O'Hare T, Rittenberg M B
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USA.
J Immunol Methods. 1998 Sep 1;218(1-2):161-7. doi: 10.1016/s0022-1759(98)00130-6.
A rapid and convenient method for measuring affinity constants (K(A)) of IgG antibody-hapten complexes is described. A key advantage of this method is its suitability for quantification of both low and high affinity interactions. A comparison is made of the K(A)s of the low affinity anti-phosphocholine (PC) antibody T15 (K(A) = 2.9 x 10(5) M(-1)) and five heavy chain complementarity determining region 2 (HCDR2) mutant antibodies expressed as IgG2b transfectants. As a demonstration of the general applicability of the technique, colchicine binding to the high affinity monoclonal IgG2a antibody C44 (K(A) = 1.5 x 10(9) M(-1)) is measured also; thus the assay is applicable over a four-log range of affinities. The assay, based upon use of fixed Staphylococcus aureus Cowan I strain cells as an adsorbent for antibody-radiolabeled antigen complexes, is conducted over a range of hapten concentrations at constant antibody concentration. The K(A) is obtained by Scatchard analysis.
本文描述了一种快速便捷的测量IgG抗体-半抗原复合物亲和常数（K(A)）的方法。该方法的一个关键优势在于其适用于定量低亲和力和高亲和力相互作用。对低亲和力抗磷酸胆碱（PC）抗体T15（K(A)=2.9×10⁵ M⁻¹）以及作为IgG2b转染子表达的五种重链互补决定区2（HCDR2）突变抗体的K(A)进行了比较。作为该技术普遍适用性的一个例证，还测量了秋水仙碱与高亲和力单克隆IgG2a抗体C44（K(A)=1.5×10⁹ M⁻¹）的结合；因此该测定法适用于四个对数级的亲和力范围。该测定法基于使用固定的金黄色葡萄球菌考恩I株细胞作为抗体-放射性标记抗原复合物的吸附剂，在恒定抗体浓度下于一系列半抗原浓度范围内进行。通过Scatchard分析获得K(A)。
