Determination of concentration and binding affinity of antibody fragments by use of surface plasmon resonance.
An assay method using a surface plasmon resonance (SPR) biosensor has been developed that allows quantitative measurement of the specific antibody concentration in crude materials. By injecting non-labeled antibody samples onto a biosensor surface on which antigen was immobilized at high densities, the concentration of active antibodies can be accurately measured. To clarify applicability of this method to pharmacokinetic studies, the concentration of active antibodies in mouse plasma was measured for 4 h after injection of antibodies in mice. Although this period of measurement might be insufficient for determining the pharmacokinetics of blood pool clearance, this method has some advantages over conventional methods in measurement of single-chain antibody fragment (scFv) concentrations. Using the SPR biosensor, scFv and antibodies without epitope tag peptides were easily detected in real time, requiring as little as 20 mul of blood sample. Moreover, from the apparent dissociation rate in the dissociation phase of the sensorgrams, we could identify whether the antibody fragments existed as bivalent or monovalent in animal blood. We also evaluated the antigen binding activity of the scFvs against human CD47 and found scFvs had slightly weak affinity to their antigen (K(D), about 10 nM) compared with F(ab')2 and Fab' fragments (K(D), about 3-4 nM). This assay method promises to be a convenient tool for quality control, screening, and simple pharmacokinetic analysis of antibody fragments and other recombinant proteins not having epitope tags.
Kikuchi
Yasufumi
Y
Chugai Pharmaceutical Co. Ltd., Fuji-Gotemba Research Laboratories, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. kikuchiysf@chugai-pharm.co.jp
Uno
Shinsuke
S
Nanami
Masahiko
M
Yoshimura
Yasushi
Y
Iida
Shin-ichiro
S
Fukushima
Naoshi
N
Tsuchiya
Masayuki
M
D000818:Animals; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D006801:Humans; D007128:Immunoglobulin Fragments / Q000097:blood* / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000097:blood* / Q000276:immunology*; D051379:Mice; D016513:Mice, SCID; D020349:Surface Plasmon Resonance / Q000379:methods*
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region