Separation of Lactoferrin from Human Saliva Using Monolithic Disc |
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Authors: | Zitka Ondrej Skalickova Sylvie Krizkova Sona Vlkova Marcela Adam Vojtech Kizek Rene |
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Institution: | 1.Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, 613 00, Brno, Czech Republic ;2.Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, 616 00, Brno, Czech Republic ;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 ;4.Department of Clinical Immunology and Allergology, Anne’s University Hospital, Masaryk University, Pekarska 53, 656 91, Brno, Czech Republic ; |
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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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