The level of antibodies against ASFV-specific protein p54 antibodies in serum was measured using an indirect ELISA (developed by our lab). The detailed process was described in the previous article [39 (link)]. Briefly, the ELISA plates (Corning, New York, NY, USA) were coated with purified p54 protein (1 μg·mL−1) which was expressed in prokaryotic expression system and was blocked with 5% skimmed milk. The serum sample (S) and positive control (P) were added to the ELISA plates and incubated for 1 h at room temperature (RT). The horseradish peroxidase (HRP)-labeled sheep anti-pig IgG (CWBIO, Haimen, China) used for second antibody was incubated for 1 h at RT. The chromogenic reaction began with the addition of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (SeraCare, Delaware, USA) and ended with 2 M sulfuric acid (BEIJING SHIJI, Beijing, China). The optical density (OD) values at 450 nm were read by iMarkTM Microplate Reader (BIO-RAD, Hercules, CA, USA). The ratio of S/P above 0.25 is recognized as a positive sample.
3 3 5 5 tetramethylbenzidine tmb substrate
Manufactured by LGC
Sourced in United States
3,3′,5,5′-tetramethylbenzidine (TMB) substrate is a chromogenic substrate commonly used in enzyme-linked immunosorbent assays (ELISA) and other colorimetric detection methods. It undergoes an enzymatic reaction that produces a blue colored product, which can be measured spectrophotometrically.
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Lab products found in correlation
Imark microplate reader, by Bio-Rad (1 mentions)
Elisa plate, by Corning (1 mentions)
Prism v8, by GraphPad (1 mentions)
Microtiter plate, by Thermo Fisher Scientific (1 mentions)
Superblock, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti mouse immunoglobulin g igg, by Southern Biotech (1 mentions)
Tmb stop solution, by LGC (1 mentions)
El 312, by Agilent Technologies (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg antibody, by Jackson ImmunoResearch (1 mentions)
5 protocols using 3 3 5 5 tetramethylbenzidine tmb substrate
1
ELISA for ASFV Protein p54 Antibodies
2
SARS-CoV-2 S Protein ELISA Assay
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Microtiter plates (96-well; Thermo) were coated with 1 µg/mL S-2P protein (Genscript) corresponding to the S protein of the Wuhan-Hu-1 virus. After overnight incubation at 4 °C, plates were washed four times with PBS/0.05% Tween-20 and blocked for 1.5 h at 37 °C (SuperBlock-Thermo). After washing, 5-fold serial dilutions of mouse serum were added (assay diluent: 0.05% Tween-20 and 5% goat serum in PBS). Plates were incubated for 2 h at 37 °C, washed and horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) (Southern Biotech) was added at a 1:20,000 dilution (S-2P) in assay diluent. Plates were incubated for 1 h at 37 °C, washed, and bound antibody was detected with a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (SeraCare). After incubation for 10 min at room temperature, the reaction was stopped by adding a TMB stop solution (SeraCare) and the absorbance was measured at 450 nm. Titers were determined using a four-parameter logistic curve fit in Prism v.8 (GraphPad Software, Inc.) and defined as the reciprocal dilution at approximately optical density450 = 1.0 (normalized to a mouse standard on each plate).
3
Quantitative Antibody Binding Assay
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Env protein containing a C-terminal StrepTag was bound in wells of a 384-well plate, which was previously coated with streptavidin (Thermo Fisher Scientific) at 2 μg/mL and blocked with PBS containing 4% (wt/vol) whey protein, 15% normal goat serum, 0.5% Tween 20, and 0.05% sodium azide. Proteins were incubated at room temperature for 1 hr and washed with PBS and 0.1% Tween 20. mAbs were then added in serial dilutions beginning at 25 μg/mL. Antibodies were incubated at room temperature for 1 hr and washed, and binding was detected with goat anti-human horseradish peroxidase (HRP; Jackson ImmunoResearch) and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Sera Care Life Sciences).
4
Quantification of C4BP Binding to LigA/B
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Microtiter plates were coated with 1 μg of each LigA or LigB fragment overnight at 4°C in PBS (130 mM NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4). After blocking with 3% BSA, C4BP (1μM—0.0156 μM, 2 fold serial dilution) was then added to the plates for 1 h at 37°C. Between each step, plates were washed with PBS-T three times. Subsequently, mouse monoclonal anti-C4BP antibodies (1:2000) (EMD Millipore) recognizing C4BP α- and β-chains were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (1:2000) (Invitrogen) was used as secondary antibody. After washing three times with PBS-T, 100 μl of 0.2 mg/ml 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories) was added to each well. Finally, after a 10 min-incubation the microtiter plates were read at 630 nm using an ELISA plate reader (Biotek EL-312). Each value represents the mean ± SE of three independent experiments, each performed in triplicate.
5
Western Blot Analysis of Recombinant Proteins
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Sf-9 cells were infected with rFCoV-I or rFCoV-II at different MOI values, and cell pellets from different post-infection times were lysed for Western blotting analysis. The expressed recombinant proteins were resolved by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked using 5% (w/v) non-fat dry milk for two hours at room temperature, followed by incubation with an anti-histidine mouse monoclonal antibody (mAb) (Invitrogen srl, Milan, Italy) at room temperature. After washes in PBS-Tween 20, each membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, PA, USA). The membranes were developed with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate (Kirkegaard & Perry Laboratories, MD, USA).
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