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9 protocols using streptavidin peroxidase polymer ultrasensitive

1

Epitope Correlation of SARS-CoV-2 Antibodies

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Competition enzyme-linked immunosorbent assay (ELISA) was performed to explore the epitope correlation of two antibodies. Briefly, the first antibody at the concentration of 2 μg/mL was coated on plates (BEAVER) and incubated at 4 °C overnight. Then, excess antibodies were washed away by PBS and blocked by 3% skim milk. SARS-CoV-2 S1 protein (Sino Biological) was biotinylated using an EZ-Link™ Sulfo-NHS-LC-LC-Biotin kit (ThermoFisher), followed by mixing with 50 μg/mL of the second competition antibodies or PBS blank control. After incubation at 37 °C for 1 h, plates were washed three times with PBS and the diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma) was added (1:2000) subsequently. Then the plates was incubated at 37 °C for 1 h again. TMB Single-Component Substrate Solution (Solarbio) was used to detect the S1 binding with the coated first antibodies. The absorbance at 450 nm was measured in an Infinite M200 PRO Multimode Microplate Reader (TECAN). Competitive percentage of two antibodies was calculated with reference to the PBS blank control.
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2

SARS-CoV-2 Antigen-Antibody Binding Assay

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For experiments involving the competitive binding of antibodies to SARS-CoV-2 RBD or S trimer, recombinant hACE2-Fc protein was first biotinylated using EZ-Link Sulfo-NHS-Biotin (ThermoFisher) as the instruction described. SARS-CoV-2 RBD (Sino Biological), S trimer (AcroBiosystems), mutated RBDs (Sino Biological), and mutated S trimers (AcroBiosystems) were coated onto High Binding ELISA 96-Well Plate (BEAVER). In order to obtain an optimized hACE2-Fc concentration for this experiment, the concentration-dependent binding of biotinylated hACE2-Fc to coated SARS-CoV-2 antigens was measured by performing a conventional receptor-binding ELISA. The 80% maximal effective concentration (EC80) of biotinylated hACE2-Fc was calculated by the four-parameter nonlinear fitting. Antibodies were serially diluted in 1% BSA (Sigma) and added 50 μL into the antigen coated plates. Biotinylated hACE2-Fc at EC80 concentration was subsequently pipetted into. After incubation at 37 °C for 1 h, plates were washed four times with PBST and incubated with 100 μL of 1:2000 diluted Ultrasensitive Streptavidin-Peroxidase Polymer (Sigma). After further washing, 100 μL TMB was added, followed by detection of the bound hACE2 in the microplate reader. Four-parameter nonlinear regression fitting in GraphPad Prism Version 9.0.0 was applied for result analysis.
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3

SELEX Aptamer Screening for MS Protein

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After 10 iterative rounds of SELEX, the archived aptamer populations from rounds R2, R4, R6, R7, R8, R9, and R10 were amplified through PCR using 5′ biotinylated forward primer and rA-containing reverse primer, followed by strand separation using denaturing urea-PAGE as described previously.40 (link) These ssDNA pools of different rounds were checked for their binding to MS by aptamer-linked immobilized sorbent assay (ALISA). For this, 500 ng of purified MS protein was coated onto a Nunc MaxiSorp 96-well plate (Thermo Scientific) overnight at 4°C followed by blocking with 5% BSA at RT. The biotinylated aptamers pool of the archived population from each round was heated at 92°C for 10 min, cooled on ice, and brought to RT. A total of 100 ng of biotinylated aptamer pool was then added to individual wells pre-coated with MS. Streptavidin-Peroxidase Polymer Ultrasensitive (Sigma) was then added at 1:2,000 dilution followed by washing with SB to remove unbound streptavidin-horseradish peroxidase (HRP) conjugate, and finally TMB (3, 3′, 5, 5′ tetramethylbenzidine) (BD OptEIA) was added. The reaction was stopped by 5% sulfuric acid followed by absorbance measurement at 450 nm.
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4

Western Blot Analysis of Cellular Proteins

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Cell lysates containing equal amounts of protein (15–35 µg, measured using BCA protein assay kit; Pierce) or proteins eluted from Neutravidin beads were mixed with reducing or non-reducing sample buffer (see above) and loaded on 10% SDS-PAGE gels and transferred to nitrocellulose membranes, and probed using antibodies against Src (L4A1; Cell Signaling 1∶1000), EGFR (C74B9 Cell Signaling 1:1000), or anti-cysteine sulfenic acid (Millipore Sigma; 07–2139; which specifically detects dimedone-conjugated cysteine residues) or with streptavidin peroxidase polymer ultrasensitive (1:10,000; Sigma). Primary antibodies were probed with rabbit or mouse-specific secondary antibodies conjugated with HRP (Cell Signaling) and detected by enhanced chemiluminescence (Pierce) using an Amersham 600 SE Imaging System. Western blot band densities were quantified using ImageQuant TL (v8.1.0.0).
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5

Quantifying SARS-CoV-2 Antibody Neutralization

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The rACE2 displacement assay was performed as previously described (33 (link), 34 ), except assay plates were Maxisorp (ThermoFisher Scientific). Briefly, the 384-well plates were coated with RBD (supplied by NRC), blocked with BSA in PBS-T, incubated with 1:10 or 1:40 sera dilutions, then with biotinylated ACE2 (supplied by Jim Rini, University of Toronto), and treated with Streptavidin-Peroxidase Polymer Ultrasensitive (Sigma-Aldrich). Incubation with ELISA Pico Chemiluminescent Substrate and reading on the EnVision were performed as in direct detection. All values were normalized to sample-free blanks on the same plate. The resulting relative ratios (RR) were converted to IU/mL using the WHO International Standard 20/136 as a calibrant (33 (link)) and the following formula: log2(IU/mL at sample dilution d)=RR/(−0.2308)+5.7898+log2(d). Neutralization threshold was determined for the 1:10 dilution as 2×MAD (median absolute deviation) from the mean of blanks.
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6

SARS-CoV-2 Spike Protein ELISA

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The snELISA assay was performed as described13 with the indicated antigens and ACE2. All wash steps included four washes in 200 μL PBST. RBD 319–541 or spike was adsorbed onto 96‐well high‐binding polystyrene Greiner Bio‐One plates (Thermo Fisher Scientific, #655061) at 100 or 200 ng/well, respectively, in 50 µL PBS and incubated overnight at 4°C. Plates were washed, then blocked for 1–1.5 h at room temperature with 200 μL 3% bovine serum albumin (BSA; BioShop Canada Inc., SKI400.1). After washing, serum or plasma was added to the plate at the indicated concentrations in 50 µL 1% BSA in PBST (final concentration) and incubated for 1 h. Wells were washed and incubated with 50 µL of biotinylated recombinant ACE2 as indicated for 1 h. After washing, wells were incubated with 44 ng Streptavidin‐Peroxidase Polymer, Ultrasensitive (Sigma‐Aldrich, S2438) for 1 h. The resultant signal was developed and quantified with TMB‐ELISA in an identical manner to the colorimetric direct ELISAs. Due to day‐to‐day variations in signal, all optical density at 450 nm (OD450) values were normalised to the OD450 of the well without serum or antibody for each sample. All values are expressed as ratios.
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7

Peptide Immobilization on Collagen Films

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Peptides in 5 mg/ml stock solution in 0.01 M AcOH were diluted to 5 μg/ml in PBS (pH 7.4) unless stated otherwise (for concentration studies, peptides were diluted to between 0.1 and 10 μg/ml as indicated). 100 μl of peptide solution was added to wells containing collagen films and incubated for 30 min in the dark at room temperature. Preliminary experiments indicated that incubation of peptide with a collagen scaffold for 30 min was optimal for passive absorption of a similar peptide (L. Mullen, PhD Thesis, University of Cambridge, 2010), and this incubation time was adopted for the experiments described here. Wells were then placed under a long-wavelength UV lamp (Blak-Ray B100AP, 365 nm wavelength) for 2–60 min, and routinely for 5 min, as indicated. Negative control wells were covered in aluminium foil to restrict UV exposure. Following UV treatment, wells were washed 3 times 2 min with 200 μl citrate buffer (pH 3) containing 1 mg/ml BSA and three times with 200 μl of PBS containing 1 mg/ml BSA. 100 μl of Streptavidin-Peroxidase Polymer Ultrasensitive (Sigma #S2438) diluted in PBS to 1:10000 were added to the wells and incubated 45 min at room temperature. After washing four times with 200 μl of PBS containing 1 mg/ml BSA, 100 μl of TMB substrate was added, the reaction stopped with 100 μl of 2.5 M sulphuric acid and A450 was measured.
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8

Immunohistochemical Analysis of VEGF-A in Brain Capillary Endotheliocytes

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IHC studies were performed on 20 patients with DRE for the analysis of VEGF-A content in brain capillary endotheliocytes. Biopsies of the temporal lobe were fixed with 10% paraformaldehyde in 0.1 M sodium phosphate buffer, dehydrated in a standard manner, and embedded in paraffin. IHC reactions were performed on paraffin-embedded 5–7 μm thick slices of the brain temporal lobe biopsies according to the standard protocol. The VEGF-A antibody (Anti-VEGF Receptor 2 antibody [SP123] Abcam, Boston, MA, USA) was used as a primary antibody. For visualization, the Streptavidin-Peroxidase Polymer Ultrasensitive system (Streptavidin-Peroxidase Polymer, Ultrasensitive, Product Number: S 2438, Sigma-Aldrich, St. Louis, MO, USA) and DAB chromogen (3,3′-Diaminobenzidine tetrahydrochloride hydrate, Product Number: D5637, Sigma-Aldrich, USA) were used. The sections were counterstained with Gill’s hematoxylin, and embedded in Bio Mount HM synthetic embedding medium (Bio Mount HM Mounting medium, Catalog number: 05-BMHM100, BIO-OPTICA Milano, Italy). Additionally, reactions lacking primary antibodies were performed to ensure the specificity of the observed staining. Sections were analyzed with a light microscope (Zeiss Mirax Midi BF/FL Fluorescence Slide Scanner Imaging System, Carl Zeiss MicroImaging GmbH., Jena, Germany).
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9

Quantitative Binding Assay for NGF-TrkA Interaction

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Recombinant human TrkA-Fc chimera (125 ng/50 μl; R&D Systems, 175-TK) was coated onto NUNC F96 Maxisorp ELISA plates (Sigma-Aldrich) at 4 °C overnight. The plate was washed 3x in PBST and 2x PBS and then blocked for 1 hr at room temperature with BlockerTM 1% casein in PBS (Thermo Scientific #37528), followed by 4x washes with PBST and 2x PBS. Thirty ng recombinant human β-NGF (R&D Systems, 256-GF/CF) was mixed with 50 µl peptide (Isogenica or Alta Biosciences, UK) at appropriate concentration in 0.1% casein in PBS/0.1% Tween. NGF/peptide solutions (55 µl/well) were transferred into the NUNC Maxisorp plate and incubated for 40 minutes at room temperature with 50 µl/well 1:200 diluted biotinylated anti-human β-NGF antibody (R&D Systems, 256-GF/CF) in 0.1% casein in PBS/0.1% Tween. The plate was washed 4x in PBST and 2x PBS. Streptavidin-HRP (Sigma-Aldrich; Streptavidin-Peroxidase Polymer, Ultrasensitive, #2438; 1:1000 dilution, 50 µl/well) in 0.1% casein/PBST was incubated on the plate for 30 minutes at room temperature followed by 4x washes with PBST and 2x PBS. TMB reagent (Thermo Scientific #34028; 50 µl/well) was incubated on the plate for 5–20 minutes, quenched with 50 µl/well 0.5 M H2SO4 and the absorbance read at 450 nm.
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