Piezoelectric quartz crystal based label-free analysis for allergy disease

This work presents a piezoelectric (Pz) quartz crystal based label-free quantification of total IgE and allergen-specific IgE in human sera for allergy testing. An evaluation of the different brands of crystals was first initiated with respect to variability in mass sensitivity, frequency measurement reliability and stability, and surface roughness. Thereafter, for total IgE quantification. a direct assay format was adopted. By means of thioctic acid (TA) and coupling reagents, anti-human IgE antibodies were immobilized on AT-cut Pz crystals (10 MHz). The modified crystals could detect serum IgE directly corresponding to a downward frequency shift. The results showed that silver-coated crystals as compared with their gold-coated counterparts provided approximately 1.5 times higher mass detection sensitivity for total IgE in the range of 5-300 IU/ml with a linear regression line, y = 1.8957 x + 1.5603, R2 = 0.995. For the detection of allergen-specific IgE, a sandwiched assay format was used. As the allergen-modified sensor surface captured various classes of associated antibodies (IgE, IgG, etc) and interfering serum proteins as well, the initial frequency shift downwards caused by sera sample incubation would not be proportional to specific IgE levels. Thus, following sample incubation, a second incubation step with secondary anti-human IgE was added to recognize IgE from other bound substances. The frequency shift after secondary antibody binding reflected the amount of allergen-specific IgE proportionally. Compared with 10 MHz crystals, the 20 MHz counterparts provided approximately four times higher mass detection sensitivity for allergen specific IgE in the range of 0.15-17.5 IU/ml with a linear regression line, y = 50.525 x + 107.777, R2 = 0.954. Total IgE and allergen specific IgE assay results of real patients' sera using the Pz sensors agreed well with those obtained by commercially available test kits with correlation coefficient 0.96-0.98. The possibility of regenerating the quartz crystals for further re-use was also dealt with.     

An automated multiplex specific IgE assay system using a photoimmobilized microarray

An automated microarray diagnostic system for specific IgE using photoimmobilized allergen has been developed. Photoimmobilization is useful for preparing microarrays, where various types of biological components are covalently immobilized on a plate. Because the immobilization is based on a photo-induced radical cross-linking reaction, it does not require specific functional groups on the immobilized components. Here, an aqueous solution of a photoreactive poly(ethylene glycol)-based polymer was spin-coated on a plate, and an aqueous solution of each allergen was microspotted on the coated plate and allowed to dry in air. Finally, the plate was irradiated with an ultraviolet lamp for covalent immobilization. An automated machine using these plates was developed for the assay of antigen-specific IgE. Initially, the patient serum was added to the microarray plate, and after reaction of the microspotted allergen with IgE, the adsorbed IgE was detected by a peroxidase-conjugated anti-IgE-antibody. The chemical luminescence intensity of the substrate decomposed by the peroxidase was automatically detected using a sensitive charge-coupled device camera. All the allergens were immobilized stably using this method, which was used to screen for allergen-specific IgE. The results were comparable with those using conventional specific IgE. Using this system, six different allergen-specific IgE were assayed using 10μL of serum within a period of 20min.     

Measurement of IgG-blocking antibodies: development and application of a radioimmunoassay.

A radioimmunoassay for measuring blocking antibodies has been developed. We used the ragweed antigen E system to show that the same blocking antibodies (IgG) measured by inhibition of antigen-induced leukocyte histamine release were precipitated in the binding assay (r8 = 0.96, p less than 0.001), thus validating a widely applicable technique for measuring blocking antibodies. Binding of phospholipase-A (Phos-A), the major allergen in honey bee venom, was also shown to correlate significantly with inhibition of histamine release. Hymenoptera (insect) hypersensitivity was used as a model to demonstrate application of the binding assay. Sera obtained from patients undergoing whole body extract therapy contained negligible amounts of specific blocking antibodies. Significantly higher blocking antibody titers to both whole honey bee venom and Phos-A were measured in sera drawn from patients immunized with whole venom. The use of the binding radioimmunoassay should facilitate management of allergic disease processes in which blocking antibodies are thought to be protective.     

IgE production after four routes of injections of Japanese cedar pollen allergen without adjuvant: crucial role of resident cells at intraperitoneal or intranasal injection site in the production of specific IgE toward the allergen

The production of specific IgE antibodies directed toward cedar pollen correlates well with the onset of allergic rhinitis; but the mechanisms of allergen recognition as nonself and Ig class switch to IgE by the immune system are still not fully understood. In the present study, we injected cedar pollen into mice through 4 different routes (intranasal (i.n.), intraperitoneal (i.p.), intravenous (i.v.), and subcutaneous (s.c.)) without adjuvant 1 to 3 times, and determined time-dependent changes in the total and specific serum IgE levels compared with those in the serum levels of other isotype Igs. After an i.p. or i.n. injection of allergen into the mice, they produced a 1.5-to 1.7-fold increase in total IgE, but none in IgG, IgM, or IgA antibodies in their serum, whereas an i.v. or s.c. injection of allergen was inactive as an inducer of total IgE antibodies. Upon a 2nd (s.c.) injection of the allergen into the i.p. or i.n. sensitized mice, a large amount of allergen-specific IgE antibodies was found in the serum. In the case of i.v. or s.c. sensitized mice, however, they produced total, but not specific, IgE antibodies; and a 3rd (s.c.) injection of the allergen resulted in a large amount of specific IgE antibodies in the serum. These results imply that resident cells at the i.p. or i.n. injection site may play a crucial role in the efficient production of total and specific IgE antibodies toward the allergen.     

The 1st step initiation essential for allergen‐specific IgE antibody production upon the 2nd step: Induction of non‐specific IgE+ small B cells containing secondly‐sensitized allergen‐specific ones in mice firstly‐sensitized with an allergen

There was a significant amount of non‐specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)‐specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain‐, but not mite feces‐ or pollen‐specific IgE⁺ cells and an i.n. injection of papain induced papain‐specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces‐specific IgE⁺ cells in the lymph nodes nor mite feces‐specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen‐specific IgE⁺ small B cells in the lymph nodes on Day 10, when non‐specific IgE Ab titers reached a peak in the serum, implying induction of related allergen‐specific IgE⁺ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen‐specific IgE Abs in the serum. These results indicate that when firstly‐sensitized non‐specific IgE⁺ small B cells in mouse lymph nodes include some secondly‐sensitized allergen‐specific ones, mice produce IgE Abs specific for the secondly‐injected allergen.

     

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.     

The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29–31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acid-long defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more α-arabinofuranosyl residues with some β-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked β-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.     

A rapid enhanced chemiluminescent immunoassay for allergen-specific IgE antibodies

An enhanced chemiluminescent immunoassay is described for the measurement of allergen-specific IgE antibodies. The assay is demonstrated for pollen from four grass species. A comparison was made between results obtained by this method and those obtained by the radioallergosorbent (RAST) procedure; a high degree of correlation (r = 0.95) was found for D. glomerata specific IgE. The assay is rapid and can be carried out in under 1 hour. The advantages of the luminescent assay as compared with the RAST procedure are discussed.     

Nasal Sensitization with Ragweed Pollen Induces Local-Allergic-Rhinitis-Like Symptoms in Mice

Recently, the concept of local allergic rhinitis (LAR) was established, namely rhinitis symptoms with local IgE production and negative serum antigen-specific IgE. However, the natural course of LAR development and the disease pathogenesis is poorly understood. This study investigated the pathophysiology of mice with allergic rhinitis that initially sensitized with ragweed pollen through the nasal route. Mice were nasally administrated ragweed pollen over consecutive days without prior systemic immunization of the allergen. Serial nasal sensitization of ragweed pollen induced an allergen-specific increase in sneezing, eosinophilic infiltration, and the production of local IgE by day 7, but serum antigen-specific IgE was not detected. Th2 cells accumulated in nose and cervical lymph nodes as early as day 3. These symptoms are characteristic of human LAR. Continual nasal exposure of ragweed pollen for 3 weeks resulted in the onset of classical AR with systemic atopy and adversely affected lung inflammation when the allergen was instilled into the lung. Fcer1a ^−/− mice were defective in sneezing but developed normal eosinophilic infiltration. Contrary, Rag2 ^−/− mice were defective in both sneezing and eosinophilic infiltration, suggesting that T cells play a central role in the pathogenesis of the disease. These observations demonstrate nasal allergen sensitization to non-atopic individuals can induce LAR. Because local Th2 cell accumulation is the first sign and Th2 cells have a central role in the disease, a T-cell-based approach may aid the diagnosis and treatment of LAR.     

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.     

A quantitative analysis of cedar pollen-specific immunoglobulins in nasal lavage supported the local production of specific IgE, not of specific IgG

Many studies have proved the relevance of local immune responses, rather than systemic immunity, to the pathogenesis of allergic rhinitis. Indeed, allergen-specific B lymphocyte undergoes class switching to IgE in situ. However, the relative contribution of in situ production to the amount in serum is still ambiguous. Here, a quantitative comparison of the local concentration of allergen-specific IgE with the systemic concentration was explored for the estimation. Among seasonal rhinitis patients, total and Japanese cedar pollen (JCP)-specific IgE, IgA and IgG antibodies were quantified in nasal lavage fluid (NLF) and serum with the time-resolved fluorescence immunosorbent assay. Although the total amounts of IgE and IgG classes in the NLF, which were apparently passive discharge from the mucosal tissue, were smaller and variable, the relative proportions of JCP-specific antibodies could be quantitatively compared between NLF and serum or between subjects. The proportions of specific IgE in the NLF were remarkably higher than in serum (average 13.2-fold) in most subjects, which strongly supported the predominant in situ production of the specific IgE and subsequent dilutions in the systemic circulations. Similar but smaller values were obtained for IgA (average 3.7-fold). In contrast, the specific proportions of IgG in the NLF were surprisingly consistent with serum (average 1.0-fold), suggesting that the specific IgG was mostly produced in the downstream lymphoid organs. The local productions of specific IgE would encourage the topical therapies and the usage of the NLF for the diagnosis of allergic rhinitis.     

Immunoregulatory function of specific IgG. I. Results in nonatopic subjects

Nonatopic subjects immunized with short ragweed developed both reaginic IgE and blocking IgG antibodies to the antigen. Administration of 250 microgram-specific ragweed IgG antibodies 1 week before, simultaneously, or up to 2 weeks after start of immunization with ragweed extract abrogated the production of anti-ragweed reaginic antibodies which was measured by skin reactivity. Formation of specific ragweed IgG antibodies was unaffected by IgG treatment. These results indicate selective regulation of an immune response in humans by IgG.     

Effects of Nasal Corticosteroids on Boosts of Systemic Allergen-Specific IgE Production Induced by Nasal Allergen Exposure

BackgroundAllergen exposure via the respiratory tract and in particular via the nasal mucosa boosts systemic allergen-specific IgE production. Intranasal corticosteroids (INCS) represent a first line treatment of allergic rhinitis but their effects on this boost of allergen-specific IgE production are unclear.AimHere we aimed to determine in a double-blind, placebo-controlled study whether therapeutic doses of an INCS preparation, i.e., nasal fluticasone propionate, have effects on boosts of allergen-specific IgE following nasal allergen exposure.MethodsSubjects (n = 48) suffering from grass and birch pollen allergy were treated with daily fluticasone propionate or placebo nasal spray for four weeks. After two weeks of treatment, subjects underwent nasal provocation with either birch pollen allergen Bet v 1 or grass pollen allergen Phl p 5. Bet v 1 and Phl p 5-specific IgE, IgG_1–4, IgM and IgA levels were measured in serum samples obtained at the time of provocation and one, two, four, six and eight weeks thereafter.ResultsNasal allergen provocation induced a median increase to 141.1% of serum IgE levels to allergens used for provocation but not to control allergens 4 weeks after provocation. There were no significant differences regarding the boosts of allergen-specific IgE between INCS- and placebo-treated subjects.ConclusionIn conclusion, the application of fluticasone propionate had no significant effects on the boosts of systemic allergen-specific IgE production following nasal allergen exposure.

Trial Registration

http://clinicaltrials.gov/ {"type":"clinical-trial","attrs":{"text":"NCT00755066","term_id":"NCT00755066"}}NCT00755066     

Determination of Specific Class E Immunoglobulins to Bet v 1 Birch Allergen by the Immuno-PCR Method

The aim of the work is the development of a method of detection of specific class E immunoglobulins (IgE) to the main Bet v 1 birch allergen based on immuno-PCR (iPCR). The recombinant Bet v 1 allergen was obtained in E. coli cells. Its ability to bind to specific IgE was confirmed by enzyme-linked immunosorbent assay (ELISA) using previously characterized sera of individuals with an allergic reaction to birch pollen and control sera in individuals, in which the reaction to this allergen is absent. Based on the obtained recombinant protein, the method of iPCR analysis of specific IgE to Bet v 1 was developed. It was demonstrated that iPCR sensitivity is comparable to ELISA sensitivity, and the titration curves of specific sera in iPCR (unlike those in ELISA) demonstrate a linear dependence; this makes the developed method preferable for quantitative estimation of specific IgE in sera as compared with ELISA.     

Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy

Type I allergy is characterized by the development of an initial Th2-dependent allergen-specific IgE response, which is boosted upon a subsequent allergen encounter. Although the immediate symptoms of allergy are mainly IgE-mediated, allergen-specific T cell responses contribute to the late phase as well as to the chronic manifestations of allergy. This study investigates the potential of costimulation blockade with CTLA4Ig and an anti-CD154 mAb for modifying the allergic immune response to the major timothy grass pollen allergen Phl p 5 in a mouse model. BALB/c mice were treated with the costimulation blockers at the time of primary sensitization to the Phl p 5 allergen or at the time of a secondary allergen challenge. Costimulation blockade (CTLA4Ig plus anti-CD154 or anti-CD154 alone) at the time of sensitization prevented the development of allergen-specific IgE, IgM, IgG, and IgA responses compared with untreated but sensitized mice. However, costimulation blockade had no influence on established IgE responses in sensitized mice. Immediate-type reactions as analyzed by a rat basophil leukemia cell mediator release assay were only suppressed by early treatment but not by a costimulation blockade after sensitization. CTLA4Ig given alone failed to suppress both the primary and the secondary allergen-specific Ab responses. Allergen-specific T cell activation was suppressed in mice by early as well as by a late costimulation blockade, suggesting that IgE responses in sensitized mice are independent of T cell help. Our results indicate that T cell suppression alone without active immune regulation or a shifting of the Th2/Th1 balance is not sufficient for the treatment of established IgE responses in an allergy.     

Measurement of absolute amounts of antigen-specific human IgE by a radioallergosorbent test (RAST) elution technique.

A technique for the absolute quantification of antigen-specific human IgE is described. It employs elution of a calculable amount of antigen-specific IgE from an allergosorbent-antibody complex by means of alkaline pH treatment, followed by measurement of the IgE content of the eluate with a modified radioimmunosorbent test (RIST). With this method IgE antibody directed against the benzylpenicilloyl determinant of penicillin (BPO) was measured quantitatively in sera from seven penicillin allergic patients. IgE specific for ragweed antigen E was measured in sera from 33 ragweed allergic patients. Values obtained for IgE anti-BPO ranged from 19 to 1806 ng/ml and comprised from 1.3 to 27.5% of total serum IgE. Values of IgE anti-antigen E ranged from 9 to 1807 ng/ml, comprising from 3 to 84% of total serum IgE. Excellent correlation (r = 0.99; p less than 0.001) was obtained for both antigen systems between values determined by the RAST elution technique and by simple RAST assay with interpolation from a reference serum of known specific IgE content as determined by the elution technique may be needed only for primary standardization of reference sera.     

Humoral and cellular cross-reactivity between Amb a 1, the major ragweed pollen allergen, and its mugwort homolog Art v 6

Ragweed and mugwort are closely related weeds that represent the major cause of pollen allergy in late summer. Concomitant sensitization and clinical cross-reactivity frequently occur in subjects who are coexposed to both pollen species, and have implications for diagnosis and specific immunotherapy. Molecules involved in this cross-reactivity might be Amb a 1, the major ragweed pollen allergen, and Art v 6, a highly homologous allergen from mugwort. Therefore, we investigated the IgE and T cell response to Art v 6 of 60 weed pollen-allergic patients and assessed its immunological cross-reactivity with Amb a 1. Results of ELISA inhibition experiments suggested that both allergens are largely cross-reactive, but Amb a 1 possesses more IgE epitopes than Art v 6. In patients with IgE to both allergens, Amb a 1-induced T cell lines and clones responded weakly to Art v 6. Moreover, Art v 6-induced T cell lines responded stronger to Amb a 1. T cell epitope mapping of Art v 6 revealed that it contains only a few cross-reactive epitopes, which is opposed to the multiple T cell-activating regions present in Amb a 1. In summary, Amb a 1 can elicit more diverse allergen-specific IgE and T cell responses than Art v 6 and dominates the cross-reactivity with its homolog. Nevertheless, Art v 6 can act as a primary sensitizing allergen in areas with high mugwort pollen exposure, and consequently may facilitate sensitization to Amb a 1 by epitope cross-recognition of T and B cells.     

Ragweed in the Czech Republic

During the last years, a well documented expansion ofragweed (Ambrosia artemisiifolia L., Ambrosia trifida L.) over the Mediterranean andtemperate Europe has been in progress. The currentdistribution of ragweed plants in the Czech Republicis summarized and the ragweed pollen concentration asmonitored by 12 pollen stations in the country isdiscussed. The present situation in the ragweed pollensensitization among children and adults with pollenallergy in Brno is described. So far no dangerousexpansion of ragweed plants in our country has beenobserved. Ragweed pollen concentration is occasionallysignificant in the Brno station only, other pollenstations are reporting insignificant amounts ofragweed pollen during August-September periods,although there has been a steady increase in ragweedpollen concentration in the Prague area over the lastfive years. Skin prick tests and/or specific IgEmeasurements with ragweed allergen were performed on94 children with pollen allergy in the Brno region in1995 and on 206, 210 and 229 adult allergic patientsin 1995, 1996 and 1997, respectively. Positive skinreaction or positive specific IgE to ragweed was foundin 22% children and in 25% (1995), 19% (1996) and 25% (1997) adults with pollen allergy. It isconcluded that ragweed does not seem to represent anyimminent major threat to the allergic population inthe Czech Republic until now, however, it remains apotentially very dangerous allergen.     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.