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Received on April 11, 2005
Accepted on July 8, 2005
Automation and Analytical Techniques |
1 Infection and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany
2 Infections and Cancer Epidemiology, International Agency for Research on Cancer (IARC), Lyon, France
3 MediGene AG, Martinsried, Germany
4 Natural and Medical Sciences Institute (NMI), University of Tübingen, Reutlingen, Germany
* To whom correspondence should be addressed. E-mail: t.waterboer{at}dkfz.de.
Background: More than 100 different human papillomaviruses (HPVs) can cause proliferative diseases, many of which are malignant, such as cervical cancer. HPV serology is complex because infection and disease lead to distinct type-specific antibody responses. Using bead-based technology, we have developed an assay platform that allows the simultaneous detection of antibodies against up to 100 in situ affinity-purified recombinant HPV proteins.
Methods: Twenty-seven HPV proteins were expressed as glutathione S-transferase fusion proteins and affinity-purified in one step by incubation of glutathione-displaying beads in bacterial lysate. Beads were sorted into spectrally distinct sets, each carrying one particular antigen, and then mixed, incubated with serum, and differentiated in a flow cytometer-like analyzer (xMAP; Luminex Corp). Antibodies bound to the antigens were detected via fluorescent secondary reagents. We studied 756 sera from 2 case-control studies of cervical cancer.
Results: Glutathione S-transferase fusion proteins bound with high affinity to glutathione-displaying beads (Kd = 6.9 x 10-9 mol/L). The dynamic range of multiplex serology covered 1.5 orders of magnitude, and antibodies were detected at serum dilutions >1:1 000 000. Imprecision (median CV) was 
5.4%, and assay reproducibility was high (R2 = 0.97). Results on clinical samples showed high concordance with ELISA (
= 0.846), but multiplex serology exhibited increased detection of weak antibody responses. Antibodies to the E6 oncoproteins of the rare HPV types 52 and 58 were associated with cervical cancer (P <0.001).
Conclusion: Multiplex serology enables antibody analyses of large numbers of sera against up to 100 antigens in parallel and has the potential to replace ELISA technology.
The following articles in journals at HighWire Press have cited this article:
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  T. Senger, M. R. Becker, L. Schadlich, T. Waterboer, and L. Gissmann Identification of B-Cell Epitopes on Virus-Like Particles of Cutaneous Alpha-Human Papillomaviruses J. Virol., December 15, 2009; 83(24): 12692 - 12701. [Abstract] [Full Text] [PDF]
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 T. Waterboer, R. Neale, K. M. Michael, P. Sehr, M. N. C. de Koning, S. J. Weissenborn, F. Sampogna, D. Abeni, A. C. Green, J. N. Bouwes Bavinck, et al. Antibody responses to 26 skin human papillomavirus types in the Netherlands, Italy and Australia J. Gen. Virol., August 1, 2009; 90(8): 1986 - 1998. [Abstract] [Full Text] [PDF]
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 L. Gao, A. Michel, M. N. Weck, V. Arndt, M. Pawlita, and H. Brenner Helicobacter pylori Infection and Gastric Cancer Risk: Evaluation of 15 H. pylori Proteins Determined by Novel Multiplex Serology Cancer Res., August 1, 2009; 69(15): 6164 - 6170. [Abstract] [Full Text] [PDF]
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S. Syrjanen, T. Waterboer, M. Sarkola, K. Michael, M. Rintala, K. Syrjanen, S. Grenman, and M. Pawlita Dynamics of human papillomavirus serology in women followed up for 36 months after pregnancy J. Gen. Virol., June 1, 2009; 90(6): 1515 - 1526. [Abstract] [Full Text] [PDF]
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L. Gao, M. N. Weck, A. Michel, M. Pawlita, and H. Brenner Association between Chronic Atrophic Gastritis and Serum Antibodies to 15 Helicobacter pylori Proteins Measured by Multiplex Serology Cancer Res., April 1, 2009; 69(7): 2973 - 2980. [Abstract] [Full Text] [PDF]
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 B. Dondog, G. M. Clifford, S. Vaccarella, T. Waterboer, D. Unurjargal, D. Avirmed, S. Enkhtuya, F. Kommoss, N. Wentzensen, P. J.F. Snijders, et al. Human Papillomavirus Infection in Ulaanbaatar, Mongolia: A Population-Based Study Cancer Epidemiol. Biomarkers Prev., July 1, 2008; 17(7): 1731 - 1738. [Abstract] [Full Text] [PDF]
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R. Z. Rizk, N. D. Christensen, K. M. Michael, M. Muller, P. Sehr, T. Waterboer, and M. Pawlita Reactivity pattern of 92 monoclonal antibodies with 15 human papillomavirus types J. Gen. Virol., January 1, 2008; 89(1): 117 - 129. [Abstract] [Full Text] [PDF]
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K. Andersson, T. Waterboer, R. Kirnbauer, K. Slupetzky, T. Iftner, E.-M. de Villiers, O. Forslund, M. Pawlita, and J. Dillner Seroreactivity to Cutaneous Human Papillomaviruses among Patients with Nonmelanoma Skin Cancer or Benign Skin Lesions Cancer Epidemiol. Biomarkers Prev., January 1, 2008; 17(1): 189 - 195. [Abstract] [Full Text] [PDF]
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 D. A.M. Heideman, T. Waterboer, M. Pawlita, P. Delis-van Diemen, I. Nindl, J. A. Leijte, J. M.G. Bonfrer, S. Horenblas, C. J.L.M. Meijer, and P. J.F. Snijders Human Papillomavirus-16 Is the Predominant Type Etiologically Involved in Penile Squamous Cell Carcinoma J. Clin. Oncol., October 10, 2007; 25(29): 4550 - 4556. [Abstract] [Full Text] [PDF]
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G. M. Clifford, H.-R. Shin, J.-K. Oh, T. Waterboer, Y.-H. Ju, S. Vaccarella, W. Quint, M. Pawlita, and S. Franceschi Serologic Response to Oncogenic Human Papillomavirus Types in Male and Female University Students in Busan, South Korea Cancer Epidemiol. Biomarkers Prev., September 1, 2007; 16(9): 1874 - 1879. [Abstract] [Full Text] [PDF]
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 A. J. Johnson, R. C. Cheshier, G. Cosentino, H. P. Masri, V. Mock, R. Oesterle, R. S. Lanciotti, D. A. Martin, A. J. Panella, O. Kosoy, et al. Validation of a Microsphere-Based Immunoassay for Detection of Anti-West Nile Virus and Anti-St. Louis Encephalitis Virus Immunoglobulin M Antibodies Clin. Vaccine Immunol., September 1, 2007; 14(9): 1084 - 1093. [Abstract] [Full Text] [PDF]
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 P. Sehr, M. Pawlita, and J. Lewis Evaluation of Different Glutathione S-Transferase-Tagged Protein Captures for Screening E6/E6AP Interaction Inhibitors Using AlphaScreen(R) J Biomol Screen, June 1, 2007; 12(4): 560 - 567. [Abstract] [PDF]
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 M. R. Karagas, H. H. Nelson, P. Sehr, T. Waterboer, T. A. Stukel, A. Andrew, A. C. Green, J. N. Bouwes Bavinck, A. Perry, S. Spencer, et al. Human papillomavirus infection and incidence of squamous cell and basal cell carcinomas of the skin. J Natl Cancer Inst, March 15, 2006; 98(6): 389 - 395. [Abstract] [Full Text] [PDF]
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 M. Schmitt, I. G. Bravo, P. J. F. Snijders, L. Gissmann, M. Pawlita, and T. Waterboer Bead-Based Multiplex Genotyping of Human Papillomaviruses J. Clin. Microbiol., February 1, 2006; 44(2): 504 - 512. [Abstract] [Full Text] [PDF]
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 J. Dillner Toward "Serolomics": Papillomavirus Serology Is Taking a Technologic Lead in High-Throughput Multiplexed Antibody Analysis Clin. Chem., October 1, 2005; 51(10): 1768 - 1769. [Full Text] [PDF]
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