Tetramethylbenzidine tmb

Plates were coated overnight at 4°C with 4 μg/mL of goat polyclonal anti-rat IgM or IgG antibodies (supplied by Jackson ImmunoResearch Laboratories). After several washing steps, saturation with PBS/5% BSA was performed for 2 hours at room temperature. Diluted samples and isotype control (rat IgM, IgG1, IgG2a, and IgG2b supplied by BD Biosciences), used to determine the standard curves, were incubated for 90 minutes at 37°C. HRP-conjugated anti-rat IgM, IgG1, IgG2a, or IgG2b (supplied by Bethyl Laboratories) was incubated for 90 minutes at 37°C to allow specific detection of rat immunoglobulin isotypes. Tetramethylbenzidine (TMB; BD Biosciences) was used as HRP chromogenic substrate before the reaction was stopped by addition of H₂SO₄. Optical density at 450 nm was determined using a Tecan Spark plate reader.

Plates were coated with 100 ng/well of SARS-CoV-2 RBD protein (RayBiotech, Norcross, GA, USA) overnight at 4 °C. Plates were washed twice with PBS-T (0.05% Tween 20; Sigma-Aldrich, St. Louis, MO, USA) and blocked with PBS containing 8% FBS (HyClone, Logan, UT, USA) for 2 h at room temperature (RT). Serially diluted (4-fold, starting with 1:40) sera samples were added and incubated for 1 h at RT. This was followed by a 1 h incubation with HRP-conjugated goat anti-mouse IgM or IgG antibodies (Southern Biotech, Birmingham, AL, USA). 3,3′,5,5′ Tetramethylbenzidine (TMB, BD Biosciences, San Jose, CA, USA) was added to the well for 5 min. Then, stop solution was added (Thermo Fisher Scientific, Waltham, MA, USA), and the absorbance was read at 450 nm by a BioTek Cytation 7 plate reader. Binding endpoint titers were determined using a cutoff value determined by the mean of the negative controls (noninfected control-fed or AHCC-fed samples) + 3 x standard deviation (SD).

The 96-well microtiter plates (Nunc, Maxisorp) were coated after optimization with 0.1 μg recombinant human proteins, Dsg1 (MyBioSource MBS2010418), Dsg3 (MyBioSource MBS2010322), BP180 (MyBioSource MBS2012222), desmocollin 3 (MyBioSource MBS637447), desmoplakin (MyBioSource MBS2031176), rPpSP32, or a control protein (recombinant form of the CFP32 protein of Mycobacterium tuberculosis) (8 (link)) in a carbonate-bicarbonate buffer (0.1 M, pH 9.6) at 4°C overnight. The plates were then washed 3 times with PBS-T and saturated with PBS-T containing 4% BSA for 1 hour. After several washes, the plates were incubated with decreasing concentrations of rPpSP32 (from 2 to 0.0002 μg) for 1 hour at 37°C. After several washes, the plates were incubated in the presence of human sera with anti-PpSP32 (donors living in endemic areas of ZCL and naturally exposed to P. papatasi bites) or anti-CFP32 antibodies (patients with confirmed tuberculosis based on clinical symptoms, chest radiography, and sputum smear microscopic examination) diluted at 1:100 for 2 hours at 37°C. The specific binding of the conjugate to the solid phase was revealed by the addition of the chromogenic substrate, tetramethylbenzidine (TMB) (BD Biosciences). After stopping the reaction by the addition of H₂SO₄, the reading was carried out using a spectrophotometer (Awareness Technology, Inc.) at 450 nm wavelength.