Abstract
In an attempt to identify B cell specific antigens, we have generated a mouse monoclonal antibody, R2D6, which is directed against plasma membranes of rat pancreatic B cells but against no other pancreatic cells. R2D6 crossreacted with mouse and guinea pig B cells, but not with human or dog. The B cell specificity of R2D6 was utilized in fluorescence-activated cell sorting to prepare highly enriched separate populations of viable pancreatic islet B cells and A cells. R2D6 also recognized adrenal chromaffin cells, secretory cells in the anterior pituitary, and the myenteric plexus of the gastrointestinal tract. Trypsin, chymotrypsin, papain, ficin, and pronase had no effect on R2D6-binding to dissociated rat islet cells. However, neuraminidase treatment of intact cells reduced R2D6-binding by 75%. The antigen recognized by R2D6, Ag(R2D6), could be quantitatively extracted from rat islets by dichloromethane/methanol (2:1) and, after drying, was soluble in methanol alone as well as in phosphate-buffered saline. When the dichloromethane/methanol extract (DME) was bound to polyvinylchloride microtiter plates, antigenic activity was retained and remained insensitive to pronase. In this solvent-extracted form, antigenic activity was totally destroyed by neuraminidase. Therefore, sialic acid is either an integral part of, or is related sterically to the binding site (epitope) for R2D6. In high performance thin-layer chromatographs of the DME, developed in 60:40:9 chloroform/methanol/2.5 N ammonia, Ag(R2D6) migrated with a relative mobility (Rf) of 0.54 +/- 0.07 (n = 3), which was a position nearly coincident with the purified brain ganglioside, GD1a. The antigen bound to DEAE-Sephacel, was not inactivated by mild treatment with base (which hydrolyzes phospholipids) and eluted in ganglioside fractions upon C18 Sep-Pak and upon silicic acid chromatography. Hence, the solubility characteristics, enzyme sensitivities, and behavior of Ag(R2D6) in four chromatography systems are consistent with its identification as a ganglioside.