Preparation and characterization of biotinylated red blood cells

Biotinylation of intact mammalian red blood cells (RBC) was performed either by attachment to the amino groups by means of biotin N‐hydrosuccinimide ester (NHS‐biotin) or by oxidation of the induced aldehyde groups of the RBC membrane by biotin hydrazide. Comparison of these different procedures showed that biotinylation by NHS‐biotin provided the highest cell recovery (> 90%), the binding of approximately 1000 biotin molecules/cell (on mouse RBC) and the 24 h survival in circulation was unaffected. In contrast, biotin hydrazide produced cell recovery in the 5‐30% range (depending on the extent of oxidation), an undetectable number of molecules of biotin/cell and negligible 24 h survival. Among the NHS derivatives of biotin, further studies were performed on those containing a spacer arm of 2.2 nm (22 A) [sulphosuccinimidyl‐6‐(biotinamido)‐hexanoate]. In vitro this derivative was similar to, or better than, the NHS‐biotin in terms of cell recovery and the number of molecules/cell. In vivo this derivative showed a 24 h circulation survival similar to that of NHS‐biotin. Unfortunately, biotin bound with such a spacer arm is lost after a few days of RBC circulation, probably due to plasma biotinidase. Possible applications of biotinylated RBCs include the in vivo measurement of RBC volume, the RBC survival and the delivery of enzyme and antigens.