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Updated: Jun 13, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
[Generation and characterization of monoclonal antibody against human PIK3IP1]
Ying Chang1, Dong Xu, Yao-yao Chen
1Department of Immunology, Peking University School of Basic Medical Sciences, Peking University Center for Human Disease Genomics, Beijing 100191, China.
Aim:
To obtain monoclonal antibody against PIK3IP1 for further study of the structure and biological function of PIK3IP1 protein.
Methods:
BALB/c mice were immunized with recombinant GST-PIK3IP1(62-168), Hybridoma cell lines secreting monoclonal antibodies against PIK3IP1 were screened by regular cell fusion and subcloning approach. The specificities of the monoclonal antibody was determined by ELISA, Western blot and Immunofluorescence assay.
Results:
One hybridoma cell line (5C6) stable in secreting specific monoclonal antibody was successfully obtained. The subclass of IgG belonged to IgG1. The ascite titers of this monoclonal antibody reached 1:10(7). It could specifically bind to recombinant GST-PIK3IP1(62-168); protein and overexpressed PIK3IP1 and variant PIK3IP1-v1 proteins proved by Western blot. This antibody failed to react with E.coli lysates and glutathione S transferase (GST). At the same time, endogenous PIK3IP1 was not detected using 5C6 antibody. Immunofluorescence results revealed that overexpressed PIK3IP1 and variant PIK3IP1-v1 protein located in cytoplasm and distributed in fleck manner.
Conclusion:
Monoclonal antibody against PIK3IP1 with high titer and specificity has been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of PIK3IP1.
