Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity

OBJECTIVE: To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. ANIMALS: 8 na?ve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). PROCEDURES: 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. RESULTS: In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.     

Identification of bovine serum albumin as an IgE-reactive beef component in a dog with food hypersensitivity against beef

IgE-reactive beef components were examined by an immunoblot analysis using a serum from a dog with food hypersensitivity against beef. The immunoblot analysis revealed a distinct band at approximately 66 kDa and a faint band at approximately 50 kDa. The immunoblot analysis for serum IgE reactivity to bovine serum albumin (BSA) also revealed a positive band at 66 kDa. Serum IgE reactivity to the 66-kDa protein of beef was diminished by pre-incubating the serum sample with BSA. Furthermore, a positive reaction to BSA was detected in intradermal testing in the dog. These results clearly indicated that BSA was an IgE-reactive beef component in the dog with food hypersensitivity against beef.     

Production of monoclonal antibodies specific for native equine IgE and their application to monitor total serum IgE responses in Icelandic and non-Icelandic horses with insect bite dermal hypersensitivity

Immunoglobulin E forms a minor component of serum antibody in mammals. In tissues IgE is bound by FcvarepsilonRI receptors on the surface of mast cells and mediates their release of inflammatory substances in response to antigen. IgE and mast cells have a central role in immunity to parasites and the pathogenesis of allergic diseases in horses and other mammals. This paper describes the production of several novel monoclonal antibodies that detect native equine IgE in immunohistology, ELISA and Western blotting. An antigen capture ELISA to quantify equine IgE in serum has been developed using two of these antibodies. The mean serum IgE concentration of a group of 122 adult horses was 23,523ng/ml with a range of 425-82,610ng/ml. Total serum IgE of healthy horses was compared with that of horses with insect bite dermal hypersensitivity (IBDH) an allergic reaction to the bites of blood feeding insects of Culicoides or Simulium spp. IBDH does not occur in Iceland where Culicoides spp. are absent, but following importation into mainland Europe native Icelandic horses have an exceptionally high incidence of this condition. In the present study Icelandic horses with IBDH had significantly higher total IgE than healthy Icelandic horse controls (P<0.05). By contrast in horses of other breeds the difference in total serum IgE between those affected with IBDH and healthy controls was not statistically significant. Total serum IgE was also monitored in a cohort of Icelandic horses prior to import into Switzerland and for a period of 3 years thereafter. High levels of serum IgE were present in all horses at the start of the study but dropped in the first year after import. Thereafter the total serum IgE remained low in Icelandic horses that remained healthy but rose significantly (P<0.05) in those that developed IBDH. These results support the conclusion that IBDH is a type I hypersensitivity response to insect allergens but indicate that IBDH in Icelandic horses may have a different pathogenesis from the same condition in other breeds.     

Equine insect bite hypersensitivity: immunoblot analysis of IgE and IgG subclass responses to Culicoides nubeculosus salivary gland extract

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses caused by IgE-mediated reactions to bites of Culicoides and sometimes Simulium spp. The allergens causing IBH are probably salivary gland proteins from these insects, but they have not yet been identified. The aim of our study was to identify the number and molecular weight of salivary gland extract (SGE) proteins derived from Culicoides nubeculosus which are able to bind IgE antibodies (ab) from the sera of IBH-affected horses. Additionally, we sought to investigate the IgG subclass (IgGa, IgGb and IgGT) reactivity to these proteins. Individual IgE and IgG subclass responses to proteins of C. nubeculosus SGE were evaluated by immunoblot in 42 IBH-affected and 26 healthy horses belonging to different groups (Icelandic horses born in Iceland, Icelandic horses and horses from different breeds born in mainland Europe). Additionally, the specific antibody response was studied before exposure to bites of Culicoides spp. and over a period of 3 years in a cohort of 10 Icelandic horses born in Iceland and imported to Switzerland. Ten IgE-binding protein bands with approximate molecular weights of 75, 66, 52, 48, 47, 32, 22/21, 19, 15, 13/12 kDa were found in the SGE. Five of these bands bound IgE from 50% or more of the horse sera. Thirty-nine of the 42 IBH-affected horses but only 2 of the 26 healthy horses showed IgE-binding to the SGE (p<0.000001). Similarly, more IBH-affected than healthy horses had IgGa ab binding to the Culicoides SGE (19/22 and 9/22, respectively, p<0.01). Sera of IBH-affected horses contained IgE, IgGa and IgGT but not IgGb ab against significantly more protein bands than the sera of the healthy horses. The cohort of 10 Icelandic horses confirmed these results and showed that Culicoides SGE specific IgE correlates with onset of IBH. IBH-affected horses that were born in Iceland had IgGa and IgGT ab (p< or =0.01) as well as IgE ab (p=0.06) against a significantly higher number of SGE proteins than IBH-affected horses born in mainland Europe. The present study shows that Culicoides SGE contains at least 10 potential allergens for IBH and that IBH-affected horses show a large variety of IgE-binding patterns in immunoblots. These findings are important for the future development of a specific immunotherapy with recombinant salivary gland allergens.     

Putative salivary allergens of the cat flea, Ctenocephalides felis felis.

The cat flea, Ctenocephalides felis felis, is the major initiator of flea bite hypersensitivity in dogs. Previous analyses of whole extracts of the flea and flea salivary secretions have failed to identify the allergens responsible. We dissected >2000 salivary glands from adult female fleas, extracted them into buffered saline containing protease inhibitors and fractionated the extract using gel permeation HPLC. Dogs were classified as hypersensitive to fleas (flea-feeding positive, FF+) or insensitive (flea-feeding negative, FF-) using a provocative test with live fleas. The allergenicity of the components of the salivary gland extract was tested by intradermal injection of samples of the column eluates. Dogs were also injected intradermally with a sample of whole salivary gland extract, and with histamine as a positive control. Negative control injections consisted of eluate from the column collected prior to fractions containing any protein. The skin of FF- dogs either did not respond or had a minimal response (a bleb approximately 2 mm larger than the injection blebs at the negative control injection sites) to all fractions and to the whole extract; histamine control injections produced positive responses (defined as wheals 5 mm greater than the blebs at the negative control injection sites) in all dogs. The skin of three of the nine FF+ dogs reacted positively to injection of a fraction containing protein/s with apparent MW 40k. Five other FF+ dogs reacted positively to the fractions containing proteins with apparent MW 12-8k. A single dog responded with very large, red wheals to injection of both the approximately MW 40k and MW12-8k fractions. These findings suggest that proteins with apparent MW 40k and MW 12k-8k are important in flea bite hypersensitivity. This work also supports a previous finding that mice which had been exposed to flea bites had antibodies to proteins with approximately MW 40k that were detected in salivary secretions of the flea.     

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch).

We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.     

Monoclonal anti-IgE antibodies in the diagnosis of dog allergy

The availability of anti-dog IgE monoclonal antibodies has enabled development of highly specific ELISA assays for measuring antigen-specific IgE in dog serum. In this article the authors propose criteria for evaluation of these monoclonals and demonstrate that some anti-human IgE monoclonals recognize dog IgE. Combinations of two or more monoclonal antibodies can enhance assay sensitivity; for example, a mixture of DE38.HRPO and 4F4.HRPO conjugates detect total dog IgE in the range 10–10000 ng/mL. Results are reported from nine clinical studies conducted in Europe, Japan and Australia involving more than 400 dogs in which serologic IgE determinations performed using the CMG IMMUNODOT strip system for house dust mites, storage mites, flea, grass pollens and moulds were compared with immediate skin test findings. House dust mites were identified as the common major dog allergen throughout these areas although regional reactions to food allergens were observed. These results confirm that the CMG IMMUNODOT system is a valuable and reliable diagnostic test for dog allergy.     

Immunodominant proteins of Sarcocystis cruzi bradyzoites isolated from cattle affected or nonaffected with eosinophilic myositis

Sarcocystis cruzi sarcocysts were isolated from eosinophilic myositis (EM)-affected and nonaffected bovine hearts. Isolates were ruptured and used to prepare a bradyzoite antigen extract from each heart. The nonaffected heart from one newborn calf contained no apparent sarcocysts when examined histologically and was used to prepare Sarcocystis-negative control antigen. Blood samples were taken from the heart approximately 20 minutes after slaughter. Serum was obtained and evaluated, using a radioimmunoassay to measure Sarcocystis-specific IgG and IgE titers. Sarcocystis cruzi extract from a heart without EM lesions was used for antigen in the radioimmunoassay. Sarcocystis-specific IgG titer ranged between 1:1,280 and 1:2,560 in EM-affected cattle and was 1:640 in non-affected cattle. Sarcocystis-specific IgE titer ranged between 1:640 and 1:1,280 in EM-affected and nonaffected cattle. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein (western) immunoblot analysis were used to compare antigen extracts and serum samples from EM-affected vs nonaffected cattle. Twenty protein bands, ranging from approximately 22 to 215 kD, were detected consistently on bradyzoite blots probed with anti-bovine IgG after incubation with serum samples. Seven of these bands, 37, 44, 53, 57, 94, 113, and 215 kD, were also detected consistently on bradyzoite blots probed with monoclonal anti-bovine IgE. One additional band, 61 kD, was detected consistently on bradyzoite blots probed for IgE, but was seldom recognized when probed for IgG. Sixteen protein bands were evident in silver-stained gels of S cruzi-negative, newborn calf antigen, but none were recognized by antisera on western blots.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domain mapping and comparative binding features of eight dog IgE-specific reagents in ELISA, immunoblots, and immunohistochemistry.

Eight dog IgE-specific reagents including monoclonal and polyclonal antibodies (Ab) and a cross-reactive alpha chain of the human high affinity IgE receptor were mapped to recombinant fragments of the second (IgEf2) and third/fourth (IgEf3/4) domains of the dog IgE heavy chain. In ELISA, five out of eight reagents reacted to solid-phase bound IgEf2, of which two polyclonal Ab bound in addition to IgEf3/4. All Ab which recognized at least one recombinant IgE fragment, also bound to IgE in ELISA, immunoblots, and immunohistochemistry. In contrast, only one monoclonal Ab, that did not bind to the recombinant IgE fragments, reacted with immunoblots of serum and immunohistochemistry. The alpha chain could only be applied to ELISA with serum IgE. Furthermore, there was a wide range of heat-lability of binding reactions. Comparative analysis of available dog IgE-specific reagents enables more in-depth functional studies on IgE-mediated phenomena in dogs, and helps to further establish the dog as an animal model for allergy research.     

Allergy diagnosis in companion animals: clinical experience with the basophil activation model

Animal allergy diagnosis is based mainly on clinical history, skin tests and, at least for dogs, specific IgE antibodies. The quality of anti-canine IgE antibodies is variable and monoclonal antibodies have been recently characterized. The allergen panel tested in humans and in dogs is similar except for flea and for Staphylococcus. Allergen-induced basophil activation may be measured by the release of mediators such as histamine and leukotriene C4 and by the expression of the CD63 marker on basophil membrane. This latter method is based on the flow cytometric analysis of leukocyte suspensions after double anti-IgE FITC, anti-CD63 PE labelling of human basophils, and has been validated for aero-allergens, food allergens, venoms and several drugs for human allergy diagnosis. After having demonstrated that, in the dog, anaphylactic anti- bodies were capable of binding to human basophil high-affinity receptors for IgE, we went up a flow cytometric method for animal allergy diagnosis based on passive sensitization of human basophils. Prelim- inary results obtained by this method for allergens such as house dust mite or pollen were very encouraging. This method is faster and less expensive than the methods based on mediator release but is still dependent on the availability of fresh human leukocytes. This method may represent a new sensitive and specific method for animal allergy diagnosis.     

Comparison between IMMUNODOT tests and the intradermal skin test in atopic dogs

This study evaluated a new perspective in the diagnosis of dermatitis in dogs with signs suggestive of allergic skin disease. The results obtained with CMG IMMUNODOT tests using the technique of allergen-specific strip tests, as employed for human allergy diagnosis, were compared with those obtained by the intradermal skin test (IDST). Forty-eight cases completed the diagnostic evaluation, which included IDST, flea-control program, exclusion of sarcoptes and, for some cases, a 1- to 2-month stabilization period on a restricted protein source diet and testing the serum in the presence of allergen-specific IgE and total IgE. The most common disorders included house and storage dust mites, allergic dermatitis and flea-allergic dermatitis together with atopy. This was confirmed serologically. In the case of positive IDST to pollens, Aspergillus spp. and cat epithelium, CMG IMMUNODOT strip tests were negative. A total of 25% of cases were considered to be primarily associated with food hypersensitivity, but only 4% were confirmed serologically. This study emphasizes the value of CMG IMMUNODOT tests as a support in the diagnosis of dog allergy.     

Porcine IgE in the context of experimental food allergy: purification and isotype-specific antibodies

Measurement of allergen-specific immunoglobulin E (IgE) is a common practice in the investigation of allergy. It has not been possible to measure porcine IgE due to unavailability of anti-porcine IgE. This study was undertaken to purify and characterize porcine IgE from sera of allergic pigs, identify heterologous anti-IgE reactive with pig IgE and to use purified heavy (H) chain of porcine IgE to generate rabbit anti-IgE. A four-step process for the purification of porcine IgE is reported using ammonium sulphate precipitation, Protein G affinity chromatography, DEAE cellulose anion-exchange chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain IgE H chain. The resultant IgE was evaluated for purity using SDS-PAGE and immunoreactivity was detected by Prausnitz-Küstner (PK) tests and passive cutaneous anaphylaxis with the allergen, crude peanut extract, used to induce experimental allergy. Cross-reactivity with anti-mouse and anti-human IgE antibodies were confirmed in western blot and enzyme-linked immunosorbent assays (ELISA). The H chain of IgE was excised from SDS-PAGE gels and used to develop rabbit anti-porcine IgE antisera. Antiserum obtained from rabbits immunized with porcine IgE, as well as heterologous murine and human-specific anti-IgE, induced reverse cutaneous anaphylaxis in pig skin and detected allergen-specific IgE in ELISA but did not react with IgG H chain in western blots. These results confirm allergy-associated bioactivity of porcine IgE and describe both homologous and heterologous anti-pig IgE suitable for use in allergen-specific and other assays. This will enhance utility of pig allergy models and provide an additional measure of type-2 immune response in pigs.     

Further purification and characterisation of horse IgE

Horse IgE was isolated from a serum pool collected from foals naturally infected with endoparasites. The serum was precipitated with ammonium sulfate, delipidated with dextran sulfate and further purified by gel filtration, anionic exchange, immunosorption or preparative polyacrylamide gelelectrophoresis. By these methods IgE could be isolated at a purity of 81%. The sera from rabbits immunized with the purified horse serum fractions were tested using reversed passive cutaneous anaphylaxis and an enzyme linked immunosorbent assay (ELISA). By the ELISA method cross reaction of rabbit anti horse IgE sera to human, mouse and rat myeloma IgE was demonstrated. Rat myeloma IgE also served to monitor the production of antibodies to horse IgE in rabbits.     

Allergic pulmonary and ocular tissue responses in the absence of serum IgE antibodies (IgE) in an allergic dog model

Allergen-specific serum IgE may be insensitive as a marker for IgE-mediated reactions at the mucosal level. Five of six atopic beagle dogs developed high ovalbumin (OVA)-specific serum IgE levels after sensitization. This study aimed to show that these dogs still express allergen-specific IgE at the pulmonary and ocular mucosal levels and in the skin even when corresponding serum IgE was below the detection limit. When serum IgE levels were negative, all dogs exhibited allergic reactions at the tissue level. Specifically, they displayed positive ocular reactions after an ocular OVA challenge. After airway challenge with aerosolized OVA, five out of six animals reacted with decreased compliance and increased resistance of the lungs. Furthermore, an eosinophilia in the bronchoalveolar lavage fluid (BALF) was observed. Four weeks after the last exposure to OVA, IgE-positive BALF cells were seen in all animals. Six weeks on, all dogs still displayed positive skin reactions to OVA. This indicates that not only skin testing but also detection of ocular and pulmonary allergic tissue reactions including cell-bound IgE in BALF can serve as more sensitive and lasting surrogate markers of hypersensitivity in the allergic dog model than detection of allergen-specific serum IgE levels.     

IgE-reactivity to major Japanese cedar (Cryptomeria japonica) pollen allergens (Cry j 1 and Cry j 2) by ELISA in dogs with atopic dermatitis

The present study investigated IgE-reactivity to two major Japanese cedar (Cryptomeria japonica, C. japonica) pollen allergens (Cry j 1 and Cry j 2) in dogs with atopic dermatitis by use of a fluorometric ELISA. The serum samples from 27 dogs that showed IgE-sensitivity to crude C. japonica pollen allergen by ELISA were tested for specific IgE to the two major allergens. All 27 dogs had anti-Cry j 1 IgE, and 10 (37%) had anti-Cry j 2 IgE. Inhibition of binding of dog specific IgE to crude C. japonica pollen allergen was carried out by addition of Cry j 1. When serum samples containing anti-Cry j 1 IgE but no anti-Cry j 2 IgE were incubated with Cry j 1, specific IgE binding to crude C. japonica pollen allergen was almost abolished. These findings suggest that Cry j 1 is a major allergen in dogs.     

Evaluation of serum obtained from atopic dogs with dermatitis attributable to Malassezia pachydermatis for passive transfer of immediate hypersensitivity to that organism

OBJECTIVE: To determine the functionality of canine anti-Malassezia IgE via the passive transfer of immediate hypersensitivity localized to the skin (ie, cutaneous anaphylaxis) from atopic dogs with dermatitis attributable to overgrowth of Malassezia pachydermatis (Malassezia dermatitis [MD]) to healthy recipient dogs by use of the Prausnitz-Küstner (P-K) technique. ANIMALS: 7 clinically normal dogs, 32 atopic dogs with MD, serum from 11 atopic dogs with MD, and 3 healthy dogs without prior sensitization to M pachydermatis. PROCEDURE: Serum from atopic dogs with MD was used for P-K tests in 3 clinically normal recipient dogs. Serial dilutions of untreated, heat-inactivated, IgE-absorbed, and bovine serum albumin (BSA)-absorbed (control) aliquots of serum were injected ID in triplicate for dermal sensitization. Twenty-four, 48, and 72 hours later, a crude extract of M pachydermatis was injected ID into the sites used for sensitization injections, and immediate hypersensitivity reactions were graded on a 4-point scale. RESULTS: Untreated serum caused P-K reactivity beginning 24 hours after passive sensitization and persisting through 72 hours (titers, 1:32 to 1:64). Heat inactivation and IgE-absorption of serum eliminated P-K reactivity, whereas treatment of serum with BSA did not. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of P-K test results supports the passive transfer of cutaneous anaphylaxis by anti-Malassezia IgE and indicates it is functional in type-1 hypersensitivity reactions of atopic dogs with MD. Reduction or blockade of anti-Malassezia IgE in atopic dogs with MD may provide better clinical control of the disease.     

Demonstration of Pasteurella-specific immunoglobulin E in bovine serum

On the basis of recent observations that immunoglobulin (Ig) E antibodies specific for bacterial antigens occur in the serum of persons with chronic respiratory tract disease, we used bovine epsilon chain-specific antiserum to investigate the possibility that IgE antibodies are induced in cattle infected with Pasteurella. Using enzyme-linked immunosorbent assay and Western blotting techniques, we studied bovine sera to detect and quantitate the presence of IgE antibodies specific for antigens of Pasteurella. Immunoglobulin E antibodies reactive with whole formalinized P haemolytica, potassium thicyanate, and saline solution extracts were detected in serum of calves with bronchopneumonia, feedlot steers with interstitial pneumonia, as well as nonaffected penmates, and adult dairy cows. The role of parenteral vaccination in eliciting an IgE response was examined in healthy calves; vaccination with a Pasteurella bacterin failed to induce an IgE response. Adsorption studies were done to demonstrate the specificity of the antibodies for Pasteurella. Enzyme-linked immunosorbent assay absorbance values were significantly decreased by adsorption with P haemolytica, whereas adsorption with other gram-negative bacteria only moderately decreased serum absorbance values. To begin identification of the antigen(s) to which the IgE binds, Western blotting of P haemolytica extract with sera from calves with bronchopneumonia was done. A dense band of protein (approximately 60,000 daltons) reacted strongly with IgE in the highest titer sera. These results indicate that Pasteurella-specific IgE antibodies are not readily induced by parenteral vaccination, but can be found in serum of some cattle, possibly induced by existing or previous infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoglobulin E antibodies to pollens augmented in dogs by virus vaccines

An inbred "atopic dog colony" was established to study the effect of viruses on immunoregulation of immunoglobulin (Ig) E antibodies. Dogs were selected for high skin reactivity to grass and weed pollens. Their offspring were inoculated with pollen extracts in alum immediately after routine vaccinations (attenuated live-virus vaccines for canine distemper and infectious canine hepatitis, and a killed bacterin for Leptospira). Heat labile antipollen IgE antibodies were measured by passive cutaneous anaphylaxis. Pups vaccinated for canine distemper before being given pollen extracts had many more IgE antibodies than did their control littermates who were not vaccinated until after the last pollen extract injection. This may be a natural example of the "allergic break-through phenomenon."     

Cloning and sequencing of a cDNA expressing a ribosomal P0 peptide from Culicoides nubeculosus (Diptera)

Insect bite dermal hypersensitivity (IBH) is an allergic dermatitis of horses caused by bites of Culicoides spp. and sometimes Simulium spp. The aim of the investigation presented here was to identify allergens causing IBH. A cDNA library expressing recombinant Culicoides nubeculosus proteins was screened using affinity-purified serum from an IBH-affected horse. Screening of the library resulted in identification of one immunoreactive clone. The sequence of the cDNA insert was determined and revealed a 600 bp insert with an open reading frame coding for a 78 amino acid long protein, called rCul n 1. Analysis of the deduced amino acid sequence revealed an identity of 67-78% to the C-terminal part of the 318 amino acid long ribosomal P0 protein from other Diptera. Furthermore, the 38 C-terminal amino acids displayed an identity of 57% with the C-terminal part of the acidic ribosomal protein P2 from Aspergillus fumigatus. The cDNA insert was subcloned and expressed as a [His]6-tagged protein in Escherichia coli and purified using Ni2(+)-chelate affinity chromatography. The 10kDa recombinant Cul n 1 protein bound the affinity-purified antibody fraction used for screening the expression library. Determination of IgE and IgG levels against rCul n 1 by ELISA in sera from 19 IBH-affected and 18 Swiss control horses and in sera from eight control horses living in Iceland showed no significant differences between the three groups of horses (median IgE levels = 60, 49 and 44 relative ELISA units, respectively). rCul n 1 did not induce sulfidoleukotriene (sLT) release from peripheral blood leukocytes of IBH-affected horses (N = 5), although sLT release was induced with the Culicoides whole body extract.     

Binding of canine anaphylactic antibodies on human basophils: application to canine allergy diagnosis

Abstract Using the technique of human basophil passive sensitization, as employed for human allergy díagnosis, we checked the ability of canine anaphylactic antibodies to sensitize human basophils. Therefore, by sensitizing human basophils with sera taken from dogs allergic to house dust mite, we demonstrated basophil activation as measured by alcian blue staining. Basophil activation was inhibited by heating dog sera at 56 °C for 6 h and by a human myeloma IgE. Basophil activation was also shown by histamine and leukotriene (LTC4) release. These results indicate canine anaphylactic antibodies bind to human basophil IgE receptors and also that they are IgE. The three methods described here for measuring basophil activation may lead to díagnostic methods applicable to canine allergy díagnosis. Resumen Mediante el método de la sensibilización pasiva de basófilos humanos como se utiliza para el díagnóstico de la alergia humana, evaluamos la capacidad de los anticuerpos anafilácticos caninos de sensibilizar basófilos humanos. Asi, sensibilizando basófilos humanos con suero extraido de perros con alergia al ácaro del polvo, demostramos la activación de basófilos medíante la tinción de Azul de Alcián. Se inhibió calentando suero canino a 56 °C durante 6 h. y por IgE de mieloma humano. La activación de los basófilos se mostró también por la liberación de histamina y leucotrieno (LTC4). Estos resultados indican que los anticuerpos caninos anafilácticos se unen a los receptores de IgE en basófilos humanos y también que son IgE. Los tres métodos descritos aqui para medir la activación de basófilos pueden llevar a métodos de díagnóstico aplicables al díagnóstico de la alergia canina. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Union de anticuerpos anafilácticos caninos a basófilos humanos: aplicacion al díagnóstico de alergia canina). Veterinary Dermatology 1996; 7 : 185–91.] Résumé Utilisant une technique de sensibilisation passive de basophiles humains, employee pour le díagnostic allergologique chez l'homme, nous avons testé la capacité des anticorps anaphylactiques canins à sensibiliser des basophiles humains. Ainsi, par sensibilisation de basophiles humains avec des sérums provenant de chiens allergiques aux acariens de la poussière de maison, nous avons démontré l'activation des basophiles mesurée par coloration au bleu alcian. Celle-ci est inhibée par des sérums canins chauffés à 56 °C pendant 6 heures et par un myélome IgE humain. L'activation des basophiles a été aussi démontrée par libération d'histamine et de leucotriénes (LTC4). Ces résultats prouvent la présence d'anticorps anaphylactiques canins fixés à des récepteurs IgE de basophiles humains et que ceux-ci sont des IgE. Les trois méthodes décrites ici pour mesurer l'activation des basophiles peuvent être utilisées pour le díagnostic allergologique chez le chien. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Fixation d'anticorps anaphylactiques canins sur des basophiles humains). Veterinary Dermatology 1996; 7 : 185–91.] Zusammenfassung Mit der Technik der passiven Basophilensensibilisierung beim Menschen, wie man sie für die Allergiedíagnose beim Menschen anwendet, untersuchten wir die Möglichkeit, menschliche Basophile durch kanine anaphylaktische Antikörper zu sensibilisieren. Dazu wurden humane Basophile mit Sera von Hunden sensibilisiert, die allergisch auf Hausstaubmilben reagierten. Dabei demonstrierten wir eine Basophilenaktivierung, die durch Elsässerblau-Färbung gemessen werden konnte. Der Vorgang wurde verhindert durch Erhitzen der Hundesera auf 56 °C für 6 Stunden und durch humanes Myelom-IgE. Basophilenaktivierung wurde auch durch Histamin-und Leukotrien(LTC4)-Ausschüttung gezeigt. Diese Ergebnisse zeigen, daß kanine anaphylaktische Antikörper sich an humane basophile IgE-Rezeptoren binden und auch IgEs darstellen. Die drei hier beschriebenen Methoden zur Messung der Basophilenaktivierung können zu einer díagnostischen Methode führen, die für die Diagnostik kaniner Allergie anwendbar ist. [Sainte-Laudy, J., Prost, C. Binding of canine anaphylactic antibodies on human basophils: application to canine allergy díagnosis (Die Bindung von anaphylaktischen Antikörpern des Hundes an Basophile Zellen des Menschen: Anwendung für die Allergiedíagnose beim Hund). Veterinary Dermatology 1996; 7 : 185–91.]