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Updated: Jun 8, 2026
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
[Preparation and identification of a monoclonal antibody against recombinant human L-type pyruvate kinase N]
Ting-Ting Liao1, Chen-Yang Zhang, Xiao-Mei Jiang
1Department of Immunology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To prepare and identify the monoclonal antibody (mAb) against pyruvate kinase N terminal (PK-N).
Methods:
BALB/C mice were immunized with immunogen PK-N-GST-tag. Then the spleen cells were isolated and fused with SP2/0 cells. After several rounds of detecting and cloning, the hybridoma cell strains secreting anti-PK-N mAb were obtained. Its specificity was evaluated with ELISA and Western blot, and the titer, immunoglobulin subtype and affinity of the mAb were measured.
Results:
Two cell strains of hybridoma, 2B2E4G and 2C6F5, were obtained. The hybridoma cell strains secreting anti-PK-N mAb belonged to IgG2b subtype, with a mAb titer in ascetic fluid of 1 : 409600 and 1 : 102400, respectively. Their affinity reached 3.54 x 10(8) L/mol and 2.72 x 10(8) L/mol, respectively, as determined by ELISA. Western blot demonstrated that the mAb could specifically recognize the immunogen and the natural cell lysis protein. The cell immunohistochemistry proved that the antibody could recognize human L type pyruvate kinase expressed in the plasma of HL-7702 cell strain and paraffin slice of hepatoma.
Conclusion:
The success in anti-PK-N mAb preparation provides a foundation for further studies into glycolysis in normal condition and metabolic diseases.
