Identification and Clinical Significance of Allergenic Molecules of Cat Origin

Freeze-dried extracts from cat dander and the corresponding rabbit antibodies were used for establishing the CIE reference pattern for cat dander extracts. Anti-Cat Ag 1 and anti-cat albumin were used for identification of the corresponding antigens. CRIE on sera from selected groups of American and Danish cat-allergic patients demonstrated antigen-specific IgE binding to 10 of 15 cat dander antigens (Cat Ag 1 being the major allergen). Only minor differences were found between the two groups. Four of these allergens were serum proteins. Variable amounts of many of the 10 allergens were measured by QIE in saliva, serum, urine and three cat pelt extracts. However, extremely wide ranges for content of the serum allergens and the non-serum allergens were found. This was exemplified by an albumin/cat Ag 1 ratio between 1 and 400, smallest in cat dander. Immunoabsorption using anti-cat dander, anti-cat albumin and anti-Cat Ag 1 indicated that the anti-cat dander, anti-cat albumin, and the anti-Cat Ag 1 absorbed approximately 90%, 25%, and 56%, respectively, of the dander RAST activity, and 87%, 11%, and 45%, respectively, of the saliva RAST activity, confirming the major importance of Ag 1. It is concluded that cat allergenic extracts should contain only modest amounts of serum albumin and other serum-derived antigens and that any relevant standardization must include quantification of at least Cat Ag 1 and cat albumin.     

Cat allergen content of commercial house dust extracts: Comparison with dust extracts from cat-containing environment

Eighteen lots of house dust extract from nine commercial sources (obtained as weight per volume or protein nitrogen unit per cubic centimeter) were analyzed for cat allergen content by direct quantitative immunoelectrophoresis after concentration. Cat allergen 1 was measurable (greater than 0.3 units) in 11 extracts with a mean (range) of 5.8 (1.3 to 31.0) U/gm of source material. Cat albumin was measurable (greater than 2.4 units) in 12 extracts with a mean (range) of 53.4 (11.5 to 319.7) U/gm. In order to evaluate whether the cat allergen 1 content is a significant contribution to the allergenic activity of the extract, 17 cat-allergic subjects were tested by prick test with a purified preparation of cat allergen 1. The mean (range) concentration that produced a 3 mm wheal was 0.01 (0.0013 to 1.33) U/ml. Therefore, the commercial house dust extracts studied, when these extracts were diluted to a concentration commonly used for prick testing, would frequently contain enough cat allergen 1 to produce strong prick test reactions in cat-allergic subjects. It is difficult to justify the use of such commercial dust extracts as diagnostic reagents. For comparison purposes, nine dust samples from an apartment housing two cats were similarly analyzed. Cat allergen 1 was measurable in seven samples with a mean (range) of 23.8 (1.8 to 64.3) U/gm. Cat albumin could be measured in all nine samples with a mean (range) of 32.3 (0.16 to 70.8) U/gm. The average amount of cat allergen 1 that could be washed off the surface of the cats was 270 units. Large reservoirs of cat allergen 1 were present.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgE antibody to cat allergens in an allergic population.

Clinical cat sensitivity and IgE antibody to cat allergens were studied in 200 consecutive patients who presented themselves for an allergy evaluation. Fifty-nine percent of the patients had no evidence of allergy to cats (group 1). Twenty-three percent of the patients had positive histories of allergy to cats and a positive prick test to cat epithelial extracts (group 2). The remaining 18% had either positive prick tests or positive histories alone (groups 3 and 4). The group of patients with strong evidence of allergy to cats (group 2) was on an average younger and had a higher incidence of asthma than did group 1 patients. Significant binding of IgE antibody to various insolubilized allergen preparations was defined as that binding which exceeded the 95% confidence interval for group 1 patients. Significant binding of IgE antibody to insolubilized cat allergen 1 was present in 65%, 15%, and 10% of groups 2, 3, and 4 patients, respectively. Significant binding of IgE antibody to insolubilized cat pelt extract was present in 28%, 11%, and 0% of group 2, 3, and 4 patients, respectively. Data for IgE antibody binding to insolubilized cat serum and cat albumin revealed no significant difference between the groups. It is concluded that cat allergen 1 is an important allergen in patients with strong evidence of allergy to cats.     

Monoclonal antibody based radioimmunoassay for the quantitation of the main cat allergen (Fel d I or Cat-1)

A two-site solid-phase radioimmunoassay has been developed for the quantitation of the main cat allergen, Fel d I or cat allergen 1. The assay is based on two different monoclonal antibodies which recognize different epitopes on the Fel d I molecule; one antibody (C5/24) was immobilized on the solid phase and the other (C5/8) was labeled with 125I, being the allergen molecule sandwiched between them. The assay is specific for the Fel d I molecule and sensitive enough to detect as little as 0.25 ng/ml of allergen. The Fel d I RIA was compared with a radial immunodiffusion technique for the determination of allergen levels in several cat extracts and a good quantitative correlation was found. The same good correlation was found when the results obtained with the Fel d I RIA were compared with the determination of the total allergenic activity by RAST inhibition. The results indicate that the MAb RIA could be very useful in the standardization of allergenic extracts from feline origin.     

Immunochemical and physicochemical characterization of commercial Altemaria extracts: a model for standardization of mold allergen extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.     

Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.     

Studies on the biochemical structure of the major cat allergen Felis domesticus I.

The major cat allergen, Fel d I, was purified to homogeneity from cat dander extract by sequential mAb affinity chromatography and HPLC size exclusion. The purity and allergenic activity of the preparation was demonstrated by different techniques such as HPLC, RAST inhibition, skin prick tests and CIE/CRIE. Fel d I showed a mol. wt of about 35,000 by HPLC gel filtration and of 18,000 by SDS-PAGE, confirming that it is a non-covalently linked dimer. However, SDS-PAGE analysis under reducing conditions as well as labelling experiments with 14C-iodoacetamide of 2-ME-reduced Fel d I showed that each mol. wt 18,000 monomer is comprised of two covalently S-S bound polypeptides with apparent mol. wt. of 4000 (alpha-chain) and 14,000 (beta-chain). Reduction and alkylation of Fel d I obliterated most of its allergenic activity, as determined by RAST inhibition and immunoblotting, suggesting that most of the IgE-binding sites are conformational. On the other hand, treatment of Fel d I by N-glycanase under reducing and non-reducing conditions indicated the presence of N-linked oligosaccharides in the beta-chain. Carbohydrate analysis data of the whole Fel d I molecule showed the presence of a relatively high carbohydrate content (approximately 20%). RAST inhibition experiments of native and deglycosilated allergen suggest that most IgE epitopes are located in the protein moiety of the molecule. However, the deglycosilated allergen showed a 2-4 fold reduction in its inhibition capacity of RAST as compared to the native allergen, suggesting that carbohydrates could have some role in keeping the active conformation of those epitopes. The N-terminal amino acid sequence of the beta-chain (20 residues) and most of the alpha-chain (40 residues) were determined. Both chain sequences showed no homology with other known protein sequences.     

A double-blind,placebo-controlled immunotherapy dose-response study with standardized cat extract

BACKGROUND: Allergen immunotherapy with doses of cat extract containing approximately 15 microg of the major allergen, Fel d 1, have been proved clinically effective in several double-blind, placebo-controlled studies. However, the maintenance doses used in allergy practice in the United States are often considerably less than this proven dose. OBJECTIVE: The purpose of this investigation was to determine whether maintenance immunotherapy with cat dander extract containing 0.6 microg or 3.0 microg of Fel d 1 was more effective than placebo and similar in efficacy to treatment with extracts containing 15.0 microg Fel d 1, immunologic parameters being used as the outcome. METHODS: Twenty-eight cat-allergic patients were randomly entered, 7 in each group, into a double-blind, placebo-controlled comparison of the immunologic response to treatment with placebo or cat dander extract containing 0.6 microg, 3.0 microg, or 15.0 microg of Fel d 1. Maintenance doses were achieved in 8 visits over a period of 4 weeks through use of a cluster regimen; each subject then received 1 weekly maintenance injection before posttreatment measurements were made. The response to immunotherapy was assessed before immunotherapy and after the first weekly maintenance injection. Studies included responses to titrated skin prick tests to cat extract and an unrelated allergen and serum allergen-specific IgE and IgG4. Titrated nasal challenges were performed with cat extract; measurement of mRNA and secreted cytokines (IL-4, IL-5, and IFN-gamma) was done at 6 hours. Serum cytokines (IL-4, IL-5, IFN-gamma) were measured, and flow cytometric analysis of intracellular cytokines (IL-4, IL-5, IFN-gamma) was performed. Cat allergen-stimulated lymphocyte proliferation was performed with measurement of cytokines in the supernatant (IL-4, IL-5, IFN-gamma). RESULTS: All 28 subjects completed the study. Significant and dose-dependent differences were encountered in the titrated skin prick tests (P =.008), the cat-specific IgG(4) (P =.01), and the reduction in CD4+/IL-4+ PBMCs on flow cytometry (P =.03). There were no significant differences between placebo and cat dander extract containing Fel d 1 0.6 microg. Both extracts containing 3.0 microg and 15.0 microg produced significant decreases in skin prick test sensitivity (P =.02 and P =.002, respectively). The extracts containing 3.0 microg and 15.0 microg produced significant increases in cat-specific IgG4 (P =.01 and P =.006, respectively). Only the 15.0-microg-per-dose extract produced a significant reduction in the percent of CD4+/IL-4+ PBMCs (P =.003). CONCLUSION: In this double-blind, placebo-controlled study, a maintenance dose of cat dander extract containing 15.0 microg Fel d 1 produced the most consistent immunologic response. A dose of 3.0 microg reduced skin prick test sensitivity and increased cat-specific IgG4 but did not reduce the circulating CD4+/IL-4+ PBMCs, a change that is likely related to the clinically significant response to allergen immunotherapy. These findings suggest that a maintenance dose of 15.0 microg of Fel d 1 is most apt to be clinically effective for allergen immunotherapy.     

Immunotherapy with cat- and dog-dander extracts. II. In vivo and in vitro immunologic effects observed in a 1-year double-blind placebo study

The effects of immunotherapy on the skin prick test, allergen-specific IgE, and IgG in 39 patients (19 adults and 20 children), treated with partially purified cat- or dog-dander extracts or placebo for 1 year, were studied by use of a double-blind protocol. IgG levels were measured by three different assays: IgG RAST, IgG4 RAST, and Staph A IgG1, 2, and 4. The skin prick test reaction decreased continuously in the allergen-treated patients, the decrease being the first sign of an immunologic effect of the therapy. Allergen-specific IgE levels increased during the first 9 months in both children and adults. The RAST activity during the last 3 months continued to rise for the children, whereas it declined for the adults. IgG levels measured by all three methods demonstrated an increase in the allergen-treated patients and no increase in the placebo-treated patients. The children developed higher values on IgG RAST and IgG4 RAST than the adults. IgG RAST correlated negatively with IgE levels in the cat allergen-treated group. No correlation between skin prick test results, IgE levels, and IgG levels was found, nor was there any correlation between these parameters and the patients' own subjective evaluation or the allergen bronchial challenge test. In summary, the expected change in skin prick test reaction and allergen-specific IgE and IgG levels was found. The children tended to be more immunologically active than the adults.     

Mapping of cat albumin using monoclonal antibodies: identification of determinants common to cat and dog.

Cat and dog albumins from commercial extracts were used to produce monoclonal antibodies (MoAb). Anti-cat albumin MoAb recognized both cat and dog albumin equally, as did anti-dog albumin MoAb; this confirms cross-reactivity between cat and dog. The MoAb were separated into two groups according to their epitopic specificity; they recognized two overlapping epitopes of cat albumin. Furthermore, by competitive inhibition of radio-allergosorbent test (RAST), it was shown that one MoAb group inhibited significantly the binding of human IgE antibodies (from a pool of 13 patients allergic to both cats and dogs) to insolubilized cat or dog extracts. These observations suggest that murine anti-cat or anti-dog MoAb and human IgE antibodies recognize identical or closely related determinants on cat and dog albumin.     

Cross-allergenicity between extracts of hair from different dog breeds and cat fur

Skin tests and RAST determinations with breed-specific dog allergen extracts and a cat allergen preparation were made on forty-four atopic patients divided into three groups. Group 1 were twenty dog-owning atopic patients without clinical signs of dog sensitivity. Group 2 contained twenty-one patients with a clinical history that suggested allergy to dogs, and Group 3 contained ten atopic patients who were sensitive to cats. In neither the in vivo nor the in vitro tests was there any evidence for dog breed specificity, nor was dog albumin found to be a major allergen in the population studied, though a few individuals showed strong RAST activity to albumin. Furthermore, a cat fur extract inhibited the reaction between dog hair and anti-dog serum, and a dog hair extract inhibited the reaction between cat fur extract and anti-cat serum.     

Maxisorp

For determination of allergen-specific IgE in cell culture supernatants and other highly diluted IgE preparations a radioallergosorbent test (RAST) based on high adsorption polystyrene test tubes has been developed ("Maxisorp RAST"). Cladosporium herbarum extract was used as a model allergen but timothy grass pollen, house dust mite and dog dander showed similar results. The test showed specificities of both allergen and immunoglobulin isotype and significant correlations (r = 0.67-0.88) with established RAST procedures were found. Based on immunosorbent-purified allergen-specific IgE the estimated sensitivity was within the order of 150-300 pg allergen-specific IgE per ml. The within-assay variation was 4-9% and the inter-assay-variation 17-29%. The Maxisorp RAST is useful as an inhibition assay for quantitating allergenic activity down to 0.1 biological units/ml of allergen extracts.     

Comparison of the antigenic and allergenic properties of three types of bovine epithelial material.

The antigenic and allergenic characteristics of three bovine epithelial extracts, dander, skin scrapings and whole skin, were compared using IgE-ELISA inhibition and SDS-PAGE with immunoblotting. Cow dander extract was shown to contain more allergenic activity than skin scrapings or whole skin extracts which were needed in about three times higher amounts than cow dander extract to induce the same degree of inhibition in ELISA. Skin scrapings and whole skin extracts contained more high-molecular weight components than dander extract. These components were at least partly serum-derived and reacted often with the IgG but not with the IgE of both the cow-asthmatics and their control subjects. The antigenic characteristics of the low-molecular weight components as well as the allergenic qualities of these three epithelial preparations were generally similar. Using the sera of 49 cow-asthmatic farmers, two major allergens were detected at 20 and 22 kD in all three extracts. Our results show that the highest amount of allergenic material and all the essential allergens are present in cow dander extract. In addition, the normally non-allergenic high molecular weight components are detected in low concentrations in dander extract. Therefore it is concluded that cow dander extract is the best alternative for allergen purification and allergen extract preparation.     

Characterization of the main IgE-binding components of cat dander

Cat dander extract (CDE) was fractionated by preparative isoelectric focusing (pIEF). All the fractions showed allergenic activity, but the most acidic ones (pH range 3.3-4.3) had the highest specific activity. These fractions also had the highest cat allergen 1 content and a partial antigenic identity as determined by fused rocket immunoelectrophoresis. Immunoblotting of the pIEF fractions and the crude CDE after analytical IEF showed the presence of native proteins with IgE-binding ability all along the pH range 3.3-6.2. However, when the IgE-immunoprecipitated components of the most acidic pIEF fractions were focused in the presence of urea, a neat band was found at pH 3.9. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of CDE proved the presence of, at least, 9 polypeptides with affinity to IgE, those with molecular weights of 18,000 and 22,000 daltons being the most active ones. These polypeptides presumably belong to cat allergen 1 since they were found in the most acidic pIEF fractions as well as in the crossed immunoelectrophoresis arc corresponding to this allergen. Furthermore, we found a group of active proteins with molecular weights of 10,000, 22,000, 25,000, 50,000 and 65,000 daltons (cat albumin) and with isoelectric points between 3.3 and 6.2.     

Identification of allergens in dog dander extract. I. Clinical and immunological aspects of allergenicity activity

Allergenic activity of mixed dog dander extract was compared with dog serum albumin by skin testing, RAST testing and RAST inhibition. Whereas albumin is a potent allergen, additional lower molecular weight allergens determined by sucrose density gradient centrifugation and immunoelectrophoresis were found. Identification of the most potent allergens and standardization of extract used for testing and treatment is a necessary prerequisite in understanding dog dander hypersensitivity.     

Isolation and characterization of cat allergens

A crude (saline soluble) extract of cat skins capable of eliciting a strong positive prick skin test in cat sensitive individuals was fractionated on Sephadex G200. Active fractions were pooled and successively fractionated on isoelectric focusing gradients of pH 3–5 and pH 4–5. Allergenic activity was localized in two peaks with mean isoelectric points of 4.1 and 4.35 respectively. On immunoelectrophoresis the allergen with pI = 4.35 was associated with a protein which was subsequently found to be immunologically indistinguishable from serum albumin and to have a molecular weight of 69,000 daltons. The allergen with pI = 4.1 migrated in the α₂-region on immunoelectrophoresis and had a mean molecular weight of 55,000 daltons. This allergen was isolated and analysed for amino acid and carbohydrate content. A combined extract of both allergens coupled to microcrystalline cellulose and used in a RAST procedure readily distinguished between two groups of individuals classified as skin test positive and skin test negative to cat allergen.     

Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy

BACKGROUND: Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. OBJECTIVE: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. METHODS: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. RESULTS: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris, and its immune reactivity closely resembled that of its natural counterpart. CONCLUSION: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein.     

Skin prick test in the diagnosis of dog dander allergy A comparison of different extracts with clinical history, provocation tests and RAST

Skin prick tests (SPT) were carried out on 164 asthmatic children using dog dander allergen preparations (A, B1, B2 and D) from three manufacturers. Bronchial and/or conjunctival provocation tests with allergen A were performed on all patients, as well as the Phadebas® RAST (allergen e5). There was a close agreement between the clinical history and the SPT with allergens A and D. The provocation tests and RAST gave results in good agreement with the SPT reactions to all allergens except B1. The overall agreement between the SPT reactions to the different allergens was good. However, significant differences were observed in the allergenic activity of the different preparations and between different batches from the same manufacturer (allergens B1 and B2).     

Allergenicity and cross-reactivity of cat and dog allergenic extracts

This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophore-tic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. Binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized. If these observations confirmed that CAT-1 is a major allergen in cat-sensitive subjects, they also showed a different profile of reactivity in patients sensitive to cats only and those sensitive to both cats and dogs, which could have diagnostic and therapeutic implications.     

Antigens and allergens from the common house dust mite Dermatophagoides pteronyssinus: Part I. Demonstration of multiple allergens by immunochemical and biologic analyses

The complexity of a highly allergenic water-soluble extract of the common house dust mite Dermatophagoides pteronyssinus has been investigated by use of the preparative fractionation techniques of ultrafiltration, gel permeation chromatography, and isoelectric focusing, and the allergenic properties of the resultant fractions are assessed by skin prick tests, RAST, and a histamine-release assay. In addition, two analytical approaches were used to identify allergens-radioimmunoelectrophoresis and a new technique for detecting IgE-binding proteins separated by isoelectric focusing and transferred onto a nitrocellulose membrane by transverse electrophoresis. These various techniques for allergen detection revealed differences in the number and distribution of allergens in the crude mite extract. The data obtained are consistent with the conclusion that allergenic activity of D. pteronyssinus extracts is due to the existence of a number of antigenically distinct allergens and not due to a single, major allergen.