Testing for IgG4 against foods is not recommended as a diagnostic tool: EAACI Task Force Report.

Serological tests for immunoglobulin G4 (IgG4) against foods are persistently promoted for the diagnosis of food-induced hypersensitivity. Since many patients believe that their symptoms are related to food ingestion without diagnostic confirmation of a causal relationship, tests for food-specific IgG4 represent a growing market. Testing for blood IgG4 against different foods is performed with large-scale screening for hundreds of food items by enzyme-linked immunosorbent assay-type and radioallergosorbent-type assays in young children, adolescents and adults. However, many serum samples show positive IgG4 results without corresponding clinical symptoms. These findings, combined with the lack of convincing evidence for histamine-releasing properties of IgG4 in humans, and lack of any controlled studies on the diagnostic value of IgG4 testing in food allergy, do not provide any basis for the hypothesis that food-specific IgG4 should be attributed with an effector role in food hypersensitivity. In contrast to the disputed beliefs, IgG4 against foods indicates that the organism has been repeatedly exposed to food components, recognized as foreign proteins by the immune system. Its presence should not be considered as a factor which induces hypersensitivity, but rather as an indicator for immunological tolerance, linked to the activity of regulatory T cells. In conclusion, food-specific IgG4 does not indicate (imminent) food allergy or intolerance, but rather a physiological response of the immune system after exposition to food components. Therefore, testing of IgG4 to foods is considered as irrelevant for the laboratory work-up of food allergy or intolerance and should not be performed in case of food-related complaints.

Diisocyanate conjugate and immunoassay characteristics influence detection of specific antibodies in HDI-exposed workers.

BACKGROUND: The structural characteristics of diisocyanate chemical protein antigens vary depending upon the methods of production, and may influence diisocyanate antigen immunoassays. The impact of different antigen preparation methods on immunoassay sensitivity, specificity, and predictive value for identifying workers with diisocyanate asthma (DA) has not been systematically evaluated. OBJECTIVE: Evaluate the influence of preparation methodology of hexamethylene diisocyanate human serum albumin (HDI-HSA) conjugates on the performance of specific antibody assays for identifying workers with confirmed HDI asthma. METHODS: Asthmatic reactions to HDI exposure were assessed in 80 autobody shop workers by specific inhalation challenge (SIC). HDI-specific IgE and IgG in serum were measured by RAST and ELISA with seven different HDI-HSA conjugates prepared in liquid phase with monomeric or polymeric HDI, or vapour-phase monomeric HDI. The HDI : HSA substitution ratios were determined by mass spectrometry. RESULTS: DA was confirmed by SIC in 23 subjects. The maximal sensitivity for detecting specific IgE among workers with positive SIC results was higher with RAST and with polymeric vs. monomeric HDI-albumin conjugates (21.7% vs. 8.7%) with a generally high specificity (>or=95%). HDI-HSA specific IgG antibody was also detected in 22-43% of HDI asthmatics depending upon the conjugate used. The specificity of specific IgG varied from 88% to 96%, and it was higher for monomeric (vs. polymeric) HDI-albumin conjugates with low (vs. high) substitution ratios. CONCLUSION: The test performance of specific IgE and IgG immunoassays for identifying a positive SIC response varied with different HDI-HSA conjugates. Standard test antigens and common immunoassays must be used to minimize inter-laboratory variability.

Substitution of Pichia pastoris-derived recombinant proteins with mannose containing O- and N-linked glycans decreases specificity of diagnostic tests.

BACKGROUND: Recombinant proteins from Pichia pastoris need to be fully evaluated before used as diagnostic tools. OBJECTIVE: The objective of this study was to investigate whether glycosylation by P. pastoris interferes with the specificity of diagnostic tests. METHODS: An autoantigen involved in Wegener's disease (protease 3) and 2 major inhalant allergens from grass pollen (Dac g 5) and house dust mite (Der p 1) were produced as recombinant molecules in P. pastoris. O-linked glycans on Dac g 5 were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The immune reactivity of the recombinant proteins was compared to that of their natural counterparts by ELISA and a radio-allergosorbent test (RAST) as well as by ELISA and RAST inhibition. RESULTS: In contrast to the non-glycosylated natural allergen, recombinant Dac g 5 was shown to carry at least 2 small mannose-containing O-glycans. We showed that both these O-glycans and the N-linked glycans on recombinant protease 3 and recombinant Der p 1 were recognized in ELISA by IgG antibodies in sera of healthy individuals. These IgG responses were closely correlated. The natural autoantigen and allergens were not recognized by IgG antibodies from healthy subjects. The carbohydrate nature of the epitopes recognized by IgG on the recombinant proteins was confirmed by inhibition studies with mannose and yeast mannan. IgE recognition of yeast glycans was observed in 2 out of 9 positive sera from patients with allergic bronchopulmonary aspergillosis. CONCLUSION: Production of recombinant molecules in yeast (or moulds) can introduce IgG-binding glycans that negatively affect the specificity of diagnostic tests.

Precision and accuracy of commercial laboratories' ability to classify positive and/or negative allergen-specific IgE results.

BACKGROUND: Accurate and reliable evaluation of the presence or absence of allergen-specific IgE is important in the differential diagnosis of allergic disease. A variety of different commercial tests are available for this purpose. There are few data available to judge how the results of these different tests compare with one another in everyday use. OBJECTIVE: To examine prospectively the extent of comparability among specific IgE results from different laboratories. METHODS: Six diagnostic laboratories employing five different methods to assay specific IgE were selected. Aliquots from 26 serum samples that contained variable levels of IgE specific to 17 common aeroallergens were sent in triplicate to each study laboratory during a 6-week time period. Results were reported numerically and by class scores and then compared by examining their concordance using Kendall's W nonparametric statistical test. In addition, cut-off values were compared by a best agreement analysis using reported results. Reproducibility was determined using precision profiles based upon the coefficient of variation among triplicates for each allergen across the range of reported results. RESULTS: In all, 7,813 tests were analyzed. Concordance among different assays in commercial use with one exception was not good. This was particularly true around the cut-off region where most assays demonstrated high imprecision. The Pharmacia CAP System used by two different laboratories demonstrated highly comparable results with good precision. Some assays were reproducible but not accurate. Others were neither reproducible nor accurate. CONCLUSIONS: The results of this study indicate that not all commercial laboratories/assays for specific IgE provide reproducible and accurate data. Significant potential for misdiagnosis was detected for some reported results. Methods were identified that do give sensitive, accurate, and reproducible results.

Airborne and dietary allergens in atopic eczema: a comprehensive review of diagnostic tests.

Aeroallergens and food allergens are relevant eliciting factors of atopic eczema. This article focuses on the methods used for diagnosis in patients with suspected allergy to airborne or dietary allergens and who are suffering from atopic eczema. In addition to classical tests of IgE-mediated hypersensitivity (intracutaneous or in vitro testing), the role of provocation procedures is described. For aeroallergens, the atopy patch test yields the most specific results with regard to clinical history as compared with classical methods. For food allergens and pseudoallergic reactions to additives, this holds true for the double-blind, placebo-controlled food challenge. The methods and their limitations are discussed from a practical point of view.

Evaluation of IgG RAST FEIA for the assay of venom-specific IgG antibodies during venom immunotherapy.

BACKGROUND: Successful venom immunotherapy (VIT) in Hymenoptera allergy is usually associated with a strong increase in venom-specific serum IgG antibodies (sIgG). METHODS: We evaluated a new commercial test for the assay of sIgG (Pharmacia CAP Systemtrade mark IgG RAST(R) FEIA; FEIA), in comparison with a conventional ELISA technique. Sera from 40 bee- and 40 Vespula-allergic patients were analyzed by FEIA and ELISA before and 3 months after starting VIT. RESULTS: The correlation between sIgG obtained with the two methods was significant: r = 0.862, p<0.0001 for bee venom (BV), r = 0.861, p<0.0001 for Vespula venom (VV). The geometric mean values obtained with FEIA were higher for both venoms (BV p = 0.03; VV p<0. 01). A highly significant increase (p<0.0001) was observed during VIT with both methods. This increase was concordant in 93% of VV- and 90% of BV-treated patients. Intra- and interassay relative coefficients of variation were below 10% for FEIA. CONCLUSION: IgG RAST FEIA is a reproducible and sensitive method for the assay of venom-specific sIgG.

[Evaluation of the quality of 2 techniques for measuring IgE: the Pharmacia CAP System (Pharmacia & Upjohn Diagnostics) and Alastat (DPC-France)]. / Evaluation de la qualité de deux techniques de dosage d'IgE: Pharmacia CAP System (Pharmacia & Upjohn Diagnostics) et Alastat (DPC-France).

The reliability of results given by specific IgE dosages technology is conditioned by the quality of the technology used. Analytical comparison of two techniques (Pharmacia CAP System and Alastat-DPC) shows important behaviour differences in the evaluation of the precision/reproducibility of results and in dilution tests. The Pharmacia CAP System demonstrates qualities comparable to those awaited from any immunodiagnostic test. The Alastat techniques shows high imprecision and the dilution tests make us wonder about calibration and/or ability of the support used to fix all epitopes.

The screening RAST: is it a valid concept?

Dr. William King in 1982 advocated the use of a "miniscreen" panel of six antigens to cost-effectively initiate allergy testing. In a study of 100 consecutive patients, we found that a "midiscreen" of nine antigens was more sensitive and efficient and more accurately identified negative responders. However, the miniscreen was also effective if adjusted for regional antigen differences.

Levels of eosinophil cationic protein and myeloperoxidase from chronic middle ear effusion in patients with allergy and/or acute infection.

BACKGROUND AND OBJECTIVE: Allergy may play a role in the middle ear inflammation that leads to otitis media with effusion. The purpose of this study was to determine whether an elevated mediator correlated with the patient's disease and thus could be used to differentiate allergy vs. infection as the cause of the middle ear inflammation. METHODS: WE evaluated 57 individuals with otitis media with effusion, 32 with persistent effusion but no recent acute infection, 14 with recent infection and purulent otitis media with effusion, and II healthy subjects. The mediator activity of eosinophils and neutrophils in effusion was studied in patients characterized as having allergy by positive intradermal skin test results and positive radioallergosorbent test results. Eosinophils were characterized by measurement of eosinophil cationic protein in the effusion. Neutrophils were characterized by measurement of myeloperoxidase in the effusion. The levels of eosinophil cationic protein and myeloperoxidase in patients with and without allergy were correlated to patient history. RESULTS: Significantly elevated levels of both eosinophil cationic protein and myeloperoxidase indicated that inflammation in the ear of patients with otitis media with effusion was characterized by a pronounced involvement of both eosinophils and neutrophils. Eighty-nine percent of all patients with disease had allergy. A higher ratio of myeloperoxidase to eosinophil cationic protein in patients with purulent otitis media with effusion indicated that in patients with a superimposed acute infection, neutrophil activity was increased even further. The level of eosinophil cationic protein was elevated only during the effusion of patients with allergies as compared with controls (p < 0.01). Among 29 cases of nonpurulent otitis media with effusion, 96.5% had allergic immune-mediated disease proved by skin testing, which was related clinically to their ear disease. Eighty-nine percent (89.6%) of these patients had eosinophil cationic protein levels greater than 10 microgram/L. CONCLUSION: Middle ear eosinophil cationic protein may be used as a marker of related allergy.

Standardization of allergenic extracts by basophil histamine release.

BACKGROUND: Allergenic extracts are standardized by using the ID50EAL (Intradermal Dilution for 50 mm sum of Erythema Determines the Allergy Unit) skin test technique to assign allergy units to reference preparations. When new batches of extracts are manufactured, they are compared with the reference by RAST inhibition or other in vitro techniques. This study was designed to determine whether basophil histamine release might be an additional useful in vitro test for the standardization of allergenic extracts. METHODS: Basophil histamine release, skin tests, and RAST were compared using several different allergenic extracts at many different strengths. Allergy units were calculated using skin test results and basophil histamine release. RESULTS: Basophil histamine release, skin tests, and RAST correlated well (P < .01). Basophil histamine release-derived allergy units and skin test-derived allergy units were highly correlated (P < .01). CONCLUSION: Basophil histamine release appears to be a useful tool for the standardization of allergenic extracts.

Specific IgE determination using the CAP system: comparative evaluation with RAST.

The CAP system's main contribution is its solid phase, which consists of a cellulose polymer activated within a capsule (the ImmunoCAP). This solid phase can bind more protein to it, and, in addition, the conditions of reaction seem to make the system more sensitive at detecting antibodies to certain antigens. It is therefore important to assess the new analytical and diagnostic performance in comparison with previous systems. In this context, we studied the reliability and comparison with the RAST and with skin tests carried out on 144 pediatric patients. Skin tests and specific IgE for radioimmunological RAST (radio-allergosorbent test) and for the fluoroimmunological CAP system were performed on all the patients. The RAST/CAP correlation quotients for the different allergens tested varied between 0.971 and 0.991. Diagnostic sensitivity increased for all the allergens studied and specificity remained unchanged. The system provides reliable results, with better diagnostic capacity than RAST, but it must be quantified for each allergen because its results are not interchangeable.

Standardization of pollen allergens of Parthenium hysterophorus and selection of an in-house reference extract.

A standardized in-house reference extract from the pollen of Parthenium hysterophorus, which is responsible for the high incidence of allergic rhinitis in India, was generated and examined by skin test, radio-allergosorbent test inhibition and isoelectric focusing. Parthenium reference allergen discs and positive reference serum were also generated. These reference reagents could not only be used for the quantitation of Parthenium-specific IgE in the sera of rhinitis patients but also for the evaluation of allergenic activity (relative potency and lot-to-lot variation) of different batches of Parthenium pollen.

Standardized extracts.

Until recently, physicians practicing allergy have not had the benefit of using allergenic extracts with potencies expressed in uniform, meaningful units. Technologic advances have provided accurate and reproducible methods for qualitative and quantitative analyses of these products, and assignment of meaningful units can be accomplished by using a threefold serial dilution titration skin test and appropriate in vitro methods. Although the perfect method for standardization of allergenic extracts has not yet been developed and some confusion has been created in the process, significant improvement in the quality of allergenic extracts has resulted. Collaborative efforts of regulatory agencies, manufacturers, specialty organizations, and clinicians should continue to produce standardized extracts that offer significant advantages to physicians practicing allergy.

In vitro testing methodologies. Evolution and current status.

Almost 25 years after the development of the radioallergosorbent test, in vitro technology has become firmly established as an outstanding diagnostic tool for physicians treating the allergic patient. The American Academy of Otolaryngic Allergy (AAO), in its most current position statement, endorses the modified RAST as reproducible, sensitive, and specific. A new organization called the American In Vitro Allergy and Immunology Society (AIVAIS) has been formed to promote and monitor the use of these tests. Both societies suggest that in vitro tests should be part of a complete clinical evaluation by a well informed physician knowledgeable about the contraindications and limitations of in vitro technology. Such physicians should be capable of incorporating this information into responsible, judicious, and effective management of allergic patients. Only after the correct diagnosis has been made can appropriate treatment be started. Whichever route you choose, you and your patients can benefit from the use of in vitro allergy testing.

Comparative evaluation of RAST and MAST-CLA for six allergens for the diagnosis of inhalant allergic disease in 232 patients.

The aim of this study was to compare a recent multiple allergosorbent chemiluminescent assay (MAST-CLA) with the RAST for the diagnosis of inhalant allergic disease in 232 patients with rhinitis and/or bronchial asthma. As judged by concordance of clinical history, skin prick tests to a range of six allergens common to our geographic area, and by nasal provocation tests, 70 patients were non-allergic and 162 allergic: 70 to grasses, 46 to mites, four to mugwort, eight to pellitory, and 34 were sensitive to several allergens. In our patient sample that, among other things, comprises subjects with fairly rare monoallergies, MAST-CLA testing gave results which closely corresponded to positive RAST for the allergens studied, and demonstrated a close correlation with the diagnosis of inhalant-specific allergy. Our results showed that, for overall allergens, MAST-CLA was lightly less sensitive and more specific than RAST (the two in vitro tests gave an identical overall efficiency).