Allergen-specific IgE Antibodies In Serum Research Articles

RAST, Inmunoblot, Immunocap and ISAC in food allergy

skin tests; Sensitization; IgE-mediated allergy; allergenic extract. After the discovery of IgE, technological advances have provided new laboratory tools for the quantification of allergen-specific IgE antibodies in serum and on the surface of basophils-mast cells. In vitro testing offers numerous advantages: accurate quantitation, lack of drug interference, safety, and long-term storage of samples. Quantitative immunoassays for IgE antibodies can be an adjunct to skin testing. The allergen reagent in solid phase (allergosorbent) or liquid is the main component of the assay that confers specificity to the IgE antibody test. It is the most complex and highly variable reagent in IgE antibody assays. The choice to use diagnostic recombinants on a single rather than multiple platforms is made on a case-by-case basis (considering prior history and clinical profile) and in an allergen-dependent manner. Although most food allergies are limited to a small number of possible triggers, these foods are very complex when evaluating their allergenic potential. The possibility of fractionating the allergen and understanding some of its components as potentially important to define the risk of clinical reaction, cross-reactivity, or persistence of allergy, opened a new era in the field of allergy, called molecular allergy. The identification of the allergenic component responsible for the reactions is an important tool to confirm the information and severity of the symptoms, natural history of the disease, possibility of cross-reactivity and clinical symptoms (allergy markers).

T2-high asthma phenotypes across lifespan.

In adults, personalised asthma treatment targets patients with type 2 (T2)-high and eosinophilic asthma phenotypes. It is unclear whether such classification is achievable in children. To define T2-high asthma with easily accessible biomarkers and compare resulting phenotypes across all ages. In the multicentre clinical All Age Asthma Cohort (ALLIANCE), 1125 participants (n=776 asthmatics, n=349 controls) were recruited and followed for 2 years (1 year in adults). Extensive clinical characterisation (questionnaires, blood differential count, allergy testing, lung function and sputum induction (in adults)) was performed at baseline and follow-ups. Interleukin (IL)-4, IL-5 and IL-13 were measured after stimulation of whole blood with lipopolysaccharide (LPS) or anti-CD3/CD28. Based on blood eosinophil counts and allergen-specific serum IgE antibodies, patients were categorised into four mutually exclusive phenotypes: "atopy-only", "eosinophils-only", "T2-high" (eosinophilia + atopy) and "T2-low" (neither eosinophilia nor atopy). The T2-high phenotype was found across all ages, even in very young children in whom it persisted to a large degree even after 2 years of follow-up. T2-high asthma in adults was associated with childhood onset, suggesting early origins of this asthma phenotype. In both children and adults, the T2-high phenotype was characterised by excessive production of specific IgE to allergens (p<0.0001) and, from school age onwards, by increased production of IL-5 after anti-CD3/CD28 stimulation of whole blood. Using easily accessible biomarkers, patients with T2-high asthma can be identified across all ages delineating a distinct phenotype. These patients may benefit from therapy with biologicals even at a younger age.

Prevalence of and risk factors for increased serum levels of allergen-specific IgE in a population of Norwegian dogs.

BackgroundThe importance of different allergens in association with IgE production and canine atopic dermatitis (CAD) has been poorly studied and few studies exist on factors influencing allergen-specific IgE antibodies in serum. The aim of this cross-sectional study was to investigate the prevalence of elevated IgE levels to different environmental allergens in Norwegian dogs with a suspicion of CAD. The secondary aim was to identify risk factors associated with elevated serum levels of allergen-specific IgE.ResultsThe study sample consisted of serum from 1313 dogs of 161 different breeds. All samples were submitted for serologic IgE-testing (Fc epsilon R1 alpha-based ELISA) based on suspicion of CAD. Overall, 84.3% of the dogs had elevated IgE levels to one or more of the allergen(s). The predominant allergens amongst the positive results were the indoor allergens (Acarus siro 84.0%, Dermatophagoides farinae 80.2%, Tyrophagus putrescentiae 79.9%). Sheep sorrel was the most commonly encountered outdoor allergen (40.0%). Only 2.6% of the dogs with elevated IgE levels were positive to flea saliva.The test results varied significantly depending on when the serum samples were taken. Samples taken during summer and autumn more often came out positive than samples taken during winter and spring. Geographical variations were also demonstrated. A greater proportion of females than males had positive test results, and more females than males tested positive to outdoor allergens. The mean age was significantly higher in the dogs testing positive than amongst the dogs testing negative. The allergen-specific IgE levels varied with breed. The boxer was the only breed with a significantly higher proportion of positive test results compared to the other breeds. Boxers also had a higher prevalence of elevated IgE levels to outdoor allergens, whereas the Rottweiler had a higher prevalence of elevated IgE levels to indoor allergens compared to the other breeds.ConclusionsIgE hypersensitivity was most often associated with indoor allergens. Outdoor allergens were of minor importance and IgE reactivity to flea saliva was rare. Breed differences in allergen-specific IgE levels were identified. Season of sampling, and the dogs’ geographical localisation, sex and age also affected the results of the IgE analysis.

Elevated Expression and Genetic Association Links the SOCS3 Gene to Atopic Dermatitis

In a systematic analysis of global gene-expression patterns, we found that SOCS3 messenger RNA was significantly more highly expressed in skin from patients with atopic dermatitis than in skin from healthy controls, and immunohistochemical analysis confirmed a similar elevation of SOCS3 protein. Furthermore, we found a genetic association between atopic dermatitis and a haplotype in the SOCS3 gene in two independent groups of patients (P<.02 and P<.03). These results strongly suggest that SOCS3, located in a chromosomal region previously linked to the disease (17q25), is a susceptibility gene for atopic dermatitis.

Validation of a New ImmunoCAP TM Test for Detecting Allergen-Specific IgE to Two Spotted Spider Mite

RATIONALE: Two spotted spider mite (TSM) is one of the most prevalent allergens in South Europe and Far East Asian countries, however, there has been no commercial kit available for detecting IgE antibodies to TSM. The aim of this study was to validate a new ImmunoCAPTM test for detecting allergen-specific IgE to TSM in serum. METHODS: 131 subjects with allergic rhinitis and/or asthma sensitized to TSM, and 60 non-atopic healthy controls were enrolled. Skin prick test was performed with home made TSM extract and skin reactivity was presented as allergen to histamine (A/H) ratio. ELISA coated with homemade TSM extract, and ImmunoCAPTM (Pharmacia Diagnostics AB, Sweden) were applied to measure allergen-specific IgE antibodies in serum. For confirming binding specificity, ELISA and ImmunoCAP inhibition tests were done with additions of TSM and D. pteronyssinus allergens. RESULTS: The percentage of patients with allergen-specific IgE by ELISA and ImmunoCAP were 28% vs. 50% among ≥2+ A/H responders and 50% vs. 62% among ≥3+ A/H responders, i.e. the higher skin reactivity the better correlation. Positive correlation was found between ImmunoCAP and ELISA (r=0.61, p<0.05). ImmunoCAP inhibition tests showed significant dose dependent inhibitions with addition of TSM, while minimal inhibitions with D. pteronyssinus. CONCLUSIONS: A new ImmunoCAP test for detecting allergen-specific IgE antibodies to TSM in serum was superior to ELISA system, and can be used for screening TSM sensitive patients in clinical practice.

Immunological characteristics of heterodimeric recombinant Fel d 1

Rationale Exposure and sensitization to domestic cat ( Felis domesticus) is a significant risk factor for developing allergic rhinitis and asthma. Earlier studies have shown that majority of the cat allergic patients develop allergic responses against major cat allergen Fel d 1. Several attempts have been made to produce of recombinant (r) Fel d 1 as heterodimer. We have recently shown that baculovirus expression system is able to produce rFel d 1 that has compatible characteristics to nFel d 1. Methods In the present study an immunological characterization of rFel d 1 in comparison to the purified nFel d 1 and cat allergen extract (IMP Fel d, ALK-Abello') was performed by use of IgE inhibition, lymphocyte proliferation and basophile histamine release assay(s). Results In inhibition assay against cat allergen extract, natural and rFel d 1 were able to inhibit 87% of the binding of cat allergen specific serum IgE antibodies. The basophile histamine release responses with rFel d 1 and nFel d 1 showed to be in line with the responses given by the cat allergen extract. Comparable T-cell responses towards natural and rFel d 1 was obtained in lymphocyte proliferation assay and when the response of Fel d 1-specific T-cell lines was investigated even though nFel d 1 seemed to be recognized more frequently. Conclusions These results show that heterodimeric rFel d 1 elicits immunological responses that are comparable to nFel d 1. The present study demonstrates that recombinant allergen(s) with well defined biochemical and immunological characteristics are feasible for development of therapeutic vaccines.

The Wiskott-Aldrich syndrome gene as a candidate gene for atopic dermatitis.

Atopic dermatitis is a multifactorial disorder probably caused by environmental factors in combination with susceptibility genes. The clinical similarity between atopic dermatitis and the eczema manifestation in patients with Wiskott-Aldrich syndrome made the previously identified WAS gene in chromosome sub-band Xpl 1.23 an interesting candidate gene for atopic dermatitis. We studied linkage and association to the WAS gene region using four polymorphic microsatellite markers in 406 Swedish families with at least two siblings affected with atopic dermatitis (in total 1514 individuals). In the analyses, we studied two qualitative traits: atopic dermatitis and elevated allergen-specific serum IgE antibodies, and one quantitative trait, a severity score of atopic dermatitis. We found that the marker MAOB gave positive linkage with a maximum lod score of 1.68 (p<0.05) to the severity score of atopic dermatitis. Association could not be seen to atopic dermatitis nor to elevated allergen-specific serum IgE antibodies in this region using the transmission disequilibrium test. Our results indicate that either the WAS gene or another gene in the area contributes to the severity of atopic dermatitis.

Detection of Multiple Allergen-specific IgEs on Microarrays by Immunoassay with Rolling Circle Amplification

First described in 1967, the radio allergo sorbent test (RAST) has been the standard technique for measuring allergen-specific IgE antibodies in serum (1). An updated version of the RAST test, termed CAP (Pharmacia), has been introduced (2). In clinical practice, CAP results must be interpreted with care. The diagnostic performance of CAP varies in an allergen-specific manner, and CAP scores do not always correlate with clinical severity (3)(4). CAP sensitivity, specificity, and positive predictive values agree well with skin prick tests (SPTs) for house dust mites and grasses, but poorly with tests for cat dander and peanuts (5). Microarray technology potentially offers advantages in diagnostic applications such as allergy testing because the amount of reagent required, and thus the cost per assay, is greatly reduced (6). This approach has been difficult to reduce to practice, however, because the extremely small volumes (∼0.5–5 nL) of sample used to create spots on these microarrays require extremely sensitive methods of analyte detection (7). We have used rolling circle amplification (RCA) (8) for the detection of antibody bound to antigen (9). In this “immunoRCA”, the 5′ end of a RCA primer is attached to an antibody; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of circular DNA sequence copies that remain attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes. ImmunoRCA, therefore, represents a novel approach for signal amplification of antibody-antigen recognition events on microarrays. ImmunoRCA can detect IgE in a format using high-density microarrays of anti-human IgE printed on glass slides by a pin-tool type microarraying robot (9). Here, we describe the production of microarrays of multiple allergens and demonstrate the utility of these microarrays in combination with immunoRCA to simultaneously detect …

Atopy in children of families with an anthroposophic lifestyle

Increased prevalence of atopic disorders in children may be associated with changes in types of childhood infections, vaccination programmes, and intestinal microflora. People who follow an anthroposophic way of life use antibiotics restrictively, have few vaccinations, and their diet usually contains live lactobacilli, which may affect the intestinal microflora. We aimed to study the prevalence of atopy in children from anthroposophic families and the influence of an anthroposophic lifestyle on atopy prevalence. In a cross-sectional study, 295 children aged 5-13 years at two anthroposophic (Steiner) schools near Stockholm, Sweden, were compared with 380 children of the same age at two neighbouring schools in terms of history of atopic and infectious diseases, use of antibiotics and vaccinations, and social and environmental variables. Skin-prick tests were done for 13 common allergens, and we took blood samples from children and their parents for analysis of allergen-specific serum IgE-antibodies. At the Steiner schools, 52% of the children had had antibiotics in the past, compared with 90% in the control schools. 18% and 93% of children, respectively, had had combined immunisation against measles, mumps, and rubella, and 61% of the children at the Steiner schools had had measles. Fermented vegetables, containing live lactobacilli, were consumed by 63% of the children at Steiner schools, compared with 4.5% at the control schools. Skin-prick tests and blood tests showed that the children from Steiner schools had lower prevalence of atopy than controls (odds ratio 0.62 [95% CI 0.43-0.91]). There was an inverse relation between the number of characteristic features of an anthroposophic lifestyle and risk of atopy (p for trend=0.01). Prevalence of atopy is lower in children from anthroposophic families than in children from other families. Lifestyle factors associated with anthroposophy may lessen the risk of atopy in childhood.

Age of entry to day nursery and allergy in later childhood

Infections in early childhood may prevent allergies in later life. If this hypothesis is true, early exposure to childcare outside the home would protect against atopy by promotion of cross infections. We investigated whether children who attend a nursery at a young age have a lower rate of atopy and fewer allergies than children who attend from an older age. In a cross-sectional study carried out in 1992-93, we examined 2471 children in three age-groups (5-7, 8-10, and 11-14 years) from the towns of Bitterfeld, Hettstedt, and Zerbst in eastern Germany. The children's parents answered a questionnaire about allergies and symptoms, attendance at day care, and related factors. Sensitisation was assessed by skin-prick tests and measurement of allergen-specific IgE antibodies in serum. In 669 children from small families (up to three people), the prevalence of atopy was higher among children who started to attend day nursery at an older age than in those who started to attend at a younger age (p<0.05). Compared with children who first attended at age 6-11 months, the adjusted odds ratios for a positive skin-prick test were 1.99 (95% CI 1.08-3.66) for children who attended at age 12-23 months and 2.72 (1.37-5.40) for those who attended at age 24 months and older. In 1761 children from large families (more than three people), age of entry to day nursery had no effect on atopy. Our findings accord with the hypothesis that early infection may protect against allergies in later life.

Distinct patterns of cow's milk allergy in infancy defined by prolonged, two‐stage double‐blind, placebo‐controlled food challenges

The clinical manifestations of cow's milk allergy (CMA) are highly variable, and challenges usually identify only immediate, IgE mediated reactions. To clearly identify CMA of immediate and delayed types using a two-stage, double-blind, placebo-controlled food challenge (DBPCFC), and to prospectively compare the clinical history and analyses of specific IgE antibodies to milk in predicting outcome of DBPCFC. A total of 69 patients (33 girls, 36 boys) were recruited for study based on a history highly suggestive of CMA and resolution of symptoms on a bovine protein-free diet. After skin-prick tests (SPTs) and search for allergen-specific serum IgE antibodies by enzyme allergosorbent test (EAST), a two-stage DBPCFC was performed over several days. Of 16 patients (mean age 36.9 months) classified as probable immediate reactors based on the history, 10 (62.5%) had a positive DBPCFC with similar patterns to historical adverse reactions (< or = 2 h after milk exposure). The other 53 (77%) patients (17.3 months) had a history of probable delayed type CMA presenting with predominantly gastrointestimal symptoms from 2 h and up to 6 days after milk exposure. Of these, 15 (28.8%) had a positive DBPCFC, again with a symptom pattern similar to the history. Sensitivity/specificity of SPT was similar to that of EAST for both immediate (70/83% and 62/83% respectively, NS) or delayed (0/97% and 0/97%) CMA confirmed by DBPCFC. Using our two-stage, prolonged DBPCFC, we clearly identified two groups of children with CMA, reflecting different pathogenesis of either immediate-type IgE-dependent, or delayed-type IgE-independent allergy. Although useful in immediate reactors, IgE antibody determination cannot predict the outcome of DBPCFC in delayed reactors. A thorough clinical history was the most helpful tool to predict the type of response in challenge positive patients.

Discrepancies between the skin test and IgE antibody assays: Study of histamine release, complement activation in vitro, and occurrence of allergen-specific IgG

Intracutaneous skin tests (STs) and RAST with the common allergens, grass pollen, house dust mite, and cat dander, were performed on 660 adult patients. In 117 patients (18%), we found 140 discordances (7%) in a total number of 1980 ST and RAST combinations. In agreement with studies in the literature, >80% of the discordances consisted of positive skin reactions without detectable allergen-specific IgE antibodies in serum. The percentages of discordant results were similar for the three allergens. Reproducibility of both the RAST and the ST was evaluated in the discordant group. Repetition of the routine RAST procedure elicited results similar to those in the first test in 81% ( 105 130 ). A second ST elicited identical results in 89% ( 47 53 ). In addition to the routine IgE antibody assay, sera of patients with a positive ST but without detectable IgE antibodies were tested in two other RAST systems: (1) a RAST with allergen extracts from the same production batch as the ST reagents, and (2) the Pharmacia RAST. In spite of having a clearly positive ST, sera from 68 (80%) of 85 patients remained completely negative in all three RAST systems. Histamine release (HR) in vitro from washed leukocytes was studied in 35 patients with a reproducible positive ST and negative RAST results with serum. Interpretation of this test was possible in 28 patients. In 82% ( 23 28 ) of these patients, clearly detectable HR was found with the relevant allergen extract. A role of IgE in the skin reactions and HR tests was confirmed by positive RAST results with IgE that was affinity purified from serum of seven of these patients. Allergen-specific lgG4 antibodies are unlikely to be implicated, since no antibodies against grass pollen and house dust mite were detectable in sera of these patients. Only 18% of the patients with an unexplained skin reaction with cat dander have detectable IgG4 antibodies, but these antibodies were found in a similar frequency in a nonallergic, ST negative control group. Low total IgG responses precluded false negative RAST results caused by competition of IgG antibodies with IgE antibodies. There were no significant differences in the degree of complement activation in vitro by house-dust extracts between healthy control subjects, nonallergic patients, and patients with unexplained skin reactivity. It is concluded that a high proportion of the positive skin reactions with common inhalant allergens, which are not accompanied by a positive RAST, are probably caused by IgE antibodies that are not detectable in serum with any of the RAST procedures. Escape from detection in the RAST is most likely explained either by very low IgE antibody titers or by specificity for allergenic components that are only present in low concentrations in the allergenic extract.

Dog Dander Allergens

Cat allergen-specific serum IgE antibodies were detected in 27 (71%) of a group of 38 dog dander-sensitive patients. In 4 (44%) of the cases the dual reaction could be explained by the presence of IgE antibodies to cross-reacting serum proteins. In a larger group the binding of dog dander-specific IgE antibodies could be inhibited by cat epithelium allergens. Two allergenic components of dog dander were separated by chromatography on Sephadex G-75. One of the allergens was closely related to a purified major cat epithelium allergen. The other allergen was apparently dog-specific and showed no cross-reactivity with cat allergens. Both components were present in the dander of dachshund, Airedale terrier, poodle and boxer. The dog dander is the preferable source of dog epithelium allergens provided that the existence of relations to other animals is considered.

An Immunoperoxidase Method for the Demonstration of Allergen‐Specific Ige in Serum

A solid-phase immunoenzymatic technique for the detection of allergen-specific IgE antibodies in serum is described. The binding of such antibodies to allergen insolubilized on cyanogen bromide-activated paper discs was detected by a subsequent two-step procedure involving the use of rabbit antihuman IgE and goat antirabbit IgG coupled with peroxidase. A solution of 3-3' diaminobenzidine and hydrogen peroxide, employed as the color indicator system, turned the discs dark brown in positive cases. Discs carrying 11 different inhalant allergens were tested with sera containing allergen-specific IgE antibodies. Agreement between the results of this technique and the RAST was seen in 85% of 310 tests performed. In the clinical practice of otorhinolaryngologic allergy, this test may prove to be an important laboratory adjunct ot clinical history and diagnostic skin test in the identification of the incriminated inhalant allergens.