Setting up of immunoassay for salivary carbonic anhydrase isoenzyme 6 (CA6), a putative biomarker for dental caries prediction

Karies er et stort globalt helseproblem som rammer 60-90% av skolebarna og nesten alle voksne. Anslagsvis 2 millioner nordmenn har ubehandlet karies. Tilskudd av individuell karies er assosiert med spyttkvalitet, spesielt på mengden spytt karboanhydrase isoenzym 6 (CA6). CA6-konsentrasjoner har 500 ganger variasjon fra en pasient til en annen. CA6-konsentrasjon er assosiert med karies utvikling men disse studier er småskala eller tverrsnitts (bare en tidspunkt). For å tydeliggjøre videre effekten av CA6 på kariesutvikling, har vi mottatt prøver fra en stor oppfølgingsstudie der pasienter har donert spytt og deltatt i tannlege undersøkelser under 8 år. Vi har satt opp et anlegg for å måle CA6-konsentrasjoner i spytt. Neste skal vi måle CA6-konsentrasjoner av oppfølgingsstudien og sammenligne konsentrasjonene med kariesutvikling til pasientene. Dette ville være første gang spyttproteinkonsentrasjoner er assosiert med kariesutvikling i en storstilt oppfølgingsstudie. Samtidig bestemmer vi den daglige og månedlige stabiliteten i spyttkvalitet. Vi har også utviklet to spesifikke monoklonale antistoffer mot CA6 som skal brukes i fremtidige forskningsprosjekter.

We have established immunoassay facility to measure concentrations of human salivary CA6. We have produced two monoclonal antibodies to human CA6 using purified human salivary CA6 provided by a collaborator. We have received REK and NSD approvals to collect saliva samples for determining the optimal time of day and methodology to collect saliva samples for CA6 measurements. We have received REK and NSD approvals plus samples from a collaborator to determine monthly intra-individual variation in salivary CA6 concentrations during a 1-year follow-up. We have agreed to a collaboration where we will receive saliva samples of a 18-year follow-up. In these samples we determine gender and age variation of CA6. Of these samples we will be able to determine also the association of CA6 concentration with caries increment. We have received REK and NSD approvals for this project.

Project summary Caries is a major oral health problem affecting 60-90% of school children and nearly all adults. If left untreated caries will progress and decrease dramatically oral-health related quality of life. Accurate caries prediction would enable efficient personalized dental care according to individuals’ needs and, thus, decrease costs to society and health disparities. Unfortunately, the existing caries prediction tools have shown only marginal clinical utility. Mice genetically modified to lack salivary carbonic anhydrase isoenzyme VI (CA6) have 75% lower caries increment than the wild-type mice. The CA6 concentration in human saliva show wide inter-individual variation from 0.1 mg/L to 16 mg/L. Therefore we plan on including patients' salivary CA6 concentration in caries prediction multivariable model to accurately predict caries increment of an individual. We will apply for ethical approval, collect saliva samples, purify CA6 from saliva, produce monoclonal antibodies and use them to set up an immunoassay for salivary CA6 concentrations. The monoclonal antibodies are needed later on A) to produce a commercial lateral flow rapid test for classifying patients into low and high risk for caries according to salivary CA6 concentrations and B) in large scale studies on salivary CA6 concentrations. Meanwhile we produce the monoclonal antibodies we will use commercially available CA6 assay kit to optimize saliva collection methodology for CA6 measurements and determine salivary CA6 concentrations in patients of different ages and genders. Once we have our own immunoassay set up and know the optimal saliva collection method we will use for the main project: i) to determine optimal cut-off point for CA6 concentrations to classify patients into high and low risk for caries, ii) validate the rapid. We will also launch an application for multivariable caries prediction.