Coupling of C3b to erythrocytes by disulfide bond formation: preparation of EC3b for hemolytic and complement receptor assays

J Immunol Methods. 1983 Dec 30;65(3):277-83. doi: 10.1016/0022-1759(83)90122-9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / physiology
  • Binding, Competitive
  • Complement Activation
  • Complement C3b / immunology
  • Complement C3b / metabolism*
  • Disulfides / metabolism
  • Epitopes
  • Erythrocytes / immunology
  • Erythrocytes / metabolism*
  • Hemolysis*
  • Humans
  • Rabbits
  • Receptors, Complement / analysis*
  • Receptors, Complement / immunology
  • Rosette Formation / methods
  • Sulfhydryl Compounds

Substances

  • Antibodies
  • Disulfides
  • Epitopes
  • Receptors, Complement
  • Sulfhydryl Compounds
  • Complement C3b