129位丝氨酸磷酸化α-突触核蛋白DELFIA检测方法的建立及初步应用

doi:10.3969/j.issn.1006-7795.2021.05.013

摘要/Abstract

摘要： 目的建立解离-增强镧系荧光免疫分析 (dissociation enhanced lanthanide fluorescent immunoassay,DELFIA)检测129位丝氨酸磷酸化α-突触核蛋白的方法及初步应用。方法使用体外蛋白重组技术纯化得到人源α-syn蛋白单体(α-syn),用Polo 样激酶催化α-syn获得磷酸化α-syn单体(phosphorylated α-syn monomer,p-α-syn),用4-氧代壬烯醛制备α-syn聚集体(aggregates of α-syn,OW)及磷酸化α-syn聚集体(aggregates of p-α-syn,OP)。使用Western blotting法和间接ELISA方法验证本实验室前期制备的识别α-syn蛋白N端的多克隆抗体SN16和识别129位丝氨酸磷酸化α-syn蛋白的单克隆抗体C140S。使用SN16作为捕获抗体,C140S作为检测抗体的双抗夹心DELFIA法建立检测OP的标准曲线,并检测Thy1-α-SYN转基因小鼠血浆内OP的变化。结果考马斯亮蓝染色和Western blotting法检测结果显示体外纯化的人源α-syn蛋白单体(17 000)纯度高,并通过催化获得了p-α-syn、OW和OP纯蛋白,均可用作建立标准曲线的标准抗原。Western blotting和酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)结果显示SN16 对α-syn、p-α-syn、OW、OP这4种形式的蛋白均可识别,C140S可以识别p-α-syn和OP,说明SN16和C140S可以用作双抗夹心法的配对抗体。间接DELFIA检测结果显示C140S对OP的识别效率更好。双抗夹心DELFIA检测结果显示,SN16-C140S作为配对抗体检测标准抗原的范围为:0.625~20 ng/mL,并建立了检测OP的标准曲线。Wt及Tg组小鼠血浆中OP在12月龄均较6月龄有所增加(P<0.001),Tg组6月龄小鼠血浆中OP显著高于同月龄Wt组(P<0.001),Tg组12月龄小鼠血浆OP较Wt组有增高的趋势,但差异无统计学意义(P>0.05)。结论本文建立的SN16-C140S配对的双抗夹心DELFIA体系可以检测Thy1-α-SYN转基因小鼠血浆中聚集的磷酸化α-syn变化情况。

关键词: 帕金森病, α-突触核蛋白, 磷酸化α-突触核蛋白, 解离-增强镧系荧光免疫分析

Abstract: Objective To establish an dissociation enhanced lanthanide fluorescent immunoassay (DELFIA) method for detection of 129 serine phosphorylated α-synuclein. Methods Human α-syn protein monomer (α-syn) was purified by in vitro protein recombination technique. Phosphorylated α-syn monomer (p-α-syn) was obtained by using pololike kinase to catalyze α-syn. Aggregates of α-syn (OW) and aggregates of p-α-syn (OP) were prepared by 4-oxo-nonenal. Western blotting and indirect ELISA were used to verify the polyclonal antibody SN16 and monoclonal antibody C140S which recognized the N-terminal of α-syn protein and phosphorylated S129-α-syn. The standard curve for the detection of OP was established by sandwich DELFIA by using SN16 as capture antibody and C140S as detection antibody, and the changes of OP in the plasma of Thy1-α-SYN transgenic mice was detected. Results Coomassie brilliant blue staining and Western blotting showed that the purity of human α-syn protein monomer (17 000) was high, and p-α-syn, OW and OP were obtained by catalysis in vitro, which could be used as standard antigens for establishing standard curve. Western blotting and ELISA results showed that SN16 could recognize α-syn, p-α-syn, OW and OP, and C140S could recognize p-α-syn and OP, indicating that SN16 and C140S could be used as paired detecting antibodies. Indirect DELFIA results showed that C140S had better recognition efficiency for OP. Sandwich DELFIA showed that the range of antigen was 0.625 ng/mL to 20 ng/mL by using SN16-C140S as the paired antibody. The standard curve of identifying OP was established. The content of OP in 12-month-old mice plasma of Wt and Tg groups was higher than that of 6-month-old mice (P<0.001). The level of OP in 6-month-old Tg mice plasma was significantly higher than that of the same month-old mice in Wt group (P<0.001). The level of OP in the plasma of 12-month-old Tg mice was higher than that of Wt group, but there was no statistical difference (P>0.05). Conclusion Sandwich DELFIA method was established to detect aggregated OP in the plasma of Thy1-α-SYN transgenic mice by using SN16-C140S antibody.

Key words: Parkinson's disease, α-synuclein, phosphorylated α-synuclein, dissociation enhanced lanthanide fluorescence immunoassay (DELFIA)

中图分类号:

R742.5

高歌, 查旭, 刘伟进, 杨慧. 129位丝氨酸磷酸化α-突触核蛋白DELFIA检测方法的建立及初步应用[J]. 首都医科大学学报, 2021, 42(5): 776-782.

Gao Ge, Zha Xu, Liu Weijin, Yang Hui. Establishment and preliminary application of DELFIA detection method for 129 serine phosphorylated α-synuclein[J]. Journal of Capital Medical University, 2021, 42(5): 776-782.

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