Cotinine direct elisa kit

For older children, urine was collected in laboratory specimen jars. For infants, cotton balls in the infant’s diaper were used to collect urine and then centrifuged to collect the urine from the cotton balls. Urine cotinine was measured by ELISA (CalBiotech Cotinine Direct ELISA Kit). Creatinine was measured using a colorimetric assay (Cayman Chemical Co. Ann Arbor, MI, USA) as per manufacturer’s instructions. A cutoff of 30 ng/mg cotinine/creatinine or above was used as an indicator of passive smoking [15 (link),16 (link)].

Human fetal lung tissue samples were collected as part of a prenatal tissue retrieval program sponsored by the National Institute of National Child Health and Development, the University of Maryland Brain and Tissue Bank for Developmental Disorders (Baltimore, MD), and the Center for Birth Defects Research (University of Washington; Seattle, WA). The study was designated an institutional review board (IRB) exempt protocol by the University of Missouri-Kansas City Pediatric IRB, Partners Human Research Committee IRB, and the Colorado Multiple Institutional Review Board (COMIRB). Due to sample de-identification, limited maternal and fetal phenotypic characteristics were available for each sample including gestational age and sex. Sample sex was previously confirmed based on paired gene expression data (Kho et al., 2016a (link)) by classifying samples as female or male based on their expression of X- and Y-chromosome genes. The age of samples was determined using estimated days post-conception. Intrauterine cigarette smoke exposure (based on placental cotinine concentration) (Vyhlidal et al., 2013 (link)) is a well-known confounder of fetal lung development and was directly measured in the samples using the Cotinine Direct ELISA kit (Calbiotech, Spring Valley, CA). Unmeasured confounders were accounted for using the RUV method described below in miRNA profiling.

For the objective assessment, the concentration of cotinine in urine was measured using an ELISA method, the Calbiotech Cotinine Direct ELISA kit. The procedure was performed according to the instructions of the manufacturer. Briefly, 10 µL of standards and samples were transferred into the microtiter plate. Thereafter, 100 µL of enzyme conjugate was added to all wells. The whole plate was briefly mixed and subsequently incubated for 60 min at room temperature. After incubation, the content of the plate was discarded, and the plate was 6 times washed using distilled water in a volume of 350 µL/well. Then, 100 µL of substrate reagent was added to all wells, and the covered plate was incubated again at room temperature for 30 min. After incubation, the reaction was stopped using 100 µL of Stop reagent. The absorbance was measured at wavelengths of 450 nm and 650 nm.

Human fetal lung tissue samples were collected as part of a prenatal tissue retrieval program sponsored by the National Institute of National Child Health and Development (NICHD), the University of Maryland Brain and Tissue Bank for Developmental Disorders (Baltimore, MD, USA), and the Center for Birth Defects Research (University of Washington; Seattle, WA, USA), as previously described [37 (link)]. The study was designated an institutional review board (IRB) exempt protocol by the University of Missouri-Kansas City Pediatric IRB, Partners Human Research Committee IRB, and the Colorado Multiple Institutional Review Board (COMIRB).
Due to de-identification, only estimated post-gestational age, IUS, and sex were available for each sample. We assessed IUS with placental cotinine concentration (Cotinine Direct ELISA kit, Calbiotech, Spring Valley, CA, USA), using an assay our group previously demonstrated had high sensitivity and specificity (AUC = 0.88), relative to self-reported maternal cigarette smoking status [36 (link)]. The samples are a subset of previously published work profiling human fetal lung gene expression [66 (link)], in which we classified sample sex as male or female based on their expression of X- and Y-chromosome genes in Affymetrix Human Gene 1.0 ST microarrays.

Calbiotech Cotinine Direct ELISA Kit (Calbiotech Inc., El Cajonm, CA, USA) was used to measure the cotinine concentrations at Centre of Arctic Health and Molecular Epidemiology, Aarhus University, Denmark. Levels were given in ng/mL and the LOD was 1.0 ng/mL. If the values were below the LOD, 0.5 ng/mL was used for the statistical analysis.