For serum samples, BPI antibody reactivity was determined by anti-human BPI ELISA kit (ORGENTEC Diagnostika GmbH). Positive reactivity threshold was established as mean reactivity of healthy controls (n=16) plus two standard deviations (>6 units/ml). To measure anti-P. aeruginosa IgG titers, ELISA plates (R&D Systems) were coated with 10 μg/ml P. aeruginosa lysate (repeated freeze-thaw cycles of overnight PA14 culture, provided by Berwin Laboratory, Dartmouth) for 2hr at 37°C. PA14 strain, a reported marker for P. aeruginosa antibody in this study, exhibited conserved epitopes across PAO1 and three new patient-isolate strains (data not shown). After blocking, sera (1:1000) were incubated for 1hr, and reactivity was determined using goat anti-human peroxidase labeled IgG F(ab)’2 (1:50000, Bio-Rad) followed by the substrate (R&D Systems). Absorbance (450 nm) was determined using ELISA reader (Epoch, BioTek).
Elisa plates
Manufactured by R&D Systems
Sourced in United States
ELISA plates are a type of laboratory equipment used in enzyme-linked immunosorbent assays (ELISA) to detect and measure specific proteins or other molecules in a sample. The plates are typically made of polystyrene and have multiple wells, each of which can be coated with a specific capture antibody or antigen. Samples are added to the wells, and the target analyte, if present, will bind to the coated surface. After a series of washing and incubation steps, a detection antibody is added, which binds to the captured analyte and produces a measurable signal, allowing for the quantification of the target molecule.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by Bio-Rad (1 mentions)
Mouse laminin, by Thermo Fisher Scientific (1 mentions)
Eclia kit, by Roche (1 mentions)
Msd 5 plex angiogenesis panel 1 human kit, by MSD (1 mentions)
Tmb substrate, by R&D Systems (1 mentions)
Nickel coated plates, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by R&D Systems (1 mentions)
Synergy htx plate spectrophotometer, by Agilent Technologies (1 mentions)
Wash buffer, by R&D Systems (1 mentions)
3 m naoh, by Merck Group (1 mentions)
13 protocols using elisa plates
1
Quantifying Anti-BPI and Anti-P. aeruginosa Antibodies
2
Enzyme-Linked Immunosorbent Assay (ELISA)
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The ELISA plates were purchased from R&D Systems (Minneapolis, MN). The goat anti-human IgG HRP-linked antibody was purchased from Abcam (Cambridge, MA). The blocking buffer was purchased from Bio-Rad (Bio-Rad, Hercules, CA).
3
Biofilm Formation Assay for P. aeruginosa
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The biofilm formation abilities of P. aeruginosa strains were assessed using the 96-well plate biofilm assay, as previously described (22 (link)). In brief, P. aeruginosa overnight LB cultures were centrifuged, and the cell pellets were washed and resuspended in fresh LB. The optical density at 600 nm (OD600) of each culture was determined, and the culture concentration was adjusted to have 1 × 107 bacteria per ml in LB by converting the OD to an estimated number of CFU per milliliter using a standard curve. For biofilm formation on protein-coated surfaces, ELISA plates (catalog no. DY990; R&D Systems, Minneapolis, MN, USA) were coated overnight at room temperature with 10 μg/ml mouse laminin (catalog no. 23017015; Thermo Fisher Scientific, Waltham, MA, USA) or 1% BSA in phosphate-buffered saline (PBS). The plates were subsequently blocked with 1% BSA, followed by fluid aspiration and air drying before concentration-adjusted P. aeruginosa cultures were added. Adjusted cultures were grown at 37°C for 18 h. The plates were subsequently washed and stained with 0.1% crystal violet (CV). The plates were washed to remove excess dye and air dried before the addition of 30% acetic acid to solubilize the CV. The solubilized CV was quantified in a plate reader at 550 nm.
4
GD3 Antibody Detection by ELISA
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ELISA plates (R&D Systems) were coated with GD3 before incubation with diluted serum samples to detect anti-GD3 antibodies (16 (link)).
5
Profiling Angiogenic Biomarkers
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Samples for biomarker analyses were collected on Cycle 1 Day 1 (predose), Cycle 1 Day 24 (72 h post-dose), and 14 days after the end of treatment. Soluble VEGFR2, VEGF, and CSF1 levels were measured using multiplex enzyme-link immunosorbent assay (ELISA) plates from R&D Systems (Minneapolis, MN), and placental growth factor (PlGF) levels were measured using an electrochemiluminescence immunoassay (ECLIA) kit from Roche Diagnostics (Mannheim, Germany).
6
Quantifying Pro-Angiogenic Factors in Samples
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Concentrations of pro-angiogenic factors in patient and PDX samples were quantified using two ELISA platforms. The electrochemiluminescence detection with Meso Scale Discovery (MSD) V-PLEX Angiogenesis Panel 1 Human Kit (VEGF-A/-C/-D, PlGF, bFGF/FGF2, sflt1/VEGFR1, Tie2) was performed according to the manufacturer’s protocol (MSD, Hercules, CA, USA). In addition, human pro-angiogenic factors VEGFA, PlGF and angiopoietin1 and mouse VEGFA and PlGF were analyzed with specific ELISA plates according to the manufacturer’s protocol (R&D Systems, Inc, Minneapolis, MN, USA). After thawing, samples were handled as described above and centrifuged briefly at 2000× g for 2 min at 4 °C. The supernatants were incubated on MSD V-plex or the specific ELISA plates. MSD V-plex plates were washed and read using SECTOR Imager 2400 software (MSD) and the ELISA (R&D Systems, Inc) on a plate reader Victor 1420 Multilabel Counter, (Wallac/PerkinElmer Life Sciences, Turku, Finland) at 450 nm with wavelength correction at 570 nm.
7
Measuring CFH Binding to Polysialic Acid Nanoparticles
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CFH-His (1 μg/mL; Abcam, Cambridge, UK, or Cloud Clone, Katy, TX, USA) was bound to nickel-coated plates (Thermo Fisher Scientific, Waltham, MA, USA) via overnight incubation at RT. CFH wild-type and Y402H polymorphism fragments (FH6,7/hIgG1Fc and FH6,7 Y402H/hIgG1Fc; 1 μg/mL each), generated as previously described [49 (link),50 (link)], were bound to ELISA plates (R&D Systems, Minneapolis, MN, USA) via overnight incubation at RT. Negative controls included PBS and recombinant human IgG-Fc (R&D Systems). Coated wells were washed and then PolySia-NPs (0.3–10 mg/mL) or blank NP negative control were incubated for 2 h, followed by washing, biotinylated PEG antibody (0.5 μg/mL; Abcam, Waltham, MA, USA) incubation for 1 h, washing, and streptavidin-HRP (R&D Systems) incubation for 20 min. PEG was then visualized using TMB substrate (R&D Systems) reaction, stopped (R&D Systems), and read at 490 nm absorbance using a BioTek Synergy HTX plate spectrophotometer. Four independent experiments were performed and data were combined for presentation here.
8
ELISA analysis of IVIg binding
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IVIg was purchased from Sichuan Yuanda Shuyang Pharmaceutical Company (> 98% IgG, Batch No.: 201908143B, 100 mg/mL) and this batch was produced before the break of COVID-19 (08/2019). ELISA was performed using streptavidin-coated 96-well ELISA plates (Cat: 45,360, R&D Systems) according to the manufacturer's instructions [14 (link)]. Briefly, 100 μL of biotinylated peptides (1 μg/mL) was added to each well, covered with adhesive strip, and incubated overnight at room temperature. After three washes (350 μL/well of wash buffer), 100 μL of 1/10 dilution of IVIg or serum of healthy donors was added. After 2 h at room temperature, three aspirations/washes were performed. Then HRP-coupled anti-Fc of human IgG (1:10,000 in PBS) was added to each well. After 1 h at room temperature, chromogen solutions A and B were added. Finally, 3 to 5 min later, a stop solution was added and the absorbance was measured at 405 nm using a microplate reader. Positive and negative antigen wells were used as controls. There were 6 replicates for each sample and 10 replicates for each control.
9
Cytokine Production in RAW264.7 Cells
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RAW264.7 cells (1 × 106/well) were cultured in a 6-cell plate overnight, and the cells were treated with a range of E9OAEE concentrations (6.25, 12.5, 25, 50 µg/mL) for 2 h and then stimulated with LPS (1 µg/mL) for 24 h. Cell culture supernatants were collected and added into ELISA plates for the determination of PGE2 (R&D Systems, Minneapolis, USA), TNFα (BIOSTER, Wuhan, China), IL6 (BIOSTER, Wuhan, China), and IL-1β (BIOSTER, Wuhan, China) following the respective manufacturer's instructions. Each sample was tested in triplicate.
10
Measuring Anti-IsdB Antibody Titers by ELISA
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To measure anti-IsdB antibody titers, ELISA plates (R&D Systems) were coated with 5 μg/ml avidin in PBS overnight at 4°C. The plates were then washed and blocked in PBS plus 1% BSA for 2 h at RT. Biotinylated IsdB protein (generously provided by Edward Schwarz, URMC) was incubated (5 μg/ml) in the plate for 2 h at RT. The plates were then washed, and patient serum was added and incubated for 1 h at RT. The ELISA was developed as described above.
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