Antibody ELISAs were carried out as previously described (17 (link)), with 50 µl per dilution used. In brief, 96 well high-binding plates (Corning) were coated with 0.1 µg S or N protein in PBS and incubated overnight at 4°C. PBS-0.1% Tween 20 was used to wash plates 3 times, and between all subsequent steps. Plates were blocked with 2% (w/v) BSA in PBS-0.1% (v/v) Tween 20 for 1 hr at room temperature (RT). Serum was diluted 1:40 and incubated for 1 hr at RT. HRP-conjugated anti-human secondary antibodies were added for 1 hr at RT. For combined anti-IgG, IgA and IgM (IgGAM) the antibodies came in a combined pre-diluted form from The Binding Site (EACONJ654). The individual constituent HRP-labelled secondary antibodies used in this are polyclonal rabbit anti-human IgG (1:16,000), polyclonal rabbit anti-human IgA (1:2000) and polyclonal rabbit anti-human IgM (1:8000). Individual immunoglobulin isotypes were detected using HRP-conjugated monoclonal antibodies: mouse anti-human IgM (clone AF6, 1:2000), mouse anti-human IgG1 (clone MG6.41, 1:3000), mouse anti-human IgG3 (clone MG5.161, 1:1000). All monoclonal antibodies were produced at the University of Birmingham. Plates were developed for up to 20 minutes using 100 µl TMB Core (Bio-Rad) and the reaction was stopped with 50µl 0.2M H2SO4. Optical density (OD) was read at 450 nm using a SpectraMax ABS Plus plate reader.
Tmb core
Manufactured by Bio-Rad
TMB Core is a laboratory reagent used as a substrate for the detection of horseradish peroxidase (HRP) in enzyme-linked immunosorbent assays (ELISA). It is a chromogenic substrate that produces a blue-colored product when oxidized by HRP. The intensity of the color can be measured using a spectrophotometer, allowing for quantitative analysis of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg h l hrp conjugate, by Bio-Rad (2 mentions)
High binding 96 well plate, by Corning (1 mentions)
Litesizer 500 instrument, by Anton Paar (1 mentions)
High binding plates, by Greiner (1 mentions)
Spectramax plus 384 microplate reader, by Molecular Devices (1 mentions)
F1804, by Merck Group (1 mentions)
Infinite m1000 pro microplate reader, by Tecan (1 mentions)
Hrp conjugated swine anti rabbit, by Agilent Technologies (1 mentions)
Elast amplification kit, by PerkinElmer (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
7 protocols using tmb core
1
SARS-CoV-2 Antibody ELISA Protocol
2
Quantification of Antibody Conjugation and miRNA Encapsulation in PLGA Nanoparticles
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Purified NPs were characterized by DLS and zeta potential measurements on a Litesizer 500 instrument (Anton Paar) before and after antibody conjugation, and the reported intensity-based hydrodynamic diameter is the average of three measurements. miRNA encapsulation was quantified using an OliGreen assay. During NP synthesis, all filtrate containing unencapsulated miRNA was concentrated and analyzed alongside a standard curve of known miRNA concentration.
Antibody loading on the NPs was quantified using a solution-based ELISA modified from a previously published protocol.48 (link) IgG-Co-NPs, IgG-34a-NPs, N1-Co-NPs, N1-34a-NPs, bare miR-Co-NPs, or bare miR-34a-NPs were incubated with 10 μg/mL horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies for 1 h at room temperature. Unbound secondary antibodies were removed through centrifugation and the samples were suspended in 3% bovine serum albumin in phosphate-buffered saline (PBS). The samples were then developed in 3,3′,5,5′-tetramethylbenzidine (TMB) solution (TMB core; Bio-Rad) for 15 s before the reaction was stopped with 2 mM sulfuric acid. The absorbance was then measured at 450 nm on a Synergy H1 plate reader and compared to a standard curve of known HRP-IgG concentration to calculate the quantity of IgG or Notch1 antibodies conjugated per mg of PLGA.
Antibody loading on the NPs was quantified using a solution-based ELISA modified from a previously published protocol.48 (link) IgG-Co-NPs, IgG-34a-NPs, N1-Co-NPs, N1-34a-NPs, bare miR-Co-NPs, or bare miR-34a-NPs were incubated with 10 μg/mL horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies for 1 h at room temperature. Unbound secondary antibodies were removed through centrifugation and the samples were suspended in 3% bovine serum albumin in phosphate-buffered saline (PBS). The samples were then developed in 3,3′,5,5′-tetramethylbenzidine (TMB) solution (TMB core; Bio-Rad) for 15 s before the reaction was stopped with 2 mM sulfuric acid. The absorbance was then measured at 450 nm on a Synergy H1 plate reader and compared to a standard curve of known HRP-IgG concentration to calculate the quantity of IgG or Notch1 antibodies conjugated per mg of PLGA.
3
Quantifying SARS-CoV-2 Spike Antibodies
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Antibodies to near-full-length trimeric viral spike glycoprotein(4 , 5 ), were detected by ELISA. High-binding plates (Greiner Bio-One) were coated with spike glycoprotein (1 μg/ml) and blocked with with Stabilcoat solution (Sigma Aldrich) before test serum was added at 1:40 or 1:50 and diluted 5-fold down the plate. HRP-labelled mouse monoclonal anti-human IgG, IgA IgM, IgG1–4 secondary antibodies, generated at the University of Birmingham (available from Abingdon Health Ltd), were added individually or combined and HRP activity detected using TMB core (Bio-rad).
4
ELISA for Anti-EtpE-C Antibody Detection
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The wells of a 96-well flat-bottom microtiter plate (Nunc MaxiSorp) were coated with 2 μg each of rEtpE-C and BSA in coating buffer (14 mM Na2CO3 and 34 mM NaHCO3 [pH 9.6]). The coated wells were blocked with 1% BSA in PBS for 1 h at 37°C. Serial 2-fold dilutions of preimmune or anti-EtpE-C serum from rabbits or 1:3,200 dilutions of sera from sham- or rEtpE-C-vaccinated dogs were incubated for 1 h at 37°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (KPL) or anti-dog IgG (KPL) for 1 h at 37°C. A TMB core+ (Bio-Rad) substrate solution was used to develop reactions for 10 to 15 min, and reactions were stopped by addition of 50 μl of 0.2 M sulfuric acid. Absorbance was measured at 450 nm in a SpectraMax Plus 384 microplate reader (Molecular Devices, San Jose, CA).
5
ELISA-based cell surface localization of GPR10
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Cells transfected with N-terminal FLAG- or cMyc-tagged GPR10 constructs were tested for cell surface localization of the receptor with ELISA. A day after transfection, cells were fixed with 3.7% paraformaldehyde for 15 min at room temperature and washed three times with PBS. Subsequently, non-specific binding sites were blocked with 3% non-fat dry milk in 50 mM Tris-PBS pH 7.4 (blocking buffer) for 1 h at room temperature. Next, cells were incubated with a either a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, F1804) or anti-cMyc antibody (Millipore, CBL434, dilution 1:1000 in blocking buffer) overnight at 4 °C followed by triple washing with PBS and incubation with goat anti-mouse IgG(H + L)-HRP conjugate (Bio-Rad Laboratories, 172-1011) (1:1250 in 1.5% non-fat dry milk in 50 mM Tris-PBS pH 7.4) for 2 h at room temperature. Finally, plates were washed three times with PBS and a high performance chromogenic substrate, 3,3´,5,5´̵tetramethylbenzidine (TMB CORE+, Bio-Rad Laboratories, BUF062) was used to detect HRP activity. The reaction was terminated with 0.5 M H2SO4 and absorbance by the colour reaction product at 450 nm was determined using Tecan Infinite M1000 PRO microplate reader.
6
SARS-CoV-2 Antibody Binding Assay
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Plates were coated as above. Plates were washed three times with PBS-0.1% Tween 20 – this wash step was carried out between all subsequent steps. Blocking was carried out for 1 hr at RT with 2% BSA in PBS-0.1% Tween 20. Test serum was heat inactivated at 56°C for 30 minutes, before being diluted 1 in 5 with 2% BSA supplemented with 5 mM calcium chloride and 5 mM magnesium chloride. 50 µl was added to the antigen-coated plate and incubated for 1hr at 37°C. After washing, 50 µl COVID negative normal human serum (same source used throughout all assays, containing no detectable S or N specific antibodies as measured by IgGAM ELISA) at a dilution of 1:40 (in 2% BSA plus 5 mM calcium chloride and 5 mM magnesium chloride) was added to each well for 1hr at RT. 100µl of rabbit anti-C1q FITC antibody (Invitrogen PA5-16601) at a 1:200 dilution in PBS-0.1% Tween 20 was added and incubated at 37°C for 1 hr. HRP conjugated swine anti-rabbit (Dako P0399) at a 1:2000 dilution was then incubated for 1 hr. The assay was amplified using the Perkin Elmer ELAST amplification kit as per manufacturer’s instructions, with an optimised dilution of streptavidin, 1:800, incubated for 20 minutes. Plates were developed using 100 µl TMB Core (Bio-Rad) for 10 minutes, before being stopped with 50 µl 0.2MH2SO4. OD was measured as described above.
7
Quantitative FLAG-tagged HTR2C Expression
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HEK293 cells seeded onto poly-d -lysine coated 96-well plates (40,000 cells per well) were transfected with 5 ng per well of FLAG-tagged HTR2C constructs as described above. The day after transfection, the cells were fixed with 3.7% paraformaldehyde in PBS for 15 min at room temperature and washed three times with PBS. Subsequently, nonspecific binding sites were blocked with 3% non-fat dry milk in 50 mM Tris-PBS pH 7.4 (blocking buffer) for 1 h at room temperature. Cells were incubated with a mouse monoclonal anti-FLAG antibody (Sigma-Aldrich, F1804), diluted 1,000× in blocking buffer overnight at 4 °C. Next, cells were washed three times with PBS and incubated with goat anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad Laboratories, 172-1011) (1:1,250 dilution in 1.5% non-fat dry milk in 50 mM Tris-PBS) for 2 h at room temperature. Finally, cells were washed three times with PBS and the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB CORE+, Bio-Rad Laboratories, BUF062) was used to detect HRP activity. The reaction was stopped with 0.5 M H2SO4 and absorbance at 450 nm was quantified using a Tecan Infinite M1000 PRO microplate reader.
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