Development of an Efficient ELISA Protocol for Measurement of SARS-CoV-2 Spike-Specific IgG in Human Plasma and Serum Samples

doi:10.21203/rs.3.pex-2194/v1

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Development of an Efficient ELISA Protocol for Measurement of SARS-CoV-2 Spike-Specific IgG in Human Plasma and Serum Samples

https://doi.org/10.21203/rs.3.pex-2194/v1

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published 01 Jun, 2024

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Here, we present an optimized enzyme-linked immunosorbent assay (ELISA) protocol for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific immunoglobulin G (IgG) in plasma or serum samples. The protocol offers a rapid and reliable method, delivering results in less than 6 hours. On day 1, 96-well plates are coated with a concentration of 5 µg/mL of SARS-CoV-2 spike protein in 0.1 M sodium hydrogen carbonate and left to incubate overnight at 4 °C. Subsequently, the plates undergo thorough washing and are blocked with a buffer containing bovine serum albumin for one hour at room temperature. Diluted samples are added to the wells, followed by a two-hour incubation period. After another round of washing, a horseradish peroxidase-labeled antibody directed against the crystallizable fragment region of IgG is added, and the plates are incubated for one hour. The plates are then washed and developed using 3,3′,5,5′-tetramethylbenzidine-substrate for 30 minutes. The reaction is stopped by the addition of 1 N sulfuric acid. The optimized protocol demonstrated excellent performance, exhibiting a specificity of 99.5% and a sensitivity of 98.1% for the detection of SARS-CoV-2 spike-specific IgG in human plasma or serum samples.

Immunology

SARS-CoV-2 spike

ELISA

IgG

In 2019, the novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, causing the widespread coronavirus disease 2019 (COVID-19) pandemic¹. At the beginning of the pandemic, laboratory resources, including tests to measure SARS-CoV-2 antibody (Ab) responses, became limited and were primarily allocated to facilities involved in the immediate management of the health crisis². To enable us to carry out SARS-CoV-2 research specifically relevant for human immunodeficiency virus (HIV) diagnostic and the community of HIV infected individuals, we developed our own direct enzyme-linked immunosorbent assay (ELISA) protocol for the detection of SARS-CoV-2 spike specific immunoglobulin G (IgG) in serum or plasma samples. In a nutshell, the protocol involves coating 96-well plates with SARS-CoV-2 spike protein in sodium hydrogen carbonate on the first day, followed by overnight incubation at 4 °C. Subsequently, the plates are washed and blocked for one hour at room temperature. After diluting the samples, they are added to the plate and incubated for a duration of 2 hours. Afterwards, the plate is washed, and an horseradish peroxidase (HRP)-labeled detection Ab specific to the crystallizable fragment (Fc) region of human IgG is added, followed by another one-hour incubation period. The plates are washed again, and 3,3′,5,5′-tetramethylbenzidine (TMB)-substrate is added for development, which is halted using sulfuric acid.

In order to optimize the ELISA protocol, we systematically evaluated and adjusted several key assay parameters, including the coating concentration of soluble SARS-CoV-2 spike, assay development time, and dilutions of the detection Ab and human plasma samples. Initially, the draft protocol employed a coating concentration of 10 μg/ml of SARS-CoV-2 spike, a 10-minute assay development time, and a detection Ab dilution of 1:40,000. While these conditions already allowed for the distinction of SARS-CoV-2 spike reactive and non-reactive human plasma samples, low sample dilutions of 1:80 or less were required to separate a sample with medium reactivity from the negative control (Fig. 1A). To improve the ELISA protocol, essential assay parameters were subsequently optimized, and improved conditions were implemented as protocol modifications for subsequent refinements. First, we performed a titration experiment using 0.3-10 μg/ml of SARS-CoV-2 spike and a sample dilution of 1:100 (Fig. 1B). This experiment demonstrated that a coating concentration of 5 μg/ml of SARS-CoV-2 spike is sufficient to secure the detection of highly reactive samples without signal loss (Fig. 1B). Next, we tested development times of 10, 20, and 30 minutes and found that a development time of 30 minutes provided the optimal signal-to-noise ratio (Fig. 1C). Next, we assessed the ideal dilution of the detection Ab and determined that dilutions of 1:5,000 and 1:10,000 generated signals above the detection limit for SARS-CoV-2 IgG+ plasma samples, while a dilution of 1:40,000 resulted in poor signal resolution (Fig. 1D). Consequently, we chose a detection Ab dilution of 1:20,000 for the final protocol. Finally, by titrating the plasma samples using the optimized ELISA protocol, we determined that a sample dilution of 1:200 achieved the most optimal sample resolution (Fig. 1E).

To evaluate the optimized ELISA protocol for the measurement of SARS-CoV-2 spike specific IgG, we investigated its suitability for the analysis of human serum samples (Fig. 1F). For that purpose, matching plasma and serum samples of 9 individuals containing SARS-CoV-2 spike-specific IgG were tested in parallel using negative plasma and serum controls, respectively. The results showed comparable signals between plasma and serum samples after background subtraction, indicating that the ELISA protocol is applicable for analyzing both types of biospecimens. In order to assess the assay's sensitivity, we analyzed plasma samples (n=51) from individuals with known SARS-CoV-2 spike-IgG titers, as determined by an in-vitro diagnostics certified commercial ELISA (Euroimmun-Anti-SARS-CoV-2-ELISA (IgG)), using our in-house ELISA (Fig. 1G). In the self-developed ELISA, 50 out of 51 plasma samples exhibited reactivity, resulting in an assay sensitivity of 98.1%. Further, the results of both ELISAs were positively correlated (r = 0.9180, p < 0.0001). Lastly, assay specificity was determined to be 99.5% using serum samples (n = 185) collected in 2018 prior to the SARS-CoV-2 pandemic (Fig. 1H). In summary, based on these findings, we conclude that the established protocol enables efficient and reliable detection of SARS-CoV-2 spike-specific IgG in both human plasma and serum samples.

Commercially available

• Sodium hydrogen carbonate (NaHCO_3,0.1 M, pH 8.6) Sigma-Aldrich, CAT# 401676

• Phosphate-buffered saline (PBS), Carl Roth, CAT# 9150.1

• Sulfuric acid (1 N), Sigma-Aldrich, CAT# SIG-258105-1L-PC

• Tween 20, Sigma-Aldrich, CAT# P9416

• 1-Step Ultra-TMB substrate, Thermo Scientific, CAT# 34028

• Bovine serum albumin (BSA), Roche Diagnostics, CAT# 10735086001

• Soluble stabilized SARS-CoV-2 Wuhan-Hu-1 spike protein produced by plasmid transfection into FreeStyle
293-F cells (plasmid: CAT# NR-52394, BEI Resources; ready-made protein: CAT# NR-52397, BEI Resources)

• Sheep serum, Sigma-Aldrich, CAT# S22-100ML

• Goat anti-human IgG (Fc) cross-absorbed-HRP (detection Ab), Sigma-Aldrich, CAT# SAB3701283-1mg,
RRID:AB_2936313

Self-prepared buffers

Stock solutions of PBSA and PBSA-S should be kept sterile and stored at 4 °C.

• 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.

Machinery

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:

- 10-20µl, TipOne, CAT# S1120-3710

- 20-200 µL, Biosphere, CAT# 70.1189.215

- 100-1000µl, TipOne, CAT# S1122-1730

• Single use reservoir, Integra, CAT# 4312

• Plates:

- 96-well cell culture 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

• GraphPad Prism 9 (version 9.5.0), Dotmatics

It is imperative for all experimenters to adhere to regulations concerning necessary precautions, personal protective equipment, laboratory sample handling, and country-specific regulations. The ELISA should be conducted at room temperature, unless stated otherwise, and without any shaking. Throughout the incubation periods, it is essential to seal the plate with a plate sealer. To optimize efficiency, utilize a multichannel pipette and a reservoir whenever feasible. Similarly, for time-saving purposes, prepare sample dilutions in cell culture plates rather than 1.5 ml reaction vessels.

Day 1

Antigen immobilization

1. Prepare a 5 µg/mL final concentration of SARS-CoV-2 spike protein by diluting it in 0.1 M sodium hydrogen
carbonate.

2. Add 100 µl of the diluted protein per well of a 96-well ELISA plate.

3. Incubate the plate overnight at 4 °C.

Day 2

Blocking of ELISA plates

If an automated plate washer is not available, you can use a multichannel pipette for plate washing. In that case, remove the washing buffer by flicking the plate after each wash and gently dry the plate on a paper towel before proceeding.

4. Wash the ELISA plate 4 times with 400 µl of PBS-T5 using an automated plate washer and blot it dry.

5. Add 360 µl PBSA-T1 per well.

6. Incubate the plate for 1 h at room temperature.

Addition of samples

When handling human clinical samples (steps 7, 9, and 11), use a biosafety cabinet. For a project involving serum and plasma samples from SARS-CoV-2 convalescent individuals, we found that a dilution of 1:200 provides the best sample resolution.

7. During step 6, vortex the samples and dilute in PBSA-S in a 96-well cell culture plate.

8. Wash the ELISA plate 4 times with PBS-T5 and blot it dry.

9. Add 100 µl of sample dilution to each well of the ELISA plate.

10. Incubate the ELISA plate for 2 h at room temperature.

Addition of detection Ab

11. Wash the ELISA plate 6 times with PBS-T5 and blot it dry.

12. Dilute the detection Ab 1:20,000 in PBSA.

13. Add 100 µL of the diluted detection Ab to each well.

14. Incubate for 1 h at room temperature.

Development and measurement

15. Wash the ELISA plate 6 times with PBS-T5 and blot it dry.

16. Develop by adding 100 µL of 1-Step Ultra-TMB substrate.

17. Incubate the plate in the dark for 30 min at room temperature.

18. Stop the reaction by adding 100 µl of 1 N sulfuric acid to each well.

19. Read the absorbance of each well at 450 nm with reference wavelength 570 nm using a plate reader.

If the quality of the assay does not meet the expectations, please refer to the following suggestions regarding common sources of errors that may occur during the conduction of ELISAs.

Weak or no signal

• Reagent issues: ensure reagents are warmed up to room temperature before use; check expiration dates and
proper storage conditions; recalculate all prepared dilutions, especially for the detection Ab.

• Samples too diluted: perform sample titrations; check calculations for sample dilutions for errors.

• Coating Ab not bound to assay plate: confirm the use of an ELISA plate for coating, not a tissue culture plate.

• Ab coating damaged: avoid scratching of the well bottom during pipetting; check plate washer settings and
adjust height of needles if necessary.

• Measurement of incorrect wavelength: adjust plate reader absorbance settings to the correct wavelength.

Too high signal

• Insufficient washing: increase number of washes.

• Incubations too long: use a timer to adhere to incubation times.

• TMB exposed to light: store TMB protected from light at all times.

Inconsistent results

• Edge effects: ensure plate sealers are fully attached to prevent evaporation.

• Sample carry-over: change pipette tips between samples; avoid touching the ELISA plate with the pipette; use
a plate sealer.

• Irregular washing: clean the dispenser of the plate washer.

Poor data reproducibility

• Variations in incubation times between plates: keep incubation times consistent across different assay plates.

• Inconsistent sample dilutions: use the same sample dilutions for all experiments.

The execution of the ELISA according to the protocol typically takes approximately 5 h 30 min (excluding overnight incubation), with a hands-on time of around 1 h 20 min. These estimates assume that preparations for subsequent steps are carried out concurrently with ongoing incubations.

The SparkControl software of the Tecan Spark plate reader will generate an excel sheet containing the measurement results. The plate reader accurately detects optical density (OD) values between 0 and 4. In cases where the signal exceeds the detection limit, the plate reader displays "over" instead of a specific value. The ELISA assay readings are specifically measured at 450 nm, and it is essential for all assay values to fall within the designated detection range. If any values surpass the upper limit, it is necessary to repeat the ELISA with higher dilutions of the samples to prevent signal saturation. To determine the background signal of the assay plate, the reference wavelength of 570 nm is measured. It is crucial for these reference values to remain below 0.01. After subtraction of the reference wavelength signal from the sample signals, assay results are analyzed by GraphPad Prism 9. To ensure the validity of the assay, certain criteria must be met: a strong positive control should yield an OD of ≥1.5, while the negative control should not exceed an OD of ≤0.3. A sample is considered positive for SARS-CoV-2 spike IgG if its value exceeds the negative control signal by at least two-fold.

1. Tan, W. et al. A Novel Coronavirus Genome Identified in a Cluster of Pneumonia Cases - Wuhan, China 2019-2020. China CDC Wkly 2, 61-62 (2020).

2. American Society for Microbiology. Supply shortages impacting COVID-19 and non-COVID testing. (2021). https://asm.org/Articles/2020/September/Clinical-Microbiology-Supply-Shortage-Collecti-1. Accessed: June 23, 2023

The mentioned reagents, which include the vector pCAGGS containing the SARS-related coronavirus 2, Wuhan-Hu-1 spike glycoprotein gene (soluble, stabilized) (CAT# NR-52394) and the plasmid set for anti-SARS coronavirus human monoclonal Ab CR3022 (CAT# NR-53260), were produced under HHSN272201400008C and acquired through BEI Resources, NIAID, NIH. The research described in this work received support from the Foundation University Medicine Essen through the Grant (2020 4699 119) awarded to CBK.

Download PDF

Journal Publication

published 01 Jun, 2024

Read the published version in MethodsX →

Version 1

posted

You are reading this latest preprint version

Development of an Efficient ELISA Protocol for Measurement of SARS-CoV-2 Spike-Specific IgG in Human Plasma and Serum Samples

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Reagents

Commercially available

Self-prepared buffers

Assay controls

Equipment

Machinery

Consumables

Software

Procedure

Day 1

Antigen immobilization

Day 2

Blocking of ELISA plates

Addition of samples

Addition of detection Ab

Development and measurement

Troubleshooting

Weak or no signal

Too high signal

Inconsistent results

Poor data reproducibility

Time Taken

Anticipated Results

References

Acknowledgements

Associated Publications

Status:

Journal Publication

Version 1

Privacy Policy

Terms of Service

Development of an Efficient ELISA Protocol for Measurement of SARS-CoV-2 Spike-Specific IgG in Human Plasma and Serum Samples

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Reagents

Commercially available

Self-prepared buffers

Assay controls

Equipment

Machinery

Consumables

Software

Procedure

Day 1

Antigen immobilization

Day 2

Blocking of ELISA plates

Addition of samples

Addition of detection Ab

Development and measurement

Troubleshooting

Weak or no signal

Too high signal

Inconsistent results

Poor data reproducibility

Time Taken

Anticipated Results

References

Acknowledgements

Associated Publications

Status:

Journal Publication

Version 1

Privacy Policy

Terms of Service

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