Estimation of the relative affinity of B cell receptor by flow cytometry

Abstract

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.