A radioimmunoassay-based method for measuring the true affinity of a monoclonal antibody with trace amounts of radioactive antigen: illustration with the products of a cell-free protein synthesis system.
This communication describes a novel highly sensitive method for measuring the affinity of a monoclonal antibody for its antigen. It is based on a radioimmunoassay in which the antigen is labeled with radioactivity. It is therefore particularly well adapted to the study of trace amounts of radiolabeled polypeptide chains produced either in vivo, or in vitro by a cell free protein synthesis system or by chemical radiolabeling. It offers several advantages over previously described methods. Though making use of insolubilized antibody, it does measure the true affinity constant of the monoclonal antibody in solution for the antigen. It can be used even when the antigen is present at concentrations far below the dissociation constant of the antibody/antigen complex. It does not require the antigen or the antibody to be purified. In most cases, it requires no sophisticated equipment. This method could be easily adapted to the determination of the equilibrium constant of any type of protein/ligand system.
Friguet
B
B
Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, Paris, France.
Fedorov
A
A
Serganov
A A
AA
Navon
A
A
Goldberg
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000941:Antigens / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002474:Cell-Free System; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D014176:Protein Biosynthesis; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D012270:Ribosomes / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014367:Tryptophan Synthase / Q000032:analysis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D046911:Macromolecular Substances; D010446:Peptide Fragments; D013462:Sulfur Radioisotopes; D014367:Tryptophan Synthase