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.     

Low molecular weight allergens of the pollen of Parietaria officinalis

The allergenic composition of a low mol. wt fraction of the pollen extract of Parietaria officinalis (PO) was investigated. Fraction C, that was eluted after oxytocin (mol. wt 1040) when the pollen extract was gel filtered on Sephadex or on Biogel, was cross-reactive in the RAST with the major allergen P015 and was capable of eliciting histamine release from leukocytes of sensitive donors. RAST inhibition (RAST I) analysis of the eluate of gel filtration on Sephadex G-10 revealed several peaks of IgE binding activity. Analysis of fine specificity of response of individual patients carried out by skin-prick tests and by RAST I, revealed individual patterns of reactivity, indicating that allergens contained in fraction C were minor allergens.     

Demonstration of a partially cryptic epitope of the major cat allergen Fel d 1: consequences for mAb-based standardization of cat extracts

BACKGROUND: Antiallergen mAbs that do not recognize clinically important isoforms have been described, raising the question of the selection of mAbs for quantifying major allergens in order to standardize allergenic extracts. This question is even more critical if mAbs can discriminate between different forms of allergen molecules with the same amino acid sequence. OBJECTIVE: We sought to demonstrate that an anti-Fel d 1 mAb was able to discriminate between two forms of the major cat allergen independently of its amino acid sequence and to determine the relative importance and stability of both forms in various cat extracts. METHODS: Anti-Fel d 1 mAbs were raised in mice and characterized. By using two of these mAbs, a two-site ELISA was developed to quantify Fel d 1 in mass units. RESULTS: One of the anti-Fel d 1 mAbs developed was shown to specifically recognize a particular form of Fel d 1. A two-site ELISA with this mAb to capture Fel d 1 was able to quantify the allergen specifically in this form. It was then shown that (1) the quantitative importance of this form of Fel d 1 could vary from one cat extract to another, (2) Fel d 1 was converted into this form under certain conditions, and (3) both converted and unconverted forms of Fel d 1 may bear IgE epitopes that are specific. CONCLUSION: Although the present study emphasizes the issue of selecting mAbs that are not too specific to standardize allergenic extracts, it also demonstrates that very specific mAbs can be of interest, especially to verify the stability of allergens in extracts, since this stability might have clinical implications.     

The cat lipocalin Fel d 7 and its cross‐reactivity with the dog lipocalin Can f 1

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat‐sensitized Swedish patients and elucidated its allergenic activity and cross‐reactivity with the dog lipocalin Can f 1. Sixty‐five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross‐reactive antisera. Fel d 7 is a common allergen in a Swedish cat‐sensitized population that cross‐reacts with Can f 1, and may contribute to symptoms in cat‐ but also in dog‐allergic patients.     

Rational design of hypoallergens applied to the major cat allergen Fel d 1

BACKGROUND: Allergen-specific immunotherapy is the only treatment for allergic disease providing long-lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy. OBJECTIVE: The aim of this study was to provide a rational design of hypoallergen candidates for immunotherapy by using structural information and knowledge of B and T cell epitopes of an allergen. METHODS: The three-dimensional structure of the major cat allergen Fel d 1 was systematically altered by duplication of selected T cell epitopes and disruption of disulphide bonds. Seven Fel d 1 derivatives were generated and screened for allergenic reactivity in comparison with recombinant Fel d 1 in competition-ELISA. The allergenicity was further evaluated in basophil activation experiments and T cell reactivity was assessed in a lymphoproliferation assay. RESULTS: Three out of seven Fel d 1 derivatives, with two duplicated T cell epitopes and one or two disulphide bonds disrupted, were carefully evaluated. The three derivatives displayed a strong reduction in allergenicity with 400-900 times lower IgE-binding capacity than recombinant Fel d 1. In addition, they induced a lower degree of basophil activation and similar or stronger T cell proliferation than recombinant Fel d 1. CONCLUSION: By a rational approach, we have constructed three Fel d 1 hypoallergens with reduced IgE-binding capacities and retained T cell reactivities. This strategy may be applied to any well-characterized allergen to improve immunotherapy for allergic patients.     

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.     

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.     

High-dose allergen exposure leads to tolerance

Reports of decreased sensitization to cat allergen (Fel d 1) among individuals living with a cat or subjects exposed to high-dose cat allergen may be explained by the development of a form of high-dose tolerance resulting from natural exposure to an inhalant allergen. Although the epidemiological data regarding the relationship between exposure and sensitization to Fel d 1 are conflicting, the ability for high-dose Fel d 1 to induce a characteristic nonallergic immune response with a distinctive serum antibody profile has been established. Definition of this modified T-helper (Th)2 response to cat allergen, coupled with the renewed interest in regulatory T cells within the immunology field, has provided an avenue for exploring the mechanism by which IgE antibody-mediated responses are controlled. There is mounting evidence to suggest that the modified Th2 response is a variation of the allergic response and that the modified Th2-allergic axis is influenced by allergen dose and genetics. This article discusses putative immune mechanisms of tolerance within the context of an allergen-specific system. The relevance of high-dose allergen exposure and alternate factors such as endotoxin to the development of tolerance is considered. Fel d 1 exhibits unique molecular and immunological characteristics that may contribute to its tolerogenic properties. Major T-cell epitopes of Fel d 1 that preferentially induce regulatory factors have been defined. Furthermore, hightiter IgE antibody responses associated with atopic dermatitis are characterized by a defect in the T-cell repertoire that is specific to these epitopes. Identification of Fel d 1 epitopes that induce interleukin-10 may provide new targets for treatment.     

Purification of Par j I, the major allergen of Parietaria judaica pollen

A component of Parietaria judaica pollen extract, previously identified as the major allergen, then reported as Pj10 and hereafter denominated Par j I has been isolated by a combination of 65% ammonium sulphate salt precipitation and gel filtration and an Ultrogel AcA54 column. The purified allergen appeared essentially homogeneous on gel filtration HPLC. The mol. wt of Par j I was estimated by electrophoretic and chromatographic techniques. All results gave values in a range from 13 K to 25 K. Analysis in SDS-PAGE under reducing conditions revealed a broad band corresponding to a mol. wt of 10 K, which retained allergenicity when tested with a patients serum pool. CIE and CRIE patterns of the isolated Par j I displayed the two precipitating lines already reported as those exhibiting the highest IgE-binding ability. Par j I showed a specific allergenic activity about 10-fold higher than that of the whole extract and was demonstrated to be the major allergen of Parietaria judaica as assessed in 25 sensitive human sera.     

Structure of the major cat allergen Fel d I in different allergen sources: an immunoblotting analysis with monoclonal antibodies against denatured Fel d I and human IgE.

In this paper we show the reactivity of monoclonal antibodies (mAbs) and human IgE with Fel d I from different allergen sources in reduced SDS-PAGE immunoblots. By SDS-PAGE analysis of affinity-purified 125I-Fel d I, a 14- to 20-kD band was found, which dissociated under reducing conditions into a 4- to 5-kD chain (chain 1) and a 11- to 15-kD chain (chain 2). In initial immunoblotting experiments with mAbs against Fel d I however, only chain 1 was detected, while the mAbs lost activity upon reduction of Fel d I. Therefore mAbs were raised against reduced and alkylated Fel d I. Two of the four mAbs to 'denatured' Fel d I that were obtained did react with chain 2 on an immunoblot under reducing conditions; the other two reacted with chain 1. The mAbs did not react with native Fel d I. With these mAbs and human IgE, differences between allergen source materials in blot patterns of Fel d I were detected. A variable molecular weight for the protein stained with mAb antichain 2 was found, and occasionally the presence of a 12-kD band stained with mAb antichain 1. Human IgE strongly bound to chain 1 of Fel d I, while only 2 out of 6 sera gave a strong reaction with chain 2. The additional 12-kD band was also recognized by human IgE. In a competitive radioimmunoassay with mAb antichain 1, differences in levels of 'denatured' Fel d I between commercial extracts were quantitated. In vitro 'denatured' Fel d I was generated under high pH conditions. The reactivity of human IgE with this 'denatured' Fel d I was demonstrated in indirect RAST experiments with mAb antichain 1. We conclude that mAb antichain 1 recognizes a form of Fel d I that is not detected by mAb antinative Fel d I, but does react with human IgE.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

Monoclonal antibodies against Olea europaea major allergen: allergenic activity of affinity-purified allergen and depleted extract and development of a radioimmunoassay for the quantitation of the allergen.

Several monoclonal antibodies (MAbs) were raised against Olea europaea pollen-extract components. Two of these antibodies, named OL 2 and OL 7, recognize two nonoverlapping, nonrepeating epitopes on the olive-allergen Ole e I, as demonstrated by different techniques. The allergen was purified in a single step by MAb-based affinity chromatography, and the allergen revealed a band at molecular weight 20 kd as well as a minor band at 18 kd on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The contribution of allergen Ole e I to the allergenic activity of O. europaea pollen extracts was determined from the effect of allergen depletion by affinity chromatography on skin reactivity and a histamine-release test. The removal of allergen caused a large reduction in the activity of the preparation in 25 monospecific olive-allergic patients. In agreement, the affinity-purified allergen demonstrated a similar response when it was compared with the whole extract in these assays. The results indicated that Ole e I is by far the most important olive-pollen allergen. A two-site solid-phase radioimmunoassay was developed for the quantitation of the allergen Ole e I in mass units. The assay was based on the MAbs, OL 2 and OL 7, and had a detection limit in the nanogram range. A good correlation was found between allergenic activity, as determined by RAST inhibition, and allergen content in 18 olive-pollen extracts. This result indicates that the assay can be a good alternative to RAST inhibition for the standardization of O. europaea extracts.     

Heterogeneity of a major allergen from olive (Olea europea) pollen.

Studies were carried out in order to confirm and extend knowledge of the physico-chemical properties of an allergenic material found in the pollen of the olive tree (Olea europea). The sera from 88% of patients who were sensitive to olive pollen contained IgE that reacted with a 19,000 Mr component and many also reacted to a 17,000 Mr band as shown by SDS-PAGE immunoblotting. A monoclonal antibody (OL-1) produced to the 19,000 Mr component also reacted with the 17,000 Mr band, and with bands at 21,000 and 41,000 under non-reduced conditions. HPLC separation followed by SDS-PAGE and immunoblotting of the fractions indicated that the allergen fraction from which the 19,000 and 17,000 components were derived had a mol. wt between 50 and 60 kD. Isoelectricfocusing followed by immunoblotting and development with (OL-1) indicated heterogeneity of the allergen with respect to pI values. Two of the strongest components of the six identified which reacted with (OL-1) had pIs of about 5.0 and 6.0 confirming the published data. The study therefore showed that olive pollen contains a number of allergenic components, with various mol. wts and pI values, with some epitopes in common, which may in the native state be bound together or aggregated.     

Evidence for a Fel d I-like molecule in the "big cats" (Felidae species).

In this study, we investigated the cross-reactivity pattern of IgE and IgG4 antibodies to the major feline allergen, Fel d I. We studied the IgE and IgG4 response of 11 cat-allergic patients against Fel d I-like structures in eight members of the Felidae family: ocelot, puma, serval, siberian tiger, lion, jaguar, snow leopard, and caracal. Hair from these "big cats" was collected, extracted, and used in a RAST system and histamine-release test. By means of a RAST-inhibition assay with affinity-purified Fel d I from cat dander, it was established that, in the Felidae species, a Fel d I equivalent is present that reacts with IgE and IgG4 antibodies. We found that all patients had cross-reacting IgE antibodies to seven of the Felidae tested; no IgE antibodies reactive with the caracal were found. Eight of 10 patients with IgG4 antibodies directed to cat dander also had IgG4 antibodies directed to several Felidae species, including the caracal. However, the correlation between the IgE and the IgG4 antibody specificity was low, indicating that, in the case of Fel d I IgE and IgG4, antibodies do not necessarily have the same specificity.     

Investigation of the allergenic activity of isolated fractions of a standardized partly purified whole mite body (Dermatophagoides pteronyssinus) extract

Twelve fractions of a molecular weight range of 1.35-670.00 kilodaltons (kD) were isolated from a biologically standardized partly purified whole mite body extract (Dermatophagoides pteronyssinus) by preparative size exclusion high-performance liquid chromatography. The allergenic activity and the antigen and allergen patterns of the isolated fractions were investigated by RAST, RAST inhibition and crossed (radio)immunoelectrophoresis (CIE/CRIE). By CRIE, each of the fractions showed allergen patterns, mostly of different compositions. Each fraction showed allergenic activity of different degrees by RAST inhibition. The highest allergenic activity could be measured by RAST inhibition with fractions which contained predominantly the major allergens Der pI and PY as detected by CRIE. Also proteins of higher molecular weights (greater than 158 kD) showed IgE-binding capacities. Nearly all antigens detected by CIE could be identified as allergens using CRIE.     

Fel d I allergen: skin and or saliva?

To determine the relative importance of saliva and sebaceous glands as sources of Fel d I allergen, we compared Fel d I levels at the base and tip of the hair in areas presenting more or less sebaceous glands and areas licked more or less frequently. The amount of Fel d I was significantly higher at the base than the tip of the hair. Further, it was strongly correlated with the density of sebaceous glands. This study demonstrated that the most abundant source of Fel d I allergen is cat skin.     

Purification and characterization of Lep d I, a major allergen from the mite Lepidoglyphus destructor

A major allergen of the storage mite Lepidoglyphus destructor (Lep d I) has been purified by affinity chromatography using an anti-Lep d I monoclonal antibody. The purity of the protein obtained by this procedure was assessed by reverse-phase HPLC. Lep d I displayed a molecular weight of 14 kD on SDS-PAGE under non-reducing conditions, and 16 kD in the presence of a reducing agent. Analytical IEF revealed a little charge microheterogeneity, showing three bands with pIs 7.6-7.8. Purified Lep d I retained IgE-binding ability, as proved by immunoblotting experiments after SDS-PAGE and RAST with individual sera from L. destructor-sensitive patients. Results from the latter technique demonstrated that 87% of L. destructor-allergic patients had specific IgE to Lep d I, and a good correlation between IgE reactivity with L. destructor extract and Lep d I was found. In addition, RAST inhibition experiments showed that IgE-binding sites on Lep d I are major L. destructor-allergenic determinants, since Lep d I could inhibit up to 75% the binding of specific IgE to L. destructor extract; on the other hand, Lep d I did not cross-react with D. pteronyssinus allergens.     

Allergenic relevance of Cupressus arizonica pollen extract and biological characterization of the allergoid

BACKGROUND: Cupressaceae (cypress) pollens can cause pollinosis in winter. However, the lack of specific commercial extracts combined with the early pollination period of cypress trees make a precise diagnosis difficult. The need for a reliable and effective cypress extract for diagnostic and therapeutic purposes is increasingly felt. METHODS: Mixed or single Cupressus arizonica, lusitanica and sempervirens pollen extracts precipitated with ammonium sulfate (PPT) were compared by direct RAST, RAST inhibition and SDS-PAGE techniques. The major allergen of C. arizonica (Cup a 1), purified by anion exchange chromatography, was checked by immunoblotting experiments before chemical modification, in parallel with a C. arizonica extract, with potassium cyanate (KCNO) to obtain a monomeric allergoid. The allergoid extract was characterized for its biological, chemico-physical and immunological features by RAST inhibition, SDS-PAGE and ELISA assays. RESULTS: Direct RAST, RAST inhibition, and SDS-PAGE data indicated that the PPT C. arizonica pollen extract showed the most allergenic potential, and it can be considered representative of the Cupressus spp. Immunoblotting data confirmed Cup a 1 as a major allergen. RAST inhibition and ELISA showed that modified PPT C. arizonica extract had less IgE reactivity than the native, non-modified extract, while preserving the immunogenic capacity typical for an allergoid. Finally, the SDS-PAGE profile of Cup a 1 allergoid was similar to native Cup a 1 allergen, suggesting the modified C. arizonica extract shows the characteristics of a monomeric allergoid. CONCLUSIONS: The PPT C. arizonica pollen extract shows good in vitro diagnostic potential and its chemically modified form offers the features of a monomeric allergoid. It might therefore lend itself to the development of a product to be administered by the sublingual or oromucosal route for immunotherapy of individuals with cypress pollinosis.     

A chimeric human-cat Fcgamma-Fel d1 fusion protein inhibits systemic, pulmonary, and cutaneous allergic reactivity to intratracheal challenge in mice sensitized to Fel d1, the major cat allergen

Co-aggregation of FcepsilonRI with FcgammaRIIb can block FcepsilonRI-mediated reactivity and Fc gamma:allergen chimeric proteins, by co-crosslinking FcgammaRIIb to allergen-specific IgE bound to the FcepsilonRI can block allergen-specific reactivity. We evaluated whether a human cat chimeric fusion protein (GFD) composed of part of the human Ig G1 Fc fused to the major cat allergen (Fel d1) would function as allergen immunotherapy while not inducing acute allergic reactivity in mice sensitized to Fel d1. Injection of GFD 6 h prior to Fel d1 challenge acutely blocked systemic and skin reactivity to Fel d1 challenge while mice given subcutaneous immunotherapy with GFD at days 37, 38, and 39 showed inhibition of systemic, lung, and cutaneous reactivity to Fel d1 2 weeks later. GFD immunotherapy did not induce systemic reactivity. Overall, the Fcgamma-Fel d1 chimeric fusion protein blocked Fel d1-induced IgE-mediated reactivity but did not induce in vivo mediator release on its own; suggesting that this approach using allergen combined with Fc gamma1 so as to achieve inhibitory signaling may provide an enhanced form of allergen immunotherapy.     

The aerodynamic characteristics of cat allergen

To further characterize airborne cat allergen and a newly established cat challenge facility, airborne Fel d I levels and particle size distributions were studied in both the cat challenge room and home environments under different conditions of ventilation and physical activity. In the cat room, there has been a dramatic and continued rise in the concentration of airborne Fel d I since the room was established. No differences in total airborne Fel d I levels or particle size distribution were detected under widely differing rates of ventilation (40 air changes per hour vs 8 ac/hr vs < 1 ac/hr). Likewise, altering ventilation had little effect on the clearance of airborne antigen after disturbance. Significant increases in allergen levels were detected, however, after simply allowing the cats to leave their holding cage and move about the room. Fel d I levels in homes ranged from 2-468.5 ng/m3, similar to the levels seen in the cat room without disturbance. Fel d I particle size distribution was very consistent in both homes and the cat room with the majority of airborne Fel d I being detected on particles > 17 microm. Although very little allergen (< 15%) was detected on particles < 4 microm, this important fraction was present under all conditions. We conclude that airborne cat allergen resides primarily on relatively large particles, that a small but consistent fraction is found on very small particles, and that neither allergen levels or particles size distribution are significantly influenced by ventilation.