Cotinine elisa

All serum samples were divided into aliquots and frozen at -80°c within 2 hours of blood draw. Peripheral blood mononuclear cells were separated and stored in liquid nitrogen on the day of blood draw. Analysis was performed in bulk on the day of thawing. Samples were not subject to repeated freeze thaw cycles.
Anti-EBNA-1 IgG titres were measured using a commercially available ELISA (DiaSorin; Salugia, Italy). Current smoking status was assessed using a commercially available cotinine ELISA (Calbiotech; California, USA), a concentration of 3.08ng/ml was used to define current smokers [25 (link)]. Serum 25-OHvD levels were measured using liquid chromatography-tandem mass spectrometry (Department of Clinical Biochemistry, Royal London Hospital, Whitechapel). Serum 25-OHvD was deseasonalised to the day of sampling according to standard methods [26 (link)].
DNA was isolated from whole blood prior to genotyping using the Illumina immunochip [27 (link)]. The immunochip provides SNP data for all SNPs associated with MS and a number of other autoimmune diseases in GWAS; SNP data for the MS-associated alleles was isolated for each individual and analysed.

Nicotine-exposed pups were obtained from pregnant dams who were exposed to chronic, episodic nicotine through drinking water starting on embryonic day (E)5. This results in fetal exposure by the placental circulation in utero, and through breast milk after birth. Nicotine at a concentration of 0.08 mg/ml of tap water was mixed with 10 mg/ml of saccharin for palatability. The nicotine-saccharin water mixture was prepared fresh and replaced every 3 d. This dose of nicotine produced plasma cotinine (a stable metabolic by-product of nicotine) levels in the pups ranging from ∼50–100 ng/ml; however, there were samples at each age where cotinine levels were higher than the measurable limit of the assay (Cotinine ELISA, CalBiotech). This range of plasma cotinine levels is similar to what we have previously observed in neonates exposed to chronic, sustained nicotine via an osmotic minipump set to deliver a nicotine dose of 6 mg/kg/d (60–92 ng cotinine/ml plasma; Powell et al., 2015 (link)). These cotinine levels are also comparable to what is found in the plasma of human infants born to mothers who are considered moderate smokers (Berlin et al., 2010 (link)). Control pups were obtained from pregnant dams who were given saccharin water only.