Crossed Radioimmunoelectrophoresis Research Articles

Cross-reactivity between storage and dust mites and between mites and shrimp

Many patients have sensitivities to multiple species of storage and house dust mites. It is not clear if this is because patients have multiple sensitivities to species-specific mite allergens or if these mites share many cross-reacting allergens. Our objective was to further define the cross-allergenicity between several species of storage and house dust mites using crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), immunoblotting, and ELISA. CIE and CRIE reactions revealed that storage mites shared two cross-antigenic molecules and one of these bound IgE in a serum pool from mite allergic patients. Antibody in anti-sera built to each species of mite recognized many SDS-PAGE resolved proteins of other mite species and this suggested the potential for other cross-reactive allergens. Among patient sera, IgE bound to many different proteins but few had IgE that bound to a protein with common molecular weights across the mite species and this suggested mostly species-specific allergens. Antiserum built to each mite species precipitated one protein in shrimp extracts that bound anti-Der p 10 (tropomyosin) and IgE in the serum pool. Anti-Der p 10 showed strong binding to shrimp tropomyosin but very little to any of the mite proteins. ELISA showed the mite extracts contained very little tropomyosin. The storage and dust mites investigated contain mostly species-specific allergens and very small amounts of the pan-allergen tropomyosin compared to shrimp and snail.

Characterization of allergens of Anisakis simplex.

Anisakis simplex is an intestinal parasite of sea mammals. The larvae infect crustaceans, cephalopods and fish. Humans may consume A. simplex third stage larvae (L3) when eating infected raw or under-cooked fish. Consumed larvae cause an inflammatory reaction when they penetrate the digestive mucosa. The larvae or their secretory/excretory products can sensitize humans and induce an immunoglobulin E (IgE)-mediated allergic reaction. This parasite is now being implicated in numerous cases of allergic reactions after eating fish. The purpose of this study was to evaluate the allergenicity of proteins present in an extract of the third stage larva. Rabbit antiserum raised to A. simplex somatic extract (L3) was reacted by crossed immunoelectrophoresis (CIE) with the same somatic extract. Crossed radioimmunoelectrophoresis (CRIE) was also performed by incubating CIE gels first in the sera of 13 individuals with positive immunoCAP to A. simplex and then in radiolabeled anti-human IgE. Twelve to 16 antigen-antibody precipitin peaks were visualized on Coomassie blue stained CIE gels in which somatic extract was reacted with somatic-antiserum. Autoradiography of CRIE gels showed that 18 different proteins bound IgE in patient sera. Individual patients had serum IgE directed at two to 10 different allergens. Five of these allergens were recognized by >/=50% of the patients. No allergen was recognized by every patient and no patient had serum IgE directed at all 18 allergens. Somatic extracts of A. simplex L3 larva contain a large number of allergenic molecules and there is significant variability between patients in their sensitivity and reactivity to these allergens.

AirborneAlternariaSpores: Potential Allergic Sensitizers in Saudi Arabia

In order to investigate the role of airborne Alternaria spp. in the sensitization of individuals having respiratory allergy symptoms, particularly bronchial asthma, an aerobiologic and clinical (diagnostic) study was conducted at several centers in Saudi Arabia. Airborne Alternaria spores were studied at four different centers in Riyadh, Jeddah and Al-Khobar, including three years at one site. Skin prick tests (SPT) were conducted on a total of 616 allergic individuals attending allergy clinics at six different hospitals in Abha, Qassim, Gizan, Jeddah, Hofuf and Makkah regions. Alternaria spores constituted between 1.9%-9.6% of the total fungal air spora, and the maximum concentration exceeded 5x10 2 spores per m3 of air in Jeddah, followed by 4.9x102 spores per m3 in Al-Khobar. Mean weekly and monthly concentration of Alternaria fluctuated, reaching up to 150 spores per m 3 and 60 spores per m 3, respectively. Quantitative regional variations were also recorded. IgE-mediated SPT reactivities conducted on allergic patients resulted in an overall 21.6% positive reactions, showing mild, moderate and strong wheal and erythema. The findings reveal that Alternaria spores are prevalent as a major component in the outdoor environment of the Kingdom, with peaks in April and October. The overall 21.6% positive SPT reactions indicate the sensitization level or cross-reactive sensitivity to Alternaria allergens. The study suggests that airborne Alternaria can be a potential allergic sensitizer in susceptible individuals and can be a risk factor in sensitized individuals with symptoms of bronchial asthma and allergic rhinitis in the Kingdom of Saudi Arabia.

In-house reference (IHR) preparation of Candida albicans allergen extract. A standardized extraction procedure.

A standardized, controlled procedure for preparation of an in-house reference (IHR) preparation of an allergen extract of Candida albicans is described. The procedure, based on previous studies of allergens of C. albicans, is designed to yield a maximum of allergens in optimum extraction conditions and to provide a reference preparation for further extract production. The SDS-PAGE, IgE-immunoblotting, and crossed radioimmunoelectrophoresis (CRIE) analyses showed that the procedure is reproducible with acceptable batch-to-batch variation. The variation in the content of the most important allergens, namely, proteins with molecular weights of 46, 29, and 27 kDa in the pooled final batches, is acceptable (coeff. of variation < 15%), although in the intermediate batches of different strains, the coefficient of variation may occasionally exceed 20%. A comparison with other C. albicans allergen preparations used in our previous studies is also presented. The resulting extract can be used as a reference in further extract production and also in experimental in vitro and in vivo studies.

Allergenic characterization of Tyrophagus putrescentiae using sera from occupationally exposed farmers.

Allergic sensitivity to storage mites is common in farmers and others who work in environments where hay and grain are handled, stored, or processed. The clinical importance of storage mite-induced allergy in these individuals is generally unknown in the United States. Tyrophagus putrescentiae is a common storage mite, the allergenicity of which has not been characterized. The purpose of this study was to characterize the allergenicity of Tyrophagus putrescentiae. Crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) were performed to characterize the allergens of this mite using the sera from 24 occupationally exposed farmers RAST-positive to T. putrescentiae. CRIE analysis of T. putrescentiae extract utilizing individual farmers' sera identified 14 allergens. The number of allergens that bound IgE in the individual sera ranged from 5 to 11. Sensitivity to the different allergens varied both within and between patients but 100% of the farmers' sera recognized the same five allergens precipitated by CIE. Fifty percent of patients' sera recognized one additional. Forty-two and 21% of the sera had IgE binding to six and seven of the allergens, respectively. The results of this study showed that farmers who were occupationally exposed to storage mites had serum IgE specific for many potent allergens from T. putrescentiae. Persons who are exposed to stored product mites in occupational settings or by consumption of food containing these mites are therefore at risk of sensitization and allergic reaction. Increased awareness of sensitivity to this mite in the United States' population is needed.

Latex allergy diagnosis: in vivo and in vitro standardization of a natural rubber latex extract.

For the diagnosis of IgE-mediated (immediate) hypersensitivity to natural rubber latex (NRL), skin prick testing with extracts of latex gloves has been widely used, but such extracts are difficult to standardize. The present study aimed to produce on an industrial scale an NRL extract from freshly collected NRL and to evaluate, calibrate, and standardize the extract by both in vivo and in vitro testing. The source material, latex of the rubber tree, Hevea brasiliensis (clone RRIM 600), was frozen immediately after collection in Malaysia and shipped in dry ice to Stallergènes SA, France. Protein and allergen profiles were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), and crossed radioimmunoelectrophoresis (CRIE). Allergen quantification was effected by RAST inhibition. The capacity of the preparation to elicit immediate hypersensitivity reactions in vivo was measured by skin prick testing in 46 latex-allergic patients and 76 nonallergic control subjects. SDS-PAGE and immunoblot profiles of the extract and an NRL standard (E8) provided by the US Food and Drug Administration were almost identical, disclosing several distinct IgE-binding proteins with apparent molecular weights of 14, 20, 27, 30, and 45 kDa, conforming to reported molecular weights of several significant NRL allergens. An arbitrary index of reactivity (IR) of 100 was assigned to the extract at 1:200 dilution (w/v), having a protein content of 22 micrograms/ml. Skin prick testing of latex-allergic patients and controls using the extract at 100 IR revealed 93% sensitivity, 100% specificity, 100% negative predictive value, and 96% positive predictive value. In conclusion, a skin prick test reagent for diagnosis of type I NRL allergy was successfully standardized. The reagent was demonstrated to contain most, if not all, of the currently known clinically significant NRL allergens, and it showed high sensitivity and specificity.

Cloning of two distinct cDNAs encoding parvalbumin, the major allergen of Atlantic salmon (Salmo salar).

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin beta (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal sl). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal sl was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST- and ELISA-inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA-library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the beta-lineage of parvalbumins and include the entire coding region.

Identification of IgE-Binding Egg White Proteins: Comparison of Results Obtained by Different Methods

The binding of IgE to egg white proteins was investigated for 34 sera from adults with a positive case history and/or positive RAST towards egg, and the impact of experimental conditions on IgE binding in commonly used methods was studied. Radioimmunoblotting after SDS-PAGE of both reduced and unreduced egg white extracts showed complex reaction patterns. The results were confirmed by crossed radioimmunoelectrophoresis (CRIE). Radio dot immunobinding was used to investigate the effect of treatment of allergens for SDS-PAGE and to evaluate the other methods. As a conclusion, the use of combinations of at least two methods is recommended for the identification of IgE-binding egg white proteins. Of the 34 sera, 18 reacted with ovotransferrin, 13 with ovomucoid, 11 with ovalbumin and 5 with lysozyme. The amounts of IgE bound to ovalbumin and lysozyme were generally lower than the amounts bound to ovotransferrin and ovomucoid.

Characterization of Chenopodiales (Amaranthus retroflexus, Chenopodium album, Kochia scoparia, Salsola pestifer) pollen allergens.

Pollen extracts of the four taxonomically related weeds, Amaranthus retroflexus (Ama r), Chenopodium album (Che a), Kochia scoparia (Koc s), and Salsola pestifer (S. kali) (Sal p), were characterized by various methods including crossed immunoelectrophoresis (CIE), crossed radioimmunoelectrophoresis (CRIE), and SDS-PAGE immunoblotting. The allergen profiles were determined by CRIE and SDS-PAGE IgE immunoblotting. CRIE detected from one to four important allergens, while SDS showed up to four bands that bound IgE from a number of patient sera. CRIE and SDS-PAGE immunoblotting did not recognize the same number of important allergens in the individual weeds, and the number of allergens detected by the two methods differed considerably, suggesting that IgE-binding epitopes may be denatured during SDS-PAGE. However, it was possible to correlate the identity of some of the important allergens detected by CRIE and SDS-PAGE immunoblotting in all four weeds.

Discrepancies between in vitro and in vivo tests for house dust mite allergy: is domestic exposure a better predictor than sensitization?

We subjected seven asthmatic children to two bronchial allergen challenges, first with an extract from the house dust mite Dermatophagoides pteronyssinus (Der p) and then Dermatophagoides farinae (Der f), or vice versa. All children had elevated specific serum IgE to both species as well as reactions by crossed radioimmuno/electrophoresis (CRIE) to both group I and II allergens from both species. Immunoabsorption and subsequent analysis by CRIE showed a considerable concentration of serum IgE with specificity for epitopes common to the two species of house dust mite. Home dust sampling established that all children were exposed to Der f and only two to Der p. On bronchial provocation tests, all responded to Der f with an immediate reaction and five with a late reaction, only three of seven showed an immediate response to Der p, with four of the seven showing a late reaction. Our data could indicate that the local allergic immune reaction in the respiratory tract is sustained by ongoing exposure, and may thus have a different species specificity than the response reflected in the serum. In conclusion, our data indicates a lack of association between in vitro and in vivo tests for house dust mite allergy, which supports the continuing need for monitoring current clinical sensitization by allergen provocation tests and by measuring domestic exposure to the corresponding allergen. Extended studies are needed to support our findings.

The allergens of dog. II. Identification and partial purification of a major dander allergen.

A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, using sera from 32 dog-allergic subjects, a number of IgE radio-staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21,000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size-exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21,000 and an isoelectric point of approximately 5.2. An additional protein-staining band could still be seen of MW of 24,000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio-immunoelectrophoresis (CRIE) and that Blands et al. [2] and Løwenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.

PCR based cloning and sequencing of isogenes encoding the tree pollen major allergen Car b I from Carpinus betulus, hornbeam

Cloning of the gene encoding the major allergen, Car b I, from Carpinus betulus (hornbeam) pollen was performed using the Polymerase Chain Reaction (PCR) to specifically amplify the gene of interest using single stranded cDNA as template. Specific primers, deduced from the aminoterminal sequence of the purified protein, were tailored to facilitate direct expression of plasmic clones, and the large fraction of positive clones obtained, revealed the presence of isogenic variation. Three clones were characterized in detail by antibody based assays and nucleotide sequencing. The recombinant allergens were shown by crossed immunoelectrophoresis (CIE) to precipitate with monospecific polyclonal rabbit antibodies raised against purified Bet v I, by crossed radioimmuno-electrophoresis (CRIE) to bind tree pollen allergic patient serum IgE, and by immunoblotting to bind murine monoclonal antibodies, raised against purified Car b I from pollen. Car b I is encoded by a 159-triplets open reading frame. The molecular masses (M r = 17272, 17355 and 17217 Da, respectively), the amino acid composition, and the aminoterminal sequence of the predicted polypeptides agree well with data obtained by analysis of the protein purified from pollen. The deduced amino acid sequences show pronounced homology (73, 75 and 74% identities respectively) to Bet v I, the major allergen from Betula verrucosa (white birch) pollen. Soluble recombinant Car b I, without a fusion partner, was produced in Escherichia coli with an immunochemical reactivity closely resembling that of the native pollen allergen. The tree pollen major allergens therefore constitute an ideal system for the study of allergenic epitopes.

IgG and IgE antibody responses to cow dander and urine in farmers with cow-induced asthma.

Cow-asthmatic farmers' and negative control subjects' IgG and IgE antibody responses to bovine epithelial antigen (BEA) and urinary antigen (BUA) were studied by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The anti-BEA IgE responses of 10 highly reactive sera were also studied by crossed radioimmunoelectrophoresis (CRIE). The relative amount of allergens common to both BEA and BUA was measured by IgE ELISA inhibition and found to be 3%. In immunoblotting the IgG reactivity of the asthmatic farmers to BEA and BUA declined along their anti-BEA IgE ELISA titres. Control subjects had IgG antibodies mainly to high molecular weight components (50-70 kD) but lacked detectable IgE responses. The IgE reactivity of the asthmatic farmers was directed to only a few components. A total of two main allergens were found in cow dander (20 and 22 kD) and one in cow urine (20 kD). The 20 kD component was shown to be the most important allergen in cow antigen extracts. In CRIE, seven reactive arcs were detected. Arcs 1, 2 and 5 were detected by all 10 sera and are 3 by six and arc 7 by seven sera.

Aspergillus fumigatus specific IgE and IgG antibodies for diagnosis of Aspergillus‐related lung diseases

IgE and IgG antibodies against Aspergillus fumigatus were detected by crossed radio immunoelectrophoresis (CRIE) on the sera of seven patients with aspergilloma, six patients with allergic broncho-pulmonary aspergillosis (ABPA) and 25 patients with extrinsic asthma with Aspergillus allergy. IgE-CRIE analysis indicated the presence of A. fumigatus-specific IgE in sera of patients with ABPA and Aspergillus asthma but not of aspergilloma patients. IgG-CRIE showed that both aspergilloma and ABPA patient sera contained high levels of circulating specific IgG antibodies in contrast to sera of Aspergillus asthma patients, which did not show detectable amounts of Aspergillus-specific IgG antibodies. Specific IgE binding could be demonstrated for the major allergens Ag-10 and AG-40 in all ABPA patients, in 80% of Aspergillus asthma patients but not in sera from aspergilloma patients. Specific IgG antibodies directed towards the major allergens could be detected in most of the aspergilloma patients, between 30-70% of the ABPA patients but not in sera from patients with Aspergillus asthma.

Group V allergens in grass pollens. II. Investigation of group V allergens in pollens from 10 grasses

In an earlier study an allergen from Phleum pratense (timothy) pollen, Phl p V, has been isolated and physicochemically characterized. In this study Phl p V and immunochemically similar components from other grass pollens (group V allergens) have been investigated using immunoelectrophoretic techniques. To study the allergenic importance of the group V allergens, the allergenic compositions of 10 grass pollen extracts were investigated in crossed radioimmunoelectrophoresis (CRIE) using 20 sera from grass pollen-allergic donors. Group V allergens were identified using monospecific rabbit antibodies raised against Phl p V, anti-Phl p V, which react with other group V allergens usually producing dense precipitates in immunoelectrophoresis. In this way group V allergens were identified in eight extracts, and when present the precipitate corresponding to the group V allergen was the dominant IgE binding precipitate. All identified group V allergens bound IgE in at least 17 of the 20 investigated sera. Monospecific rabbit antibodies raised against the group I allergen of Lolium perenne (rye grass), anti-Lol p I, do not precipitate group V allergens, indicating that there are no immunochemical similarities between group I and group V allergens. In SDS-PAGE anti-Phl p V identifies IgE-binding components with molecular weights between 26 and 33 kD. In contrast, anti-Lol p I binds to components of slightly higher molecular weight. Apparently, the group V components are allergens that are physicochemically and immunochemically distinct from group I allergens.

A computational approach to the description of individual immune responses

Detailed evaluation of the IgE and IgG-subclass immune response during immunotherapy can now be performed by crossed radio immunoelectrophoresis (CRIE). Some new concepts are introduced facilitating the handling of the vast amount of data obtained by quantitating the immune response. These concepts are "distance" between antibody responses and "immune response width". The 20 patients included in this study were pollen-allergic patients who underwent specific immunotherapy in a 3-year prospective study. It was found that the immune response during immunotherapy was restricted to IgG1 and IgG4 antibodies. The semi-quantitative CRIE analysis correlated with the RAST analysis for the IgE samples before start of immunotherapy, for the IgG1 samples at week 12, and for all the IgG4 samples. During immunotherapy the number of IgG1 antibodies directed to the different antigens increased towards 11 antigens and decreased towards six. For the IgG4 antibodies the number of reactions increased towards 15 antigens and decreased towards four. The increase is generally paralleled by an increase in quantitative immune response as well. For some of the antigens a rise in the IgE antibodies is contrasted by a fall in the IgG4 antibody response, and for other antigens the opposite was true, indicating a regulatory mechanism between the IgE and the IgG4 synthesis. The IgE immune response to a number of antigens, including the major allergens before the start of immunotherapy, was quantitatively diminished during the period of immunotherapy when IgG1 was present early (week 12) in the period, and for other antigens there was a rise in IgE without an early IgG1 antibody response. This suggests that IgG1 can have a regulating influence on the IgE synthesis. Finally, we have found that IgE antibodies with specificities not present in the samples taken before immunotherapy were formed during immunotherapy. These new IgE antibodies do not, however, seem to impair the outcome of treatment.

Purification and Characterization of &lt;i&gt;Par·&lt;/i&gt; I, Major Allergen of Parietaria officinalis Pollen

Par o I, a major allergen of Parietaria officinalis, was purified from the pollen extract. The purified allergen was obtained by ultrafiltration, Sephadex gel filtration and DE-52 ion exchange chromatography: the purified preparation yields a single band in polyacrylamide gel isoelectric focusing (PAG-IEF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, a single immunoprecipitation arc in crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a single peak in size exclusion high-performance liquid chromatography (HPLC). Par o I is a glycoprotein with a protein to carbohydrate ratio of 100:21. The molecular weight, determined by SDS-PAGE, Sephadex G-50 gel filtration and size exclusion HPLC, varied between 13.5 and 14.5 kDa according to the method employed. The isoelectric point was 4.6. The amino acid composition and the sequence of the first twelve N-terminal residues were determined. The allergenicity was assayed in vivo and in vitro. 29/29 Parietaria-allergic patients were skin positive to Par o I and possessed high level of specific serum IgE antibody as it determined by radioallergosorbent test (RAST). Par o I contained dominant epitopes for human IgE as inhibited to 85% the pollen extract RAST performed with a pool of sera of allergic patients. The RAST inhibitory activity was not abolished by deglycosylation.

Comparison of allergenic potency of five batches ofAlternaria alternata for preparation of reference standard

Five batches of 34-016 isolate were grown separately on synthetic revised tobacco medium for 28 days. Extracts of the mycelia were prepared and their biochemical and immunological properties were examined. The extracts had similar isoelectric focussing (IEF) patterns. In crossed-radioimmuno-electrophoresis (CRIE) using human atopic sera, each showed two dominant and two to three minor allergens. In direct radioallergosorbent (RAST) and RAST inhibition tests with human atopic sera and passive cutaneous anaphylaxis (PCA) tests with mouse IgE, all samples were of similar potency. In IgE immunoblots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, each extract showed strongly reactive bands at 16, 18, 30, 33 and 59–100 kDa MW. These results indicate that a reference preparation ofA. alternata suitable for standardization purposes can be obtained from any batch of 34-016 isolate.

Antigenic and allergenic determinants of ovalbumin—III. MHC Ia-binding peptide (OA 323–339) interacts with human and rabbit specific antibodies

Three analogous peptides mimicking the known primary structure of ovalbumin (OA) in the region 323–339 (namely, OA 323–339, OA 323–338 and OA 324–336) were manually synthesized by the Merrifield solid phase technique. The synthetic preparations were purified by gel filtration and ion exchange high performance liquid chromatographies. The sequence linearities were deduced from the amino acid composition prior to each stage of coupling and the amino acid sequence determination on the ultimate chain. OA 323–339 was conjugated to BSA, using carbodiimide activation of the carrier protein and the conjugated compound was further used for production of antibodies in rabbit. The results showed that the region OA 323–339 was immunogenic; it could give immunoprecipitates with rabbit anti-OA 323–339-BSA in crossed immunoelectrophoresis (CIE). It similarly could bind human specific IgE from serum pools from patients allergic to egg in crossed radio immunoelectrophoresis (CRIE). Quantitative precipitation inhibition and specific IgE inhibition were used for confirming the antigenic and allergenic activities of this region. The results led us to conclude that the region 323–339 of the OA molecule encompassed an allergenic and antigenic epitope which were recognized by human and rabbit B-cells.

Determination of IgE‐specificities in nasal secretions and sera of allergic subjects by crossed radio‐immunoelectrophoresis

Allergen-specific IgE antibodies have not only been demonstrated in the serum, but also in the nasal secretions of allergic patients by means of the radio-allergosorbent test (RAST). In this preliminary study we adapted the technique of crossed radio-immunoelectrophoresis (CRIE) to nasal secretions in order to obtain further insight into the specificity of locally collected antibodies. Based on RAST results we compared CRIE patterns in serum, nasal secretions in 13 out of 23 grass pollen allergic subjects and two out of three house dust mite allergic subjects. In the nasal secretions the same IgE specificities were found as in the corresponding serum of the individual subject and as in a reference serum pool. These data indicate that the same specificity of IgE antibody can be found in the serum and the target organ. From these data there is no evidence for a local mucosal immune response that differs in specificity from the serum response.