一种高通量自动ELISA检测SARS-CoV-2刺突蛋白IgG抗体的方法

Juliana Conkright-Fincham; Chieri Tomomori-Sato; Rich McGhee; Ella M. Leslie; Carolyn J. Beucher; Lauren E. Weems; Shigeo Sato; William B. Redwine; Kyle J. Weaver; Brandon D. Miller; Kym M. Delventhal; John J. Kary; Andrew B. Koebbe; Alexander Dean; Jessica L. Witt; Laura M. Remy; Tari J. Parmely; Chongbei  Zhao; Yan Wang; Joan W. Conaway; Jay R. Unruh

doi:10.21769/BioProtoc.4301

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A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein

一种高通量自动ELISA检测SARS-CoV-2刺突蛋白IgG抗体的方法

发布: 2022年01月20日第12卷第2期 DOI: 10.21769/BioProtoc.4301 浏览次数: 3744

评审: Juan Facundo Rodriguez AyalaLuis Alberto Sánchez VargasVikas DuhanAnonymous reviewer(s)

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Abstract

The SARS-CoV-2 pandemic and vaccination campaign has illustrated the need for high throughput serological assays to quantitatively measure antibody levels. Here, we present a protocol for a high-throughput colorimetric ELISA assay to detect IgG antibodies against the SARS-CoV-2 spike protein. The assay robustly distinguishes positive from negative samples, while controlling for potential non-specific binding from serum samples. To further eliminate background contributions, we demonstrate a computational pipeline for fitting ELISA titration curves, that produces an extremely sensitive antibody signal metric for quantitative comparisons across samples and time.

Keywords: SARS-CoV-2 (SARS-CoV-2)

Background

As SARS-CoV2 spread around the globe in 2020, the urgent need for assays to measure antibodies reactive to viral proteins became clear. Such assays are crucial as a first step for assessing immunological response to infection and vaccination. While it is clear that immunological responses are complex, with many factors other than circulating antibodies contributing to protection after infection and long-term immunity, the ability to rapidly monitor antibody responses allows for population surveillance and provides the foundation for other more complex studies.

While several methods exist for colorimetric ELISA assays (Krammer and Simon, 2020), such methods are laborious, time consuming, and require manual processing, which poses a safety risk to the researcher. We present a method based on previously developed ELISA titration protocols (Stadlbauer et al., 2020 and 2021), using a combination of semi-automated and fully automated steps for sample dilution and ELISA assay processes, to allow for higher throughput in a 384-well format with less human intervention. We have included an alternative 96-well format assay that can be performed using an automated platform or manually, using a multi-channel pipettor and hand-held 8-channel aspiration device. In addition, we present a computational method for fitting ELISA titration curves that accounts for varying background levels. We recently released a preprint utilizing this assay to survey antibodies over time in a midwestern cohort (Remy et al., 2021).

Materials and Reagents

Stericup 0.22 μm filter (MilliporeSigma, catalog number: SCGVU11RE)
384w Immulon Maxisorp ELISA plates (ThermoFisher Scientific, catalog number: 464718)
Universal plate lids (Fisher Scientific, catalog number: 5500)
P30 384w tips for Janus MDT (PerkinElmer, catalog number: 6001299)
150 mL automation reservoirs and base (Integra, catalog number: 6318)
Plastic plate seal (Greiner, catalog number: 676001)
0.3 mL Biobank tube in plate frame (Greiner, catalog number: 9766561)
Axygen 96w U-bottom plate (Axygen, catalog number: P-06-450R-C-S)
125 µL Low retention Viaflo 96/384 XYZ GripTips (Integra, catalog number: 6565)
96w PCR plate (Axygen, catalog number: PCR-96-FS-C)
96w Immulon 2 HB ELISA plates (ThermoFisher Scientific, catalog number: 3455)
0.45 µm Stericup (Sigma Millipore, catalog number: S2HVU02RE)
125 mL plastic bottle (Corning, catalog number: 431731)
Rack (plate frame) for 0.3 mL biobanking tubes (Greiner, catalog number: 976501)
125 µL Viaflo 96/384 XYZ GripTips (Integra, catalog number: 6465)
PPE appropriate for working with SARS-CoV-2 materials including: knee-length disposable lab coat/gown with sleeves that cover the entire length of the arm, safety glasses, shoe covers, face mask, disposable sleeve covers, and two pairs of nitrile gloves.
MBS Capillary Collection Tube, EDTA-2k, 100-150 µL, model #CH100ED2K (Micro Blood Science, catalog number: 001001-BX2)
Expi293F^TM cells (Gibco, catalog number: A14527)
Expi293 expression medium (Gibco, catalog number: A1435101)
HisTrap Excel^TM column (Cytiva, catalog number: 29048586)
Protease inhibitor cocktail (Sigma, catalog number: P8849)
Bovine Serum Albumin (BSA) (SeraCare, catalog number: 1900-0002)
Tween-20 (Sigma, catalog number: P7949)
Accurun 810 negative serum (SeraCare, catalog number: 2010-0020) (stored at 4°C)
SARS-CoV-2 Spike Antibody, 414-1 (AM001414) (Active Motif, catalog number: 91361) (aliquoted and stored at -20°C)
Anti-human IgG (H+L)-HRP conjugated (Promega, catalog number: W4031) (10 µL aliquots were stored at -20°C)
Normal goat serum (Jackson Immunoresearch, catalog number: 005-000-121)
1-Step Turbo TMB-ELISA substrate solution (ThermoFisher Scientific, catalog number: 34022). Store at 4°C. Warm to ambient temperature prior to use
Sulfuric Acid (J.T. Baker, catalog number: 9681-2)
PFM COVID-19 Active Symptomatic, Recovered (post-symptomatic), and Negative Serum are human serum samples collected and curated to support screening, diagnosis, and monitoring research efforts (Precision for Medicine, special order). Aliquot and store at -80°C
Blood Center samples (for our work, this was kindly provided by the Kansas City Community Blood Center)
Sodium chloride (NaCl) (EMD Millipore, catalog number: SX0420-5)
Potassium chloride (KCl) (Calbiochem, catalog number: 7300)
Sodium phosphate dibasic, anhydrous (Na₂HPO₄) (J.T. Baker, catalog number: 3828-01)
Potassium phosphate, monobasic (KH₂PO₄) (Macron, catalog number: 7100-12)
Phosphate buffered saline (PBS, see Recipes)
HisTrap wash buffer (see Recipes)
HisTrap elution buffer (see Recipes)
4× Plasma dilution buffer (see Recipes)
4× Blocking buffer (see Recipes)
Wash buffer (see Recipes)
AM1414 (20) positive control stocks (see Recipes)
AM1414 (10) positive control stocks (see Recipes)
AM1414 (2.5) positive control stocks (see Recipes)
4× HRP buffer (see Recipes)
HRP solution (see Recipes)
Stop solution (see Recipes)

Equipment

AKTA Start (Cytiva, catalog number: 29022094-ECOMPPGST) or equivalent FPLC system
Thermo Scientific Orion Star A211 pH meter (ThermoFisher Scientific, catalog number: STARA2110)
Beckman Coulter Allegra X15X centrifuge with plate carrier rotor and carrier covers (VWR, catalog numbers: BKB09823, BK392806 and BK393070)
Viaflo 96 base unit with 3 position stage and 5-125 µL 96w head (Integra, catalog numbers: 6001, 6230 and 6102)
Viaflo 96/384 base unit with 3 position stage and 5-125 µL 96w head (Integra, catalog numbers: 6031, 6230 and 6102)
LabElite decapper/scanner (Hamilton, catalog numbers: HST-193601, HST-193620 and HST-193487)
405 TS washer/dispenser with 96 pin head (Biotek, catalog number: 405TSUVSQ)
ELISA automated workstation (see Figure 1) contains the following components, which are integrated using a plate::handler II robotic arm (PerkinElmer, catalog number: HH2010100) and controlled with plate::works scheduling software (PerkinElmer: catalog number: HH2010100).
1. Stationary plate hotels (PerkinElmer, catalog number: HH20110091)
2. Automated 2× suction cup delidder (PerkinElmer, catalog number: HH20110051)
3. G3 Janus liquid handling platform with 4 PlateStaks with 4 magazines and 384w p30 MDT head (PerkinElmer, catalog numbers YJSM001 and PSS00021)
4. EL406 washer/dispenser with 192-pin head and peripump with 8-channel 10 µL tubing (Biotek, catalog number: 406PUB3)
5. Victor Nivo plate reader with 450/10 filter set (PerkinElmer, catalog numbers: HH35000000 and HH35000919)

Software

COVIDtrack custom sample tracking software (will be available on acceptance)
plate::works scheduling software (PerkinElmer)
Victor Nivo plate reader software (PerkinElmer)
Janus Winprep software (PerkinElmer)
Biotek LHC washer software
Microsoft Excel
Graphpad Prism 9
Miniconda Python version 3 (https://docs.conda.io/en/latest/miniconda.htmL)
Custom python analysis code: https://github.com/jayunruh/Jay_Serology

Procedure

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Conkright-Fincham, J., Tomomori-Sato, C., McGhee, R., Leslie, E. M., Beucher, C. J., Weems, L. E., Sato, S., Redwine, W. B., Weaver, K. J., Miller, B. D., Delventhal, K. M., Kary, J. J., Koebbe, A. B., Dean, A., Witt, J. L., Remy, L. M., Parmely, T. J., Zhao, C., Wang, Y., Conaway, J. W. and Unruh, J. R. (2022). A High-throughput Automated ELISA Assay for Detection of IgG Antibodies to the SARS-CoV-2 Spike Protein . Bio-protocol 12(2): e4301. DOI: 10.21769/BioProtoc.4301.