Identification and application of fluorescent labeled small molecule antibodies based on cell surface specific recognition
ZHANG Jing1, XIONG Ming2, LI Xiaojuan1, MA Wei2, ZHANG Dajin2, QIAO Yuanyuan2
1. Department of Radiation Diagnosis,2. Center for Basic Medical Sciences,The Sixth Medical Center of the General Hospital of PLA, Beijing 100048, China
Abstract：Objective To establish a method of identification of fluorescent-labeled small molecule antibodies based on cell surface specific recognition and to verify their applicability in detection of peripheral blood circulating tumor cells (CTCs) of esophageal cancer.Methods Phage antibodies NFC 20b and NFC 70a were obtained via high-throughput screening and enrichment using phage display technology in the early stage of this study. After fluorescence labeling, the aggregation tracer between them and esophageal cancer cells was observed in zebrafish. Small molecule antibodies were also used to detect CTCs in peripheral blood of esophageal cancer patients. Meanwhile, the expression profile of antigen proteins was analyzed using the high-throughput protein chip.Results The distribution of fluorescent labeled small molecule antibodies NFC 20b and NFC 70a could be observed in the dorsal aorta, caudal artery and caudal vein of zebrafish, so was the binding of antibodies to transplanted tumors in yolk sac. The protein chip used to screen the expression profile of the antigen protein proved that the antigen protein bound by NFC 20b was cyclin dependent kinase 2(CDK2).Conclusions Small molecule antibodies labeled by fluorescence labeling technology can be distributed to yolk sac through blood circulation, and can aggregate with and bind to transplanted tumors of esophageal cancer in yolk sac. This approach can be used to detect CTCs in peripheral blood of ESCC. The protein chip can be used to assist in identifying the antigen binding to small antibodies.
Agarwal A, Balic M, Ashry D, et al. Circulating tumor cells: strategies for capture, analyses, and propagation [J]. Cancer J, 2018,24(2): 70-77.
[3]
Lowes L E, Allan A L. Circulating tumor cells and implications of the epithelial-to-mesenchymal transition [J].Adv Clin Chem, 2018,83:121-181.
[4]
Popkov M, Rader C, Barbas C F,et al. Isolation of human prostatecancer cell reactive antibodies using phage display technology[J]. J Immunol Methods, 2004,291(1):137-151.
[5]
Siva A C, Kirkland R E, Lin B, et al. Selection of anti-cancer antibodies from combinatorial libraries by whole-cell panning and stringent subtraction with human blood cells[J]. J Immunol Methods, 2008, 330(1):109-119.
Wang S, Miller S R, Ober E A, et al. Making it new again: insight into liver development, regeneration, and disease from zebrafish research[J]. Curr Top Dev Biol, 2017,124:161-195.
Patton E E, Tobin D M. Spotlight on zebrafish: the next wave of translational research[J]. Dis Model Mech, 2019,712 (3):15.
[10]
Tseng W C, Loeb H E, Pei W, et al. Modeling niemann-pick disease type c1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds[J]. Dis Model Mech,2018, 11 (9):234.
[11]
Hu B, Niu X, Cheng L, et al. Discovering cancer biomarkers from clinical samples by protein microarrays[J].Proteomics Clin Appl,2015,9 (2):98-110.
[12]
Huang Y, Zhu H. Protein array-based approaches for biomarker discovery in cancer[J]. Genomics Proteomics Bioinformatics, 2017,15(2):73-81.
[13]
Zhang H, Yang L, Ling J, et al. Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic[J]. Natl Acad Sci, 2015,112(49):15084-15089.
[14]
Chohan T A, Qian H Y, Pan Y L, et al. Cyclin-dependent kinase-2 as a target for cancer therapy: progress in the development of cdk2 inhibitors as anti-cancer agents[J]. Curr Med Chem, 2015,22(2): 237-263.
Li S P, Guan Q L, Zhao D, et al. Detection of circulating tumor cells by fluorescent immunohistochemistry in patients with esophageal squamous cell carcinoma: potential clinical applications[J]. Med Sci Monit, 2016,22:1654-1662.
2021, Vol. 32 
