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The amount of active groups of cyanate esters and imidocarbamates present on cyanogen-bromide-activated paper discs were estimated. The protein-binding capacity of activated discs was found to be dependent on the amount of these active groups. The results suggested that monitoring of active groups on activated discs can help in preparing consistent allergosorbent for immunoassays.

Sera of 60 allergic persons and 20 healthy RAST-negative control persons were examined using immunoblotting to establish the presence of grass pollen-specific antibodies. These antibodies were depicted by an enzymatic, immunoglobulin class-specific indicator system. Seven bands of the timothy pollen extract proved to be major allergens. One of the 4 grass pollen-specific monoclonal antibodies tested (Bo 1) recognized the 7 major allergen bands. The possibility of employing monoclonal antibodies for the isolation of allergens is discussed.

Immunoglobulin E (IgE) was demonstrated by a double antibody radioimmunoassay technique (PRIST) in 5 of 17 unconcentrated fecal extracts from children. Four of the PRIST-positive extracts also had measurable levels of IgE determined by an enzyme-linked immunosorbent test (Enzygnost IgE). However, using a competitive antibody radioimmunoassay technique (RIST), IgE was found in all unconcentrated fecal extracts. The RIST-IgE levels of the extracts were higher and did not correlate to IgE measured by the other methods, nor to the IgE in serum from the same children or to the manifestation of allergy. On the other hand, 4 of the 5 children with measurable PRIST-IgE levels, and 3 of the 4 children with detectable Enzygnost IgE concentrations in the extracts, had elevated serum IgE as well as a history of allergy. Gel filtration studies indicated that IgE determined by the PRIST and Enzygnost IgE methods had been degraded to fragments of lower molecular weight than that of albumin. This study also suggests that the IgE in feces measured by the RIST method is overestimated due to the influence from nonspecific substances of high molecular weight.

The performance of ELISA to detect IgG and IgM antibodies to Aspergillus fumigatus has been evaluated in strongly precipitin-positive, weakly precipitin-positive and precipitin-negative patient sera, with immunoblot analysis as the confirmatory test. All strongly precipitin-positive sera contained increased IgG titers and showed clearly positive immunoblot patterns. Most of the weakly precipitin-positive sera contained ELISA titers within the normal range established with sera of healthy blood donors and showed normal immunoblot patterns. Increased titers of IgG and/or IgM were measured in one-sixth of the precipitin-negative patient sera. Immunoblot analysis confirmed the presence of antibodies to A. fumigatus in 55% of the precipitin-negative sera with increased antibody titers. ELISAs for A. fumigatus-specific IgG and IgM are sensitive tests for screening of patient sera. However, positive results with ELISA should be confirmed by means of immunoblot analysis.

Polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS) followed by protein staining has shown that extracts from 11 different grass pollens contained proteins with similar molecular weights to that of the allergen R7 from rye grass (Lolium perenne) pollen extract (i.e. 29,000-31,000 daltons). Western blotting and detection with polyclonal (rabbit) antibodies raised against the purified R7 (Rye I) allergen indicated that these proteins were antigenically related and their allergenic properties were demonstrated by the binding of human IgE to immunoblots. The distribution of cross-reacting antigenic determinants was further investigated by immunoblotting with 2 mouse monoclonal antibodies, R7M1 and R7M2, produced with purified R7 as the initial immunogen. The 2 monoclonal antibodies were shown to react with 'R7-like' components of grass pollen extracts other than the component from rye grass. Differences in the distribution of R7M1 and R7M2 binding were found indicating that they are directed at separate R7 epitopes.

Monoclonal antibodies against major allergens of the short ragweed pollen were produced by fusion of NS-1 cells with splenic cells from BaLB/C mice that had been immunized separately with the major allergens, AgE-B + E-C and AgK, of the short ragweed pollen. These monoclonal antibodies were detected by enzyme-linked immunosorbent assay and further characterized by immunoblot analysis using the crude extract and highly purified allergens of the ragweed pollen. Three monoclonal antibodies obtained by immunization with AgE-B + E-C, designated as 36-6, 27-2 and 48-5, reacted mainly with the beta (36-6) and alpha (27-2) subunit of AgE and both AgE and AgK (48-5), respectively. Two monoclonal antibodies obtained by immunization with AgK, 4-7 and 8-5, had a similar reactivity with AgE-B + E-C, AgK, AgK-A and AgK-B. In addition, however, antibody 4-7 also reacted with AgE-B2 as well as the 36- and the 24- to 22-kilodalton antigens of the crude extract. All 5 monoclonal antibodies were characterized as IgGl subclass. Besides, monoclonal antibody 48-5 also showed cross-reactivity to components of sage pollen. Beyond academic interests, the monoclonal antibodies described here may also be useful in clinical allergy.

Antigliadin antibodies (AGA) were analyzed by immunoblotting from the sera of 30 patients with dermatitis herpetiformis (DH) and of 13 patients with gluten-sensitive enteropathy (GSE). The results were correlated to the jejunal morphology and to the serum AGA values as quantified with an enzyme-linked immunosorbent assay (ELISA). Immunoblotting analysis disclosed a distinct pattern of AGA which was similar in patients with DH and GSE. In both diseases individual variation in IgG class AGA patterns was large, suggesting that several antigenic determinants are involved in the AGA response. The IgA class AGA pattern was clearly more homogeneous. The sera with several bands in immunoblotting had the highest AGA levels in ELISA. Strong staining in the main gliadin area (bands from 31 to 38 kd) indicated severely damaged intestinal mucosa whereas the serum of DH patients with partial villous atrophy or normal mucosa showed usually weaker staining. The immunoblotting pattern of sera from healthy controls was even weaker but was directed against the same polypeptides as in the patients suggesting a quantitative rather than qualitative difference between healthy and diseased subjects. This difference was, however, more obvious in the IgA than in the IgG class AGA pattern possibly indicating a more fundamental defect in the regulation of synthesis of IgA isotype in DH and GSE.

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 allergen composition of moulds (Alternaria alternata and Aspergillus fumigatus) and mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae) was studied by electroblotting. The allergens were separated by SDS-gPAGE and transferred to a nitrocellulose membrane by electroblotting. Specific IgE antibodies directed against mould and mite allergens were utilized as probes to detect the allergens. The bands were localized on the nitrocellulose membrane by means of enzyme- or isotope-labelled antibody. Circulating specific IgE antibodies in mould and mite sensitive patient sera were also analyzed and characterized with respect to their IgE specificity. Under appropriate experimental conditions this electroblotting technique could also be used for quantitation of individual allergens by scanning the isotope- or enzyme-stained nitrocellulose strips.