丙烯醛单克隆抗体的制备及ELISA方法的建立

Preparation of the monoclonal antibodies against acrolein and establishment of an ELISA method

目的利用丙烯醛阳性杂交瘤细胞株制备腹水型单克隆抗体，优化并建立丙烯醛的ELISA检测技术。方法采用直接人工合成法，将丙烯醛半抗原与载体耦合制备完全抗原（免疫原）。利用PEG法将免疫效价高的Balb／c小鼠的脾脏细胞与骨髓瘤细胞进行细胞融合，采用有限稀释法进行4次亚克隆，筛选得到1株能稳定分泌抗丙烯醛抗体的亚型为IgG2b的杂交瘤细胞株1G7G6，并制备腹水型抗体。在此基础上，优化并建立检测丙烯醛的间接竞争ELISA方法。结果合成适于免疫和ELISA检测的完全抗原丙烯醛-KLH（免疫原）和丙烯醛-BSA（包被原），制备的丙烯醛抗体（腹水）的效价均在8×10^4以上，与其他丙烯醛类似物交叉反应率均小于1％，特异性高。优化后丙烯醛ELISA方法的检测灵敏度（IC50）为43．2ng／ml，检测线性范围为4．4-417．2ng／ml，检测限（LOD）为1．8ng／ml。结论成功制备得到高亲和力、高效价的丙烯醛腹水型单抗，筛选优化了间接竞争ELISA检测条件，建立了检测液相中丙烯醛的特异、快速、灵敏的ELISA检测技术。

To prepare the ascitic type monoclonal antibodies （mcAbs） against acrolein based on the positive hybridoma cell strain, the acrolein complete antigen was synthesized by direct synthesis method, and used to immunize 6~8 weeks Balb/c female mice five times. Then spleen cells with high titer were selected and fused with myeloma cells by using PEG method. Through four times subcloning, the monoclonal antibody hybridoma cell 1G7G6 was obtained, which was able to produce IgG2b subtype antibody, and were used to produce ascetic type mcAbs. The titer of obtained mcAbs was up to 8×10^4, with low cross reactivity to other acrolein analogues. Based on these produced monoclonal antibodies, the indirect competitive ELISA for detecting acrolein was established and optimized. The sensitivity of the ELISA was 43.2 ng/ml （IC50）, the detection limits （LOD） was 1.8 ng/ml （IC10）, and the linear concentrations ranged from 4.4 to 417.2 ng/ml. All the results indicated that the high affinity and high specific mcAbs against acrolein has been prepared, and a rapid, sensitive and specific indirect competitive ELISA for acrolein detection is established successfully.