Enzyme-linked immunosorbent assay plates (Maxisorp, NUNC, Roskilde, Denmark) were coated with 2 μg/well of purified recombinant NPC-2 protein antigen in 0.1 M carbonate/bicarbonate buffer pH 9.6 and maintained at 37°C for 45 min. Thereafter, wells were blocked with phosphate buffered saline, pH 7.4, 0.05% (v/v) Tween 20 (PBST), containing 3% (p/v) skimmed milk, for 1 h at room temperature (RT). The plates were incubated with serum samples diluted 1:200 in PBST with 1% skimmed milk, for 2 h at RT. Duplicates of positive (sera from a confirmed COVID 19 patient), and negative controls were included in each plate. Plates were washed three times with PBST and incubated with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Thermofisher), diluted 1:5,000 in PBST, for 1 h at RT. Finally, after three additional washes with PBST, plates were incubated with peroxidase substrate (TMB-MAX, Neogen Corporation) for 5 min in the dark at RT. Reaction was stopped by adding 0.5 M H2SO4. Optical density (OD) was measured in each well at 450 nm in an ELISA microplate reader. Results were valid when OD in negative control wells was below 0.8 OD units and the OD in positive control wells was over 2.50 OD units.
Tmb max
Manufactured by Neogen
Sourced in United States
TMB-MAX is a colorimetric substrate used in enzyme-linked immunosorbent assays (ELISA) to detect and quantify the presence of target analytes. It is a ready-to-use solution that produces a blue color upon reaction with the enzyme, which can be measured spectrophotometrically.
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4 protocols using tmb max
1
SARS-CoV-2 Antibody Detection by ELISA
2
Optimized SARS-CoV-2 RBD Antibody ELISA
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A DR-ELISA was developed as previously described [2 (link), 22 (link)] with some modifications. The above- described proteins were tested: RBDHis rendered better specificity results and RBDmFc was more suitable for conjugation. Briefly, 1 ng/µl of the RBD protein (RBDHis) was used to coat 96-well plates and was incubated overnight at 4°C in 50 mM carbonate buffer, pH 9.6. After washing the wells with PBS, 0.05% Tween 20 (PBST) using a manual washer, a blocking step was performed with StabilZyme SELECT Stabilizer (SurModics, Inc.) for 1 h at room temperature (RT). The plate was incubated with serum samples diluted 1:5 in PBST with 2.5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at RT. Duplicates of positive (pool of human SARS-CoV-2 positive sera) and negative (dilution buffer) controls were included in each plate. The wells were washed as described above and incubated with the HRP-conjugated RBDmFc protein for 30 min at RT. Finally, after a washing step as above mentioned, the plate was incubated for 15 min with the substrate (TMB-MAX, Neogen Corporation), and the reaction was stopped by the addition of 0.5 M sulfuric acid. The absorbance was measured at 450 nm using a SpectraMax M5 plate reader (Molecular Devices, LLC). Results were presented as S/P, defined as: (sample OD – negative control OD)/(positive control OD – negative control OD) × 10.
3
Serological Detection of Mycobacterial Antibodies
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The methodology was developed by INGENASA (Madrid, Spain). The principles of double recognition ELISA (DR-ELISA) was developed with recombinant protein (MPB83) serving both as coating antigen and conjugated form (horseradish peroxidase (HRP) labelled), the signal capture. The results were read by ELISA spectrophotometry and 0.3 was chosen for cut-off OD.
This assay is able to recognize not only IgGs but also other immunoglobulins, such as IgMs.
Briefly, the recombinant MPB83 expressed in baculovirus system was used as antigen to coat 96-well microtitre plates (25ng/well). After an incubation at 4 o C, the wells were blocked and a 1/200 dilution of serum in 0.05% Tween, 0.35M NaCl in Phosphate-Buffered Saline (PBS) was incubated for one hour at RT. Bound antibodies were detected by incubation with MPB83-HRP one hour at RT and subsequent addition of the substrate (TMB-MAX, Neogen Corporation, KY, USA).
This assay is able to recognize not only IgGs but also other immunoglobulins, such as IgMs.
Briefly, the recombinant MPB83 expressed in baculovirus system was used as antigen to coat 96-well microtitre plates (25ng/well). After an incubation at 4 o C, the wells were blocked and a 1/200 dilution of serum in 0.05% Tween, 0.35M NaCl in Phosphate-Buffered Saline (PBS) was incubated for one hour at RT. Bound antibodies were detected by incubation with MPB83-HRP one hour at RT and subsequent addition of the substrate (TMB-MAX, Neogen Corporation, KY, USA).
4
SARS-CoV-2 N Protein ELISA Protocol
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A DR-ELISA was developed as previously described (Venteo et al., 2012 (link)). Briefly, the N protein was used to coat 96-well plates and incubated overnight at 4 °C in carbonate buffer, pH 9.6. After washing the wells with phosphate buffered saline pH 7.4 with 0.05% Tween 20 (PBST), a blocking step was performed with StabilZyme® SELECT Stabilizer (SurModics, Inc.) for 1 h at room temperature (RT). The plate was incubated with serum samples diluted 1:5 in PBST with 2.5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) for 30 min at RT. Duplicates of positive (rabbit polyclonal antibody to N protein of SARS-CoV-2 produced in-house) and negative (dilution buffer) controls were included in each plate. The wells were washed as described above and incubated with the HRP-conjugated N protein for 30 min at RT. Finally, after a washing step, the plate was incubated for 15 min with the substrate (TMB-MAX, Neogen Corporation), and the reaction was stopped by addition of 0.5 M sulfuric acid. The absorbance was measured at 450 nm using a SpectraMax M5 plate reader (Molecular Devices, LLC).
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