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Biomedical subjects

W M Becker

Publications and source records attributed to W M Becker.

At least 91 records · Page 5Linked to original sources
PubMed1989

[Monoclonal antibodies in allergy research].

Allergen-specific monoclonal antibodies (MoAb) are of growing importance in allergen standardization, especially in setting up assays to measure quantitatively allergen content in therapeutic extracts and to establish secondary standards. MoAb are useful in measuring allergenic indoor pollution and in monitoring allergen clearance in cases of recommended allergen avoidance. In applied research MoAb are valuable tools in allergen purification providing primary standards. As a source for developing MoAb of the second generation they open new perspectives.

Allergens↗
PubMed1989

[Extrinsic allergic alveolitis: IgG subclass reactivities and monoclonal antibodies against Micropolyspora faeni and Aspergillus fumigatus].

IgG-subclass reactivities to Micropolyspora faeni of farmer's lung patients and exposed persons were investigated. Additionally, specific IgG and IgE antibodies of patients suffering from IgE-mediated allergy and extrinsic allergic alveolitis to Aspergillus fumigatus and allergic bronchopulmonary aspergillosis (ABPA) were examined. Antibody reactivities of patients were compared with reactivities of monoclonal antibodies raised against M. faeni and A. fumigatus, respectively. The results indicate a special importance of specific IgG2 reactivity for serologic diagnosis. Monoclonal antibodies raised against M. faeni and A. fumigatus recognize components which induce antibody production in patients as well. Reactivities of monoclonal antibodies with different batches of A. fumigatus extract show the variability of these antigen preparations and illustrate the need for standardization.

Alveolitis, Extrinsic Allergic↗
PubMed1989

[Immunoprint technic: a new method for planned production of allergen-specific monoclonal antibodies].

Working on the isolation of allergens from crude pollen extracts we were looking for a simple method to produce monoclonal antibodies (mab) specific for a major allergen of cultivated rye (S. cereale) with an isoelectric point (pl) of 5.9. We used immunization with allergen isolated from nitrocellulose blots. Allergen was obtained by isoelectric focusing (IEF) of a rye pollen extract followed by immunoprint and detection of allergen bound to nitrocellulose by a monoclonal antibody (mab). Fusion with myeloma cell line P3X63Ag8.U1 led to production of 7 different mab specific for the protein with pl 5.9. The method seems to be a simple way to produce mab specific for a certain component of a crude protein mixture as a pollen extract.

Allergens↗
PubMed1989

[Farmer's lung: IgG-subclass reactivities against thermophilic actinomycetes].

IgG subclass reactivities of patients suffering from farmer's lung, healthy family members and blood donors were tested for specific binding to Micropolyspora faeni extract by means of immunoblot techniques (IEF-Print, Western Blot). In patients' sera IgG1 and IgG2 had shown the strongest reactivities while IgG3- and IgG4-reactivities were not found in all cases. Strong IgG2-reactivities against acidic proteins (pl 3-5) of M. faeni seem to distinguish between patients and exposed or not exposed controls, respectively.

Blotting, Western↗
PubMed1988

A common epitope on major allergens from non-biting midges (Chironomidae).

A synthetic peptide corresponding to sequence 91-101 of the Chironomus thummi thummi haemoglobins (Chi t I) components III and IV was used to investigate binding and cross-reactivity with polyclonal human IgE and rabbit IgG antibodies and murine IgG1 subclass monoclonal antibodies (MABs). The synthetic peptide reacted with antibodies from all three mammals. The specificity of the reaction, especially that with IgE antibodies was shown by dose dependent inhibition with native Chi t I component III. Epitope(s) reacting with these antibodies were also found in haemoglobins from 14 of the 15 chironomid species analyzed. The synthetic peptide III/IV 91-101 enabled the identification of an important antigenic/allergenic determinant of the broadly distributed insect family Chironomidae. The antigenic potency of this synthetic peptide as shown by testing with human IgE, rabbit IgG and mouse MABs, and the widespread occurrence of the epitope in an identical or homologous sequence and/or superficial location, qualifies the peptide for therapeutic applications in medicine.

Allergens↗
PubMed1988

Analysis of rye pollen (Secale cereale) allergens using patients' IgE, immunoprint, Western Blot and monoclonal antibodies.

Rye pollen (Secale cereale) was investigated by Immunoprint and Western Blot analysis. Using Immunoprint, more than 30 proteins could be shown to be allergens in terms of their IgE binding, and 9 were determined as major allergens. In Western Blot 17 allergens were discovered, 3 of them major allergens. 4 monoclonal antibodies raised against a pollen extract of 6 grasses showed cross-reactivity to rye pollen, thus indicating common epitopes. 1 of 3 monoclonal antibodies produced against an extract of rye pollen was only reacting to this pollen. Looking at the overall binding pattern of the 7 monoclonal antibodies, they showed partial identity to patients' IgE in Immunoprint and Western Blot. The possibility of defining epitopes using preparative isoelectric focusing and monoclonal antibodies is discussed.

Antibodies, Monoclonal↗
PubMed1987

Epitope mapping of major insect allergens (chironomid hemoglobins) with monoclonal antibodies.

Monoclonal antibodies (m ABs) were raised against the physically and chemically well-defined insect allergen, component CTT III from Chironomus thummi thummi (CTT) hemoglobin. With these m ABs, we observed epitopes present in as many different hemoglobin (Hb) molecules from the widespread insect family Chironomidae as possible. These Hbs were identified as important allergens in previous studies. Twenty m ABs from seven different clones were tested for specificity against the CTT Hbs III, IIIa, IV, and I to X, as well as against Hbs from 14 other chironomid species; m ABs 1 to 3 were found to be specific for an epitope that was expressed on the CTT III component as well as in Hbs from three other species. m ABs 5 to 7 and 15 recognized an epitope expressed in CTT III, CTT IIIa, CTT IV, and in Hbs from 13 other species. m ABs 8 to 14 and 16 to 20 demonstrated heteroclitic behavior, that is, they bound CTT IIIa better than the immunogen CTT III. Reactivity of m AB 6 with a clinically relevant epitope was demonstrated by up to 15% inhibition of antibody binding by human IgE from sensitized patients. Our results demonstrate that clinically relevant allergenic epitopes can be localized on different molecules with m ABs. This could be important in the development of immunotherapy protocols with highly purified or fragmented allergens.

Allergens↗
PubMed1986

Analysis of allergen components in grass pollen extracts using immunoblotting.

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.

Animals↗
PubMed1985

Isolation of the specific mRNA coding for a CALLA-like protein and partial characterization of its cell-free translational products.

In a cell-free translation directed mRNA from the human lymphoblastoid cell line KM3, common ALL-Antigen (CALLA)-like proteins were detected immunologically. For immunoprecipitation a combination of three monoclonal antibodies and a rabbit heteroantiserum with anti-CALLA specificity were used. Employing a cocktail of two anti-CALLA monoclonal antibodies CALLA directing mRNA could be purified by specific polysomal immunoadsorption. The CALLA-like protein defined in the cell-free products of total mRNA and mRNA derived from polysomes showed a Mr of 95,000 daltons. Additional CALLA-like proteins could be identified in the cell-free translational products when mRNA from polysomes precipitated by the anti-CALLA heteroantiserum was used. The calculated Mr of these proteins were 80,000 97,000 and 135,000 daltons.

Animals↗
PubMed1985

Investigation of the glyoxysome-peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy.

Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function. We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A-gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.

Alcohol Oxidoreductases↗
PubMed1985

Isolation of serine:glyoxylate aminotransferase from cucumber cotyledons.

Serine:glyoxylate aminotransferase, a marker enzyme for leaf peroxisomes, has been purified to homogeneity from cucumber cotyledons (Cucumis sativus cv Improved Long Green). The isolation procedure involved precipitation with polyethyleneimine, a two-step ammonium sulfate fractionation (35 to 45%), gel filtration on Ultrogel AcA 34, and ion exchange chromatography on diethylaminoethyl-cellulose, first in the presence of pyridoxal-5-phosphate, and then in its absence. The enzyme was purified approximately 690-fold to a final specific activity of 34.4 units per milligram. Electrophoresis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gels revealed two polypeptide bands with apparent molecular weights of approximately 47,000 and 45,000. Both polypeptides coeluted with enzyme activity under all chromatographic conditions investigated, both were localized to the peroxisome, and both accumulated in cotyledons as enzyme activity increased during development. The two polypeptides appear not to be structurally related, since they showed little immunological cross-reactivity and gave rise to different peptide fragments when subjected to partial proteolytic digestion. Antiserum raised against either the denatured enzyme or the 45,000-dalton polypeptide did not react with any other polypeptides present in a crude cotyledonary homogenate. The purified enzyme also had alanine:glyoxylate aminotransferase activity, but was about twice as active with serine as the amino donor.

Journal Article↗
PubMed1983

Identification of inflammatory cell phenotypes in human oral carcinomas by means of monoclonal antibodies.

Monoclonal antibodies reacting with human T cell sub-populations, Langerhans cells and macrophages were used to examine the quantitative distribution of immune-competent cells in normal oral mucosa and invasive oral carcinomas. Both immunofluorescent and immunoperoxidase procedures were applied. In normal oral epithelia, the dominant immune-reactive cell was the Langerhans cell, positive for OKT 6 and expressing HLA-DR gene products (OKIa1+). Many intra-epithelial non-epithelial cells (non-keratinocytes), belonged to the lymphocyte system carrying the suppressor/cytotoxic phenotype (OKT 8+). This lymphocyte sub-population was also the most prominent cell type in the normal mucosal stroma. The quantitative evaluation of immune-competent cells in squamous cell carcinomas revealed elevated numbers of all the inflammatory cell sub-populations investigated (suppressor/cytotoxic lymphocytes, helper/inducer lymphocytes, Langerhans cells, macrophages) compared with the normal oral mucosa. There was a striking increase in suppressor/cytotoxic lymphocytes (OKT 8+) and in cells of the macrophage system, including Langerhans cells (OKIa1+, OKM 1+, OKT 6+). In the stroma distant to the tumour complexes, many helper/inducer lymphocytes (OKT 4+) were also observed.

Antibodies, Monoclonal↗
PubMed1983

Purification and characterization of hydroxypyruvate reductase from cucumber cotyledons.

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and 50% saturation), affinity chromatography on Cibacron Blueagarose, and ion-exchange chromatography on DEAE-cellulose. HPR was purified 541-fold to a final specific activity of 525 +/- 19 micromoles per minute per milligram of protein. Enzyme homogeneity was established by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was 91 to 95 kilodaltons, approximately double the apparent subunit molecular weight of 40,500 +/- 1,400. With hydroxypyruvate as substrate, the pH optimum was 7.1 and K(m) values were 62 +/- 6 and 5.8 +/- 0.7 micromolar for hydroxypyruvate and NADH, respectively. With glyoxylate as substrate, the pH optimum was 6.0, and the K(m) values for glyoxylate and NADH were 5700 +/- 600 and 2.9 +/- 0.5 micromolar, respectively. Antibodies to HPR were raised in mice (by the ascites tumor method) and in rabbits, and their monospecificity was demonstrated by a modified Western blot immunodetection technique.

Journal Article↗
PubMed1982

Subcapsular thymic lymphoblasts expose receptors for soy bean lectin.

In the course of analysing thymic cellular constituents concerning the expression of lectin receptors and defined lymphocyte differentiation antigens, respectively, a subpopulation of thymic lymphocytes was detected exposing both peanut lectin (PNL) and soy bean lectin (SBL) receptors on their surfaces, while the majority of cortical thymic lymphocytes were found to be PNL positive, but SBL negative. This subset of SBL+PNL+thymic lymphocytes in the adult populates the area beneath the thymic capsule, forming a narrow rim only, while in perinatal thymus a corresponding subcapsular cell layer comprises almost one third of the thymic tissue and in early ontogenesis the vast majority of thymic lymphoid cells are PNL+SBL+. By ultrastructural analysis of double labelled cell suspensions as well as of cells separated by affinity chromatography, it could be shown that PNL/SBL+ cells frequently exhibit morphological features of lymphoblastic cells. Thus this subcapsularly located subset seems to represent a compartment of proliferating immature cells, descended from bone marrow and foetal liver prethymic cells, respectively, and giving rise to PNL/SBL- cortical thymic lymphocytes.

Animals↗