Characterization of monoclonal antibodies against human red blood group cells antigens by laser backscattering

Abstract

A problem in immunohematology is to define the antibody quality which is related to its affinity expressed by the equilibrium constant. The activity of an antibody can be measured by the strength of its interaction, related to the adhesive energy exchanged during RBC agglutination which depends on the antigen–antibody liaison strength. To estimate this adhesive energy, two methods are used in this paper. Firstly, the dissociation behaviour of suspended RBC agglutinates was analysed by laser backscattering intensity (r) in a Couette flow. Backscattered intensity issued from shear‐induced mechanical dissociation is recorded and submitted to a numerical process to obtain the energy parameter (E_D). Secondly, a modification of this technique is proposed for measuring specific binding energy. Samples were exposed to increasing shear stress, and backscattered intensity was recorded. A constant increase of this intensity with raising shear stress was observed, pointed to a progressive dissociation of RBC agglutinates into smaller ones. Considering that complete dissociation of agglutinates is only approached asymptotically it is assumed that the final break‐up of doublets (two‐cell agglutinates) is produced at a critical shear stress (τ_C) reflecting the work done to breaking‐up the molecular bridges between both adjacent cells. This shear stress is defined by the extrapolation of the linear part of the curves [r–log τ] to the backscattered signal (r₀) corresponding to the complete dispersion of RBCs. These approaches permit to define the specific surface adhesive energy (Γ) by using the Derjaguin relation and to assess the functional characterization of specific immunoglobulins. In conclusion, two parameters characterizing monoclonal antibody agglutination properties, E_D and Γ, were estimated by laser backscattering methods, which could be very useful for antibodies quality control.