Sandwich elisa

The concentration of IgA and/or IgM in SMG, GW, CC, and feces were determined by a sandwich ELISA (Bethyl Laboratories Inc., Montgomery, TX, United States), as previously explained (36 (link)). Absorbance was quantified on a microplate photometer (Labsystem Multiskan, Helsinki, Finland) and data were interpolated by Ascent v.2.6 software (Thermo Fisher Scientific) according to the respective standard curves. The Ig content in SMG, CC and feces was normalized by the total protein concentration, which was determined using the Pierce-660 nm ready-to-use Protein Assay Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions.

Total immunoglobulin IgG, IgA, IgM isotypes levels were measured in serum samples by sandwich ELISA (Bethyl Laboratories, Montgomery, TX, USA), according to manufacturer’s instructions. In total, serum samples from 534 mice were measured. Also, six additional traits were calculated as linear combinations of the log-transformed and standardized individual immunoglobulin isotype values (IgA, IgG and IgM)^{44 (link)}. For general immunoglobulin production capacity, we calculated total immunoglobulin AGM as (log(IgA) + log(IgG) + log(IgM)). The efficacy of class switching was defined by AG (log(IgA) + log(IgG)), while the ratio of class-switch to non-class-switch immunoglobulins was measured as AG/M ((log(IgA) + log(IgG)) − log(IgM)). Isotype-specific class switching was calculated by A/M (log(IgA) − log(IgM)) and G/M (log(IgG) − log(IgM)). The opposite direction on the two isotypes was captured by A/G (log(IgA) − log(IgG)).

Immunoglobulin G and M (IgG and IgM) concentrations in serum and supernatants from non-stimulated spleen cells were determined using a sandwich ELISA (Bethyl Laboratories, Montgomery, TX, United States) following the manufacturer’s instructions. Data were analyzed with Ascent v.2.6 software (Thermo Fisher Scientific, S.L.U, Barcelona, Spain) using the respective standard curves. Changes in immunoglobulin concentrations are represented considering the SED group values as 100%.

The animals were weighed 3 times weekly on a digital scale and the abatacept dose adjusted to current body weight. Blood glucose was measured three times per week for the first two weeks and subsequently once per week on Biosen 5040. Blood glucose samples were taken in non-fasted mice before dosing. HbA1c% was assessed every three weeks starting one week after streptozotocin.treatment. HbA1c% was analysed on a Cobas 6000. At baseline and during weeks 6, 10 and 14 of diabetes the mice were placed individually for 16 hours in metabolic cages (Techniplast) for the collection of urine. The total amount of urine was weighed and a sample of 20 μL was taken for analysis of urine albumin and pre-diluted 20:380 with buffer containing 50 mM TBS pH 8.0, 1% BSA, 0.05% tween20. Samples were analysed using a sandwich ELISA from Bethyl Labs with some modifications. Upon sample collection the urine was diluted 20-fold in kit sample diluent prior to storage at -20°C. 24h albumin excretion rate was calculated from the 16h collection. Mouse creatinine was analysed with HPLC-UV from the same samples. One way ANOVA (multiple testing) was performed on albumin excretion rate and albumin/creatinine ratio.

The concentrations of IgG, IgM and IgA in MLNL culture supernatants, GW, SMG or CC homogenates were quantified by a sandwich ELISA (Bethyl Laboratories Inc., Montgomery, TX, USA). After assay development as previously described^{76 (link)}, absorbance was measured on a microplate photometer (Labsystems Multiskan, Helsinki, Finland) and data were analysed by Ascent v.2.6 software (Thermo Fisher Scientific, S.L.U, Barcelona, Spain) according to the respective standard curves. The immunoglobulin content in SMG and CC was normalized by total protein concentration which was measured using the Pierce-660 nm ready-to-use Protein Assay Reagent (Thermo Fisher Scientific). Changes in each Ig concentration are expressed considering the SED group mean value as 1.