Direct calibration ELISA: a rapid method for the simplified determination of association constants of unlabeled biological molecules.
We present a novel method for the rapid determination of association constants. The method is based on the direct calibration of an enzyme-linked immunosorbent assay (dcELISA) and does not require any external calibration. It combines kinetic and equilibrium binding experiments and can be performed on a single microtiter plate. The absorbance data are evaluated by several linearized plots without the need for sophisticated computations. The dcELISA has been used to analyze the binding of a monoclonal antibody, OKT9, to its cognate antigen, the human transferrin receptor, and yielded an association constant of Ka = 2.2 x 10(9) l/mol and a complex formation rate constant of kc = 2.7 x 10(-4) s-1. A 26% larger association constant was obtained with a radioimmunoassay (RIA)-based Scatchard analysis using 125I-labeled OKT9. By quantifying the binding of the same iodinated antibody with the dcELISA we were able to verify that the iodination modifies the binding properties of the antibody. The dcELISA thus appears to be superior to all methods requiring covalent modifications. In principle, the direct calibration method can also be combined with all other solid phase assays. It should thus expand their scope in quantifying the binding properties of biologically important molecules.
Fuchs
H
H
Institut für Klinische Chemie und Biochemie, Virchow-Klinikum, Humboldt-Universität zu Berlin, Germany.
Orberger
G
G
Tauber
R
R
Gessner
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011863:Radioimmunoassay; D011990:Receptors, Transferrin / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011990:Receptors, Transferrin