Journal: MethodsX
Article Title: An efficient ELISA protocol for measurement of SARS-CoV-2 spike-specific IgG in human plasma and serum samples
doi: 10.1016/j.mex.2024.102596
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Article Snippet: , Self-prepared buffers and solutions Stock solutions of PBSA and PBSA-S should be kept sterile and stored at 4 °C. • 0.1 M sodium hydrogen carbonate (pH 8.6) • 1 N sulfuric acid • PBS with 0.05 % Tween 20 (PBS-T5) • PBS with 5 % BSA (PBSA) • PBSA with 0.01 % Tween 20 (PBSA-T1) • PBSA with 20 % sheep serum (PBSA-S) Assay controls • Positive control: SARS-CoV-2 IgG high serum or plasma sample. If not available, a positive control can be generated by dilution of SARS-CoV-2 spike specific Ab, e.g. CR3022 (expression plasmid set used for production in FreeStyle 293-F cells: CAT# NR-53260, BEI Resources; ready-made Ab: CAT# NR-52481, BEI Resources, RRID:AB_2936472), in non-reactive serum or plasma. • Negative control: serum or plasma drawn from healthy donors before 2019. Equipment Utilizing a plate washer minimizes variation in assay results and is the preferred method over manual washing of ELISA plates using a multichannel pipette. • Class II biological safety cabinet, Thermo Scientific, CAT# 51025411 • Eppendorf Research plus pipettes: ○ 30 – 300 µL, 12-channel, CAT# 3125000060 ○ 2 – 20 µL, 1-channel, CAT# 3123000039 ○ 20 – 200 µL, 1-channel, CAT# 3123000055 ○ 100 – 1,000 µL, 1-channel, CAT# 3123000063 • Plate washer HydroSpeed, Tecan, CAT# 30054550, RRID:SCR_023457 • Plate reader Spark, Tecan, CAT# 30086376, RRID:SCR_021897 Consumables • Pipette tips with filter ○ 10-20 µl, TipOne, CAT# S1120-3710 ○ 20-200 µL, Biosphere, CAT# 70.1189.215 ○ 100-1000 µl, TipOne, CAT# S1122-1730 • Single use reservoir, Integra, CAT# 4312 • Plates: ○ 96-well plates, polystyrene, transparent, U-bottom, Greiner Bio-One, CAT# 650101 ○ 96-well ELISA plates, polystyrene, transparent, F-bottom, MaxiSorp, Nunc, Thermo Scientific, CAT# 442404 • 50 mL tubes, Greiner Bio-One, CAT# 227261 • EASYseal plate sealer, transparent, Grainer Bio-One, CAT# 676001 Software • SparkControl (version 3.1), Tecan, included with Plate washer • GraphPad Prism 9 (version 9.5.0), Dotmatics.
Techniques: Enzyme-linked Immunosorbent Assay, Software, Saline, Produced, Plasmid Preparation, Transfection, Sterility, Positive Control, Generated, Expressing, Negative Control, Transferring
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