Lambris J D, Scheiner O, Schulz T F, Alsenz J, Dierich M P
J Immunol Methods. 1983 Dec 30;65(3):277-83. doi: 10.1016/0022-1759(83)90122-9.
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.
我们描述了一种通过将C3偶联到硫醇活化的红细胞上来制备C3包被红细胞的新方法。该过程包括三个步骤。首先，用N-琥珀酰亚胺基3-(2-吡啶二硫基)丙酸酯(SPDP)处理绵羊红细胞，将3-(2-吡啶二硫基)丙酰基残基引入膜蛋白中。其次，用胰蛋白酶或CoVF、Bb酶裂解C3以获得暴露SH基团的C3b(C3b-SH)。最后，通过硫醇/二硫键交换将C3b-SH偶联到硫醇活化的红细胞(TA-E)上，形成TA-EC3b缀合物。用KSCN灭活的血清和纤溶酶处理TA-EC3b制备C3d包被的E。通过研究TA-EC3b或TA-EC3d与表达C3b(CR1)和C3d(CR2)受体的细胞之间的花环形成以及用抗CR1和抗CR2抗体以及C3-绵羊E膜蛋白复合物对其的抑制作用，我们发现TA-EC3b和TA-EC3d分别仅与CR1和CR2结合。此外，如通过溶血和直接结合试验所测试的，TA-EC3b像EAC1423b一样结合因子B和H。TA-EC3用于补体受体和溶血试验的优点是制备方法简单且TA-EC3具有普遍适用性。
