Coupling of C3b to erythrocytes by disulfide bond formation: preparation of EC3b for hemolytic and complement receptor assays.
We describe a new method of preparing C3-coated erythrocytes by coupling C3 to thiol-activated erythrocytes. The procedure involves three steps. Firstly, sheep erythrocytes were treated with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to introduce 3-(2-pyridyldithio) propionyl residues into membrane proteins. Secondly, C3 was cleaved with trypsin or CoVF, Bb enzyme to obtain C3b exposing the SH group (C3b-SH). Finally, the C3b-SH was coupled to the thiol-activated erythrocytes (TA-E) through thiol/disulfide exchange to form the TA-EC3b conjugate. E coated with C3d was prepared by treating TA-EC3b with KSCN inactivated serum and plasmin. Studying the rosette formation between TA-EC3b or TA-EC3d and cells expressing C3b (CR1) and C3d (CR2) receptors and the inhibition thereof with anti-CR1 and anti-CR2 antibodies as well as with C3-sheep E membrane protein complexes, we found that TA-EC3b and TA-EC3d bound exclusively to CR1 and CR2, respectively. In addition, TA-EC3b like EAC1423b bound factors B and H as tested by hemolytic and direct binding assays. The advantage of TA-EC3 for complement receptor and hemolytic assays are the simplicity of the preparation method and the general applicability of the TA-EC3.
D000818:Animals; D000906:Antibodies / Q000502:physiology; D001667:Binding, Competitive; D003167:Complement Activation; D003179:Complement C3b / Q000276:immunology / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D000939:Epitopes; D004912:Erythrocytes / Q000276:immunology / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D011817:Rabbits; D011951:Receptors, Complement / Q000032:analysis* / Q000276:immunology; D012397:Rosette Formation / Q000379:methods; D013438:Sulfhydryl Compounds
D000906:Antibodies; D004220:Disulfides; D000939:Epitopes; D011951:Receptors, Complement; D013438:Sulfhydryl Compounds; D003179:Complement C3b