Detection of IgE- and IgG-binding proteins after electrophoretic transfer from polyacrylamide gels.

An electrophoretic technique for transferring proteins to a nitrocellulose membrane has been applied to proteins separated in polyacrylamide gels by isoelectric focusing, and gradient, SDS and 2-dimensional electrophoresis. Allergenic proteins were then identified by successive incubation of the transfer membrane with serum from allergic individuals and with 125I-labelled anti-human IgE, followed by autoradiography. Alternatively, IgG binding proteins were detected using 125I-labelled protein A. The application of these methods was illustrated with cereal grain proteins and sera from individuals with bakers' asthma and coeliac disease. This approach allowed the easy comparison of allergens important for different patients in a single source, and of allergens present in different but cross-reacting sources.

Simultaneous detection of IgE binding to several allergens using a nitrocellulose 'polydisc'.

The use of nitrocellulose for allergen disc preparation permits several different allergens to be applied as separate spots to the same disc for simultaneous evaluation with one serum sample (50 microliter). To achieve this, an immunological application method involving anti-human IgE and the peroxidase-anti-peroxidase procedure must be used with a substrate that produces an insoluble product. The suitability of the polydiscs for routine clinical evaluation of several allergens at once was demonstrated by testing the sera of seven allergic bakers with discs containing extracts of flour or pollen from wheat, cereal rye, or rye grass.

Allergen discs prepared from nitrocellulose: detection of IgE binding to soluble and insoluble allergens.

Nitrocellulose discs, 6 mm in diameter, are suggested as an alternative to cyanogen bromide-activated paper for the coupling of allergens in radio- or enzyme-linked assays to estimate allergen-specific IgE in serum. The preparation of allergen discs with nitrocellulose is simple, involving 3 steps: (a) drying allergen extract onto disc, (b) soaking discs in a 3% solution of bovine serum albumin, and (c) washing out the buffer. Very similar results (r = 0.94) were obtained using either paper or nitrocellulose discs for radioallergosorbent testing of sera from 15 bakers using allergens from mites, ryegrass pollen or wheat grain. The amount of protein (as radiolabelled albumin) actually bound to either type of disc or microtitre trays was similar, and low (5% of the protein applied for each media). However, when the protein was applied to nitrocellulose in 1% KOH up to 70% of it was bound. This solvent permitted better evaluation of IgE binding to insoluble allergens such as glutenin, which proved to be the most allergenic wheat-grain fraction tested by this method for a group of 9 bakers.

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii.

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2(t) and T2, and was considered as part of the same allele at the Glu-D (t) 1locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2(t) and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an omega-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of omega-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F(2)grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii ( Glu-D (t) 1 and Gli-D (t) 1) segregated as alleles of the Glu-D1 and Gli-D1loci of bread wheat. A new locus encoding the omega-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively.

Identification of species of fish by gradient-gel electrophoresis.

An electrophoretic method is described for distinguishing between fish fillets according to their protein composition. Thaw fluid (4 microL) was applied to one of 14 sample positions of a precast gel, containing a gradient of polyacrylamide of either 2.5 to 27% or 3 to 40%. All reagents and gels are commercially available in ready-to-use form. Either gel provided a distinction between any of the 42 fish types, but the 3 to 40% gel gave better identification because of its superior molecular-sieving properties. Reproducible electrophoretic patterns were obtained for different samples of the same fish type, but small differences were shown for fish of widely different origin, for example Australian and New Zealand ling.

Rapid (ten-minute) pore-gradient electrophoresis of proteins and peptides in Micrograd gels.

Precast gradient gels of short migration length (25 mm) have been developed to provide rapid electrophoretic separation without loss of resolution. These Micrograd gels have been prepared in gel ranges (conventional and unique) to match pore-gradient electrophoresis conditions to proteins/peptides ranging in size from several hundreds to millions. The Hylinx Micrograd gel combines an extreme gel range (6 to 48% polyacrylamide) with a novel crosslinker to provide sieving of polypeptides, and pore-limit electrophoresis of the smallest proteins (e.g. insulin monomer). All gel ranges (such as 3 to 30%) provide zone sharpening in routine analysis of conventional protein mixtures (e.g. serum) within 10 min electrophoresis at 200 to 300 volts. The gels are thin (1 mm) and thus stain quickly, but the gel cassette is of conventional overall width (83 mm), thus fitting many apparatus designs and accommodating 12 samples. The gels are finding valuable use in screening applications, requiring the electrophoretic analysis of many samples, and in cases where a rapid answer is needed, such as monitoring protein purification. The gels have proved particularly useful, in-house, for the latter application in developing Gradipore's new large-scale preparative electrophoresis system, the Gradiflow.

IgE antibodies to wheat flour components. Studies with sera from subjects with baker's asthma or coeliac condition.

Sera from two subjects with baker's asthma and six patients with coeliac condition were examined for the presence of IgE antibodies with specificities for wheat flour components. Sera were studied using the radioallergosorbent test (RAST) together with whole flour and thirteen purified and partially purified flour fractions. IgE antibodies to a number of flour components were demonstrated in the allergic bakers' sera, but the strongest reactivities were observed with wheat albumins and globulins. A more detailed examination of the flour water-soluble proteins using RAST inhibition methods demonstrated that albumins were more reactive with the allergic sera than the globulins. Apart from the results with the water-soluble proteins, the two sera showed a different pattern of reactivity with the other flour preparations. No IgE antibodies to whole flour or any of the flour components, including A gliadin, were found in the coeliac sera. Failure to detect wheat gluten- or gliadin-specific IgE antibodies indicates that IgE-mediated reactions are not important in the pathogenesis of coeliac condition. Levels of total IgE in the sera from the coeliac subjects were elevated and, with two of the sera, some success was achieved in identifying the allergens responsible for the elevation. We propose that elevation of serum IgE may frequently occur in coeliac condition and may arise due to an increased uptake of antigens via the damaged intestinal mucosa.

Relationships between plants relevant to allergy.

This article gives background information on the taxonomic relationships between plants, for clinicians, patients and students of allergy. A list is provided of plants considered potentially allergenic either by inhalation of pollen or by ingestion of grain products. Distributions in Australia of plant species important in assessing pollen-related allergy, and the range of foodstuffs that contain cereal-grain protein, are also indicated. Particular emphasis is placed on the grasses because of their importance as producers of both grain and pollen.

Our obsession with high resolution in gel electrophoresis: does it necessarily give the right answer?

Poor resolution of protein zones in an electrophoretic pattern may not necessarily be the result of poor technique. The example is given of the 'streak material', extracted from wheat flour, now recognised to be aggregated subunits of glutenin. The size distribution of the aggregated glutenin 'streak' is the key to elucidating the functional properties of wheaten dough. A stepped-layer gel technique has been devised to quantitate the proportions of aggregated glutenin in specific size groupings.

A rapid (less than 10 minute) electrophoresis method for identification of wheat varieties.

Conventional procedures for electrophoretic identification of grain samples according to variety are too slow to permit checking at the time of delivery. The method described permits electrophoretic identification within an hour. It involves extraction of gliadin proteins from crushed grain with 6% urea solution or ethylene glycol, cathodic electrophoresis for 9 min at 300 V in a Micrograd gel (MG 315 from Gradipore Ltd, Sydney, Australia) using sodium lactate buffer (pH 3.1), and staining in Gradipore (at about 50 degrees C). Distinction between a set of Australian varieties was similar to that obtainable with the Australian Standard Procedure.

Electrophoresis of small proteins in highly concentrated and crosslinked polyacrylamide gradient gels.

A high concentration (40%) of acrylamide plus N,N'-methylenebisacrylamide combined with a high level of crosslinking (12.5%) yielded clear gels capable of restricting the passage of small proteins. This gel composition was chosen in preference to other combinations, in particular those producing opaque gels which have larger pore sizes and which provide a reduced sieving effect. Gradient gels were prepared in which the gel concentration rose from 3 to 40% and the degree of crosslinking increased from 4 to 12.5%. Such gels were suitable for fractionating crude, unreduced, and uncharacterized extracts containing proteins ranging in molecular size from 10,000 to several million daltons under conditions where all proteins are retained on the gel even after prolonged electrophoresis. The gels yielded zones which were of improved sharpness and resolution compared with gels of lower concentration and degree of crosslinking, and can be used to provide an estimate of molecular size. Examples of the use of HX gradient gels included both anodic and cathodic electrophoresis at pH 8.3 and 3.1, respectively, of serum and cereal-grain proteins and a partial enzymic hydrolysate of serum albumin.

Hypersensitivity to inhaled flour allergens. Comparison between cereals.

Radioallergosorbent testing (RAST) of sera from subjects sensitized to wheat and rye flour indicated that there is significant reaction with seed extracts of 12 cereals (wheat, durum wheat, triticale, cereal rye, barley, rye grass, oats, canary grass, rice, maize, sorghum and Johnson grass). Results were evaluated in terms of taxonomic relationships and of the electrophoretically determined protein composition of the cereal extracts. RAST uptakes were uniformly low in sera from four rhinitic bakers, yet were significantly above the levels for non-allergic and cord sera. Much higher RAST uptakes were obtained with sera from four asthmatic bakers when tested with wheat and its close relatives, but there was still reasonably high reactivity with more distantly related cereals. RAST inhibition experiments indicated in a more direct way the extent of cross-reactivity between grain extracts of wheat, rye, barley and oats. One baker had a history of more severe attacks of breathlessness following inhalation of rye flour compared with wheat flour. This was confirmed by bronchial challenge testing, but the comparison was not obviously consistent with the results of prick testing or estimation of histamine released from his leucocytes. The results as a whole suggested that the bran layers of cereal grains are at least as allergenic as flour.

Multifunctional apparatus for electrokinetic processing of proteins.

New equipment (the "Gradiflow") has been designed and constructed to provide efficient large-scale preparative fractionation of macromolecules, based on charge and/or size differences, as well as the concentration of macromolecules and electrodialysis. Examples of its capability are the separation of a mixture of haemoglobin (50 mg) from bovine serum albumin (50 mg) within 15 min (based on charge differences at pH 6.8), the purification of phycoerythrin from a crude extract on the basis of size, and the fractionation of serum proteins into two discrete size classes.

Rapid and automated characterisation of seed genotype using Micrograd electrophoresis and pattern-matching software.

New precast microgels are described for use in quickly identifying seed of cereal varieties by determining protein composition within an hour. For example, gliadin proteins are extracted from crushed wheat grain, wheatmeal or flour with ethylene glycol (centrifugation not necessary) and 5 microliters extract is applied to a Micrograd gel (3-15% gel gradient) for ten minutes' electrophoresis at 300 volts in sodium lactate buffer (pH 3.1). Alternatively, precast gels are available for SDS gel electrophoresis for examining a different aspect of grain composition as a means of identification. To further expedite identification, software packages have been developed to match the protein pattern for an unknown sample against those of authentic samples, thus to provide quick and definite identity, based on electrophoretic banding, densitometer scan, HPLC profile, multiple antibody reaction or RFLP pattern (PatMatch program). Furthermore, the program WhatWheat offers advice on the best combination of methods to use for a specific task of identification.

Distinction between genotypes of Lupinus species by sodium dodecyl sulphate-gel electrophoresis and by capillary electrophoresis.

Effective distinction was achieved among a wide range of lupin grain samples by either sodium dodecyl sulphate (SDS)-gel electrophoresis or capillary electrophoresis, based on grain-protein composition. Capillary electrophoresis was faster (< 1 h) and provided slightly greater distinction between the samples. On the other hand, SDS-gel electrophoresis could provide a greater through-put of samples in a 24 h period. Either technique could be used successfully to distinguish between lupin species and cultivars for taxonomic analysis or seed identification.

Preparative affinity membrane electrophoresis.

Using the patented Gradiflow system in conjunction with newly developed affinity membranes, the suitability of an electrokinetic technique for affinity fractionation was investigated. Blue dextran incorporated into an affinity membrane was used to deplete a solution of horse serum of albumin, with the result that a majority of serum proteins were enriched tenfold relative to albumin. The technique, when fully developed, would offer some advantages over affinity chromatography, since to a degree it is possible to control which components of the sample are presented to the affinity matrix. Furthermore, the technique would extend the capabilities of the already multifunctional Gradiflow system.

Two-dimensional analysis of gliadin proteins associated with quality in durum wheat: chromosomal location of genes for their synthesis.

Two-dimensional electrophoresis was used to fractionate the gliadin proteins from the endosperm of durum wheat. The increased resolution of the system, as compared with single-dimensional analysis, accentuated the heterogeneity of the proteins. This resolution, coupled with the use of aneuploid lines of the cultivar 'Langdon', permitted identification of the chromosomes controlling synthesis of the major protein components. Homoeologous Group 1 chromosomes controlled omega- and gamma-gliadin synthesis and the Group 6 chromosomes 6A and 6B controlled alpha- and beta-gliadins. Chromosome 1B was primarily responsible for the two groups of protein-polypeptides associated with strong or weak gluten characteristics of durum wheat. However, some of these proteins were controlled by chromosome 1A. In the beta-gliadin region several hybrid bands, whose chromosomal control was not identified by electrophoresis alone, were specified primarily by genes on chromosome 6B, although chromosome 6A was also involved. Control of some other hybrid bands could not be determined. Chromosomes in Groups 2, 3, 4, 5 and 7 were not implicated in the synthesis of the gliadin proteins of durum wheat.

The diversity of allergens involved in bakers' asthma.

Sera from 35 individuals with suspected allergies to inhaled flour were screened for the presence of immunoglobulin E (IgE) specific for wheat-flour proteins. Sera from nine asthmatic bakers with high wheat RAST scores were selected for further study with the aim of purifying the allergen(s) involved in bakers' asthma and related conditions. However, each of the different techniques applied--ion exchange chromatography, preparative isoelectric focusing and the electrophoretic transfer, or 'Western blotting' technique, showed that serum IgE from different individuals have markedly different specificities and bind to numerous wheat proteins. When three purified wheat proteins were tested--wheat germ agglutinin; a fraction purified using a concanavalin-A affinity column and a putative trypsin inhibitor--all were identified as allergens for some but not all of the allergic bakers.

Immunoglobulin E antibodies to ingested cereal flour components: studies with sera from subjects with asthma and eczema.

The specificity of immunoglobulin E for different cereal grain proteins was investigated using sera from twenty paediatric patients with asthma and/or eczema. Close correlations were observed between radioallergosorbent test values for grain extracts of wheat, rye and barley, and, to a lesser extent, oats. Of the different wheat four fractions tested, the globulins and glutenins consistently bound higher levels of IgE than the gliadins and albumins. This is in contrast both with bakers' asthma (an allergy to inhaled flour where the albumins are important allergens) and with coeliac disease (in which gliadin is the most toxic fraction). Partial digestion of the flour proteins largely removed their ability to bind IgE. An analytical technique of identifying allergens after gel isoelectric focusing demonstrated that many different flour proteins were involved.