Establishment and preliminary application of DELFIA detection method for 129 serine phosphorylated α-synuclein

doi:10.3969/j.issn.1006-7795.2021.05.013

Abstract

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)

CLC Number:

R742.5

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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URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2021.05.013

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2021/V42/I5/776

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