For MnPV VLP-ELISA, 96-well plates (Polysorp, Nunc) were coated overnight at 4°C with 1 µg/ml VLPs in 50 mM carbonate buffer pH 9.6 and blocked the next day with casein blocking buffer (0.2% casein in PBS, 0.05% Tween 20). Plates were subsequently incubated 1 h at room temperature with three-fold dilutions of the sera in casein blocking buffer. Plates were washed and bound specific Mastomys IgG was detected with a HRP-conjugated goat anti-mouse IgG antibody (heavy+light chain; Promega, Mannheim, Germany) diluted 1∶10,000 in blocking buffer. Color development was performed by addition of 0.1 mg/ml tetramethylbenzidine (Sigma, Steinheim, Germany) and 0.006% H2O2 in 0.1 M sodium acetate pH 6 (100 µl/well). After 8 min, the enzyme reaction was stopped with 50 µl of 1 M sulfuric acid per well and the absorbance was measured in an automated microplate reader (Labsystems Multiskan; Thermo Fisher Scientific, Waltham, USA) at a wavelength of 450 nm. Antibody titer represents the last reciprocal serum dilution above blank.
Labsystems multiskan
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Labsystems Multiskan is a versatile microplate reader designed for a wide range of quantitative and qualitative assays. It is capable of absorbance measurements across a broad wavelength range, making it suitable for various photometric applications in life science research and clinical diagnostics.
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6 protocols using labsystems multiskan
1
ELISA for Mastomys VLP Antibodies
2
ELISA for S-ED Protein Quantification
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Ninety-six-well plates were coated with 100 μL per well of 100 ng of rabbit polyclonal anti-S-ED serum in 50 mM carbonate/bicarbonate buffer (pH 9.6) and incubated overnight (ON) at 4 °C. After blocking 1 h at 37 °C with 2% (w/v) non-fat milk in PBS, plates were incubated with 1:2 serial dilutions of S-ED internal standard or samples (culture supernatants, harvests, different purification fractions) for 1 h at 37 °C. At the same time, the following controls were tested in each run: (1) a reactive blank and (2) a sample blank. Then, plates were incubated with biotinylated rabbit anti-S-ED antibodies diluted 1:1500 for 1 h at 37 °C. Afterwards, streptavidin–horseradish peroxidase (HRP) conjugate (Sigma) diluted 1:2.000 was added to the wells. Finally, plates were incubated for 13 min with substrate solution (0.5 mg.mL−1 o-phenylenediamine, 0.5 μl.mL−1 H2O2 30 vol in 50 mM phosphate citrate buffer). Absorbance was measured at 492 nm with a microtiter plate reader (LabSystems Multiskan, Thermo Fisher Scientific). Between every step, plates were washed six times with 0.05% (v/v) Tween-20 in PBS (PBS-T). Dilutions of tested samples and antibodies were prepared in PBS-T containing 0.2% (w/v) non-fat milk.
3
ELISA for Anti-Snake Venom Antibody
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Microplates of 96-well (Sarstedt, Germany) were sensitized with 100 µL/well of the heterologous proteins in the serial 1:2 dilution from 1 to 0.008 µg/mL and incubated in a humid chamber at 4 °C for 18 h. Crude venom and GST protein were used as controls under the same conditions. Subsequently, blocking was performed with PBS containing 1% bovine serum albumin (BSA) for 30 min. After blocking, the addition of 1:200 (7.5 µg) of polyclonal IgG anti-C. iheringi venom antibody diluted in PBS + 1% BSA at 37 °C for 1 h. Subsequently, the microplates were incubated with a peroxidase-conjugated anti-rabbit IgG antibody (1:5000) at 37 °C for 45 min. After this, a revelation solution of OPD (ortho-phenylenediamine) was added (1 mg of OPD, 2 mL of citrate/phosphate buffer, and 1 µL of hydrogen peroxide). Then, the microplates were statically incubated, in the dark, at 24 °C for 15 min, and sulfuric acid (H2SO4) 2 N was used to stop the reaction and the plate was read in an ELISA reader (Labsystems Multiskan, Thermo Fisher Scientific, Waltham, MA, USA) at 492 nm.
4
SARS-CoV-2 RBD Antibody Detection ELISA
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Ninety-six-well plates were coated with 100 µL per well of 136.3 ng of purified RBD in 50 mM carbonate/bicarbonate buffer (pH 9.6) and incubated overnight (ON) at 4 °C. After blocking 1 h at 37 °C with 5% (w/v) non-fat milk in PBS, plates were incubated with 1:40 diluted serum or plasma sample for 1 h at 37 °C. At the same time, the following controls were tested in each run: (1) a blank reactive control (BRC); (2) a negative sample control (prepandemic serum); and (3) an antigen-free control (without RBD) and a 1:40 positive control during the sample incubation step (named control A). Then, plates were incubated with a 1:3225 diluted rabbit anti-human IgG-HRP (Santa Cruz Biotechnology) for 1 h at 37 °C. Finally, plates were incubated for 13 min with substrate solution (0.5 mg/mL o-phenylenediamine, 0.12% (v/v) H2O2 in 50 mM phosphate citrate buffer). Absorbance was measured at 492 nm with a microtiter plate reader (LabSystems Multiskan, Thermo Fisher Scientific). Between every step, plates were washed six times with PBS, 0.05% (v/v) Tween-20 (PBS-T). Dilutions of tested samples and antibodies were prepared in PBS-T containing 0.2% (w/v) non-fat milk.
The same procedure was applied for the iELISA employing stressed RBD sample treated with 100 mM DTT as coating antigen.
The same procedure was applied for the iELISA employing stressed RBD sample treated with 100 mM DTT as coating antigen.
5
Quantitative ELISA for Pig Antibodies
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96-well Maxisorp ELISA plates (Biolegend, UK) were coated overnight at 4°C with pre-optimized concentrations of ISU-1 full length S protein (1 µg/ml). Two-fold dilutions of serum, either collected pre-inoculation (day 0) or collected post-mortem, starting from 1/20 in PBS-Tween plus Marvel (4%) were applied. Binding of antibodies was detected with goat anti-pig IgG (Fc-specific) conjugated to horseradish peroxidase (AA141P, Biorad, Watford, UK), used at optimal dilution. 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Scientific Pierce) was added and the optical density values were read for each well at dual wavelengths (450 nm and 630 nm) using a Labsystems Multiskan plate reader (Fisher Scientific, Loughborough, UK). The quantities of antibody were determined as the reciprocal value of the dilution that gave the first reading above the cut-off value (end-point titer). Cut-off values were determined as mean blank ODs plus 2-fold standard deviations. Starting dilutions were 1:20 for serum, 1:16 for BAL and 1:2 for nasal swab respectively.
6
Enzyme-Linked Immunosorbent Assay for Anti-OVA Antibodies
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Plasma total anti-OVA IgG and anti-OVA IgM were determined using enzyme-linked immunosorbent assay procedures. Ninety-six-well microtiter plates were coated with 50 μL of 20 μg•mL -1 OVA in carbonate coating buffer and incubated overnight at 4°C. Nonspecific binding was blocked with PBS supplemented with 10% fetal bovine serum and incubated for 1 h at 37°C. After washing three times with PBS-Tween 20, 50 μL of plasma was added at a dilution of 1:20 (IgG) or 1:200 (IgM) in a diluting buffer of PBS/1% fetal bovine serum and incubated for 1 h at 37°C. Plates were washed again, and 50 μL of horseradish peroxidase-rabbit anti-mouse IgG or horseradish peroxidase-goat anti-mouse IgM (Life Technologies, Frederick, MD), diluted 1:800 or 1:400, respectively, in diluting buffer, was added. Plates were incubated again and then washed. Plates were incubated for 20 min in 50 μL of a 1:1 mixture of 3, 3′, 5,5′ tetramethylbenzidine (TMB) and hydrogen peroxide (TMB Substrate Reagent Set; BD Biosciences, San Jose, CA). Lastly, 25 μL of stop solution (sulfuric acid) was added, and plates were read at 450 nm on a spectrophotometric plate reader (Labsystems Multiskan; Fisher Scientific, Pittsburgh, PA). Plasma anti-OVA IgG or IgM was quantified as the difference in optical density at 450 nm from the preinjection time point.
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