ELISA was performed to determine the amount of viral nucleoprotein (NP) expression in the culture supernatant as previously described with modifications [14 (link)]. Briefly, 100 ng/well of rabbit-anti-SFTSV serum (Cat#PAB27171, Abnova, Taipei City, Taiwan) was coated in 96-well ELISA plates for overnight incubation at 4 °C, followed by blocking with 2.5% FBS plus 2.5% FBS in PBS with Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. After washing, the culture supernatants collected at 1, 3, or 5 dpi were transferred to the ELISA plates accordingly (50 μL, incubated at room temperature for 2 h), followed by intensive wash and addition of 50 μL/well mouse-anti-SFTSV-NP serum (in-house preparation), the secondary goat-anti-mouse horseradish peroxidase (HRP) antibody (Invitrogen, Carlsbad, CA, USA), the 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Invitrogen), and the stop solution (0.1M HCl). Subsequently, the optical density of each well was read at 450 nm (OD450) using VICTOR 3 multi-label plate reader (PerkinElmer, Inc., Waltham, MA, USA).
Tmb 3 3 5 5 tetramethylbenzidine solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
TMB (3,3',5,5'-tetramethylbenzidine) solution is a common colorimetric substrate used in enzyme-linked immunosorbent assays (ELISA) and other applications. It is a chromogenic substrate that undergoes a color change upon enzymatic reaction, producing a blue-colored product. The solution is used to detect and quantify the presence of specific analytes in a sample.
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Lab products found in correlation
Victor3 multilabel plate reader, by PerkinElmer (2 mentions)
Tween 20, by Merck Group (2 mentions)
Polyclonal anti mouse ig hrp, by Agilent Technologies (2 mentions)
Rbd or s1 protein, by Sino Biological (1 mentions)
96 well flat bottom plate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
Multiskan fc microplate photometer, by Thermo Fisher Scientific (1 mentions)
Model infinite 200 pro, by Tecan (1 mentions)
Carbonate bicarbonate coating buffer, by Merck Group (1 mentions)
Pvc elisa microplate, by Corning (1 mentions)
Dynamis, by Thermo Fisher Scientific (1 mentions)
Anti clumping agent, by Thermo Fisher Scientific (1 mentions)
Peroxidase affinipure f ab 2 fragment goat anti human igg h l hrp, by Jackson ImmunoResearch (1 mentions)
Cd forticho, by Thermo Fisher Scientific (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Avantor (1 mentions)
Infinite 200 pro plate reader, by Tecan (1 mentions)
9 protocols using tmb 3 3 5 5 tetramethylbenzidine solution
1
SFTSV Nucleoprotein Quantification by ELISA
2
SARS-CoV-2 Antibody Detection Assay
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To detect the serum or BALF antibody levels, serum or BALF samples were collected from the immunized mice following euthanasia via CO2 inhalation. The 96-well plate was coated with purified RBD or S1 protein (Sino Biological) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) overnight at 4 °C. Serum or BALF samples were added each well and incubated for 2 hours at RT. After washing each well three times with PBS-T, RBD or S1 protein-specific antibodies in serum or BALF samples were detected with mouse IgG2a, IgG1 (BD, New Jersey, USA; 1:1,000 dilution), total IgG (Invitrogen, Massachusetts, USA; 1:1,000 dilution) or IgA (Invitrogen, Massachusetts, USA; 1:1,000 dilution) antibodies. All the detection antibodies were conjugated with horseradish peroxidase (HRP). After final washing, the plates were developed using 3,3’,5,5’-tetramethylbenzidine (TMB) solution (Invitrogen, Massachusetts, USA). Reactions were stopped using 1N sulfuric acid within 5 minutes. The optical density (OD) was determined using a spectrometer at a wavelength of 450 nm.
3
ELISA for SARS-CoV-2 Nucleocapsid Protein Quantification
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ELISA was performed to determine the amount of viral N protein expression in the culture supernatant of SARS-CoV-2-infected cells using a similar approach as previously described [22 (link)]. Briefly, 500 ng/well of mouse-anti-SARS-CoV-N monoclonal antibody (clone 14E7A11A8) was coated in 96-well ELISA plates for overnight incubation at 4 °C, followed by blocking with 2.5 % FBS plus 2.5 % FBS in PBS with Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) for 2 h at 37 °C. After washing, the infectious culture supernatants were transferred to the ELISA plates accordingly (50 μL, incubated at room temperature for 2 h), followed by intensive wash and addition of another 50 μL/well rabbit anti-SARS-CoV-N polyclonal antibody (1:4000), the secondary goat-anti-rabbit horseradish peroxidase (HRP) antibody (1:1500, Invitrogen, Carlsbad, CA, USA), the 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Invitrogen), and the stop solution (0.1 M HCl). Subsequently, the optical density of each well was read at 450 nm (OD450) using VICTOR 3 multi-label plate reader (PerkinElmer, Inc., Waltham, MA, USA). The rabbit anti-SARS-CoV-N monoclonal antibody showed good cross-reactivity against SARS-CoV-2-N [17 (link),23 (link)].
4
Quantifying HSP70 Binding Dynamics
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A total of 150 ng of purified NRLLLTG peptide was diluted in 0.05 M sodium carbonate buffer (pH 9.6) and coated onto a flat-bottom 96-well plate (Thermo). After overnight incubation, the plate was washed with PBST 3 times and blocked with PBST containing 1% BSA. Each well was washed with PBST, and wild type HSP70 or S385D/S400D mutant protein was added in a dose-dependent manner. After washing with PBST, the primary antibody against HSP70 and the secondary antibody were incubated sequentially. 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Thermo) was used to detect the bound HSP70, and 1 N H2SO4 was used for developing and stopping the reaction. The absorbance was measured at 450 nm with a 620 nm reference, and all experiments were repeated three times.
5
SARS-CoV-2 RBD and N Protein ELISA
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96-well plates were coated with 100 ng/well His-tagged SARS-CoV-2 receptor binding domain (RBD) or nucleocapsid protein (N) and incubated at 4 °C overnight. Plates were washed with 0.05% Tween-20 in PBS (PBST) once and blocked for 1 h at room temperature. Mouse sera were 4-fold diluted starting from 1:40 and incubated with the protein-coated plates for 1 h at 37 °C. Uninfected mouse serum and previously in-house raised anti-RBD/anti-N mouse sera were included as negative and positive control, respectively. Plates were washed for three times with PBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) for 1 h at room temperature. After incubation, plates were washed for six times with PBST followed by adding 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Thermo Fisher Scientific) for colour development. Plate development was terminated by adding equal volume of 0.3 N H2SO4. Absorbance at 450nm was measured with a Thermo Scientific Multiskan FC Microplate Photometer.
6
Whole Cell and Recombinant Protein ELISA
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For whole cell ELISA, bacteria were grown on BHI or GC plates for 16 h. Cells were harvested and resuspended in PBS at an OD600 of 0.2. Microtiter plate wells were filled with 50 μl of the bacterial suspension and dried at room temperature overnight in the laminar flow cabinet. After the bacteria in the dried wells were heat-killed for 1 h in 56°C. For recombinant protein ELISA, wells of plates were coated with 100 ng of purified recombinant MsrA/B protein in 100 μl of coating buffer (0.5 M carbonate/bicarbonate buffer, pH 9.6) for 1 h at room temperature. All ELISAs were performed with mouse pre-immune or MsrA/B immunized sera, and secondary antibody as specified in the results [polyclonal anti-mouse Ig HRP (Dako) or IgG1, IgG2a, IgG2b, IgG3, or IgM HRP (Thermofisher Scientific)]. The substrate TMB (3,3′, 5,5;-tetramethylbenzidine) solution (Thermofisher Scientific) was used as per manufacture's instruction. Equal amount of 1 N hydrochloric acid was added to stop the reaction. Absorbance was read in a TECAN Model Infinite 200 Pro plate reader at 450 nm.
7
Quantitative Cytokine Profiling by ELISA
8
ELISA Assay for Antibody Quantification
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Dynamis (catalog no. A2661501), CD FortiCHO (catalog no. A1148301), and anti-clumping agent (catalog no. 0010057AE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). For ELISA method, Invitrogen™ Nunc MaxiSorp™ flat-bottom 96-well plates (catalog no. 44-2404-21) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Protein A (catalog no. GSZ02201) was purchased from GenScript (Piscataway, NJ, USA). Peroxidase AffiniPure F(ab′)2 fragment goat anti-human IgG (H + L) HRP (catalog no. 109-036-088) was purchased from Jackson Immuno Research Laboratories (West Grove, PA, USA). ABTS (Diammonium 2,2′-azinobis[3-ethyl-2,3-dihydrobenzothiazole-6-sulfonate]) was ordered from VWR (Tuas, Singapore). TMB (3,3′,5,5′-tetramethylbenzidine) solution (catalog no. 34028) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
9
ELISA Detection of Bacterial Antigens
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Bacteria were grown on BHI or GC plates for 16 h. Cells were harvested and resuspended in PBS at an optical density at 600 nm of 0.20. Microtiter plate wells were filled with 50 μl of the bacterial suspension and dried at room temperature overnight in the laminar flow cabinet. After the bacteria in the dried wells were heat killed for 1 h at 56°C, the wells were washed and ELISA was performed with MAb 6E4 at a dilution of 1:64. Secondary antibody (polyclonal anti-mouse Ig HRP; P044701; Dako) was used at a dilution of 1:2,000. The substrate TMB (3,3′,5,5-tetramethylbenzidine) solution (ThermoFisher Scientific) was used per the manufacturer’s instructions. Equal amounts of 1 N hydrochloric acid were added to stop the reaction. Absorbance was read in a Tecan model Infinite 200 Pro plate reader at 450 nm.
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