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首页> 外文期刊>Biological chemistry >Immunolabeling of CD3-Positive Lymphocytes with a Recombinant Single-Chain Antibody/Alkaline Phosphatase Conjugate
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Immunolabeling of CD3-Positive Lymphocytes with a Recombinant Single-Chain Antibody/Alkaline Phosphatase Conjugate

机译：重组单链抗体/碱性磷酸酶结合物对CD3阳性淋巴细胞的免疫标记

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摘要

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigenbinding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.
机译：G3（3）是一种针对人T淋巴细胞CD3抗原的新型鼠类单克隆抗体，可用于分析淋巴样恶性肿瘤。我们已经生产并表征了具有抗体G3（3）抗原结合特异性的重组比色免疫偶联物。使用原始杂交瘤细胞作为抗体可变重链（VH）和可变轻链（VL）基因的来源，组装了编码单链抗体可变片段（scFv）的基因。为了产生与细菌碱性磷酸酶融合的可溶性scFv，将嵌合基因引入原核表达载体。 DNA测序和蛋白质印迹分析表明，从诱导的重组细菌中回收的可溶性免疫偶联物的完整性。 scFv / AP蛋白具有双重功能，并且与亲本G3（3）抗体的免疫反应性相似。流式细胞仪和免疫染色实验证实，scFv / AP蛋白的活性与亲本抗体的活性相比具有优势。 scFv / AP偶联物在T细胞表面与CD3抗原结合，并通过其酶活性直接检测。因此，这种新颖的融合蛋白作为免疫诊断试剂具有潜在的应用。

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