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.     

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.     

Immunoglobulin E-mediated hypersensitivity in food-producing animals.

Type I hypersensitivity has been described as a cause of allergic reactivity to inhalants, injectables, endoparasites, and ectoparasites in food animal species. In addition, IgE is credited with showing some host-sparing effect when produced in response to certain gastrointestinal and other parasites. Recently, the sophistication of diagnostic procedures has increased with the elucidation of epsilon heavy chain sequences, expressed protein, development of chimeric IgE antibodies, and production of species-specific anti-IgE reagents. Application of ELISA and Western blotting has replaced the passive cutaneous anaphylaxis test for demonstration of antigen-specific IgE in serum. Regulation of the IgE response is complex, and its dependence on induction of T helper cell type 2 cytokines is now established. The next frontier in IgE research, as for many inherited diseases, lies in understanding the genetic make-up of the animal and which genes are important in controlling the IgE response.     

Serodiagnosis of canine herpesvirus infection--development of an enzyme-linked immunosorbent assay and its comparison with two improved methods of serum neutralization test

An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.     

A reagin-like antibody in horse serum II. Anti-human IgE induced reversed cutaneous anaphylaxis-like responses in horse skin

Fc specific anti-human IgE serum induced prolonged reversed cutaneous anaphylaxis (RCA)-like reactions in horse skin. Morphologically and histologically, these reactions resembled passively induced late cutaneous anaphylaxis responses in human skin, but differed from reversed passive Arthus responses induced in horse skin using anti-horse IgG serum. The induction of RCA-like responses in horse skin by anti-human IgE indicates shared Fc antigenic determinants on human IgE and a horse homocytotropic or reagin-like antibody.     

Local and systemic immune responses to Echinococcus granulosus in experimentally infected dogs

Local and systemic immune responses were studied in six dogs experimentally infected with the dog/sheep tapeworm Echinococcus granulosus. All dogs developed similar IgG antibody response to parasite antigens. In contrast, IgE and IgA responses differed widely. No relationship between IgA responses and parasite burden at the end of the infection were observed. Further, clear differences in the anti-parasite IgA response in serum as compared with specific IgA forming cells in mesenteric lymph nodes were observed within the same dog. An inverse association of anti-parasite IgE and parasite load seemed to be present, with the strongest IgE response in the one dog that had no worms in the intestine at the end of the experiment. No differences were observed in the numbers of intestinal mast cells and goblet cells among all infected dogs. However, the dog with no detectable parasite load had a marked reduction of detected mast cells in the submuscular and muscular layer of the mucosa. Our data give new insight into the immune response of dogs during E. granulosus infection and provide information that may be useful for the rational design of vaccines for the control of hydatid disease.     

Production of polyclonal antisera against feline immunoglobulin E and its use in an ELISA in cats with experimentally induced asthma

Serum samples from six cats with experimentally induced asthma were used to purify feline IgE using gel filtration and affinity chromatography. The resultant IgE, evaluated for purity by immunoelectrophoresis (IEP) and reactivity by Prausnitz-Kustner (PK) testing, was used to develop polyclonal rabbit anti-feline IgE antisera. Using reverse cutaneous anaphylaxis (RCA), the antisera were determined to be specific for feline IgE. The polyclonal rabbit anti-feline IgE antiserum was then validated in an allergen-specific ELISA. Serum samples from an additional five asthmatic cats sensitized with Bermuda grass allergen (BGA) were evaluated prior to sensitization, after parenteral sensitization, and after aerosol sensitization and challenge. A significant increase in serum BGA-specific IgE was noted over time.     

Humoral immunity in the prepatent primary infection of dogs with Echinococcus granulosus

Twenty-one parasite-naive dogs were infected with 60,000 protoscolices of Echinococcus granulosus. Transformation of peripheral lymphocytes was investigated before and 29 days after the infection, immunoglobulin concentration and anti-hydatid fluid protein (HFP) titers in serum and feces before and at 35 days of infection, skin reactivity to HFP at 36 days, and characteristics of the parasites at 40 days. The infection caused a significant depression of the spontaneous, lipopolysaccharide-stimulated, and purified protein derivative-stimulated blastogenesis. Responses to phytohemagglutinin were unchanged and reactivity to concanavalin A was enhanced with the infection. Only the concentrations of IgG and IgA in the serum and IgA in the feces increased significantly after infection. Fifteen (71%) dogs produced significant serum titers of anti-HFP hemagglutinins but copro-antibodies were detectable in only 3 dogs at minimum titers. Titers were abolished by treatment with 2-mercaptoethanol. The serum of 11 (52%) dogs transferred passive cutaneous anaphylaxis to guinea pigs but none transferred skin reactivity to pups or rabbits. Five and 1 (but not 0.2) micrograms of HFP caused skin reactivity in 4 parasite-naive dogs. Nineteen (90.5%) infected dogs reacted significantly to skin inoculation of 0.2 microgram of HFP at 0.5 hours and 13 (62%) at 6 hours. The 7 dogs with the highest anti-HFP serum titers or the greatest skin reactivity at 6 hours had significantly less mature or fewer tissue parasites, respectively, than the 7 dogs with the smallest responses. Since there was evidence that the specific immunity was still developing at the time of the study, these results indicate that immunological diagnosis of, and artificial immunization against, canine echinococcosis are feasible.     

Immunologic responses against hydrolyzed soy protein in dogs with experimentally induced soy hypersensitivity

OBJECTIVE: To assess whether dogs with experimentally induced type I hypersensitivity against soy protein would respond to soy hydrolysate and develop cutaneous or gastrointestinal tract reactions after intradermal and oral challenge exposure. ANIMALS: 12 na?ve Beagle pups (9 sensitized and 3 control dogs). PROCEDURE: 9 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization period, serum concentrations of soy-specific IgE were determined and an intradermal test was performed to confirm the dogs were sensitized against soy protein. An intradermal challenge test and an oral challenge test with native and hydrolyzed soy protein were conducted on 6 sensitized and 2 control dogs. RESULTS: High serum concentrations of soy-specific IgE and positive results for the intradermal test were observed for the 9 sensitized dogs after completion of the sesitization process. Sensitized dogs challenge exposed with hydrolyzed soy protein had a reduced inflammatory response after intradermal injection and no clinical response after an oral challenge exposure, compared with responses after intradermal and oral challenge exposure with native soy protein. CONCLUSIONS AND CLINICAL RELEVANCE: Soy-sensitized dogs did not respond to oral administration of hydrolyzed soy protein. Thus, hydrolyzed soy protein may be useful in diets formulated for the management of dogs with adverse reactions to food.     

Evaluation of a novel enzyme-linked immunosorbent assay for serological diagnosis of porcine proliferative enteropathy

A specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the porcine pathogen Lawsonia intracellularis was developed and evaluated using sera from na?ve, naturally infected as well as experimentally infected pigs. On the basis of 37 serum samples collected from experimentally infected pigs and 62 serum samples from naturally infected pigs the sensitivity of the ELISA was calculated to 98.0%. The specificity of the test was 99.3%, calculated on the basis of 273 serum samples collected in six herds free of L. intracellularis after medicated eradication. The novel ELISA was a specific and sensitive method for detecting specific antibodies, and may be a good alternative to the existing serological tests for L. intracellularis. It may be usable for diagnosis of proliferative enteropathy and for determination of a herd's epidemiologic status.     

Enzyme-linked immunosorbent assay for the detection of Salmonella enteritidis infection in chickens

An ELISA was developed and tested for its ability to detect antibodies against Salmonella enteritidis in chickens. Various features of the ELISA were evaluated and optimized. The outer membrane protein antigens selected by use of the protein immunoblotting method made the assay specific and sensitive. The assay was evaluated in chickens experimentally infected with S enteritidis. Blood samples collected at weekly intervals after experimental infection with S enteritidis were analyzed by ELISA. Results of the ELISA were compared with those of conventional serum plate and microagglutination tests. The ELISA was more sensitive and specific in the detection of S enteritidis infection than the other 2 conventional tests.     

Establishment of a quantitative ELISA for the measurement of allergen-specific IgE in dogs using anti-IgE antibody cross-reactive to mouse and dog IgE

As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable to measure absolute quantity of serum antigen-specific IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantification of antigen-specific IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize canine IgG. The reactivity of CRE-DM to canine IgE was also confirmed by an inhibition ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In order to know whether canine IgE specific to an allergen could be quantitatively measured with an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, a standard curve was created by using concentration-predetermined Cry j 1-specific monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-specific IgE in dogs that were experimentally sensitized to Japanese cedar pollen could be calculated and determined in ng/ml. The specificity of the Cry j 1-specific IgE ELISA using CRE-DM was also confirmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate was 97.0%. Reproducibility of the ELISA in three independent assays was determined using groups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml. Intra- and inter-assay reproducibility was determined with coefficient of variation ranging between 3.1-5.2% and 2.2-8.0%, respectively. These results demonstrated that the ELISA utilizing CRE-DM was a specific, reliable and robust new laboratory test that could quantify absolute amount of antigen-specific IgE in canine serum. The ELISA will serve as a useful tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments as well as during seasonal fluctuation of allergen exposure.     

Enzyme-linked immunosorbent assays for detecting antibody to Rickettsia australis in sera of various animal species

New endemic areas of spotted fever-like rickettsial disease have been found in south-eastern Australia (Gippsland, Victoria and Flinders Island, Tasmania). The rickettsia responsible is currently unknown although it may be Rickettsia australis. To investigate serological evidence of rickettsial exposure in various wild animal species, a competitive ELISA was developed which detected antibodies to R. australis. It was based on inhibition of an indirect ELISA detecting antibody to R. australis in guinea pig sera. Pre- and post-infection sera from 2 dogs, 2 rabbits, 5 mice and 6 rats, experimentally infected with R. australis, were tested by competitive ELISA. The results showed that all pre-infection sera were negative and all post-infection sera positive for antibody to R. australis. To test the utility of the competitive ELISA for detecting natural rickettsial infection in non-laboratory animals, 51 dog sera, negative for rickettsial antibody by immunofluorescence (IF) and 20 IF positive dog sera (collected from various locations on the east coast of Australia) were tested. Compared to the IF test the competitive ELISA was 90% sensitive and 96% specific. This new test has potential for detecting antibody to R. australis in the sera of different wild animal species.     

Lack of detection of circulating skin-specific IgE autoantibodies in dogs with moderate or severe atopic dermatitis

Human patients with atopic dermatitis (AD) commonly exhibit IgE reactivity to cutaneous self-antigens. The presence of serum IgE autoantibodies appears to correlate with disease severity, and it is suspected to reflect or contribute to tissue damage. The objective of this study was to determine whether IgE autoantibodies specific for cutaneous antigens could be detected in the serum of dogs with AD. Serum was collected from 19 dogs with untreated moderate to severe AD and four specific-pathogen free (SPF) dogs. Indirect immunofluorescence was performed using normal canine skin collected at four different locations (concave ear, nose, medial thigh and lateral thorax), while Western immunoblotting was done using normal canine ear pinna epidermal and dermal extracts and reducing conditions. In both methods, IgE was detected using a monoclonal antibody specific for heat stable epitopes of canine IgE. At 1:10 dilution, specific IgE autoantibodies against cutaneous autoantigens were not detected, with either method, in AD and SPF canine sera. Either IgE autoreactivity is not associated with moderate to severe AD in dogs, or the methods employed herein were not sensitive enough to permit IgE autoantibody detection.     

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.     

Enzyme-linked immunosorbent assay for measurement of allergen-specific IgE antibodies in canine serum

A micro-ELISA, using horseradish peroxidase-conjugated anti-canine IgE and polystyrene microtitration wells for detection of allergen-specific IgE in canine serum, was developed. Specificity of anti-canine IgE was confirmed by reversed cutaneous anaphylaxis evaluations, gel-precipitation reactions, immunoelectrophoresis, immunoaffinity chromatography, and heat inactivation. Individual allergen blanks were used to account for variable nonspecific binding among various allergens, and results were normalized using 4 reference sera. Coefficients of variation for intra-assay and interassay variability ranged from 0.77 to 5.66% and 3.15 to 9.83%, respectively. Results observed with wells coated with mixtures of various allergen extracts yielded results approximately equal to results (average) of wells containing individual components. Agreement between ELISA and skin test results ranged from 43 to 64%, depending on allergen used.     

Efficacy of a modified polymerase chain reaction assay for detection of Ehrlichia canis infection.

Detection of Ehrlichia canis in acutely infected and convalescent dogs is important for effective treatment and control. However, accurate detection has been difficult to achieve, in part because dogs that have been treated therapeutically often remain seropositive for extended periods. A new method, polymerase chain reaction (PCR) assay using biotinylated E. canis-specific primers (PCR-BP), was developed for detection of E. canis. Four dogs experimentally infected with E. canis by intravenous inoculation of whole blood from carrier dogs and 2 naturally infected convalescent carriers were used to compare the specificity and sensitivity of the new method with that of microscopy/blood smear evaluation, serologic test, and conventional PCR assay using E. canis-specific primers. In experimentally infected animals, infection was detected as early as 7 days post-exposure using PCR-BP. Although the 2 naturally infected dogs were positive by serologic test and PCR-BP, both were negative by conventional PCR. Results suggest that the new method is a sensitive assay for detection of E. canis infection. In addition, results were obtained more rapidly than with other PCR-based assays.     

Immunologic responses of calves to aerosolized antigen: humoral response to ovalbumin

The humoral response of cattle to ovalbumin (OA), a nonenvironmental well-defined antigen, was studied. During 9 weeks of aerosolization, weekly serum and nasal secretion concentrations of immunoglobulin (Ig)G1, IgG2, IgM, IgA, and IgE were determined by enzyme-linked immunosorbent assay (ELISA) for OA specific antibody. Data from 3 calves given aerosol OA were compared and contrasted with data from 3 calves given aerosol saline solution and 1 calf given parenteral OA. The presence of cytotropic (skin sensitizing) antibody was evaluated during weeks 6 and 9 by direct skin testing with OA. A humoral response was induced in all 3 calves given aerosol OA. Serum IgG1 and IgG2 titers reached a maximum of 64,000 and 2,000, respectively, in calves given aerosol OA compared with 521,000 and 16,000, respectively, in the calf given parenteral OA. The ELISA did not detect an OA-specific IgM response. In contrast, all 3 calves given aerosol OA had serum IgA concentrations that increased to a peak by week 9. The mean IgA absorbance value for the 3 calves given aerosol OA was slightly greater than 5 times that of the calf given parenteral OA. Similarly, nasal secretions from calves given aerosolized OA had absorbance values that were 15-fold greater than that from the calf given parenteral OA. Calves given aerosol OA had antigen-specific IgE responses during weeks 6 to 8. The ELISA results were compared with results of passive cutaneous anaphylaxis tests. The presence of skin-sensitizing antibody was indicated by positive skin tests in the calves given aerosol OA and the calf given parenteral OA by week 9.     

Generation and characterization of murine monoclonal antibodies specific for bovine immunoglobulin E

Monoclonal antibodies were produced against serum-derived bovine immunoglobulin E (IgE). Culture supernatants of hybridomas were initially screened by enzyme-linked immunosorbent assay (ELISA). Supernatant-derived antibodies were concentrated and further characterized using ELISA, reverse cutaneous anaphylaxis, immunohistochemical staining, and immunoblotting of IgE-containing samples separated by SDS-polyacrylamide gel electrophoresis (PAGE). Eight monoclonal antibodies showed specificity for bovine epsilon immunoglobulin heavy chain. Two antibodies (E2 and E32) reacted in immunoblots of SDS-PAGE of serum IgE under reducing conditions. Additionally, E2, E5, and E32 detected epsilon chain in serum separated by SDS-PAGE and then renatured. Antigen-specific IgE was detected in Western blots by E5 and E32. Immunoperoxidase staining of IgE-containing cells in mesenteric lymph node sections was detected with E5, E21 and E32. All eight antibodies produced positive reverse cutaneous anaphylaxis reactions in calf skin. All functioned well in ELISA as a plate-sensitizing reagent for quantitation of total IgE; E5 and E32 worked well as a primary antibody in antigen-specific IgE assays. These antibodies will be useful in research applications and in diagnostic assays.     

Probable occurrence of IgE in the adult domestic fowl (Gallus domesticus) after horse serum stimulation

Radioimmunosorbent techniques using antisera to human immunoglobulin E (IgE) showed the presence of high levels (up to 15,840 U/ml) of IgE in serum and plásma from fowls immunised by repeated intraperitoneal injections of horse serum. These results were confirmed by passive cutaneous anaphylaxis and heat lability tests.