David Scheinberg

Rapid screening of monoclonal antibodies: New ‘microstick’ radioimmunoassay

A new system for assaying monoclonal antibodies consisting of an 8 x 12 array of sticks which fits into a 96-well microtiter plate is described. Tests using virus specific monoclonal antibodies and virus proteins demonstrated sensitivity equivalent to the conventional microtiter plate assay. Antibody production, antigen specific antibody, and immunoglobulin isotypes could be measured under sterile conditions directly in the original fusion mixture wells and with much greater rapidity than with the microtiter plate assay.

A microradioiodination method for detection of nanogram amounts of proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

A procedure is described for the rapid analysis of nanogram quantities of protein resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins are first radioiodinated by the chloramine T method, acid precipitated after addition of a visible marker protein and collected on a nitrocellulose filter. The region of the filter containing protein is excised and the proteins are then extracted from the filter into electrophoresis sample buffer.

Transformation-related proteins associated with kirsten sarcoma virus

Abstract The protein composition of a defective woolly monkey leukemia virus pseudotype of Kirsten sarcoma virus (KiSV) has been characterized. These noninfectious particles contain KiSV RNA, reverse transcriptase, and the major core protein, p28, of the helper woolly monkey leukemia virus. Instead of the usual complement of helper virus proteins, these defective particles include several polypeptides previously not identified in RNA tumor viruses. Two proteins, gp110 and gp55, of 110,000 and 55,000 daltons, were glycoproteins. The third protein, p20, of 20,000 daltons, was a phosphoprotein, as was gp110. We show by immunoprecipitation that the two glycoproteins were common to a variety of different rodent viruses. In contrast, the 20,000-dalton protein appeared to be specific to the KiSV. All three proteins were associated with cell membranes. Analysis of the processing and release of these proteins from rat cells revealed that gp55 had a markedly short half-life of 20 min whereas gp110 and p20 had long half-lives similar to those of most cell membrane proteins. The presence of gp110 and p20 in particles was dependent upon transformation and independent of helper virus infection. Both gp110 and p20, but not gp55, showed a variety of alterations following transformation of cells by Kirsten sarcoma virus. The amount of gp110 in transformed cells increased both in cell plasma membranes and in the medium. In addition, gp110 became phosphorylated and accessible to lactoperoxidase radioiodination. The alterations of gp110 with respect to the cell membrane were independent of the transforming agent since we observed the same results in BALB/3T3 cells transformed by an RNA virus (Kirsten), a DNA virus (SV40), and a chemical carcinogen (methylcholanthrene). Thus, gp110 is a transformation-related cell surface marker. P20 showed similar changes upon transformation: The amount of p20 increased dramatically in membranes; in addition, p20 appeared as a double band, became phosphorylated, and was shed into the medium. However, p20 was not accessible to lactoperoxidase radiodination. The immunological specificity and many of the biochemical cheracteristics of this protein were similar to the properties ascribed to the Kirsten sarcoma virus src gene product.