Towards an indirect screening technique facilitating detection of cellular populations bearing specific cell surface markers.
We describe and demonstrate a technique for detection of cell surface antigens, with potential use in tissue antigen research. Briefly, a small volume of wetted chromatographic beads (specifically, 100 μL of Nickel-nitriloacetic acid (NTA) agarose) was bound to small quantities (specifically, ∼0.1-0.2 μg) of a single-chain Fv antibody recognizing the Class I MHC heavy chain antigen. The beads were then used to capture and detect cells bearing this antigen, through SDS polyacrylamide gel electrophoresis of boiled bead-cell complexes. A key feature of this method is its use of "signal amplification." Although the antibody-mediated binding and immobilization of intact cells involves relatively small numbers of antibodies, and cells, what is being detected is simply the presence of cells. Each bound cell contains a number of abundant proteins that are present in millions of copies per cell, and the abundance of these proteins ensures that they are detectable on SDS-PAGE, signaling cell-binding. As few as 10(10) -10(12) scFv antibodies immobilized on 100 μL of Ni-NTA beads are thus enough for the trapping of enough cells to allow visualization of their abundant proteins. Conceptually, this method could be easily developed and applied to detection of cells bearing any other cell specific antigen.
Arora
Sandeep K
SK
Dept. of Immunopathology, Postgraduate Institute of Medical Education and Research , Chandigarh 160012, India.
Sharma
Rohit
R
Kaur
Gagandeep
G
Bhoria
Preeti
P
Sharma
Maryada
M
Luthra-Guptasarma
Manni
M
D000937:Antigen-Antibody Reactions; D000954:Antigens, Surface / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D015415:Biomarkers / Q000032:analysis / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007158:Immunologic Techniques; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000276:immunology
D000954:Antigens, Surface; D015415:Biomarkers; D057127:Single-Chain Antibodies