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Separation of Lactoferrin from Human Saliva Using Monolithic Disc

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Abstract

In this study, we optimized method for the isolation and detection of lactoferrin from human saliva using 3 mm short monolithic disc. We optimized the conditions for separation as flow rate 4 mL min−1 and ionic strength of effluent as 2 M·NaCl. We estimated limit of detection of whole method, which was hyphenated to the Bradford’s assay, down to 100 ng mL−1. The purity of the isolated fractions was verified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and recovery of isolation was found to be 51 % using minimally processed sample of saliva. Further, we tested the optimized method on group of healthy volunteers (n = 7). We were able to distinguish between the healthy subjects and subject suffering from celiac disease, which reported at least 2.5× higher level of lactoferrin in comparison to healthy ones. The results were correlated with standard enzyme-linked immunosorbent assay (ELISA) kit with obtained correlation coefficient R 2 = 0.8446. Analysis of lactoferrin in saliva by monolithic disc and subsequent offline photometric detection is faster and cheaper method compared to ELISA commercial kit. The total analysis of one sample takes <20 min.

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Acknowledgments

Financial support from the grants CEITEC CZ.1.05/1.1.00/02.0068 and NanoBioTECell GA CR P102/11/1068 is highly acknowledged.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czech Republic

    Ondrej Zitka, Sylvie Skalickova, Sona Krizkova, Vojtech Adam & Rene Kizek

  2. Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00, Brno, Czech Republic

    Ondrej Zitka, Sona Krizkova, Vojtech Adam & Rene Kizek

  3. Department of Veterinary Ecology and Environmental Protection, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42, Brno, Czech Republic

    Ondrej Zitka

  4. Department of Clinical Immunology and Allergology, Anne’s University Hospital, Masaryk University, Pekarska 53, 656 91, Brno, Czech Republic

    Marcela Vlkova

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  1. Ondrej Zitka
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  2. Sylvie Skalickova
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  3. Sona Krizkova
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  4. Marcela Vlkova
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  5. Vojtech Adam
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Correspondence to Rene Kizek.

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Zitka, O., Skalickova, S., Krizkova, S. et al. Separation of Lactoferrin from Human Saliva Using Monolithic Disc. Chromatographia 76, 611–619 (2013). https://doi.org/10.1007/s10337-013-2459-x

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  • DOI: https://doi.org/10.1007/s10337-013-2459-x

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