Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.
Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.
Fägerstam
L G
LG
Pharmacia Biosensor AB, Uppsala, Sweden.
Frostell
A
A
Karlsson
R
R
Kullman
M
M
Larsson
A
A
Malmqvist
M
M
Butt
H
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D015374:Biosensing Techniques; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D015683:Gene Products, gag / Q000276:immunology*; D015488:HIV Antigens / Q000276:immunology*; D016655:HIV Core Protein p24; D008969:Molecular Sequence Data; D013499:Surface Properties; D014758:Viral Core Proteins / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015683:Gene Products, gag; D015488:HIV Antigens; D016655:HIV Core Protein p24; D014758:Viral Core Proteins