抗人FXYD3单克隆抗体的制备与初步鉴定

Preparation and Preliminary Identification of Monoclonal Antibody Against Human FXYD3

制备抗人FXYD3单克隆抗体(McAb)并鉴定其生物学特性,以固相合成法获得的FXYD3合成多肽与载体蛋白KLH偶联后免疫BALB/c小鼠,采用杂交瘤技术制备抗人FXYD3 McAb。用ELISA、Western Blot、免疫组化技术对抗人FXYD3McAb的亚型、效价、亲和力及特异性进行鉴定。筛选出两段较好的优势抗原表位,分别为NDLEDKNSPFY和KFGQKSGHH-PGETPPLITPGSA。获得四株可稳定分泌抗人FXYD3 McAb的杂交瘤细胞株02A12C4、02F1E8、02E6B10与03A10E11,亚型鉴定重链均为IgG1,轻链为kappa链。间接ELISA法测定效价均在为1∶104以上。其中02F1E8效价为1∶105以上,亲和常数为3.11×108L/mol。免疫组化分析显示了FXYD3均匀分布于肝癌细胞与人胰腺癌Bxpc-3细胞系细胞的胞膜上。结果成功制备了四株抗人FXYD3 McAb,其中02F1E8纯度好、效价高及特异性强。为进一步研究FXYD3在肿瘤组织及细胞系中的表达及临床意义奠定了基础。

To prepare and preliminary identify the monoclonal antibody（McAb） against human FXYD3, dominant epitopes of FXYD3 were analyzed by software BioSunV1.0, two peptides were synthesized and conjugated with KLH. BALB/C mice were immunized with KLH-conjugated peptide to prepare McAb by using hybridoma technique. The immunoglobulin subtype, titer, affinity and specificity of obtained anti-human FXYD3 McAb were evaluated with ELISA, Western blot and immunohistochemistry respectively. It is resulted that four celllines of hybridoma were obtained named 02A12C4, 02F1E8, 02E6B10 and 03A10Ell, which were identified that the heavy and light chains were all IgG1 and K, respectively, with better titer than 1：104. The titer of 02F1E8 was 1 ： 105, the affinity was 3.11 × 10^8 L/tool. The cell immuno-histochemistry proved that the McAbs could recognize the human FXYD3 located in the cell membrane of mammary adenocarcinoma and pancreatic carcinoma cellline Bxpc-3. It is concluset that success of four anti-human FXYD3 McAbs preparation with 02F1E8 of high titer and specificity provides the basis for further study of FXYD3 expression and Clinical Significance in tumor tissues and celllines.