ELISA kits to assess serum DSG2 level were purchased from RayBio® (Catalog number: ELH-DSG2.; U.S.A.), according to the user manual. Briefly, reagents and samples were prepared as instructed, serum samples diluted to 1:1, and the standard was diluted to concentrations of 4000, 1600, 640, 256, 102.4, 40.96, 16.38, and 0 pg/ml. Then, 100 μl of each standard and sample were added to appropriate wells and incubated at room temperature for 2.5 h. Plates were washed four-times using a microplate washer (Thermo Fisher Scientific) and 100 μl Biotin-antibody (1×) added to each well and incubated at room temperature for 1 h, followed by a further four washes using the microplate washer. Prepared streptavidin solution (1×, 100 μl) was added to each well and the plates incubated at room temperature for 45 min. After washing, 100 μl of TMB One-Step Substrate Reagent was added to each well and then incubated at room temperature for 30 min. Finally, 50 μl of stop solution was added to terminate the reaction, and optical density (OD) values read at wavelengths of 450 and 590 nm using a plate microplate reader (Thermo Fisher Scientific). OD values were converted into concentration using the standard curve and then multiplied by the dilution factor. Two replicates of each serum sample were analyzed and mean values calculated.
Microplate washer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Finland
The Microplate Washer is a laboratory instrument designed to automate the washing process of microplates. It is used to remove unwanted substances from the wells of a microplate, which is a common step in various analytical and experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Spark 10m plate reader, by Tecan (2 mentions)
Microplate plate reader, by Thermo Fisher Scientific (1 mentions)
Multifunctional microplate reader, by Thermo Fisher Scientific (1 mentions)
Xyz3050 dispensing platform, by BioDot (1 mentions)
Cm4000 guillotine cutter, by BioDot (1 mentions)
Mouse igg elispot basic kit alp, by Mabtech (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
5 protocols using microplate washer
1
Quantitative Serum DSG2 Measurement
2
ELISA for Mouse Serum IgG Detection
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The peptide OT-II (peptide OVA 23–339, InvivoGen) was diluted at 2 μg/mL in PBS, and 50 mL was coated overnight at 4°C in a 96-well plate (Maxisorp Nunc Immunoplate, 442404). Plates were washed using a Thermo Scientific microplate washer (5165040). After three washes with 100 μL of PBS-Tween (0.05%), plates were blocked with PBS-BSA (3%) for 1 h. Mouse serum was serially diluted in PBS and incubated for 1 h (50 mL/well) at RT. After five washes with PBS-Tween (0.05%), a goat anti-mouse immunoglobulin G (IgG) (H + L) secondary antibody linked to horseradish peroxidase (JIR 115-035-062) diluted at 1:2,500 in PBS-Tween (0.05%) was added for 1 h. After five washes, 50 mL of transmembrane domain substrate was distributed per well. The enzymatic reaction was stopped after 70 s by addition of 50 mL of H2S04 (1 M), and plates were read at 450 nm with a Tecan Spark 10M plate reader.
3
ELISA-based Antibody Detection Assay
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The antigens (either RBD or ADDomer) were diluted at 1 μg/mL in PBS, and 50 μL was coated overnight at 4°C, in a 96-well plate (Maxisorp NUNC Immunoplate, #442404). Plates were washed using a ThermoScientific Microplate washer (#5165040). After three washes with 100 μL of PBS-Tween 0.05%, plates were blocked with PBS-BSA 3% for 1 h. Mouse serum was serially diluted in PBS and incubated for 1 h (50 μL/well) at room temperature. After five washes with PBS-Tween 0.05%; a goat anti-mouse IgG (H + L) secondary Ab linked to horseradish peroxidase (JIR 115-035-062) diluted at 1:2,500 in PBS-Tween 0.05% was added for 1 h. After five washes, 50 μL of transmembrane domain substrate was distributed per well. The enzymatic reaction was stopped after 70 s by addition of 50 μL of H2S04 (1 M), and plates were read at 450 nm with a TECAN Spark 10M plate reader.
4
Quantitative biomarker detection assay
5
Evaluating Specific B-cell Response to rSMEV Vaccine
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The Enzyme-Linked Immunospot (ELISPOT) experiment was performed for evaluating the specific B-cell response in mice. First, the rSMEV vaccine protein solution (10 μg/mL) was coated on the ELISPOT plate and incubated overnight at 4 °C. Then the plate was washed for 5 times by Microplate washer (Thermo Fisher, FI-01620 Vantaa, Finland) and blocked with 200 μl RPMI-1640 medium (Sigma Aldrich, St. Louis, UA) containing 10% fetal calf serum (FCS) (Sigma) for 30 minutes at 37 °C. After the plate was washed by PBS containing 0.05% Tween 20, the vaccine protein and mice splenocytes suspension prepared (2×105) were added into the ELISPOT plate3. Three repeat wells were set for each sample and the plate was incubated for 20 hours in a 37 °C humidified incubator with 5% CO2. After the plate was washed for 5 times, the biotinylated detecting antibody (anti-mouse IgG) was added and the plate was incubated for 2 hours at room temperature. After the plate was washed, the Streptavidin-ALP was added and incubated at room temperature for 1 hour. The BCIP/NBT solution (MlBio) was added and incubated for chromogenic reaction. The plate was rinsed and dried, the antibody secreting cell (ASC) spot were counted using EliSpot Reader (AID, D 72479, Germany). In this experiment work, the Mouse IgG ELISpot BASIC kit (ALP) (3825-2A, Mabtech, Stockholm, Sweden) was adopted for the ELISPOT experiment.
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