Identification of crustacea allergens by crossed radioimmunoelectrophoresis

Crossed immunoelectrophoresis (CIE) detected 18 precipitating antigens in extracts of shrimp. Of these antigens, crossed-line immunoelectrophoresis (CLIE) of shrimp extract demonstrated that 5 cross-reacted with crayfish, 3 with lobster and 1 with crab extract. Allergens present in the shrimp CIE plates were identified by crossed radioimmunoelectrophoresis (CRIE) using sera from 6 study subjects who were skin-test and RAST positive to shrimp extract. Of the 7 allergens detected, 3 (precipitins 1, 3 and 6) reacted with most of the 6 sera tested from shrimp-sensitive subjects. Precipitins 1 and 6 appear to be common crustacea allergens (present in shrimp, crayfish, lobster and crab) whereas precipitin 3 may be a specific allergen since it is present only in shrimp.     

Allergy to guinea pigs: II Identification of specific allergens in guinea pig dust by crossed radio-immunoelectrophoresis and investigation of the possible origin

An extract of dust from the air-vent filters of a room housing guinea pigs was analysed by quantitative immunoelectrophoretic procedures and compared with extracts of various materials derived from guinea pigs. Crossed radio-immunoelectrophoresis (CRIE) of the dust, performed with sera from twenty asthmatic patients who were positive by skin testing and RAST to guinea pig extracts, identified fourteen IgE-binding constituents. Although responses varied, most sera reacted with lour particular allergens, antigens 2. 3, 10 and SI. The numbers of allergens recognized by individual patients correlated with the RAST score, but not with total scrum IgE. All seventeen dust constituents detected by crossed immunoelectrophoresis (and all four major allergens), were also present in extracts of guinea pig dander, fur. saliva and urine; several of these components were absent in an epithelial extract, and there were even less in preparations of shaved pelt, serum or faeces. None of the dust extract antigens were detected in materials used in animal husbandry, dust samples from rooms without guinea pigs, or a D. pteronyssinus extract. These findings suggest that inhalant allergens may be derived predominantly from material shed from the guinea pig coat after contamination with saliva, and possibly to a lesser extent, urine.     

The allergens of Epicoccum nigrum Link. I. Identification of the allergens by immunoblotting.

Two atmospheric isolates of Epicoccum nigrum (EN) were grown under sporulation conditions. Dialyzed extracts of spores, (greater than 95% pure) and pure mycelia were used for skin testing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. By skin testing, 49 of the 126 atopic patients were found to be sensitive to EN in St. Louis, Mo., and Corpus Christi, Texas, combined. On immunoblotting, which was performed on 17 sera, 44 bands (12.3 to 119.0 kd) were detected; six were unique to spore, four were unique to mycelium, and 34 were common to both. No single band bound IgE from all sera. The most frequent band corresponding to 42 kd occurred in 11 sera. Five other bands were recognized by more than one half, whereas the remainder bound fewer sera. All skin test-positive patients had positive immunoblots; the number of bands recognized varied from three to 25. Spore or mycelium-specific, as well as common bands were recognized by 13 of 17 sera. Two sera recognized only spore and mycelium-specific bands. Only spore-specific bands were bound by two sera. No strain differences were detected. The binding patterns were comparable in the sera from both St. Louis, Mo., and Corpus Christi, Texas. These data suggest that EN is a significant allergen in urban communities. Allergenic proteins occur in both spore and mycelium, suggesting that both must be included in the reagents for skin testing and immunotherapy.     

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

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

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.     

Identification of allergens in extract of horse hair and dandruff by means of crossed radioimmunoelectrophoresis.

Sera from 26 patients and 4 normals were examined for specific IgE binding to antigens of extract of horse hair and dandruff by means of CRIE. 22 of the patients were RAST- and intracutaneous-positive to horse extract. 4 more of the patients were RAST-negative to horse allergens, but showed allergies to extract of allergens from sources other than horse. The remaining four sera from controls were RAST-negative to horse and had no history of allergy. Antigens of horse hair and dandruff showed a significantly higher degree of binding to specific IgE in the sera from the first group of patients than was the case for the two other groups. A linear correlation between specific IgE binding in RAST and in CRIE was found for the first group of patients. On the basis of these results the major allergens of the examined extract of horse hair and dandruff were identified.     

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.     

Detection of Alternaria allergens by crossed radioimmunoelectrophoresis

Allergens involved in sensitivity to Alternaria have not been well defined. We used crossed radioimmunoelectrophoresis (CRIE) to study antigens in a crude A. alternata extract, which were capable of binding IgE in human serum. Sera from 35 patients sensitive to Alternaria, 10 not sensitive to Alternaria, and five normal controls were examined. CRIE with hyperimmune rabbit sera demonstrated 22 antigens in the Alternaria extract. After exposure of CRIE gels sequentially with patient serum and 125I-labeled anti-human IgE, autoradiography demonstrated that three of the antigens bound IgE in sera of Alternaria-sensitive subjects. Our results suggest that multiple allergens are involved in A. alternata sensitivity.     

Analysis of tobacco leaf allergens by crossed radioimmunoelectrophoresis

The reactivity of eleven ‘tobacco smoke sensitive’ and eight ‘non-sensitive’ individuals to tobacco leaf allergens was tested by Crossed Radioimmunoelectrophoresis (CRIE). All nineteen study subjects had IgE antibodies to tobacco leaf antigens as measured by Radioallergosorbent Test (RAST) and seventeen of the nineteen individuals were atopic. Of the thirty-seven tobacco leaf precipitins detected by Cross Immunoelectro-phoresis (CIE), three were identified as allergens by CRIE. All nineteen subjects reacted to at least one of the three allergens detected. However, neither the intensity nor the incidence of reactivity with any of the three allergens correlated with smoking or ‘smoke sensitivity’.     

Identification of novel allergens of Aspergillus fumigatus using immunoproteomics approach

BACKGROUND: Approximately 20% of the world's asthmatics are suffering from Aspergillus fumigatus (Afu)-induced allergies. The characterization of specific IgE-inducing allergens in allergic aspergillosis patients is fundamental for clinical diagnosis and for immunotherapy. METHODS: Immunoproteomics combined with mass spectrometric analysis was used to identify proteins of third-week culture filtrate (3wcf) potentially responsible for Afu-specific IgE immunoreactivity, using pooled sera from Afu-sensitized asthmatics. Their allergenic potential was also tested against patients with allergic bronchopulmonary aspergillosis (ABPA), by two-dimensional (2-D) gel electrophoresis immunoblotting of 3wcf proteins with individual sera from such patients. This helped us to establish a set of candidate allergens, which could be explored further for diagnostic application in allergic aspergillosis asthmatics including ABPA. RESULTS: Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and/or de novo sequencing by MS/MS analysis of the protein spots from 2-D gels led to the identification of a total of 16 allergens of Afu. Eleven of them are being reported as allergens for the first time and five had been reported earlier. Putative isoforms of the proteins Asp f 13 and chitosanase have been observed for the first time. When studied for reactivity of these proteins among patients with ABPA using their individual sera, these patients exhibited sensitization although the pattern was varying. Taken together, these proteins could thus be considered as potential allergens even among patients with ABPA. Three of these proteins viz. the hypothetical protein (# spot no. 5), extracellular arabinase (# spot no. 6) and chitosanase (# spot no. 11) could be major allergens with specific IgE immunoreactivity with six out of eight patients' sera. CONCLUSIONS: The immunoproteomic approach applied to the analysis of culture filtrate proteins resulted in the identification of several candidate allergens, many of them novel, contributing to the catalogue of Afu allergenic proteins, which would facilitate improved serodiagnosis for allergic aspergillosis. In addition, the immunoreactivity of these proteins observed among the patients with ABPA may be potentially useful for its serodiagnosis and opens up further opportunities for the development of personalized immunotherapeutics for patients with ABPA.     

Antigens and allergens from the common house dust mite Dermatophagoides pteronyssinus. Part II. Identification of the major IgE-binding antigens by crossed radioimmunoelectrophoresis

Crossed immunoelectrophoresis demonstrated 51 antigens in a water-soluble extract of the house dust mite Dermatophagoides pteronyssinus. Autoradiography demonstrated that 11 of the antigens bound IgE antibodies in the sera of house dust mite-allergic patients. IgE antibodies in 23 different sera reacted with from one to eight antigens. On the basis of Lowenstein's (1978) definition of a "major allergen," two of the antigens, numbers one and 36 would be described as "major allergens." Apart from antigen number 36 that has already been demonstrated to be an important allergen in patients allergic to D. pteronyssinus, the clinical importance of the other 10 IgE-binding antigens has yet to be assessed. Reasons against use of the term "major allergen" and its replacement with the title "clinically important allergen" are advanced.     

Studies on Alternaria allergens. I. Isolation of allergens from Alternaria tenuis and Alternaria solani.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.     

Identification, quantitation, and purification of cockroach allergens using monoclonal antibodies

A panel of murine IgG monoclonal antibodies (MAbs) was raised against German cockroach (CR) (Blattella germanica) extract and selectively screened to identify MAb directed against allergen(s) recognized by IgE antibodies. Sera from 28 CR-allergic patients were used as sources of IgE antibodies to detect allergens "presented" by the MAb. Four clones (10A6, 3G12, 8F4, and 1D4) produced MAb to allergen(s) that bound IgE antibodies. Quantitative radioimmunoassays were used to compare levels of the MAb-defined allergens in CR extracts. MAb 10A6 reacted with a cross-reacting allergen that was detected in 9/14 CR species, including Blattella, Periplaneta, Blatta, Leucophea, and Supella spp, at concentrations of 100 to 10,000 U/ml. In contrast, MAb 3G12, 8F4, and 1D4 were Blattella specific. The allergen defined by MAb 8F4 was purified by MAb affinity chromatography and size-exclusion by high-performance liquid chromatography. It is a 36 kd heat-sensitive protein, isoelectric point, 5.2 to 5.4. Allergen 10A6 was partially purified by isoelectric focusing and high-performance liquid chromatography. It is a heat-stable, acidic protein (isoelectric point 3.15). Based on comparison of their properties with properties of previously described CR allergens, the allergens defined by MAb 10A6 and 8F4 have been provisionally designated Blattella germanica allergen I (Bla g I) and Blattella germanica allergen II (Bla g II), respectively. Assays of six commercial CR skin test extracts demonstrated a 200-fold difference in Bla g I levels (4.7 to 1085 U/ml) and only two extracts that contained detectable Bla g II (248 and 324 U/ml). The results demonstrate that MAb can be used to identify and define CR allergens and that the strategy of the use of MAb as a first step in allergen analysis and purification can be very effective, especially for poorly characterized allergen extracts.     

The allergens of Schistosoma mansoni: I. Initial separation and comparative assay

Ten antigen fractions were prepared from adult Schistosoma mansoni by extraction into borate-buffered saline, precipitation at pH 4.6 and separation on Sephadex G-100. The allergic activity of these antigens was assayed by a modified Prausnitz—Kustner type reaction in rats; this test system was found to be sensitive and consistent, allowing differences in allergenicity between antigens to be accurately assessed.

Skin-reactivity was detected in both acid-soluble and acid-insoluble fractions. Specific allergenicity was located in peak 3 of a G-100 separation of the acid-soluble fraction and in peaks 1 and 2 of a G-100 separation of the acid-insoluble fraction suggesting that the allergens of S. mansoni were of at least two types: (1) a protein of mol. wt above 150,000 precipitated at pH 4.6, and (2) a protein of mol. wt 20–30,000 remaining in solution at this pH.

It is suggested that both these allergens are glycoproteins. Non-specific histamine-releasing agents were found in peak 1 of the G-100 separation of the acid-soluble material.

     

Identification of Parietaria judaica pollen allergens

Parietaria judaica pollen allergens, fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 52 sera collected in Australia and Sicily from P. judaica pollen-allergic patients. IgE-binding pollen components transferred to nitrocellulose were detected by reaction with 125I-anti-human IgE and autoradiography. Nine pollen components, ranging in molecular weight (MW) from approximately 10,000 to 80,000 daltons, bound IgE antibodies but the two fastest migrating components sometimes each separated into two very closely migrating bands. The faster of the two components exhibiting doublet formation (MW approximately 10,000 daltons) showed by far the highest frequency of IgE binding, being recognised by 50 of the 52 sera examined. Although patients' IgE reaction patterns to P. judaica allergens were heterogeneous, the degree of heterogeneity was much less than that observed with house dust mite and other pollen extracts studied by electrophoretic transfer analysis. Results with gradient gel-nitrocellulose transfer experiments which showed no IgE-binding components with MWs less than 70,000 daltons, and comparisons of our electroblotting results with crossed radioimmunoelectrophoresis results of others, suggested that the doublet proteins with MWs of approximately 10,000 probably bind to higher MW proteins in P. judaica pollen extracts.     

The effect of dry heat on mite, cat, and dog allergens

Background Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens.
Methods Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60°, 80°, 100M20°, and 140°C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA, Results For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120°C, allergen levels were reduced to < 1 % of control. Der p 2 was more heat stable, requiring 140°C for 30-60 min to achieve >99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast. Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140°C.
Conciusions The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens: however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens.     

Studies on Alternaria allergens: I. Establishment of the radioallergosorbent test for measurement of Alternaria allergens

The ability to covalently couple Alternaria allergens to macrocrystalline cellulose particles has permitted not only the measurement of IgE antibodies to Alternaria in patient serums but also the identification of allergenic fractions from crude Alternaria extracts. Crude aqueous Alternaria extracts from 3 commerical suppliers were coupled to cellulose but failed to bind more than 5% of total radioactive counts (TRC) when reacted with serums from highly sensitive patients. Fractionation of a commercial extract through Sephadex G-25 showed that almost all allergenic activity was located in a protein and carbohydrate-containing peak eluting at the column void volume. These fractions were pooled and coupled to cellulose to yield a RAST polymer which produced up to 20% TRC binding when tested with serums from over 100 Alternariasensitive patients, and only up to 1% TRC binding with 17 nonallergic serums. The study of commercial Alternaria extracts by chromatographic and RAST inhibition techniques showed that present extracts are neither qualitatively or quantitatively comparable.     

Photo-inactivated allergens I. Preparation, physicochemical and biological properties

A method is described for the preparation of inactive atopic allergens by irradiation with ultraviolet light. From an analysis of the absorption and fluorescence emission spectra, and with the aid of synthetic model compounds, it is concluded that the allergenic determinant of lysine-sugar conjugation is extremely sensitive to ultraviolet light. Considerable reduction of allergenicity in vitro (diminished capacity to inactivate human complement in fluid phase) and in vivo (impressive reduction of skin-reactivity in atopic patients and, by passive transfer, in monkeys) was demonstrated. Only slight modification of the antigenic characteristics was detected.