Nuevos conceptos en la fabricación de extractos de veneno de himenópteros / New concepts in the manufacture of Hymenoptera venom extracts

A lo largo del siglo XX se produjeron importantes avances clínicos en el tratamiento de la alergia a himenópteros. En un principio se utilizaron extractos de cuerpo completo de abeja como diagnóstico y tratamiento de pacientes sensibles a este insecto, más tarde se describió que la utilización de extractos de veneno de himenópteros era mucho más eficaz que los cuerpos completos y, por último, la aplicación de técnicas de biología molecular ha permitido ensayar nuevos tipos de tratamientos con proteínas recombinantes, cuyo perfil de seguridad y eficacia es similar al de los extractos de veneno. Todos estos avances clínicos han hecho cambiar de una manera significativa los criterios de fabricación de los extractos alergénicos en general y en particular el de los extractos de himenópteros, no solo por el ingrediente activo utilizado en la producción de extractos (cuerpo completo, veneno o proteína pura) o por los procesos de estandarización y de control de calidad empleados, sino también por la aplicación a los sistemas de producción de requerimientos farmacéuticos como las Normas de Correcta Fabricación (GMP's), que garantizan el control de las instalaciones, equipos, documentación, validación de los procesos y, por tanto, la consistencia de los productos para uso clínico (AU)

Group 5 determination in Pooideae grass pollen extracts by monoclonal antibody-based ELISA. Correlation with biologic activity.

A solid-phase, monoclonal antibody-based ELISA was set up to quantitate group 5 allergens in pollen extracts of wild and cultivated Pooideae grasses. The method was able to evaluate group 5 concentration in mass units with a sensitivity in the ng/ml range and a practical working range of 1-100 ng/ml. The group 5 ELISA was compared with rocket immunoelectrophoresis for determination of allergen levels in several Phleum pratense extracts, and a very good quantitative correlation was found (r = 0.98; P < 0.0001). A highly significant correlation (r > 0.8) was also obtained in comparing allergenic potency determined by RAST inhibition to group 5 content in several wild and cultivated grass species. The results proved the usefulness of the method in the standardization of Pooideae pollen extracts employed in diagnosis and treatment.

Epitope mapping of beta-lactam antibiotics with the use of monoclonal antibodies.

In order to evaluate the antigenic contribution of different regions of the penicillin molecule, monoclonal antibodies were raised against amoxicillin-protein conjugates and their specificities analysed in detail. A random sample of the clones produced was analysed by a quantitative inhibition-ELISA, using, as inhibitors, monomeric conjugates of the following antibiotics to butylamine (BA), amoxicillin (AX), ampicillin (AMP), benzylpenicillin (BP) and the nuclear part of these, 6-aminopenicillanic acid (6-APA); and different parts of the following molecules: N-(p-hydroxyphenyl)-glycine (PHPG), N-phenylglycine (NPG), phenylacetic acid (PA) and thiazolidine (TIAZ). The results showed that 92% of the antibodies recognized an epitope in which the side chain was a major constituent, although with variable contributions from other regions of the molecule. There was a high degree of crossreactivity with aminopenicillins, but low or absent crossreactivity with BP. None of the antibodies recognized the thiazolidine ring or the conjugated nuclear region of the penicillins. Finally, one antibody seemed to recognize, equally, all the different structures tested. The possible relevance of these results to penicillin allergy is discussed.

The main allergen of Olea europaea (Ole e I) is also present in other species of the Oleaceae family.

Three major pollen allergens from Fraxinus excelsior, Ligustrum vulgare and Syringa vulgaris belonging to the Oleaceae family were purified. Monoclonal antibodies previously raised against the main allergen of Olea europaea (Ole e I) were used for their purification by affinity chromatography. The three new purified allergens were able to bind human IgE from serum of olive-allergic patients in a way analogous to Ole e I. Crossed radioimmunoelectrophoresis of the four allergens, using anti-olive extract rabbit serum, showed a unique immunoprecipitation arc with the same characteristics. The four purified proteins had similar molecular weights on SDS-PAGE and the N-terminal sequences for the first 20 amino acids were identical. Furthermore, the concentration of the allergens could be determined using a two-side solid phase assay previously developed for the allergen Ole e I. Our results indicate that the four purified proteins share, to a great extent, antigenic and allergenic epitopes leading to cross-reactivities which could cause common clinical manifestations. We propose for the newly purified allergens the nomenclature of Fra e I, Lig v I and Syr v I.

Monoclonal antibodies against human IgE. Identification of an epitope sharing properties with the high-affinity receptor binding site.

Three monoclonal antibodies to human IgE are described. One of them recognizes an epitope located within a region of 76 amino acids that has been shown to contain the Fc epsilon RI binding site. That epitope is shown to be susceptible to heating and to alkylation of cysteines involved in inter heavy chain bonds, but not to their reduction alone. In addition, this monoclonal antibody, although having a high affinity for free IgE, is unable to bind Fc epsilon RI-linked IgE. Based on these results, we discuss the possibility that the antibody recognizes the Fc epsilon RI binding site of the IgE molecule.

[Purification of fibronectin using gelatin derivatives]. / Purificación de fibronectina mediante derivados de gelatina.

This work compares two distinct methods, column and batch, for the purification of fibronectin, from different plasma fractions, using gelatin and one of its derivates (Hemoce, Behring). The yields of both techniques at quantitative as well as qualitative levels (levels of immunologically active fibronectin), are evaluated. The data indicate that better conservation of immunological immunological characteristics is obtained with the use of gelatin derivatives (Hemoce). The plasma fraction does not have significant influence on the process yields.