A one-step method for determining the maximum number of bound antibodies, and the affinity and association rate constants for antibody binding.
OBJECTIVE
A reliable analysis of antibody binding may lead to more successful selection of the optimal antibodies. The most important parameters are affinity (equilibrium dissociation constant, Kd), the number of antigen sites on the cells (Bmax) and the on (ka) and off (kd) rate constants of binding. The affinity and the number of cellular binding sites are usually determined by equilibrium binding experiments and subsequent Scatchard analysis. The on and off rate constants are determined by kinetic binding experiments. However, it is necessary to perform two to three different types of experiment in order to determine these parameters.


METHODS
We have developed an alternative one-step method based on a kinetic binding experiment and a mathematical description of antibody binding to antigen. The method was compared with kinetic and equilibrium binding methods.


RESULTS
The results obtained using two different cell lines were in good agreement with results obtained with Scatchard analysis and kinetic binding experiments.


CONCLUSIONS
An alternative one-step method for determination of parameters describing binding of antibodies to antigens on cells has been developed. The method gives reliable estimates of affinity and number of antigens and in addition gives information on the kinetics of binding.
Dahle
Jostein
J
Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway. jostein.dahle@rr-research.no
Krogh
Cecilie
C
Melhus
Katrine B
KB
Kaalhus
Olav
O
Larsen
Roy H
RH
Stokke
Trond
T
D000465:Algorithms; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000494:pharmacology*; D058846:Antibodies, Monoclonal, Murine-Derived; D000951:Antigens, Neoplasm / Q000737:chemistry; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D006801:Humans; D007700:Kinetics; D015233:Models, Statistical; D008962:Models, Theoretical; D000069283:Rituximab; D013997:Time Factors
D000906:Antibodies; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D000069283:Rituximab