A method for the generation of monoclonal antibodies against rare cell‐surface molecules

Abstract

Monoclonal antibodies provide a powerful tool for the identification and analysis of novel cell‐surface molecules. We present here a method for antigen preparation and an immunization protocol that facilitates generation of mAb reactive with cell‐surface molecules of low abundance and/or low antigenicity. The procedure involves isolation and extensive fractionation of cell‐surface and detergent‐soluble extracellular‐matrix molecules prior to immunization. Cell‐surface proteins on intact tissue are biotin‐labeled using a reagent that does not penetrate cells. Avidin affinity chromatography is then used to purify these biotinylated molecules. Size‐exclusion HPLC is used to separate these surface molecules on the basis of apparent molecular mass. Finally, immunization with antigen coupled to keyhole‐limpet hemocyanin is combined with long‐term booster immunizations to generate a hyperimmune response resulting in high‐affinity IgG. A test application of this approach was aimed at the generation of mAb against cell‐surface molecules of approximately 135 kDa in the developing chicken retinotectal system. Immunochemical analyses using antibodies produced by this approach which showed restricted patterns of tissue staining reveal that mAb were generated against all previously identified immunoglobulin superfamily molecules of this size in this system. Furthermore, we produced many additional antibodies that labeled retinotectal tissue in novel staining patterns. In the two cases analyzed in detail, these new patterns reflect the distributions of previously uncharacterized members of the immunoglobulin superfamily. The success of this initial study suggests that this method may represent a broadly applicable approach towards the preparation of extensive libraries of antibodies against cell‐surface molecules expressed on cells from numerous sources.

Early expression of a novel radial glia antigen in the chick embryo

“…Monoclonal Antibody Production mAb 3CB2 was obtained while developing a project aimed at identifying and characterizing molecules involved in the interaction between growing axons and their environment (De la Rosa et al, 1990;Kayyem et al, 1992). Briefly, surface molecules in E8 chick optic tecta were selectively biotinylated, isolated by avidinaffinity chromatography, and further fractionated by HPLC.…”

Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer.

“…The monoclonal antibody 10-22A8 used in these experiments was generated using a procedure previously described (Kayyem et al, 1992b). In brief: to isolate cell surface molecules from developing neural tissues, intact tecta from 1,000 embryonic day 8 (ES) chicken embryos (obtained from Lake View Farm, Lake View, CA) were dissected.…”

“…Neogenin protein was purified from posthatching day 1 (PI) chicken brains following a procedure described earlier (Kayyem et al, 1992b). After ultracentrifugation (100,000 g, 5 h, 4°C) the supernatant from 200 brains was gravity loaded onto a 1 ml affinity column at a flow rate of approximately 50 rnl/h at 20°C.…”

Nr‐CAM expression in the developing mouse nervous system: Ventral midline structures, specific fiber tracts, and neuropilar regions

“…Nr-CAM binds to several molecules, including axonin-1/TAG-1 (Suter et al, 1995;Lustig et al, 1999;Fitzli et al, 2000), contactin/F11 (Morales et al, 1993;Volkmer et al, 1996;Faivre-Sarrailh et al, 1999), RPTP␤ (Milev et al, 1996;Sakurai et al, 1997), and Nr-CAM itself (Mauro et al, 1992). Nr-CAM was cloned originally from chick (Grumet et al, 1991;Kayyem et al, 1992) and, subsequently, was cloned from rat (Davis et al, 1996), human (Lane et al, 1996;Wang et al, 1998), and mouse (Moscoso and Sanes, 1995;current study). Nr-CAM is highly conserved among species, suggesting that its biological functions also are conserved.…”