Analysis of binding of monoclonal antibody to a malarial peptide by surface plasmon resonance biosensor and integrated rate equations.
Using biosensor technology and integrated rate equations, we have developed procedures to determine the kinetic parameters and equilibrium affinity constant of Ag-Ab interactions. The Ag used in these studies was a peptide that represents the major B cell epitope of the circumsporozoite protein of Plasmodium falciparum, a promising malaria vaccine candidate Ag. Measurements of association and dissociation rate constants of this peptide with the mAb 2A10 were determined by fitting integrated rate equations to binding data obtained with a BIAcore surface plasmon-resonance biosensor. We examined whether accurate estimates of initial velocity and final equilibrium levels of binding of Ab to peptides can be obtained using these methods, and whether kinetic rates and equilibrium constants obtained with systematic variation of the experimental parameters conform to a simple bimolecular model of binding. We found that initial velocity was approximately first order with respect to Ab concentration. When we used a series of four sensor cells with different peptides loads, however, we found that the initial velocity of binding appeared to be nearly independent of peptide concentration. Equilibrium analyses yielded dissociation constants of approximately 3 x 10(-7) M. Integrated rate treatment of biosensor data supports a critical examination of the assumptions on which the binding models are based and suggests a need to refine such models. Nevertheless, it provides a powerful quantitative tool for assessing the Ag-Ab binding reaction.
Wohlhueter
R M
RM
Biotechnology Core Facility Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333.
Parekh
K
K
Udhayakumar
V
V
Fang
S
S
Lal
A A
AA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000937:Antigen-Antibody Reactions; D000953:Antigens, Protozoan / Q000737:chemistry / Q000378:metabolism*; D007158:Immunologic Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D010963:Plasmodium falciparum / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D010455:Peptides