Tetramethyl benzidine tmb solution

Supernatants were collected from stimulated DCs (16 hrs), and cytokine production was measured by ELISA according to standard protocol. Capture and detection antibody pairs for IL-6 (MP5-20F3 and MP5-32C11), IL-10 (JES5-2A5 and SXC-1), and IL-12 (C15.6 and C17.8) were purchased from BD Biosciences. Recombinant mouse IL-6 (R&D), mouse IL-10 (R&D), and mouse IL-12 (Peprotech) were used as standards. Cytokine-antibody complexes were visualized by the addition of Tetramethyl Benzidine (TMB) solution (Life Technologies), and color development was stopped by the addition of TMB stop solution (Life Technologies). Absorbance at 492 nm was measured on a microplate reader (Biotek).

Binding of recombinant DPP4 to MERS-CoV spike clamp protein was determined via ELISA. Recombinant DPP4 was diluted to 2 µg/mL in PBS, and 50 µL was coated on a Nunc MaxiSorp™ high protein-binding capacity 96 well ELISA plate overnight at 4 °C. Plates were blocked with 150 µL/well of 5% KPL Milk Dilutent/Blocking solution concentrate (SeraCare, Milford, MA, USA) in PBS with 0.05% Tween-20 for 30 min at room temperature. Next, serial dilutions of MERS-CoV or SARS-CoV-2 spike protein, or a control protein, were added and incubated for 1 h at 37 °C. Plates were then washed three times with PBS with 0.05% Tween-20 before incubation with 2 µg/mL of an antibody towards the clamp domain, HIV1281 [57 (link)]. Plates were washed as before and incubated with a goat anti-human HRP secondary antibody (1:2000 dilution, Sigma Aldrich) for 1 h at 37 °C. Plates were washed a final time before the binding was revealed by the addition of tetramethylbenzidine (TMB) solution (Life Technologies) for 5 min. Reactions were stopped by the addition of 1 M sulfuric, acid and absorbance read at 450 nm. Absorbance was plotted using Graphpad Prism software version 8 using a one-site specific binding model.

Quantitative ELISA for FH, C7, C9, or plasminogen binding by CspA proteins was performed similarly to that previously described [78 (link)]. For FH binding, one microgram of BSA (negative control; Sigma-Aldrich) or FH from human (ComTech, Tyler, Texas), mouse (MyBiosource, San Diego, CA)[81 (link)], horse, or quail was coated onto microtiter plate wells. For FH binding, one hundred microliters of increasing concentrations (0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 μM) of histidine-tagged DbpA from B. burgdorferi strain B31 (negative control) or a CspA variant or mutant, including CspA_B31, CspA_PKo, CspA_ZQ1, or CspA_B31L246D was then added to the wells. Mouse anti-histidine tag (Sigma-Aldrich, St. Louis, MO; 1:200) and HRP-conjugated goat anti-mouse IgG (1: 1,000x) were used as primary and secondary antibodies, respectively, to detect the binding of histidine-tagged proteins. The plates were washed three times with PBST (0.05% Tween 20 in PBS), and 100 μL of tetramethyl benzidine (TMB) solution (ThermoFisher) were added to each well and incubated for five minutes. The reaction was stopped by adding 100 μL of 0.5% hydrosulfuric acid to each well. Plates were read at 405 nm using a Tecan Sunrise Microplate reader. To determine the dissociation constant (K_D), the data were fitted by the following equation using GraphPad Prism software (Version 7, La Jolla, CA).