Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-1 00-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [ '2sI]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten a-methyl-D-mannopyranoside contained at least 15 prominent bands which bound ['*'I] -Con A.
A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability t o secrete antibody which could bind t o both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able t o precipitate a single ['251]-labeled CHO surface component of Q265,OOO daltons.
In order t o produce monoclonal antibodies against various cell surface Con