IgE-binding and cross-reactivity of a new 41 kDa allergen of codfish

Background:   A 41-kDa IgE-reactive protein (p41) was purified from raw cod extract. This protein is homologous to an aldehyde phosphate dehydrogenase (APDH). The present study aims to evaluate the IgE-binding and the cross-reactivity of this protein in 13 patients allergic to codfish.
Methods:   IgE binding of sera from 13 patients allergic to codfish was tested by Sepharose RIA and by Western blot.
Results:   Among the 13 patients, only 4 had specific IgE to APDH detected by APDH-Sepharose RIA. The two patients who had the highest level of specific IgE to human APDH also had a class 5–6 CAP-RAST IgE level to codfish, but two other patients with a class 5 had a negative APDH-Sepharose IgE-RIA. Relative content of APDH was higher in extracts of commercial nonfrozen fish, compared to pre rigor mortis , post rigor mortis and frozen commercial codfish. A high homology of codfish APDH was found with the corresponding human enzyme. A significant inhibition of APDH-Sepharose by human and, to a lesser extent, by rabbit APDH was observed. Western blot of APDH codfish extract showed two bands at 41 and 36 kDa, respectively.
Conclusions:   We have characterized a new allergen from codfish, which had a high level of homology in different species. The p41 relative content of extracts from nonfrozen codfish was higher than in the other samples assessed.     

Demonstration of distinct allergens by means of immunological methods. Comparison of crossed radioimmunoelectrophoresis, radioallergosorbent test and in vivo passive transfer test

An immunosorbent column was prepared containing a purified major allergen fraction from codfish (DS 22) covalently coupled to agarose. Sera from patients allergic to codfish were run through the column at pH 7.2. After extensive washing, the IgE retarded in the column was eluted with a buffer at pH2.5. The original sera and fractions from the chromatography experiments were examined by means of crossed immunoelectrophoresis (CIE), crossed radioimmunoelectrophoresis (CRIE), radioallergosorbent test (RAST) and in vivo passive transfer (PK) tests using the DS 22 from codfish and a crude codfish extract. The experiments demonstrated that the crude extract contained a minor codfish allergen (antigen-17-cod) which was distinct from DS 22. RAST was the most convenient technique for the identification of fractions containing allergenic activity. The PK tests served to prove the biological activity in vivo. CIE/CRIE were superior to RAST and PK tests regarding their ability to identify distinct allergens. Full agreement was found between results using different techniques including the immunosorbent experiments. Some of the radiostaining in CRIE, however, was misleading due to coprecipitation of DS 22 in several precipitates in the CIE preparations of the crude codfish extract.     

Common antigenic and allergenic determinants on codfish proteins detected with mouse monoclonal IgG and human IgE antibodies

The antigenic and allergenic profiles of codfish extract have been examined and a comparison made of the specificities of the determinants defined by mouse monoclonal antibodies and human IgE antibodies. By gel electrophoresis, codfish extract was found to comprise a heterogeneous mixture of proteins, in which the principal component and allergen was Gad cI (allergen M). Using monoclonal antibodies and sera from human cod-allergic subjects as immunological probes, common antigenic and allergenic determinants were demonstrated on some codfish proteins. It was also established that, although two monoclonal antibodies recognized the same determinant on Gad cI, there was no cross-reactivity between this determinant and those specified by IgE antibodies in the sera of cod-allergic patients. The specificity of IgE populations directed against Gad cI was found to vary from patient to patient, and was indicative of the existence of two types of allergenic determinants: those unique to a particular allergen and those shared by other proteins in the extract. These studies promote speculation regarding the relative immunogenicity of antigenic and allergenic determinants and the size and diversity of the IgE repertoire, given the potential immunogenicity of the entire protein surface.     

Identification of a 36‐kDa olive‐pollen allergen by in vitro and in vivo studies

BACKGROUND: Ole e 1 has been considered the major allergen of olive (Olea europaea) pollen. Some other relevant allergens (Ole e 2, 3, 4, and 6) have been recently described. This work aimed to study the IgE-binding frequency of a 36-kDa protein from O. europaea pollen in a large population of olive-allergic patients, its allergenic reactivity in vivo, and its presence in olive pollens of different origin, as well as in other relevant allergenic pollens. METHODS: Identification of IgE-binding components from O. europaea pollen extracts was elucidated by inhibition of SDS-PAGE immunoblotting using recombinant profilin (Ole e 2) and Ole e 1 molecules. The IgE-binding frequency of the 36-kDa protein was estimated by Western blot in a sample of 120 sera from olive-allergic patients. The cutaneous test with the 36-kDa protein was performed by intradermoreaction in allergic patients and control subjects. RESULTS: Exactly 83% of the sera from O. europaea-allergic patients recognized a protein with an apparent molecular weight of 36 kDa, under reducing conditions. It was detected by sera from monosensitized and polysensitized patients, showing a higher IgE frequency than the major allergen Ole e 1 (59%) and the minor profilin (Ole e 2) allergen (27%). Similar reactivity rates (79%) was found by intradermal test. Extracts from olive pollens collected in California presented a much higher amount (around 16-fold on average) of the 36-kDa protein than those from pollens of Spanish origin. The presence of similar allergens was detected only in closely related species (Syringa, Fraxinus, Ligustrum), and not in other common allergenic pollens. CONCLUSIONS: The 36-kDa protein constitutes a major allergen for olive-sensitized patients, but it is not equally represented in O. europaea pollens of different origins.     

Isolation and characterization of proteic allergens in refined peanut oil

Allergic reactions to peanut oil are very much debated, even if the responsibility of peanut oil has been evoked in several cases of adverse reactions, including death related to severe asthma. The aim of the present study was to investigate the presence of allergenic proteins in peanut oil. Proteins were extracted from commercial refined peanut oil, with a relative content in the order of 0.1–0.2 μg per g of oil, and molecular sizes ranging from 14 up to 76 kDa in SDS-PAGE. Eight protein bands were systematically observed in crude, neutralized and refined oils, with a molecular mass ranging from ≈ 14 to 76 kDa, including one at 18 kDa which was identified by Western blot performed with serum from two allergic patients. The protein extract gave positive IgE-RIA with patient sera, positive in vitro leucocyte histamine release tests and positive skin-prick tests in allergic patients. The allergenic protein was purified by HPLC and [¹²⁵I] iodide-labelled. It had an isoelectric point at 4.5 in isoelectrofocusing. In conclusion, we have demonstrated the presence of allergenic proteins in crude and refined peanut oil. These proteins are the same size as two allergens previously described in peanut protein extracts.     

Molecular and immunological characterization of cytochrome c: a potential cross-reactive allergen in fungi and grasses

Background:  Recombinant allergens are required for component-resolved diagnosis/therapy of allergic disorders. The study was aimed to express and characterize an allergenic protein from Curvularia lunata and study its cross-reactivity.
Methods:  A clone encoding a 12-kDa protein screened from the cDNA library of C. lunata was sequenced and expressed in pET22b+ vector. The purified protein was characterized by biophysical and immunological methods.
Results:  The sequence of gene encoding a 12-kDa protein showed homology to cytochrome c. It was expressed in Escherichia coli yielding 0.5 mg protein/l culture and designated as Cur l 3. The absorption and circular dichroism spectrum of Cur l 3 were similar to horse cytochrome c and the protein reacted with cytochrome c antibody. ELISA with different fungal-positive patients' sera showed ≥ 3 times specific IgE to Cur l 3 compared with healthy controls. Mice anti-Cur l 3 reacted with tropical and temperate grass extracts. Protein also reacted with grass-positive patients' sera. In vitro stimulation of peripheral blood mononuclear cells from C. lunata , fungi or grass-positive patients with it released significant levels of Th2 cytokines. In vivo testing of this protein in allergic patients showed marked positive skin reactivity in 60% fungal and 43% grass-positive cases. Cross inhibition assays (EC₅₀) demonstrated allergenic cross-reactivity of Cur l 3 with fungi and grasses.
Conclusions:  Cytochrome c, a major allergen from C. lunata was cloned, sequenced and expressed. It was identified as a cross-reactive allergen among fungi and grasses and has potential for clinical application.     

The spectrum of allergens in ragweed and mugwort pollen

Ragweed and mugwort are important allergenic weeds belonging to the Asteraceae or Compositae plant family. Pollen of mugwort is one of the main causes of allergic reactions in late summer and autumn in Europe and affects about 10-14% of the patients suffering from pollinosis. Ragweed pollen represents the major source of allergenic protein in the United States, with a prevalence of about 50% in atopic individuals. In Europe, ragweed allergy is now rapidly increasing particularly in certain areas in France, Italy, Austria, Hungary, Croatia, and Bulgaria. Amb a 1 and Art v 1, the major allergens of ragweed and mugwort, respectively, are unrelated proteins. Amb a 1 is an acidic 38-kDa nonglycosylated protein. The natural protein undergoes proteolysis during purification and is cleaved into a 26-kDa alpha chain, which associates noncovalently with the beta chain of 12 kDa. The two-chain form seems to be immunologically indistinguishable from the full-length molecule. Art v 1 is a basic glycoprotein comprising two domains: an N-terminal cysteine-rich, defensin-like domain and a C-terminal proline/hydroxyproline-rich module. The proline/hydroxyproline-rich domain was recently shown to contain two types of glycosylation: (1) a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core substituted by a variable number (5-28) of alpha-arabinofuranose residues forming branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses, and (2) single and adjacent beta-arabinofuranoses linked to hydroxyproline. As described for other pollen, ragweed and mugwort pollen also contain the pan-allergen profilin and calcium-binding proteins, which are responsible for extensive cross-reactivity among pollen-sensitized patients.     

The effects of gastric digestion on codfish allergenicity

BACKGROUND: In a recent murine study, we showed that impaired gastric digestion supports the induction of fish allergy by protecting the digestion-sensitive major allergen parvalbumin and thus enhancing its sensitizing properties. OBJECTIVE: The aim of the present study was to investigate whether impairment of peptic degradation might also play a role in the effector phase of codfish allergy. METHODS: The resistance of cod proteins to digestion by simulated gastric fluid was assessed in vitro . Gastric solutions with pH values ranging from 1.25 to 5.0 were prepared, and the influence of the pH on protein degradation was evaluated by means of SDS-PAGE and IgE immunoblotting. The allergenic potency of digested and undigested cod extract was further characterized in RAST inhibition and basophil histamine release experiments. RESULTS: The digestion experiments revealed that codfish proteins were degraded within 1 minute under physiologic gastric conditions. An only marginal pH shift from 2.5 to 2.75 abrogated completely the digestion of cod allergens. In RAST inhibition experiments digested cod extracts showed a reduced IgE-binding capability that was dependent on the digestion time. Moreover, peptic fragments expressed a 10,000 times reduced allergenic potency, as evaluated on the basis of histamine release from human basophils. CONCLUSION: Codfish allergens have a grossly reduced ability to trigger an intestinal allergic reaction when they are physiologically degraded. Impairment of the physiologic digestion might thus lower the threshold levels of a food allergen in sensitized patients.     

Identification and characterization of the major allergens of buckwheat

BACKGROUND: Buckwheat (BW) has been recognized as a common food allergen in Korea, Japan, and other countries. Until now, serologic findings of BW food-allergic patients and its major allergenic components have not been clarified. In this study, we analyzed the serologic findings of BW food allergy and characterized its major allergenic components. METHODS: Nineteen BW-allergic subjects with symptoms after BW ingestion and 15 asymptomatic control subjects with positive skin prick test to BW were recruited. BW-specific IgE was measured with the Pharmacia CAP kit. Allergenic components of BW were analyzed by IgE immunoblotting, periodate oxidation, two-dimensonal PAGE, and sequencing of N-terminal amino acids. RESULTS: From the BW-allergic patients and asymptomatic controls, the sensitivity (100%), specificity (53%), and negative (100%) and positive predictive values (73%) of Pharmacia CAP specific IgE for diagnosis were estimated. The prevalence of IgE binding to 24-kDa (pI 8.3), 16-kDa (pI 5.6), and 9-kDa (pI 5.0/ 6.0) allergens was higher than 50% in BW-allergic and asymptomatic subjects. However, the specific IgE to split 19-kDa (pI 6.5/7.0) allergens were more specifically found in BW-allergic patients than in asymptomatic subjects (78% vs 7%). N-terminal amino-acid sequences of 19-kDa and 16-kDa allergens showed moderate and weak homology to the 19-kDa globulin protein of rice and alpha-amylase/trypsin inhibitor of millet, respectively. The N-terminus of the 9-kDa isoallergens were not different from each other and were identified as the reported trypsin inhibitors of BW. Attenuation of the IgE binding to the 9-kDa allergen was found with periodate oxidation. CONCLUSIONS: The allergens of 24, 19, 16, and 9 kDa are strong candidates to be major allergens, and the 19-kDa allergen was relatively specific for BW-allergic patients. Moreover, measurement of BW-specific IgE and the features of immunoblotting should be very useful tools in the diagnosis of BW allergy.     

Identification of cyclophilin as an IgE-binding protein from carrots

BACKGROUND: Plant food allergies have been associated with pollenosis, although most of the causative allergens are as yet undefined. It is important to elucidate the properties of plant food allergens in order to minimize a patient's risks in food selection. The purpose of the present study was to examine and characterize the IgE-binding proteins in carrots as possible allergens by using patients' sera. METHOD: IgE-binding proteins in carrot extract were screened by an immunoblot technique using sera of patients with atopic dermatitis (selected based upon a case history of food allergies). An allergenic protein was purified from carrot extract by chromatographic procedures. The N-terminal amino acid sequence of allergenic protein was determined and subjected to a computer homology search. Cross-reactivity between carrot and birch allergens was examined by immunoblotting. RESULTS AND CONCLUSION: A unique, approximately 20-kDa allergenic protein that reacted with about 14% of patients' sera was isolated and characterized. The N-terminal amino acid sequence of the purified protein was found to be homologous with those of plant cyclophilins. This allergen exhibited a peptidyl-prolyl cistrans isomerase activity, which was inhibited by the conjugation of cyclosporin A. These properties of the allergenic protein isolated from carrot identified it as a cyclophilin, a possible plant food allergen. No cross-reactivity between this 20-kDa carrot allergen and Bet v 7, a birch pollen cylcophilin, was observed.     

Isolation and purification of two immunodominant antigens from Nocardia brasiliensis.

Two immunogenic proteins from a crude extract of Nocardia brasiliensis were purified to homogeneity. A 61-kDa protein (P61) was isolated from a 50% ammonium sulfate precipitate in two steps. Initially, P61 was obtained by electroelution in a 10% nondenatured preparative polyacrylamide gel electrophoresis (PAGE). In a second step, the eluate from the nondenatured gel was run in a 12% sodium dodecyl sulfate (SDS) preparative polyacrylamide gel. After elution, a single band was demonstrated by SDS-PAGE and Western blot (immunoblot). Also, a 24-kDa immunogenic protein (P24) was isolated by gel filtration in a Sephadex G-100 column and then by electroelution in a 12% nondenatured polyacrylamide gel. In a previous paper, we showed by Western blot assays that these proteins are recognized by the sera of mycetoma patients and not by sera from mycobacterial-infected or healthy individuals. We consider these proteins to be good candidates for the study of the host-parasite relationship in nocardial infections. The possible clinical application of these purified antigens in a serological diagnosis is discussed.     

Codfish allergy in adults. Specific tests for IgE and histamine release vs double-blind, placebo-controlled challenges

Background At present, several in vitro tests for immunoglobulin E (lgE)-mediated food allergy are available. An estimation of the diagnostic accuracy of the various tests used in predicting clinical sensitivity to codfish in a well-characterized allergic material is necessary. Objectives To compare the diagnostic value of four specific IgE tests, and histamine release from basophils (HR) in identifying clinical type I allergy to codfish. As a true diagnosis, double-blind, placebo-controlled food challenges (DBPCFC) were employed. Methods Eight clinically codfish-allergic adult patients were investigated together with 30 codfish-tolerant control subjects for evidence of codfish-specific reactivity by Phadebas RAST® (PHA). Pharmacia CAP System RAST® (CAP), Magic® Lite (ML) and HR. To characterize the diagnostic properties of a freshly prepared raw codfish extract, experiments were conducted employing an in-house radioallergosorbent test (RAST). the Maxisorp RAST (MAXI) and HR. Finally, protein profile and IgE-reacting allergens were detected by means of sodium dodecyl sulfate-polyacrylamide gel elcctrophoresis (SDS-PAGE) and immunoblotting. Results The sensitivities of HR with commercial extract and the three commercially available specific IgE analyses were 0.83 and 1.00 respectively. Specificities were 1.00 (H R) and 0.87-1.00 (specific IgE tests). Ereshly prepared codfish extracts improved the sensitivity of HR. SDS-PAGE revealed ～29 bands (< 14.3-200 kDa) including a band of 12-13 kDa. and in immunoblotting 18 sera identified 17 IgE-binding bands. The protein migrating at 12-13 kDa was identified in the fresh codfish extract tested with gen from all clinical codfish allergies, while no significant reaction was seen in the control subjects. Conclusion Based on the small number of adult patients included in our study, the in vitro assays with commercial and fresh extracts have high sensitivity and are acceptable for screening for codfish allergy. Specificity of Phadebas. CAP. and our in-house RAST was less than unity, whereas ML and strong binding of IgE to a 12-13kDa protein completely matches DBPCFC results, and thus seems sufficient for establishing the diagnosis.     

Antigens and Allergens in Birch Pollen Extract

More than 70% of the total allergenic activity of a birch pollen (BP) extract was detected within the first 30 min of extraction. Fractionation of the BP extract by gel filtration and analysis of the eluted antigens by a fused rocket immunoelectrophoresis revealed at least three antigens with molecular weights of about 29 000, and 17 000-10 000, corresponding to antigens Nos. 7-8 and No. 2, respectively, in crossed-immunoelectrophoresis (CIE) and in crossed-radioimmuno-electrophoresis (CRIE). Gel isoelectrofocusing of the pooled allergenic fractions revealed two major protein bands with pI's around 5.6 and 5.7, probably corresponding to antigens Nos.7-8 and No. 2, respectively. Antigens Nos. 7-8 were thermoresistant, while antigen No. 2 was thermolabile. The allergenic activity was determined by prick skin testing and by the RAST inhibition method. More than 90% of the allergenic activity in the fractions was located in the protein peak C (mol. wt. 10 000-17 000) containing antigens 7-8. About 30% of the total allergenic activity of the extract (1:10 w/v) was recovered in the peak C fractions, and only less than 0.5% outside these fractions. Higher allergenic activity was obtained for the peak B fractions (mol. wt. 29 000) by skin prick testing than by the RAST. Peak B contained allergens (antigen 2) distinct from those of peak C by the CRIE and by the RAST. The allergenic material in the low molecular weight fractions of peak D (mol. wt. 2000-5000) was allergenically similar to that of peak C in the RAST. Only weak and even negative skin reactions were observed with the peak D fractions in allergic subjects.     

The isolation, characterization and cloning of a globin-like, host-protective antigen from the excretory-secretory products of Trichostrongylus colubriformis.

An 18-kDa component from the excretory-secretory (ES) products of adults of Trichostrongylus colubriformis was isolated and characterized, and was shown to induce 60-84% protection of guinea pigs from challenge infection following a single intraperitoneal injection. Amino-terminal sequence analysis of gel-purified protein enabled oligonucleotides to be synthesized and used to screen a lambda gt10 cDNA library made from young adult worm mRNA, and to synthesize full-length clones from cDNA using the polymerase chain reaction (PCR). The full-length clones coded for a 20-kDa precursor protein of 173 amino acids which had a strongly hydrophobic leader sequence of 15 residues. The mature protein sequence of 158 amino acid residues was rich in charged amino acids (32%), including 8 oppositely charged pairs of amino acids. The protein sequence contained no half-cystine residues and no potential N-glycosylation sites. Unlike 2 other fully characterized ES components which are expressed only in the parasitic stages, mRNA coding for the 20-kDa component was present in both the parasitic and free-living stages of T. colubriformis. The parasite protein had approximately 20% identity with globins from human and from the larvae of the insect Chironomus thummi thummi. The homology included the invariant distal histidine and phenylalanine, and a number of other residues highly conserved in globins.     

Antacids and dietary supplements with an influence on the gastric pH increase the risk for food sensitization

Background Elevation of the gastric pH increases the risk for sensitization against food allergens by hindering protein breakdown. This can be caused by acid‐suppressing medication like sucralphate, H2‐receptor blockers and proton pump inhibitors, as shown in recent murine experimental and human observational studies. Objective The aim of the present study was to assess the sensitization capacity of the dietary supplement base powder and of over‐the‐counter antacids. Methods Changes of the pH as well as of protein digestion due to base powder or antacids were measured in vitro. To examine the in vivo influence, BALB/c mice were fed codfish extract with one of the acid‐suppressing substances. Read‐out of antibody levels in the sera, of cytokine levels of stimulated splenocytes and of intradermal skin tests was performed. Results The pH of hydrochloric acid was substantially increased in vitro by base powder as well as antacids in a time‐ and dose‐dependent manner. This elevation hindered the digestion of codfish proteins in vitro. A significant increase in codfish‐specific IgE antibodies was found in the groups fed codfish combined with Rennie^® Antacidum or with base powder; the latter also showed significantly elevated IgG1 and IgG2a levels. The induction of an anaphylactic immune response was proven by positive results in intradermal skin tests. Conclusions Antacids and dietary supplements influencing the gastric pH increase the risk for sensitization against allergenic food proteins. As these substances are commonly used in the general population without consulting a physician, our data may have a major practical and clinical impact. Cite this as: I. Pali‐Schöll, R. Herzog, J. Wallmann, K. Szalai, R. Brunner, A. Lukschal, P. Karagiannis, S. C. Diesner and E. Jensen‐Jarolim, Clinical & Experimental Allergy, 2010 (40) 1091–1098.     

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I)

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith apples were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients’ sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Fürthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity. Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.     

The group III allergen from the house dust mite Dermatophagoides pteronyssinus is a trypsin-like enzyme.

Faecally enriched extracts of Dermatophagoides pteronyssinus were shown to contain a trypsin-like enzyme which was allergenic. Chromatofocusing studies revealed the presence of nine major isoforms in D. pteronyssinus, with pI in the range 4 to greater than 8, but only two (range 4-5) in D. farinae. Trypsin isolated from D. pteronyssinus by benzamidine-Sepharose 6B affinity chromatography and gelfiltration was found to be a 31-kDa protein which was enzymatically similar to both invertebrate and vertebrate trypsins. The N-terminal sequence obtained (IVGGEXALAGEXPYQISL) was identical to that reported for the mite allergen Der p III and showed homology with crayfish trypsin and Der f III from D. farinae. Mite trypsin underwent autolysis and the N-terminal sequences of two fragments were found to be ALAGEXPYQI and NNQVXGI respectively. Both showed homology with crayfish trypsin, and the former sequence was identical to residues 7-18 of the native enzyme and Der p III. All isoforms of mite trypsin were showed to be allergenic by radioallergosorbent assay and further studies indicated that the trypsin degradation products were also allergenic. The enzyme was compared with other mite allergens and the rank order of allergenic potency was shown to be: whole mite extract greater than Der p I greater than trypsin. However, all sera from a panel of mite allergic individuals showed IgE reactivity to trypsin, comparable to that seen using whole mite extract and Der p I. These data indicate that mite trypsin is a major allergen corresponding to the previously described allergen, Der p III.     

Molecular analysis of allergenic proteins in bovine dander

An analytic procedure was established to characterize bovine dander proteins with allergenic properties. The proteins from dander extract were separated by size-exclusion gel filtration, and the fractions were studied with SDS-PAGE followed by immunoblotting. An 11-kDa allergen was found in the same gel filtration fractions as 20- and 22-kDa allergens, and this suggests that the 11-kDa allergen is a dirtier in its native form. Our method also detected two separate 22-kDa allergens. The primary structure of the major bovine dander allergen (BDA20) was also studied. A protein sequencer was used to determine the amino acid sequences of enzymatically cleaved peptides. The homology searches revealed that BDA20 is not a previously known bovine protein.     

Culture filtrate antigens and allergens of<Emphasis Type="Italic"> Epicoccum nigrum</Emphasis> cultivated in modified semi-synthetic medium

Epicoccum nigrum (EN) is an important fungal allergen for nasobronchial allergy. Fungal extracts should contain all the relevant allergen components from spores, mycelium and culture medium for the purpose of allergy diagnosis and therapy. EN extract from spore-mycelial mass has been standardized, but the culture filtrate (CF) allergens of EN have not been studied as EN grows poorly in synthetic medium. The objective of the present study was to obtain a standard CF extract of EN by cultivating the source material in a modified semi-synthetic medium and to compare this with the EN cellular extract. Sabouraud's medium containing yeast extract (50 mg/l) was filtered using 10-kDa cut-off membrane and the lower molecular mass media components were used to cultivate EN. The CF obtained after removing the spore-mycelia was dialyzed to remove media components. The CF extract was characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. It was compared with EN spore-mycelial extract by enzyme-linked immunosorbent assay (ELISA), ELISA inhibition and by intradermal testing on allergy patients. The CF extract of EN resolved into 30 protein bands on SDS-PAGE. About 27 IgG bands were detected using anti-EN rabbit antibodies and 12 IgE bands by EN-sensitive pooled patients' sera. Periodate modification of CF proteins showed that the carbohydrate moieties are not important for IgE binding. Protein components of 26, 34 and 43 kDa were recognized as the major CF allergens. Three different batches of CF extract required 7.5-9 ng of self protein for 50% inhibition of binding to anti-EN rabbit antibodies in ELISA. Intradermal testing with CF extract showed comparable allergenic potency to standardized EN spore-mycelial extract, although it contained some allergenic proteins in higher amounts as compared to the spore-mycelial extract. In summary, the semi-synthetic medium has been suitably modified for obtaining EN CF antigens. This medium can be an important substitute for producing potent CF allergens of fungi that grow poorly in synthetic medium. The EN CF extract elicited good allergenic reactivity and may be used for allergy diagnosis along with spore-mycelial extract.     

Characterization of immunoglobulin G and its subclass response to Indian kala-azar infection before and after chemotherapy

Serologic parameters of kala-azar were evaluated by Western blot analysis. Sera from kala-azar patients with confirmed diagnoses were screened for immunoglobulin G (IgG) and IgG subclass-specific reactivity against Leishmania donovani membrane antigen (LAg). Heterogeneous LAg-specific IgG reactivity with numerous proteins with molecular masses ranging from 18 to 190 kDa was observed. Though the individual band patterns were varied, seven polypeptides of approximately 31, 34, 51, 63, 72, 91, and 120 kDa were immunoreactive with all the sera tested from kala-azar patients. The band patterns of the immunoblots of sera from patients after treatment and clinical cure with sodium antimony gluconate revealed a decrease in the frequency of the bands. Still, recognition of the 63- and 120-kDa bands was 100%, and the 55- and 91-kDa fractions were recognized in 93% of the sera from cured individuals. Among the IgG subclasses, IgG1 reacted with the greatest number of polypeptides. The 63-kDa protein was again detected by all of the IgG subclasses of all the sera tested. Other fractions recognized by the subclasses of more than 70% of the serum samples included those of 47, 51, 55, and 78 kDa. Following treatment, 63- and 51-kDa bands were the most reactive with the IgG subclasses. LAg-associated cross-reaction with other reference human antisera revealed a mild reactivity of the 63-kDa polypeptide with some of the serum samples from leprosy, malaria, typhoid, tuberculosis, and healthy controls. Western blot analysis of LAg entrapped in liposomes, strong vaccine candidates against experimental visceral leishmaniasis, revealed a more restricted band pattern. The 63-kDa fraction revealed by all pre- and posttreatment sera showed almost negligible levels of cross-reaction with sera from patients with other diseases or from healthy controls. These observations provide insight into induced immunity during kala-azar infection for future application.