The relevance of microbial allergens to the IgE antibody repertoire in atopic and nonatopic eczema

BACKGROUND: A propensity to microbial skin infections has been reported in atopic ("high IgE") and nonatopic ("low IgE") forms of eczema. However, the relationship between antimicrobial IgE antibodies and nonatopic disease is unclear. OBJECTIVE: We examined the relevance of microbial allergens to the allergen-specific IgE antibody repertoire in patients with atopic dermatitis. METHODS: Patients with IgE levels of less than 150 IU/mL were stratified according to sensitivity (n = 22) or no sensitivity (n = 27) to 11 common food allergens and aeroallergens. The prevalence and titers of antimicrobial IgE antibodies were compared with those of patients (n = 36) with increased total IgE levels (>150 IU/mL). Skin-derived serum chemokines were also analyzed. RESULTS: Patients with low IgE levels showed decreased disease severity, increased age of onset, a striking female predominance, and a distinct distribution of skin lesions. High titer IgE antibodies (sum of 8 bacterial and fungal allergens = 29.8 +/- 32.6 IU/mL) and multisensitization specific for microbial allergens was characteristic of patients with high IgE levels, with an overall 84% positivity; however, antimicrobial IgE antibodies comprised 3% or less of allergen-specific IgE antibodies. By contrast, antimicrobial IgE antibodies were detected in only 20% of patients with low IgE, and titers were negligible, irrespective of sensitization to common allergens. These patients were monosensitized, and exclusive microbial sensitivity was uncommon (10%). Patients with low IgE with no sensitivity to common allergens had lower levels of serum macrophage inflammatory protein 3alpha compared with their sensitized counterparts. CONCLUSION: Antimicrobial IgE antibodies are uncommon in patients with atopic dermatitis with low IgE levels. CLINICAL IMPLICATIONS: Hypersensitivity to microbial allergens is an unlikely trigger for eczematous eruptions in patients with low IgE levels.     

Evaluation of new system for the detection of IgE antibodies (CAP) in atopic diseases

An evaluation of a newly developed IgE antibody assay system (CAP) was carried out. There was a clear correlation between IgE antibody titers measured by CAP single and RAST (= 0.642 to 0.979). It turned out that CAP single is more sensitive than RAST and non-specific adsorption of IgE immunoglobulin to the solid phase was assumed to be less in CAP than in RAST. Pathogenic allergens diagnosed either clinically or by in vitro assay systems were compared. The sensitivity and specificity of the CAP system were 94.2% and 87.3%, respectively. CAP multi which binds groups of multiple allergens on the solid phase, was examined for the screening of hypersensitivity to categories of allergens. Statistical sensitivities of CAP multi resided between 63.9% to 86.2%, while the specificities were 98% to 100%, indicating that CAP multi is useful for the exploration of causative allergens. Phadiatop, which fixes multiple inhalant allergens, showed a sensitivity of 89.6%. The specificity of phadiatop was 93.9%, when examined for intrinsic bronchial asthma patients, and 91.2% for normal subjects indicating that phadiatop is more useful than total IgE measurement for the screening of atopic trait.     

Specific IgE and IgG antibody-binding patterns to recombinant cockroach allergens

BACKGROUND: The specificity of serum antibody responses to different cockroach allergens has not been studied. OBJECTIVE: We sought to quantitate serum IgE and IgG antibodies to a panel of purified cockroach allergens among cockroach-sensitized subjects. METHODS: IgE antibodies to recombinant cockroach allergens (rBla g 1, rBla g 2, rBla g 4, rBla g 5, and rPer a 7) were measured in sera containing IgE antibodies to Blattella germanica extract (n = 118) by using a streptavidin CAP assay and a multiplex flow cytometric assay. Specific IgG antibodies were determined by using radioimmunoprecipitation techniques. RESULTS: Specific IgE antibodies measured by means of CAP assay and multiplex assay were strongly correlated ( r = 0.8, P < .001). The sum of IgE antibodies (in international units per milliliter) against all 5 allergens equated to IgE antibodies to cockroach extract. Although the prevalence of IgE antibodies was highest for rBla g 2 (54.4%) and rBla g 5 (37.4%), patterns of IgE antibody binding were unique to each subject. Surprisingly, only 16% of cockroach-sensitized subjects with IgE antibodies to house dust mite exhibited IgE antibody binding to cockroach tropomyosin (rPer a 7). Specific IgE antibodies were associated with increased IgG antibody levels, although detection of IgG in the absence of IgE was not uncommon. CONCLUSION: The techniques described offer a new approach for defining the hierarchy of purified allergens. IgE antibodies directed against 5 allergens constitute the majority of the IgE antibody repertoire for cockroach. Such distinct patterns of IgE-IgG responsiveness to different cockroach allergens highlight the complexity of B-cell responses to environmental allergens.     

人IgE工作标准血清的制备与标定

本工作用单向免疫扩散法和反向被动血凝法筛选并收集高IgE健康人混合血清,制备供体外测定用的IgE作标准血清。混合血清无乙型肝炎表面抗原,定量分装为每安瓿0.5ml,经冷冻干燥后,4℃保存。参照 WHO code 69/204(10,948IU/ml)IgE 国际参考制剂,以纸片放射免疫吸附试验,用四剂量法标定其中总IgE的含量。标定结果是2,680IU/ml,并对血清分装量进行测定,各安瓿间血清湿重误差 ±2.12％;血清干重误差 ±1.81％;冻干血清溶解时间范围 1分 5秒～1分16秒。本次制备的IgE工作标准血清批号为861A。本法不仅保证标准化,且可用于其他类免疫球蛋白的标定,有普遍意义。     

Development of a multiplex bead-based assay for detection of hepatitis C virus

Hepatitis C virus (HCV) infection is a major burden to public health worldwide, affecting approximately 3% of the human population. Although HCV detection is currently based on reliable tests, the field of medical diagnostics has a growing need for inexpensive, accurate, and quick high-throughput assays. By using the recombinant HCV antigens NS3, NS4, NS5, and Combined, we describe a new bead-based multiplex test capable of detecting HCV infection in human serum samples. The first analysis, made in a singleplex format, showed that each antigen coupled to an individual bead set presented high-level responses for anti-HCV-positive reference serum pools and lower-level responses for the HCV-negative pools. Our next approach was to determine the sensitivity and specificity of each antigen by testing 93 HCV-positive and 93 HCV-negative sera. When assayed in the singleplex format, the NS3, NS4, and NS5 antigens presented lower sensitivity values (50.5%, 51.6%, and 55.9%, respectively) than did the Combined antigen, which presented a sensitivity of 93.5%. All antigens presented 100% specificity. These antigens were then multiplexed in a 4-plex assay, which resulted in increased sensitivity and specificity values, performing with 100% sensitivity and 100% specificity. The positive and negative predictive values for the 4-plex assay were 100%. Although preliminary, this 4-plex assay showed robust results that, aligned with its small-sample-volume requirements and also its cost- and time-effectiveness, make it a reasonable alternative to tests currently used for HCV screening of potentially infected individuals.     

Development and validation of a third generation allergen-specific IgE assay on the continuous random access IMMULITE 2000 analyzer

In vitro determination of allergen-specific IgE (sIgE) represents an important aid in the diagnosis and treatment of allergy. Improvements in laboratory methodology--the development of second, and now third generation assays for sIgE--have brought about major advances in speed, convenience, performance, and in the standards for judging performance. In this study, following the NCCLS I/LA20-A guidelines, we evaluated the analytical performance of a quantitative chemiluminescent enzyme immunoassay for sIgE using the continuous random access IMMULITE 2000 system. Defining features of this "third generation" sIgE assay include a true zero calibrator with a detection limit and functional sensitivity of 0.1 and 0.2 kU/L, respectively. The use of liquid allergens allows for complete automation, fast binding kinetics between IgE and the natural allergenic protein conformations, and a time-to-first-result of 65 min. Stable reagents and the low nonspecific signal associated with the liquid allergens and centrifugal wash technique permit extension of the measuring range to 0.1-100 kU/L, based on lot-specific, factory-calibrated master curves standardized to the WHO 75/502 reference standard. The assay demonstrated good precision and linearity over its measuring range. Relative to a first generation RIA (mRAST, from Hycor), clinical sensitivity, specificity, and concordance were 88%, 92%, and 90%, respectively (n = 812). Quantitative comparisons to a second generation assay yielded a linear regression relationship of IMMULITE 2000 = 0.99 (Pharmacia FEIA) + 1.99 kU/L, r = 0.859 (n = 169).     

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E

BACKGROUND: The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. OBJECTIVE: To test the performance of this allergen microarray in a serological analytical study. METHODS: Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen (Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. RESULTS: The microarray has a good dynamic range similar to the CAP/RAST system. Microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/RAST. CONCLUSION: A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. Microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.     

Usefulness of AlaSTAT, a new method for the measurement of IgE]

AlaSTAT is an enzyme-immunoassay method for the measurement of allergen specific IgE antibodies. This method has a special feature in that carbohydrate, nucleic acid and fat as well as protein allergens can be used as antigens. In this study, IgE antibodies were measured by the use of AlaSTAT kits which were able to detect IgE antibodies to protein or nucleic acid allergens, and the results were compared to those of skin tests and RAST. 1367 samples from 479 patients were examined with 20 allergens. The capacity in the AlaSTAT system was larger than that in the RAST system. The correlation coefficient between AlaSTAT and RAST was 0.90 (p less than 0.01) and the correspondence rate was 90.7%. When the rice allergen was used in RAST, a nonspecific reaction was observed at high concentrations of total IgE. However, no such reaction was observed in AlaSTAT, even with total IgE of 10,000 IU/ml. The sensitivity, specificity and correspondence rate to skin tests was 71%, 87% and 75% respectively. The specificity of AlaSTAT seemed to be slightly higher than that of RAST, but the sensitivity and the correspondence rate were similar to those of RAST. From these results, AlaSTAT appears to be a good or better method for the measurement of IgE antibodies in comparison with RAST.     

Contribution of dust mite and cat specific IgE to total IgE: relevance to asthma prevalence

BACKGROUND: The prevalence of asthma is strikingly different in some Westernized countries: approximately 20% in New Zealand and approximately 8% in northern Sweden. OBJECTIVE: We investigated differences in total IgE and in the prevalence of wheezing related to the observation that high exposure to dust mite allergens induces high titers of IgE antibodies. METHODS: Two age-matched, population-based cohorts-1155 children in New Zealand (224 sera) and 3431 children (797 sera) in the Norrbotten area of Sweden-were studied. Sera were assayed for total IgE and specific IgE antibodies to relevant allergens. RESULTS: The mean total IgE among wheezing children was higher in New Zealand than Sweden (218 IU/mL vs 65.2 IU/mL; P < .001). In addition, the prevalence of high titer specific IgE antibody (> or =50 IU/mL) was greater among the wheezing children in New Zealand compared with Sweden (35.7% vs 13.0%; P < .001). Specific IgE antibody to mite in New Zealand was significantly related to high total IgE (> or =200 IU/mL; r = 0.47; P < .001), whereas the IgE antibody response to cat allergens did not make a significant contribution to high total IgE in either country. CONCLUSION: The quantity of IgE antibody produced to dust mite provides a possible explanation for the higher total IgE levels found in children in New Zealand and may help to explain the differences in prevalence and severity of asthma between these 2 countries. CLINICAL IMPLICATIONS: Specific IgE antibody responses to dust mite and cat allergens may contribute differently to total serum IgE and to the prevalence of allergic disease.     

Atopic profile of inner-city asthma with a comparative analysis on the cockroach-sensitive and ragweed-sensitive subgroups

Background: Inner-city asthma is well known for its high risk of mortality. To better understand urban asthma, we examined clinical characteristics and aeroallergen sensitivities of 592 of 680 consecutive urban Chicago residents with asthma.Methods: A total of 227 male and 453 female subjects who met the criteria for the study were registered. A comprehensive clinical evaluation was followed by allergy skin testing (prick and intradermal testing) with. 10 groupings (5 indoor and 5 outdoor) of common aeroallergens. Serum total IgE and selective antigen-specific IgE levels, including cockroach-specific IgE, were routinely measured. A total of 592 (196 male and 396 female) subjects with an average age of 35 years were, skin tested_ The average duration of asthma was 12.6 years, and 31 % of the population was receiving corticosteroids.Results: Aeroallergen sensitivity was noted in 85%, and 94 subjects (15%) were nonallergic. House dust sensitivity (76%) was most prevalent, distantly followed by sensitivity to cockroach (48%), ragweed (4_5%), other weeds (42%), cat (40%), and dust mite (24%). The average number o f aeroallergen sensitivities detected was 4 of 10 groupings of both indoor and outdoor allergens. Twenty percent of subjects were allergic to only indoor allergens, whereas 4% were allergic to outdoor allergens only. Serum IgE was 245 ± 17.3 IU/ml (geometric mean + SEM), and 74% of 444 serum samples assayed showed IgE antibody levels greater than or equal to 100 IU/mI. A cockroach-sensitive subgroup (283 subjects) had longer duration of asthma (p, < 0.0001) and fewer additional aeroallergen sensitivities (p < 0.0001) than the ragweed-sensitive subgroup (264 subjects).Conclusion: The results indicate that a great majority (85%) of inner-city Chicago residents with asthma have atopic asthma, as demonstrated by highly elevated IgE levels and multiple aeroallergen sensitivities. Sensitivity to indoor allergens is more prevalent than sensitivity to outdoor allergens. The subjects with cockroach-sensitive asthma appear to be a distinctive subgroup characterized by chronicity, and elevated serum IgE antibody levels with fewer aeroallergen skin test sensitivities.     

Evaluation of a new molecular assay for detection of human immunodeficiency virus type 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA.

BACKGROUND: Rapid, sensitive, specific, and cost-effective screening of donated blood to prevent transmission of infectious agents remains challenging. In recent years, incorporation of nucleic acid testing for HIV-1 and HCV RNA improved blood safety by reducing the window period between infection and serologic detection. For HBV infection, this window period with most serologic assays is 50-60 days. Adding a nucleic acid test (NAT) for HBV DNA with existing NATs for HIV-1 and HCV RNA would further improve blood safety and blood screening efficiency. OBJECTIVE: To evaluate the Procleix Ultrio Assay for simultaneous detection of HIV-1 and HCV RNA and HBV DNA and corresponding discriminatory assays. STUDY DESIGN: The performance of these assays, which utilize the same technology and assay format as the Procleix HIV-1/HCV assay, was determined using relevant clinical specimens and analytical sensitivity and specificity panels. RESULTS: The Procleix Ultrio Assay demonstrated specificity of > or =99.5% in healthy donor blood specimens and in plasma containing potentially interfering substances or other blood-borne pathogens. Assay sensitivity demonstrated >95% detection of 100copies/mL, 30IU/mL, and 15IU/mL for HIV-1 and HCV RNA, and HBV DNA, respectively. The assay detects all known HIV-1 subtypes and HCV and HBV genotypes and is highly reproducible. Statistical analysis using receiver operating characteristic plots demonstrated wide analyte cutoff values for each assay associated with assay specificity and sensitivity of > or =99.5%. CONCLUSIONS: In this investigational study, the Procleix Ultrio Assay sensitivity and specificity were similar to existing NATs used in blood-bank settings to detect HIV-1 and HCV RNA and provided equivalent sensitivity and specificity for detection of HBV DNA. Using this combination assay, blood safety may be improved and the multiplex format enhances blood screening efficiency. The throughput capability of this assay is compatible with large volume processing and the chemistry is adaptable to full automation.     

Correlation of the turbo-MP RIA with ImmunoCAP FEIA for determination of food allergen-specific immunoglobulin E

It has been reported that in vitro measurement of food-specific IgE can be used to accurately predict food allergy and reduce the risk associated with double-blinded placebo-controlled food challenges (DBPCFC). Our objective was to assess the performance characteristics of the Hycor Turbo-MP quantitative radioimmunoassay for food-specific IgE and to determine this method's comparability to another assay, the Pharmacia ImmunoCAP fluorescence enzyme immunoassay (FEIA). The dynamic range of the Turbo-MP assay is 0.05 to 100 IU/ml, compared to 0.35 to 100 IU/ml for the FEIA. Performance characteristics of the Turbo-MP assay (ie, reproducibility of the calibration curve, within-run precision, total precision, parallelism, and linearity) were determined using samples from the Hycor serum bank. The precision (CV) of IgE calibrator replicates was <10%. The total precision (CV) of the Turbo-MP assay ranged from 8.8% to 18.4% for specific IgE concentrations between 0.28 to 31.4 IU/ml. Testing of serial dilutions of sera with IgE specificities for egg white, cow's milk, codfish, wheat, peanut, and soybean showed that the assay is linear over the entire dynamic range. Serial dilution data (slopes of 1.01 to 1.10) showed parallelism to serial dilutions of the IgE calibrator (slope of 0.96). The Turbo-MP and FEIA methods were both used for quantitative assays of food-specific IgE in 457 serum samples obtained from a clinical reference laboratory. Comparison of specific IgE results by the Turbo-MP and FEIA methods for 6 major food allergens exhibited a slope of 0.99 (0.92 to 1.03) with a correlation coefficient of 0.81.     

Comparison of a new specific IgE assay Quidel allergy screen to skin prick test, intradermal test, and RAST

Quidel Allergy Screen (QAS), an enzyme-linked immunosorbent assay, has been developed for measuring IgE antibodies against 9 allergens (HD 1, HD 2, Mite 1, Mite 2, Japanese cedar, ragweed, cat dander, sweet vernal grass, and egg white) at the same time. To determine whether this assay is useful in screening allergen-specific IgE antibody, we compared the titers of IgE antibodies against the 9 allergens measured by QAS to the obtained in skin prick tests, in intradermal tests and by RAST in 93 atopic asthmatics and 25 normal subjects. We found a good agreement between the reactivity of skin prick tests and the reactivity of QAS. There was a significant correlation between the threshold doses of intradermal tests and the titers of QAS. We also found a good agreement between the reactivity of RAST and the reactivity of QAS, and a strong correlation between the titers of RAST and the titers of QAS. Thus, it is concluded that QAS is useful in screening IgE antibodies against multiple allergens simultaneously.     

Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors

Background Peanut is a most common and potent food allergen. Many peanut allergens have been characterized using, in particular, IgE-binding studies.
Objectives We optimized an in vitro functional assay to assess the capacity of peanut allergens to degranulate humanized rat basophilic leukaemia cells, RBL SX-38 cells, after sensitization by serum IgE from peanut-allergic patients. We thus compared the activity of the main peanut allergens, i.e. Ara h 1, Ara h 2, Ara h 3 and Ara h 6, purified from roasted peanut.
Methods Sera of 12 peanut-allergic patients were collected and total and peanut-specific IgE were measured. They were used to sensitize RBL SX-38 cells and the degranulation was induced by incubation with ranging concentrations of a whole peanut protein extract or of purified peanut allergens. The mediator release was quantified by the determination of β-hexosaminidase activity in the supernatant. The intensity of the degranulation was expressed as maximum release and as EC50, corresponding to the dose of allergen that induced 50% of the maximum release.
Results For each serum, only 10 IU/mL of human IgE was necessary to sensitize the cells and obtain an optimal degranulation. With all the allergens, the release was positively correlated with the concentration of allergen-specific IgE in the serum used to sensitize the cells. The medians of EC50 obtained for Ara h 2 and Ara h 6 were 2.1 and 2.8 p m , respectively, while they were much higher for Ara h 3 and Ara h 1 (65 and 150 p m , respectively).
Conclusion The RBL SX-38 release assay proved to be sensitive, specific and reproducible. It allowed the comparison of the degranulation potential of different peanut allergens. For all the sera tested, Ara h 2 and Ara h 6 were more potent than Ara h 1 or Ara h 3.     

QUANTITATION OF HEPATITIS B VIRUS DNA IN PLASMA USING A SENSITIVE COST-EFFECTIVE “IN-HOUSE” REAL-TIME PCR ASSAY

Background: Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an “in-house” real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV. Materials and Methods: The “in-house” real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the “in-house” assay’s capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. Results: The lower limit of detection of this “in-house” HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this “in-house” assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this “in-house” HBV real-time assay correlated well with the commercial artus HBV RG PCR assay (r = 0.95, P < 0.0001). Conclusion: This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.     

Relationship between extremely low total serum IgE levels and rhinosinusitis.

BACKGROUND: The few studies examining clinical manifestations in adults with serum IgE levels less than 2.0 IU/mL provide conflicting information. OBJECTIVE: To examine self-reported respiratory disease in women with total serum IgE levels less than 2.0 IU/mL to further elucidate previous reports of an association between IgE deficiency and chronic rhinosinusitis. METHODS: In a geographically based cohort of 626 pregnant women, total serum IgE levels were measured using a standard assay with a lower limit of detection of 2.0 IU/mL. Sera with IgE levels less than 2.0 IU/mL were assayed again using a low IgE protocol with a detection limit of 0.02 IU/mL. RESULTS: Twenty-one individuals (3.4%) were found to have IgE levels less than 2.0 IU/mL. On repeated assay, 20 of these individuals with available clinical data were found to have detectable IgE levels ranging from 0.5 to 2.1 IU/mL (geometric mean, 1.2 IU/mL). None of these individuals with low IgE levels had physician-diagnosed sinusitis compared with 19.3% (113/585) of those with IgE levels of 2.0 IU/mL or greater (P = .03). Physician-diagnosed asthma was also less prevalent (1/19, 5.3%) in the low IgE group compared with 20.6% in those with higher IgE levels, but this was not significant (P = .14). The low IgE group reported a higher prevalence of hay fever symptoms than the remaining cohort (31.6% vs 24.4%; P = .43) but had less physician-diagnosed hay fever (5.3% vs 15.8%; P = .34). CONCLUSIONS: Low serum IgE levels were relatively common in these pregnant women. In contrast to previous studies, a low IgE level was not associated with chronic rhinosinusitis.     

Enzyme immunoassay of specific human IgE to purified cows’ milk allergens

An immunometric enzyme immunoassay for specific immunoglobulin E (IgE) against five purified cows’ milk allergens, β‐lactoglobuHn, α‐lactalbumin, bovine serum albumin, lactoferrin and whole casein fraction, has been developed. Allergens were immobilized on microtitration plates. After incubations with sera from allergic patients, specific IgE bound to the plastic were detected using a monoclonal anti‐human IgE antibody labelled with acetylcholinesterase. Quantitative determinations were made by comparison with a dose‐response curve obtained under the same conditions with standard total IgE. A quantification limit of 0.08 IU ml^‐1 can thus be obtained with a coefficient of variation of lower than 5%. This allows specific IgE determinations of clinical significance in sera from allergic patients at a dilution of at least 1/10 (i.e. in a few μl of serum) with good precision and reproducibility. The determinations of specific IgE in sera from 11 patients allergic to cows’ milk showed an excellent correlation of this assay with clinical data.     

Comparison of VIDAS Stallertest and Pharmacia CAP assays for detection of specific IgE antibodies in allergic children

In vitro determination of specific IgE antibodies in serum is the most frequently used method, besides the skin test, for diagnosing allergies. Standardized and reproducible assays of specific IgE antibodies contribute to the quality of diagnosis and treatment of allergic disease. This study compared the results and performance characteristics of the Pharmacia CAP system and a new specific IgE method using the VIDAS Stallertest (manufactured by bioMériux). To evaluate their clinical efficiency, the results of the CAP and VIDAS Stallertest assays were compared with skin prick test (SPT) results. After allergic patients completed SPTs, serum samples were collected and CAP and VIDAS Stallertest assays were performed to determine specific IgEs for Dermatophagoides farinae, D. pteronyssinus, cockroach, and alternaria. For egg and milk, we measured only the correlation between the 2 in vitro assays. When SPT was used as a reference standard, the sensitivity and specificity of the CAP assay was a little higher in respect to all inhalant allergens. There were significant correlations between the results of VIDAS Stallertest and CAP assays for IgE antibodies to inhalant and food allergens. This study indicates that the VIDAS Stallertest and Pharmacia CAP assays are feasible and replicable for measuring allergen-specific IgE.     

Development of a functional in vitro assay as a novel tool for the standardization of allergen extracts in the human system

BACKGROUND: Biochemical and immunochemical methods used for batch control of allergen extracts rely on the binding of IgE molecules to allergens. They do not measure the ability of a protein to induce type I allergic reactions. Therefore, a biological assay was established that is based on the cellular mechanisms of allergies in order to assess the cross-linking capacity of allergens. METHODS: Rat basophilic leukaemia cells were transfected with cDNA coding for the human high affinity IgE receptor chains. The surface expression of the IgE-binding alpha-chain was detected by FACS analysis and the functional integration of the 'humanized' receptors into the signal transduction cascade was addressed by intracellular calcium mobilization. Mediator release was measured in response to human IgE and a variety of cross-linking allergen preparations. RESULTS: Several clones were obtained that were able to bind allergen-specific human IgE. The results of the biological assay were compared with those obtained by immunochemical methods. The biological assay was used to determine the potency of allergen extracts, including highly diluted products that cannot be analysed by conventional methods. CONCLUSION: A stable 'humanized' basophil cell line was established that will be a useful tool for the standardization and batch control of allergen extracts. Because of its high sensitivity, it can also be used to detect minute quantities of potentially allergenic proteins, e.g. in processed foods. In addition, the test may support the development of novel allergy vaccines, such as recombinant hypoallergenic molecules.