Soluble Gal1 was determined using an in-house Enzyme-Linked Immunosorbent Assay (ELISA) as described (34 (link)). In brief, high-binding 96-well microplates (Costar; Corning) were coated with capture Ab (2 μg/mL purified rabbit anti-human Gal1 polyclonal IgG) in 0.1 M sodium carbonate, pH 9.5. After incubation for 18 h at 4 °C, wells were rinsed three times with wash buffer (0.05% Tween-20 in PBS) and incubated for 1 h at room temperature with blocking solution (2% Bovine Serun Albumin (BSA) in Phosphate buffer saline (PBS)). Samples and standards (100 μL) were diluted in 1% BSA–Tween-20 and incubated for 18 h at 4 °C. Plates were then washed and incubated with 100 ng/mL biotinylated detection Ab (purified rabbit anti-human Gal1 polyclonal IgG) for 1 h. Plates were rinsed three times before adding 0.33 μg/mL HRP-labeled streptavidin (Sigma-Aldrich) for 30 min. After washing, 100 μL 3,3’,5,5’-Tetramethylbenzidine (TMB) solution (0.1 mg/mL tetramethylbenzidine and 0.06% H2O2 in citrate–phosphate buffer, pH 5.0) was added to plates. The reaction was stopped by adding 2N H2SO4. Optical densities were determined at 450 nm in a Multiskan MS Microplate Reader (Thermo Fisher Scientific). A standard curve ranging from 2.5 to 160 ng/mL human rGal1 was run in parallel.
96 well high binding microplates
Manufactured by Corning
Sourced in United States
The 96-well high-binding microplates are a laboratory equipment product designed for a variety of assays and experiments. These microplates feature a high-binding surface that enhances the attachment of biomolecules, such as proteins, enzymes, and antibodies, to the plate wells. The plates are typically used in applications that require the efficient immobilization of these molecules for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Microtiter plate reader, by Agilent Technologies (2 mentions)
96 deep well plate, by Corning (2 mentions)
Stop solution, by Solarbio (2 mentions)
Multiskan ms microplate reader, by Thermo Fisher Scientific (1 mentions)
Hrp labeled streptavidin, by Merck Group (1 mentions)
Opteia, by BD (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Cell death detection elisa kit, by Roche (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Tmb substrate solution, by Solarbio (1 mentions)
Hrp conjugated goat anti mouse igg, by Abcam (1 mentions)
Spectramax 190, by Molecular Devices (1 mentions)
Hrp conjugated anti mouse igg, by Abcam (1 mentions)
Igg subclass specific antibodies, by Abcam (1 mentions)
Tmb substrate solution, by Thermo Fisher Scientific (1 mentions)
Vcsm13, by Agilent Technologies (1 mentions)
9 protocols using 96 well high binding microplates
1
Quantifying Soluble Galectin-1 by ELISA
2
ELISA for HIV-1 Env and S. mitis Antibodies
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High-binding 96-well microplates (Costar, Lowell, MA) were coated with the purified HIV-1 HXBc2 Env protein (2 μg/ml) or S. mitis lysate antigens (2 μg/ml) prepared in 0.2 M sodium carbonate/bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Wells were washed with PBS containing 0.05% Tween-20 (PBS-T) and blocked with 1% BSA in PBS-T for 2 hr. Serum or saliva samples were diluted 1:3 and added at 2- to 3-fold serial dilutions in PBS-T containing 0.1% BSA and plates were incubated for 1 hr at room temperature. After another washing step, horseradish peroxidase, HRP-conjugated rat anti-mouse IgA (clone 11-44-2), IgG1 (clone SB77e) or IgG2a (clone SB84a) (Southern Biotech, Birmingham, AL) was added at 1:4000, 1:5000, 1:5000 dilution, respectively and incubated for 1 hr. A substrate solution containing tetramethylbenzidine (KPL, Gaithersburg, MD) was added and colour development was stopped using 1 m HCl. Optical density data were recorded as absorbance at 450 nm. OD values twice above the background were considered positive and values between 0.01 and 1.0 were used to determine the amount of antibody produced and adjusted according to the dilution factor to determine the OD value of the undiluted saliva and serum samples.
3
ELISA for Detecting Antibody Responses
4
Quantifying Serum MPO-DNA Complexes in SLE
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Serum MPO-DNA complex levels of SLE patients and HCs were measured as previously reported18 (link). In brief, high-binding 96-well microplates (Corning) were incubated overnight at 4 °C with a mouse anti-human MPO antibody (clone 4A4; Bio-Rad) diluted in coating buffer (Cell Death Detection ELISA kit; Roche). Following blocking with 1% bovine serum albumin (cat# A2153; Sigma) in phosphate-buffered saline (PBS), plates were incubated at room temperature with 10% human serum in blocking buffer, washed, and then 10 × anti-DNA-POD (Clone MCA-33, Cell Death Detection ELISA kit; Roche) in blocking buffer was added. After the incubation, TMB substrate (KPL) was added, and absorbance was measured at 450 nm after the addition of the stop reagent (Wako). Serum MPO-DNA complex “high” patients were defined as those whose optical density values were two standard deviations higher the mean of HCs.
5
SARS-CoV-2 S-ECD Binding Assay
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Individual colonies of the ninety-six clones selected from the fourth round of biopanning were inoculated into 1 mL of SB containing 50 μg/mL of carbenicillin in 96-deep-well plates (Axygen, Union City, CA, USA) and incubated at 37 °C for 5 h. Subsequently, 1010 pfu of VCSM13 phages (Agilent) were added to the plates and incubated overnight at 37 °C. The plates were centrifuged at 2000× g, and the phage supernatant was used for ELISA. Briefly, 96-well high binding microplates (Corning, Corning, NY, USA) were coated with 0.1 μg of SARS-CoV-2 S-ECD (Sino#40589-V08B1) or 3% BSA in PBS and incubated overnight at 4 °C. After blocking using 3% (w/v) BSA in PBS, the plates were incubated with 100 μL of phage supernatant at 37 °C for 2 h. After three rounds of washing with PBS containing 0.05% (v/v) Tween 20 (PBST), horseradish peroxidase (HRP)-conjugated anti-hemagglutinin (HA) antibody (1:3000; Bethyl Laboratories, Montgomery, TX, USA) was added and incubated at 37 °C for 1 h. Colorimetric detection was performed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific, Waltham, MA, USA). The reaction was terminated by the addition of 1 M H2SO4, and absorbance was measured at 450 nm using a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA).
6
Quantifying RVFV Gn and Gc Antibodies
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To determine the RVFV Gn- and Gc-specific IgG, IgG1, or IgG2a titer in mice, RVFV Gn or Gc proteins were diluted in carbonate–bicarbonate buffer and coated in the 96-well high-binding microplates (Corning, NY, United States) with 0.2 μg/well at 4°C overnight. The plates were then blocked with PBS containing 2% BSA at 37°C for 1 h. After three rinses with PBST (PBS containing 0.2% Tween-20), serially diluted sera of mice in dilution buffer (PBST containing 0.2% BSA) were incubated with RVFV Gn or Gc proteins coated in the microplates at 37°C for 1 h. After that, the microplates were rinsed three times with PBST and incubated with the secondary antibodies at 37°C for 1 h, which target mouse IgG, IgG1, or IgG2a (Abcam, Cambridge, United Kingdom). After microplates rinsed three times, the TMB substrate solution (Solarbio, Beijing, China) was added in the microplates for 6 min at room temperature and the reaction was terminated by stop solution (Solarbio, Beijing, China). The optical density (OD) was measured at 450/630 nm (OD450/OD630; SPECTRA, Molecular Device, San Jose, CA, United States). We defined the endpoint titers as the reciprocal of the highest serum dilution, the OD value of which was 2.1-fold higher than that of the negative control.
7
LASV GPC-specific IgG Antibody Assay
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For LASV GPC-specific IgG assays, 96-well high-binding microplates (Corning, NY, USA) were coated with 1 µg/mL LASV rGPe protein in carbonate–bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. Then, the plates were blocked for 1 h at 37 °C in PBS containing 2% BSA and washed with PBST (PBS +0.1% Tween-20). Mice sera serially diluted in dilution buffer (PBS with 0.2% BSA) were added to the plates and incubated for 1 h at room temperature (RT). HRP-conjugated goat anti-mouse IgG (Abcam, UK) was diluted 10,000 times and added to the plates; then, the plates were incubated for 1 h and washed with PBST. The assay was developed for 10 min at RT in the dark using 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Solarbio, Beijing, China) and the reaction was halted by 50 µL stop solution (Solarbio, China), followed by measurement of emission at 450 nm (SPECTRA MAX 190, Molecular Device, San Jose, CA, USA). The endpoint titer was defined as the highest reciprocal serum dilution that yielded an absorbance ≥2.1-fold that of negative control serum values.
8
SARS-CoV-2 RBD Antibody Detection
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For the detection of RBD-specific antibodies in the serum, BAL, nasal wash and tracheal wash, RBD protein was coated onto 96-well high binding microplates (Corning) at 4 μg/mL overnight at 4°C. After washing three times with PBST, the plates were blocked with PBS containing 3% skim milk (Bio-Rad) at 37°C for 1 hour. Again, the plates were washed with PBST three times and pat dried. Twofold serially diluted samples were added and incubated for 90 min. After washing five times, HRP-conjugated anti-mouse IgG (1:10000, Abcam), IgA (1:2000, Abcam) or IgG subclass-specific antibodies (1:10000, Abcam) were used as secondary antibodies. After the addition of ABTS, the absorbance signal was read at 405 nm using a microplate reader. The endpoint titer was determined as the reciprocal of the highest dilution showing an absorbance value that was 2-fold greater than the background level.
9
Phage ELISA for CADM1 Binding
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Single colonies were inoculated into 1 mL of SB media containing 50 μg/mL of carbenicillin in 96-deep-well plates (Axygen, Union City, CA, USA) and incubated at 37 °C overnight. Then, 1012 pfu/mL helper phages (VCSM13; Agilent, Santa Clara, CA, USA) and 70 μg/mL of kanamycin were added and incubated overnight at 37 °C. The plates were centrifuged at 4000 rpm, and the supernatant was applied for phage ELISA. The 96-well high-binding microplates (Corning, Corning, NY, USA) were coated overnight with 0.1 μg of MF-hCADM1 and MF-mCADM1 in phosphate-buffered saline (PBS) at 4 °C. After blocking with 3% (w/v) BSA in PBS, the plates were incubated with 100 μL of phage supernatant at 37 °C for 2 h. After washing three times with PBS containing 0.05% (v/v) tween 20 (PBST), HRP-conjugated anti-hemagglutinin (HA) antibody (1:3000; Bethyl Laboratories, Montgomery, TX, US) was added and incubated at 37 °C for 1 h. Colorimetric detection was performed by adding 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) as the chromogenic substrate. The reactions were stopped by the addition of 2 M of sulfuric acid (H2SO4), and the absorbance was read at 450 nm using a microtiter plate reader (Bio-Tek Instruments, Winooski, VT, USA).
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