Tetramethylbenzidine tmb

To determine Nb6-pFc binding with Nsp9, a modified ELISA was designed and performed as previously described [10 (link)]. The recombinant Nsp9 was diluted with 0.01 M phosphate-buffered saline (PBS) and coated into an ELISA plate (Nunc, Denmark) at 400 ng/well overnight at 4 °C. After washing with PBS’T (5% of Tween-20 in 0.01 M PBS) three times, the plates were blocked with 200 μL of 2.5% dry milk (BD, USA) in PBS’T for 1 h at room temperature. Then, different dilutions of Nb6-pFc, Nb53-pFc, and His-Nb6 (stored from earlier studies) [20 (link)] were added to the plate as the first antibody to incubate for 1 h at 37 °C. After washing again, the rabbit anti-camel IgG sera (1:2000) were added and incubated for 1 h at 37 °C. Then, the peroxidase-goat anti-rabbit IgG (H+L) (Jackson, USA) were added and incubated for 1 h at 37 °C. After a second washing, the single-component substrate solution tetramethylbenzidine (TMB) (Solarbio, China) was added to produce a color reaction. Finally, 3 M H₂SO₄ was used to stop the reaction, and OD values were read at 450 nm (OD_450 nm) by an automated ELISA plate reader.

adalimumab (Humira, 40 mg/0.8 ml) was purchased from the manufacturer (Vetter Pharma-Fertigung GmbH & Co. KG, Germany), and etanercept (Enbrel, 25 mg) was purchased and used as received from the provider (Pfizer, New York, NY, USA). Recombinant human TNF-α (Peprotech) was used to capture the adalimumab or etanercept by being immobilized on the solid phase surface of ninety-six-well plates (Greiner, Germany). A 5% solution of nonfat dried milk (Dingguo Changsheng Biotechnology, China) was dissolved in phosphate-buffered saline (PBS) (Dingguo Changsheng Biotechnology) for sealing the solid phase surface of each well. 0.5% Tween-20 (Damao Chemical Reagent Factory, China) was added into PBS to prepare a wash solution. Horseradish peroxidase (HRP)-goat anti-human IgG (H + L) conjugate (ABclonal Technology, Woburn, MA, USA) was used for detecting adalimumab or etanercept. Tetramethyl benzidine (TMB) (Solarbio, China) was used as the substrate solution, and 2 mol/L hydrogen chloride (Sinopharm Chemical Reagent, China) were prepared in the laboratory to be used as a stop solution.

The samples were diluted in PBS to a final volume of 100 μL and coated overnight at 4 °C in a 96-well ELISA plate (Corning, USA). After three times wash with PBST (200 μL/well), the plate was blocked with PBST + 5%BSA (200 μL/well) at 37 °C for 2 h. The plate was washed with PBST (200 μL/well) three times and the anti-tag mAb (1:10,000) (Beyotime, China) was added (100 μL/well). After incubation at 37 °C for 1 h, the plate was washed with PBST (200 μL/well) three times, and Horseradish Peroxidase (HRP)-conjugated Goat anti mouse mAb (1:10,000) (Beyotime, China) was added (100 μL/well). After incubation at 37 °C for another 1 h, the plate was washed with PBST (200 μL/well) three times and the reaction was finished using tetramethylbenzidine (TMB) (Solarbio, China) and stopped with H₂SO₄ (2 M). Using a Synergy LX multi-mode reader (BioTek, USA), the OD₄₅₀ value were detected. Each sample in the assay was implemented in triplicate.

Whole-cell lysates were generated in 1 mL of ice-cold lysis buffer (1× Tris-buffered saline, 1.5%Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, protease inhibitor cocktail, and 1mM PMSF). After centrifugation (12,000 g, 20min, 4°C), the supernatants were collected for western blotting. Protein concentration was determined with Bio-Rad Dc protein assay solution. Then protein samples were loaded onto SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked and then incubated with primary antibodies specific for ICAM-1, MCP-1, Sp1 and GAPDH at 4°C overnight and then with secondary antibodies labeled with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG at room temperature. The signals were detected using enhanced chemiluminescence reagents (Thermo Scientific).
To measure the MCP-1 production in the transfected RAW264.7 cells, the cell culture medium was collected for ELISA. The antibody against MCP-1 was used as the primary antibody after blocking. After horseradish peroxidase (HRP)-conjugated secondary antibody incubation and washing, 3,3′,5,5′-Tetramethylbenzidine (TMB) (Solarbio Life Science, Beijing, China) substrate solution was used for enzyme reaction. After sufficient color developments add stop solution and read the optical density at 450 nm with ELX800 Bio-Tek.