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Utvärdering av ett nyutvecklat ELISA-kit för analys av fritt kalcidiol: samt dess korrelation med totalt kalcidiol i serum (LC-MS/MS)
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
2017 (Swedish)Independent thesis Basic level (professional degree), 10 credits / 15 HE creditsStudent thesis
Abstract [sv]

Studier har på senare tid visat att D-vitaminbrist är ett vanligare tillstånd än vad som tidigare antagits. Det är det proteinbundna pro-hormonet kalcidiol i serum som analyseras när D-vitaminstatus bedöms. Den största fraktionen av kalcidiol är bundet till plasmaproteiner, men en mycket liten fraktion finns fritt i plasma. Det senaste decenniet har forskningen visat intresse för den fria fraktionen, som möjligen kan vara mer biologiskt relevant för bedömning av D-vitaminstatus. En Enzyme-Linked Immunosorbent Assay (ELISA) metod har lanserats som kan mäta den fria mängden kalcidiol i serum. Syftet med denna studie var att jämföra den nya ELISA-metoden med en tidigare studies resultat, där proteinbundet kalcidiol mättes med metoden LC-MS/MS. I studien deltog 46 individer, 20 män och 26 kvinnor. Medel och standardavikelsen för total mängd kalcidiol var 74,2 nmol/l respektive 18,8 nmol/l och för fritt kalcidiol 5,0 pg/mL respektive 1,4 pg/mL. Resultatet visade att metoden hade en total imprecision (CV%) mellan dubbelproverna på 6,0 %. Den totala imprecisionen för hög kontroll; 26,6 % och låg; 17,8 %. Determinationskoefficienten mellan LC-MS/MS och ELISA var svag, R2 = 0,114. Den svaga korrelationen kan ha orsakats av den mindre spridningen av kalcidiolkoncentrationer i den undersökta gruppen, samt få antal undersökta individer. Vid manuell ELISA är drift och pipetteringsfel inte ovanligt, eftersom analysen omfattar flera manuella steg. Detta kan minimeras med automatisering. Studier av olika sjukdomsgrupper, med varierande D-vitaminstatus krävs och ytterligare analyser av fritt och proteinbundet kalcidiol behövs för att påvisa relevansen med att mäta fritt kalcidiol som en markör för D-vitaminstatus.

Abstract [en]

Studies have recently shown that vitamin D deficiency is a more common condition than previously assumed. It is the protein-bound pro-hormone calcidiol in serum that is analysed when the vitamin D status assessed. The largest fraction of calcidiol is bound to plasma proteins, but a very small fraction calcidiol circulate free in plasma. The last decade’s researches have had an interest in the free fraction which can possibly be more biologically relevant for the assessment of vitamin D status. An Enzyme-Linked Immunosorbent Assay (ELISA) method is introduced that can measure the amount of free calcidiol in serum. The purpose of this study was to compare the new ELISA method with a previous studies result, where in protein bound calcidiol was measured by LC-MS/MS. Study participants were 46 individuals, 20 men and 26 women. The average and standard deviation for total calcidiol was 74.2 nmol/L and 18.8 nmol/L and for free calcidiol it was 5.0 pg/mL and1.4 pg/mL. The results showed that the method had a total imprecision (CV%) between two samples of 6.0 %. The total imprecisionen for the high control was 26.6 % and for the low control it was 17.8 %. The coefficient of determination between the LC-MS/MS and ELISA was weak, R2 = 0.114. The weak correlation might have been caused by the small spread of calcidiol concentrations in the group studied, as well as the limited number of individuals sampled. In a manual ELISA are drift and pipetting errors not unusual, because the analysis includes several manual steps and can be minimized with automation of the method. Studies of various disease groups, with varying vitamin D status and more analyses of free and protein bound calcidiol are needed to establish the relevance of measuring free calcidiol as a marker of vitamin D status.

Place, publisher, year, edition, pages
2017. , 25 p.
Keyword [en]
Free vitamin D, calcidiol, ELISA, imprecision, LC-MS/MS
Keyword [sv]
Fritt vitamin D, kalcidiol, ELISA, imprecision, LC-MS/MS
National Category
Clinical Laboratory Medicine Biomedical Laboratory Science/Technology
Identifiers
URN: urn:nbn:se:lnu:diva-60137OAI: oai:DiVA.org:lnu-60137DiVA: diva2:1068104
External cooperation
Avdelningen för klinisk kemi och transfusionsmedicin, Länssjukhuset i Kalmar
Subject / course
Biomedical Laboratory Science
Educational program
Biomedical Laboratory Science Programme, 180 credits
Supervisors
Examiners
Available from: 2017-02-08 Created: 2017-01-24 Last updated: 2017-02-08Bibliographically approved

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