Allergens of mammalian origin. V.

Eight commercial cat dander extracts and two pelt extracts derived from mongrel and Siamese cats were compared. Cat allergen 1 and cat albumin were measured by radial immunodiffusion. Allergenic activity was evaluated by prick test and a modified radioallergosorbent test. In the latter, the dilution of each extract that produced 50% inhibition of binding of IgE antibodies to insolubilized cat allergen 1 (RAST 1) and insolubilized cat serum (RAST 2) was determined. The total non-dialysable solid content of the extracts did not correlate with any other parameter. Cat allergen 1 content determined by radial immunodiffusion correlated with average prick test results in ten cat-sensitive subjects and with RAST 1 activity. Cat albumin content correlated weakly with RAST 2 activity but not with any other measure of allergenic activity. Absorption of each extract with the γ-globulin fraction of rabbit antiserum to cat allergen 1 significantly reduced prick test reactivity and RAST 1 activity, but not RAST 2 activity. These results indicate that cat allergen 1 is an important allergen in cat dander extracts and its measurement may be used to standardize the allergenic activity of such extracts.     

Allergy to mice. I. Identification of two major mouse allergens (Ag 1 and Ag 3) and investigation of their possible origin

An extract of dust from the outlet filters of a mouse isolator was used as a basis for determining the source of inhalant allergens for subjects sensitive to this species. The antigenic components, identified by crossed immunoelectrophoresis (XIE), were compared to those found in extracts of other mouse-derived source materials, i.e. urine, fur, dander and saliva. Of the eight dust components, one (Ag 1) was identified as antigenically identical to the major urinary pre-albumin whilst the others were detected in fur, and to a lesser extent dander and saliva. None of the dust antigens was detected as a component of food or bedding.
Crossed radio-immunoelectrophoresis (XRIE), performed using sera from a group of fifteen mouse-allergic subjects (positive by RAST to mouse extracts), identified seven of the dust antigens as IgE-binding components. Antigens 1 and 3 were reactive with all the sera tested and have, therefore, been termed the'major'allergens. Varied responses were obtained to the other'minor'antigens.
Ag 1 (urinary pre-albumin) and Ag 3 were detected in all samples of mouse dust studied. RAST and RAST inhibition also indicated the presence of urinary prealbumin. These findings suggest that the major mouse inhalant allergens may be derived predominantly from urine and secretions originating in the skin and present on the fur.     

Allergy to rabbits

Quantitative immunoelectrophoretic techniques have been used to study the antigenic components found in extracts of dust collected from rabbit housing areas. To determine the possible source of these antigens, comparisons have been made to rabbit saliva, urine, fur and dander. Specific antisera for the rabbit extracts were raised in guinea pigs. One major component of the dust (Ag Rl) was also found in large amounts in saliva, slightly less in fur and in only minimal amounts in urine and dander. Crossed radioimmunoelectrophoresis (XRIE) of the dust, performed with sera from 14 rabbit allergic individuals who were RAST positive to rabbit saliva, urine and dust identified four IgE-binding constituents. Individual responses varied but all sera reacted with Ag Rl, identifying this as a major rabbit allergen. Dust RAST inhibition studies with rabbit dust, saliva and urine indicated saliva to be closely related to the dust. Ag Rl is a glycoprotein which appears to be very heterogeneous in nature. It produced a broad biphasic precipitin peak on immunoelectrophoresis and eluted from Sephacryl S-200 gel filtration over the molecular weight range 30-50 Kd, although a molecular weight of 17 Kd was indicated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gradient gel electrophoresis. The RAST inhibition results and the antigenic similarity of saliva to the dust suggest this to be the most likely source of the major rabbit allergen, Ag Rl.     

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.     

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.     

A comparative study of the allergens of cat urine, serum, saliva, and pelt

In direct RAST analyses of sera from 43 individuals with a history of cat allergy, 39.5% were positive to cat pelt, 37.5% to cat saliva, and 12% each to cat urine and serum. The cat pelt and saliva extracts contained allergen 1, but cat serum and cat urine collected by bladder puncture had no detectable levels of this allergen. A crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis analysis failed to reveal any allergen in urine or serum that was not also present in the saliva or pelt preparations, although urine had two allergens not present in serum. When serum from a patient who was direct RAST positive to cat pelt, serum, saliva, and urine was tested by crossed radioimmunoelectrophoresis, it was determined that a total of six allergens were detectable in cat pelt, three in cat urine, and six in cat serum. Since cat serum contains no detectable cat allergen 1, it may be concluded that at least seven allergens derived from the cat are capable of binding to IgE antibody in humans.     

RAST with animal dander, urine, saliva and serum

Binding of specific IgE antibodies from the sera of patients allergic to animals was investigated by direct RAST, using the animal's dander, urine, saliva or blood serum as insolubilized allergens. In allergy to rat, mouse, guinea pig, dog, cat or horse, the RAST results with the excretions of a particular animal were mutually well correlated. RAST with the animal blood serum was positive less often, and only in cases of a positive dander RAST. It is concluded that a RAST with animal dander precludes the use of other animal products.     

Affinity purification of a major and a minor allergen from dog extract: serologic activity of affinity-purified Can f I and of Can f I-depleted extract

Most dog-allergic patients react to a major 25 kd component on sodium dodecyl sulfate blots, Can f I (Ag 13). We initially raised monoclonal antibodies (Cf-3 and Cf-2) reactive with IgE-binding components distinct from Can f I. After a slight modification, we immunized other strains of mice and produced monoclonal antibodies coded Cf-1a and Cf-1b reactive with Can f 1. We affinity purified the allergens, Can f I and "dog allergen 2" with Cf-1a and Cf-2 ascites, respectively, and house dust-rich dog dander. Comparison of purified Can f I with dog saliva in RAST demonstrated that Can f I is a potent allergen for most dog-allergic patients (average response, 70%). After depletion of dog saliva of Can f I, a slightly lower contribution for Can f I was found, but the overall results supported the conclusion that Can f I is a major allergen in dog saliva. Comparison of purified dog allergen 2 with dog dander in RAST demonstrated that dog allergen 2 is less important for dog-allergic patients (average response, 23%). We radiolabeled the purified allergens and developed assays to measure Can f I and dog allergen 2 in allergen extracts and dust samples. Dog saliva was a strong allergen source, dog urine and feces contained very little of the allergens, and both allergens were found to a variable degree in the nine dog breeds tested.     

Allergy to guinea pigs: I Allergenic activities of extracts derived from the pelt, saliva, urine and other sources

Guinea pig-sensitive patients with asthma and rhinitis were skin test positive to extracts of several materials derived from guinea pigs. A radioallergosorbent test (RAST) was developed to measure serum IgE specific for the dander, urine, saliva and also for dust from the air-vent filters of a room housing guinea pigs. A strong correlation was found between positive skin test reactions, and raised serum IgE to these extracts. Furthermore, the relative allergenic potency of extracts was similar when determined by skin-prick testing and by inhibition of the RAST to guinea pig dust. Non-guinea pig-derived extracts such as the hay, sawdust and diet had negligible activity in skin testing and RAST inhibition; and preparations of Dermatophagoides pteronyssinus, house dust and rat dust did not inhibit the RAST for guinea pig room dust. The guinea pig dust, dander, fur, urine and saliva were the more potent extracts; while whole pelt, faces and serum were considerably less active. Extracts from different sexes were not appreciably different in potency. The results of skin testing. RAST and RAST inhibition suggest cross-allergenicity between the various extracts. Although material shed from the pelt may have been derived from saliva, or even urine, allergenic activities of urinary and salivary preparations were found to be less than those of the dander, fur or dust. This suggests that allergens have become concentrated on the pelt.