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...

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Bibliographic Details
Published in:PloS one 2018-02, Vol.13 (2), p.e0192506-e0192506
Main Authors: Zhang, Weikai, Li, Zhitao, Wang, Zihua, Yue, Chunyan, Zheng, Hui, Li, Ren, Zhou, Mingxing, Hu, Zhiyuan, Wei, Zewen, Li, Qin
Format: Article
Language:English
Subjects:
Affinity
Animals
Antibodies, Monoclonal - immunology
Antigens
Biology and Life Sciences
Biomedical engineering
Blood cells
Blood circulation
Breast cancer
CD45 antigen
Cell fusion
Cloning
Erythrocytes
Female
Glycosylation
HEK293 Cells
Humans
Laboratories
Leukocyte Common Antigens - immunology
Leukocytes
Life sciences
Medicine and Health Sciences
Mice
Mice, Inbred BALB C
Monoclonal antibodies
Nanomaterials
Neoplastic Cells, Circulating
Penicillin
Proteins
Research and Analysis Methods
Studies
Tumor cells
White blood cells
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0192506