Estimation of the relative affinity of B cell receptor by flow cytometry.
We have developed a simple method using flow cytometry to estimate the relative affinity of B cell receptor (BCR) possessing the hapten-binding activity. Bovine serum albumin (BSA) was conjugated with a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP) and biotin (NP-BSA-bio). The interaction between NP-BSA-bio and anti-NP monoclonal antibodies (mAb) was studied as a model of the BCR reaction by surface plasmon resonance (SPR) using a biosensor chip immobilized with mAbs through anti-Fc antibody (Ab). The relative affinity of these mAbs was estimated on the basis of resonance units for the binding of NP(0.5)-BSA-bio(21) relative to that of NP(7.4)-BSA-bio(21) expressed as a ratio (NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21)). In combination with streptavidin (SA)-R-phycoerythrin (PE), we measured the binding of NP-BSA-bio to BCR by flow cytometry and found that a high number of biotin molecules was necessary to improve the sensitivity of detection of the bound NP-BSA-bio without steric hindrance in the NP-BCR interaction. We demonstrated that the ratio of the mean fluorescence intensity (MFI) of NP(0.5)-BSA-bio(21)/NP(7.4)-BSA-bio(21) at a concentration of 10(-8) M could be used as a practical measure of the affinity. This method is expected to be useful for the study of affinity maturation on the cellular level.
Shimizu
Takeyuki
T
Research Institute for Biological Science (RIBS), Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.
Oda
Masayuki
M
Azuma
Takachika
T
D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity; D001710:Biotin / Q000032:analysis; D002462:Cell Membrane / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006241:Haptens / Q000737:chemistry / Q000276:immunology*; D006825:Hybridomas; D009596:Nitrophenols / Q000276:immunology*; D010648:Phenylacetates; D011947:Receptors, Antigen, B-Cell / Q000276:immunology* / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D006241:Haptens; D009596:Nitrophenols; D010648:Phenylacetates; D011947:Receptors, Antigen, B-Cell; C026242:4-hydroxy-5-nitrophenyl acetic acid; D001710:Biotin