Multiscan ex elisa reader

ELISA was used to determine serum IgG ACPA concentrations and to test ACPA cross-reactions. The biotinylated, citrulline- or arginine-containing filaggrin19, vimentin, collagen II and the multi-epitope peptides were added at 1 µg/mL to the Medisorp plates (Greiner Bio-One GmbH, Kremsmünster, Germany) pre-coated with 5 µg/mL Neutravidin (Pierce Biotechnology, Rockford, IL, USA) overnight, at 4 °C, and then the plates were incubated for 1 h at 37 °C. EBNA2, α-enolase and fibrin β peptides were coated directly to Maxisorp (Nunc) plates at 2.5 µg/mL. After washing, the plates were blocked with 150 mM NaCl and 2% BSA containing PBS for 1 h, at 37 °C.
To determine the specific ACPA concentrations in sera a calibration curve was prepared from the affinity-purified ACPA as standard, in a two-fold dilution series, starting from 0.5 mg/mL. Serum samples were diluted 1:100. Dilution buffer was 2 M NaCl, 2% BSA in PBS. In cross-reaction assays, ACPAs were diluted to 0.1 mg/mL. Sera/ACPA samples were incubated overnight at 4 °C, shaking. After washing, the plates were incubated with 1:15,000 dilution of rabbit anti-human IgG HRP (H+L) (Southern biotech, Birmingham, AL, USA) for 1 h at 37 °C. Signal was developed with TMB substrate (Sigma-Aldrich, St. Louis, MO, USA) and the reaction was stopped with 2 N H₂SO₄. Plates were read at 450 nm with THERMO Multiscan EX ELISA-reader.

The biofilm production of the S. aureus ATCC 25923 strain was studied in 96-well plates using TSB broth in the presence of compounds. The overnight cultures were diluted to an OD of 0.1 at 600 nm and then measured to each well with the exception of the medium control wells, and compounds were pipetted individually at ¼ MIC concentration. The final volume was 200 μL in each well. The samples were incubated at 30 °C for 48 h with gentle agitation (100 rpm). Then, the medium was removed, and the plate was washed with tap water to discard unattached cells. 200 μL CV (0.1% [v/v]) was added to the wells and incubated for 15 min at room temperature. CV was removed from the wells and the plate was washed again with tap water. 200 μL 70% ethanol was transferred to each well and the biofilm formation was determined by measuring the OD at 600 nm using Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, CT, USA). The anti-biofilm effect of compounds was determined as the percentage (%) of decrease in biofilm formation. The assay was repeated a minimum of three times. The results were calculated using a t-test and p-values of <0.001 were considered significant.

MTT assay was performed in 96-well flat-bottomed microtiter plates as described before. Briefly, a 10 mM concentration stock solution in DMSO was prepared for each compound. These were diluted in 100 μL of McCoy’s 5A medium. Subsequently, 1 × 10⁴ (cytotoxicity assay) or 6 × 10³ (antiproliferative assay) T-cell mouse lymphoma cells in 100 μL of medium were added to each well, except for the medium control wells.
The adherent human fibroblast cells were seeded in 100 μL of EMEM medium overnight before each assay. Two-fold serial dilutions of the compounds (0.19–100 µM) were prepared in separate plates, then transferred to the plates containing the adherent cell lines.
The culture plates were further incubated at 37 °C for 24 h (cytotoxicity assay) or 72 h (antiproliferative assay); at the end of the incubation period, 20 μL of MTT solution (from a 5 mg/mL stock) was added to each well. After incubation at 37 °C for 4 h, 100 μL of SDS solution (10% in 0.01 M HCI) was added to each well and the plates were further incubated at 37 °C overnight. The cell growth was determined by measuring the optical density at 540 nm (ref. 630 nm) with a Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). IC₅₀ values were calculated by variable slope nonlinear regression using the log(inhibitor) vs. normalized response of GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, CA, USA).

Briefly, Nunc Maxisorp ELISA plates were coated with the immunogens, recombinant sGP or GP protein, blocked with PBS + 1% BSA and incubated with sera or cell culture supernatant. After a washing step, appropriate dilution of HRP-conjugated sheep anti-mouse Ig was added to the wells and incubated for 1 hour at room temperature (RT) following by a washing step. The reaction was revealed with TMB one component substrate (RD-Biotech). The reaction was stopped with 20% H₂SO₄ and the optical density (OD) measured by a Multiscan EX ELISA reader (ThermoFischer) at 450 nm & 620 nm. To evaluate the specificity of the generated mAbs, ELISA were also using GP and sGP from different EBOV viruses.

The effects of increasing concentrations of the compounds on cell growth were evaluated in 96-well flat-bottomed microtiter plates. The 2-fold serial dilutions of the isolated constituents were made starting with 100 μM. Then, 6 × 103 human colonic adenocarcinoma cells in 100 μL of the medium (RPMI-1640) were added to each well, except for the medium control wells. Culture plates were incubated at 37 °C for 72 h; at the end of the incubation period, 20 μL of MTT (thiazolyl blue tetrazolium bromide) solution (from a 5 mg/mL stock solution) was added to each well. After incubation at 37 °C for 4 h, 100 μL of sodium dodecyl sulfate (SDS) solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were further incubated at 37 °C overnight. Cell growth was determined by measuring the optical density (OD) at 540 nm (ref 630 nm) with a Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). Inhibition of cell growth was expressed as IC₅₀ values defined as the inhibitory dose that reduces the growth of the cells exposed to the tested compounds by 50%. IC₅₀ values and the SD of triplicate experiments were calculated by using GraphPad Prism software version 5.00 for Windows with nonlinear regression curve fit (GraphPad Software, ver. 9.0.1, San Diego, CA, USA; www.graphpad.com, accessed on 15 March 2022).