Walter Weichel

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS.

BACKGROUND The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibodys gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). OBJECTIVES pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. STUDY DESIGN AND RESULTS Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. CONCLUSIONS Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.

Sorting of Rare Cells

Positive selection on the basis of a particular phenotype is the most efficient way for the isolation of cells occurring at frequencies of below 1% in a given population. As one of the most powerful methods for positive selection, fluorescence-activated cell sorting (FACS) has been used for the enrichment and isolation of rare cells ever since its introduction (Assenmacher et al., 1990). Examples include immunoglobulin switch variants (Radbruch et al., 1980, Dangl et al., 1982), MHC mutants (Holtkamp et al., 1981, Weichel et al., 1985), and transfectants (Kavathas et al., 1984, Hombach et al., 1988). Such cells occur at frequencies of less than 10−2down to 10−8 in some cases. In general, rare cells were isolated in several rounds of cell sorting and expanding the enriched cells between the sorts in vitro; thereby gradually increasing the frequency of variants until isolation of rare cells and cloning became possible. Under optimal conditions, enrichment rates of up to 103 per sort could be achieved, but the number of sorted cells was usually too low to allow immediate resorting for purification of the rare cells. Naturally, such experiments can only be performed with transformed cells, adapted to continuous growth in culture. Although this approach still is the method of choice in many experimental situations, additional parameters are available in flow cytometry today and new methods for fast and efficient enrichment of cells prior to FAGS sorting have been introduced, substantially facilitating the isolation of rare cells. Here we describe a combined protocol for preselection of rare cells by magnetic cell sorting followed by fluorescence activated enrichment or even isolation of the same cells in one experiment. However, the FACS sorting part of this procedure also applies for enrichment problems where MACS enrichment is not possible, for instance the isolation of rare cells expressing green fluorescent protein as a marker.