The study children participated in the Diabetes Prediction and Prevention (DIPP) birth cohort study in Finland. In this study, all newborn infants born at three University hospitals in Finland are first screened for HLA-DQB1 alleles that affect the risk of developing type 1 diabetes, and the children with increased HLA-defined risk are enrolled in the follow-up as described previously . The follow-up includes visits to the study clinic every 3–12 months when serum samples are taken for the screening of disease-associated islet autoantibodies (islet cell antibodies, antibodies to insulin, glutamic acid decarboxylase 65 and the protein tyrosine phosphatase-related IA-2 antigen). Serum samples have been stored frozen at −70°C.
The case children included all 95 children who had turned positive for two or more of the autoantibodies associated with type 1 diabetes (54 had also developed clinical type 1 diabetes) in the Tampere DIPP centre and regularly followed from birth. They were born between 1997 and 2007 (67% were boys); the median age at the first detection of autoantibodies was 3.0 years (range 1.4–8.1 years) and at the diagnosis of type 1 diabetes was 6.3 years (range 1.4–13.2 years). The first autoantibody-positive samples were selected for the analysis of influenza A virus antibodies. One to two autoantibody-negative nondiabetic control participants (n = 186) were selected pairwise for each case child and matched for calendar time of birth (±1 month), sex, date of sampling (±1 month) and HLA-defined risk of type 1 diabetes. No case or control participants were from birth cohorts that received influenza A vaccination in the national programme and no influenza vaccinations were reported in questionnaires during the follow-up.
All children had written parental consent and the Ethics Committee of Tampere University Hospital had granted approval for the DIPP study. The study was carried out in accordance with the principles of the Declaration of Helsinki.
Serum IgG class antibodies to influenza A virus were analysed using an enzyme immunoassay. The wells of a microtitre plate (Immunoplate II; Nunc, Roskilde, Denmark) were coated overnight at room temperature with 50 μl of influenza A antigen (3 μg/ml per well of concentrated viral matrix and nucleoproteins of Beijing strain; Virion Serion, Wurzburg, Germany; product code BA1231VS), then washed four times with washing buffer (PBS + 0.5% Tween) and blocked by 0.1% BSA in PBS for 30 min at room temperature. Serum was incubated for 1 h at 37°C in 1/200 dilution in PBS + 1% BSA + 0.5 mol/l NaCl + 0.05% Tween 20, followed by washings and incubation of polyclonal rabbit anti-human IgG conjugate (P0214, Dako, Glostrup, Denmark) in 1/3,000 dilution for 30 min at 37°C. Finally, tetramethylbenzidine substrate (OPD-tablet, citrate buffer, H2O2) was added and allowed to react for 15 min in room temperature. The reaction was stopped by adding 100 μl/well of 1 N H2SO4, and the results were read at 490 nm in a spectrophotometer (Victor2 Perkin Elmer Wallac 1420 Multilabel Counter, Waltham, Massachusetts, USA). Samples from case and corresponding control children were tested blindly in the same test run. The antibody results are given as EIU (enzyme immunoassay units), which are calculated using the following formula: (absorbance of the tested sample – absorbance of negative control sample)/(absorbance of positive control sample – absorbance of negative control sample) × 100. An EIU value of 10 was used as a cut-off for seropositivity. The assay conditions were optimised using paired sera from ten patients with confirmed acute influenza A virus infection (collaboration with T. Ziegler, National Institute for Health and Welfare, Helsinki, Finland). These sera showed a clear increase in median IgG EIU levels between acute and convalescent sera (from 52 to 170 EIU; p < 0.001).
IgG prevalence in cases and controls was analysed using conditional logistic regression (Stata 12.1. statistical program, StataCorp, College Station, Texas, USA, and R version 3.1.2., https://www.r-project.org/). A p value of less than 0.05 was set as an indicator of statistical significance. Sample size was estimated by a priori power calculations based on results from previous publications.