Generation of a monoclonal antibody recognizing the heavily glycosylated CD45 Protein and its application on identifying circulating tumor cells

Here, we provide direct evidence that using recombinant proteins expressed in eukaryotic cells as antigen is a practical way to generate monoclonal antibodies (mAbs) against heavily glycosylated proteins. Heavily glycosylated proteins are typically difficult targets for mAb generation, being limited by unsatisfactory affinity and low specificity. Using the heavily glycosylated CD45 protein as an example, we demonstrate the entire process of expressing the protein in eukaryotic cells and using it as an antigen to generate CD45-targeting mAbs in mice. The mAbs generated showed robust affinity and specificity, which are crucial factors for differentiate circulating tumor cells from white blood cells in human breast cancer patient samples. Only 1 cell fusion and 2 cyclic sub-cloning steps were necessary before mAbs with satisfactory performance were obtained.本研究提供直接证据表明，以真核细胞（eukaryotic cells）表达的重组蛋白（recombinant proteins）作为抗原（antigen），是制备靶向高度糖基化蛋白的单克隆抗体（monoclonal antibodies，mAbs）的可行途径。高度糖基化蛋白通常是单克隆抗体制备的难点靶点，其制备过程常受限于亲和力不佳与特异性不足的问题。本研究以高度糖基化的CD45蛋白为范例，完整展示了在真核细胞中表达该蛋白，并将其作为抗原在小鼠体内制备靶向CD45的单克隆抗体的全过程。所获得的单克隆抗体展现出优异的亲和力与特异性，而这正是在人类乳腺癌患者样本中区分循环肿瘤细胞与白细胞的核心要素。仅需一轮细胞融合与两轮循环亚克隆步骤，即可获得性能符合要求的单克隆抗体。