The effect of temperature on the binding kinetics and equilibrium constants of monoclonal antibodies to cell surface antigens.
The effect of temperature on the kinetic association and dissociation binding parameters, and equilibrium constants of four monoclonal antibodies to the murine Ly-2.1 and Ly-3.1 antigens has been studied using flow cytometry. All four monoclonal antibodies were conjugated to FITC and their association to, and dissociation from, the surface of murine thymoma cells was observed at 15 sec intervals, at temperatures between 1 and 37 degrees C. The initial association rate constant and the dissociation rate constant for each antibody at each temperature were calculated from graphs of the first-order reactions and it was demonstrated that an increase in temperature caused an increase in both association rate and dissociation rate of the antibodies. Generally the increase in association rate with temperature was less than the increase in dissociation rate. Differences between antibodies to the same antigen (Ly-2.1) suggest that changes in membrane fluidity were not solely responsible for the changes in association rate. However, the equilibrium constants (Keq) did not always show a simple relationship of increasing temperature causing decreasing values for Keq. For one antibody the highest value for Keq was seen at 17 degrees C rather than at 37 degrees C and differences in Keq between individual antibodies were greater at 1 degree C than at 37 degrees C. Kinetic rate constants are usually measured at 4 degrees C or room temperature, therefore for antibodies under consideration for in vivo use, measurements at 37 degrees C are more appropriate.
Johnstone
R W
RW
Department of Pathology, University of Melbourne, Parkville, Victoria, Australia.
Andrew
S M
SM
Hogarth
M P
MP
Pietersz
G A
GA
McKenzie
I F
IF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000915:Antibody Affinity / Q000502:physiology; D000937:Antigen-Antibody Reactions / Q000502:physiology*; D000950:Antigens, Ly / Q000378:metabolism*; D002460:Cell Line; D007700:Kinetics; D051379:Mice; D013696:Temperature
D000911:Antibodies, Monoclonal; D000950:Antigens, Ly