Immunochemical and physicochemical characterization of commercial Altemaria extracts: a model for standardization of mold allergen extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.     

Shared allergenic and antigenic determinants in Alternaria and Stemphylium extracts

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p < 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibition using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibition, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.     

Immunochemical partial identity between two independently identified and isolated major allergens from Alternaria alternata (Alt-I and Ag 1)

Partially purified preparations of Alt-I, the main allergenic fraction of Alternaria alternata isolated by Yunginger, and of Ag 1, shown in crossed radioimmunoelectrophoresis (CRIE) to be the dominating major allergen of A. alternata (Løwenstein, Nyholm), were compared by tandem crossed immunoelectrophoresis (CIE), RAST inhibition, and the CRIE-related technique, single radial radioimmunodiffusion (SRRID). The two allergen preparations showed reaction of identity in tandem-CIE and indistinguishable specific IgE binding in CRIE and SRRID, regardless of antibodies and serum pools used. In RAST inhibition, the relative potencies of the allergen preparations and of the crude extracts correlated well with their Alt-I/Ag 1 content as estimated by rocket immunoelectrophoresis. Moreover, all inhibition curves were parallel, confirming identical IgE binding by Alt-I and Ag 1 with the serum pools used. A second preparation of Alt-I, isolated from another strain of Alternaria, showed reaction of partial identity with Ag 1 in tandem-CIE, indicating that different variants of Alt-I (Ag 1) may exist in different strains of A. alternata.     

Modification of house dust mite allergens by monomethoxypolyethylene glycol. Allergenicity measured by in vitro and in vivo methods

In animal models, allergen modification by coupling to monomethoxypolyethylene glycol (mPEG) molecules can reduce allergenicity of the extract and makes the allergen capable of suppressing boosted IgE response. To investigate in a human system the degree of attenuation implied by a mPEG modification of a house dust mite (Dermatophagoides pteronyssinus) extract, 55 adults with asthma caused by house dust mites were tested by skin prick test (SPT) and histamine release assay (HR). RAST inhibition was performed on sera from 6 additional patients. Modified extract containing 0.42 mmol mPEG/g protein was used for the analyses. In order to get the same response of the two extracts when assessed by HR and SPT, a median increase in concentration of 10-fold of the mPEG-modified extract compared to the unmodified extract was needed. Interindividual variation was limited. Sixty-four to 72% needed a dose increase within +/- half a decade from this value. In 42-49% of the patients, results from SPT and HR deviated less than half a decade. The relative potency of the modified extract as measured by RAST inhibition was reduced to 17-78% (mean 39%). Reduced allergenicity would by itself mean less side effects in immunotherapy. When planning such therapy it is important to know that mPEG modification reduces the allergenicity to a similar extent in a majority of patients.     

Banana allergy in patients with immediate-type hypersensitivity to natural rubber latex: Characterization of cross-reacting antibodies and allergens

Background: An association between allergy to latex and banana has been reported. Even though cross-reacting IgE antibodies have been demonstrated, in no study has the existence of structurally similar allergens been confirmed. In the present study banana allergy was studied in a large series of patients with latex allergy. Specific IgE antibodies were characterized for cross-reactivity and compared with pollen RAST results. Latex and banana extracts were investigated for common antigens and allergens. Methods: Latex-, banana-, and pollen-specific (birch, timothy, mugwort) IgE were measured in 47 sera from patients with latex allergy. Thirty-one patients were skin prick tested with banana and questioned for possible reactions after eating bananas. Several RAST inhibition and immunospot inhibition studies were used to characterize cross-reacting IgE antibodies. Structurally similar antigens and allergens were evaluated with crossed-line immunoelectrophoresis and crossed-line radioimmunoelectrophoresis, respectively. Results: Latex RAST results were positive in 31 (66%) and banana RAST results were positive in 26 (55%) of the 47 sera. Of the 31 latex RAST–positive sera, 25 (81%) were also banana RAST–positive. Results from latex RAST correlated significantly with results from banana RAST (p < 0.001), but not with those from pollen RAST (p > 0.05). Banana skin prick test results were positive in 11 (35%) of the 31 patients tested. Symptoms after eating bananas were reported by 16 (52%) of the 31 patients. In inhibition studies the binding of IgE antibodies to solid-phase banana and to several latex preparations was inhibited by latex and banana, respectively. In crossed-line immunoelectrophoresis at least one antigen from banana fused with an antigen from latex, which also bound IgE antibodies in autoradiography (crossed-line radioimmunoelectrophoresis). Conclusions: Patients with latex allergy have symptoms caused by banana and show positive skin test and specific IgE test results. Cross-reacting IgE antibodies were confirmed by several inhibition techniques. For the first time, a structurally similar antigen/allergen was demonstrated. (J ALLERGY CLIN IMMUNOL 1994;93:990-6.)     

Measurement of the potency of pollen extract by radioimmunoprecipitation.

We describe a novel procedure for standardization of allergen extracts using radioiodinated crude extracts. Radiolabeled extracts were reacted with immunoglogulin E (IgE) antibody, and the resulting complexes precipitated by anti-IgE. Experiments with ragweed pollen, grass pollen, honeybee venom, and Alternaria extracts revealed that 1% to 14% of the radioactivity was precipitated, these percentages ranged from 2 to 20 times blank values. Binding of labeled ragweed extracts to IgE antibody could be inhibited by unlabeled extracts and linear inhibition responses could be derived. The potencies of 11 short ragweed extracts were ranked by their inhibitory capacities in such an IgE radioimmunoprecipitation (RIP) test and compared to potencies determined by radioallergosorbent test (RAST) inhibition; these were related, rs = +0.97 (p less than 0.001). Labeled extracts were also reacted with IgG antibodies, and the resulting complexes were precipitated by anti-IgG. From 1% to 34% of the radioactivity associated with the various extracts was precipitated by anti-IgG, and these percentages were from 2 to 20 times blank values. The reaction of IgG antibody with radiolabeled pollen extracts could be inhibited by unlabeled extract, and in the case of short ragweed the measurements of potencies using IgG and IgE antibodies were correlated, rs = +0.96 (p less than 0.001), suggesting that similar determinants were recognized by both IgG and IgE classes. The RIP procedure provides an alternative to the RAST for the standardization of allergy extracts and eliminates the need for affinity chromatography-purified antibody to IgE.     

Antigens and allergens in Dermatophagoïdes farinae mite. I. Immunochemical and physicochemical study of two allergenic fractions from a partially-purified Dermatophagoïdes farinae mite extract.

The fractionation of a partially-purified extract of Dermatophagoïdes farinae mite culture has been undertaken by gel filtration. Two fractions were isolated. One, P25, is a protein-rich fraction with mol. wt about 25,000. The other, GP8, is a polysaccharide-rich fraction with mol. wt around 8,000. By crossed immunoelectrophoresis, we detected eleven antigens in the partially-purified dialysed D. farinae extract (Df 80d) as well as in P25 fraction. One of them, ag 11, seems the most important allergen since in crossed-radio immunoelectrophoresis experiments it displays the faster radiostaining, implying that it binds the greatest part of the mite-specific IgE present in a pool of sera from mite-sensitive patients. By crossed-line immunoelectrophoresis, we demonstrated the absence of ag 11 in GP8, in which only ag 5 and ag 6 were identifiable. By radioalloergosorbent tests (RAST), it was found that P25- and GP8- coated paper discs can fix specific IgE induced in the majority of D. farinae sensitive patients. Defining a 'major allergen' as an allergen to which the majority of sensitive patients develop specific IgE, both P25 and GP8 do appear to contain at least one major allergen. By RAST inhibition method, using Df 80d as a solid phase, the allergenic activity of P25 appeared as slightly higher than that of Df 80d, whereas GP8 displayed a very weak inhibitor capacity. Thus, the allergic specificity of GP8 differs from that of Df 80d or P25.     

Lipid transfer protein: a pan-allergen in plant-derived foods that is highly resistant to pepsin digestion

BACKGROUND: Lipid transfer proteins (LTPs) are stable and highly conserved proteins of around 10 kD. They have recently been identified as allergens in fruits of the Rosaceae family. OBJECTIVE: The aim of this study was to investigate whether the highly conserved structure of LTPs justifies a designation as a true pan-allergen, and to study the role of protein stability in allergenicity. METHODS: Thirty-eight patients with a positive skin prick test to Rosaceae fruit extracts were characterized by interviews and skin prick tests. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified natural and recombinant LTPs. To address the role of protein stability in the allergenicity of LTP, fruit extracts and LTPs were digested with pepsin. RESULTS: IgE antibodies to Rosaceae LTPs cross-reacted with a broad range of non-Rosaceae vegetable foods. Inhibition studies with purified natural and recombinant LTPs confirmed the role of LTP in this cross-reactivity. Many of the patients with this type of cross-reactive IgE antibodies had a clinical food allergy. In contrast to the typical birch Rosaceae cross-reactive patients, the oral allergy syndrome was frequently accompanied by more severe and systemic reactions. IgE reactivity to LTP was shown to be resistant to pepsin treatment of the allergen. CONCLUSION: LTP is a true pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.     

Expression of birch pollen-specific IgE-binding activity in seeds and other plant parts of birch trees (Betula verrucosa Ehrh.).

Dot immunoblotting of crude extracts of various aerial parts of birch trees, using patient serum rich in birch pollen IgE, showed IgE-binding activity in leaves, buds, twigs, seeds, bark, and old male catkins. Seed extracts analysed by SDS-PAGE, electroblotting to nitrocellulose and immune detection using isotope-conjugated anti-IgE verified the presence in seeds of an IgE-binding protein of an approximate molecular weight of 12 kD, distinct from the major allergen (molecular weight 17 kD) of Betula verrucosa pollen. The allergen of birch seeds was readily leachable from the seeds. Many of the birch plant part extracts were active in RAST inhibition using birch pollen RAST discs, but showed low potency relative to the allergenicity of birch pollen allergens.     

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.     

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.     

Production of an international reference standard alternaria extract. I. Testing of candidate extracts

As part of a program to establish international standards of selected allergens, 6 coded extracts of Alternaria were assessed in 6 laboratories by immunochemical, biochemical and physicochemical procedures. Direct RAST, RAST inhibition, quantitative skin tests and leukocyte histamine release were used to assign relative orders of potency to the 6 extracts. The composition and major allergen content was tested by thin-layer isoelectric focusing and quantitative immunoelectrophoresis (crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis). Three laboratories determined the quantity of purified allergens in each of the preparations. In addition, source materials were sent to an expert Alternaria taxonomist for independent identification. The results showed considerable variation with respect to total allergenic potency and content of individual allergens. Source materials could not be confirmed as Alternaria in some instances. Based on fulfillment of written specifications and assay results, extract No. 6 was recommended by the Alternaria Working Group as the candidate international standard to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies.     

Allergenicity of the cat flea (Ctenocephalides felis felis)

Adult fleas, spent and unspent culture media were extracted and the radio-atlergosor-bent test (RAST) performed with sera of 48 cat flea skin test-positive individuals from the Tampa Bay area of Florida. Sixteen sera (33.6%) had a positive RAST to the cat flea extract prepared in our laboratory [1.7-11.4% of the total counts (TC) added]. Six of the 16 sera (12.5%) also contained specific IgE to allergens in thespent medium (0.8-3.3% TC). The allergen composition and strength were studied by RAST inhibition of two commercial cat flea extracts and compared with our in-house flea extract. The results demonstrated similar allergen compositions and different potencies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the in-house flea extract showed several Coomassie blue-stained bands (10–85 kD). SDS-PAGE immunoblols revealed five IgE-binding bands at 34, 35, 39, 54 and 60 kD. Flea allergens were quantified in eight house dust samples using RAST inhibition assays and expressed as RAST inhibition units; five of these samples contained detectable levels. Cat flea allergens may contribute to the allergenicity of house dust in areas of heavy llea infestation.     

Glycoprotein allergens in pollen of timothy. V. Significance of the carbohydrate moiety for the immunological activity of a basic glycoprotein allergen

When the allergen was oxidized with periodate the size of its precipitate in rocket immunoelectrophoresis (RIE) was reduced. Incubation of the allergen with various glycosidases did not significantly affect its precipitation in RIE. The binding of human IgE and IgG to allergen bound to nitrocellulose seemed not to be affected by incubation with alpha-L-fucosidase, beta-D-xylosidase, beta-D-galactosidase or periodate oxidation. Incubation with alpha-D-mannosidase reduced the binding of IgG to the allergen but not its binding of IgE. Periodate oxidation did not significantly affect the IgE binding of the allergen in radioallergosorbent test (RAST) inhibition. When RAST discs of the allergen were incubated with alpha-D-mannosidase, alpha-D-galactosidase, Arachis hypogaea lectin and concanavalin A, the IgE binding to the discs was slightly reduced. Pretreatment of the discs with the monoclonal antibody FMC-A9 against ryegrass pollen allergens and beta-D-xylosidase did not reduce their IgE binding. L-Arabinose, D-mannose, methyl-alpha-D-mannopyranoside, D-galactose, D-glucose or the monoclonal antibody FMC-A9 did not inhibit the binding of IgE to RAST discs of the allergen. After incubation of the allergen with pronase, it did not form a precipitate in RIE and its IgE-binding ability in RAST and RAST inhibition was almost completely lost.     

Allergenic cross-reactions among legume foods--an in vitro study

The specific IgE binding by protein extracts of 11 food legumes, including soybean, was examined by RAST and RAST inhibition. Sera from 15 peanut-sensitive patients were, with very few exceptions, positive in the RAST to all the legumes. RAST-inhibition testing of each extract against RAST discs of the other legumes indicated considerable cross-reactivity of IgE binding between the legumes. Cross-allergenicity was demonstrated to be most marked between the extracts of peanut, garden pea, chick pea, and soybean. The results have important implications for selection of effective hypoallergenic diets and for the diagnosis of patients hypersensitive to foods.     

Immunochemical characterization of a high molecular weight basic allergen (HMBA) of rye grass (Lolium perenne) pollen

A high molecular weight basic allergen (HMBA) was isolated from the mixture of non-dialysable components of the aqueous extract of defatted rye grass pollen by a combination of gel filtration and isoelectrofocussing. HMBA, a glycoprotein of mol. wt 56,800 (17% carbohydrate) contained all naturally occurring amino acids. A hyperimmune rabbit anti-HMBA serum gave only a single precipitin band with the crude extract of the rye grass pollen in crossed immunoelectrophoresis. Thus. it was concluded that HMBA was a unique and highly purified antigen. The allergenicity of HMBA was revealed by its ability to elicit immediate skin reactions in grass allergic patients. Moreover, all patients' sera tested had IgE antibodies to HMBA detectable by direct RAST with HMBA allergosorbent discs, These observations indicated that HMBA was a major allergenic constituent of rye grass pollen. Treatment of HMBA by 6 M guanidine HC1 led to a significant reduction in its ability to combine with human IgE antibodies. The treatment also resulted in the complete loss of allergenicity (i.e. inability to elicit PCA reactions with a murine reaginic antiserum to HMBA) and antigenicity (inability to form precipitins with rabbit anti-HMBA): hence, it would appear that the allergenic and antigenic determinants of HMBA are ‘conformational’.     

Allergens in the white and yolk of hen's egg. A study of IgE binding by egg proteins

The radioallergosorbent test (RAST) was used to compare the IgE binding of egg white and yolk, and allergenic proteins were detected by immunoelectrotransfer ('Western blotting'). The main allergens were found in egg white, but for a large proportion of the egg-sensitive patients, yolk contained specific IgE-binding constituents. For blood sera from 36 patients, there was a positive correlation between the results of RAST for egg white and for yolk. Lysozyme was found to be an allergen for some patients. The effect of heating on the allergenicity of egg white was examined and the allergenicity of hen egg white was compared with that of a duck egg. The allergens in yolk were associated with each of the three yolk fractions, and several of the proteins in the low-density lipoprotein fraction bound IgE.     

Purification and characterization of Par o 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.     

Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test

The potencies of 11 commercial extracts of short ragweed pollen were analyzed by skin test end point titration and compared to potency as measured in vitro: (1) by the radioallergosorbent test (RAST), (2) by AgE concentrations, and (3) by protein nitrogen determinations (PNU). RAST potency was determined by the capacity of the extract to inhibit the binding of IgE antibody to solid-phase allergen in the first step of the RAST, and it was expressed as the quantity of extract needed for 50 per cent inhibition of binding. Potencies determined by skin testing in 7 patients were strongly related among the various patients in spite of a 1,000-fold difference in the patients' sensitivity to the extracts. Similarly, potency as measured by RAST inhibition showed about a 1,000-fold difference among the extracts, and the RAST potencies were strongly related to potency as measured by skin testing. Purified antigens derived from short ragweed inhibited binding of IgE antibodies, but the slopes of the inhibition curves were significantly less than those produced by crude or partially purified short ragweed. AgE concentration and PNU concentrations also were correlated with skin test potency, but the range of potencies was less than that found by RAST inhibition. Measurement of the potency of extracts by RAST inhibition should prove useful as a general procedure for the standardization of allergy extracts.     

Measurement of alum precipitated allergen extracts by RAST inhibition methods

The feasibility of measuring the allergenic activities of alum precipitated pollen extracts by means of the radioallergosorbent test (RAST) was investigated. Different batches of aqueous and alum precipitated birch and mixed grass pollen extracts were used as antigens. The alum precipitates could be readily dissolved with 0.1 m EDTA (pH 9.0) without any loss in allergenicity. No significant differences in RAST activities of undissolved and dissolved alum precipitated allergen extracts could be detected in terms of ability to inhibit the binding of allergen specific IgE to solid-phase allergens.