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Antibody variable-region sequencing as a method for hybridoma cell-line authentication

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Abstract

Cross-contamination and misidentification of various cell lines is a widespread problem that can lead to spurious scientific conclusions. DNA fingerprinting is a powerful identification technique, which can be effectively used for the authentication of human cell lines. In contrast to human cancer cell lines, little attention has so far been given to establishing authentication practices for hybridoma cell lines. Since the majority of hybridomas stem from inbred animals, they have high genetic uniformity, which reduces the applicability of DNA fingerprinting. In the present study, we propose antibody variable-region sequencing as a method of choice for hybridoma cell-line authentication. This method focuses on the most diverse characteristic of hybridoma cell lines and thereby achieves a very high discriminatory power. The sequencing of light-chain variable regions has proven to be especially suitable for routine use because of its high success rate. Two other possible authentication methods, random amplified polymorphic DNA analysis and two-dimensional gel electrophoresis, were also examined. Compared to these and other methods that can be used for discrimination between hybridoma cell lines, variable-region sequencing has many advantages, most notably those of a very high discriminatory power, insensitivity to changes in experimental conditions, simple data analysis, and accessibility to most laboratories.

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References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Bennett D (1965) The karyotype of the mouse, with identification of a translocation. Proc Natl Acad Sci U S A 53:730–737

    Article  CAS  Google Scholar 

  • Chatterjee R (2007) Cell biology. Cases of mistaken identity. Science 315:928–931

    Article  CAS  Google Scholar 

  • Dirks WG, Drexler HG (2005) Authentication of scientific human cell lines: easy-to-use DNA fingerprinting. Methods Mol Biol 290:35–50

    CAS  PubMed  Google Scholar 

  • Drexler HG, Dirks WG, Matsuo Y, MacLeod RA (2003) False leukemia-lymphoma cell lines: an update on over 500 cell lines. Leukemia 17:416–426

    Article  CAS  Google Scholar 

  • Fernandes DL (2005) Demonstrating comparability of antibody glycosylation during biomanufacturing. Eur Biopharm Rev Summer 2005:106–110

    Google Scholar 

  • Gartler SM (1968) Apparent Hela cell contamination of human heteroploid cell lines. Nature 217:750–751

    Article  CAS  Google Scholar 

  • Gilbert DA, Reid YA, Gail MH, Pee D, White C, Hay RJ, O, Brien SJ (1990) Application of DNA fingerprints for cell-line individualization. Am J Hum Genet 47:499–514

    CAS  PubMed  PubMed Central  Google Scholar 

  • Guo HR, Zhang SC, Tong SL, Xiang JH (2001) Analysis of three marine fish cell lines by rapd assay. In Vitro Cell Dev Biol Anim 37:430–433

    Article  CAS  Google Scholar 

  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98

    CAS  Google Scholar 

  • Hampe J, Nurnberg P, Epplen C, Jahn S, Grunow R, Epplen JT (1992) Oligonucleotide fingerprinting as a means to identify and survey long-term cultured B cell hybridomas and T cell lines. Hum Antibodies Hybridomas 3:186–190

    Article  CAS  Google Scholar 

  • Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Honjo T, Alt FW (1995) Immunoglobulin genes, 2nd edn. Academic, New York

    Google Scholar 

  • Jentsch I, Adler ID, Carter NP, Speicher MR (2001) Karyotyping mouse chromosomes by multiplex-FISH (M-FISH). Chromosome Res 9:211–214

    Article  CAS  Google Scholar 

  • Lacroix M (2008) Persistent use of “false” cell lines. Int J Cancer 122:1–4

    Article  CAS  Google Scholar 

  • Lee JY, Lee CH, Shim SH, Seo HK, Kyhm JH, Cho S, Cho YH (2002) Molecular cytogenetic analysis of the monoblastic cell line U937. Karyotype clarification by G-banding, whole chromosome painting, microdissection and reverse painting, and comparative genomic hybridization. Cancer Genet Cytogenet 137:124–132

    Article  CAS  Google Scholar 

  • Lery X, LaRue B, Cossette J, Charpentier G (2003) Characterization and authentication of insect cell lines using RAPD markers. Insect Biochem Mol Biol 33:1035–1041

    Article  CAS  Google Scholar 

  • Liyanage M, Coleman A, du Manoir S, Veldman T, McCormack S, Dickson RB, Barlow C, Wynshaw-Boris A, Janz S, Wienberg J, Ferguson-Smith MA, Schrock E, Ried T (1996) Multicolour spectral karyotyping of mouse chromosomes. Nat Genet 14:312–315

    Article  CAS  Google Scholar 

  • MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG (1999) Widespread intraspecies cross-contamination of human tumor cell lines arising at source. Int J Cancer 83:555–563

    Article  CAS  Google Scholar 

  • Manniello A, Aresu O, Parodi B, Romano P, Iannotta B, Rondanina G, Viegi L, Ruzzon T (1993) The cell line data base and the new catalogue: detailed information on 2650 human and animal cell lines. Cytotechnology 11(Suppl 1):S130–S133

    Article  CAS  Google Scholar 

  • Masters JR, Thomson JA, Daly-Burns B, Reid YA, Dirks WG, Packer P, Toji LH, Ohno T, Tanabe H, Arlett CF, Kelland LR, Harrison M, Virmani A, Ward TH, Ayres KL, Debenham PG (2001) Short tandem repeat profiling provides an international reference standard for human cell lines. Proc Natl Acad Sci U S A 98:8012–8017

    Article  CAS  Google Scholar 

  • Nardone RM (2007) Eradication of cross-contaminated cell lines: a call for action. Cell Biol Toxicol 23:367–372

    Article  Google Scholar 

  • Nelson-Rees WA, Flandermeyer RR, Hawthorne PK (1974) Banded marker chromosomes as indicators of intraspecies cellular contamination. Science 184:1093–1096

    Article  CAS  Google Scholar 

  • Nelson-Rees WA, Daniels DW, Flandermeyer RR (1981) Cross-contamination of cells in culture. Science 212:446–452

    Article  CAS  Google Scholar 

  • O’Brien SJ, Kleiner G, Olson R, Shannon JE (1977) Enzyme polymorphisms as genetic signatures in human cell cultures. Science 195:1345–1348

    Article  Google Scholar 

  • Parson W, Kirchebner R, Muhlmann R, Renner K, Kofler A, Schmidt S, Kofler R (2005) Cancer cell line identification by short tandem repeat profiling: power and limitations. FASEB J 19:434–436

    Article  CAS  Google Scholar 

  • Perkins M, Theiler R, Lunte S, Jeschke M (2000) Determination of the origin of charge heterogeneity in a murine monoclonal antibody. Pharm Res 17:1110–1117

    Article  CAS  Google Scholar 

  • Tanke HJ, Wiegant J, van Gijlswijk RP, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J (1999) New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet 7:2–11

    Article  CAS  Google Scholar 

  • Thammana P (1994) Characterization of a deletion in the P3 myeloma heavy chain V region sequence in the non-secreting cell line NS1. Mol Immunol 31:77–78

    Article  CAS  Google Scholar 

  • Tonegawa S (1988) Nobel lecture in physiology or medicine—1987. Somatic generation of immune diversity. In Vitro Cell Dev Biol 24:253–265

    Article  CAS  Google Scholar 

  • Wang Z, Raifu M, Howard M, Smith L, Hansen D, Goldsby R, Ratner D (2000) Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3¢ to 5¢ exonuclease activity. J Immunol Methods 233:167–177

    Article  CAS  Google Scholar 

  • Witmer PD, Doheny KF, Adams MK, Boehm CD, Dizon JS, Goldstein JL, Templeton TM, Wheaton AM, Dong PN, Pugh EW, Nussbaum RL, Hunter K, Kelmenson JA, Rowe LB, Brownstein MJ (2003) The development of a highly informative mouse Simple Sequence Length Polymorphism (SSLP) marker set and construction of a mouse family tree using parsimony analysis. Genome Res 13:485–491

    Article  CAS  Google Scholar 

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Acknowledgments

We would like to thank Prof. Dr. M. Dolinar for insightful discussions, Dr. G. Kosec and L. Bojič for assistance with the 2D electrophoresis, and Dr. C. P. Berrie for critical reading of the manuscript. We also thank G. Wöstemeyer who programmed the RAPD-primer generator. This work was funded by Slovenian Research Agency grants L6-6006 and P4-0176 and Ph.D. grants for S.K., M.K., and A.C.V. The work of S.M. was supported by DiaMed AG, Cressier, Switzerland.

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Authors and Affiliations

  1. Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Šlajmerjeva 6, 1000, Ljubljana, Slovenia

    Simon Koren, Miha Kosmač, Anja Colja Venturini, Sendi Montanič & Vladka Čurin Šerbec

  2. Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, 1000, Ljubljana, Slovenia

    Simon Koren, Miha Kosmač & Vladka Čurin Šerbec

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  1. Simon Koren
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  2. Miha Kosmač
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  3. Anja Colja Venturini
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  4. Sendi Montanič
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  5. Vladka Čurin Šerbec
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Correspondence to Vladka Čurin Šerbec.

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Simon Koren and Miha Kosmač contributed equally to this work.

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253_2008_1386_MOESM1_ESM.doc

Table S1 Light-chain variable-region sequence identity matrix for 21 out of the 39 cell lines used in the present study. Multiple sequence alignments and sequence identity matrix calculations were performed using the BioEdit program. The conditional formatting option of the Microsoft Office Excel program was used to quickly identify pairs of sequences with identities higher than 0.96 (depicted in bold). Note that the VL of the suspected contaminated cell line, 6.1, was shown to be completely identical to that of cell line 1.1 (DOC 72 kb)

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Koren, S., Kosmač, M., Colja Venturini, A. et al. Antibody variable-region sequencing as a method for hybridoma cell-line authentication. Appl Microbiol Biotechnol 78, 1071–1078 (2008). https://doi.org/10.1007/s00253-008-1386-5

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  • DOI: https://doi.org/10.1007/s00253-008-1386-5

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