Bärbel Fahlbusch

The in vitro activity of lectin I from mistletoe (ML I) and its isolated A and B chains on functions ofmacrophages and polymorphonuclear cells

We investigated the effects of ML I and its isolated chains, A and B, in regard to selected functions of phagocytes (human granulocytes, paraffin-oil stimulated M phi from guinea pigs). On these cells, ML I has no cytotoxic effect between 10(-14) and 10(-8) (trypan blue exclusion and ethidium bromide exclusion). Over the same concentration range, ML I and B chain diminish the negative surface charge of M phi and agglutinate M phi at concentrations greater than or equal to 2 X 10(-8) M (ML I) and greater than or equal to 3 X 10(-7) M (B chain), respectively. The diminishing of the negative surface charge shows two peaks, indicating the existence of two types of receptors on the M phi surface with different affinities for sugar-binding sites. Moreover, the B chain shows a third peak at higher concentrations (3 X 10(-8) M) that could be inhibited by D-galactose (greater than or equal to 10(-4) M). In comparison, the A chain reduces the surface charge at concentrations over 3 X 10(-7) M, but D-galactose has no effect on this. By means of the agarose droplet test, the spontaneous migration of M phi is inhibited in the sequence ML I much greater than B chain greater than A chain. The phagocytic activity of human leukocytes tested with a radiometric phagocytosis technique reveals an increasing effect only for the B chain; ML I and the A chain have no effect. Perhaps the mechanism of the described activities of ML I and its B chain are comparable with the action of lymphokines activating M phi.

Purification and Characterization of the Major Allergen from Apple and Its Allergenic Cross-Reactivity with Bet v 1

The major allergen from apple extract was concentrated by anion exchange chromatography and further purified by reverse-phase HPLC. A distinct peak with a high degree of homogeneity was obtained. The isolated protein has a MW of 18 kD and specific IgE-binding capacity (immunoblotting, IgE-binding inhibition). N-terminal amino acid analyses of the allergen allowed 37 cleavages and showed 67.6% identity to Bet v 1, the major allergen of birch pollen. Enzyme immunoassay inhibition studies with serum of birch/apple-allergic patients showed that besides cross-reacting structures to Bet v 1, apple-specific IgE antibodies could exist. Monoclonal antibodies (mAbs) were raised against the 18-kD allergen from apple and characterized by means of immunoblotting and ELISA. Only three of the eight studied mAbs reacted with Bet v 1.

Detection of crossreactive determinants in grass pollen extracts using monoclonal antibodies against group IV and group V allergens.

Monoclonal antibodies (MoAb) to defined allergens of P. pratense were raised. Five of them were selected for detailed studies by means of immunoblotting after SDS–PAGE and IEF of extracts from P. pratense and L. perenne. Three antibodies (1D11, 3B2, 2D1) recognize structures with mol. wt of 29 and 34 kD and pI of 4.4‐7.7, corresponding to group V allergens. Two other MoAbs (2D8, 3C4) are directed against strong basic structures with a mol. wt of 50 kD and pI of 7.7‐9.9 according to group IV allergens. The specificity of antibodies was supported by direct ELISA with purified group V and IV allergens. The isolated allergens were characterized before by SDS‐PAGE and CIE and that allergenicity was detected with sera of patients with allergic rhinitis. Using our selected MoAbs crossreactive epitopes on group V and IV allergens have been excluded. Our antibodies have been used to detect crossreactivity in 14 grass pollen extracts. The evaluation of the pollen extracts has been performed by enzyme immunoassay (EIA) inhibition. One MoAb (3C4) is able to recognize group IV allergens in all grass species analysed whereas the MoAb 2D8 seems to identify group IV structures in selected grasses only. Binding to conserved structures of group V has been proved for MoAb 1D11. Other group V specific MoAbs (2D1, 3B2) identify similar, however incomplete, spectra. These results have been confirmed also by the dot immunobinding assay.

Analysis of Human T Cell Clones Reactive with Group V Grass Pollen Allergens

Twenty-seven T cell clones (TCC) reactive with group V allergens of Phleum pratense (Phlp V) were established from the peripheral blood of 3 patients allergic to grass pollen. Twenty-four TCC showed the helper cell phenotype CD3+, CD4+, CD8-; the remaining clones were CD3+CD4-CD8+. T cell recognition of Phlp V was exclusively HLA-DR restricted. Many of the Phlp V reactive TCC (19 of 27; 70%) were stimulated additionally by other group V allergens isolated from Lolium perenne, Poa pratensis, and Dactylis glomerata. These data indicate the existence of cross-reacting T cell epitopes among group V allergens of different grasses. The Phlp V triggered cytokine production demonstrated in 13 out of 24 CD4+ TCC a Th2-like pattern (high interleukin 4/gamma-interferon ratios) suggesting group V allergens as important targets of grass pollen specific IgE.

A two‐site monoclonal antibody ELISA for the quantification of group V allergens in grass extracts

A two‐site solid‐phase enzymimmunoassay was used for the quantification of group V allergen in grass pollen extracts in mass units. The assay is based on the monoclonal antibodies (MoAbs), 1D11 and 3B2 which recognize different epitopes on the standard Phl p V. The MoAb‐ELISA is very sensitive (15 100ng ml Phl p V)and highly specific for group V allergens. Six pollen extracts of different grasses demonstrated parallel binding curves. The group V content ranged between 73 and 673 μg/ml in the extracts. The International Standard of Phleum pratense (IS 82 520) contains 400 μg/nil Phl p V. A good correlation was observed between the group V content and RAST inhibition, with the exception of Poa pratensis.

Monoclonal antibody immunoassay for quantitative analysis of group V allergens in grass pollen extracts

A two‐site monoclonal antibody (MoAb) ELISA has been developed for the quantification of the Phleun pratense major allergen, Phl p V. The assay is based on two MoAbs which recognize different non‐overlapping epitopes on the Phi p V molecule; one antibody (1D11) was immobilized on the solid phase and the other (3B2) was biotinylated. An affinity‐purified Phi p V preparation (purity of 95%) was used as standard. The assay has a sensitivity of 10 ng/ml of allergen and is suitable for the detection of group V allergen in aqueous grass extracts. The specificity of the assay was investigated with 14 grass pollen and five non‐grass pollen extracts. Different levels of group V allergen were delected in extracts of grasses, but not in non‐grasses. The assay gives a good correspondence with allergenic activity of extracts as determined by ELISA inhibition using serum pool of allergic patients. The results indicate that the two‐site MoAb ELISA could be very useful in the standardization of allergenic extracts from grass pollen.

Application of reversed-phase high-performance liquid chromatography in the purification of major allergens from grass pollen

Group 1 and 5 allergens of different grasses possess similar physicochemical parameters (molecular weight, pI) and therefore separation with conventional chromatographic methods (gel filtration, ion exchange chromatography) is difficult or impossible to achieve. In this paper we describe the isolation of biologically active group 1 and 5 allergens from extracts of Lolium perenne and Phleum pratense by means of reverse phase chromatography on HPLC. The chromatograms showed very different retention times for group 1 (Rt 19.1-20.5 min) and group 5 (Rt 24.3-26.3 min) containing fractions. In addition, this technique is suitable for the separation of group 5 allergens into 5a and 5b subgroups and for the estimation of the amounts of allergen (groups 1 and 5) in the different extracts.

Characterization of Murine Monoclonal Antibodies to Phospholipase A2 and Mellitin from Bee Venom

9 murine monoclonal antibodies (MoAbs), 7 to phospholipase A2 (Pla2; Api m I) and two to mellitin (Api m III) have been produced. Specificities were demonstrated using immunoblotting and enzyme immunoassays (ELISA). Antibodies specific for Pla2 were characterized in detail. Epitope mapping of Pla2-specific MoAbs identified three independent binding regions. 5 selected MoAbs bound to one of these sites compete with specific human IgE. Two of MoAbs characterized are suitable tools to quantify Pla2 in a two-site binding assay.

Interaction of Lotus-tetragonolobus lectin (LTL) and an MIF-like factor with guinea-pig macrophages I. Effects on macrophage migration inhibition and receptor-binding studies

Purified Lotus-tetragonolobus lectin (LTL) was studied for its interaction with guinea-pig peritoneal macrophages in the migration-inhibition and spreading-inhibition assay. LTL was found capable of inhibiting macrophage migration in a dose-response relationship similar to that of an MIF-life factor present in rat Zajdela-hepatoma ascites, whereas macrophage spreading was less affected. Mitogenic activity of the fucolectin could be excluded. Both the LTL and MIF-like factor seem to come into action by means of alpha-L-fucopyranosyl residues-containing macrophage surface receptors. Both substances act synergistically in inhibiting migration of macrophages. Since binding studies with 125I-LTL demonstrated competition with the MIF-like factor, it is suggested that the latter and LTL share a common surface receptor, and that optimal occupation densities are required for the realization of the inhibitory effect. Possibly, LTL acts in a monomeric form in the migration inhibition assay.

Isolierung von Lieschgrasallergenen (Phleum pratense) mit Hilfe monoklonaler Antikörper

Fur das Studium des Mechanismus der Auslosung und Unterhaltung einer Allergie sowie des Mechanismus der Desensibilisierung konnen gereinigte Allergene wert volle Hilfsmittel sein. Fur die Therapie IgE-vermittelter allergischer Erkrankungen ist die Stimulierung von spezifischen Ts-Zellen, die zur Suppression der antigenspezifischen IgE-Antikorper fuhren, denkbar und wunschenswert. Eine andere hypothetisch-therapeutische Moglichkeit besteht in der Kreuzreaktion der zellgebundenen IgE-Antikorper mit univalenten Haptenen an Stelle der naturlichen multivalenten Allergene. Um solche Haptene herstellen zu konnen, mussen die Allergendeterminanten genau charakterisiert und verfugbar sein [2].