Elisa max deluxe

The conditioned medium of the treated cells was used to measure the concentration of IL-1β and TNF-α. The measurement was conducted according to Human IL-1β ELISA Kit (Elisa Max Deluxe, 437004; BioLegend, San Diego, CA, USA) and Human TNF-α ELISA Kit (Elisa Max Deluxe, 430204; BioLegend, San Diego, CA, USA) protocol. The treatments used in this experiments were as follows: (1) normal control; (2) BJ cells + DMSO 1% (vehicle control); (3) UV-induced BJ cells (positive control); (4) UV-induced BJ cells + CA 6.25 µg/ml; and (5) UV-induced BJ cells + CA 25 µg/ml (Laksmitawati et al., 2017 (link); Widowati et al., 2016 (link); Widowati et al., 2019a (link); Lister et al., 2020 (link); Widowati et al., 2021 ).

Detection of total / overall amounts of IFNγ and IL2 in human plasma was conducted using ELISA MAX Deluxe sets from Biolegend, Germany: IFNγ (#430106) and IL2 (#431806). The manufacturer’s Avidin-horseradish peroxidase conjugate was replaced by PolyHRP80 streptavidine conjugate (#SP80C, SDT Reagents, Germany) in order to achieve a tenfold lower limit of detection. Lower limit of detection (Background + 3x S.D.) was generally at 2 – 5 pg/ml for IFNγ and IL2, respectively. Initial dilution of control and test samples was performed described as follows: negative control (NaCl) 1/5, 1^st positive control (SEB) 1/2500 for IFNγ and 1/500 for IL2, 2^nd positive control (CEFT) 1/50 for IFNγ and 1/25 for IL2, test samples (HBcAg and HBsAg) 1/5. If a test sample’s absorbance value fell outside the maximum standard curve range, these samples were subsequently retested with a tenfold higher dilution, e.g. 1/5 → 1/50, 1/500 → 1/5000, 1/2500 → 1/25000. A seven-point standard curve from 1 – 64 pg/ml IFNγ or IL2 was used for quantitation. Standards, controls and test samples were measured in duplicate. The samples were analyzed using Magellan software (version 6.5) equipped on a Tecan M200 plate reader.

Fresh blood was collected from the hearts of each group (N = 3–4), and serum was collected by centrifugation at 875.17× g for 15 min at 4 °C. Inflammatory cytokines, TNF-α, which are produced in periodontal inflammatory tissues, were analyzed in serum by enzyme-linked immunosorbent assay (ELISA) using ELISA MAX Deluxe (BioLegend, San Diego, CA, USA) in each well of a microplate. After four washes with PBS-T, the collected serum was diluted 10-fold with PBS and reacted with standards diluted to 4.4, 15.6, 62.5, 250, and 1000 pg/mL for 2 h at room temperature. After four washes with PBS-T, the biotin-conjugated detection antibody was reacted for 1 h at room temperature. For further signal amplification, streptavidin was reacted with PBS-T for 30 min at room temperature after four washes with PBS-T. After five washes with PBS-T, the chromogenic substrate was allowed to react for 15 min at room temperature to develop color. To stop the chromogenic reaction, 100 μL of 2 N sulfuric acid was added, and the absorbance at 450 nm was measured using a microplate reader SH-1000 Lab (Corona Electric Co., Hitachinaka, Japan). All tests were performed in two wells per individual, the serum concentrations of TNF-α were quantified, and the concentrations were set to 0 if the results were below the detection limit.