Microimmunofluorescence Using Terasaki Plates and Direct Plate Freezing Method—Rapid and Reliable Screening System of Hybridomas

Abstract

Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at -80 C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly-L-lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.There are several methods for screening hybridomas; e.g., immunofluorescence staining (3), radioimmunoassay (RIA) (1), cytotoxic assay, enzyme-linked immunosorben t assay (ELISA) (2, 5) and red cell adherence assay (11). The advantage of using immunofluorescence for hybridoma screening is that it reveals stable results and elucidates the specificities of antibodies against subcellular components, whereas a common binding assay such as RIA, can merely provide information about reactivity of the antibodies against whole cells. However, the conventional immunofluorescence method has the disadvantages that it requires many slides of target cells or tissues and quite a large amount of hybridoma culture supernatant. Several methods have been reported to improve these difficulties (3,7,8).We report here our improved methods for screening hybridomas which consist of attachment of target cells on Terasaki plates and immunofluorescence performed on them. Our microassay method is clearly superior to the conventional methods and is useful for hybridoma screening.