Monoclonal antibody TOP 71
Springer Online Journal Archives 1860-2000
Abstract Highly purified tonoplast and plasma-membrane vesicles isolated from roots of Lepidium sativum L. (garden cress) were used as a starting material for generating a monoclonal antibody against plant tonoplast. Tonoplast vesicles were isolated by discontinuous-sucrose-gradient centrifugation followed by free-flow electrophoresis. The deglycosylated tonoplast fraction was used to generate monoclonal antibodies by immunization of Balb/c-mice and by fusion of their β-lymphocytes with the mouse cell line X 63 Ag 8.653. Using plasma membrane purified by two-phase partitioning and freeflow electrophoresis to define the negative signal in screening, and purified tonoplast to define the positive signal in screening, a monoclonal antibody (TOP 71) was obtained which recognized a tonoplast protein of 71 kDa by immunoblotting in cress-root membrane fractions. Two-dimensional gel electrophoresis, affinoblotting and binding to concanavalin A showed that the TOP 71-antigen was a glycosylated protein and had an isoelectric point (pI) of 4.5. The TOP 71-antigen was found in the different tissues of organs of several higher plants (Glycine max L., Curcurbita pepo L., Zea mays L.) where it did not cross-react with the purified plasma-membrane fractions of these plants. Additionally, TOP 71 recognized its antigen in microsomal fractions of two lower plants (Chara globularis Thuili., Matteucia struthiopteris Tod.).
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