Search

Purification of tumour-associated antigenic material from the ascites sarcoma cells was attempted by extraction with 3 M KCl and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The antigenic activity was assayed in the macrophage-electrophoretic-mobility (MEM) test. Extraction of whole tumour cells with 3 M KCl results in preparations with low antigenic activity which on SDS-PAGE show a very heterogenous composition of more than 20 protein bands. On comparison of the antigenic activity of different subcellular fractions obtained by differential centrifugation of the homogenate, the antigenic activity could be alloted to the cell membrane fraction. With this cell membrane fraction, three extraction media -- 3 M KCl, 2% Triton X-100, and 5% sodium cholate -- were tested for their ability to selectively extract the antigenic membrane proteins. The Triton X-100 treatment solubilized the greatest amount of membrane protein. Both Triton X-100 and sodium cholate, however, produced very heterogeneous protein extracts. In contrast, 3 M KCl selectively extracted three membrane components: a glycoprotein of high molecular weight and two low-molecular-weight carbohydrate-free proteins. Removal of the KCl in the presence of Triton X-100 precipitates the carbohydrate-free proteins, while the glycoprotein remains in solution. Testing the components of the KCl-extracts in the MEM-test after isolation by preparative SDS-PAGE revealed antigenic activity only with the glycoprotein component.

Tumor-bearing mice show a cellular sensitization to fetal antigens as detected by the macrophage electrophoretic mobility (MEM) test. Sensitization is independent of etiological and histological tumor factors. An identical state of sensitization can be found after application of chemical carcinogens (N-methyl-N-nitrosourea, benz(a)pyrene or 7,12-dimethylbenz(a)anthracene) before the tumors have developed. The results indicate that the effect is dose-dependent.

After passing normal mouse spleen cells through columns of a weakly basic ion-exchanger, the percentage of cells with B cell-typical electrophoretic mobility (EPM) is 90% after fractionation at pH 8.0 and 10--20% at pH 5.0. However, the characterization of fractionated cells with regard to theta-antigen as well as graft-versus-host reaction shows that separation of T- and B-lymphocytes had not occurred. After incubation of the separated cells for 2 h at 37 degrees C the EPM returned to that of unseparated cells. It appears that the net charge of T- and B-lymphocytes is changeable depending on pH, temperature and separation material.

Calf serum beads coated with antigen-antibody complexes were used as cellular immuno-adsorbents to separate mouse T-lymphocytes with a purity of more than 90%. The beads coated by means of glutaraldehyde could be used at least three times without loss of cell-binding capacity.