Clinical evaluation of a new in-vitro assay for specific IgE, the immuno CAP system

The Pharmacia CAP system is a new assay for specific IgE characterized by a new solid phase (hydrophilic polymer encased in a capsule). The CAP results were compared to those of the Phadebas IgE RAST and skin-prick tests (SPT) performed in 145 subjects suffering from rhinitis and/or asthma with eight common inhalant allergens (total number of tests = 1160). Concording CAP/RAST results were found in 91% of the tests. The CAP was positive in 78% and the RAST in 65% of the positive SPT. Conversely, the CAP was negative in 90.6% and the RAST in 96.4% of the negative SPT. A pattern negative SPT, negative RAST and positive CAP' was found in 56 tests (40 subjects): in four such subjects, the CAP positivity was confirmed by a positive IgE crossed radioimmunoelectrophoresis. Three borderline positive results were found among 240 negative controls (serum from cord blood or non-atopics). These data indicate that compared with SPT the CAP system is (a) more sensitive than the Pharmacia RAST and (b) does not seem less specific.     

A computerized micro-ELISA assay for allergen-specific IgE antibodies

A computer-normalized micro-ELISA assay employing horseradish peroxidase and polystyrene microtiter plates (IP assay) for allergen-specific IgE antibodies is described. The specificity of the assay was confirmed by heat inactivation and multiple absorption experiments. Individual allergen blanks were used to account for the variability in the nonspecific binding among various allergens. The results obtained in milliunits of absorbance were normalized by a computer protocol using four reference sera. The coefficients of variation for the intraassay and interassay reproducibility ranged from 1.49 to 9.33% and 7.11 to 15.19%, respectively. Direct correlations between the results of the IP and the RAST assays (Absorbance vs. bound radioactivity) were excellent; the values for correlation coefficients for six pollens (June grass, Bermuda grass, Short ragweed, English plantain, Oak and Box elder), house dust mite, and two epidermal (cat and dog) allergens ranged from 0.88 to 0.99. Correlation between the two assays was lacking for the Alternaria and Penicillium mold allergens (r values: Alternaria 0.32, Penicillium 0.5). For sera from patients with a positive history and positive skin tests challenged with 11 allergens cited above, the IP assay detected low levels of specific IgE in many cases where the RAST assay was negative (from 10% for June grass to 48% for Alternaria and Penicillium mold antigens). The RAST positivity accompanied by the IP negativity, by contrast, was rarely seen. The superior sensitivity of the IP assay for specific IgE antibodies was achieved by lowering the background "noise" in the IP assay. This was attributed to the following factors: (1) use of individual allergen blanks; (2) 1:5 dilution of the serum sample that minimizes interference from high levels of total IgE antibodies and from allergen-specific IgG antibodies; (3) nonporous nature of the polystyrene immunosorbent surface; and (4) the use of a computer data reduction system to normalize the assay results.     

Experimental studies on red cell-based assays for total IgE and allergen-specific IgE

The potential of red cell-based assays for IgE and allergen-specific IgE has been examined using mouse monoclonal anti-human IgE antibodies and chimaeric human IgE anti-4-hydroxy-3-iodo-5-nitrophenacetyl. Experiments concerned with developing a red cell IgE antibody-capture assay for allergen-specific IgE have pointed to the advantages of presenting the allergen on a second agglutinable red cell.     

Hydrocortisone enhances allergen-specific IgE production by peripheral blood mononuclear cells from atopic patients with high serum allergen-specific IgE levels.

BACKGROUND: Although there is convincing evidence that human B cells can be induced to produce IgE by a combination of interleukin 4 (IL-4) and hydrocortisone (HC) in atopic subjects, it is still uncertain if this performs the same functions in allergen-specific IgE synthesis. OBJECTIVE: This study was designed to investigate the differences of IgE regulation between atopics and nonatopics, interactions of HC with IL-4, and the correlation between in vitro total IgE, allergen-specific IgE synthesis and serum IgE levels. METHODS: Peripheral blood mononuclear cells (PBMCs) from 16 atopic asthma patients sensitive to Dermatophagoides farinae and seven nonatopic controls were cultured with IL-4 and/or HC. Total IgE and D. farinae-specific IgE in culture supernatant were measured by ELISA and FAST. RESULTS: IL-4 increased total IgE synthesis in PBMCs from both atopics and nonatopics, whereas, HC had this effect only in some atopics who showed spontaneous IgE production in vitro. HC acted synergistically with IL-4 in total IgE synthesis. Their effects were more remarkable in cases with lower total serum IgE levels. PBMCs from eight of 16 atopics produced D. farinae-specific IgE in vitro either spontaneously or by IL-4 and/or HC. HC had more profound effects than IL-4 in these patients. They also showed higher total IgE synthesis by HC, and higher specific serum IgE levels than the others. IL-4 and/or HC did not induce any D. farinae-specific IgE synthesis by PBMCs from nonatopics. CONCLUSION: HC had a more profound effect than IL-4 on the induction of D. farinae-specific IgE synthesis in atopic patients with high serum allergen specific IgE levels. Further studies to determine the causes of these effects, such as the presence of long lived allergen specific B cells as the result of the priming effect of IL-4 in vivo, may be needed.     

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.     

Evaluation of allergen-specific IgE antibodies by a newly developed mast allergy system

MAST, which stand for multiple antigen simultaneous test, uses enzyme-linked anti-human IgE and chemiluminogenic substate to determine IgE. This system is characterized by simultaneous analysis of multiple allergen items, up to 35, together with total IgE determination. We evaluated usefulness of this MAST system using 191 serum samples obtained from patients with bronchial asthma, allergic rhinitis and/or atopic dermatitis. It was found that there were statistically significant correlations between IgE antibody quantification by MAST and RAST in 24 out of 35 allergen items, with correlation coefficients more than r = 0.60. These included Dermatophagoides farinae and pteronyssinus, Japanese cedar pollen, orchard grass, Alternaria, Candida as inhalant allergens; egg white, milk, soybeans, wheat and rice as food allergens. It was also evaluated how many allergen-specific IgE antibodies could be detected in one serum sample. More than six allergen-specific IgE antibodies were simultaneously detected in 33% of 191 cases, indicating the importance of multiple-allergen analysis. These results indicate the clinical usefulness of the MAST allergy system in detecting IgE antibodies in allergic subjects.     

Characterization of polyclonal allergen-specific IgE responses by affinity distributions

Polyclonal IgE responses have been previously characterized by allergen-specific antibody levels and by identification of amino acid sequences related to immunodominant epitopes. However, the binding affinities related to these antibody families are not well known. Using sera from donors with known sensitivities to ragweed or house dust mite allergens, we studied the binding reactions between the purified allergens Amb a 1 and Der p 1 and allergen-specific IgE's by determining affinity distribution functions. The distributions of binding affinities only exhibited a few dominant reactions indicated by peaks in an affinity distribution display. In all the donors tested, there were two dominant peaks and in 2/3 of the cases there was a third peak for both Amb a 1 and Der p 1. We further characterized the polyclonal interactions between IgE and Der p 1 by inhibiting the specific binding of IgE using peptide fragments known to be constituents of Der p 1 epitopes. Each peptide inhibited only a single peak in the affinity distributions. It would appear that the peaks in the affinity distribution represent antibodies directed to single epitopes. These results suggest that in our atopic population the response is surprisingly uniform. The bulk of the IgE response (70-80%) is of high affinity (10(8)-10(11) M(-1)) and directed towards a few epitopes. The relative affinities towards epitopes seem to be determined by the structure of the epitope and not variations of individuals' immune responses.     

Longitudinal trends of total and allergen-specific IgE throughout childhood

Background:  The development and the quantitative relationship between allergen-specific IgE (S-IgE) responses and total IgE (T-IgE), during childhood and adolescence have not been described and understood in detail. The objective of this study was to describe and compare the longitudinal trends of serum levels of S-IgE and T-IgE during childhood.
Methods:  We analysed data from participants in the MAS birth cohort study at 2, 5, 7 and 10 years of age ( n  = 273) and at 1, 3, 5, 6, 7, 10 and 13 years ( n  = 84). Total-IgE and the overall level of specific-IgE against nine locally relevant airborne and food allergens were determined by FEIA (ImmunoCAP). Allergic rhino-conjunctivitis and asthma were ascertained by questionnaires.
Results:  Longitudinal patterns of T-IgE levels from age 1 to 13 years were highly heterogeneous (declining, flat or increasing with different profiles). From 5 years of age, logarithmic (log₁₀) transformed values of T-IgE and of S-IgE levels tend to follow a parallel trend, so that their relation remained constant throughout school age. A flat trend of T-IgE vs a constantly increasing trend of T-IgE was associated with a low or, respectively, high rate of wheezing at 13 years of age.
Conclusions:  Beginning at the age of 5 years, total serum IgE levels in children from an industrialized country evolved in parallel with overall S-IgE levels. Therefore, variations in T-IgE levels at school age closely reflect variations in overall S-IgE levels. Further studies are required to strengthen the biological and clinical implication of this novel finding.     

Establishment and characterization of a murine model for allergic asthma using allergen-specific IgE monoclonal antibody to study pathological roles of IgE

Allergen-specific IgE has long been regarded as a major molecular component of allergic asthma. Although IgE plays a central role in the early asthmatic response, its roles in the chronic phase, such as the late asthmatic response, airway hyperresponsiveness (AHR), and airway remodeling (goblet cell hyperplasia and subepithelial fibrosis) have not yet been defined well. In this study, we investigated the hypothesis that chronic responses could be induced by IgE-dependent mechanisms. BALB/c mice passively sensitized with an ovalbumin (OVA)-specific IgE monoclonal antibody (mAb) were repeatedly challenged with intratracheal administration of OVA. The first challenge induced early phase airway narrowing without any late response, but the fourth challenge caused not only an early but also a late phase response, AHR, and goblet cell hyperplasia. Macrophages, lymphocytes and neutrophils, but not eosinophils, were significantly increased in the lung 24 h after the fourth challenge. Interestingly, levels of OVA-specific IgG1 in serum increased by multiple antigen challenges. A C3a receptor antagonist inhibited the late asthmatic response, AHR, and infiltration by neutrophils. In contrast, no late response, goblet cell hyperplasia, inflammatory cells, or production of IgG1 was observed in severe combined immunodeficient mice. On the other hand, seven challenges in BALB/c mice induced subepithelial fibrosis associated with infiltration by eosinophils. In conclusion, the allergic asthmatic responses induced by passive sensitization with IgE mAb can provide a useful model system to study the pathological roles of IgE in acute and chronic phases of allergic asthma.     

Inhibition of allergen-specific IgE reactivity by a human Ig Fcgamma-Fcepsilon bifunctional fusion protein

BACKGROUND: Coaggregating FcepsilonRI with FcgammaRII receptors holds great potential for treatment of IgE-mediated disease by inhibiting FcepsilonRI signaling. We have previously shown that an Fcgamma-Fcepsilon fusion protein, human IgG-IgE Fc fusion protein (GE2), could inhibit FcepsilonRI-mediated mediator releases in vitro and in vivo. OBJECTIVE: We sought to test whether GE2 was capable of blocking mediator release from FcepsilonRI cells sensitized with IgE in vivo or in vitro before exposure to GE2, a critical feature for GE2 to be clinically applicable. METHODS: GE2 was tested for its ability to inhibit Fel d 1-induced mediator release from human blood basophils from subjects with cat allergy, human lung-derived mast cells, human FcepsilonRIalpha transgenic mice sensitized with human cat allergic serum, and rhesus monkeys naturally allergic to the dust mite Dermatophagoides farinae. RESULTS: Basophils from subjects with cat allergy and lung mast cells degranulate when challenged with Fel d 1 and anti-IgE, respectively. GE2 itself did not induce mediator release but strongly blocked this Fel d 1- and anti-IgE-driven mediator release. GE2 was able to block Fel d 1-driven passive cutaneous anaphylaxis at skin sites sensitized with human serum from subjects with cat allergy in human FcepsilonRIalpha transgenic mice, but by itself, GE2 did not induce a passive cutaneous anaphylaxis reaction. Finally, GE2 markedly inhibited skin test reactivity to D farinae in monkeys naturally allergic to this allergen, with complete inhibition being observed at 125 ng. CONCLUSION: GE2 is able to successfully compete for FcepsilonRs and FcgammaRs on cells presensitized in vitro and in vivo and lead to inhibition of IgE-mediated reactivity through coaggregation of FcepsilonRI with FcgammaRII.     

Monoclonal antibodies to three distinct epitopes on human IgE: their use for determination of allergen-specific IgE

Three different monoclonal antibodies (MAb) against human immunoglobulin E have been obtained which specifically bind to human myeloma and polyclonal IgE. The antibodies showed high avidities for soluble IgE (0.7 X 10(9) to 3.3 X 10(9) M-1). These MAb defined three distinct epitopes on IgE. A mixture of these antibodies in combination with an 125I-labelled anti-mouse Kappa chain MAb has been used to measure allergen-specific IgE. This determination was performed by a solid-phase radioimmunoassay using allergen extracts coated to either chemically activated paper discs or to polyvinyl chloride wells. This method is 4-10 times more sensitive than other previously reported procedures. A similar technique has also been applied to detect individual allergens in immunoblots of allergen extracts.     

Clustering of fungal allergen-specific IgE antibody responses in allergic subjects

Statistical analysis of specific IgE immune responses to a panel of nine allergens from 2,094 patients demonstrated significant clustering between Aspergillus fumigatus, Alternaria alternata, and Cladosporium herbarum, not explained by crossreactive elements. This may represent a linked, high responder status to these fungal antigens in the allergic population.     

Amplification of the enzyme-linked immunosorbent assay for measuring allergen-specific IgE and IgG antibody

The amplified enzyme-linked immunosorbent assay (a-ELISA) has been successfully applied to the measurement of allergen-specific IgE and IgG antibodies. Data presented indicate a high positive correlation among measurements of IgE-specific antibodies to ragweed antigen E using the a-ELISA, RAST and radiometric assay of Zeiss. Similarly, a high correlation between allergen-specific IgG antibodies measured by the a-ELISA and Farr assay was also observed. Data presented indicate that the measurement of serum IgE antibodies in the linear region of the ELISA titration plot is unaffected by competition with IgG antibodies. The assay is sensitive, reliable, and without risk to laboratory personnel. It is capable of measuring both IgE and IgG antibodies in patients using the same assay system.     

The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum

BACKGROUND: For the detection of allergen-specific IgE in serum, IgE-binding assays such as the radioallergosorbent assay (RAST) are commonly used. In this study, the applicability and sensitivity of the stripped basophil histamine release bioassay was investigated and compared to the RAST. METHODS: Basophils were stripped of their IgE by an acidic buffer, sensitized by human serum and stimulated by allergen with or without interleukin (IL)-3. The histamine release was determined by fluorometric analysis. RESULTS: We showed that for enhancement of the maximal histamine release and the sensitivity of the stripped basophil assay, the priming cytokine IL-3 can be added to the basophils simultaneously to the stimulus. Preincubation of the cells with IL-3, as described in other studies, was not necessary. The bioassay can be used to study the specificity of IgE-mediated reactions. Basophils sensitized by serum absorbed to a particular allergen did not respond to this allergen anymore. This method is very suitable to study cross-reactivity between allergens. The results obtained in the bioassay were comparable to those obtained in the RAST. Using the RAST, lower concentrations of allergen-specific IgE were detected than in the bioassay. However, sera containing IgE against minor allergenic components were negative in the RAST, but strongly positive in the basophil assay. CONCLUSIONS: The stripped basophil histamine release bioassay is useful to complement and extend serological detection of allergen-specific IgE. Especially with sera containing IgE against minor components, this assay is more suitable than the RAST. Furthermore, in this assay, the dependency of IgE and of allergen-specific IgE in reactions can be studied in more detail.