For competitive ELISA used in epitope mapping of mAbs, 2 μg/mL recombinant RBD-his (Sino Biological, Beijing, China) was added in 384-well plates and incubated at 4 °C overnight. 50 μg/mL mAbs per well were added. The plates were incubated at 37 °C for 1 h and then washed. Biotinylation of mAbs (the top 20 neutralizing Abs and 81A11, previously reported SARS-CoV CR302224 (link)) were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) according to the manufacturer’s protocol and purified using MINI Dialysis Unit (ThermoFisher, 69576). 500 ng/mL biotinylated mAbs were added to each well, and the plates were incubated at 37 °C for 1 h. ALP-conjugated streptavidin (Mabtech, Sweden, 3310-10) was added at 1:1000, followed by an incubation of 30 mins at 37 °C. For the quantification of bound IgG, PNPP (Thermo Fisher) was added at 1 mg/mL and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).
Multiskan go fluoro microplate reader
Manufactured by Thermo Fisher Scientific
The MultiSkan GO fluoro-microplate reader is a compact and versatile instrument designed for fluorescence-based experiments. It can accurately measure fluorescence signals in standard 96- and 384-well microplates. The MultiSkan GO provides consistent and reliable results, making it a suitable choice for a wide range of laboratory applications.
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2 protocols using multiskan go fluoro microplate reader
1
Competitive ELISA for Epitope Mapping
2
SARS-CoV-2 Spike Protein Binding Assay
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Recombinant spike protein 1 (S1) of SARS-CoV-2 (2 μg/ml, Sino Biological, Beijing, China) were coated on 384-well plates (Corning Costar), and incubated at 4 °C ovnernight. Then the plates were blocked with the blocking buffer (PBS with 5% BSA) at 37 °C for 1 h, and subsequently added with serially diluted monoclonal antibodies (10-fold dilutions, from 10 μg/ml to 10 pg/ml). After 30 min incubation at 37 °C, the plates were washed 5 times and incubated with goat anti-human IgG (H + L) antibody conjugated with ALP (Thermo Fisher, a18808, 1:5000) for 30 min at 37 °C. For the quantification of bound IgG, 1 mg/ml PNPP (Thermo Fisher) was added and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).
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