Od donations took place, which means that at least 92 of donors
Od donations took place, which means that at least 92 of donors agreed to be included. From this sample cohort, 50 men and 50 women from each 10-year age span period, between 20 and 70 years of age, were randomly included. As few blood donors above 60 years of age, only 16 women and 40 men were included in the age group 60?0 years. In total, 456 controls were examined during the same time period as the patients. From this cohort, two age- and gender-matched controls were randomly extracted for each patient in this study.Statistical analysesBlood samples were drawn from patients and the serum was separated and kept frozen at -20 until analyzed. Analysis of anti-GnRH antibodies was carried out by an ELISA slightly modified on the basis of the results described in previous studies [8,16]. The wells of micro titer plates were coated with human GnRH (L7134, Sigma, St Louis, MO, USA) for an overnight incubation at 4 and PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27196668 thereafter the plastic wells were blocked with 0.5 fish gel solution (G7765, Sigma) in PBS containing 0.05 Tween20 (PBS-T). Serial dilutions of patient serum (1/100, 1/500 and 1/2500 in PBS-T) were then added to the plates and incubated for 2 h at room temperature (RT) and overnight at 4 . After rinsing with PBS-T, deposition of autoantibodies directed to GnRH was detected using biotinylated rabbit anti-human IgM (673211, MP Biomedicals, Solon, OH, USA) or IgG antibodies (ab7159, ABcam, Cambridge, MA, USA) appropriately diluted in PBS-T. After another incubation for 2 h at RT, the plates were washed and the bound, biotinylated antibodies detected by alkaline phosphatase-conjugated streptavidin (405211, Biolegend, San Diego, CA, USA), incubated for 1 h at RT. To develop a color reaction a phosphatase substrate kit (37620, Pierce,The data were analyzed using the statistical software package SPSS for Windows?(Release 20.0; IBM). All variables were analyzed for normal distribution by Kolmogorov-Smirnov test. Group-wise differences between patients and controls were tested by using the unpaired Student’s t-test and, when normality was rejected (antibody levels), the Mann-Whitney U-test was used. Fisher’s exact test was used for dichotomized variables. Correlations were calculated by Spearman rank correlation test. Values are expressed as mean ?standard deviation (SD) or median, interquartile range (IQR). Binary logistic regression analysis was performed to test for an association between the presence of antibodies against GnRH (dependent variable) and clinical findings in patients. Independent PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27864321 variables were age (years), gender, type of diabetes, duration of disease (years), BMI (kg/m2), HbA1c (mmol/mol), esophageal and gastric dysmotility, secondary complications, and symptoms, examined as univariate analysis. No presence of antibodies, symptoms or pathological findings was used as ��-Amanitin site reference. P < 0.05 was considered statistically significant.Berntorp et al. BMC Research Notes 2013, 6:329 http://www.biomedcentral.com/1756-0500/6/Page 4 ofResultsPatient characteristicsmajority of patients reported some sort of symptoms related to the gastrointestinal tract and food intake (Table 2).Antibodies against gonadotropin-releasing hormoneThirty-nine patients with diabetes mellitus (27 females), age 52.4 ?12.5 years, were included. All the patients were insulin-treated, and were in acceptable metabolic control (Table 1). Esophageal dysmotility was more common (69 ) than gastroparesis (18 ). Retinopathy was the most common diabetes compl.