Browse > Article
http://dx.doi.org/10.7841/ksbbj.2016.31.1.33

Live Cell Detection of Monoclonal Antibody Light and Heavy Chain mRNAs using Molecular Beacons  

Jeong, Seunga (Division of Bioengineering, Incheon National University)
Rhee, Won Jong (Division of Bioengineering, Incheon National University)
Publication Information
KSBB Journal / v.31, no.1, 2016 , pp. 33-39 More about this Journal
Abstract
Developing the method for the selection of animal cell line producing therapeutic monoclonal antibody (mAb) is invaluable as its market is rapidly growing. Although the quality of produced mAb is as important as quantity, however there is no method developed for the selective screening of cell lines on the basis of both quantity and quality. From recent reports, the ratio of light and heavy chain mRNAs of mAb in the cell is a key parameter for the indication of product quality. Therefore, it is obvious that developing the novel method that can detect both light and heavy chain mRNAs in single live cell will provide unprecedented opportunities in bio-industry. Here, we have constructed oligonucleotide probes, molecular beacons for the detection of light or heavy chain mRNAs, respectively, in the live cells producing mAbs. Both beacons showed increased fluorescent intensity after transient transfection of plasmid expressing mAbs analyzed by fluorometer. Flow cytometric analysis clearly demonstrated that both molecular beacons can simultaneously detect the expression of light and heavy chain mRNAs of mAb in the same cell. The technique described in the thesis provides the new direction and concept for developing the method for the smart selection of cell lines producing recombinant proteins including therapeutic mAbs.
Keywords
Monoclonal antibody; Cell line; Anti-apoptosis; mRNA; Molecular beacon; Recombinant protein;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Browne, S. M. and M. Al-Rubeai (2007) Selection methods for high-producing mammalian cell lines. M. Trend Biotechnol. 9: 425-432.
2 Lee, C. J., G. Seth, J. Tsukuda, and R. W. Hamilton (2009) A clone screening method using mRNA levels to determine specific productivity and product quality for monoclonal antibodies. Biotechnolol. Bioeng. 102: 1107-1118.   DOI
3 de la Cruz Edmonds, M. C., M. Tellers, C. Chan, P. Salmon, D. K. Robinson, and J. Markusen (2006) Development of transfection and high-producer screening protocols for the CHOK1SV cell system. Mol. Biotechnol. 34: 179-190.   DOI
4 Fussenegger, M., J. E. Bailey, H. Hauser, and P. P. Mueller (1999) Genetic optimization of recombinant glycoprotein production by mammalian cells. Trends Biotechnol. 17: 35-42.   DOI
5 Wurm, F. M. (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22: 1393-1398.   DOI
6 De Maria, C. T., V. Cairns, C. Schwarz, J. Zhang, M. Guerin, E. Zuena, S. Estes, and K. P. Karey (2007) Accelerated clone selection for recombinant CHO cells using a FACS-based high-throughput screen. Biotechnol. Prog. 23: 465-472.   DOI
7 Dorai, H., B. Csirke, B. Scallon, and S. Ganguly (2006) Correlation of heavy and light chain mRNA copy numbers to antibody productivity in mouse myeloma production cell lines. Hybridoma. 25: 1-9.   DOI
8 Barnes, L. M. and A. J. Dickson (2006) Mammalian cell factories for efficient and stable protein expression. Curr. Opin. Biotechnol. 17: 381-386.   DOI
9 Andersen, D. C. and L. Krummen (2002) Recombinant protein expression for therapeutic applications. Curr. Opin. Biotechnol. 13: 117-123.   DOI
10 Han, S. X., X. Jia, J. L. Ma, and Q. Zhu (2013) Molecular beacons: A novel optical diagnostic tool. Arch. Immunol. 61: 139-148.   DOI
11 Tyagi, S. and F. R. Kramer (1996) Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14: 303-308.   DOI
12 Antony, T. and V. Subramaniam (2001) Molecular beacons: Nucleic acid hybridization and emerging applications. J. Biomol. Struct. Dyn. 19: 497-504.   DOI
13 Deiman, B., C. Schrover, C. Moore, D. Westmoreland, and P. van de Wiel (2007) Rapid and highly sensitive qualitative real-time assay for detection of respiratory syncytial virus A and B using NASBA and molecular beacon technology. J. Virol. Methods. 46: 29-35.
14 Liu, B., X. Yang, K. Wang, and W. Tan (2010) Quantitative detection of p21 mRNA in tumor cells based on molecular beacon. Acta Chim. Sinica. 68: 1303-1307.
15 Nitin, N., P. J. Santangelo, G. Kim, S. Nie, G. Bao (2004) Peptide linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucleic Acids Res. 32: e58.   DOI
16 Perlette, J. and W. Tan (2001) Real-time monitoring of intracellular mRNA hybridization inside single living cells. Anal. Chem. 73: 5544-5550.   DOI
17 Li, Y., X. Zhou, and D. Ye (2008) Molecular beacons: An optimal multifunctional biological probe. Biochem. Biophys. Res. Commun. 373: 457-461.   DOI
18 Monroy-Contreras, R. and L. Vaca (2011) Molecular beacons: Powerful tools for imaging RNA in living cells. J. Nucleic Acids. 2011: 741723.
19 Sum, S. S., D. K. Wong, M. F. Yuen, H. J. Yuan, J. Yu, C. L. Lai, D. Ho, and L. Zhang (2004) Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA. J. Clin. Microbiol. 42: 3438-3440.   DOI
20 Hadjinicolaou, A. V., G. A. Farcas, V. L. Demetriou, T. Mazzulli, S. M. Poutanen, B. M. Willey, D. E. Low, J. Butany, S. L. Asa, K. C. Kain, and L. G. Kostrikis (2011) Development of a molecular-beacon-based multi-allelic real-time RT-PCR assay for the detection of human coronavirus causing severe acute respiratory syndrome (SARS-CoV): A general methodology for detecting rapidly mutating viruses. Arch. Virol. 156: 671-680.   DOI
21 Tsourkas, A., M. A. Behlke, Y. Xu, and G. Bao (2003) Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons. Anal. Chem. 75: 3697-3703.   DOI
22 Santangelo, P. J., B. Nix, A. Tsourkas, and G. Bao (2004) Dual FRET molecular beacons for mRNA detection in living cells. Nucleic Acid Res. 32: e57.   DOI
23 Rhee, W. J., P. J. Santangelo, H. Jo, and G. Bao (2008) Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons. Nucleic Acid Res. 36: e30.   DOI