Immune responses to OMVs from A. baumannii strains 19606, 17961, 17978, and LAC-4 were measured using G1 (IgG2b) and G2 (IgG1) fraction polyclonal IgGs from pre-immune and terminal bleed sera by ELISA. Briefly, each of the four OMVs (0.38 μg) was coated in wells of a microtiter plate (Corning, Glendale, AZ) in PBS by incubating at 4°C for 72 h. Wells were blocked with 5% (w/v) skim milk in PBS for 1 h at 37°C. Subsequently, 10-fold serial dilutions of fractionated serum IgGs (100 μL/well in PBS) were incubated in wells for 1 h at room temperature. Wells were washed 3× with PBST (PBS, pH 7.4 containing 0.05% (v/v) Tween-20) followed by incubation with a 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated anti-llama IgG (Cat#A160-100P, Bethyl Laboratories, Montgomery, TX) in PBST for 1 h at room temperature. Wells were washed as above and developed with 50 μL of High Sensitivity 3,3′,5,5′-tetramethylbenzidine (TMB) ELISA Substrate (Abcam, Cambridge, UK) for 5 min followed by the addition of 50 μL of 1 M phosphoric acid. The absorbance was measured at 450 nm.
Tmb elisa substrate
Manufactured by Abcam
Sourced in United States
TMB ELISA Substrate is a chromogenic substrate used in enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of analytes. It contains 3,3',5,5'-Tetramethylbenzidine (TMB) as the active ingredient, which undergoes an enzymatic reaction to produce a colored product. The intensity of the color is proportional to the amount of the target analyte present in the sample.
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Lab products found in correlation
Synergy h1 microplate reader, by Agilent Technologies (2 mentions)
Prism 5, by GraphPad (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Microtiter plate, by Corning (1 mentions)
Spike rbd antibody, by Sino Biological (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Ni coated plates, by Thermo Fisher Scientific (1 mentions)
Versamax elisa microplate reader, by Molecular Devices (1 mentions)
Polystyrene microplates, by Thermo Fisher Scientific (1 mentions)
Non fat dried milk, by Merck Group (1 mentions)
Albumin from chicken egg white, by Merck Group (1 mentions)
Cd40 pe, by BioLegend (1 mentions)
Quant ittm picogreendsdna reagent, by Thermo Fisher Scientific (1 mentions)
Mouse il 12p70 elisa kit, by Dakewe (1 mentions)
Mouse il 1β elisa kit, by Dakewe (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
11 protocols using tmb elisa substrate
1
Evaluating OMV Immunogenicity in Acinetobacter Strains
2
RBD-loaded Hydrogel: Protocol for Vaccine Delivery
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Hydrogels were prepared the same way as described in the “PNP Hydrogel Preparation” section and were loaded with 10 µg RBD. After the mixing step, the vaccine‐loaded hydrogels or bolus controls were placed in a 50 °C incubator on top of a shaker at 200 rpm to agitate. At each timepoint, 20 µl of the bolus samples were extracted and mixed with PBS at a 1:50 dilution for analysis with ELISA. Similarly, 20 µl of the gel samples were extracted and mixed with PBS in two syringes at a 1:50 dilution. For analysis with ELISA, 96‐well Maxisorp plates (Thermo Fisher) were coated with 50 µl of the diluted samples overnight at 4 °C. Plates were then blocked with 1% bovine serum albumin (BSA in 1X PBS) for 1 h at RT. Spike RBD antibody (Sino Biological 40592‐MP01) was added at a 1:2000 dilution and incubated on blocked plates for 2 h at RT. IgG Fc‐HRP goat‐anti‐mouse secondary antibody (Invitrogen A16084) was then added at a 1:10000 dilution (in 1% BSA) for 1h at RT. Plates were developed with TMB substrate (TMB ELISA Substrate (High Sensitivity), Abcam). The reaction was stopped with 1 M HCl. Plates were analyzed using a Synergy H1 Microplate Reader (BioTek Instruments) at 450 nm. Data was normalized to the day 0 timepoint for the gel and bolus groups, respectively.
3
ELISA Characterization of S-Protein Antibodies
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The ELISA assay was performed following the protocols published previously46 . The S-protein trimer was immobilized in a 96-well plate (0.2 μg/well) overnight at 4°C. The proteins were serial diluted (1:2) and applied to each well. HRP-labeled goat anti-human IgG was used at 1:1,000 dilution and the color development was with TMB ELISA Substrate (Abcam, ab171523) The experiment was performed in duplicate and the background subtraction was done by subtracting the corresponding binding signals obtained with BSA-immobilized wells. Affinity constant was calculated with GraphPad Prism 6.0 (GraphPad Software, Inc.).
4
Influenza Vaccine IgG Antibody Titer Quantification
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Serum IgG antibody titers for the influenza vaccine were measure using an ELISA. Ni‐coated plates (Thermofisher) were coated with HA (Sino Biological) at 2.5 μg/ml in PBS for 1 h at 25°C and blocked with PBS containing 1% BSA for 1 h at 25°C. First, serum was diluted 1:250 and then four‐fold serial dilutions were performed up to 1:4,096,000 dilution. Titrations were added to plates and after 2 h at 25°C, goat–anti‐mouse IgG Fc‐HRP (1:10,000, Invitrogen, A16084) was added for 1 h at 25°C. Plates were developed with TMB substrate (TMB ELISA Substrate [High Sensitivity], Abcam). The reaction was stopped with 1 M HCl. The plates were analyzed using a Synergy H1 Microplate Reader (BioTek Instruments) at 450 nm. Endpoint titers were defined as the reciprocal of the highest serum dilution that gave an optical density above 0.1 and normalized to a pooled control used with each reading.
5
Quantification of gp140 Antibody Titers in Rabbit Sera
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An ELISA assay was used to quantify the gp140 antibody binding titers in rabbit sera as described previously (van Diepen et al., 2018 (link)). Briefly, 96-well Maxisorb® microtiter ELISA plates (NunC) were coated with 10 ng/well of the soluble gp140 trimers (van Diepen et al., 2019 ). Duplicate wells were incubated with serum three-fold serially diluted from 1:10. Following secondary incubations with swine anti-rabbit IgG-HRPO conjugate (DakoCytomation) and detection with TMB ELISA substrate (Abcam®), the reaction was stopped after 10 min with 1N H2SO4. The absorbance signals at 450 nm were measured using a VersaMax ELISA Microplate Reader (Molecular Devices, Sunnyvale). Antibody end-point titers were defined as the last dilution to give a signal above the matching pre-bleed (1:10) ELISA signal. GraphPad Prism 5.0 software was used to present the end-point titres against bleeds time points (weeks). The V1V2-specific antibodies in sera collected at week 0 (pre-immune) and week 22 (2 weeks after the final protein inoculation) were evaluated as described above except that 500 ng/well of CAP256 SU V1V2 scaffold protein was used as a capture antigen.
6
ELISA for VLP Antibody Detection
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The VLP concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific). Ninety-six-well polystyrene microplates (Nunc) were coated with 200 ng VLP diluted in PBS (pH 7.4) at 4°C overnight. The VLP-coated wells were blocked with 300 μL PBS containing 2% nonfat dried milk (Sigma-Aldrich) for 2 hours at room temperature. The sera were diluted 1:200 in the blocking solution, applied in duplicate, and incubated at 37°C for 1 hour on a shaker. After incubation, the plates were washed three times with PBS–Tween washing solution. Peroxidase-conjugated goat anti-human immunoglobulin G (H and L, Abcam) was diluted 1:5000 and added to each well. The plates were incubated at 37°C for 1 hour and then washed as described previously. One hundred microliters TMB ELISA substrate (Abcam) was added to each well and incubated at room temperature for 15 minutes in the dark. The reaction was stopped by adding 100 μL 1 M sulfuric acid to each well. Readings were taken at 450 nm in an automated microplate reader (Bio Hit) with a 690 nm filter used as reference.
7
Murine Dendritic Cell Activation Assay
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DMEM (C11995500BT), Fetal bovine serum qualified Australia (10099141C) and Trypsin-EDTA (0.25%), phenol red (25200072) were purchased from Gibco; CD11c-FITC (117306), CD86-PerCP (105026), CD80-APC (104714) and CD40-PE (124610) were purchased from Biolegend; Goat Anti-Mouse IgG Antibody (HRP) (ab98762), Goat Anti-Mouse IgA alpha chain (HRP) (ab97235), Goat Anti-Mouse IgG1 heavychain (HRP) (ab97240), Goat Anti-Mouse IgG2a heavy chain (HRP) (ab97245), TMB ELISA Substrate (ab171523) and 450nm stop solution for TMB substrate (ab171529) were purchased from Abcam; Penicillin-Streptomycin Solution100X (C0222), and BSA, FranctionV (ST023) were purchased from Beyotime; Mouse IFN-gamma ELISpot PLUS kit (HRP) (3321-4HPW-2), Mouse TNFa ELISA kit (1217202), Mouse IL-1β ELISA kit (1210122), and Mouse IL-12p70 ELISA kit (1211202) were purchased from Dakewe; GM-CSF (315-02-10ug) and IL-4 (214-14-20ug) were purchased from PEPROTECH; Quant-iTTM PicoGreendsDNA Reagent (P7581) was purchased from Thermo Scientific; 2019-nCoV Protein RBD (DRA36) was purchased from novoprotein; Albumin from chicken egg white (A5503) was purchased from sigma.
8
Soluble CAP256 Env Binding ELISA for Antibody Titers
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To assess Env binding antibody titers in rabbit sera, a matching soluble CAP256 Env binding ELISA was performed as described earlier (80 (link)). In short, Nunc MaxiSorp flat-bottom 96-well plates (Sigma) were coated with 10 ng/well of soluble, trimeric Env. Rabbit sera were used in the primary incubation in a serial dilution range starting at 1:10. Anti-rabbit IgG-horseradish peroxidase (HRP) (1:5,000, Roche) was used for detection with TMB ELISA substrate (Abcam). The reaction was stopped after 10 min with 1 N H2SO4. The ELISA signal was analyzed using a VersaMax ELISA microplate reader (Molecular Devices), which subtracted absorbance values at 540 nm from values at 450 nm. ELISAs for the whole time course and each group were performed at the same time on duplicate plates. Duplicate data points were averaged and fitted to a four-parameter logistic regression curve (4PL curve) in GraphPad Prism 5.0. Antibody endpoint titers were calculated from 4PL curves with the threshold set as 4PL curve minimum + standard error of minimum for each time point. Data were plotted as the mean ± SEM for the whole group. ELISAs for binding to CAP256 SU V1V2 loop scaffold for week 0 and 2 weeks after the final MVA inoculation or protein boost were performed in a similar fashion, but wells were coated with 500 ng/well of protein.
9
Quantification of Serum Immunoglobulins in Naïve and Immunized Mice
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For the quantification of serum immunoglobulins in naïve mice, MaxiSorp 96-well plates (Thermo Fisher Scientific, 442404) were coated with anti–mouse isotype-specific antibodies at 0.5 µg/mL in a carbonate/bicarbonate coating buffer (100 mM, pH 9.6). For the determination of antigen-specific antibodies in sera from immunized mice, the plates were coated with 20 µg/mL of NP(27)-BSA (bovine serum albumin; Biosearch Technologies, N-5050H). The plates were incubated at 4 °C overnight, washed with PBS containing 0.05% Tween-20 (PBST), and then blocked with PBST containing 1% BSA for 1 h at room temperature. After three washes with PBST, the plates were incubated with diluted serum samples for 2 h at room temperature. Following another three washes with PBST, the plates were incubated with HRP-conjugated goat anti-mouse isotype-specific antibodies (SouthernBiotech; IgM: 1021-05; IgG: 1015-05; IgG1: 1071-05; IgG2b: 1091-05; IgG2c: 1078-05; and IgG3: 1101-05). After a final three washes, TMB ELISA Substrate (Abcam, ab171522) was added to detect HRP enzymatic activity. The reactions were then quenched by the addition of Stop Solution for TMB Substrate (Abcam, ab171529), and the absorbance was read at 450 nm on the Promega GloMax Multi+ Detection System.
10
Quantifying Mouse Immunoglobulin Levels
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Sera collected from WT and BC-CosmcKO mice were titrated and added in duplicate into 96-well plates (Corning) precoated with polyvalent goat antibody against mouse immunoglobulins (IgM, IgG1, IgG2b, IgG2b, IgG3, IgA from Southern Biotech), and followed by HRP-conjugated goat anti-mouse IgG (Southern Biotech and Fisher Scientific) and then TMB ELISA substrate (Abcam). Absorbance was measured at 450 nm with a Multiskan Spectrum spectrophotometer (Thermo Scientific). The mouse immunoglobulin concentration was calculated from a curve constructed using mouse immunoglobulin standard as listed: IgM, IgG1, IgG2b, IgG2b, IgG3, IgA (Southern Biotech).
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