Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA

doi:10.3390/app12199868

HFCAS OpenIR

	Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA
	Zhu, Zhu 1,2,3; Wang, Han 2; Wang, Li 2; Wei, Zhou 3; Zheng, Zhiming2 ; Wang, Peng1,2
	2022-10-01
发表期刊	APPLIED SCIENCES-BASEL
通讯作者	Zheng, Zhiming(zhengzhiming2014@163.com) ; Wang, Peng(pengwang@ipp.ac.cn)
摘要	To detect residual pre-proinsulin (PPI) in recombinant human insulin production, an analytical method based on double-antibody sandwich ELISA was developed in this study. The BALB/c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained using the conventional cell fusion technique and ELISA screening. We purified the antibody using a Protein G gel column and identified its purity by SDS-PAGE. The sandwich ELISA was used to explore the pairing effect, and the specificity of the paired antibody was determined. We selected a paired antibody with relatively good specificity to establish sandwich ELISA, constructed a quantitative curve, and evaluated the accuracy and sensitivity of the method. Six anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer value. The sandwich ELISA method was established with P5 for plating and P2 as detection antibodies. The linear range of the quantitative curve of PPI by sandwich ELISA was 0.645 to 82.5 pg/mL, the recovery was 95% and the detection limit was 3.06 pg/mL. In this study, we prepared six anti-PPI monoclonal antibodies and established the sandwich ELISA method to detect PPI in process control and product release control for recombinant human insulin production.
关键词	recombinant human insulin pre-proinsulin monoclonal antibody sandwich ELISA
DOI	10.3390/app12199868
关键词[WOS]	PURIFICATION PROCESS ; DIABETES-MELLITUS ; INSULIN ; OBESITY
收录类别	SCI
语种	英语
资助项目	Major Projects of Science and Technology of Anhui Province[202103a06020003] ; China National Key Research and Development Program[2019YFA0904300] ; China National Key Research and Development Program[2019YFA0904304]
项目资助者	Major Projects of Science and Technology of Anhui Province ; China National Key Research and Development Program
WOS研究方向	Chemistry ; Engineering ; Materials Science ; Physics
WOS类目	Chemistry, Multidisciplinary ; Engineering, Multidisciplinary ; Materials Science, Multidisciplinary ; Physics, Applied
WOS记录号	WOS:000866632300001
出版者	MDPI
引用统计
文献类型	期刊论文
条目标识符	http://ir.hfcas.ac.cn:8080/handle/334002/129326
专题	中国科学院合肥物质科学研究院
通讯作者	Zheng, Zhiming; Wang, Peng
作者单位	1.Univ Sci & Technol China, Sci Isl Branch, Grad Sch, Hefei 230026, Peoples R China 2.Chinese Acad Sci, Hefei Inst Phys Sci, Hefei 230031, Peoples R China 3.Hefei Tianmai Biotechnol Dev Co Ltd, Hefei 230061, Peoples R China
推荐引用方式 GB/T 7714	Zhu, Zhu,Wang, Han,Wang, Li,et al. Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA[J]. APPLIED SCIENCES-BASEL,2022,12.
APA	Zhu, Zhu,Wang, Han,Wang, Li,Wei, Zhou,Zheng, Zhiming,&Wang, Peng.(2022).Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA.APPLIED SCIENCES-BASEL,12.
MLA	Zhu, Zhu,et al."Efficient Detection of Pre-Proinsulin by Double Antibody Sandwich ELISA".APPLIED SCIENCES-BASEL 12(2022).