[Preparation and identification of a monoclonal antibody against recombinant human L-type pyruvate kinase N].
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.


CONCLUSIONS
The success in anti-PK-N mAb preparation provides a foundation for further studies into glycolysis in normal condition and metabolic diseases.
Liao
Ting-Ting
TT
Department of Immunology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Zhang
Chen-Yang
CY
Jiang
Xiao-Mei
XM
Wei
Da-Peng
DP
Zhang
Chong-Jie
CJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011770:Pyruvate Kinase / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D011770:Pyruvate Kinase