Jun 14, 2020
MERS-CoV Spike Glycoprotein (GP) – ELISA Jenner Clinical SOP Template
- Mustapha Bittaye1,
- Sarah Gilbert1,
- Katie Ewer1,
- Teresa Lambe1
- 1The Jenner Institute, University of Oxford
- Coronavirus Method Development Community
Protocol Citation: Mustapha Bittaye, Sarah Gilbert, Katie Ewer, Teresa Lambe 2020. MERS-CoV Spike Glycoprotein (GP) – ELISA Jenner Clinical SOP Template. protocols.io https://dx.doi.org/10.17504/protocols.io.bgr6jv9e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 22, 2020
Last Modified: July 20, 2020
Protocol Integer ID: 37406
Keywords: IgG, immunoglobulin G, MERS-CoV, COVID-19, Coronavirus, 2019-nCoV
Abstract
This Standard Operating Procedure (SOP) describes the techniques used to measure the titres of total human immunoglobulin G (IgG) responses against a full length recombinant clamped MERS-CoV spike glycoprotein (GP) in human plasma and serum samples. The measured antibody titres will serve to indicate the humoral immune response to a candidate ChAdOx1 MERS vaccine encoding the full length spike GP of the MERS-CoV in human subjects.
This assay must be performed in accordance with the principles of the International Conference on Harmonisation Tripartite Guideline on Good Clinical Practice and the Human Tissue Act. Informed consent must be in place for samples to be tested in this assay.
Note
All staff employed by University of Oxford who work on outbreak pathogen vaccine trials including clinicians or visiting scientists working in the lab, must follow this protocol.
Attachments
Guidelines
This assay must be performed in accordance with the principles of the International Conference on Harmonisation Tripartite Guideline on Good Clinical Practice and the Human Tissue Act. Informed consent must be in place for samples to be tested in this assay.
INTRODUCTION
One of the primary readouts for assessing the protective humoral immunity against this candidate vaccine is the measurement of total IgG responses to MERS-CoV spike GP in the blood of vaccinated volunteers. This will be done as described in this SOP using an in-house ELISA that includes a standard curve derived from a pool of the volunteers’ sera containing highly concentrated anti-MERS IgG.
SCOPE
This SOP applies to all studies sponsored by the University of Oxford that are conducted by the Jenner Institute where measurement of total IgG against the candidate ChAdOx1 MERS vaccine is required, for which ELISA is the primary readout.
DEFINITIONS/ABBREVIATIONS
ELISA = Enzyme-Linked ImmunoSorbent Assay.
MERS-CoV = Middle East Respiratory Syndrome Coronavirus.
ChAdOx1 MERS = replication-deficient simian adenovirus vector ChAdOx1, expressing a codon-optimised coding sequence for the full-length spike GP of the MERS-CoV isolate Camel/Qatar_2_2014 (GenBank, accession number KJ650098.1), including a 32 amino acid N-terminal tissue plasminogen activator leader sequence.
RELATED DOCUMENTS
J284 Standardised ELISA set-up
ELISA analysis template.xls
ELISA Record sheet.pdf
REFERENCES
ICH Harmonised Tripartite Guideline for Good Clinical Practice
MHRA Guidelines ‘Good Clinical Practice for Clinical Laboratories
TRAINING
Record the level of training required by users
| Training type | Required (tick at least one box) | |
| Read and Understand | YES | |
| Attend face to face training based on SOP | YES | |
| Competency assessment | YES | |
| List other specific training types if required |
Read and understand training means that the user will be sufficiently trained to perform the activity described after reading the SOP and having any questions answered by the author or other trained individual.
Attend face to face training based on SOP training means that the user must read and understand the SOP, but must also attend face to face training. This enables additional discussion to be undertaken and clarification where necessary. The trainer is able to emphasise specific requirements that may differ from standard practice. This may often be performed as a group session.
Competency assessment requires the user to read and understand the SOP and also to receive face to face training (usually 1:1 or small groups). Specific practical aspects of the procedure will be covered and the individual must be able to demonstrate competency in all aspects prior to being signed off by the trainer:
Materials
MATERIALS
TWEEN® 20Sigma AldrichCatalog #P7949
Blocker™ Casein in PBSThermo FisherCatalog #37528
Clear Flat-Bottom Immuno Nonsterile 96-Well Plates, 400μL, MaxiSorpThermo FisherCatalog #442404
Dulbecco’s PBS (without calcium magnesium)Sigma AldrichCatalog #D8537
Anti-Human IgG (γ-chain specific)−Alkaline Phosphatase antibody produced in goatSigma AldrichCatalog #A3187
Pierce™ Diethanolamine Substrate Buffer (5X)Thermo Fisher ScientificCatalog #34064
4-Nitrophenyl phosphate disodium salt hexahydrateSigma AldrichCatalog #N2765
Fisherbrand™ Polypropylene Microcentrifuge TubesFisher ScientificCatalog #11558232 (FB74031)
Corning™ Costar™ Sterile Disposable Reagent ReservoirsFisher ScientificCatalog #10320551
Water (sterile-filtered BioReagent suitable for cell culture)Sigma AldrichCatalog #W3500
DPBS powder no calcium no magnesiumThermo Fisher ScientificCatalog #21600069
Additional reagent required:
- MERS-CoV clamped spike GP (0.5 mg/mL)
Equipment:
- Fridge (+4°C)
- Freezer (-20°C and -80°C)
- Vortexer
- Eppendorf Racks
- Pipettes including 8- or 12-well multi-channel and automatic multi-channel
- Pipetteboy and stripettes
- Bio-tek ELx800 Microplate Reader with Gen5 ELISA software
- Timer
- Sufficient tips for pipettes. 0.1-10 μl, 2-20 μl, 20-200 μl, 100-1000 μl
- Safety Glasses
- PBS-Tween Immunowash
Buffers and solutions:
- DPBS Use for coating plates with MERS-CoV Spike Glycoprotein.
- DPBS/T (10 L) (DPBS with 0.05 % (v/v) Tween ) for washing plates. Dissolve one DPBS powder tub in 10 L deionised water (15.0 MΩ setting). Add 5 mL Tween-20 . Shake and return to the ELISA plate wash station.
- Blocking and Dilution buffer Blocker Casein in PBS.
- Anti-human IgG Alkaline phosphatase 1:1000 dilution in casein. 6 µL in a final volume of 6 ml (6 mL per plate).
- Developer Dilute 5 X Diethanolamine (DEA) substrate buffer to 1 X in sterile-filtered, Bioreagent Water. Add one 20 mg pNPP tablet per 20 ml. 10 ml is required per plate (minimum that can be made up is 20 ml). Make up just before use, wrap in foil and vortex well.
Safety warnings
All staff employed by University of Oxford who work on outbreak pathogen vaccine trials including clinicians or visiting scientists working in the lab, must follow this protocol.
Senior immunologist is responsible for training new members of staff to be competent with this SOP and ensuring that sample receipt is conducted according to this SOP:
Please refer to Safety Data Sheets (SDS) for health and environmental hazards.
Before start
Day 1 – Coating ELISA plates on the bench
Day 1 – Coating ELISA plates on the bench
Print off a new ELISA record sheet for each experiment (accessed from: X:\KEwer\7. ELISA\Templates and protocols).
Number the experiment with the next experiment number and fill this in with the required information throughout the experiment.
Calculate the number of Nunc Immuno ELISA plates required (One plate: 23 samples and an internal control in triplicate in addition to the standard curve in duplicate).
Thaw an aliquot of MERS-CoV clamped spike GP (typically 0.5 mg/ml stock solution kept at -80 °C and prepare as shown in the table below. Calculate accordingly if stock protein concentration differed).
| Number of plates to coat | Volume of Spike GP (0.5 mg/ml stock solution) | Volume of DPBS | |
| 1 | 10 µl | 4.99 ml | |
| 2 | 20 µl | 9.98 ml | |
| 3 | 30 µl | 14.97 ml | |
| 4 | 40 µl | 19.98 ml | |
| 5 | 50 µl | 24.95 ml | |
| 6 | 60 µl | 29.94 ml |
Coat plates with 50 µl/well of MERS-CoV Spike clamped GP at 1.0 μg/ml in DPBS).
Cover plates in clingfilm and store overnight for 18:00:00 at 4 °C . Note time of coating and record on the experiment record sheet.
Day 2 – Blocking plates
Day 2 – Blocking plates
Thaw required serum samples (up to 23 samples per plate) at Room temperature for at least 01:00:00 .
Flick off coating solution into the sink.
Safety information
Wear eye protection.
Wash plates 6x in DPBS/T using the handheld plate washer and tap dry on paper towel between washes. After the final wash, invert plates and tap firmly. Leave plates on the bench for 2 min and tap firmly on a paper towel one more time to remove residual liquid from the wells
Block plates with 100 µL casein per well.
Stack plates (with an empty blank plate on top). Leave for 01:00:00 at Room temperature . Note time of blocking and record on the experiment sheet.
During blocking prepare serum samples and reference standard dilutions as in section below.
Day 2 – Sample/standard preparation
Day 2 – Sample/standard preparation
Vortex the thawed samples thoroughly and dilute in casein at 1:500 (5 µL sample diluted in 2495 µL casein and vortexed). All sample dilutions MUST be done using a minimum of 5 µL sample .
Add 50 µL diluted sample to each well (each sample in triplicate). Store diluted serum Overnight in fridge in case repeat of samples at higher dilution is necessary.
The standard curve is a two-fold dilution series with an initial dilution of 1:100 from the positive standard pool. To avoid multiple freeze-thaw of the positive standard pool, prepare aliquots of 1:100 working standards in casein to a final volume of 600 µL (6 µL pool sample in 594 µL casein ) and store in -80 °C freezer, where it is stable for up to a year.
All standard dilution is done using Eppendorf tubes labelled Tube1 – Tube10.
To run 2 plates prepare standards as follows: Add 520 µL of the 1:100 working standard to the first tube (Tube1). Add 260 µL casein to the rest of the tubes (Tube2 – Tube10). Transfer 260 µL from Tube1 to Tube2 (mix 15 times). Transfer 260 µL from Tube2 into Tube3 and continue across the rest of the tubes. Discard 260 µL from the last tube (Tube10).
Make a 1:800 dilution of the standard serum pool in casein to use for the plate internal control. (eg 175 µL casein + 25 µL positive standard pool per plate). Add to wells D12, E12 and F12.
If one plate is assayed, refreeze the remaining 1:100 working standard serum and reuse in subsequent assays. This can be freeze-thawed up to three times if required.
Day 2 – Plating out
Day 2 – Plating out
After 1 h incubation for blocking, flick off blocking solution into the sink and tap dry.
Add 50 µL of the thoroughly vortexed diluted samples (in triplicate) and the blank control (casein only) as shown in the plate layout below using an automatic pipette if available. Without adding the standards and internal control, cover plate with an empty blank plate and repeat step if multiple plates are prepared.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
| Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Internal control (1:800) | |
| Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Internal control (1:800) | |
| Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Internal control (1:800) | |
| St 1:100 | St 1:200 | St 1:400 | St 1:800 | St 1:1600 | St 1:3200 | St 1:6400 | St 1:12800 | St 1:25600 | St 1:51200 | Blank | Blank | |
| St 1:100 | St 1:200 | St 1:400 | St 1:800 | St 1:1600 | St 1:3200 | St 1:6400 | St 1:12800 | St 1:25600 | St 1:51200 | Blank | Blank |
Plate Layout
Add 50 µL of the 1:100 Standard as shown in the plate layout to all plates first then followed by 1:200 and so on. Add the internal control immediately after adding the 1:800 standard then followed by the rest of the standards.
Note
Use an automatic pipette if available for transferring the standards.
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
| Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Internal control (1:800) | |
| Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Internal control (1:800) | |
| Sample 13 | Sample 14 | Sample 15 | Sample 16 | Sample 17 | Sample 18 | Sample 19 | Sample 20 | Sample 21 | Sample 22 | Sample 23 | Internal control (1:800) | |
| St 1:100 | St 1:200 | St 1:400 | St 1:800 | St 1:1600 | St 1:3200 | St 1:6400 | St 1:12800 | St 1:25600 | St 1:51200 | Blank | Blank | |
| St 1:100 | St 1:200 | St 1:400 | St 1:800 | St 1:1600 | St 1:3200 | St 1:6400 | St 1:12800 | St 1:25600 | St 1:51200 | Blank | Blank |
Plate Layout
Incubate plate at Room temperature for 02:00:00 with a blank plate on top.
Day 2 – Secondary Antibody
Day 2 – Secondary Antibody
After 2 h incubation with samples, wash plates 6x in DPBS/T using the handheld plate washer as previously described.
Add 50 µL detection antibody to each well.
Incubate plate at Room temperature for 01:00:00 with a blank plate on top.
Day 2 – Development and plate reading
Day 2 – Development and plate reading
Prepare the pNPP substrate (development buffer) as shown in the table below. Make up the substrate during the antibody incubation, at least 00:30:00 prior to development to allow adequate time for the pNPP tablet to dissolve and protect from light until use (see section 'Materials').
| Number of plates to develop | Number of pNPP tablets required | Volume of 5X DEA buffer required | Volume of H20 | |
| 1 | 1 | 4 ml | 16 ml | |
| 2 | 1 | 4 ml | 16 ml | |
| 3 | 2 | 8 ml | 32 ml | |
| 4 | 2 | 8 ml | 32 ml | |
| 5 | 3 | 12 ml | 48 ml | |
| 6 | 3 | 12 ml | 48 ml |
After 1 h incubation with secondary antibody, wash plates 6x in DPBS/T using the handheld plate washer as previously described.
Add 100 µL development buffer to each well. Add the development buffer to the internal control column (Column 12) immediately after the Column containing standard 4 (Column 4) and then continue from Columns 5 – 11.
Turn the plate reader on and log in to the computer.
Open the Gen5 software, create new experiment using the protocol template (MERS STANDARDISED ELISA_SAMPLE ANALYSIS TEMPLATE) located in this folder (X:\Vaccine\10. MERS-CoV\1. MERS001_Oxford\12.0 Immunology\8. ELISA\Data and analysis\MERS001 STANDARDISED ELISA_SAMPLE ANALYSIS DATA).
Wipe dry the bottom of the plate with clean tissue paper and place the plate in the plate reader.
Start taking readings of the plate at OD405 after 00:10:00 and repeat readings until the control readings and curve parameters fit expected values.
Expected result
- internal control OD (wells D12, E12, F12) should be around 0.9-1.1 (1300-2300 EU)
- St 1:100 wells (G1 and H1) should be 3.5-3.8
- St 1:800 wells (G4 and H4) should be close to the values in the internal control wells (D12, E12, F12).
- Blank wells should be <0.2
- Standard curve reaches 4 parameter ranges below:
| Parameters | A | B | C | D | R^2 | |
| Ideal range | 0.01-0.2 | 1-1.3 | 5-13 | 4.5-6 | >0.996 |
Export the plate data, standard curve and curve results to Excel. Record time of reads. Save the Gen5 Experiment and the Excel worksheet in the corresponding experiment folder. Trial specific assay data must be stored in the corresponding clinical trial folder on the X drive or for blinded studies use X:\KEwer\7.ELISA\ 14.Confidential Immunology Data.
Day 2 – Data Analysis
Day 2 – Data Analysis
Analyse the data in the Excel sheet “ELISA analysis template” (accessed from: X:\KEwer\7. ELISA\Templates and protocols). All blue-shaded fields in the worksheets MUST be completed or confirmed as correct.
Fill in the assay information and plate layout in the blue boxes in the “Plate layouts and raw data” page, then paste the plate layout, plate data and parameters (A,B,C,D and R2) into the “plate layout and data” sheet.
Check for outliers (“high CV” on Results page) and take average of duplicates instead of triplicates if necessary. On the Raw data, highlight outlying well in yellow, calculate the average of the other two wells and type this into the yellow outlier cell.
The ELISA units for each sample are on the Results sheet.
Individual plate data and QC can be seen on the individual plate sheets.
The sheet will indicate if a sample dilution is too high or low. If the sheet indicates that sample dilution is “TOO LOW” at 1:100, the sample is negative and can be assigned an arbitrary value of 1.
If the sheet indicates that sample dilution is too low at 1:500 repeat at 1:100. For samples that are too high at 1:500 repeat them at higher dilution e.g. 1:1000, 1:2000 or more. When analysing samples that have been retested at 1:100 or higher dilutions such as 1:1000, change the dilution in the blue boxes on the individual plate pages (“plate 1”, “plate 2”, “plate 3” etc.)
If further dilutions from the 1:500 dilutions are not required then the tubes can be discarded.
Mark samples with values greater than 225 ELISA units as positive.
Required: Add all assay parameter data for each experiment to a designated QC workbook.
Required: QC all ELISA data for clinical trials prior to publication.