Quantikine enzyme linked immunosorbent assay elisa kit

Blood samples were collected at baseline and after 8 and 16 weeks of treatment (Figure 1a). Serum levels of total IgE were measured using electrochemiluminescence. Serum TARC levels were assessed using a chemiluminescent enzyme immunoassay. The number of eosinophils was counted by an automatic analyzer method. Serum IL-4 and IL-13 levels were measured using a cytokine-specific ProQuantum Immunoassay kit (Invitrogen, Waltham, MA, USA), IL-22 by a Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minnesota, MN, USA), and IL-31 by the DuoSet ELISA kit (R&D Systems) according to the manufacturers’ protocols.

Murine macrophage RAW264.7 cells were cultured up to ~80% confluency in complete Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% antibiotic cocktail (penicillin/streptomycin, Sigma-Aldrich, Germany) at 37°C in 5% CO₂. The cells were collected and seeded at a density of 1 × 10⁶ cells/well in a 24-well cell culture plate (SPL Life Science, Korea) for 24 h. Prior to treatment, Lactobacillus strains were lysed by treating the bacterial pellets with lysozyme and sonication, and the lysates were heated at 80°C for 30 min to avoid bacterial growth. About 1 μg/mL of LPS (Sigma-Aldrich, Germany), 100 μg/mL of bacterial lysate, or the combination of LPS and lysate was used treated to cells. Culture supernatants were collected after 24 h of incubation, and IL-10 concentration was determined with IL-10 Quantikine enzyme-linked immunosorbent assay (ELISA) Kit (R&D systems, USA). ELISA results were read with Epoch microplate spectrophotometer (BioTek instruments, USA) at 450 nm wavelength. The inhibition of LPS-induced nitrite production by each lysate was measured by incubating 100 μL of cell culture supernatant and 100 μL of Griess reagent (Sigma-Aldrich, USA) for 10 min at 25°C. The results were analyzed at 540 nm wavelength.

CSF LCN2 was quantified using the human LCN2/NGAL (Neutrophil Gelatinase-Associated Lipocalin) Quantikine Enzyme-linked Immunosorbent Assay (ELISA) Kit from R&D according to the manufacturer’s instructions (R&D Systems, Inc. Minneapolis, MN). CSF samples were diluted 1:2. Inter- and intra-assay coefficients of variation were below 12 and 10%, respectively. The limit of quantification was 0.052 (52 pg/ml) and the limit of detection was 0.023 (23 pg/ml). Total-Tau, phospho-Tau (p-Tau), and amyloid beta 42 were quantitatively measured using ELISA kits from Fujirebio (Fujirebio, Ghent, Belgium). QAlb (CSF albumin/serum albumin*10³) was measured by local neurochemistry laboratories (cohort 1 and cohort 4) according to standard methodology. Test performers were blind to clinical information and clinical investigators vice versa.

Collagen gels treated with collagenase (treated) or PBS (control) for 60 min were laminin-coated, seeded with 3 × 10⁵ LESCs, and incubated in CnT-7 medium at 37 °C for 3 days. Subsequently, culture supernatants were collected on ice, centrifuged at 10,000 × g for 10 min at 4 °C, sampled, and analysed for human IL-6 expression using the Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer’s instructions. The expression of IL-6 was then normalised for the amount of total protein present in the supernatants, and expressed as pg mL⁻¹ µg⁻¹ of total protein. Both conditions were assayed three independent times (n = 3).