Liao Ting-Ting, Zhang Chen-Yang, Jiang Xiao-Mei, Wei Da-Peng, Zhang Chong-Jie
Department of Immunology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2010 Jul;41(4):571-4.
To prepare and identify the monoclonal antibody (mAb) against pyruvate kinase N terminal (PK-N).
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.
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.
The success in anti-PK-N mAb preparation provides a foundation for further studies into glycolysis in normal condition and metabolic diseases.
制备并鉴定抗丙酮酸激酶N端（PK-N）的单克隆抗体（mAb）。
用免疫原PK-N-GST标签免疫BALB/C小鼠。然后分离脾细胞并与SP2/0细胞融合。经过几轮检测和克隆，获得分泌抗PK-N mAb的杂交瘤细胞株。用ELISA和Western blot评估其特异性，并测定mAb的效价、免疫球蛋白亚型和亲和力。
获得两株杂交瘤细胞株，2B2E4G和2C6F5。分泌抗PK-N mAb的杂交瘤细胞株属于IgG2b亚型，腹水mAb效价分别为1:409600和1:102400。ELISA测定其亲和力分别达到3.54×10⁸L/mol和2.72×10⁸L/mol。Western blot表明mAb能特异性识别免疫原和天然细胞裂解蛋白。细胞免疫组化证明该抗体能识别HL-7702细胞株血浆和肝癌石蜡切片中表达的人L型丙酮酸激酶。
抗PK-N mAb制备成功为进一步研究正常状态下的糖酵解及代谢性疾病奠定了基础。
