Using an enzyme-linked immunosorbent assay (ELISA) and limited dilution methods to screen and clone antigen-specific hybridoma cells is extremely time-consuming and labor-intensive. This work features a simple and rapid cell surface fluorescence immunosorbent assay (CSFIA), designed for the detection and isolation of antigen-specific hybridoma clones. In this assay, antigens are first anchored to the hybridoma cell surface through a dual-functioning molecular Oleyl-PEG4000-NHS. Specific antibodies secreted from hybridoma cells are then captured by the antigens on the cell surface. Positive hybridoma cells are stained using a fluorescently labeled anti-mouse IgG-Fc antibody. After the addition of a methylcellulose semisolid medium, positive clones are easily picked using a pipet. These positive cell clones can be used to produce monoclonal antibodies after direct expansion. Using this method, positive hybridoma clones against both malachite green and porcine epidemic diarrhea virus are selected with high efficiency. Compared to the ELISA-based method, the CSFIA-based method achieved the capability of isolating >2-fold more hybridoma clones in <25% of the corresponding processing time. In brief, the CSFIA-based method is highly efficient and inexpensive with a simple and direct operation, which is an excellent candidate method for antigen-specific positive clone isolation in a monoclonal antibody preparation.

Monoclonal antibodies are used extensively in flow cytometry to identify subpopulations of cells differing in surface antigens. Conventional studies on living cells do not allow analysis of internal antigens, because antibody molecules do not pass through an intact plasma membrane. It is important for developmental studies on Dictgostelium discoideum that not only surface but also internal antigens be analysed. Here techniques are reported that make possible such studies by permeabilising cells with mild detergent treatments using digitonin. Over the past ten years there has been a revolution in culture cells that it is possible to permeabilise them so the understanding of the differentiation of lymphocyte that antibodies have access to internal antigens for flow subpopulations. The isolation of monoclonal antibodies cytometric measurements (14). that recognise antigenic determinants exposed at the In this report, studies that optimise techniques for surface of blood cell subpopulations and their applica-analysing internal antigens of D. discoideurn slug cells tion in flow cytometry has hastened this understanding are described. With mild detergent treatments, it is pos-(15). We have developed techniques for disaggregating sible to analyse internal antigens of unfixed cells. The the cells of the cellular slime mold Dictyostelium discoz-techniques outlined here are simple and rapid. We use deum slug, which is one of the simplest multicellular digitonin to permeabilise unfixed cells and to demoneukaryotes, and analysing them by flow cytometry (16). strate the effectiveness of this technique using two preOur studies centre on the differentiation of this organ-spore-specific internal antigens. ism, to produce two predominant cell types, the prespore and prestalk cells (16). In this way, cell surface antigens MATERIALS AND METHODS that identify prespore (9) or prestalk (10) cells have been Materials discovered.Digitonin (80% pure) was from Calbiochem. DeterIn recent work antigens that are cell type-specific but gents Triton X-100 and Nonidet P-40 were from Sigma. not found at the cell surface have been discovered (Alex-Monoclonal antibodies MUD1 (71, MUD3 (171, and ander et al., submitted). While internal antigens can be MUD102 (Smith, unpublished) raised against D. discoistudied after sorting the cells on the basis of cell surface markers, it would be more convenient to be able to detect internal antigens, directly in mixed-cell popula-'This research was supported by a NH & MRC (Australia) p a n t to tions, in the Same way that cell surface antigens are KLW. RLB was on sabbatical leave from San Francisco State studied by flow cytometry. Since antibodies cannot pen-u~~~~~r e p r i n t requests to Keith Williams, School of Biological etrate intact flow cytometry precludes such studies. However, it has been shown in tissue 2109.