Immuno-isolation of vesicles using antigenic sites either located on the cytoplasmic or the exoplasmic domain of an implanted viral protein. A quantitative analysis.
In this study, we present a new general approach for immuno-isolation: a foreign integral membrane protein, the G-protein of vesicular stomatitis virus (VSV), is implanted into the plasma membrane for subsequent immuno-isolation. A quantitative analysis was accomplished using the erythrocyte plasma membrane as a model system. The virus was artificially bound to the membrane via a lectin and subsequently fused at low pH. Vesicles of two opposite orientations were prepared from erythrocytes with fused G-protein. Right-side-out and inside-out vesicles expose the exoplasmic and the cytoplasmic domains of the G-protein on their surfaces respectively. In immuno-isolation experiments antibodies against each of the domains of the G-protein were used. Vesicles were presented to an immunoadsorbent (ImAd) consisting of a solid support with appropriate antibodies bound to its surface. Two commonly used immunoadsorbents prepared from either polyacrylamide beads or fixed Staphylococcus aureus cells were compared and found to have identical immuno-isolation efficiencies. It was possible to control and quantitate the amount of implanted antigen. Therefore, we were able to show that the critical antigen density required for immuno-isolation is 50 G molecules/micron2 plasma membrane surface area for both types of vesicle/antibody couples. This analysis showed that vesicles presenting either the cytoplasmic or the exoplasmic domain of the G-protein are immuno-isolated with the same efficiency.
D000818:Animals; D000954:Antigens, Surface / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D002458:Cell Fractionation / Q000379:methods*; D006224:Cricetinae; D004910:Erythrocyte Membrane / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology; D006801:Humans; D007163:Immunosorbent Techniques; D008561:Membrane Fusion; D008562:Membrane Glycoproteins; D013997:Time Factors; D014721:Vesicular stomatitis Indiana virus / Q000276:immunology / Q000378:metabolism*; D014759:Viral Envelope Proteins; D014764:Viral Proteins / Q000276:immunology* / Q000378:metabolism
D000954:Antigens, Surface; D000956:Antigens, Viral; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014764:Viral Proteins