The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigensRat lymphoid cells have been labeled with sodium 3H-borohydride after periodate oxidation. The labeled glycoproteins were solubilized in detergent and analyzed by fluorography after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Major bands were found at 150 000, 95 000 and 25 000 apparent mol.wt. for thymocytes; a t 170 000 and 95 000 mol.wt. for T lymphocytes and at 200 000 mol.wt. for B lymphocytes. Bone marrow cells showed a diffuse band at 100 000 mol.wt. with relatively minor bands around 150 000 mol.wt. With t h e exception of the 95 000 mol. wt. bands, all these glycoproteins bound t o lentil lectin.Using monoclonal or monospecific antibodies in immunoprecipitation and o n antibody affinity columns, each of these glycoprotein bands was identified as a previously defined lymphocyte differentiation antigen. The bands at 150 000 mol.wt. o n thymocytes, at 170 000 on T lymphocytes, at 200 000 on B lymphocytes, and at 130 000 t o 150 000 on bone marrow cells all consist of a leukocyte-common antigen, which has previously been shown to be present o n leukocytes but not on other tissues. At least a part of the 95 000 mol.wt. band on thymocytes, T lymphocytes and bone marrow cells is the W3/ 13 antigen previously shown t o be o n mature T lymphocytes, polymorphonuclear cells, and in brain. The 25 000 mol.wt. band of thymocytes is the Thy-1 antigen.Similar experiments were carried out o n thymocytes labeled with lZSI by the lactoperoxidase method. An intense band at 150 000 mol. wt. was identified as the leukocyte-common antigen b y immunoprecipitation. A labeled band, which did not bind t o lentil lectin, was immunoprecipitated at 95 000 mol. wt. with W3/13 antibody. Rat Thy-1 antigen was not labeled with lZ51.
Three fractions of rye-grass (Lolium perenne) pollen extract have been isolated by preparative isoelectric focusing (i.e.f.) and characterized in terms of physicochemical and immunochemical properties. The purified components were designated 'R7' and 'R14' on the basis of their positions in relation to other rye-grass pollen extract components on SDS/polyacrylamide-gel electrophoresis and their apparent molecular masses were assessed as 31 and 11 kDa respectively. On i.e.f., R14 split into two components, one acidic (pl 5.0) and one basic (pl 9.0), termed 'Rl4a' and 'Rl4b' respectively, and R7 focused at pl 5.8. R7 and R14a were shown to be allergenic by skin-prick test and all three components were recognized by rye-grass-pollen-specific human IgE. On SDS/polyacrylamide-gel electrophoresis and i.e.f., R7 behaved in a manner identical with that shown by an authentic sample of Rye I and gave an amino acid analysis similar to published data Immunochemistry 3, 91-100] for Rye group-I isoallergens; the amino acid sequence of the first 27 N-terminal amino acids was also determined. Physicochemical analysis revealed that R14a was equivalent to Rye II and 14b to Rye III. Preparative i.e.f. followed by gel-permeation chromatography proved to be a rapid and efficient method for purifying the allergenic components of Rye I (R7), Rye II (Rl4a) and Rye III (Rl4b) from rye-grass pollen extract.
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.
Using a combination of immunoprecipitation and polyacrylamide gel electrophoresis techniques, patterns of antibody responses to a number of components in rye pollen extract have been studied in hyperimmune mouse sera. Common laboratory strains of mice were investigated, including AKR, C3H/He, SwR, Ist, Asn, Balb/c, C57BL10, DBA2 and BD1: F1 hybrids. All strains responded vigorously to the immunisation protocol used (rye pollen extract/alum followed by a booster dose in aqueous media) when tested with non-discriminating assay procedures such as haemagglutination or passive cutaneous anaphylaxis directed against the whole rye pollen extract. However, markedly variable antibody responses were observed between strains and between murine and human sera when considered in relation to individual rye extract components. Particularly noteworthy was the relatively poor response of all mice to the low molecular weight rye antigen component (11,000 daltons). This material was readily detected by human sera derived from pollenosis patients. Notable also was the fact that no single rye component was detected by all mice sera. These results illustrate the complex serological response induced by heterogeneous antigen mixtures, such as grass pollen extracts.
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