The hemolytic activities of the parental and recombinant viruses were performed according to previously published methods57 (link). In brief, the allantoic fluids of the rescued viruses were clarified by centrifugation (4 °C, 20 min, 500 g). Supernatant fluids of different NDVs were diluted to the same hemagglutination unit/ml (HAU/ml) based on the HA titer. The diluted virus sample 0.5 ml was mixed with 1 ml of 1% suspension of chicken erythrocytes. After incubated on ice for 20 min with occasional shaking, the erythrocytes were sedimented by centrifugation at 500 g for 3 min, washed with phosphate-buffered saline (PBS) and resuspended in the same buffer (0.5 ml). After incubation for 60 min at 37 °C, tubes were then centrifuged at 200 g for 5 min. Supernatant fluids were then transferred to 96-well plates and their hemoglobin contents were measured as absorbance at 549 nm with a Spectramax M5 ELISA reader (Molecular Devices). As for the positive control, 0.03 M NH4OH was added to the assay and for the negative control, the PBS was used. The hemolytic values for all the viruses were expressed as percentages of the values for rNDV-SG10-HN571 at 28 HAU/ml, which were considered to be 100%.
Spectramax m5 elisa reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax M5 ELISA reader is a multi-mode microplate reader designed for absorbance, fluorescence, and luminescence measurements. It provides accurate and reliable data for a range of assays, including ELISA, cell-based, and biochemical applications.
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11 protocols using spectramax m5 elisa reader
1
Hemolytic Activity of Recombinant NDVs
2
Neuraminidase Activity Assay for NDV
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The NA activity was determined using a modification of an assay described previously (Potier et al., 1979 (link)). Vero cell monolayers were infected with NDVs at 0.1 MOI. At 24 hpi, the cells were incubated at 37°C for 30 min with 30 μl of substrate mix (one volume of 325 mM 2-N-morpholinoethanesulfonic acid [MES; pH 6.4], two volumes of 0.5 mM 2′-(4-methylumbelliferyl)-α-D -N-acetylneuraminic acid [MUN; Sigma], and three volumes of 10 mM calcium chloride) per well. The reaction was terminated using 0.014 M sodium hydroxide in 83% (v/v) ethanol. Fluorescence intensity was measured at an emission wavelength of 450 nm and an excitation wavelength of 360 nm with a Spectramax M5 ELISA reader (Molecular Devices). The NA assay investigating HN at the protein level was performed with monolayers of Vero cells transfected with the same expression plasmids utilized in HAd assay.
3
Cytokine Profiling of Myeloid Cells
4
Biofilm Formation Assay for E. piscicida
5
Quantifying SARS-CoV-2 RBD Antibodies in Vaccinated Mice
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To determine the total IgG antibodies against the vaccine mCuMVTT-RBM as well as mRNA-1273 in sera of vaccinated mice, ELISA plates (96 well half-area ELISA plates; Costar, Corning, Catalog # 3690) were coated with SARS-CoV-2 RBD (wildtype) at a concentration of 1μg/ml overnight at 4°C. ELISA plates were washed with PBS-0.01% Tween and blocked using 100μl PBS-Casein 0.15% for 2h in RT. Sera from vaccinated mice was serially diluted 1:3 starting with a dilution of 1:20 and incubated for 2h at RT. After washing with PBS-0.01%Tween, goat anti-mouse IgG conjugated to Horseradish Peroxidase (HRP) (Jackson ImmunoResearch, West Grove, Pennsylvania) was added at 1/1000 and incubated for 1h at RT ELISA was developed with tetramethylbenzidine (TMB), stopped by adding equal 1 M H2SO4 solution. OD450 was measured using the SpectraMax M5 ELISA reader (Molecular Devices, Catalog # M5) and read at OD450 nm or expressed as Log OD50. Detecting RBD-specific IgGs against mutated RBDs was carried out in a similar way. Detecting antibody response from mice injected with different doses of mRNA-1273 (10μg, 1μg and 0.1μg) was done in a similar set up.
6
Virus-Induced Cell Hemoglobin Release Assay
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Confluent monolayers of Vero cells in 24-well plates were infected with 0.1 MOI of virus. Cells were washed with cold PBS at 24 hpi and then overlaid with a 2% (v/v) suspension of CRBCs at 4°C for 30 min (Li et al., 2004 (link)). Unbound CRBCs were washed with ice-cold PBS and the CRBCs bound to the virus-infected cells were lysed with an RBC lysis solution (17mM Tris-HCl, 0.145 M NH4Cl). The released hemoglobin was measured with a Spectramax M5 ELISA reader at 549 nm (Molecular Devices, Sunnyvale, CA, United States). The HAd assay investigating HN at the protein level was performed with monolayers of Vero cells transfected using 0.5 μg each of pCI-HN plasmids (pCI-SGHN, pCI-BJHN, pCI-LaHN, pCI-HBHN and pCI-YuHN) as described previously.
7
Quantifying CCL3 and CCL5 in Liver
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CCL3 and CCL5 were measured in liver lysates using the mouse CCL5/RANTES DuoSet ELISA (DY478-05, R&D systems, Minneapolis, MN, USA) and the mouse CCL3/MIP-1 alpha DuoSet ELISA (DY450-05, R&D systems, Minneapolis, MN, USA). Absorbance was measured at a wavelength of 450 nm using the SpectraMax M5 ELISA reader (Molecular Devices, San José, CA, USA).
8
Hemolytic Activity Assay for Rescued Viruses
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The hemolytic activities were performed as described previously (Bratt and Clavell, 1972 (link)). Briefly, the allantoic fluids of the rescued viruses were centrifuged with 500 × g for 20 min, and the supernatants were diluted to the same hemagglutination unit/ml (HAU/ml). Five hundred microliters of the diluted virus sample was mixed with 1 ml of 1% CRBC suspension and incubated on ice for 20 min. Then the CRBCs were centrifuged at 500 × g for 3 min, washed and resuspended with 0.5 ml PBS. After 1 h incubation at 37°C, tubes were centrifuged for 5 min at 200 × g. Subsequently, supernatant fluids were added to 96-well plates and the hemoglobin contents were measured at 549 nm with a Spectramax M5 ELISA reader (Molecular Devices). Positive and negative controls were 0.03 M NH4OH and PBS, respectively. The hemolytic values for each virus were expressed as percentages of the values for parental virus rSG10 and rLaSota at 28 HAU/ml, which were considered to be 100%.
9
Fluorimetric Neuraminidase Activity Assay
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The NA activity was determined with a fluorimetric procedure, as described by Potier et al.37 (link), with some modifications. Vero cell monolayers in 96-well plates were inoculated with NDVs at an MOI of 0.1 PFU/cell. At 24 hpi, the cells were washed with cold PBS and overlaid with 30 μl of substrate mix (one volume of 325 mM2-N-morpholinoethanesulfonic acid [MES; pH 6.4], two volumes of 0.5 mM 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid [MUN; Sigma, St. Louis, MO, USA], and three volumes of 10 mM calcium chloride) per well, to give a final concentration of 100 μM MUN in the assay. Cells were incubated at 37 °C for 30 min with shaking, and the reaction was terminated by the addition of 0.014 M sodium hydroxide in 83% (vol/vol) ethanol. The fluorescence intensity was measured at an excitation wavelength of 360 nm and an emission wavelength of 450 nm with a Spectramax M5 ELISA reader (Molecular Devices). The NA assay at the protein level was performed with monolayers of Vero cells transfected using the same expression plasmids utilized in the HAd assay.
10
Quantifying Interleukin-1β in Mouse Serum
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The ELISA was used for the quantification of interleukin 1 beta in the serum of HFD mice. The mouse IL1β/IL-1F2 (# DY401-05, R&D Systems®) DuoSet® ELISA DEVELOPMENT SYSTEM kit was used as standard. 50 μL of serum was added to 96 well half- area ELISA plate (# 3690, Costar, Corning), which had been coated overnight at RT according to kit instructions. The ELISA was performed according to the kit instructions. For development, 50 μL of development solution (4.75 mL citrate buffer (30 mM), 0.25 mL 3,3′,5,5′Tetramethylbenzidine (TMB), and 25 μL H2O2 (30%)) was added to each well, and developed at room temperature for 20 min. After the incubation step, the reaction was stopped with 1 M H2SO4 (50 μL/well). OD450 was measured using the SpectraMax M5 ELISA reader (# M5, Molecular Devices) at a wavelength of 450 nm. OD540 was measured for wavelength correction.
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