TITLE: MERS-CoV Spike Glycoprotein (GP) – ELISA Jenner Clinical SOP Template
AUTHORS: Mustapha  Bittaye, Sarah Gilbert, Katie Ewer, Teresa Lambe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">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. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-style:italic;">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.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [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).
?. [Day 1 – Coating ELISA plates on the bench]
Number the experiment with the next experiment number and fill this in with the required information throughout the experiment.
?. [Day 1 – Coating ELISA plates on the bench]
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).
?. [Day 1 – Coating ELISA plates on the bench]
Thaw an aliquot of MERS-CoV clamped spike GP (typically 0.5 mg/ml stock solution kept at  and prepare as shown in the table below. Calculate accordingly if stock protein concentration differed).   ABC1  Number of plates to coat    Volume of Spike GP (0.5 mg/ml stock solution)    Volume of DPBS  2  1  10 µl4.99 ml3  2  20 µl9.98 ml4  3  30 µl14.97 ml5  4  40 µl19.98 ml6  5  50 µl24.95 ml7  6  60 µl29.94 ml
-80 °C
ABC1  Number of plates to coat    Volume of Spike GP (0.5 mg/ml stock solution)    Volume of DPBS  2  1  10 µl4.99 ml3  2  20 µl9.98 ml4  3  30 µl14.97 ml5  4  40 µl19.98 ml6  5  50 µl24.95 ml7  6  60 µl29.94 ml
?. [Day 1 – Coating ELISA plates on the bench]
Coat plates with 50 µl/well of MERS-CoV Spike clamped GP at  in DPBS).
?. [Day 1 – Coating ELISA plates on the bench]
Cover plates in clingfilm and store overnight for  at . Note time of coating and record on the experiment record sheet.
4 °C
?. [Day 2 – Blocking plates]
Thaw required serum samples (up to 23 samples per plate) at  for at least  .
4 Room temperature
?. [Day 2 – Blocking plates]
Flick off coating solution into the sink.
Wear eye protection.
?. [Day 2 – Blocking plates]
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
?. [Day 2 – Blocking plates]
Block plates with  per well.
[casein]
?. [Day 2 – Blocking plates]
Stack plates (with an empty blank plate on top). Leave for  at . Note time of blocking and record on the experiment sheet.
4 Room temperature
?. [Day 2 – Blocking plates]
During blocking prepare serum samples and reference standard dilutions as in section below.
?. [Day 2 – Sample/standard preparation]
Vortex the thawed samples thoroughly and dilute in casein at 1:500 ( diluted in  and vortexed). All sample dilutions MUST be done using a minimum of .
[sample]
[ casein]
[sample]
?. [Day 2 – Sample/standard preparation]
Add  to each well (each sample in triplicate). Store diluted serum  in fridge in case repeat of samples at higher dilution is necessary.
[diluted sample]
?. [Day 2 – Sample/standard preparation]
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  (  in ) and store in  freezer, where it is stable for up to a year.
600 µl
[pool sample]
[casein]
-80 °C
?. [Day 2 – Sample/standard preparation]
All standard dilution is done using Eppendorf tubes labelled Tube1 – Tube10. To run 2 plates prepare standards as follows: Add  of the 1:100 working standard to the first tube (Tube1). Add  to the rest of the tubes (Tube2 – Tube10). Transfer  from Tube1 to Tube2 (mix 15 times). Transfer  from Tube2 into Tube3 and continue across the rest of the tubes. Discard  from the last tube (Tube10).
520 µl
[casein]
260 µl
260 µl
260 µl
?. [Day 2 – Sample/standard preparation]
Make a 1:800 dilution of the standard serum pool in casein to use for the plate internal control. (eg   +  per plate). Add to wells D12, E12 and F12.
[casein]
[positive standard pool]
?. [Day 2 – Sample/standard preparation]
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]
After 1 h incubation for blocking, flick off blocking solution into the sink and tap dry.
?. [Day 2 – Plating out]
Add  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.   ABCDEFGHIJKL1  Sample 1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  2  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  3  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  4  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)5  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)6  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)7  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  8  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
50 µl
ABCDEFGHIJKL1  Sample 1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  2  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  3  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  4  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)5  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)6  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)7  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  8  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
?. [Day 2 – Plating out]
Add  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.   ABCDEFGHIJKL1  Sample 1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  2  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  3  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  4  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)5  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)6  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)7  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  8  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
50 µl
Use an automatic pipette if available for transferring the standards.
ABCDEFGHIJKL1  Sample 1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  2  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  3  Sample
  1    Sample
  2    Sample
  3    Sample
  4    Sample
  5    Sample
  6    Sample
  7    Sample
  8    Sample
  9    Sample
  10    Sample
  11    Sample
  12  4  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)5  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)6  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)7  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  8  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
?. [Day 2 – Plating out]
Incubate plate at  for  with a blank plate on top.
-80 Room temperature
?. [Day 2 – Secondary Antibody]
After 2 h incubation with samples, wash plates 6x in DPBS/T using the handheld plate washer as previously described.
?. [Day 2 – Secondary Antibody]
Dilute anti-human IgG antibody alkaline phosphatase 1:1000 in casein (see section 'Materials').
?. [Day 2 – Secondary Antibody]
Add  to each well.
[detection antibody]
?. [Day 2 – Secondary Antibody]
Incubate plate at  for  with a blank plate on top.
-80 Room temperature
?. [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  prior to development to allow adequate time for the pNPP tablet to dissolve and protect from light until use (see section 'Materials').  ABCD1  Number of plates to develop       Number of pNPP tablets required       Volume of 5X DEA buffer required       Volume of H20  2  1    1  4 ml16 ml3  2    1  4 ml16 ml4  3    2  8 ml32 ml5  4    2  8 ml32 ml6  5    3  12 ml48 ml7  6    3  12 ml48 ml
ABCD1  Number of plates to develop       Number of pNPP tablets required       Volume of 5X DEA buffer required       Volume of H20  2  1    1  4 ml16 ml3  2    1  4 ml16 ml4  3    2  8 ml32 ml5  4    2  8 ml32 ml6  5    3  12 ml48 ml7  6    3  12 ml48 ml
?. [Day 2 – Development and plate reading]
After 1 h incubation with secondary antibody, wash plates 6x in DPBS/T using the handheld plate washer as previously described.
?. [Day 2 – Development and plate reading]
Add  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.
[development buffer]
?. [Day 2 – Development and plate reading]
Turn the plate reader on and log in to the computer.
?. [Day 2 – Development and plate reading]
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).
?. [Day 2 – Development and plate reading]
Start taking readings of the plate at OD405 after  and repeat readings until the control readings and curve parameters fit expected values.
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 Standard curve reaches 4 parameter ranges below:  ABCDEF1  Parameters    A    B    C    D  R^22  Ideal range  0.01-0.2  1-1.3    5-13    4.5-6    &gt;0.996
?. [Day 2 – Development and plate reading]
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]
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.
?. [Day 2 – Data Analysis]
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.
?. [Day 2 – Data Analysis]
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.
?. [Day 2 – Data Analysis]
The ELISA units for each sample are on the Results sheet.
?. [Day 2 – Data Analysis]
Individual plate data and QC can be seen on the individual plate sheets.
?. [Day 2 – Data Analysis]
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.
?. [Day 2 – Data Analysis]
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.)
?. [Day 2 – Data Analysis]
If further dilutions from the 1:500 dilutions are not required then the tubes can be discarded.
?. [Day 2 – Data Analysis]
Mark samples with values greater than 225 ELISA units as positive.
?. [Day 2 – Data Analysis]
Required: Add all assay parameter data for each experiment to a designated QC workbook.
?. [Day 2 – Data Analysis]
Required: QC all ELISA data for clinical trials prior to publication.
?. [Day 2 – Development and plate reading]
Wipe dry the bottom of the plate with clean tissue paper and place the plate in the plate reader.