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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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.
Simon Koren and Miha Kosmač contributed equally to this work.
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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
- 2D electrophoresis
- Cell-line contamination