Standard steps of a traditional sandwich ELISA were followed, with the key exception of use of identical clone antibodies for both capture and detection antibodies. A commercially available purified anti-human CCL20 (MIP-3α) antibody, Clone L2412F11 (described above), was coated in 96-well plates as a capture antibody, followed by incubation with CCL20LD test specimens at different concentrations or in unknown quantities from serum of mice that had been injected with CCL20LD. The same clonal anti-human CCL20 antibody was biotin-labeled and incubated (details below) and subsequently used as the detection antibody. The addition of streptAvidin-HRP conjugate and substrate TMB (3,3′5,5’-Tetramethylbenzidine) yielded a yellow color, with a λ-max of 450 nm for evaluation with a plate reader (Bio-Rad, Hercules, California). Please be noted, the blocking buffer, diluent, Avidin-HRP, substrate TMB, and stop solution are all purchased from Invitrogen (Thermo-Fisher Scientific. Waltham, MA).
Tmb substrate
Manufactured by Bio-Rad
Sourced in United States, Denmark
TMB substrate is a colorimetric substrate used in enzyme-linked immunosorbent assay (ELISA) and other immunoassays to detect and quantify analytes. When the enzyme-linked detection system is added, it catalyzes the conversion of the TMB substrate, resulting in a blue-colored product that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Maxisorp, by Thermo Fisher Scientific (4 mentions)
Galanthus nivalis lectin, by Vector Laboratories (3 mentions)
Pierce protein free blocking buffer, by Thermo Fisher Scientific (3 mentions)
Avidin hrp, by Thermo Fisher Scientific (2 mentions)
Tmb substrate, by Thermo Fisher Scientific (2 mentions)
Stop solution, by Thermo Fisher Scientific (2 mentions)
Legendplex human th cytokine panel 12 plex, by BioLegend (2 mentions)
Anti ifn γ capture, by Bio-Rad (2 mentions)
Recombinant ifn γ standard, by Bio-Rad (2 mentions)
Streptavidin hrp, by Merck Group (2 mentions)
Igg2a, by Thermo Fisher Scientific (2 mentions)
Synergy ht, by Agilent Technologies (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Streptavidin hrp conjugate, by Bio-Rad (1 mentions)
Streptavidin hrp, by Bio-Rad (1 mentions)
Carbonate buffer, by Merck Group (1 mentions)
Ez link sulfo nhs biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Tween 20 pbst, by Merck Group (1 mentions)
Maxisorp microtitration plates, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
21 protocols using tmb substrate
1
Quantification of CCL20 by Modified ELISA
2
CCL20 Capture ELISA Protocol
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Standard steps of a traditional sandwich ELISA were followed, with the key exception of use of identical clone antibodies for both capture and detection antibodies. A commercially available purified anti-human CCL20 (MIP-3α) antibody, Clone L2412F11 (described above), was coated in 96-well plates as a capture antibody, followed by incubation with CCL20LD test specimens at different concentrations or in unknown quantities from serum of mice that had been injected with CCL20LD. The same clonal anti-human CCL20 antibody was biotinlabeled and incubated (details below) and subsequently used as the detection antibody. The addition of streptAvidin-HRP conjugate and substrate TMB (3,3′5,5'-Tetramethylbenzidine) yielded a yellow color, with a λmax of 450 nm for evaluation with a plate reader (Bio-Rad, Hercules, California). Please be noted, the blocking buffer, diluent, Avidin-HRP, substrate TMB, and stop solution are all purchased from Invitrogen (Thermo-Fisher Scientific. Waltham, MA).
3
Biotin-labelled Pgp3 ELISA Protocol
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Biotin-labelled Pgp3 was produced using the EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo Scientific). Optimised assay conditions were determined by checkerboard titrations, as previously described [9 (link)]. Maxisorp microtitration plates (Nunc) were coated with unlabelled Pgp3 with bovine serum albumin (BSA) in carbonate buffer, pH 9.6 (Sigma) at 4°C. The protein-coated wells were blocked and stabilised by dilution buffer (PBS with 0.05% Tween-20 (PBST) (Sigma) with 1% Hammersten casein (GE Healthcare)) containing 5% sucrose (Sigma). Bound protein was incubated with either Pgp3 antibody-positive or negative defibrinated plasma [20 (link)] (25μl) diluted in dilution buffer (75μl) containing BSA at 37°C. After washing with PBST, biotinylated Pgp3 was added, incubated at 4°C, washed and incubated with horseradish peroxidase (HRP)-labelled streptavidin (Thermo Scientific). Finally, HRP activity was measured with TMB substrate (Biorad).
Assay cut-off was determined by receiver operating characteristic (ROC) analysis of absorbance (450–620nm) values on 505 paediatric samples and 342 samples from GUM patients, previously used to characterize our indirect ELISA [9 (link)]. As before, we determined specificity on the 494 samples from micro-immunofluorescence (MIF) assay-negative children [9 (link)].
Assay cut-off was determined by receiver operating characteristic (ROC) analysis of absorbance (450–620nm) values on 505 paediatric samples and 342 samples from GUM patients, previously used to characterize our indirect ELISA [9 (link)]. As before, we determined specificity on the 494 samples from micro-immunofluorescence (MIF) assay-negative children [9 (link)].
4
Quantifying Antibody Responses in Immunized Mice
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The levels of IgG, IgG1, and IgG2a in the sera of immunized mice were determined by indirect ELISA as described [22 (link)]. Briefly, each well of an ELISA plate was precoated with either purified UV-DENV-2 or NS11-279 (1 μg/well). Plates were washed and blocked with 1% BSA (w/v) in PBST before 100 µL of 2-fold serial diluted sera was added. After incubation for 2 h, DENV-2 or NS11-279-specific antibodies were detected using 100 μL of HRP-conjugated goat anti-mouse IgG (1:3000 dilution, Invitrogen) and the TMB substrate (Bio-Rad). The reaction was terminated, and the absorbance was read at 450 nm. The endpoint titer was calculated as described by Wang et al. [22 (link)].
The IgG1 and IgG2a subclasses were detected using HRP-conjugated goat anti-mouse IgG1 or IgG2a antibodies (1:4000, Southern Biotech, Birmingham, AL, USA) as probes. The levels of DENV-2- or NS11-279-specific IgG1 and IgG2a were reported as the OD value, as described by Wang et al. [22 (link)].
The IgG1 and IgG2a subclasses were detected using HRP-conjugated goat anti-mouse IgG1 or IgG2a antibodies (1:4000, Southern Biotech, Birmingham, AL, USA) as probes. The levels of DENV-2- or NS11-279-specific IgG1 and IgG2a were reported as the OD value, as described by Wang et al. [22 (link)].
5
Cytokine Profiling of CD4+ T Cells
6
DENV-2 Antibody Detection ELISA
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Each well of the ELISA plates (Corning costar, USA) was coated with DENV-immune human serum at a dilution of 1:1600 overnight at 4 °C. The plates were washed and blocked with 1% BSA (w/v) in PBST before being incubated with UV-DENV-2 (1 μg/well). After 2 h of incubation, sera (at 1:2000 dilution) from each mouse were applied. The DENV antibody interaction was detected using goat anti-mouse IgG antibody conjugated with HRP (1:3000 dilution, Invitrogen, Carlsbad, CA, USA) and TMB substrate (Bio-Rad, Hercules, CA, USA). The signals were analyzed at 450 nm.
7
Quantifying Anti-IFN-α2 Auto-Abs in COVID-19
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The concentrations of auto-abs to type I IFNs in the plasma of COVID-19 patients were measured using ELISAs as previously described [34 (link)]. In brief, on the day before the experiment, 96-well plates were coated with 0.5 μg/ml of E. coli derived non-glycosylated recombinant human (rh) IFN-α2 (11101-1; R&D systems). After washing with PBS (0.05% Tween) and blocking (UKB80; KAC Co., Ltd.), patient plasma diluted 1:50 or rabbit anti-human IFN-α2 IgG as a positive control (ab193055; Abcam) was added to the plate. After washing, auto-abs captured by rh type I IFNs were detected by peroxidase-labeled anti-human IgG antibody (62-8420; Invitrogen) or anti-rabbit IgG (H+L) antibody (G-212340; Invitrogen). Color was developed by TMB substrate (1721066; Bio-Rad), and absorbance values were measured at 450 nm with a plate reader (Bio-Rad).
8
Quantifying sE2-specific Antibody Responses
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To detect sE2-specific total IgG, IgG1 and IgG2a responses in mouse serum, 96-well plates (MaxiSorp, Thermo Fisher, Waltham, MA) were coated overnight with 5 μg/mL Galanthus Nivalis Lectin (Vector Laboratories, Burlingame, CA, USA) at 4 °C. Plates were washed (PBS + 0.05% Tween 20) and coated with 200 ng/well E2 for 2 h at room temperature. After blocking with Pierce™ Protein-Free Blocking Buffer (Thermo Fisher, Waltham, MA, USA) for 1 h, serially diluted sera from mice were added to the plate and incubated for 2 h. The binding of sE2-specific antibodies was detected with 1:5000 dilutions of HRP-conjugated goat anti-mouse secondary antibodies to detect total IgG (H+L), IgG1 and IgG2a (Southern Biotech, Birmingham, AL, USA) with TMB substrate (Bio-Rad Laboratories, Hercules, CA, USA). Absorbance values at 450 nm (SpectraMax M3 microplate reader) were used to determine endpoint titers, which were calculated using curve fitting in GraphPad Prism software and defined as four times the highest absorbance value of pre-immune sera. Significance comparison was performed using a Kruskal–Wallis one-way ANOVA.
9
Antibody Isotype Profiling in Mice
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Ninety-six–well ultrahigh-binding flat-bottom microtiter plates (Immulon 4HBX) were coated overnight at 4°C with 100 μl of scFv2H7-P18F3 at 1 μg/ml in PBS. Plates were then washed three times with 200 μl of PBS and blocked for 1 hour at RT with 100 μl of PBS 1% BSA (Sigma-Aldrich). After removing the blocking solution, individual mouse sera (1/5) in PBS 1% BSA were added into the wells and incubated for 1 hour at RT. Plates were then washed three times with PBS. One hundred microliters of alkaline phosphatase–conjugated goat anti-mouse IgG1, IgG2a, IgG2b, or IgG3 (Jackson ImmunoResearch) diluted 1/4000 in PBS 1% BSA was added to each well and incubated for 45 min at RT. Plates were then washed three times with PBS, and 100 μl of TMB substrate (Bio-Rad) was added per well. Absorbance was measured at 415 nm on an iMARK microplate absorbance reader (Bio-Rad).
10
Quantifying RSV Binding to LL-37
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High-affinity binding plates were coated with 2 μg/ml LL-37 or scrLL-37 for 12 h at 4°C, washed with PBS, and incubated with 2.5% BSA for 3 h. RSV 3.25 × 105 PFU was added for 30 min at 4°C, and wells were washed with 0.02% Tween 20/PBS. For detection of bound RSV, wells were exposed to anti-RSV Ab (1:200; Goat Anti Respiratory Syncytial Virus Biotin Polyclonal 7950-0104; AbD Serotec, Kidlington, U.K.), HRP-labeled secondary Ab (1:10,000, P0449; Dako, Glostrup, Denmark), and TMB substrate (Bio-Rad). Reaction was stopped using 0.5 M sulfuric acid, and absorbance at A450 was determined using a plate reader (Synergy HT; Biotek, Winooski, VT).
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