title: A novel method for the sensitive detection of mutant proteins using a covalent-bonding tube-based proximity ligation assay.
abstract: Tumorigenesis is the cumulative result of multiple gene mutations. The mutant proteins that are expressed by mutant genes in cancer cells are secreted into the blood and are useful biomarkers for the early diagnosis of cancer. However, some difficulties exist; for example, the same gene will express different protein mutants in different patients, and early tumors secrete only small amounts of mutant protein. Thus, the presence of mutant proteins in plasma has not previously been exploited for the early diagnosis of cancer. Proximity ligation assay is a protein-detection method that has been developed in recent years and has been widely used because of its high sensitivity. However, this approach still suffers from some shortcomings that should be addressed. In this paper, we develop a covalent-bonding tube-based proximity ligation assay (TB-PLA). The limit of detection of TB-PLA for 0.001pM, and the method exhibited a broad dynamic range of up to seven orders of magnitude. Furthermore, we coupled the conformation-specific antibody PAb240 of p53 mutants to PCR tubes for TB-PLA. The assay was capable of detecting an approximately 500-fold lower concentration of mutant p53 in serum compared with sandwich ELISA. Thus, we demonstrate TB-PLA to be a highly sensitive and effective approach that is suitable for the early clinical diagnosis of cancer using the conformation-specific antibodies of protein mutants.
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Jiang
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Lixiao
authors.initials: L
authors.lastname: Zhou
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Cheng
authors.affiliation: Department of Radioimmunoassay and Clinical Laboratory, Luwan Branch of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200020, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Huixia
authors.initials: H
authors.lastname: Wang
authors.affiliation: Hangzhou Genesea Biotechnology Limited Company, Hangzhou, Zhejiang 315000, China.
authors.forename: Zhixiong
authors.initials: Z
authors.lastname: Chen
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Shi
authors.affiliation: College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China. Electronic address: cgzhu@zju.edu.cn.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Zhu
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D050505:Mutant Proteins / Q000032:analysis*; D017430:Prostate-Specific Antigen / Q000032:analysis; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
chemical_list: D050505:Mutant Proteins; D016159:Tumor Suppressor Protein p53; D017430:Prostate-Specific Antigen
keywords: Mutant p53 detection; Prostate-specific antigen; Proximity ligation assay