Laboratory standard operating procedure for detecting sporadic Creutzfeldt-Jakob disease using Real-Time Quaking-Induced Conversion (RT-QuIC) assay : version 1.0 February 2021.
Date: 2021-02-28
Institutions: European Centre for Disease Prevention and Control
Subjects: disease of the nervous system, medical research, research method, standard

TECHNICAL REPORT

Laboratory standard operating 
procedure for detecting sporadic 
Creutzfeldt-Jakob disease using Real-
Time Quaking-Induced Conversion 
(RT-QuIC) assay

Version 1.0 

February 2021

www.ecdc.europa.eu

ECDC TECHNICAL REPORT 

Laboratory standard operating procedure 
for detecting sporadic Creutzfeldt-Jakob 
disease using Real-Time Quaking-Induced 
Conversion (RT-QuIC) assay 
Version 1.0 February 2021 

 
 
This report was commissioned by the European Centre for Disease Prevention and Control (ECDC) and produced by 
the National CJD Research and Surveillance Unit (NCJDRSU), University of Edinburgh, United Kingdom.  

Authors 
Neil McKenzie, Lynne McGuire and Alison Green at the National CJD Research and Surveillance Unit, University of 
Edinburgh, United Kingdom, EH4 2XU. 

Suggested citation: European Centre for Disease Prevention and Control. Laboratory standard operating procedure 
for detecting sporadic Creuzfeldt-Jakob disease using Real-Time Quaking-Induced Conversion (RT-QuIC) assay. 
Stockholm: ECDC; 2021. 

Stockholm, February 2021 

ISBN 978-92-9498-529-3 
doi: 10.2900/271946 
Catalogue number TQ-04-21-058-EN-N 

© European Centre for Disease Prevention and Control, 2021 

Reproduction is authorised, provided the source is acknowledged 

ii 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Contents 

Abbreviations ............................................................................................................................................... iv 
Scope and principles ...................................................................................................................................... 1 
PrP expression and purification .................................................................................................................. 1 
RT-QuIC assay ......................................................................................................................................... 1 
Reagents ................................................................................................................................................. 2 
Apparatus ................................................................................................................................................ 3 
Procedures .................................................................................................................................................... 5 
Production of RecPrP protein ..................................................................................................................... 5 
Performing the RT-QuIC assay ................................................................................................................. 10 
Data analysis ............................................................................................................................................... 13 
References .................................................................................................................................................. 14 
Annex 1. ..................................................................................................................................................... 15 
Buffer recipes for recPrP purification ......................................................................................................... 15 
Annex 2. ..................................................................................................................................................... 16 
Recommended and alternate assay parameters ......................................................................................... 16 
Batch script for running an RT-QuIC on either the FLUOstar Optima or Omega ............................................ 17 
Read script parameters, RT-QuIC FLUOstar Optima ................................................................................... 18 
Read script parameters, RT-QuIC FLUOstar Omega ................................................................................... 18 
Shake script parameters, RT-QuIC FLUOstar Optima .................................................................................. 19 
Shake script parameters, RT-QuIC FLUOstar Omega .................................................................................. 20 
Annex 3. ..................................................................................................................................................... 21 
Example - unseeded and brain homogenate controls .................................................................................. 21 
Example - negative and positive 30 µL CSF samples ................................................................................... 21 

Tables 

Table 1. Reagents for production of recombinant prion protein ........................................................................... 2 
Table 2. Reagents for quantitating the amount of control seed used in the RT-QuIC assay .................................... 2 
Table 3. Reagents for performing the RT-QuIC assay ........................................................................................ 3 
Table 4. Apparatus for production of recombinant prion protein ......................................................................... 3 
Table 5. Apparatus for quantitating the amount of control seed used in the RT-QuIC assay ................................... 4 
Table 6. Apparatus for performing the RT-QuIC assay ........................................................................................ 4 
Table 7. Master mix in a single well ................................................................................................................ 10 
Table 8. Final master mix composition in a well ............................................................................................... 11 
Table 9. The final composition of a well .......................................................................................................... 11 
Table 10. Typical ranges for endpoint RFUs of two highest wells at Cycle 361 .................................................... 13 

iii 

 
 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

TECHNICAL REPORT 

Abbreviations 

Absorbance at 280 nm wavelength 
(DNA) base pair 
Brain homogenate 
Creutzfeldt-Jakob disease 
Cerebrospinal fluid 
Double-stranded DNA 
Escherichia coli 
Ethylenediaminetetraacetic acid 
Fast Protein Liquid Chromatography 
Hamster derived 
Hamster full length (residues 23-231) 
Hamster – sheep chimeric protein 
Human derived 
Human full length 
Inclusion Bodies 
Isopropyl β-D-1-thiogalactopyranoside 
Lysogeny Broth 
Microbiological Safety Cabinet 
Genotype that is homozygous for methionine at codon 129, Type 1 
Molecular-weight cut-off 
Nickel Immobilized Metal Affinity Chromatography 
Nitrilotriacetic acid 
Overnight Express Instant TB Medium 
Overnight 
Phosphate-Buffered Saline 
Prion Protein 
Prion Protein (insoluble/disease causing/‘scrapie’ form) 
PK-resistant Prion Protein 
Proteinase K (a broad spectrum Serine protease) 
Relative Centrifugal Force 
Recombinant Prion Protein 
Relative fluorescence unit 
Real-Time Quaking-Induced Conversion (assay) 
Sporadic Creutzfeldt-Jakob disease 
Sodium Dodecyl Sulfate 
Tris-Acetate-EDTA buffer 
Terrific Broth 
Thioflavin T., a solvatochromic fluorescent dye 
Variant Creutzfeldt-Jakob disease 
Weight to volume 

Abs280 
bp 
BH 
CJD 
CSF 
dsDNA 
E. coli 
EDTA 
FPLC 
Ha 
Ham FL 
Ham-Sh 
Hu 
Hum FL 
IBs 
IPTG 
LB 
MBSC 
MM1 
MWCO 
Nickel-IMAC 
NTA 
OETB 
o/n 
PBS 
PrP 
PrPSc 
PrPres 
PK 
RCF 
recPrP 
RFU 
RT-QuIC 
sCJD 
SDS 
TAE  
TB 
ThT 
vCJD 
w/v 

iv 

 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Scope and principles 

This document provides laboratories with a single protocol for producing recombinant full-length hamster prion 
protein and using it to perform the Real-Time Quaking-Induced Conversion assay (RT-QuIC), which can distinguish 
sporadic Creutzfeldt-Jakob disease (sCJD) from variant Creutzfeldt-Jakob disease (vCJD).  

This protocol describes how to produce recombinant hamster prion protein and use it to perform RT-QuIC. It is 
based on methods published by the Caughey Group[1] and the National CJD Research and Surveillance Unit at the 
University of Edinburgh, United Kingdom (NCJDRSU) [2]. 

PrP expression and purification 
Production of recombinant hamster Prion protein (Ha recPrP) uses standard techniques in molecular biology and 
affinity purification. The protein is expressed in bacteria, purified over a Nickel-IMAC column and then dialysed and 
concentrated. There are several steps during the purification which are critical to obtaining satisfactory yield, purity 
and function. These have been marked at the appropriate point in the procedural sections with the comment ‘NB’. 

The current protocol for expression of Ha recPrP requires use of the BL21 Rosetta DE3/pRSET system from 
Novagen. This is sold under license by Novagen and may not be freely shared between organisations. For this 
reason, although the Ha PrP gene can be supplied on an alternate plasmid, the gene must be excised and ligated 
into the correct bacterium and vector. Using the specified BL21 bacterium and plasmid provides good yields of high 
quality Ha recPrP. Use of other commercial bacterial expression systems may affect the final yield of protein but 
should give good quality Ha recPrP. 

RT-QuIC assay 
The Real-Time Quaking-Induced Conversion (RT-QuIC) assay is a protein aggregation assay. A buffered solution of 
recombinant PrP (recPrP) is prepared and then aliquoted out into a 96-well plate. The wells of the plate are then 
‘seeded’ in quadruplicate with either a control sample or a test sample. If the added sample contains a PrPSc seed, 
it will cause the recPrP to aggregate and make the Thioflavin T (ThT) fluoresce.  

In this assay, recPrP aggregation is promoted by shaking and heating in the presence of an sCJD seed. When 
protein, such as recPrP, aggregates in the presence of ThT, the ThT binds to the protein and the fluorescent 
properties of the dye change. The amount of aggregation can therefore be followed by exciting the ThT dye at 
450 nm and recording the fluorescence emission at 480 nm.  

The assay has 91% sensitivity and 98% speciﬁcity in detecting sCJD [3, 4]. It has been reported as being capable 
of detecting ≥ 1fg PrPSc in a sample [4]. The test is negative for vCJD. 

In the RT-QuIC assay, the control samples used are brain homogenates and the test samples are cerebrospinal 
fluid (CSF). Both control and CSF samples are run in quadruplicate (i.e. four wells allocated on the plate per 
sample) to reduce the risk of false positives biasing the test outcome.  

The control samples are: 

 
 
 
 

unseeded master mix. 
100 fg of PrP from sudden-death brain. 
100 fg of PrP from Alzheimer’s diseased brain. 
100 fg of sCJD PrPres from MM1 genotype brain. 

Control samples may be obtained by petitioning a local neuropathology laboratory. 

An example plate layout is shown in the appendix. Example traces showing the typical responses of controls and 
CSF samples are also included in the annexes. 

For the control samples, 2 µL of brain homogenate is used to seed each quadruplicate well set. Of the four control 
types, only those wells spiked with sCJD brain homogenate exhibit a large increase in relative fluorescence unit (RFU) 
during the RT-QuIC assay. The RFU of the other control wells remain low and constant throughout the whole assay. 

For the CSF test samples, 30 µL of CSF is used to seed each quadruplicate well set. CSFs are tested at 30 µL 
volumes in the first instance and are only tested further, at 15 µL and 7 µL, if the results of the 30 µL tests are 
equivocal. If a CSF sample contains a sCJD seed, the well exhibits an increase in RFU during the RT-QuIC assay. If 
the CSF sample does not contain a sCJD seed, the RFU remains low and constant throughout the whole assay. 

1 

 
 
 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

Reagents 

Table 1. Reagents for production of recombinant prion protein 

Reagent 

Acrylamide gels 
Agarose 
Antibiotic(s) appropriate for plasmid  
BL21 DE3 Rosetta E. coli competent cells 
BugBuster Master Mix  
DNA loading dye 
Dry ice or liquid nitrogen 
Glycerol, sterile 
Guanidine.HCl 
Hydrochloric acid 
Imidazole.HCl 
Lysogeny broth 
Molecular weight markers for DNA and protein gels 
NTA Superflow Ni charged resin (500ml) 
Overnight Express instant TB medium (‘OETB’ media, sufficient for 2L culture/purification run) 
Plasmid containing the gene for Ha PrP 
pRSET vector  
Rapid DNA ligation kit 
Restriction enzymes appropriate for excising the Ha PrP gene from the supplied plasmid 
SOC medium 
Sodium chloride 
Sodium hydrogen phosphate (dibasic) 
Sodium hydroxide 
Sodium phosphate 
TAE (Tris-Acetate-EDTA) buffer, 10x concentrate 
Terrific broth 
Tris 
Water ice 
Water, sterile 

Table 2. Reagents for quantitating the amount of control seed used in the RT-QuIC Assay 

Reagent 

Concentration 

Proteinase K  
N2 supplement (Invitrogen) 
Recombinant Hu PrP (as reference) 
0.1% SDS in a 1 x PBS solution  
Anti-prion protein antibody 3F4 epitope 

50 mg/ml 
1 x stock 
50 – 200 fg/µL 
0.1% SDS, 10 mM phosphate, 154 mM NaCl 
As specified by manufacturer 

Samples 

Amount 

Source Location 

Sudden-death brain  
Alzheimer’s diseased brain  
sCJD brain 

approx. 100 mg 
approx. 100 mg 
approx. 100 mg 

Frontal cortex 
Frontal cortex 
Frontal cortex 

2 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Table 3. Reagents for performing the RT-QuIC Assay 

Reagent 

Concentration 

Volume per well 

Hamster Prion Protein 
Phosphate Buffered Saline (PBS), 5x conc  50 mM phosphate 

0.3-0.5 mg/ml 

 20 – 30 µL (10 µg after spin filtration) 
20 µL 

0.1% SDS in a 1 x PBS solution  

Sodium chloride solution 
EDTA 
ThT (make 10 mM and dilute 10x) 
Water, molecular biology grade 

770 mM NaCl 
10 mM Phosphate 
154 mM NaCl 
0.1% SDS 
2 M 
100 mM 
1 mM 
- 

Not used in RT-QuIC assay, only used to 
determine recPrP concentration 

8.5 µL 
1 µL 
1 µL 
(100-30.5-recPrP–sample) µL 

Samples 

Concentration 

Volume per well 

Sudden-death brain homogenate 
Alzheimer’s diseased brain homogenate 
sCJD brain homogenate 
CSF sample 

50 fg Hu PrP/µL† 
50 fg Hu PrP/µL† 
50 fg Hu PrPres/µL† 
Neat 

2 µL 
2 µL 
2 µL 
30, 15 or 7 µL 

†Concentrations are determined using western blot. Quantitation is calculated as detailed in the section ‘Preparing the brain 
homogenate controls’. 

Prepare fresh reagent buffers as above and filter through a 0.22 µ filter. The hamster prion protein may be frozen 
and thawed, so long as it is spin-filtered through a 100 kDa molecular weight cut-off (MWCO) filter before use. 
After dilution into the master mix, the 1x PBS is pH 7.4. 

N.B. The ThT should be made up at a 10 mM concentration (e.g. 32 mg ThT/10 ml water) to allow accurate 
weighing. The ThT should be solubilised at room temperature, then passed through a 0.22 µ filter. The yellow 
solution is stored in foil at 4oC until ready for use, at which point it is diluted ten-fold in water to afford a 1mM 
solution. This 10 mM master solution should be freshly made for every RT-QuIC assay. 

Apparatus 

Table 4. Apparatus for production of recombinant prion protein 

Apparatus 

+ 4 oC fridge or cold room 
0.22 µ filter papers for degassing units and filtering solutions 
0.22 µ filters with Luer lock syringe attachments 
-20 oC freezer 
-80 oC freezer 
A packable FPLC column with >150 ml internal volume (e.g. XK 26/40 column) 
Agar plates, impregnated with appropriate antibiotic 
Amicon Ultra15 centrifugal units, 8 pk 
Autoclave tape 
Benchtop rocker/shaker/roller 
Cell spreader 
Centrifuge and rotor that can spin 6 x 50 ml Falcon tubes at 13 500 RCF 
Containers for wet ice, dry ice, liquid nitrogen and dialysis 
Cuvette(s) with UV-Vis transparency at 250 nm-700 nm 
Dialysis tubing clips  
Dialysis tubing with a 7 KDa MWCO 
Disposable culture flasks, 2.5L, non-baffled 
Disposable scalpels 
DNA gel extraction kit  
DNA ligase kit 
DNA spin-purification kit 
Eppendorfs 
Equipment for running and visualising SDS-PAGE and agarose gels 
Falcon tubes 
Filter units for degassing buffers 

3 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

Apparatus 

FPLC equipment, including computer and control software 
Glass bottles for storing sterile buffers, with capacities from 250 ml – 2 L 
Incubator shaker with shaker plate and flask clamps 
Measuring cylinders, capacities from 20 ml to 2 L 
Metal foil 
Microbiological safety cabinet Level 2 OR Bunson burner 
Microfuge 
Mini-prep kit 
PCR tubes  
Pipettes and sterile tips, ranging from 0.1 µL to 1 ml 
Plastic pipettes,  
Sharpsafe 
Spatulas  
Spectrophotometer (e.g. Nanodrop) 
Syringes, polypropylene Luer lock tips, capacities from 1 ml20 mL  
UV-light box 

Table 5. Apparatus for quantitating the amount of control seed used in the RT-QuIC Assay 

Apparatus 

Alkali resistant carboy (for disposal of samples, Eppendorfs and well plates) 
Eppendorfs, 2 ml 
Equipment and software for running, imaging and quantitating western blots 
Equipment for running and visualising SDS-PAGE 
Heating block 
Level 2 Microbiological Safety Cabinet (MBSC) inside a Category 3 laboratory 
Pipettes and sterile filter tips, ranging from 0.1 µL to 1 ml 
Scalpel 
Sharpsafe  
Sterile filters, 0.22 µ porosity, with built-in Luer-lock screw attachment 
Syringes, polypropylene Luer-lock tips, capacity 10 ml 

Table 6. Apparatus for performing the RT-QuIC Assay 

Apparatus 

Alkali resistant carboy (for disposal of samples, Eppendorfs and well plates) 
BMG Labtech FLUOstar Omega or Optima, including computer and control software 
Clear polyolefin adhesive tape for sealing 96 well plate 
Eppendorfs, 2 ml 
Falcon tubes, 15 and 50 ml 
Level 2 MBSC inside a Category 3 laboratory 
Optical flat bottom plate, 96 well 
Pipettes and sterile filter tips, ranging from 0.1 µL to 1 ml volume 
Sharpsafe (for disposal of pipette tips) 
Spin filtration units, with 100 kDa MWCO 
Sterile filters, 0.22 µ porosity, with built-in Luer-lock screw attachment 
Syringes, polypropylene Luer-lock tips, capacity 10 ml 

4 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Procedures 

Production of RecPrP protein 

Molecular biology 

The cDNA used to express the Ha recPrP is based on PRNP from Mesocricetus auratus (Golden Syrian Hamster), 
NCBI Gene ID: 101829062. Residues 23-231 are expressed. 

The gene sequence used for expression is: 

5’-
atggcgaaccttagctactggctgctggcactctttgtggctatgtggactgatgttggcctctgcaagaagcggccaaagcctggagggtggaacactggcggaa
gccgataccctgggcagggcagccctggaggcaaccgttacccacctcagggtggcggcacatggggacagccccatggtggtggctggggtcagccccatggt
ggtggctggggtcaaccccatggtggtggctggggtcagccccatggtggtggctggggtcagggaggtggcacccacaatcagtggaacaagcccagtaagcc
aaaaaccaacatgaagcacatggccggcgctgctgcggcaggggccgtggtggggggccttggtggctacatgctggggagtgccatgagcaggcccatgatg
cattttggcaatgactgggaggaccgctactaccgtgaaaacatgaaccgctaccctaaccaagtgtattaccggccagtggaccagtacaacaaccagaacaact
ttgtgcacgattgtgtcaacatcaccatcaagcagcacacagtcaccaccaccaccaagggggagaacttcacggagaccgacatcaagataatggagcgcgtgg
tggagcagatgtgtaccacccagtatcagaaggagtcccaggcctactacgatggaagaaggtccagcgcggtgctgttctcctcccctcctgtgatcctcctcatttc
ctttctcatcttcctgatggtgggatgaaggaagcctccc-3’ 

N.B. This sequence is for the Ha PrP protein alone and does not include 5’ and 3’ terminal restriction sites 
necessary for cloning into the pRSET plasmid. 

1. Excise the Ha PrP gene from the plasmid using appropriate restriction enzymes. 

Follow the instructions that come with the restriction enzymes to determine the amount of enzyme, plasmid DNA 
and buffer to use.  

2. Cut open the pRSET plasmid using the same restriction enzymes as those used to excise the PrP 
gene from the plasmid. 

Follow the instructions that come with the restriction enzymes to determine the amount of enzyme, plasmid DNA 
and buffer to use.  

3. Gel purify both the linear plasmid and the PrP gene. 

 
 
 

 

 

Prepare a 1% w/v agarose gel.  
Add DNA loading dye to the DNA samples.  
Load the molecular weight marker, the cut plasmid DNA and the excised PrP gene into separate wells on 
the agarose gel.  
Set voltage and time parameters on the electrophoresis equipment in accordance with manufacturer’s 
instructions for a DNA gel.  
Run the agarose gel until the DNA loading dye approaches the bottom of the agarose gel.  

N.B. The gene insert (approx. 800 bp) and linear plasmid (approx. 30004000 bp) may be identified by their 
molecular weight. The molecular weight lane marker should be used as a reference for this. Both lanes may 
contain one or more unexpected bands due to incomplete digestion of the plasmid DNA. Bands at the incorrect 
molecular weight should not be used. 

4. Excise the bands containing the gene insert and the linear plasmid. 

 
 

 

Lay the gel on a UV-light box. 
Wearing appropriate personal protective equipment – including a UV eye shield – take a scalpel and excise 
the bands of interest from the gel.  
Deposit each band’s gel piece(s) into an Eppendorf.  

N.B. Gel pieces can be frozen at -20 oC and stored for up to two weeks as required. 

5. Purify the DNA obtained from the bands. 

Using a commercial DNA-gel extraction kit, and in accordance with the kit instructions, isolate the DNA obtained in 
each of the excised bands by spin-purification. 

As an alternative to a commercial DNA-gel extraction kit, there are protocols available online or in the textbook 
‘Molecular Cloning – A Laboratory Manual’ by Sambrook and Russell [5]. 

5 

 
 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

6. Quantitate the amount and purity of the DNA obtained. 

 
 

Measure the absorbance of the DNA solution at 260, 280 and 320 nm. 
Calculate the amount and purity of DNA obtained using the equation below. 

dsDNA concentration (µg/ml) = (Abs260 – Abs320) x (dilution factor) x (50 µg/ml/cm) 

Mass of DNA (µg) = Concentration (µg/ml) x ml solution 

Purity =

(Abs 260 nm )
(Abs 280 nm )

N.B. The DNA is of suitable quality for further use if it has a purity ratio in the range 1.7–2.0. As a desirable yield, 
the ligation in the next step requires around 75 ng of double stranded insert and 25 ng of linear insert. 

If the purity and amount of DNA obtained from the previous purification step is inadequate, remedial ‘trouble-
shooting’ action should be attempted before progressing to the next steps. This action is usually detailed in the 
manual accompanying the DNA extraction kit. 

7. Ligate the gene into the plasmid. 

In this step the Ha PrP gene is ligated into the new plasmid. A 1:3 ratio of plasmid to insert is desirable. Follow the 
instructions provided by the manufacturer of the ligase used. 

N.B. Best practice is to store some of the plasmid obtained as a stock for transformation, should the glycerol 
bacterial stock (step 10) fail. 

8. Transform the ligation reaction product into the BL21 DE3 Rosetta E. coli-competent cells. 

Follow the instructions provided by Novagen. Alternatively, there are protocols available online or in the textbook 
‘Molecular Cloning – A Laboratory Manual’ by Sambrook and Russell [5]. 

9. Isolate a pure bacterial colony. 

 
 

 

 

Inoculate 2-5 ml of LB medium with a single stab from the bacterial colony obtained in the previous step.  
Grow this bacterial culture up overnight at 30oC and 180 rpm in the presence of an appropriate quantity of 
antibiotic to which the plasmid has resistance. 
Inoculate 500 µL of various dilutions of the overnight culture onto agar plates which contain an appropriate 
antibiotic. 
Grow these colonies in a stationary incubator overnight at 30oC. 

10. Prepare a glycerol stock for future cultures. 

 

 
 

 

Choosing an agar plate which has discrete and well-defined colonies, pick four colonies and use them to 
prepare four overnight starter cultures.  
Prepare four 50% v/v solutions of sterile glycerol in sterile water (e.g. 4 x 500 µL). 
Add an equal volume of the overnight culture to these glycerol solutions, mixing well. The relative final 
glycerol concentration is 25%. 
Store the glycerol stock at -80oC.  

N.B. Glycerol stocks are stable for several months or even years, depending on the host bacterium, and provide an 
easy way to inoculate future cultures during recPrP production runs.  

Bacterial culture 

1. Prepare a ‘starter’ culture. 

 

 

Using a sterile toothpick or pipette tip, dip the narrow end into the glycerol stock and use it to inoculate 2–15 ml 
of antibiotic impregnated TB medium.  
Grow overnight at 30oC and 180 rpm.  

2. Inoculate the ‘grow-up’ cultures. 

Use 1–3 ml of starter culture to inoculate four 500 ml Overnight Express Instant TB Medium (OETB) cultures.  
 
 
 

Add appropriate antibiotic(s) to the cultures. 
Add glycerol (final concentration = 10 ml/L) to the cultures.  
Grow the cultures at 30oC, 180 rpm in non-baffled 2.5 L sterile plastic flasks.  

N.B. Around 80–150 mg recPrP is expected from 2L of ‘grow-up’ culture. The OETB media mix does not require 
manual isopropyl β-D-1-thiogalactopyranoside (IPTG) induction by the operator.  

6 

 
 
 
 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

3. Pelleting of bacterial cells. 

 
 
 
 

Once the OETB culture is complete, pellet the bacterial cells at 3000 x RCF for 15 minutes. 
Discard the supernatant, retaining the cell pellets in the Falcon tubes. 
The Falcon tubes are convenient storage containers for subsequent lysis steps.  
Cell pellets may be stored at this stage for up to six months at -80oC.  

N.B. Expected pellet yield is 9 g/L culture under the specified growth conditions. Pellets should be orange or 
custard-yellow in colour. Expected yield is around 2g of cell pellet per Falcon tube when the freezing step has been 
reached. 

Protein purification 

1. Buffer preparation. 

 
 

Prepare the buffers as described in the annex. The pH tolerances are +/- 0.05 units. 
Filter the buffers through a 0.22 µ filter and de-gas prior to use.  

N.B. The purification can be performed at 18–20oC without detriment to the protein; however the dialysis step 
should be at 0–4oC. The water used to dilute the dialysis buffer does not have to be de-gassed. 

2. Double freeze-thawing of cells. 

 
 
 

Remove the pellets from the -80oC freezer and allow them to warm up for 15 minutes at room temperature.  
Return them to the -80oC freezer for 15 minutes. 
Remove the pellets from the -80oC freezer and allow them to warm up for 15 minutes at room temperature.  

3. Isolate the inclusion bodies. 

 
 
 

Re-suspend the pellets in Bug Buster Master Mix (15 ml/2g of cell pellet).  
Place the suspended pellets on a benchtop rocker for 30 minutes at 10 rpm.  
Pellet IBs (and cell debris) by spinning at 13 500 x RCF for ten minutes at 4oC. 

N.B. Do not vortex during this step. This has been reported anecdotally to adversely affect the final recPrP yield. 

4. First wash of the inclusion bodies. 

 
 
 

Re-suspend the IBs and debris in the same volume of Bug Buster Master Mix as was used in the previous step.  
Place the suspended IBs and debris mix on a benchtop rocker for ten minutes at low rpm.  
Pellet IBs (and cell debris) by spinning at 13 500 x RCF for ten minutes at 4oC. 

5. Second wash of the inclusion bodies. 

 

 
 
 

Using one tenth of the volume of Bug Buster Master Mix used in the previous step, dilute the Bug Buster 
Master Mix with dd.H2O to obtain a 10% Bug Buster solution.  
Re-suspend the IBs and debris in this solution. 
Place the suspended IBs and debris mix on a benchtop rocker for ten minutes at 10 rpm.  
Pellet IBs (and cell debris) by spinning at 13 500 x RCF for ten minutes at 4oC. 

N.B. At this point in the protocol, the washed inclusion bodies can be frozen at -80 oC and stored. After isolation and 
washing, the inclusion bodies should be milky-white. When frozen, they should be a dark yellow-green in colour.  

6. Buffer equilibration of the Affinity Matrix. 

The equilibration is comprised of three buffer washes. 

 
 

 
 

 
 

 
 

Add 17 ml of the Nickel NTA Superflow slurry to each of the 6 x 50 ml Falcon tubes. 
Wash the resin in each tube with 10 ml of the 6 M Guanidine denaturing buffer (‘6DB’) using a rocker at 10 
rpm for five minutes. 
Spin the 1500 RCF for five minutes to sediment the matrix. 
Wash the resin in each tube with 10 ml of the 6 M Guanidine denaturing buffer (‘6DB’) using a rocker at 10 
rpm for five minutes. 
Spin the 1500 RCF for five minutes to sediment the matrix. 
Wash the resin in each tube with 10 ml of the 6 M Guanidine denaturing buffer (‘6DB’) using a rocker at 10 
rpm for five minutes. 
Spin the 1500 RCF for five minutes to sediment the matrix. 
Decant off the supernatant and replace with enough ‘6DB’ to afford 20 ml slurry volume per Falcon tube.  

7 

 
 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

7. Lyse inclusion bodies. 

 
 
 
 
 
 

Re-suspend each inclusion body pellet in 15 ml of the 8M denaturing buffer (‘8DB’). 
Pellet the debris for ten minutes at 4oC and 13 500 RCF. 
Add the supernatants (6 x approx. 15 ml) to the equilibrated matrix slurries in the above step. 
Re-suspend the debris in a further 15 ml of the 8M denaturing buffer (‘8DB’). 
Pellet the debris for ten minutes at 4oC and 13 500 RCF. 
Add the supernatants (6 x approx. 15 ml) to the equilibrated matrix slurries in the above step. 

N.B. Remember to pre-chill the 1 x dialysis buffer in preparation for the dialysis step. 

8. Load lysate onto the nickel NTA resin. 

 
 
 

Mix the inclusion body lysates with the equilibrated NTA resin (in six Falcon tubes).  
Shake on a rocker at 10 rpm for 60 minutes at 18–20oC. 
Spin down the matrix at 1 500 RCF for five minutes at 18-20oC and discard the supernatant. 

9. Purge the FPLC system. 

Equilibrate the FPLC system with 6M Denaturing Buffer ‘6DB’ (Line A) and refolding buffer ‘RB’ (Line B). 

10. Pack the column with the nickel NTA resin. 

 
 

Pour the equilibrated NTA resin slurry into a suitable column and bed the matrix. 
Connect the column to the FPLC. 

11. Purify recPrP – wash non-PrP protein off the column (25 minutes). 

Pump isocratic 6M denaturing buffer ‘6DB’ for five minutes at 5 ml/min (approx. 0.25 column volumes) using line A. 

12. Purify PrP – refold recPrP on the column (3 hrs 35 minutes). 

 

 

Run a gradient at 5 ml/min for 200 minutes (approx. 10 column volumes), switching from the current 6M 
denaturing buffer ‘6DB’ (line A) to the renaturing buffer ‘RB’ (line B).  
Once the gradient is complete, continue pumping isocratic renaturing buffer ‘RB’ (line B) over the column 
for a further 15 minutes (approx. 0.75 column volumes).  

13. Purify PrP - Elute recPrP (1 hr 20 minutes). 

 

Once the refolding step is complete, remove line A from the 6M denaturing buffer ‘6DB’ buffer and put it 
into the Elution Buffer ‘EB’. 

N.B. Remember to purge pump A and line A of the former ‘6DB’ buffer before starting the elution. 

 

Run a gradient at 5 ml/min for 60 minutes (approx. three column volumes), switching from the current 
renaturing buffer ‘RB’ (line B) to the elution buffer ‘EB’ (line A). Collect this volume in fractions. 

N.B. The recPrP peak typically begins to elute when the buffer gradient is comprised of 50% elution buffer. The 
peak is broad (approx. 30 minutes) with little to no shoulder. Collect the middle 75% of the peak fraction-wise and 
directly into 1 x dialysis buffer at a ratio of one volume elute to two volumes of buffer. This increases final recPrP 
yield by reducing loss to spontaneous fibrillisation.  

Only once the elution has been shown to be reproducible on the instrument should the elute be collected as one 
volume into a beaker containing 1 x dialysis buffer. 

 

Once the gradient is complete, continue pumping the isocratic elution buffer at 5 ml/min over the column 
for a further 20 minutes (approx. one column volume). Collect this volume in fractions. 

N.B. The Abs280nm trace will rise slowly during the gradient since imidazole absorbs at 280 nm. The elution endpoint 
comes at the conclusion of this isocratic step. The earliest and latest eluting fractions are unsuitable for use in RT-
QuIC. 

14. Combine desired fractions and dialyse overnight. 

 

 
 

By referring to the UV trace on the FPLC, calculate which of the fractions contain the middle 75% of the 
elution.  
Combine these and filter them through a 0.22 µ filter.  
Dialyse the diluted and filtered fractions overnight against pre-chilled 1 x dialysis buffer ‘DB’ at a ratio of 
0.45L fractions to 10L dialysis buffer (approx. 20 x diluted fraction volume).  

N.B. Filtration of the recPrP solution PRIOR to dialysis is critical to preventing premature fibrillisation and a 
substantial yield loss.  

Prepare the same volume of fresh 1 x dialysis buffer so that it is pre-chilled for the next day’s buffer exchange. 

8 

 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

15. Exchange buffer. 

Exchange the dialysis buffer for fresh, pre-chilled 1 x dialysis buffer and continue dialysis for five hours. 

16. Determine recPrP concentration. 

Determine the dialysed concentration of the recPrP – for example, using Abs280nm.  
ε280nm = 62005 M-1cm-1  (1 g/L = 2.702) 
MW = 23.0603 kDa. 

17. Concentrate recPrP. 

Using spin concentrators, concentrate the recPrP to give a final concentration of between 0.3 and 0.5 mg/ml.  

18. Quality control. 

 
 
 

Determine the purity of the concentrated recPrP, for example, using SDS-PAGE and western blot.  
If substantially contaminated, do not proceed to the next step. 
RecPrP quality is determined by testing in RT-QuIC against the control brain homogenates and known 
positive and negative CSF samples. The recPrP batch passes if it produces the expected signal responses, as 
set out in the data analysis section. 

N.B. A ‘double overlapping band’ for recPrP is often observed when purifying with this method but it has no 
detrimental effect on the RT-QuIC assay. However, only very small amounts of other bands should be accepted. 

If the recPrP is substantially contaminated, a lesser yield of functional recPrP can be salvaged by performing an 
additional purification step, such as ion-exchange chromatography. 

19. Washing the resin. 

 
 
 
 

Switch both lines to denaturing elution buffer (‘DEB’). 
Pump the denaturing elution buffer at 5 ml/min over the column for 30 minutes (approx. 1.5 column volumes). 
Switch both lines to water. 
Pump the water at 5 ml/min over the column for 30 minutes (approx. 1.5 column volumes). 

20. Re-condition the resin. 

 
 
 
 
 
 
 
 

Transfer the resin from the column into six 50 ml Falcon tubes (approx. 17 ml resin/tube). 
Add demineralised water (15 ml) to each tube and spin the resin down at 3 000 RCF for five minutes. 
Wash 1 - Decant off the supernatant and wash with 5 M NaOH (15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 
Wash 2 - Decant off the supernatant and wash with 5 M NaOH (15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 
Wash 3 - Decant off the supernatant and wash with 5 M NaOH (15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 

N.B. A colour change of blue to brown may be observed during this step. 

 
 
 
 
 
 
 
 

Wash 4 - Decant off the supernatant and wash with demineralised water (15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 
Wash 5 - Decant off the supernatant and wash with demineralised water (15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 
Wash 6 - Decant off the supernatant and wash with renaturing buffer (‘RB’, 15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 
Wash 7 - Decant off the supernatant and wash with renaturing buffer (‘RB’, 15 ml). 
Spin the resin down at 3 000 RCF for five minutes. 

N.B. If storing for 1–2 weeks, store the matrix in renaturing buffer at 4oC. If storing for longer, use a 20% 
ethanol/water solution at 4oC. 

9 

 
 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

Performing the RT-QuIC assay 

Safe handling and disposal of sCJD brain homogenates 

All work involving the handling of sCJD brain homogenates should be carried out in a Class 2 MBSC in a Category 3 
laboratory. This specifically includes, but is not limited to, the seed dilutions, loading of well plates and the disposal 
of sCJD contaminated materials after the experiment is complete. Disposable pipette tips can be sealed inside a 
Sharpsafe and kept within the MBSC.  

Eppendorfs and well plates can be disposed of by soaking in 2M NaOH solution at room temperature for a 
minimum of one hour in an alkali impervious container with an airtight seal. 

Preparing the brain homogenate controls 

 
 

 

 

 
 

 

 
 

Prepare N2 buffer, as detailed in Annex 1. 
Collect frontal cortex samples from suitable brains. Around 100 mg each of sudden-death, Alzheimer’s 
diseased and sCJD brains are required. 
Prepare 10% w/v solutions of these brain samples in N2 buffer, along with a reference sample containing a 
known concentration of recombinant Hu PrP. 
Collect a 100 µL aliquot of the sCJD sample solution and using Proteinase K (PK, 2 µL, 2 mg/ml), digest it at 
37oC for one hour. The other controls are not treated with PK.  
Prepare between 1012 serial 1:10 dilutions of the PK-digested sCJD brain sample. 
Run a western blot using these dilutions against a known concentration of Hu PrP. Visualise bands using 
anti-prion protein antibody 3F4 epitope, in accordance with the manufacturer’s instructions.  
Compare the combined band densities of each digested sCJD dilution (three bands) to that of the known Hu 
PrP concentration (one band) and calculate stock concentration. 
Dilute the sCJD stock in N2 buffer as required to afford a Hu PrP concentration of 100 fg /2 µL. 
The brain homogenate controls should be aliquoted out and stored at -80oC until required. 

Preparing the master mixes 

One master mix is prepared for the brain homogenate controls and one master mix is prepared for each group of 
CSF volumes being tested (i.e. one master mix for all the samples to which 30 µL of CSF will be added, one master 
mix to which 15 µL of CSF will be added and one master mix to which 7 µL will be added.)  

CSFs are always tested at 30 µL volumes in the first instance and are only tested at 15 µL and 7 µL volumes if the 
results of the 30 µL tests are equivocal. 

 

 

Master mix for the brain homogenate control samples – Calculate enough master mix to make 17 wells (16 
control wells + 1 extra). 
Master mix for the CSF test samples – Calculate the total number of CSF samples to be analysed at each 
CSF volume as follows: 

Total number of wells for the master mix calculation = (number of CSF samples at X µL x 4) + 1. 

For each master mix, multiply each component in the table below by the total number of wells in that master mix 
to give each master mix’s composition.  

N.B. The Ha recPrP must be spin-filtered before being added to the master mix. The concentration of recPrP must 
be determined in order to calculate the correct volume of water and recPrP to add to each master mix. This 
process is detailed in the next step. 

Table 7. Master mix in a single well 

Reagent 

Volume per well 

PBS, 5x conc 
Sodium chloride solution, 2M 
EDTA, 100 mM 
1 x ThT, 1 mM (dilute from 10mM stock) 
Water, molecular biology grade 
recPrP (add last) 

20 µL 
8.5 µL 
1 µL 
1 µL 
(100 - 30.5 - recPrP – Sample) μL 
 Must be calculated – see next step. 

10 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Preparing and quantitating the recPrP 

 

 

 

 

 

Thaw the frozen recPrP solution at RT then spin filter at 3 000 x RCF and 4oC through 100 kDa MWCO spin-
filtration units. Quantitate the recPrP solution concentration as described in the following steps. (Store the 
bulk filtered recPrP at 4oC until required.) 
Take two samples of the recPrP filtrate and dilute x 10 into a solution of 1 x PBS containing 0.1% SDS 
which has been passed through a 0.22 µ filter (e.g. 30 µL of recPrP filtrate diluted into 270 µL of 1 x PBS 
containing 0.1% SDS works well.)  
Measure the absorption of these samples at 280 nm. The results should agree within the limits of the 
instrument. Repeat the sampling process if they do not. 
Perform the following calculation to determine the volume of recPrP solution to be added to each well: 
Abs280 nm x 10 = Neat Abs280 nm 
Neat Abs280 nm ÷ 2.7 = neat conc (in mg/mL) 
10 µg/neat conc (in mg/mL) = volume in L to add per well (typically 20-40 µL). 

Volume to add per well x number of wells = volume of recPrP to add to each master mix. 

Combine enough recPrP with master mixes so that the final master mix compositions match that described 
in the table below. ThT is added as the penultimate step and the master mixes are vortexed immediately 
before the recPrP is added. The recPrP is always the last component to be added to the master mixes. 
Once the recPrP has been added, master mixes should be mixed by gentle inversion x 3, then 
subsequently handled with care to reduce accidental fibrillisation. 

Table 8. Final master mix composition in a well 

Reagent 

Volume per well 

Hamster Prion Protein 
PBS, 5x conc 
Sodium chloride solution, 2M 
EDTA, 100 mM 
ThT solution in water, 1 mM 
Water, molecular biology grade 

20–30 µL (10 µg after spin filtration) 
20 µL 
8.5 µL 
1 µL 
1 µL 
(100 - 30.5 - recPrP – Sample) μL 

N.B. The ThT is best handled in darkened conditions to minimise photobleaching, such as foil-wrapped 
Eppendorfs/tubes and in an unlit MBSC. As mentioned previously, the ThT should be made up at a 10 mM 
concentration (e.g. 32 mg ThT/10 ml water) to allow accurate weighing. The ThT should be solubilised at room 
temperature, then passed through a 0.22 µ filter. The yellow solution is stored in foil at 4oC until ready for use, at 
which point it is diluted ten-fold in water to afford a 1mM solution. This 10 mM solution should be freshly made for 
every RT-QuIC assay. 

Preparing the RT-QuIC plate 

 

 

Load the RT-QuIC plate using the master mixes – i.e. Control Master Mix (98 µL) then add the control 
samples (2 µL) or Sample Master Mix (100 µL – CSF sample volume) then add the test CSFs (30 /15/7 µL). 
Once the 96-well plate has been filled with samples, seal the top of the plate using adhesive film and load it 
into the plate reader. 

Table 9. The final composition of a well 

Reagent 

Concentration 

Volume per well 

Hamster Prion Protein 
PBS, 5x conc 

Sodium chloride solution 
EDTA 
ThT solution in water 
Water, molecular biology grade 

10 µg/100 µL 
50 mM PO4
770 mM NaCl 
2 M 
100 mM 
1 mM 

- 

20–40 µL 
20 µL 

8.5 µL 
1 µL 
1 µL 
(100 - 30.5 - recPrP – Water -Sample) μL 

Samples 

Concentration 

Volume per well (control or sample) 

Unseeded blank 
Sudden-death brain homogenate 
Alzheimer’s diseased brain homogenate 
sCJD brain homogenate 
CSF sample 

1 x PBS 
50 fg Hu PrP/µL† 
50 fg Hu PrP/µL† 
50 fg Hu PrPres/µL† 
Neat 

2 µL 
2 µL 
2 µL 
2 µL 
30, 15 or 7 µL 

†Concentrations are determined using western blot. The quantitation method is detailed above in the section ‘Preparing the brain 
homogenate controls.’ 

11 

 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

N.B. The presence of blood in a CSF sample can suppress a positive response, increasing the likelihood of the 
sample returning a false negative. As a suggested limit, CSFs with red blood cell counts of ≤ 150 cell/mcL do not 
exhibit this effect. 

RT-QuIC instrument protocol 

Each ‘measurement cycle’ consists of two phases – shaking and reading. These phases differ in length depending 
on the instrument used. A batch script is used to call the shake and read scripts. Full details and examples of how 
these are written are listed in the appendix. 

Briefly, the FLUOstar Optima and Omega phases are 14 minutes of shaking followed by one minute of reading the 
plate. The 14 minutes of shaking is programmed in as 7 x 2 minute shaking cycles on the instrument. The Optima 
and Omega take slightly different times to accelerate up to the specified shaking speed. This difference results in 
the Optima having a two-minute cycle consisting of 60 seconds of shaking and 60 seconds of rest, while the 
Omega has 86 seconds of shaking and 34 seconds of rest. 

It can take up to 90 hours for a sample to give a clear positive result so the measurement cycles should be 
continued until the instrument has completed measurement cycle 361, at around 96 hours. 

The RT-QuIC assay requires the FLUOstar OMEGA to shake at 900 rpm, which is above the maximum shake speed 
available through standard software options. However, the manufacturers can modify the software settings to allow 
this higher shake speed to be reached.  

Annex 2 contains all required input parameters for instrument set-up.  

It should be noted that the high sensitivity and specificity values reported for RT-QuIC were obtained using the 
recommended settings for the FLUOStar Optima instrument. The FLUOstar Omega has been tested at the 
NCJDRSU and delivered comparable results to the Optima. The use of the recommended conditions is supported by 
a large body of sample data and deviations from this protocol may lower the quoted sensitivity and specificity 
values for detection of sCJD. 

Some laboratories may only have access to certain instruments and substrates or a limited access to CSF reference 
samples. A range of conditions for different instruments and substrates has been included in Annex 2. 

12 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Data analysis 

Samples or controls are positive if the two wells with the highest RFUs exceed +3 standard deviations of the 
negative controls within the duration of the run, or at cycle 361. The number of positive wells out of each 
quadruplicate sample set is noted, as well as the time taken to reach the positive cut-off RFU value.  

With regard to the brain homogenate controls, the unseeded, sudden-death and Alzheimer’s disease controls 
should give a flat response. Only the sCJD controls should exhibit an increase in RFU. 

Positive CSF samples frequently give between 2/4 and 4/4 positive results.  

If only one well out of the four is positive, or if the fluorescent trace rises above the background but does not 
reach the cut-off value, this could either be: 

 
 

a negative CSF sample exhibiting a stochastic effect due to local well conditions – a ‘false positive’; 
a positive CSF sample where there are lots of PrPSc seeds in the wells. In this case, all the recPrP is templated 
onto many small plaques before the plaques can bind enough ThT to give a strong positive signal. 

In cases where only one well out of the four is positive, the sample analysis should be repeated in a subsequent 
run using 15 µL CSF1. 

Table 10. Typical ranges for endpoint RFUs of two highest wells at Cycle 361 

Sample type 

Unseeded (control) 

Sudden-death brain (control) 

Alzheimer's disease brain (control) 

sCJD disease brain (control) 

30 CSF (positive sample) 

N 

16 

17 

19 

21 

57 

30 CSF (negative sample) 

126 

Mean (RFU) 

Standard deviation (RFU) 

16600 

16749 

16709 

146145 

92024 

16159 

677 

566 

753 

31264 

37972 

936 

Values calculated using the recommended conditions for the FLUOStar Omega, as described in Annex 2. 

1 NCJDRSU has subsequently found that using 7ul of CSF is unreliable. 

13 

 
 
 
                                                                    
Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

TECHNICAL REPORT 

References 

1.  Wilham JM, Orrú CD, Bessen RA, Atarashi R, Sano K, Race B, et al. Rapid end-point quantitation of prion 

seeding activity with sensitivity comparable to bioassays. PLoS Pathog, 2010. 6(12):e1001217. 

2.  Peden AH, McGuire LI, Appleford NEJ, Mallinson G, Wilham JM, Orrú CD, et al. Sensitive and Specific Detection 
of Sporadic Creutzfeldt-Jakob Disease Brain Prion Protein Using Real-Time Quaking Induced Conversion. Prion, 
2011. 5:75-75. 

3.  McGuire LI, Peden AH, Orrú CD, Wilham JM, Appleford NEJ, Mallinson G, et al. Real time quaking-induced 

conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt–Jakob disease. Annals of Neurology, 2012. 
72(2):278-285. 

4.  Atarashi R, Satoh K, Sano K, Fuse T, Yamaguchi N, Ishibashi D, et al., Ultrasensitive human prion detection in 

cerebrospinal fluid by real-time quaking-induced conversion. Nature Medicine, 2011. 17(2):175-178. 

5.  Sambrook JF, Russell DW. Molecular Cloning – A Laboratory Manual (Third Edition), Cold Spring Harbor 

Laboratory Press, 2001. 

14 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Annex 1. 

Buffer recipes for recPrP purification 

NB: the volumes given here are sufficient for ONE purification run under prescribed conditions. All 
buffers should be 0.22 µ filtered and (apart from the 20 x DIB) de-gassed before use. 

‘8DB’ Denaturing buffer 

38.5 g   Guanidine hydrochloride 

Final volume = 100 ml at pH 8.0 

‘6DB’ Denaturing buffer 

1.3 g NaH2PO4.H2O 
13.0 g Na2HPO4  
1.2 g Tris  
580.0 g Guanidine hydrochloride  

Final volume = 1 L at pH 8.0 

‘RB’ Refolding buffer 

(100mM total phosphate) 
(10mM Tris) 
(6.0 M) 

1.0 g NaH2PO4.H2O 
14.0 g Na2HPO4  
1.2 g Tris 

(100mM total phosphate) 
(10mM Tris) 

Final volume = 1 L at pH 8.0 

‘EB’ Elution buffer 

2.3 g NaH2PO4.H2O 
1.2 g Na2HPO4  
0.3 g Tris 
13.0g Imidazole hydrochloride 

Final volume = 250 ml at pH 6.0 

(100mM total phosphate) 
(10mM Tris) 
(500 mM) 

‘DEB’ Denaturing elution buffer 

2.1 g NaH2PO4.H2O 
4.4 g NaCl 
87.0 g Guanidine hydrochloride 

(100mM total phosphate) 
(500 mM NaCl) 
(6M Gu.HCl) 

Final volume = 150 ml at pH 4.0 

(Denaturing elution buffer for cleaning Ni-NTA resin only) 

20 x ‘DIB’ (20 x dialysis buffer)  

24.0 g NaH2PO4.H2O 
4.0 g Na2HPO4 

(200mM total phosphate) 

Final volume = 1000 ml at pH 5.8 

For dialysis dilute 20 x to give 10mM phosphate at about pH 6.5.  

NB: This buffer does not have to be de-gassed before use. After diluting to 1 x, remember to pre-chill 
this buffer down to 04 oC in preparation for dialysis. 

0.5 M NaOH solution 

6.0 g NaOH 

Final volume = 300 ml at pH 13 

Recipe for N2 buffer 

(10mM total phosphate) 

1 x PBS 
0.1% SDS 
1 x N2 supplement 

Volume required = 10 ml at pH 7.4. 

15 

 
TECHNICAL REPORT 

Annex 2. 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

Recommended and alternate assay parameters 

Recommended 

Others have had success with: 

Recommended 

Others have had success with: 

OPTIMA 
Ham FL 

Ham-Sh Chimeric 

OPTIMA 
Ham FL 

Hum FL 

OMEGA 
Ham FL 

OMEGA 
Ham FL 

TECAN 
Hum FL 

Reader 

Recombinant 
PrP 

CSF volume† 

30 or 15 µL 

15 µL 

5 µL 

30 or 15 µL 

20 µL 

30 µL 

5 µL 

Shake 
conditions 

Temperature 

600rpm 
60s shake/60s 
rest 
42◦C 

Criteria for 
positive 
result‡ 

Mean of two 
highest 
replicates out of 
4 >10 000 RFU 
at 90 hrs 

600rpm 
60s shake/60s rest 

750rpm  
30s shake/30s rest 

42◦C 

37◦C 

Mean of two 
highest replicates 
out of 4 >10 000 
RFU at 80 hrs 

Mean of two highest 
replicates out of 4 
>6 000 RFU at 90 
hrs 

Mean of two highest 
replicates out of 4 
>70% of baseline 
reading at 90 hrs 

900rpm 
86s shake/34s 
rest 
42◦C 

Mean of two 
highest 
replicates out of 
4 >24 000 RFU 
at 90 hrs 

900rpm 
90s shake/30s rest 

Max 
30s shake/30s rest 

42◦C 

37◦C 

Mean of two highest 
replicates out of 4 
>20 000 RFU at 90 hrs 

Average reading 
of all four wells 
>4 344 RFU at 
90 hrs 

At least two of six 
replicates >400 RFU at x 
hrs. 

† The CSF volume used in the assay is a speculative decision which balances the need for a robust and sensitive assay against the available amount of patient sample and the time taken to complete the 
measurement. The assay has a sensitivity of 91% and a specificity of 98% when 15 µL of CSF is used, as per the recommended FLUOstar Optima settings.[3]  

‡ The RFU ‘cut-off’ value used to determine whether an unknown sample is positive or negative is the mean end-point of a population of known negative samples + 3 standard deviations. The RFU value also varies 
with the ‘gain’ setting on the instrument, which is a control of the detector sensitivity. Two different research groups may therefore use the same instrument and reference samples but use a different RFU ‘cut-off’ 
limit. 

16 

 
 
 
 
 
 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Batch script for running an RT-QuIC on either the FLUOstar Optima or Omega 

17 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

Read script parameters, RT-QuIC FLUOstar Optima 

Basic Parameters 

Microplate 
Optic 
Number of multichromatics 
Positioning delay 
Filters  
Gain 
Measurement start time  
Number of flashed per well  
Orbital averaging  
Pause before plate reading 

Nunc 96 
Bottom Optic 
1 
0.2 (Flying mode unchecked) 
Excitation filter 450 and Emission filter 480 
2000 
0 
20 
(Unchecked)  
(Unchecked)  

Layout 

Groups 
Start value  
Replicates  
Reading direction  

(Unchecked)  
1, Increase 
1, Horizontal 
Snaking 

Concentrations/volumes/shaking 

Standard concentration 
Start volume 
Volume 
Shaking options 
Additional shaking 

Factor (greyed out) 
0 
Factor, 1 
Double orbital, shaking width 4mm, 150 rpm 
No shaking 

Read script parameters, RT-QuIC FLUOstar Omega 

Basic parameters 

Microplate 
Optic 
No. of Multichromatics 
Positioning delay 
Filters  
Gain 
Measurement start time  
No. of flashed per well  
Orbital averaging  
Pause before plate reading 

Nunc 96 
Bottom Optic 
1 
0.2 (Flying mode unchecked) 
Excitation filter 450 and Emission filter 480 
2000 
0 
20 
(Unchecked)  
(Unchecked)  

Layout 

Groups 
Start value  
Replicates  
Reading direction  

Concentrations/volumes/shaking 

(Unchecked)  
1, Increase 
1, Horizontal 
Snaking 

Standard concentration 
Start volume 
Volume 
Shaking options 
Additional shaking 

Factor (greyed out) 
0 
Factor, 1 
Double orbital, 900 rpm 
No shaking 

NB: Shaking at 900 rpm requires software update from BMG Labtech. 

18 

 
 
 
 
 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

Shake script parameters, RT-QuIC FLUOstar Optima 

Basic parameters 

Nunc 96 
Top Optic 
1 
0.0 (Flying mode unchecked) 
1 
Excitation 450 and Emission filter 480 
2000 

Kinetic window 1 

Microplate 
Optic 
No. of Multichromatics 
Positioning delay 
No. of kinetic windows  
Filters  
Gain 

No. of cycles 
Measurement start time  
No. of flashed per well and cycle 
Cycle time 

Basic parameters continued 

Orbital averaging  
Minimum cycle time 1 
Pause before plate reading 

Layout 

7 
0 
0 
120 

(Unchecked)  
(Greyed out) 
0 

Sample 

(Unchecked)  

1, Increase 

1, Horizontal 

Snaking 

Content 

Groups 

Start value  

Replicates  

Reading direction  

Standard concentration 

Start volume 

Volume 

Shaking options 

Additional shaking 

Shaking time  

Concentrations/volumes/shaking 

Factor (greyed out) 

0 

Factor, 1 

Double orbital, shaking width 1mm, 600 rpm 

Before each cycle 
57s 

19 

 
 
 
 
TECHNICAL REPORT 

Laboratory standard operating procedure for detecting sCJD using RT-QuIC  

Shake script parameters, RT-QuIC FLUOstar Omega 

Example plate layout 

When performing RT-QuIC on a 96 well plate with 4 control sets, there is space for 20 CSF samples.  

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

CONTROL 2 
Sudden-death 
BH 

CONTROL 2 
Sudden-death 
BH 

CONTROL 2 
Sudden-death 
BH 

CONTROL 2 
Sudden-death 
BH 

CONTROL 3 
Alzheimer’s 
Disease BH 

CONTROL 3 
Alzheimer’s 
Disease BH 

CONTROL 3 
Alzheimer’s 
Disease BH 

CONTROL 3 
Alzheimer’s 
Disease BH 

A  CONTROL 

1 
Unseeded 
Well 

B  CONTROL 

4 
sCJD BH 

CONTROL 
1 
Unseeded 
Well 

CONTROL 
4 
sCJD BH 

CONTROL 
1 
Unseeded 
Well 

CONTROL 
4 
sCJD BH 

CONTROL 
1 
Unseeded 
Well 

CONTROL 
4 
sCJD BH 

CSF  
Sample 1 
30 µL CSF 

CSF  
Sample 1 
30 µL CSF 

CSF  
Sample 1 
30 µL CSF 

CSF  
Sample 1 
30 µL CSF 

CSF  
Sample 2 
30 µL CSF 

C  CSF  

Sample 3 
30 µL CSF 

CSF  
Sample 3 
30 µL CSF 

CSF  
Sample 3 
30 µL CSF 

CSF  
Sample 3 
30 µL CSF 

CSF  
Sample 4 
30 µL CSF 

CSF  
Sample 4 
30 µL CSF 

CSF  
Sample 4 
30 µL CSF 

CSF  
Sample 4 
30 µL CSF 

CSF  
Sample 5 
30 µL CSF 

D  CSF  

Sample 6 
30 µL CSF 

CSF  
Sample 6 
30 µL CSF 

CSF  
Sample 6 
30 µL CSF 

CSF  
Sample 6 
30 µL CSF 

CSF  
Sample 7 
30 µL CSF 

CSF  
Sample 7 
30 µL CSF 

CSF  
Sample 7 
30 µL CSF 

CSF  
Sample 7 
30 µL CSF 

CSF  
Sample 8 
30 µL CSF 

CSF  
Sample 2 
30 µL CSF 

CSF  
Sample 5 
30 µL CSF 

CSF  
Sample 8 
30 µL CSF 

CSF  
Sample 2 
30 µL CSF 

CSF  
Sample 5 
30 µL CSF 

CSF  
Sample 8 
30 µL CSF 

CSF  
Sample 2 
30 µL CSF 

CSF  
Sample 5 
30 µL CSF 

CSF  
Sample 8 
30 µL CSF 

E  CSF  

Sample 9 
30 µL CSF 

CSF  
Sample 9 
30 µL CSF 

CSF  
Sample 9 
30 µL CSF 

CSF  
Sample 9 
30 µL CSF 

CSF  
Sample 10 
30 µL CSF 

CSF  
Sample 10 
30 µL CSF 

CSF  
Sample 10 
30 µL CSF 

CSF  
Sample 10 
30 µL CSF 

CSF  
Sample 11 
30 µL CSF 

CSF  
Sample 11 
30 µL CSF 

CSF  
Sample 11 
30 µL CSF 

CSF  
Sample 11 
30 µL CSF 

F  CSF  

Sample 12 
30 µL CSF 

CSF  
Sample 12 
30 µL CSF 

CSF  
Sample 12 
30 µL CSF 

CSF  
Sample 12 
30 µL CSF 

CSF  
Sample 13 
30 µL CSF 

CSF  
Sample 13 
30 µL CSF 

CSF  
Sample 13 
30 µL CSF 

CSF  
Sample 13 
30 µL CSF 

CSF  
Sample 14 
30 µL CSF 

CSF  
Sample 14 
30 µL CSF 

CSF  
Sample 14 
30 µL CSF 

CSF  
Sample 14 
30 µL CSF 

G  CSF  

Sample 15 
30 µL CSF 

CSF  
Sample 15 
30 µL CSF 

CSF  
Sample 15 
30 µL CSF 

CSF  
Sample 15 
30 µL CSF 

CSF  
Sample 16 
30 µL CSF 

CSF  
Sample 16 
30 µL CSF 

CSF  
Sample 16 
30 µL CSF 

CSF  
Sample 16 
30 µL CSF 

CSF  
Sample 17 
30 µL CSF 

CSF  
Sample 17 
30 µL CSF 

CSF  
Sample 17 
30 µL CSF 

CSF  
Sample 17 
30 µL CSF 

H  CSF  

Sample 18 
30 µL CSF 

CSF  
Sample 18 
30 µL CSF 

CSF  
Sample 18 
30 µL CSF 

CSF  
Sample 18 
30 µL CSF 

CSF  
Sample 19 
30 µL CSF 

CSF  
Sample 19 
30 µL CSF 

CSF  
Sample 19 
30 µL CSF 

CSF  
Sample 19 
30 µL CSF 

CSF  
Sample 19 
15 µL CSF 

CSF  
Sample 19 
15 µL CSF 

CSF  
Sample 19 
15 µL CSF 

CSF  
Sample 19 
15 µL CSF 

20 

 
 
 
 
 
Laboratory standard operating procedure for detecting sCJD using RT-QuIC 

TECHNICAL REPORT 

Annex 3.  

Example - unseeded and brain homogenate controls 

Example - negative and positive 30 µL CSF samples 

21 

 
 
 
 
 
 
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