Antibody-sandwich ELISA analysis of a novel blood biomarker of CST4 in gastrointestinal cancers
Authors Dou YL, Lv YL, Zhou XJ, He LF, Liu LH, Li PF, Sun YL, Wang MH, Gao MJ, Wang C
Received 15 August 2017
Accepted for publication 17 December 2017
Published 28 March 2018 Volume 2018:11 Pages 1743—1756
Checked for plagiarism Yes
Review by Single-blind
Peer reviewer comments 2
Editor who approved publication: Dr Yao Dai
Yaling Dou,1,* Yali Lv,2,* Xiaojin Zhou,3,* Linfu He,4,* Lihong Liu,2 Pengfei Li,2 Yulong Sun,3 Minghui Wang,3 Meijuan Gao,3 Chong Wang3
1Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, People’s Republic of China; 2Department of Pharmacy, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of China; 3Shanghai Liangrun Biomedicine Technology Co. Ltd., Shanghai, People’s Republic of China; 4Institute of Bioengineering, Jiangsu University, Zhenjiang, People’s Republic of China
*These authors contributed equally to this work
Background: Members of the cystatin family have increasingly been proven to be involved in several tumors, including gastric cancer (GC) and colorectal cancer (CRC). Cystatin S (CST4) was found to be upregulated at the gene expression level in GC cells, making it a potential novel biomarker for the early diagnosis of gastrointestinal cancer.
Materials and methods: Quantitative real-time polymerase chain reaction and Western blotting analysis were used to explore CST4 expression in gastrointestinal cancer tissues and cell lines. We purified CST4 recombinant protein and generated anti-CST4 monoclonal antibodies to develop an antibody-sandwich enzyme-linked immunosorbent assay (ELISA) analysis system for blood CST4 detection. The performance and clinical efficacy of the detection method were evaluated using a training set and validation set, respectively.
Results: According to the quantitative real-time polymerase chain reaction and Western blotting results, CST4-mRNA expression and protein expression were upregulated in gastrointestinal cancer tissues and cell lines. The ELISA detection system for CST4 showed significantly better sensitivities of 69.0% and 69.0% and specificities of 85.6% and 83.6% for GC and CRC, respectively, than other common clinical biomarkers, carcinoembryonic antigen, CA19-9, CA125, and CA72-4. Clinical verification experiments using GC and CRC validation sets also found distinguishable CST4 median concentrations (177.7 pg·mL-1 and 174.2 pg·mL-1 respectively) and high positive detection rates (72.3% and 88.4% respectively), further confirming the specificity and sensitivity of this method.
Conclusion: We validated the overexpression of CST4 in gastrointestinal cancer tissues and cell lines and developed an antibody-sandwich ELISA analysis system for blood CST4 detection, which exhibited high specificity and sensitivity. Novel blood biomarkers of CST4 have enormous potential in terms of clinical diagnostic value in GC and CRC.
Keywords: cystatin S, ELISA, gastrointestinal cancer, biomarker
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