Covalent immunochemical membrane labeling of viable cells with K698-T708, a simian virus 40 tumor antigen-derived peptide.
The process of covalent immunochemical linking of viable cell membranes with a Simian Virus 40 (SV40) tumor antigen-derived undecapeptide, K(698)PPTPPPEPET(708) (KT), is described. The principle applied was the reaction of the lysine residue, K 698, of the undecapeptide with the succinimidyl moiety of a heterobifunctional linker molecule, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) or sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate (sulfo-SIAB). Thereby, upon release of N-hydroxy-succinimide, the rest of the linker molecule reacts covalently with the epsilon-NH2 group of lysine. Upon release of pyridyl-2-thion or hydrogen iodide, respectively, the second reactive moiety of the linker is then ready to form a covalent bond with SH-groups of cell membrane compounds. As a result, KT is covalently linked onto the cell membrane by an -SS- or an -S-bond, respectively. Binding is prevented by treatment of the candidate cells with iodoacetamide, an SH-reactive compound. This artificial cell membrane epitope can be demonstrated by surface immunofluorescence and by binding to immunomagnetic beads loaded with PAb1605, a KT-specific monoclonal antibody. Quantitation by cytofluorimetry shows some 10(4) KT molecules bound per cell, a number that is in the range of the number of SV40 tumor antigen molecules of genuine SV40-transformed mammalian cells.
Hess
R
R
Abteilung Virologie, Institut für Medizinische Mikrobiologie & Hygiene, Freiburg, Germany.
Rau
P
P
Schwab
M
M
Paetzold
S
S
Kuther
M
M
Obert
M
M
Agostini
H
H
Haessler
C
C
Braun
D G
DG
Brandner
G
G
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D003432:Cross-Linking Reagents; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008221:Lymphoid Tissue / Q000166:cytology; D051379:Mice; D009374:Neoplasms, Experimental / Q000628:therapy; D009842:Oligopeptides / Q000737:chemistry* / Q000276:immunology; D014612:Vaccines / Q000737:chemistry
D000952:Antigens, Polyomavirus Transforming; D003432:Cross-Linking Reagents; D000939:Epitopes; C089279:K698-T708 undecapeptide; D009842:Oligopeptides; D014612:Vaccines