the development of more consistent products for clinical use.