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    Untersuchungen zur Charakterisierung der allergischen Komponenten des Katzenflohs Ctenocephalides felis felis (Bouche 1837) (2001)

    Art
    Hochschulschrift
    Autor
    Nadler, Andreas
    Quelle
    Berlin, 2001 — 116 Seiten
    Kontakt
    Institut für Parasitologie und Tropenveterinärmedizin

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35, 22, 23
    14163 Berlin
    Tel.+49 30 838 62310 Fax.+49 30 838 62323
    email:parasitologie@vetmed.fu-berlin.de

    Abstract / Zusammenfassung

    The allergic components of the cat flea Ctenocephalides felis felis were examine d by means of one- and two-dimensional electrophoresis and immunoblot.Initially, the whole-body extract of a flea was produced, followed by the preparation of t he salivary glands of female fleas, which have not sucked, from which a homogena te was produced, which was tested by using the above methods.The primary antibod y used was the serum of a cat suffering from fleabite dermatitis.The secondary a ntibody used was the commercially available lyophilisate ,,Rabbit-AntiCat" by NO RDIC".The one-dimensional electrophoresis rendered inaccurate results with respe ct to both the whole-body extract and the salivary gland extract. The proven pro tein bands had molecular weights between 13 and 190 kD.Due to the inaccuracy and the poor reproducibility of the results, the tests were extended using a two-di mensional electrophoresis. More concrete results could be expected from a two-di mensional electrophoresis.A round gel made of polyacrylamide was used for the fi rst dimension of the 2Delectrophoresis. The antigen sample ( whole-body extract and salivary gland extract ) was applied to the gel. Isoelectric focussing takes place in an electric field. For the second dimension, the same round gel is app lied to the top edge of a flat small gel of 55 cm2 . Here the electric field is positioned horizontally to the first dimension.The result of the 2D-electrophore sis is a spot pattern. The molecular mass and the isoelectric spots are determin ed using standard markers by BIO-RAD°.Using these two parameters, the different proteins of the whole-body extract and the salivary gland extract can be charact erised precisely. The protein spots become visible when using silver dye. Due to the samples different protein concentrations, 333 protein spots were detected i n the whole-body extract and 351 protein spots were detected in the more concent rated salivary gland extract. During a subsequent immunoblotting, the flea prote ins and later the flea"s salivary gland proteins were coated with the primary an tibody ( cat serum ), followed by the secondary antibody ( RAC conjugate ) and d yed with Chloro1 Naphtol.The result is yet another two-dimensional spot pattern of the proteins after reacting with both, the primary and the secondary antibodi es.The patterns are analysed by a computer. The area containing the individual s pots is scanned. Each detected spot is assigned values such as x- and y-coordina tes, quality, intensity, size, moleculare weight and isoelectric spot using a sp ecial computer program called TOPSPOT°.With respect to the IgG-conjugate used du ring the reactions, 57 protein spots were located in the whole-body extract and 4 of these proteins had a molecular weight of over 100 kD.However, after the imm unoreaction with the IgG-conjugate, 38 perfect spots were found in the salivary gland extract having molecular weights between 21.5 and 95.0 kD.The relatively l arge proteins of the flea"s salivary gland extract shown in the immunoblotting a re - as antigens - presumably responsible for the allergic reactions in cats.