IgE-positive plasma cells are present in adenoids of atopic children.

CONCLUSIONS: We demonstrated the presence of IgE(+) plasma cells in the adenoids of atopic children. Our data suggest that adenoids are capable of local production of IgE and support the role of adenoids as an inductive site for allergic reactions. OBJECTIVE: We have previously demonstrated increased numbers of IgE(+) cells in the adenoids of atopic children and also found support for an IL-4-induced class switch to IgE production in adenoids. In search of further evidence of adenoids being involved in IgE-mediated sensitization, we investigated the distribution of plasma cells and macrophages positive for IgE in adenoids. MATERIAL AND METHODS: Adenoid tissue from atopic and non-atopic children was examined using immunohistochemical markers for IgE, plasma cells (CD138) and macrophages (CD68). The distribution of positive cells was determined in the extrafollicular area and in the follicles of adenoids. Co-localization of IgE and CD138(+) plasma cells and CD68(+) macrophages was examined using immunohistochemical double-staining methods. RESULTS: Non-atopic adenoids contained no or very few IgE(+) cells. In contrast, IgE(+) cells were found in high numbers in atopic adenoids, mainly in the extrafollicular area. Higher numbers of IgE(+) plasma cells and IgE(+) macrophages were also found in the adenoids of atopic children.

Haplotypes of G protein-coupled receptor 154 are associated with childhood allergy and asthma.

RATIONALE: Allergic diseases are influenced by both genes and environment. A 70-kb haplotype block in the G protein-coupled receptor for asthma susceptibility gene (GPR154; alias GPRA) on chromosome 7p was recently identified to influence susceptibility to asthma and elevated total serum IgE levels in adults. OBJECTIVES: To assess the impact of GPR154 on childhood allergic disease, including allergic sensitization, asthma, and rhinoconjunctivitis, in study populations with diverse environmental backgrounds. METHODS: We studied farm children, Steiner school children, and two reference groups from five Western European countries in the cross-sectional PARSIFAL (Prevention of Allergy Risk factors for Sensitization In children related to Farming and Anthroposophic Lifestyle) study and a sample of children from the Swedish birth cohort study BAMSE. DNA samples from 3,113 PARSIFAL and 800 BAMSE children were genotyped for 7 GPR154 polymorphisms and haplotypes were inferred. The proportions of alleles and haplotypes (H1-H7) were compared in affected children with their healthy counterparts. RESULTS: Data indicate a global association of the haplotype block to sensitization (allergen-specific serum IgE > or = 0.35 kU/L, p = 0.022), with significant haplotype-specific associations for H1, H5, and H6. Haplotypes H1 and H5 were also significantly associated with childhood allergic asthma (p = 0.045 and p = 0.023, respectively), and H5 to asthma regardless of sensitization. A broader involvement of GPR154 in allergic diseases was further supported in allergic rhinoconjunctivitis (H3: p = 0.046). The associated haplotypes could be allocated into risk (H5/H6) and nonrisk (H1/H3) groups, a pattern supported by allelic association of single nucleotide polymorphisms (SNPs) rs324384 and rs324396. CONCLUSIONS: Our results indicate that polymorphisms and haplotypes in the haplotype block of GPR154 are associated with asthma, rhinoconjunctivitis, and sensitization in European children.

Crystallization and preliminary crystallographic data of a Fel d 1 (1+2) construct corresponding to the major allergen from cat.

The domestic cat (Felis domesticus) is one of the most important causes of allergic disease worldwide. A homologue of the major allergen Fel d 1 was created by linking the two chains that compose the protein. Fel d 1 (1+2) was expressed in Escherichia coli and subsequently purified using three chromatographic steps. Crystals of Fel d 1 (1+2) were obtained using the hanging-drop vapour-diffusion method in 22.5% PEG 3350, 0.5 M CaCl2. The crystals belong to space group P1, with unit-cell parameters a = 38.5, b = 42.9, c = 49.0 A, alpha = 70.7, beta = 80.5, gamma = 81.5 degrees , and diffract to 1.6 A resolution.

A novel adjuvant-allergen complex, CBP-rFel d 1, induces up-regulation of CD86 expression and enhances cytokine release by human dendritic cells in vitro.

Allergen-specific immunotherapy is commonly performed with allergen extracts adsorbed to aluminium hydroxide (alum). The undesirable effects associated with the use of alum, including granuloma formation at the site of injection and stimulation of T helper 2 (Th2) cytokine production, has generated interest in alternative allergen carriers, one being carbohydrate-based particles (CBPs). Here, we have investigated the in vitro effects of the recombinant major cat allergen Fel d 1 (rFel d 1) coupled to CBPs (CBP-rFel d 1) on human monocyte-derived dendritic cells (MDDCs) obtained from healthy blood donors. A majority of the CD1a(+) MDDCs internalized fluorescein isothiocyanate-labelled CBP-rFel d 1, as demonstrated by flow cytometry and confocal laser-scanning microscopy. Furthermore, an up-regulation of the expression of the costimulatory molecule, CD86, on the MDDCs was induced by CBP-rFel d 1, but not by rFel d 1 or CBPs alone. Finally, three- and fourfold increases in the release of interleukin-8 and tumour necrosis factor-alpha, respectively, were observed when MDDCs were cultured in the presence of CBP-rFel d 1. Altogether, our results indicate that the use of CBPs as an allergen carrier and adjuvant is a promising candidate for the improvement of allergen-specific immunotherapy.

Qualitative and quantitative evaluation of bird-specific IgG antibodies.

BACKGROUND: Exposure to organic dust produced by birds often gives rise to an immune response, e.g. IgG antibodies, but intense exposure can lead to high concentrations of IgG antibodies and the development of allergic alveolitis, often known as "bird fancier's lung". The aim of this study was to establish the distribution of bird-specific IgG antibodies in exposed and nonexposed individuals and compare a nonquantitative and quantitative method in evaluating raised levels of IgG antibodies. METHODS: Sera were collected in Sweden and South Africa and levels of IgG antibodies specific to pigeon, budgerigar and parrot antigens were quantified using the UniCAP system. Results were compared to the precipitation in gel assay. The IgG antibody values of symptomatic patients without precipitating antibodies (non-PP group; n = 51) and patients with precipitating antibodies (PP group; n = 34) were analyzed and compared to nonexposed asymptomatic blood donors (BD group; n = 73) and environmentally exposed pigeon breeders (n = 11). RESULTS: The IgG antibody response of the analyzed groups in Sweden and South Africa did not vary significantly from each other. IgG antibody responses were the strongest to pigeon antigens with clear increased IgG antibody levels in the PP group [geometric mean (GM) 603 mg/l] compared to the non-PP (GM 6.9 mg/l) and BD group (GM 5.0 mg/l). Threshold values, calculated as the GM value from the BD group plus 3 standard deviations (99% confidence interval), were 9.8, 10.8 and 10.0 mg/l for pigeons, budgerigars and parrots, respectively. Comparison of the two methods resulted in a good concordance with a level of agreement of 94.1% (kappa statistic = 0.83). CONCLUSIONS: The UniCAP system for the detection of bird-specific IgG antibodies is a highly reproducible, generally available, quantitative method for routine diagnostic testing and monitoring of exposed subjects with a very high level of agreement to the precipitating gel assay.

Quantification of IgE antibodies simplifies the classification of allergic diseases in 4-year-old children. A report from the prospective birth cohort study--BAMSE.

Allergic diseases are common among small children, but it is still unclear how immunoglobulin E (IgE) antibodies to ambient allergens are distributed in a population-based prospective material of children at 4 years of age. The study is based on 75% (n = 4089) of all eligible children from northern Stockholm, born between 1994 and 1996 in pre-defined geographical areas. Data on exposure and outcome were obtained by parental questionnaires when the child was 3 months and 4 years of age. Of the 92% who responded to the 4 years of age questionnaire, serum was obtained in 88% of these children for analysis of IgE antibodies performed with Pharmacia CAP system (Phadiatop and food mix fx5). An antibody level > or =0.35 kUA/l was considered as positive. A positive Phadiatop or fx5 was found in 24% of the 4 years old children. A rather poor correlation was found between the two tests (r = 0.39). Occurrence of IgE antibodies > or =3.5 kU/l for both Phadiatop and fx5 in combination could predict any suspected allergic disease [asthma, rhinitis, atopic eczema dermatitis syndrome (AEDS) and allergic reaction to food] to 97.4%. However, the presence of > or =3.5 kUA/l of Phadiatop or fx5 used as single tests only, was far less efficient to predict any allergic disease. The two mixes of airborne and food allergens were also associated, not only to the severity of the allergic disease in terms of number of organ involved, but also to the severity of recurrent wheeze, in particular in boys with a positive Phadiatop who exhibited significantly limited peak flows compared to those with a negative test. Already at the age of 4, one child in four is sensitized to an allergen as assessed by Phadiatop or food mix (fx5). The presence of IgE antibodies seems not only to predict allergic diseases in this age group, but also relates to severity of such diseases, in particular to asthma. Notable, there was a poor correlation between Phadiatop and fx5 that needs to be considered when identifying allergic diseases in young children. The study demonstrates that quantification of IgE antibodies in blood may be beneficial, not only to diagnose allergic diseases in young children, but especially to serve as a marker of severity of asthma.

Death in anaphylaxis in a man with house dust mite allergy.

Up to recently the post-mortem diagnosis of anaphylaxis has been based solely on circumstantial evidence. With the development of assays for mast cell tryptase it is now possible to verify cases of suspected anaphylaxis. Here we present one such case, which initially appeared to be due to sudden death of unknown cause. A 47-year-old farmer was found dead in his bathroom around midnight. Hospital records revealed that he had previously been diagnosed with an allergy to house dust mites. He had also had infrequent episodes of airway symptoms, nausea, hypotension and diarrhoea usually after going to bed. The forensic autopsy did not give any clue to the cause of death. Serum tryptase in post-mortem blood was found to be substantially elevated in two samples (170 and >200 microg/L). Analysis of allergen-specific IgE showed high values for Dermatophagoides pteronyssinus and farinae. High mite allergen levels were found in dust obtained from the patient's mattress. The results of the immunological tests support the assumption that he died of anaphylactic shock. The circumstances and the patient's history of previous attacks after going to bed point to the fact that exposure to mite contaminated food and/or exposure to mite allergens in bed might have caused his death.

The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family.

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.

Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1.

The domestic cat (Felis domesticus) is an important cause of allergic disease worldwide. The major cat allergen 1 (Fel d 1) has been expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Fel d 1 were obtained in 13% 2-methyl-2,4-pentanediol, 0.1 M sodium acetate pH 4.8. The Fel d 1 crystals belong to space group P2(1), with unit-cell parameters a = 43.3, b = 51.5, c = 67.7 A, and diffract to 1.9 A resolution.

Formation of disulfide bonds and homodimers of the major cat allergen Fel d 1 equivalent to the natural allergen by expression in Escherichia coli.

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

Cloning and characterisation of two IgE-binding proteins, homologous to tropomyosin and alpha-tubulin, from the mite Lepidoglyphus destructor.

BACKGROUND: The dust mite Lepidoglyphus destructor is a major source of mite allergy in European rural environments, but it also causes allergy in urban populations around the world. We have previously cloned, sequenced and expressed several allergens from L. destructor (Lep d 2, Lep d 5, Lep d 7 and Lep d 13). The aim of this study was to identify and clone additional allergens from L. destructor, and to evaluate their IgE-binding reactivities. METHODS: PCR and screening with sera from L. destructor-sensitised individuals were used to isolate new clones from a phage display L. destructor cDNA library. The complete coding sequences of the clones were determined and expressed as His(6)-tagged recombinant proteins in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE, immunoblotting and mass spectrometry. RESULTS: Two new clones, showing homology to tropomyosin and alpha-tubulin in several species, were isolated from the phage display L. destructor cDNA library. Due to its homology to group 10 dust mite allergens, the tropomyosin clone was named Lep d 10. The IgE-binding frequencies of the recombinant Lep d 10 and alpha-tubulin were 13% (18/136) and 12% (11/95), respectively, among subjects with IgE reactivity to mites and/or crustaceans. CONCLUSIONS: Two new allergens from L. destructor have been identified and can now be added to the repertoire of recombinant L. destructor allergens. In addition, both these allergens belong to highly conserved protein families and may be important for evaluation of allergenic cross-reactivity.

Heredity, pet ownership, and confounding control in a population-based birth cohort.

BACKGROUND: The association between pet ownership in childhood and subsequent allergic disease is controversial. Bias related to selection of pet exposure has been suggested as a reason for contradictory study results. OBJECTIVE: The purpose of this investigation was to elucidate how pet exposure depends on family history of allergic disease, smoking, and socioeconomic factors in a prospective birth cohort. METHODS: Parents of 4089 two-month-old children answered a questionnaire that included detailed questions about family history of asthma (maternal, paternal, and sibling), rhinoconjunctivitis, atopic eczema/dermatitis syndrome, pollen and pet allergy, smoking habits, parental occupation, and family pet ownership (cat and dog). Dust samples collected from the mothers' beds were analyzed for Fel d 1 and Can f 1 in a subgroup of the cohort. RESULTS: Cats were less frequently kept in families with parental asthma, rhinoconjunctivitis, or pet or pollen allergy (3.5% to 5.8%) than in families without parental allergic disease (10.8% to 11.8%). Dogs were less common in families with (3.3%) than in families without (5.9%) parental atopic eczema/dermatitis syndrome. Families with smoking mothers and those with low socioeconomic index kept cats and dogs more frequently. Cat allergen levels were lower in homes with than in homes without maternal pet allergy, and this tended to hold true even for homes without a cat. Cat ownership decreased from birth to 2 years of age, especially in families with parental history of allergic diseases. CONCLUSION: There seems to be a selection of pet exposure based on parental history of allergy, maternal smoking, and socioeconomic factors. This has to be taken into consideration in evaluations of risk associations between pet exposure and allergic disease in childhood.

Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mites.

We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long-term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon-gamma and interleukin-5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release.

Mast cell tryptase and hemolysis after trauma.

BACKGROUND: We have previously found increased mast cell tryptase in accidental deaths due to trauma, indicating that mast cell degranulation had occurred. The present study was designed to confirm the previous observation and to determine if tryptase release after trauma is acute or delayed. Furthermore, the importance of hemolysis and direct trauma to the mast cells was investigated. MATERIALS AND METHODS: Mast cell tryptase was measured in post-mortem blood from the femoral vein in 27 cases of death from trauma and in 27 control cases by means of a commercially available immunoassay. The trauma cases were further classified into groups with single versus multiple trauma, and groups with short survival time (i.e. death at the scene of the accident) versus longer survival time (death in hospital). In five multi-trauma deaths, blood was sampled locally from the sites of crush injury. RESULTS: The mean value of tryptase in femoral vein blood was 35.6+/-34.6 microg/l in the entire trauma group and 14.7+/-6.5 microg/l in the controls (P<0.005). In bloody liquid sampled from crush injuries, tryptase was substantially elevated in all cases, with a mean of 227+/-146 microg/l. In cases with short survival time, tryptase was significantly higher than in those who died after several hours or days in hospital (P<0.001). No statistically significant difference was seen between multi- and single-trauma cases. A correlation between hemolysis in the samples and elevated tryptase was found only in the trauma cases (P<0.05), but experimentally induced hemolysis in vitro was not found to influence the measurements. CONCLUSION: Mast cell tryptase becomes elevated in trauma deaths and this seems to be ascribable either to direct mechanical injury to tissue mast cells and/or to cell lysis. In patients initially surviving severe injuries, the effects of massive release of histamine and other mast cell mediators might be of importance for treatment strategies and prognosis.

Increased levels of IL-2 and IL-4 in stimulated adenoidal lymphocytes of atopic children.

BACKGROUND: The adenoid is involved in the defence against airway pathogens and its surface is also exposed to airborne allergens. The knowledge about reactions taking place in the lymphatic tissue of this organ is, however, limited. To elucidate the influence of atopy we investigated the cellular and cytokine profile of in vitro-stimulated adenoid lymphocytes. METHODS: Adenoid tissue cells from 13 atopic and 8 non-atopic children were cultured and stimulated with ionomycin and 4beta-phorbol 12-myristate 13-acetate. Supernatants were collected after 4 and 20 h of stimulation and interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were analysed by ELISA. Flow cytometry was used to quantify the leukocyte markers CD3, CD19, CD25 and HLA-DR. RESULTS: Increased levels of IL-2 and IL-4 but not IFN-gamma were detected in the supernatants of adenoid cell cultures from atopic children after 20 h of stimulation (p < 0.05) and a significant correlation with a positive regression between IL-2 and IL-4 was found. Atopy was also associated with a greater increase in the percentage of CD19-positive B cells after stimulation (p < 0.05). CONCLUSIONS: A difference in the reactivity of adenoidal lymphoid cells in children was observed between atopic and non-atopic subjects. Atopy was associated with an increased production of IL-2 and IL-4 as well as a more pronounced increase of B cells.

Decreased frequency of intracellular IFN-gamma producing T cells in whole blood preparations from patients with atopic dermatitis.

There have been contradictory reports on the shift in the T-cell cytokine expression pattern of peripheral blood mononuclear cells from patients with atopic dermatitis (AD); more specifically the interleukin (IL)-4 and interferon (IFN)-gamma profiles. The aim of this study was to shed further light on this contradiction by measuring the intracellular cytokines IL-4 and IFN-gamma by flow cytometry on unseparated whole blood to obtain results that, as accurately as possible, reflect the situation in circulating cells in vivo. The patient group including 64 patients with AD was compared with 18 nonatopic healthy adults. The results showed that the percentage of CD4+ T cells expressing IFN-gamma was significantly decreased (P < or = 0.001), as well as the percentage expressing IL-4 (P < 0.05) in AD patients compared with healthy controls. Furthermore, in supernatants from whole blood samples stimulated with phorbol 12-myristate 13-acetate and ionomycin, production of IFN-gamma was significantly decreased, while IL-4 production remained unchanged in AD patients compared with healthy controls. We also investigated if there was a relationship between serum IgE level and Phadiatop, a screening test for atopy, vs. the levels of IL-4 and IFN-gamma, but found no correlation with either. However, there was a significant correlation between disease severity and the level of total IgE (r = 0.67, P < 0.05). In conclusion, our results support the evidence for a decreased ability of peripheral CD4+ T cells to produce IFN-gamma among AD patients.

Carbohydrate-based particles: a new adjuvant for allergen-specific immunotherapy.

The occurrence of systemic anaphylactic side-effects in the course of allergen-specific immunotherapy has been strongly reduced by the adsorption of allergens to aluminium hydroxide, the most frequently used adjuvant in humans. Using the major timothy grass pollen allergen, Phl p 5b, in its recombinant form for immunization of mice, we demonstrate that carbohydrate-based particles (CBP) exhibit several potential advantages over aluminium-hydroxide as adjuvant for immunotherapy. Similar to alum-bound rPhl p 5b, CBP-bound rPhl p 5b induced a stronger antibody and cytokine response than unbound rPhl p 5b after subcutaneous injection in mice. The antibodies induced by CBP-bound rPhl p 5b, exhibited potentially beneficial activities as they cross-reacted with group 5 allergens from five other grass species and inhibited the binding of grass pollen allergic patients IgE to Phl p 5b. Alum-bound rPhl p 5b induced a preferential allergen-specific Th2-response characterized by high immunoglobulin G1 (IgG1) antibody levels and elevated interleukin (IL)-4 and IL-5 production in cultured splenocytes. By contrast, CBP-bound rPhl p 5b, but not rPhl p 5b alone or coadministered with CBP, induced a mixed allergen-specific T helper 1 (Th1)/Th2 immune response characterized by the additional production of allergen-specific IgG2a/b antibody responses and elevated interferon-gamma production. Conjugation of rPhl p 5b to CBP yielded a stable vaccine formulation with preserved immunogenic features of the allergen and, in contrast to alum, induced no granulomatous tissue reactions. Based on these results, CBP is suggested as a potentially useful adjuvant for specific immunotherapy of IgE-mediated allergies.

Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.