Avidin acylation prevents the complement-dependent lysis of avidin-carrying erythrocytes

Abstract

Non-covalent binding of avidin to biotinylated erythrocytes results in complement-dependent haemolysis. Biotinylated erythrocytes, as well as native cells, are not lysed by complement. Complement activation requires a tight contact between avidin and the erythrocyte membrane, since avidin does not in itself activate complement and does not inhibit lysis of sensitized sheep erythrocytes. The efficiency of haemolysis depends on avidin's surface density. When the avidin concentration in the reaction mixture is less than 15 micrograms/ml, erythrocyte lysis is not induced. However, the attachment of biotinylated antibodies to avidin-carrying erythrocytes decreases dramatically. Acylation of avidin with succinic anhydride strongly decreases its ability to induce complement-dependent haemolysis. However, the ability of avidin to cross-link the biotin-containing structures decreases after acylation. A 50% modification of avidin by succinic anhydride (pI about 7.0) allows preparation of 'immunoerythrocytes', which retain their affinity to antigen and stability in the presence of complement.

Regulation of the Complement-Mediated Elimination of Red Blood Cells Modified with Biotin and Streptavidin

“…After injection of 125 Istream for a prolonged time. However, attachment of SA induces cross-linking of biotinylated DAF and labeled b-IgG/SA/b 23 -RBC in rats, we have observed a high iodine-125/ 51 chromium-51 ratio in the lungs, an CD59, thus inactivating both regulators of complement. This leads to both opsonization of SA/b-RBC by organ containing about 30% of the total endothelial cells in the body.…”

“…Rat alveolar macrophages were obtained by bron-this leads to a significant decrease in the observed lysis (16,23). We have also shown that the ability of SA to choalveolar lavage as described (21).…”

“…More vivo fate of the complex b-IgG/SA/b 23 -RBC, where bIgG and b 23 -RBC were labeled with 125 I and 51 Cr, rerecently biotinylated RBC (b-RBC) have also been utilized in a series of in vivo experiments which traced spectively. the RBC in the bloodstream in animals (6,7) and in humans (8).…”

“…We have bio-rate experiment, radiolabeled SA was added to b-RBC to assess streptavidin-accessible biotin residues on b ntinylated rat RBC at various levels of biotinylation (generating rat b n -RBC, where n represents the micro-RBC, as described (13). According to this assay, streptavidin binding to b 0 -RBC, b 2.4 -RBC, b 7 -RBC, b 23 -RBC, molar input of biotinylating reagent) and studied the properties of these RBC as well as SA/b n -RBC, in the b 70 -RBC, b 240 -RBC, and b 700 -RBC was equal to 290, 14,000, 35,000, 90,000, 260,000, 290,000, and 510,000 circulation of rats. Unexpectedly, we observed that RBC biotinylated at relatively high levels (b 700 -RBC, molecules per RBC, respectively.…”

“…To evaluate whether immunoerythrocytes circulate in the bloodstream as a complex possessing attached b-IgG, we have injected in rats b-IgG/SA/b 23 -RBC containing 51 Cr-labeled b 23 -RBC and 125 I-labeled b-IgG. Figure 10 shows that only 25-30% of iodine-125 could be found in the plasma several hours after injection of such a complex, whereas 100% of iodine-125 circulates labeled b-IgG (mean { SD, n Å 3, P õ 0.05).…”