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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8×104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43.2 ng/ml,检测线性范围为4.4~417.2 ng/ml,检测限(LOD)为1.8 ng/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。
Objective To prepare monoclonal antibodies against ascites by using acrolein-positive hybridoma cell lines and to optimize and establish an ELISA assay for acrolein. Methods Direct synthetic method was used to prepare complete antigen (immunogen) by coupling acrolein hapten with carrier. The spleen cells of Balb / c mice with high immunopotency were fused with myeloma cells by PEG method. The subclones were subcloned 4 times by limiting dilution method, and one subtype that could stably secrete anti-acrolein antibody was IgG2b hybridoma cell line 1G7G6 and prepared ascites type antibody. On this basis, an indirect competitive ELISA for the detection of acrolein was optimized and established. Results The complete antigens acrolein-KLH (immunogen) and acrolein-BSA (immunogen) suitable for immunization and ELISA assay were synthesized. The titer of acrolein antibody (ascites fluid) prepared was above 8 × 104, Acrolein analog cross-reactivity rates were less than 1%, high specificity. The optimized detection limit of acrolein ELISA was 43.2 ng / ml. The linear range of detection was 4.4 ~ 417.2 ng / ml. The limit of detection (LOD) was 1.8 ng / ml. Conclusions Acrylic ascites-type monoclonal antibodies with high affinity and high titer were successfully prepared, and the conditions of indirect competitive ELISA were optimized. A specific, rapid and sensitive ELISA assay was developed for the determination of acrolein in liquid phase.