An indirect-ELISA was used to detect SWAP, SEA, and rSj97 specific IgG1 and IgG2. One μg per well of each antigen were used to coat a 96-well Maxisorb Nunc immunoplate (Sigma-Aldrich,) overnight. The plates were blocked with 150 μL 0.5% gelatin-PBST for 1hr at 37°C. Antigen-specific IgG1 and IgG2 were detected using sheep anti-bovine IgG1-HRP and IgG2-HRP conjugates (Bio-rad, Raleigh, NC) at 1:6000 dilution. After a final wash, the plates were developed by adding 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich, Mannheim, Germany). The reaction was stopped with 1M H2SO4 and read on a micro-plate ELISA reader (Bio-rad, Raleigh, NC) at 450nm.
Tetramethylbenzidine tmb liquid substrate
Manufactured by Merck Group
Tetramethylbenzidine (TMB) liquid substrate is a chromogenic substrate used in various immunoassay and enzyme-linked immunosorbent assay (ELISA) applications. It serves as a sensitive colorimetric detection reagent that undergoes a color change reaction when in the presence of an enzyme, typically horseradish peroxidase (HRP).
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Lab products found in correlation
Elisa microplate reader, by Bio-Rad (1 mentions)
Non animal protein blocker, by G Biosciences (1 mentions)
Horseradish peroxidase conjugated secondary goat anti human secondary igg, by Jackson ImmunoResearch (1 mentions)
Biotinylated detection antibody, by Mabtech (1 mentions)
Capture antibodies against γifn, by Mabtech (1 mentions)
Streptavidin horseradish peroxidase conjugate in pbs bsa 0.5, by Agilent Technologies (1 mentions)
Phytohemagglutinin (pha), by Merck Group (1 mentions)
Elispot plate, by Merck Group (1 mentions)
Biostack ready, by Agilent Technologies (1 mentions)
Goat anti rabbit igg hrp antibody, by Thermo Fisher Scientific (1 mentions)
El800 plate reader, by Agilent Technologies (1 mentions)
Anti mouse igg whole molecule peroxidase antibody, by Merck Group (1 mentions)
Sterile 96 well plate, by Corning (1 mentions)
Crp 8, by Merck Group (1 mentions)
Pbs tween, by Merck Group (1 mentions)
5 protocols using tetramethylbenzidine tmb liquid substrate
1
Antigen-Specific IgG1 and IgG2 Detection
2
SARS-CoV-2 Spike RBD ELISA Assay
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OC43 and NL63 RBD ELISA were carried out as previously described 80 (link),81 (link). Briefly, coating was performed by Streptavidin (Invitrogen) at 4 μg/mL in Tris-Buffered Saline (TBS) pH 7.4 for 1 h at 37°C followed by blocking with Non-Animal Protein-BLOCKER™ (GBiosciences). Then biotinylated spike RBD antigens for OC43 and NL63 were added at 1 μg/mL at 37°C for 1 h. All plasma samples were heat-inactivated before usage to minimize risk of residual virus in serum and then incubated at serial dilution followed by multiple washes and incubation with horseradish peroxidase-conjugated secondary Goat Anti-Human secondary IgG (Cat No: 109–035-008, Jackson ImmunoResearch) at 1:40,000 dilution in 3% milk at 37°C for 1 h. The resulting plate was washed and 3,3’,5,5’ -Tetramethylbenzidine (TMB) Liquid Substrate (Sigma-Aldrich) was added for optical density (OD) measurement at 405 nm after stopping the reaction with 50 μl of 1 N HCl.
3
ELISPOT Assay for Cytokine Detection
4
Indirect ELISA for Anti-rEx160 IgG
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An indirect-ELISA was used to detect anti-rEx160 IgG antibody. The antigen, 1 ng per well, was used to coat a 96-well flat bottom polystyrene High Binding plate (Corning, Acton, MA, USA) overnight at 4°C. The plates were blocked with 5% skimmed milk in PBS + 0.1% Tween 20. Antigen-specific IgG was detected using goat anti-rabbit IgG-HRP antibody (Thermo Fisher Scientific). After wash, the plates were developed by adding 3, 3', 5, 5'-Tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich). The reaction was stopped with 1 M H 2 SO 4 and read on a micro-plate ELISA reader (BioStack Ready, BioTek Instruments, Vinooski, Vermont, USA) at 450 nm.
5
Enzyme-Linked Immunosorbent Assay for CRP Detection
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The general procedures for ELISA were as follows: All ELISA analysis was carried out on a sterile, 96-well plate (Costar). 50 μl of each sample was added to each well. Plates were blocked with 3% bovine serum albumin (BSA) and washed with phosphate buffered saline (PBS)-Tween (Sigma). The primary antibody used was a monoclonal anti-C-reactive protein antibody (CRP-8, Sigma) produced in a mouse; the secondary antibody was an anti-mouse IgG (whole molecule)–peroxidase antibody produced in rabbit (Sigma). Both were used at 1:40,000 dilution. Positive and negative controls were included in analysis and all samples were tested in triplicate. The developing substrate used was 3, 3′, 5, 5′, tetramethylbenzidine (TMB) liquid substrate (Sigma). ELISA plates were left to develop for 10 min prior to addition of 2 M sulfuric acid. Plates were read at 450 nm on a BioTek EL800 plate reader using Gen5 software.
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