ELISAs were performed as previously reported (Israelow et al., 2020 (link)). In short, Triton X-100 and RNase A were added to serum samples at final concentrations of 0.5% and 0.5 mg/ml, respectively, and incubated at room temperature for 3 h before use to reduce risk from any potential virus in serum. 96-Well MaxiSorp plates (#442404; Thermo Fisher Scientific) were coated with 50 µl/well of recombinant SARS-CoV-2 S1 protein (100 µg, #S1N-C52H3; ACROBiosystems) at a concentration of 2 µg/ml in PBS and incubated overnight at 4°C. The coating buffer was removed, and plates were incubated for 1 h at room temperature with 200 µl of blocking solution (PBS with 0.1% Tween-20 and 3% milk powder). Serum was diluted 1:50 in dilution solution (PBS with 0.1% Tween-20 and 1% milk powder), and 100 µl of diluted serum was added for 2 h at room temperature. Plates were washed three times with PBS-T (PBS with 0.1% Tween-20), and 50 µl of mouse IgG-specific secondary antibody (1:10,000, #405306; BioLegend) diluted in dilution solution added to each well. After 1 h of incubation at room temperature, plates were washed three times with PBS-T. Samples were developed with 100 µl of TMB Substrate Reagent Set (#555214; BD Biosciences), and the reaction was stopped after 15 min by the addition of 2 N sulfuric acid.
96 well maxisorp plates
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The 96-well maxisorp plates are a laboratory equipment product designed for use in various experimental applications. These plates feature a high-binding polystyrene surface that is optimized for immobilizing proteins, peptides, and other biological molecules. The plates provide a consistent and reliable platform for performing assays, such as ELISA, cell-based experiments, and other binding studies. The 96-well format allows for efficient sample handling and data collection.
Automatically generated - may contain errors
Lab products found in correlation
Mouse igg specific secondary antibody, by BioLegend (1 mentions)
Tmb substrate reagent set, by BD (1 mentions)
Rat anti mouse ige, by Southern Biotech (1 mentions)
Opteia elisa set, by BD (1 mentions)
Extravidin, by Merck Group (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti human iga hrp, by Merck Group (1 mentions)
Tmb liquid substrate, by Merck Group (1 mentions)
Incomplete freund s adjuvant, by BD (1 mentions)
Complete freund s adjuvant, by BD (1 mentions)
5 protocols using 96 well maxisorp plates
1
SARS-CoV-2 S1 Protein ELISA Protocol
2
Serum Antibodies Detection by ELISA
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Blood samples were collected by cardiac puncture, centrifuged and serum was stored at −20 °C. Serum antibodies were determined by Enzyme-linked immunosorbent assay (ELISA). Total IgE concentrations were determined by sandwich-ELISA using kit BD OptEIA ELISA Set (BD, San Diego, CA, USA). OVA-specific IgE levels were determined by adding serum samples at 1/10 dilutions to plates coated with rat anti-mouse IgE (SouthernBiotech, Birmingham AL, USA). After washing, biotin-labelled OVA was added and reaction revealed using ExtrAvidin (Sigma-Aldrich, St. Louis, MO, USA) plus substrate. OVA-specific IgE serum concentrations were calculated using a Chondrex kit (Chondrex, Redmond, WA, USA) with known concentrations of OVA-specific monoclonal IgE antibody. OVA-specific IgG1 and IgG2c were measured by coating the plates with 20 µg/mL of OVA. Serum samples were added at multiple dilutions and revealed with goat anti-mouse IgG1 or IgG2a conjugated to HRP (SouthernBiotech, Birmingham, AL, USA), which also reacts with IgG2c isotype. Standard OVA-specific curves for IgG1 and IgG2c were purchased from Chondrex. All ELISA were performed in 96-well maxisorp plates (Thermo Fisher Scientific, Rochester, NY, USA).
3
Receptor-Ligand Binding Assay
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The 96-well maxisorp plates (ThermoFisher) were coated overnight with either the FcαRI or the DECTIN-1 receptors-Fc (R&D systems, Lille, France) or with IgA (for the DC-SIGN and CD71 ELISA). FcαRI and Dectin-1 concentrations began at 6 × 10 -7 mol/L and serial dilutions were performed at 1/3 whereas IgA concentration was stable at 6 × 10 -8 mol/L in PBS. After 2 h of blocking (PBS, tween 0.05%, BSA 1%), the plates were washed three times with PBS/Tween (0.05%). Either the IgA (6 × 10 -8 mol/L) or the DC-SIGN or CD71 (serial dilutions beginning at 4.8 × 10 -8 mol/L) receptors-Fc were added and incubated during 2 h. After three washes, plates were incubated either with an anti-human IgA HRP (Sigma-Aldrich) or with an anti-human DC-SIGN (Life technologies, Illkirch, France) or with an anti-human CD71 antibody (Life technologies). After three washes, plates previously incubated with the anti-human DC-SIGN or CD71 were incubated with anti-rabbit IgG coupled to HRP (Cell signaling technology). Plates were eventually incubated with TMB (Tebubio laboratories) after four last washes during 30 min maximum and the reaction was stopped with hydrochloric acid. The OD was read at 450 nm (TECAN).
4
B7-H3 Antigen ELISA Binding Assay
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Recombinant murine B7-H3 (Val29-Phe244) and human B7-H3 (Leu29-Pro245) with a C-terminal 10-His tag were obtained from R&D Systems. DS-5573, a full-sized anti-human B7-H3 afucosylated humanized antibody (150 kDa) was obtained from Daiichi Sankyo. Phosphate-buffered saline (PBS) was added for reconstitution to a final concentration of 100 μg/mL. B7-H3 antigens (3 μg/mL in PBS; 50 μL per well) were coated to 96-well maxisorp plates (Nunc) overnight at 4 °C. Plates were blocked with 3% fetal calf serum in PBS (240 μL/well) for 1 h at room temperature. A serial dilution of DS-5573a or an IgG isotype control antibody (10 μg/mL to 3.15 ng/mL) were added to the plate in duplicate and incubated for 1 h at room temperature. Plates were washed three times with washing buffer (0.05% Tween in PBS). Secondary goat anti-human Ig Fc-horse radish peroxidase-conjugated antibody (100 μL per well) was added at a 1 in 2000 dilution and incubated for 1 h at room temperature. After washing three times with washing buffer, 3,3′,5,5′-Tetramethylbenzidine (TMB) Liquid Substrate (Sigma) (100 μL/well) was added and allowed to react for 10 min at room temperature before adding stop solution (2 M H2SO4, 50 μL/well). Absorbance was read using an ELISA plate reader at 450 nm.
5
Synthesis and Characterization of p75 Peptides
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Peptides were synthesized using reagents and amino acid derivatives produced by Merck (Germany) and Fluka (Switzerland). Immunological studies were carried out with the keyhole limpet hemocyanin (KLH) solution in PBS at a concentration of 5 mg/ml (Sigma–Aldrich, USA), 25% aqueous solution of glutaric dialdehyde (Sigma, USA), Freund’s complete adjuvant, Freund’s incomplete adjuvant (Difco Laboratories, USA), goat antimouse immunoglobulin antibodies conjugated to horseradish peroxidase (Imtek, Russia), and 96-well Maxisorp plates (Nunc, Denmark). Peptides were chosen according to the sequence of the human p75 (TNR16_HUMAN) UniProtKB), and their synthesis was carried out on Wang resin, using the Fmoc/But-scheme. All peptides contained additional C-terminal glycine residue. TBTU/ DIEA method was applied for elongation of peptide chain. Cleavage of peptides from the resin was carried out in a mixture of TFA (94%), triisopropylsilane (1%), 1,2-ethane dithiol (2.5%), and water (2.5%) for 2 h. The peptides were purified by reverse phase HPLC in Phenomenex Jupiter 10 μ C18 300A 250×10 mm in an acetonitrile gradient from 10 to 70% in 0.1% TFA solution. Synthetic peptides were characterized by the data of amino acid analysis, analytical HPLC, and mass spectrometry.
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