Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay
Determination of the binding affinity of an anti-CD34 single-chain antibody using a novel, flow cytometry based assay
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DOI：
10.1016/s0022-1759(96)00227-x
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发表时间：
1997-02-28
影响因子：
2.2
通讯作者：
Anderson, WF
中科院分区：
文献类型：
--
作者：
Benedict, CA;MacKrell, AJ;Anderson, WF
从表达抗CD34单克隆抗体My10的杂交瘤构建单链抗体(scFv)。 scFv 在小鼠成纤维细胞系 NIH 3T3 中表达，并通过与蛋白质 C 末端融合的表位标签从培养物上清液中纯化。使用基于荧光的流式细胞术测定涉及表位标签的识别，将 scFv 平衡解离常数 (K-D) 确定为 2.4 x 10(-7) M，并且与天然抗体 My10 相比，与表达 CD34 的 KG-1a 细胞的结合力大约低 24 倍。这种新颖且先前未报道的用于确定抗体结合亲和力的方法与其他方法相比具有多种优势。它快速且简单，并且与直接标记抗体的方法不同，它不涉及可能干扰抗原结合的抗体可变结构域残基的共价修饰。还测定了抗CD33抗体HuG1的K-D（Caron等人（1992）人源化M195（抗CD33）单克隆抗体的生物学和免疫学特征。Cancer Res.52, 6761-6767）。该值与之前报道的通过放射性配体竞争方法确定的值非常一致，验证了该测定法在抗体亲和力测定中的用途。我们讨论了这种抗 CD34 scFv 的各种潜在应用。
A single chain antibody (scFv) was constructed from a hybridoma expressing the anti-CD34 monoclonal antibody My10. The scFv was expressed in the mouse fibroblast cell line NIH 3T3, and purified from culture supernatant via an epitope tag fused to the C-terminus of the protein. The scFv equilibrium dissociation constant (K-D) was determined to be 2.4 x 10(-7) M using a fluorescence based flow cytometry assay involving recognition of the epitope tag, and bound approximately 24-fold less avidly to CD34 expressing KG-1a cells than the native antibody My10. This novel and previously unreported method for determining antibody binding affinity offers several advantages over alternative methods. It is rapid and simple, and unlike methods that directly label the antibody, it involves no covalent modifications of antibody variable domain residues that could potentially interfere with antigen binding. The K-D for the anti-CD33 antibody HuG1 (Caron et al. (1992) The biological and immunological features of humanized M195 (anti-CD33) monoclonal antibodies. Cancer Res. 52, 6761-6767) was determined as well. The close agreement of this value and the previously reported value, determined by a radioligand competition method, validates the use of this assay for antibody affinity determination. We discuss various potential applications for this anti-CD34 scFv.