A 96-well plate (Nunc Maxisorb, Nunc, Wiesbaden, Germany) was incubated with 1:500 dilutions of monoclonal anti-IFN-γ, IL-17A, or IL-12p40 antibody for 7 d at 4°C, as previously described (Truong et al., 2017 (link)). After blocking with 5% skim milk for 1 h at room temperature, the plate was incubated with culture supernatants of transfected cells or different dilutions of recombinant IFN-γ, IL-12p40, or IL-17A, overnight at 4°C. Following incubation with biotinylated-IFN-γ, -IL-12p40, or -IL-17A antibodies, HRP-conjugated streptavidin was added and incubated for 2 h at room temperature. The substrate 3,3′,5,5′-tetramethylbenzidine (TMB, Thermo Scientific) was used as a chemiluminescent substrate and luminescence was measured in a Hybrid Microplate Reader (Epoch, BioTek Instruments, Winooski, VT, USA).
Nunc maxisorb
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc MaxiSorb is a high-binding surface 96-well microplate designed for use in enzyme-linked immunosorbent assays (ELISAs) and other protein-binding applications. The plate is constructed with a polystyrene surface that has been treated to maximize protein binding capacity.
Automatically generated - may contain errors
Lab products found in correlation
Victor x3 multilabel plate reader, by PerkinElmer (3 mentions)
Supersignal elisa femto, by Thermo Fisher Scientific (3 mentions)
Donkey anti mouse igg hrp, by Jackson ImmunoResearch (2 mentions)
Capture antibody solution, by BioLegend (2 mentions)
Hybrid microplate reader, by Agilent Technologies (1 mentions)
Fl 140, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Jackson ImmunoResearch (1 mentions)
P nitrophenyl phosphate, by Merck Group (1 mentions)
Ifn α, by Thermo Fisher Scientific (1 mentions)
Ifn β, by BioLegend (1 mentions)
Rabbit anti anxa1 antibody, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Thermo Fisher Scientific (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Il 12p70, by Thermo Fisher Scientific (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti mouse igg, by Solarbio (1 mentions)
3 3 5 5 tetramethylbenzidine tmb solution, by Solarbio (1 mentions)
13 protocols using nunc maxisorb
1
Cytokine Quantification Enzyme-Linked Immunosorbent Assay
2
Quantitative α-Synuclein ELISA in CSF
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A 384-well ELISA microplate (Nunc MaxiSorb, NUNC) was coated by overnight incubation at 4 °C with 0.1 μg/ml Syn-140 (sheep anti-α-syn polyclonal antibody) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer (PBST containing 2.5 % gelatin) for 2 h at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 was added to a final concentration of 0.05 %) was added to each well, and plates were incubated at 37 °C for 2.5 h. 11D12 (mouse anti-α-syn monoclonal antibody), diluted in blocking buffer at 1:5 K was added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody (donkey anti-mouse IgG HRP, Jackson ImmunoResearch) diluted in blocking buffer (1:20 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was carried out using serial dilutions of recombinant human α-syn in artificial CSF.
3
Quantifying α-Synuclein in Cerebrospinal Fluid
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A 384-well ELISA microplate (Nunc MaxiSorb, Nunc) was coated by overnight incubation at 4 °C with Syn-O2 (0.2 μg/ml) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer for 2 h at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 was added to a final concentration of 0.05 %) was added to each well, and plates were incubated at 37 °C for 2.5 h. FL-140 (rabbit polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted in blocking buffer at 1:1 K, was added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of goat anti-rabbit IgG HRP (Jackson ImmunoResearch) diluted in blocking buffer (1:15 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was obtained using serial dilutions of recombinant human o-α-syn in artificial CSF (50 μl/well).
4
Detecting Cowpea Viruses in Nigeria
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Samples were tested for seven cowpea viruses (CABMV, CPMMV, BCMV-BlCM, CMoV, CMV, SBMV and CYMV) already reported in Nigeria, using homologous anti-rabbit antibodies to individual virus available at the VMD Unit of IITA, Nigeria, with Antigen Coated Plate-Enzyme-linked Immunosorbent Assay (ACP-ELISA) as described in Kumar et al. (2001 (link)). About 100 mg of tissue from the leaf apex was used for virus testing in a 96- well NUNC MaxiSorb (Nunc, Denmark) ELISA plate. Alkaline phosphatase (ALP)-labelled anti-rabbit antibodies were used to detect the immobilized antigen–antibody complex, and p-nitrophenylphosphate (Sigma, Gillingham, UK) was used as substrate. After 1 h of incubation, readings were taken at absorbance of 405 nm (A405 nm) in a Multiscan Plus ELISA plate reader (Labsystems, Helsinki, Finland). Sample with ELISA reading (A405 nm) value that is at least twice (2 ×) that of the healthy control was considered as positive to virus.
5
Cytokine Profiling in Murine Lung Infection
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Bronchoalveolar lavage fluid was obtained from uninfected and infected mice at 1,3 and 7 days post-infection. Fluids were spun down to remove contaminating leukocytes and supernatants were analyzed for IFN-γ, IL-6, IL-12p70, IL-10, IL-1β, TNFα, TGF-β, IFNα, (Ebioscience, San Diego, CA, USA) and IFNβ (Biolegend, San Diego, CA, USA) following the manufacturer's instructions. ANXA1 levels in nasal swab fluid was analyzed using a homemade ELISA. Briefly, Nunc Maxisorb (NUNC) 96 well plates were coated with 50 μl of 1 μg/ml goat anti-ANXA1 antibody (Santa Cruz) in PBS overnight at 4 °C. Plates were washed and blocked with 1% BSA for 1 h. Plates were washed again and samples were added to the wells overnight at 4 °C. Wells were aspirated and washed and 50 μl of 0.5 μg/ml rabbit anti-ANXA1 antibody (Invitrogen, Carlsbad CA, USA) was added for 1 h at room temperature, followed with washing and addition of 50 μl of 1 μg/ml donkey anti-rabbit horse radish peroxidase (Santa Cruz, Dallas, TX, USA) for 1 h. Wells were washed and substrate was added for up to 15 min in the dark. Plates were read in a plate reader at 450 nm.
6
Quantifying Phosphorylated α-Synuclein in CSF
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A 384-well ELISA microplate (Nunc MaxiSorb, Nunc) was coated by overnight incubation at 4 °C with 0.5 μg/ml Syn-140 (sheep anti-α-syn polyclonal antibody) in 200 mM NaHCO3, pH 9.6 (50 μl/well). The plate was then washed with PBST and incubated with 100 μl/well of blocking buffer for 2 h at 37 °C. After washing, 50 μl of the CSF samples (thawed on ice and Tween-20 was added to a final concentration of 0.05 %) was added to each well, and plates were incubated at 37 °C for 2.5 h. PS129 (mouse anti-pS129-α-syn monoclonal antibody) diluted in blocking buffer (1:1 K) were added to the appropriate wells, and incubated at 37 °C for 2 h. Next, the plate was washed and incubated for 2 h at 37 °C with 50 μl/well of species-appropriate secondary antibody (donkey anti-mouse IgG HRP (Jackson ImmunoResearch)) diluted in blocking buffer (1:20 K). After washing, the plate was incubated with 50 μl/well of an enhanced chemiluminescent substrate (SuperSignal ELISA Femto, Pierce Biotechnology, Rockford, IL). The chemiluminescence, expressed in relative light units, was immediately measured using VICTOR™ X3 multilabel plate reader (PerkinElmer). The standard curve for the ELISA assay was obtained using serial dilutions of recombinant human p-S129-α-syn in artificial CSF (50 μl/well).
7
Vip3Aa11 Protein Binding Assay
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EPS-HD270 (2.0 mg/mL, 100 μL) was loaded into 96-well ELISA plates (Nunc Maxisorb, Thermo) and immobilized at 4 °C overnight. The ELISA plates were washed three times with Tris-buffered saline (TBS) and blocked with TBST (TBS containing 0.1% Tween-20) containing 2.0% BSA (200 μL) at 37 °C for 2 h. The plates were washed with TBST three times and incubated with different concentrations of Vip3Aa11 (0, 0.5, 1, 2, 4, 8, 16, 32 nmol/L) (100 μL) for 1 h at 37 °C. After washing with TBST buffer 3 times, anti-Vip3A antibody (TBST 1:5000 dilution, 100 μL) was added. TBST (100 μL) containing 1/10,000 HRP-conjugated goat anti-mouse IgG (Solarbio Life Sciences, Beijing, China) was incubated at 37 °C for 1 h. For each treatment, three replicates were performed. After washing, the reaction was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) solution (Solarbio Life Sciences, Beijing, China) (100 μL) for 15 min in the dark at 37 °C. The reaction was terminated with HCl (2.0 mol/L, 100 μL), and the absorbance was immediately read at 450 nm using a microplate reader. The equilibrium dissociation constant (Kd) was analyzed using Sigma-plot Software (Version 12.0).
8
ELISA-based Seroconversion Assay for Vaccine Candidates
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To assess for seroconversion to the vaccine candidates, ELISA plates (Nunc Maxisorb, Thermo Fisher) were coated with 80 ng/well of BinJ/DENV2-prME in PBS overnight at 4 °C. Plates were blocked with blocking buffer for 1 h at room temperature. The vaccinated mouse sera were initially diluted 1 : 100, then 5-fold down a 96-well plate in blocking buffer before adding 50 µL of diluted sera to the ELISA plate and incubated at 37 °C for an hour. Plates were washed four times with PBS-T and were then probed with 50 µL/well of HRP-labelled goat anti-mouse (1 : 1000, Thermo Fisher). TMBW was added after washing the plates for six times with PBS-T and relative absorbance was measured at 450 nm on a plate reader.
9
Quantification of Cytokine Levels in Cell Culture
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Concentrations of GM-CSF, IFN-gamma, and IL-10 in cell culture supernatants were measured by ELISA, according to the manufacturer’s protocol (DuoSet Elisa, R&D Systems, Abington, UK). For measurement of IL-3, ELISA plates (NUNC Maxisorb, Thermofisher Scientific, Waltham, USA) were coated with a capture anti-IL-3 antibody (Clone P8C11; 5 µg/ml in PBS) in 100 µl/well at room temperature (RT) overnight. Plates were washed three times with PBS/0.05% Tween-20, blocked with PBS containing 1% bovine serum albumin (BSA) at RT for 1 h, and washed again with PBS. Samples were preincubated with 100 µg/ml mouse IgG1, kappa isotype control antibody (MOPC21, BioXCell, Lebanon, USA) for 1 h by RT and added to the plates for 2 h by RT (100 µl/well, diluted in PBS/1% BSA). Recombinant human IL-3 (BioLegend) was diluted from 7.8 to 500 pg/ml in PBS/1% BSA and served as standard. After washing, plates were incubated with 400 ng/ml HRP-labeled detection anti-IL-3 antibody (Clone 13) (100 µl/well) for 1.5 h at RT, and color reaction was performed with TMB Substrate Solution (BioLegend) according to the manufacturer’s protocol.
10
Murine anti-neuraminidase IgG Quantification
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