Neutralising capacity of SARS‐CoV‐2 antibodies was assessed using a competitive assay. Serum samples were incubated at 125‐fold dilution for 60 min with 5 ng mL−1 biotinylated RBD in PTG. Next, 100 μL aliquots were transferred to MaxiSORP microtitre plates (Thermo Fisher Scientific, Waltham, MA, USA) coated with 300 ng mL−1 ACE2 and incubated for 60 min. The ACE2 was produced in HEK cells with a HAVT20 leader peptide, 10xhis‐tag and a BirA‐tag as described by Dekkers et al.48 . After washing five times with PBS supplemented with 0.02% polysorbate‐20 (PBS‐T), plates were incubated for 30 min with streptavidin‐poly‐HRP (M2032, Sanquin). Plates were washed five times with PBS‐T, and 100 μL of TMB substrate (100 μg mL−1) and 0.003% (v/v) hydrogen peroxide (Merck, Darmstadt, Germany) in 0.11 m sodium acetate buffer (pH 5.5) were added to each well. A total of 100 μL of 0.2 m H2SO4 (Merck) was added to stop the reaction. Absorbance was measured at 450 and 540 nm. The difference was used to evaluate RBD binding. As such, results were corrected for background signals (absence of RBD) and expressed as percentage binding relative to the uninhibited condition (% non‐inhibited signal).
Maxisorp microtitre plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States, Germany
Maxisorp™ microtitre plates are a type of laboratory equipment designed for conducting various immunoassays. They feature a high-binding surface that is optimized for the immobilization of proteins, peptides, and other biological molecules. The plates are made of polystyrene and are available in different well formats to accommodate different experimental requirements.
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Lab products found in correlation
Rpmi 1640 medium, by Merck Group (4 mentions)
Goat anti mouse and goat anti rabbit fluorescence dye labelled antibodies, by LI COR (3 mentions)
Eif4ebp1, by Cell Signaling Technology (2 mentions)
Mouse monoclonal antibody, by Abcam (2 mentions)
Donkey anti ovine igg horseradish peroxidase hrp conjugate, by Jackson ImmunoResearch (2 mentions)
Gliadin solution, by Merck Group (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Thermomax microtitre plate reader, by Molecular Devices (2 mentions)
Multiscreen 96 well filter plate, by Merck Group (2 mentions)
Hydrogen peroxide, by Merck Group (1 mentions)
H2so4, by Merck Group (1 mentions)
Skim milk, by Nacalai Tesque (1 mentions)
Microplate autoreader, by Tecan (1 mentions)
Stop reagent, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Infinite f50, by Tecan (1 mentions)
Igg mouse elisa kit, by Abcam (1 mentions)
Tetramethylbenzidine substrate, by Merck Group (1 mentions)
Goat anti mouse hrp conjugated serum, by Agilent Technologies (1 mentions)
21 protocols using maxisorp microtitre plate
1
SARS-CoV-2 Antibody Neutralization Assay
2
SARS-CoV-2 Antibody Titration by ELISA
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Antibody titres were determined by a virion‐based ELISA as previously described.18 , 24 , 25 , 26 , 27 Briefly, purified virus was immobilised on 96‐well Maxisorp microtitre plates overnight (Thermo Fisher Scientific, Waltham, MA, USA). Wells were blocked with 0.05% PBST [0.05% Tween‐20 (Sigma‐Aldrich, Saint Louis, MO, USA) in PBS] containing 5% skim milk (Nacalai Tesque, Kyoto, Japan) at 37°C for 1.5 h. Heat‐inactivated patient and pooled healthy control plasma samples at 1:200 to 1:8000 dilutions prepared in PBST with 2.5% milk were incubated at 37°C for 1 h. HRP‐conjugated goat anti‐human IgM or IgG (H+L) (Thermo Fisher Scientific) or mouse anti‐human IgG1, IgG2, IgG3 and IgG4 (Thermo Fischer Scientific) antibodies were used for detection. Reactions were developed using TMB (3,3,5,5‐tetramethylbenzidine) substrate (Sigma‐Aldrich) and terminated with Stop reagent (Sigma‐Aldrich), and absorbance was measured at 450 nm in a microplate autoreader (Tecan, Männedorf, Zürich, Switzerland).18 , 24 , 25 , 26 , 27 ELISA readings were conducted in duplicates or triplicates.
3
SARS-CoV-2 Antibody Titration by ELISA
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Anti‐RBD, spike and nucleocapsid antibody titres were quantified by ELISA, as described previously.53 , 54 , 55 In short, the viral antigens were coated overnight at 4°C on MaxiSorp microtitre plates (Thermo Fisher Scientific, Landsmeer, the Netherlands). Plasma samples were diluted 1200‐ and/or 3600‐fold in PBS supplemented with 0.1% polysorbate‐20 and 0.3% gelatin (PTG) and were subsequently incubated on the antigen‐coated plate for 1 h at room temperature (RT). After washing, 0.5 μg mL−1 of HRP‐conjugated anti‐human IgG (MH16‐1; Sanquin, Amsterdam, the Netherlands) was added in PTG and incubated for 30 min at RT, after which the plated was washed. The enzymatic conversion of TMB substrate was monitored, and absorbance was measured at 450 and 540 nm. The signals were quantified using a serially diluted calibrator consisting of a reference plasma pool of previously confirmed convalescent COVID‐19 patients that were included on each plate. This calibrator was arbitrarily assigned a value of 100 AU mL−1, and seroconversion threshold of IgG was set at 4 AU mL−1 levels as determined by using preoutbreak samples.53 , 54 Since plasma samples of donors D09 and D29 were collected after PBS dilution and the Ficoll centrifugation, ELISA results were multiplied accordingly to the predilution of the sample.
4
Scedosporium Sandwich ELISA Protocol
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For the Scedosporium sandwich ELISA (ScedELISA), wells of Maxisorp microtitre plates (10547781, Thermo Fisher Scientific, Loughborough, UK) were coated with 50 µL volumes of Protein A-purified mAb HG12 at a concentration of 3 mg/mL PBS. After incubation for 16 h at 4 °C, the wells were washed three times (5 min each wash) with PBST (PBS containing 0.05% (vol:vol) Tween-20), once with PBS for 5 min, and then given a final rinse with dH2O before air-drying at 23 °C. Antibody-coated wells were incubated at 23 °C for 1 h with 50 µL of 72 h old culture filtrates diluted 1:10 (vol:vol) with PBST (control wells incubated with YNB+G medium only diluted 1:10 (vol:vol) with PBST), after which they were given four 5 min washes with PBST. Washed wells were then incubated for 1 h at 23 °C with HG12-HRP conjugate diluted 1 in 5000 (vol:vol) in PBST, after which they were washed four times with PBST as described, given a final 5 min wash with PBS, and bound antibody visualised by incubating wells with tetramethyl benzidine (TMB) substrate solution for 30 min. Enzyme–substrate reactions were stopped by the addition of 3 M H2SO4, and absorbance values were determined at 450 nm using a microplate reader (infinite F50, Tecan Austria GmbH, Reading, UK). All incubation steps were performed at 23 °C in sealed plastic bags.
5
Indirect ELISA for TB Antibody Detection
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For detection of human antibodies specific for the proteins in the plasma samples of active TB patients, indirect sandwich ELISA was performed according to the method described previously [8 (link), 11 (link)]. The coating concentration for each antigen was 2 μg/ml in 1X PBS buffer (pH 7.4), on 96-well maxisorp microtitre plates (Nunc, Denmark, 4912). The differential absorbance, OD450/630 was read with HUMAREADER plus (Human GmBH, Germany). All the plasma samples were tested in duplicate and their average values were calculated for further analysis.
6
Quantification of Zika Virus Antibodies
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Quantification of antigen‐specific antibodies by depletion was adapted from Lemke et al., 2004. Briefly, depletion of anti‐ZIKV antibodies was done using ZIKV virion‐coated (1 × 106 virions per well) 96‐well maxisorp microtitre plates (Nunc, Roskilde, Denmark). Pooled mouse sera samples from 45 dpi were added at 1:500 and incubated for 10 min at room temperature for adsorption. A further 10 × dilution to 1:5000 was done at the 12th well to shorten the depletion process. The unbound portion was collected after 47 rounds of adsorption. ELISA analysis was performed to verify complete depletion of ZIKV‐specific antibodies. IgG concentrations were then quantified using an IgG Mouse ELISA kit (Abcam, Cambridge, UK) according to the manufacturer's protocol where serum samples were recommended to dilute at 10 000×. ZIKV‐specific IgG concentration was obtained by subtracting the total IgG concentration in ZIKV‐specific IgG‐depleted serum from the total IgG concentration in non‐depleted serum (Supplementary table 1 ).
7
ELISA for FPenvM766 Antibody Detection
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The ELISA was essentially performed as described previously [74 (link)], using lysed Vero cells previously infected with 5 PFU FPenvM766 as the plate-bound antigen. Briefly, 96-well maxisorp microtitre plates (Nunc, Naperville, IL, USA) were coated with Vero lysates in PBS- (5 ×104 cells/well) in 0.05 M carbonate–bicarbonate buffer, pH 9.6, and incubated overnight at 4°C. The sera from all of the animals, drawn at T0 and at different times p.i. (T1, T2), were then added at 1:20 dilution, and the binding was revealed using goat anti-mouse HRP-conjugated serum (1:2000 dilution; Dako) and tetramethylbenzidine substrate (Sigma). The absorbance for each well was measured at 450 nm with a microplate reader (550; Bio-Rad Laboratories, Hercules, CA, USA).
8
Immunosuppressive Factors in Cancer
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RPMI-1640 medium, foetal bovine serum, supplements as well as basic laboratory chemicals were purchased from Sigma (Suffolk, UK). Maxisorp™ microtitre plates were obtained from Nunc (Roskilde, Denmark) and Oxley Hughes Ltd (London, UK). Mouse monoclonal antibodies directed against HIF-1α, mTOR and β-actin, as well as rabbit polyclonal Antibodies against phospho-S2448 mTOR, RAGE and HRP-labelled rabbit anti-mouse secondary antibody were purchased from Abcam (Cambridge, UK). Antibodies against phospho-S65 and non-phosphorylated (total) eukaryotic initiation factor 4E binding protein 1 (eIF4E-BP1) were obtained from Cell Signaling Technology (Danvers, MA USA). Goat anti-mouse and goat anti-rabbit fluorescence dye-labelled antibodies were obtained from LI-COR (Lincoln, Nebraska USA). ELISA-based assay kits for the detection of TNFα, IL-1β, SCF and VEGF were purchased from Bio-Techne (R&D Systems, Abingdon, UK). Anti-Tim-3 mouse monoclonal antibody, its single chain variant as well as human Ig-like V-type domain of Tim-3 (amino acid residues 22–124) and human HMGB1 expressed and purified from E. coli (see below for more details) were used in our experiments.11 (link),15 (link) All other chemicals purchased were of the highest grade of purity commercially available.
9
Screening Hybridoma Supernatants by ELISA
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Hybridoma cells were produced by the method described elsewhere [24] , [25] and the supernatants were screened by enzyme-linked immunosorbent assay (ELISA) against antigens immobilized to the wells of Maxisorp microtitre plates (442404; Nunc) (50 µl per well). For antibody specificity tests, antibodies were tested against surface washings [25] prepared from replicate slant cultures of fungi. Protein concentrations, determined spectrophotometrically at 280 nm (Nanodrop, Agilent Technologies Limited, Berkshire, UK), were adjusted with PBS to produce equivalent protein concentrations for each organism. Fifty µl volumes were then used to coat the wells of microtitre plates. After incubating overnight at 4°C, wells were washed four times with PBST (PBS containing 0.05% Tween-20) and once each with PBS and dH2O and air-dried at 23°C in a laminar flow hood. The plates were stored in sealed plastic bags at 4°C in preparation for screening of hybridoma supernatants by ELISA.
10
Enzyme-Linked Immunosorbent Assay Protocol
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Nunc MaxiSorp microtitre plates were coated with 2 μg/ml recombinant protein in bicarbonate buffer overnight at 2–8 °C. Plates were washed 3 times with phosphate buffered saline (PBS) containing 0.05% Tween20 (PBST) and blocked for 1 h at 37 °C with blocking buffer (5% skimmed milk powder in PBS). Plates were incubated for 1 h at 37 °C with prediluted samples (sera collected from sheep bleeds or purified antibody preparations); washed with PBST; and incubated with a donkey anti-ovine IgG horseradish peroxidase (HRP) conjugate (Product 713-035-003; Jackson ImmunoResearch, USA) for 1 h at 37 °C. After further washing, TMB substrate was added and the reaction was stopped by the addition of stop solution before reading the optical density at a wavelength of 450 nm.
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