V(H)H (nanobody) directed against human glycophorin A: a tool for autologous red cell agglutination assays.

The preparation of a VHH (nanobody) named IH4 that recognizes human glycophorin A (GPA) is described. IH4 was isolated by screening a library prepared from the lymphocytes of a dromedary immunized by human blood transfusion. Phage display and panning against GPA as the immobilized antigen allowed isolating this VHH. IH4, representing 67% of the retrieved VHH sequences, was expressed as a soluble correctly folded protein in SHuffle Escherichia coli cells, routinely yielding approximately 100mg/L fermentation medium. Because IH4 recognizes GPA independently of the blood group antigens, it recognizes red cells of all humans with the possible exception of those with some extremely rare genetic background. The targeted linear epitope comprises the GPA Y52PPE55 sequence. Based on surface plasmon resonance results, the dissociation constant of the IH4–GPA equilibrium is 33nM. IH4 is a stable protein with a transition melting temperature of 75.8°C (measured by differential scanning calorimetry). As proof of concept, we fused HIV p24 to IH4 and used the purified construct expressed in E. coli to show that IH4 was amenable to the preparation of autologous erythrocyte agglutination reagents: reconstituted blood prepared with serum from an HIV-positive patient was readily agglutinated by the addition of the bifunctional reagent.