Immunization with purified natural and recombinant allergens induces mouse IgG1 antibodies that recognize similar epitopes as human IgE and inhibit the human IgE-allergen interaction and allergen-induced basophil degranulation

Molecular characterization of allergens by recombinant DNA technology has made rapid progress in the recent few years. In the present study we immunized mice with aluminum hydroxide-adsorbed purified recombinant major timothy grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5), dog albumin, a major animal dander allergen, and proteins with low (beta-lactoglobulin) or no (ribulose diphosphate carboxylase) allergenic potential in humans. Allergens that bind high levels of IgE in humans (Phl p 1, Phl p 5, dog albumin) induced high IgE and IgG1 levels in mice, whereas proteins with little or no allergenic activity in humans failed to induce significant IgE and IgG1 levels in mice. Continuous immunization for a period of 27 wk resulted in the production of mouse IgG1 Abs that recognized recombinant allergen fragments/epitopes defined by IgE Abs of allergic patients. As a consequence, allergen-specific mouse Abs strongly inhibited human IgE binding to the allergens and suppressed the allergen-induced histamine release from human basophils. In summary, our data indicate that 1) the allergenic potency of a protein may be related to its overall immunogenicity and 2) prolonged immunization with single purified recombinant allergens induces protective IgG Abs. The presented experimental in vivo/in vitro system allows the evaluation of Ag preparations (e.g., recombinant allergens) to be used for immunotherapy in humans.     

Immunological characterization of Asp f 2, a major allergen from Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis

The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases.     

The effect of dry heat on mite, cat, and dog allergens

BACKGROUND: Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens. METHODS: Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60 degrees, 80 degrees, 100 degrees, 120 degrees, and 140 degrees C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA. RESULTS: For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120 degrees C, allergen levels were reduced to < 1% of control. Der p 2 was more heat stable, requiring 140 degrees C for 30-60 min to achieve > 99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast, Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140 degrees C. CONCLUSIONS: The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens; however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens.     

Immunoaffinity analysis of cross-reacting allergens by protein blotting

IgE antibodies from sera having reactivity against ryegrass pollen protein allergens, wheat endosperm protein allergens and also several other cereal protein allergens were adsorbed with either ryegrass pollen or the wheat/globulin fraction immobilised on solid phases and subsequently eluted with low pH buffer. The eluted antibodies were reacted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) blots of the different allergens. Antibodies adsorbed and subsequently eluted from the two allergen sources recognised different spectra of proteins in the ryegrass pollen and cereal allergen sources and indicated the degree of immunological cross-reactivity. Intra-species cross-reactivity of IgE antibodies was demonstrated employing similar methods to those used for the pollen and cereal allergens by using a recombinant allergen from the venom of the ant Myrmecia pilosula as the immunoadsorbent protein on the solid phase.     

Allergens of Aspergillus fumigatus and Candida boidinii share IgE-binding epitopes

From an Aspergillus fumigatus complementary deoxyribonucleic acid (cDNA) library displayed on phage surface, an allergen formally termed rAsp f 3 was cloned. The open-reading frame of the cloned gene for the allergen encodes a protein of 168 amino acids with a predicted molecular mass of 18.5 kD, showing 36% identity and 58% similarity to two peroxisomal membrane proteins of Candida boidinii. Recombinant Asp f 3 was expressed as a [His]6-tagged fusion protein in Escherichia coil at yields of 30 mg/L, and was purified by Ni(2+)-chelate chromatography. In an enzyme-linked immunosorbent assay (ELISA), serum IgE antibody reactivity to rAsp f 3 could be detected in 72% of 89 individuals sensitized to A. fumigatus, demonstrating that the protein represents a major allergen of the mold. IgE specific to rAsp f 3 and the two recombinant Candida proteins was further demonstrated by IgE-immunoblot analysis. IgE binding to rAsp f 3 could be inhibited in the ELISA by adding either of the recombinant Candida peroxisomal proteins to sera containing IgE directed against Asp f 3. Taken together, these observations prove that the Asperigillus allergen and the two Candida proteins share IgE-binding epitopes.     

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies. The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein. In contrast to other E. coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E. coli total protein, the percentage varying with culture conditions. The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium. The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography. The purified recombinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations.     

Molecular cloning of German cockroach (Blattella germanica) allergens

Allergens produced by cockroaches (CRs) are an important cause of IgE antibody responses and asthma. Using molecular cloning and nucleic acid hybridization techniques, we have identified and sequenced several important allergens produced by the German CR (Blattella germanica) and studied their expression in the American CR (Periplaneta americana). Principal allergens include Bla g 2 (36-kD protein) and Bla g 4 (21-kD protein), to which 60-70% of CR-allergic patients make IgE antibodies. Bla g 2 is only expressed by B. germanica, whereas DNA encoding Bla g 4 is present in P. americana, but is not transcribed into mRNA. Sequence homology searches have identified Bla g 2 as an aspartic protease and Bla g 4 as a calycin. Other CR allergens that have been cloned include a glutathione transferase and a troponin. These studies will enable recombinant allergens to be expressed and used to investigate the role of CR allergens in asthma.     

Effects of IL-4 and IL-13 on total and allergen specific IgE production by cultured PBMC from allergic patients determined with recombinant pollen allergens

BACKGROUND: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. OBJECTIVE: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood mononuclear cells (PBMC) from pollen allergic patients and healthy control individuals. METHODS: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profilin (Bet v II) and recombinant timothy grass pollen allergens, Phl p I, Phl p II, and Phl p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. RESULTS: PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. CONCLUSION: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.     

Novel allergen structures with tandem amino acid repeats derived from German and American cockroach

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.     

Identification and cloning of prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris

Avocado, the fruit of the tropical tree Persea americana, is a source of allergens that can elicit diverse IgE-mediated reactions including anaphylaxis in sensitized individuals. We characterized a 32-kDa major avocado allergen, Prs a 1, which is recognized by 15 out of 20 avocado- and/or latex-allergic patients. Natural Prs a 1 was purified, and its N-terminal and two tryptic peptide sequences were determined. We isolated the Prs a 1 encoding cDNA by PCR using degenerate primers and 5'-rapid amplification of cDNA ends. The Prs a 1 cDNA coded for an endochitinase of 326 amino acids with a leader peptide of 25 amino acids. We expressed Prs a 1 in the yeast Pichia pastoris at 50 mg/liter of culture medium. The recombinant Prs a 1 showed endochitinase activity, inhibited growth and branching of Fusarium oxysporum hyphae, and possessed IgE binding capacity. IgE cross-reactivity with latex proteins including a 20-kDa allergen, most likely prohevein, was demonstrated, providing an explanation for the commonly observed cross-sensitization between avocado and latex proteins. Sequence comparison showed that Prs a 1 and prohevein had 70% similarity in their chitin-binding domains. Characterization of chitinases as allergens has implications for engineering transgenic crops with increased levels of chitinases.     

Ubiquitous structures responsible for IgE cross-reactivity between tomato fruit and grass pollen allergens

The simultaneous presence of IgE reactivity to tomato fruit and grass pollen allergens is evident in many patients with allergy and may be caused by cross-reactivity. Using sera from polysensitized patients with a positive enzyme allergosorbent test (EAST) result (score > 2), we tested reactivity to both allergen sources. IgE reactivity against both extracts was demonstrated in eight serum samples, and cross-reactivity was confirmed by the EAST inhibition assay. The structures responsible for this cross-reactivity were identified by Western blotting: five of the eight sera demonstrated a 16 kd protein in both extracts, which was identified as profilin. Additionally, seven of the eight sera showed IgE binding to epitopes on carbohydrate moieties, which contained alpha 1, 3 fucosylations. To determine the allergens of tomato fruit extract, we performed two-dimensional polyacrylamide gel electrophoresis blotting. We were able to demonstrate one highly concentrated and about 20 weaker proteins possessing terminal fucose residues. These are similarly found in grass pollen extracts. It is therefore postulated that the cross-reactivity is affected by profilins and similar carbohydrate determinants. If carbohydrate structures can provoke IgE cross-reactivity between phylogenetically distant species, such structures may play an important role in sensitization and mediator release. The ubiquitous nature of the IgE-binding determinants was studied by additional EAST inhibition tests with tomato allergen disks and extract from birch pollen, mugwort pollen, apple, and celery, leading to significant inhibitions among all these allergen sources. Epitopes exclusive to grass pollen and tomato have not been detected.     

Immunological and biological properties of Bet v 4, a novel birch pollen allergen with two EF-hand calcium-binding domains

We have isolated a cDNA clone coding for a birch pollen allergen, Bet v 4. The deduced amino acid sequence of Bet v 4 contained two typical EF-hand calcium-binding domains. Sequence similarities of Bet v 4 to calmodulin are primarily confined to the calcium-binding domains. However, significant sequence similarities extending outside the Ca2+-binding sites were found with a recently described group of pollen-specific allergens of Brassica and Bermuda grass. Both EF-hand domains of Bet v 4 are able to bind Ca2+, as demonstrated by 45Ca2+ blot overlay of wild type and calcium-binding deficient mutants of Bet v 4. Among pollen-allergic patients, protein-bound Ca2+ was not an absolute requirement for IgE recognition of Bet v 4. However, disruption of the carboxyl-terminal Ca2+-binding domain indicated that most IgE antibodies from allergic patients are directed against this site. IgE inhibition experiments suggested that Bet v 4 represents a highly cross-reactive pollen allergen. Pre-absorption of allergic sera with Bet v 4 drastically reduced IgE binding to proteins of similar molecular weight in pollen extracts from distantly related plant species (e.g. timothy grass, mugwort, lily) but not in extracts from plant-derived foodstuff. To test for a possible biological role in pollen germination and tube growth, we introduced recombinant Bet v 4 protein into growing lily pollen tubes by iontophoresis. As a result, cytoplasmic streaming stopped in the vicinity of the electrode tip, and a slight depolarization of the membrane voltage was measured. These effects were not observed with Ca2+-binding deficient mutants of Bet v 4. Thus, Bet v 4 and homologous proteins represent a new class of pollen-specific Ca2+-binding allergens that may have a physiological role as inhibitors of cytoplasmic streaming in outgrowing pollen tubes.     

Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.     

Studies on pulmonary and systemic Aspergillus fumigatus-specific IgE and IgG antibodies in horses affected with chronic obstructive pulmonary disease (COPD)

Inhalant exposure to Aspergillus fumigatus (Asp. f.) antigens induces marked inflammatory and immunological alterations in the lungs of horses affected with chronic obstructive pulmonary disease (COPD). In this study we investigated the role of specific allergen(s) present in Asp. f. on systemic and pulmonary IgE and IgG responses in control and COPD-affected horses, using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques. Compared with controls, horses affected with COPD had significantly higher levels of BALF IgE and IgG to somatic Asp. f. antigens as well as to the allergen 1/a (Asp. f. 1/a). Serum levels of IgE and IgG against these antigens did not differ between control and COPD-affected horses. Antigen specific IgE and IgG levels did not correlate between BALF and serum. Scanning of Asp. f. and IgE and IgG blots revealed bands that are recognised by both IgE- and IgG-specific antibodies. Additionally, all horses responded with BALF IgE- and IgG-specific for 93, 35, 31 and 23 kDa allergens, suggesting that these antigens are involved in the induction of airway IgE and IgG responses. These allergens may have the potential to be used as biomarkers for the diagnosis of Asp. f. related exacerbations of equine COPD.     

Evolution of the vertebrate H1 histone class: evidence for the functional differentiation of the subtypes

BACKGROUND: Indoor suspended particulate matter (SPM) consists of many different types of particles, the vast majority of which are less than 2.5 microm in diameter. The question arises how these particles may contribute to asthma and respiratory symptoms. One possibility is that airborne dust particles act as carriers of allergens into the airways, as several allergens have been found to be associated with inhalable airborne dust particles. OBJECTIVE: We studied the presence of three different allergens on the surface of SPM, i.e. Can f 1 (dog), Bet v 1 (birch pollen) and Der p 1 (house dust mite). We also examined the ability of diesel exhaust particulates (DEP) to attach these allergens and Fel d I (cat) in vitro. METHODS: SPM was collected on polycarbonate filters and an immunogold labelling technique was used to detect the allergens on the particles. The specimens were examined in the backscatter mode of a scanning electron microscope. The same technique was used to examine the binding of the allergens to DEP, after exposing DEP to either crude allergen extracts or partly purified allergens. RESULTS: Both Can f 1 and Bet v 1 allergens were detected on the surface of the soot particles in SPM mixtures, although to a lesser degree than previously found with Fel d 1. Der p 1 (house dust mite), however, did not show any significant binding to SPM particles. Furthermore, DEP had the ability to adsorb all four allergens in vitro, although to a varying extent. CONCLUSION: Soot particles in airborne house dust may act as carriers of several allergens in indoor air. Furthermore, DEP has the ability to bind all the four allergens investigated under aqueous conditions in vitro.     

Recombinant allergens

A great variety of recombinant plant, mite, mold, mammal, and insect allergens have been expressed in heterologous hosts (e.g., Escherichia coli), their cDNA being used as a template. The number of biologically active recombinant allergens available for experimental, diagnostic, and therapeutic purposes is increasing tremendously. Recombinant allergens have proven to be valuable tools to investigate T-cell and B-cell recognition of allergens as well as to study mechanisms of specific IgE regulation. The immunologic equivalence of many relevant recombinant allergens with their natural counterparts has been demonstrated, and the three-dimensional structures of several recombinant allergens have been described recently. As a result of extensive cross-reactivities among the relevant allergens, it appears that the number of epitopes needed for diagnosis and specific immunotherapy is less diverse than originally anticipated and might be soon covered by recombinant molecules. Recombinant allergens have been used for successful in vitro, as well as in vivo, allergy diagnosis, and work is in progress to produce recombinant allergen derivatives with reduced anaphylactic potential to improve current forms of immunotherapy.     

Facial rehabilitation by the application of osseointegrated craniofacial implants

The prevalence of allergic disease is low in Eastern Europe for reasons that are poorly understood. Our study aimed to investigate the levels of exposure to indoor allergens and living conditions among Estonian infants in relation to sensitization. Dust samples were collected during four winter months in 1993/94 from the homes of 197 infants participating in a prospective study of sensitization. Information about living conditions was collected through a home visit and interviewing the mothers when the children were 6 weeks old. Three dust samples were collected from each home; i.e., from the infant's mattress, bedroom floor, and living-room carpet. The levels of allergens were determined by ELISA with monoclonal antibodies. The highest allergen level in a home was regarded as the peak value. The peak geometric mean values (+/-SD) of Der p 1 and Der f 1 were 0.3 (0.07-1.4) microg/g dust, of Can f 1, 0.86 (0.23-3.12) microg/g dust, and of Fel d 1, 0.1 (0.01-0.9) microg/g dust. In 12 homes (9%), the peak value of house-dust mite (HDM) allergens exceeded 2 microg/g dust, with Der p 1 as the dominating allergen. Multivariate analyses indicated that high levels of HDM allergens were more common in apartments that were on the ground floor or first floor, that were heated with stoves, and/or that had a dampness problem. The mean allergen levels at home were similar in children sensitized to HDM (n=17, 0.29 vs 0.3 microg/ g dust), dog (n=5, 0.55 vs 1.06 microg/g dust, and cat (n=18, 0.21 vs 0.09 microg/g dust) and in children who were not sensitized to these allergens. Most of the sensitized children were exposed to relatively low allergen levels at home; i.e., below 1 microg/g dust. This level was exceeded in the homes of 4/17 mite-, 5/18 cat-, and 0/5 dog-sensitized children. The similar levels of the major indoor allergens in Estonia and in Scandinavia indicate that the large differences in atopy prevalence among children and young adults in the two regions are not due to differences in allergen exposure. No allergen threshold level for sensitization was identified.     

Alteration of acidic lipids in human sera during the course of pregnancy: characteristic increase in the concentration of cholesterol sulfate

Using genomic DNA from late-onset retinal degenerate and wild type Labrador Retrievers as templates and canine exon-specific oligonucleotides as primers in polymerase chain reaction, all four introns of opsin were cloned and sequenced. Dot-matrix comparisons were made for human, murine and canine introns. Selected sequences containing either intronic or coding sequences were aligned and used for phylogenetic relationship analysis. The opsin gene introns are conserved between the human, the mouse and the dog with regards to number and length. In addition there is an astonishingly high degree of sequence homology between the second and fourth introns. Introns 2(1277 bp in dog) and 4 (863 bp in dog) are 72% and 71% homologous to the human introns, and 57% and 52% homologous to the mouse introns, respectively. The coding sequence (CDS) of the dog shows 93% homology to human CDS and 88% homology to mouse CDS. A phylogenetic analysis of the intronic sequences 2 and 4 confirms the higher relatedness between dog and human than between mouse and human opsin genes. As there are good reasons to believe that the primate and rodent lineages are closer to each other than to the Canis familiaris, there must be some functional constraints on the evolution of human and dog opsins.     

The interacting effects of prices and weather on population cycles in a preindustrial community

Two recombinant [His]6-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [His]6-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The recombinant proteins were used to immunize hens. The raised and affinity-purified chicken antibodies (Ab) isolated from egg yolk exhibited specific binding to the respective recombinant canine IgE fragment (IgEf) on immunoblots and displayed high titers against the IgEf in ELISA. Immunoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enriched dog serum separated under denaturing conditions. In an ELISA for the detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog serum. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]6-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recognized cells in paraffin-embedded tissue sections of lymph nodes. Furthermore, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In conclusion, both IgEf-specific Ab recognize native dog IgE with the advantages that they are directed against different and known constant domains of the IgE molecule, and that they can be used for immunohistochemistry on paraffin-embedded tissue. The two dog IgE-specific Ab could initiate clinical research on the involvement of immediate-type hypersensitivity reactions in dogs.