Contamination indicator

Abstract

The present invention relates to contamination indicators, and the use of such indicators for monitoring decontamination in healthcare facilities.

Description

CONTAMINATION INDICATORFIELD OF THE INVENTION

[0001] The present invention relates to contamination indicators. In particular, the present invention relates to indicators to track decontamination in healthcare facilities. However, it will be appreciated that the invention is not limited to this particular field of use.

[0002] The present application claims priority from Australian Provisional Patent Application No. 2024900537 (filed 1 March 2024), the contents of which are incorporated in their entirety herein.BACKGROUND OF THE INVENTION

[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0004] The Covid-19 pandemic has emphasised the risks associated with infectious transmission and cross contamination and the subsequent requirements for efficacy, traceability and compliance associated with all aspects of decontamination. The reprocessing (cleaning, disinfection, sterilisation) of medical instruments represents a multibillion-dollar market. Current methodologies have various failure points. Test methods to ensure efficacy lack sensitivity and outcomes are often subjective. Importantly, they do not address the resistance of many pathogenic species to decontamination, which in turn can compromise the safety of patients.

[0005] The idea that a protein can act as an infectious pathogen and cause degeneration of the central nervous system is now well accepted. In 2015, a study published in Nature demonstrated the first human transmission of Alzheimer’s pathology through a medical procedure (Jaunmuktane et al. 2015, Nature 525: 247-250). Since then, multiple studies have shown independently that pathogenic protein aggregates linked to Alzheimer’s and Parkinson’s diseases bind strongly to surgical instruments and remain infectious after autoclaving (Pinder et al. 2021, J. Hosp. Infect. 108: 25-32). The risk of transmission of such proteins and the lack of surveillance associated with the reuse of surgical instruments is particularly problematic in a hospital setting (Lauwers et al. 2020, Lancet 19(10): 872-878).

[0006] There is currently no system that addresses this challenge. In the UK, the Department of Health has introduced new guidelines for protein decontamination and urged implementation of improved indicators with higher resistance and sensitivity as soon as available.

[0007] Currently, central sterilising departments rely on cleaning indicators which are placed in each washing load to determine the effectiveness of the cleaning step. However there are major deficiencies in this practice:• Current indicators consist of artificial soils which are deposited on strips of plastic, using water-soluble dyes that dissolve easily in the wash cycle (such as Getinge Wash Monitor, TOSI test strips, etc). However, such indicators cannot accurately mimic the decontamination resistance of pathogenic proteins bound to surgical steel. Such proteins resist sterilisation and remain infectious, and cross-contamination could take place.• The pass / fail decision for each load is judged manually based on a visual assessment of the indicator. The sensitivity is very poor (up to 1% of contaminant can be left undetected), and the decision is subjective, with little traceability to account for human error.

[0008] Poor sensitivity and traceability can have serious consequences. For example, The Royal Melbourne Hospital was forced to dispose of 15,000 surgical instruments and sterilise 30,000 more following the discovery that one patient had died from a prion disease.

[0009] While the binding of prion proteins bound to surgical steel wires or beads have been used to investigate the effectiveness of decontamination protocols, quantification requires the use of in vivo bioassays (Edgeworth et al. 2011 , J. Gen. Virol. 92: 718-726), protein misfolding cyclic amplification (PMCA) followed by proteinkinase K digestion, electrophoresis and western blotting (Erana et al 2020, Front. Bioeng. Biotechnol. 8: 589182; Moudjou et al. 2020, Front. Bioeng. Biotechnol. 8: 591024), or real-time quaking-induced conversion (RT-QuIC) and fluorescence (Hughson et al. 2016, PLoS Pathlog. 12(9): e1005914; Moudjou et al. 2020, Front. Bioeng. Biotechnol. 8: 591024). Such methods are time consuming, require special expertise and materials, and are not suitable for commercial use in a hospital setting.

[0010] Consequently, there is a need for decontamination indicators that are easy to use, reduce human error, and reflect the resistance of pathogenic proteins to decontamination.

[0011] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.SUMMARY OF THE INVENTION

[0012] According to a first aspect, the present invention provides a contamination indicator comprising mesh coated with fluorescence-labelled protein fibrils.

[0013] In one embodiment, the mesh is wire mesh.

[0014] In another embodiment, the wire mesh has an opening size of 0.01 mm to 0.6 mm.

[0015] In another embodiment, the wire mesh has an opening size of 0.025 mm to0.1 mm.

[0016] In another embodiment, the wire mesh has an opening size of about 0.01 mm, about 0.02 mm, about 0.03 mm, about 0.04 mm, about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.11 mm, about 0.12 mm, about 0.13 mm, about 0.14 mm, about 0.15 mm, about 0.16 mm, about 0.17 mm, about 0.18 mm, about 0.19 mm, about 0.2 mm, about 0.21 mm, about 0.22 mm, about 0.23 mm, about 0.24 mm, about 0.25 mm, about 0.26 mm, about 0.27 mm, about 0.28 mm, about 0.29 mm, about 0.3 mm, about 0.31 mm, about 0.32 mm, about 0.33 mm, about 0.34 mm, about 0.35 mm, about 0.36 mm, about 0.37 mm, about 0.38 mm, about 0.39 mm, about 0.4 mm, about 0.41 mm, about 0.42 mm, about 0.43 mm, about 0.44 mm, about 0.45 mm, about 0.46 mm, about 0.47 mm, about 0.48 mm, about 0.49 mm, about 0.5 mm, about 0.51 mm, about 0.52 mm, about 0.53 mm, about 0.54 mm, about 0.55 mm, about 0.56 mm, about 0.57 mm, about 0.58 mm, about 0.59 mm or about 0.6 mm.

[0017] In another embodiment, the wire mesh has an opening size of about 0.05 mm.

[0018] In another embodiment, the wire mesh has an opening size of about 0.043 mm

[0019] In another embodiment, the wire mesh has a wire diameter of 0.01 mm to 0.3 mm.

[0020] In another embodiment, the wire mesh has a wire diameter of 0.02 mm to 0.05 mm.

[0021] In another embodiment, the wire mesh has a wire diameter of about 0.01 mm, about 0.02 mm, about 0.03 mm, about 0.04 mm, about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.11 mm, about 0.12 mm, about 0.13 mm, about 0.14 mm, about 0.15 mm, about 0.16 mm, about 0.17 mm, about 0.18 mm, about 0.19 mm, about 0.2 mm, about 0.21 mm, about 0.22 mm, about 0.23 mm, about 0.24 mm, about 0.25 mm, about 0.26 mm, about 0.27 mm, about 0.28 mm, about 0.29 mm or about 0.3 mm.

[0022] In another embodiment, the wire mesh has a wire diameter of about 0.03 mm or about 0.04 mm.

[0023] In another embodiment, the wire mesh has: an opening size of 0.01 mm to 0.6 mm; and a wire diameter of 0.01 mm to 0.3 mm.

[0024] In another embodiment, the wire mesh has: an opening size of 0.025 mm to 0.1 mm, and a wire diameter of 0.02 mm to 0.05 mm.

[0025] In another embodiment, the wire mesh has: an opening size of about 0.01 mm, about 0.02 mm, about 0.03 mm, about 0.04 mm, about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.11 mm, about 0.12 mm, about 0.13 mm, about 0.14 mm, about 0.15 mm, about 0.16 mm, about 0.17 mm, about 0.18 mm, about 0.19 mm, about 0.2 mm, about 0.21 mm, about 0.22 mm, about 0.23 mm, about 0.24 mm, about 0.25 mm, about 0.26 mm, about 0.27 mm, about 0.28 mm, about 0.29 mm, about 0.3 mm, about 0.31 mm, about 0.32 mm, about 0.33 mm, about 0.34 mm, about 0.35 mm, about 0.36 mm, about 0.37 mm, about 0.38 mm, about 0.39 mm, about 0.4 mm, about 0.41 mm, about 0.42 mm, about 0.43 mm, about 0.44 mm, about 0.45 mm, about 0.46 mm, about 0.47 mm, about 0.48 mm, about 0.49 mm, about 0.5 mm, about 0.51 mm, about 0.52 mm, about 0.53 mm, about 0.54 mm, about 0.55 mm, about 0.56 mm, about 0.57 mm, about 0.58 mm, about 0.59 mm or about 0.6 mm; and a wire diameter of about 0.01 mm, about 0.02 mm, about 0.03 mm, about 0.04 mm, about 0.05 mm, about 0.06 mm, about 0.07 mm, about 0.08 mm, about 0.09 mm, about 0.1 mm, about 0.11 mm, about 0.12 mm, about 0.13 mm, about 0.14 mm, about 0.15 mm, about 0.16 mm, about 0.17 mm, about 0.18 mm, about 0.19 mm, about 0.2 mm, about 0.21 mm, about 0.22 mm, about 0.23 mm, about 0.24 mm, about 0.25 mm, about 0.26 mm, about 0.27 mm, about 0.28 mm, about 0.29 mm or about 0.3 mm, or any combination thereof.

[0026] In another embodiment, the wire mesh has an opening size of about 0.05 mm and a wire diameter of about 0.03 mm.

[0027] In another embodiment, the wire mesh has an opening size of about 0.043 mm and a wire diameter of about 0.03 mm

[0028] In another embodiment, the wire mesh is formed from stainless steel.

[0029] In another embodiment, the stainless steel is surgical steel.

[0030] In another embodiment, the protein fibrils are prion fibrils.

[0031] In another embodiment, the prion fibrils are alpha synuclein fibrils.

[0032] In another embodiment, the alpha synuclein fibrils are human alpha synuclein fibrils.

[0033] In another embodiment, the alpha synuclein fibrils are mouse alpha synuclein fibrils.

[0034] In another embodiment, the alpha synuclein has the amino acid sequence of any one of SEQ ID Nos 1 to 3.

[0035] In another embodiment, the protein fibrils are non-pathogenic protein fibrils.

[0036] In another embodiment, the protein fibrils are whey protein fibrils.

[0037] In one embodiment, the whey protein fibrils are whey protein isolate fibrils.

[0038] In another embodiment, the whey protein fibrils are p-lactoglobulin fibrils.

[0039] In one embodiment, the whey protein fibrils, whey protein isolate fibrils or p- lactoglobulin fibrils are formed in artificial cerebrospinal fluid.

[0040] In one embodiment, the artificial cerebrospinal fluid comprises Glucose and MgCl2.

[0041] In one embodiment, the artificial cerebrospinal fluid further comprises NaCI, KCI,NaH2PO4and NaHCO3.

[0042] In one embodiment, the artificial cerebrospinal fluid further comprises NaN3.

[0043] In one embodiment, the artificial cerebrospinal fluid comprises about 120mM NaCI, about 0.01% NaN3, about 2.5mM KCI, about 1mM NaH2PO4, about 10mM Glucose, about 1 ,3mM MgCh and about 2.7mM NaHCO3.

[0044] In another embodiment, the fluorescent label is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 647, tetramethylrhodamine (TMR) and Cyanine 5.5.

[0045] In another embodiment, the fluorescent label is Alexa Fluor 488.

[0046] In another embodiment, the fluorescent label is Alexa Fluor 647.

[0047] In another embodiment, the fluorescent label is tetramethylrhodamine.

[0048] In another embodiment, the fluorescent label is Cyanine 5.5.

[0049] In another embodiment the contamination indicator is a protein contamination indicator.

[0050] In another embodiment, the protein is from a biological contaminant.

[0051] In another embodiment, the biological contaminant is selected from the group consisting of microorganisms (including viruses, bacteria and fungi), substances produced by microorganisms, protein aggregates, protein fibrils, parasites and prions.

[0052] According to a second aspect, the present invention provides a method for producing a contamination indicator, the method comprising: depositing a solution containing fluorescence-labelled protein fibrils on a mesh; and allowing the mesh to dry.

[0053] In another embodiment, the protein fibrils are labelled with fluorescence before being deposited onto the mesh.

[0054] In another embodiment, the protein fibrils are labelled with fluorescence after being deposited onto the mesh.

[0055] In another embodiment, about a 20 pL drop of the solution is deposited on the wire mesh.

[0056] In another embodiment, the concentration of fibrils in the solution is 0.25mg / mL to 2.5mg / mL.

[0057] According to a third aspect, the present invention relates to a contamination indicator produced by the method of the second aspect.

[0058] According to a fourth aspect, the present invention provides a method for monitoring decontamination of an object, comprising positioning the contamination indicator of the first or third aspect near the object during decontamination and then measuring the amount of fluorescence on the indicator.

[0059] In another embodiment, the object is a surgical or dental instrument.BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : SDS PAGE for WT human a-synuclein (A) and WT mouse a-synuclein (B) and spectroscopy for WT human a-synuclein (C) and WT mouse a-synuclein (D).

[0061] Figure 2: Electron microscopy of fibrils before and after sonication (A), Fluorescence intensity of fibril fragments before and after amplification (B), and prominence / residence of fibril fragments before and after amplification (C).

[0062] Figure 3: Fluorescent image of steel mesh contaminated with a-syn-Alexa488 (A) and fluorescence analysis of highlighted area (B) prior to decontamination.

[0063] Figure 4: Fluorescent image of steel mesh contaminated with a-syn-Alexa488 (A) and fluorescence analysis of highlighted area (B) following decontamination by Autozyme in the Eppendorf Thermomixer, non-shaking conditions.

[0064] Figure 5: Graph showing results of decontamination experiments. Control = uncoated mesh, A488 = mesh with a-syn-Alexa488 and TMR = mesh with a-syn-TMR- Alexa647.

[0065] Figure 6: 4-12% Bis-Tris gel with PageRuler LR ladder in MOPS buffer stained with SimplyBlue followed by destaining in MilliQ H2O overnight. Imaging conducted using BioRad Chemidoc with Cy 5.5 exposure settings.

[0066] Figure 7: aCSF and LSB pH 7.0 buffered 5mg / ml WPI fibrils at day 5 and day 7 of growth, pre- and post-sonication respectively, in aCSF or LBS whilst shaking at 1000rpm at 37°C.

[0067] Figure 8: Recombinant a- synuclein fibrils at day 7 of growth in aCSF whilst shaking at 1000rpm at 37°C.

[0068] Figure 9: Recombinant a- synuclein Periodic growth measurement of Cy 5.5 labelled aCSF WPI fibrils (5mg / ml) at day 1, day 4, and day 5.

[0069] Figure 10: Graphic representations of urea resistance comparisons based off peak numbers and total peak intensities of recombinant WT human a-syn fibrils grown in aCSF (Blue) and Cy 5.5 labelled 5mg / ml WPI in aCSF buffer (Green).

[0070] Figure 11 : Comparison of meshes with opening sizes 0.034 mm, 0.043mm and 0.062 mm, and different concentrations of fluorescent-labelled WPI.

[0071] Figure 12: Imaging of a mesh with an opening size of 0.318mm showing fluorescent-labelled WPI fibrils.

[0072] Figure 13: Comparison of mesh coating methods.

[0073] Figure 14: Analysis of fluorescence profiles for drops of different Cy5-labelled WPI fibrils loaded onto a mesh with an opening size of 0.043 mm.

[0074] Figure 15: Imaging of Cy5-labelled WPI fibrils coated mesh treated with Autozyme.

[0075] Figure 16: Fluorescence profiles of Cy5-labelled WPI fibrils coated mesh treated with Autozyme.DEFINITIONS

[0076] In describing and claiming the present invention, the following terminology has been used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0077] As used herein the term “about” can mean within 1 or more standard deviation per the practice in the art. Alternatively, “about” can mean a range of up to 20%. When particular values are provided in the specification and claims the meaning of “about” should be assumed to be within an acceptable error range for that particular value.

[0078] As used herein, the term “opening size” is interchangeable with “aperture size” and “mesh size” and refers the size of the openings or holes in the mesh. It's typically measured as the distance from one wire strand to the next. The skilled person would be able to determine the opening size by measurement or by calculation based on the mesh count (the number of holes per inch / centimetre in the mesh.

[0079] As used herein, the term “wire diameter” is interchangeable with wire “gauge” or “thickness” and refers to the thickness of the wore that forms the mesh. The skilled person would be able to determine the opening size by measurement or by calculation based on the industry standards (higher numbers indicating thinner wires).

[0080] As used herein the term “artificial cerebrospinal fluid” or “aCSF” is a synthetic solution designed to mimic the properties and functions of natural cerebrospinal fluid (CSF). Artificial cerebrospinal fluid is available from commercial sources and contains salts (e.g., NaCI, KCI, CaCI2, MgCI2, MgS04, NaH2PO4, etc.), buffers (e.g., NaHCO3, NaH2PO4, Na3PO4, etc.) and glucose. The artificial cerebrospinal fluid can also contain substances that prevent the growth of contaminants, such as NaN3, penicillin, streptomycin, etc.

[0081] As used herein, the term “non-pathogenic protein” means a protein that does not possess properties or characteristics that would lead to an infectious or harmful effect on a human.

[0082] As used herein, the term “coated” means a mesh that is fully or partially covered with protein fibrils.

[0083] As used herein, the term “protein contamination indicator” means an indicator that indicated whether proteins or protein-based products are present. The proteins may be from a biological contaminant, such as microorganisms (including viruses, bacteria and fungi), substances produced by microorganisms, protein aggregates, protein fibrils, parasites and prions.

[0084] As used herein, the term “protein fibrils” means proteins that have aggregated and folded into long, fibrous structures, such as prion fibrils.

[0085] As used herein, the term “whey protein isolate” is a purified form of whey protein that contains 90% or more protein with minimal amounts of lactose, fat, and carbohydrates.

[0086] In the context of the present invention, the words “comprise”, “comprising” and the like are to be construed in their inclusive, as opposed to their exclusive, sense, that is in the sense of “including, but not limited to”.

[0087] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0088] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term ‘about’.

[0089] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).PREFERRED EMBODIMENT OF THE INVENTION

[0090] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.

[0091] The present invention relates to the use of mesh as a substrate for proteins. The mesh should have low background fluorescence, bind a sufficient load of protein, and be made of a material that has similar properties / composition of objects to be decontaminated (e.g., surgical or dental instruments).

[0092] The present invention also relates to the use of protein fibrils bound to surgical steel mesh, wherein the protein fibrils mimic the binding and resistance to decontamination of pathogenic proteins (e.g., prions). To avoid cross-contamination, protein fibrils that are not pathogenic in humans may be used. Protein fibrils may be labelled with fluorescent dyes and imaged by taking overlapping images of the mesh with a laser, with fluorescence bleaching indicating the quantity of proteins remaining on the mesh after decontamination. The decontamination indicator may also include Radio Frequency Identification (RFID) traceability for digital tracking and logging each load.

[0093] Mesh coated with fluorescence-labelled protein fibrils is (or forms part of) a contamination indicator. As protein fibrils (such as prions) strongly bind to surfaces (including metal), they are resistant to sterilization / decontamination methods which are effective against other pathogens (e.g., autoclaving or treatment with chemical disinfectants). Accordingly, protein fibrils are well suited for monitoring sterilization / decontamination, particularly for protein contaminants.

[0094] The present invention is further described by the following non-limiting examples.EXAMPLESExample 1 : Production of recombinant a-synuclein

[0095] WT human alpha synuclein10 20 30 40 50MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH 60 70 80 90 100GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL 110 120 130 140GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 1)

[0096] WT mouse alpha synuclein10 20 30 40 50MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH60 70 80 90 100GVTTVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGNIAA ATGFVKKDQM 110 120 130 140GKGEEGYPQE GILEDMPVDP GSEAYEMPSE EGYQDYEPEA (SEQ ID NO: 2)

[0097] Modified human alpha synuclein10 20 30 40 50MDVFMKGLCK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH60 70 80 90 100GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL 110 120 130 140GKNEEGAPQE GILEDMPVDP CNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 3)

[0098] To produce recombinant WT human alpha synuclein (a-syn), recombinant WT mouse a-syn and recombinant modified human a-syn, the plasmid pT7-7 (Addgene 36046) coding for SEQ ID NO: 1 , SEQ ID NO: 2 or SEQ ID NO: 3 was transformed into E. coli BL21 Rosetta (DE3, pLysS RARE) for expression in Luria-Bertani medium containing ampicillin (100 pg / mL) and chloramphenicol (34 pg / mL) at 37°C. Protein expression was induced with IPTG (1 mM) at an optical density (600 nm) of 0.5 and allowed to proceed at 18°C for 16hours with shaking. Cells were harvested with centrifugation, resuspended in cold lysis buffer (25 mM Tris, pH 8, 0.02% w / v NaN3, Complete protease inhibitor, Roche, 04693132001) and lysed using the CF cell disruptor (Constant Systems Ltd) at 20 kPSI. Lysate was supplemented with EDTA (10 mM) and incubated at 90°C for 20 min to precipitate bacterial proteins. Lysate was clarified by centrifugation (Thermo Fisher Scientific, SS-34 rotor, 19000 rpm, 30 min, 4°C). The supernatant was retained and supplemented with streptomycin sulfate (10 mg / mL, Sigma Aldrich, S6501-25G), stirred for 20 min at 4°C and then centrifuged at 19000 rpm (SS-34 rotor, 20 min, 4°C) to recover the clarified supernatant. This incubation and centrifugation steps were repeated with 20 mg / mL and 30 mg / mL of streptomycin sulfate. The clarified supernatant was then incubated with ammonium sulfate (0.4 mg / mL) for 30 min at 4°C and centrifuged at 13600 rpm, 20 min, 4°C (SS-34). The pellet was resolubilised in buffer A (20 mM Tris, pH 7.7, 0.02% w / v NaN3) and dialysed overnight at 4°C (Thermo Fisher Scientific, 68700). Dialysed solution was filtered (0.22 pm) and further purified by anion exchange chromatography using 2 HiTrap Capto Q ImpRes columns (Cytiva, 17547055). The column was equilibrated with buffer A before injecting the sample at 1 mL / min. a-syn eluted at approximately 175 mM NaCI using a linear 300 mL gradient from 0 to 1 M NaCI in buffer A (3 mL / min). Fractions containing a-syn were identified using reducing SDS-PAGE, combined, and concentrated using Amicon Ultra 15 filters (Merck, UFC901024) for size exclusion chromatography. Elution was performed using a HiLoad 16 / 600 Superdex 200 column (GE Healthcare, 28989335) equilibrated with buffer A at 1 mL / min. Purity was assessed using reducing SDS-PAGE (Figure 1A & 1 B) and samples were concentrated to > 343 pM (4.9 mg / mL). Protein concentration was determined spectroscopically at 280 nm absorbance with an extinction coefficient of 5960 M'1cnr1(Figure 1C & 1 D). Purified a-syn was aliquoted, flash frozen with liquid nitrogen and stored at -80°C. The yield was 4.2 mg / g of cell mass.Example 2: Formation of a-synuclein fibrils

[0099] To confirm the ability of recombinant human a-syn to aggregate to form fibrils, a solution of monomeric recombinant human WT a-syn (208 pM) was incubated in PBS at 45°C with shaking (500 rpm) for 72 h in the presence of a mini-stirrer. The fibrils were imaged by electron microscopy (Figure 2A, left panel). To form fibril fragments that are suitable, the fibril solution was sonicated for 15 min at 12 h then every 24 h at room temperature in a water bath (Ultrasonics, FXP 14M). The fibril fragments were also imaged by electron microscopy (Figure 2A, right panel). The ability of fibril fragments to re-aggregate to form fibrils in the presence of monomeric recombinant WT human a-syn was demonstrated by analysis of fluorescence peaks using Thioflavin (Figure 2B), with anincrease in residence showing an increase in size (Figure 2C). Samples were incubated with 30 micromolar of monomeric WT human a-Syn in PBS in the presence of 10 micromolar Thioflavin T (ThT). The fluorescence of ThT is very weak in solution but is increased significantly upon binding to the core of fibrils. The large number of binding sites along the fibrils enables the detection of fibrils with extremely high signal-to-noise ratio. In these experiments, single molecule confocal imaging (Zeiss Axio Inverted microscope equipped with single photon counters from Micro Photon Devices Sri) was used to detect fibrils in solution. A UV laser (450nm) was focused in the solution, creating a very small excitation volume (typically 1 femtoliter), and the fluorescence is measured as a high-resolution time trace. In the absence of fibrils, unbound ThT molecules are detected as a constant, weak background. When a ThT-positive fibril enters the detection volume, a peak in fluorescence is observed. The maximal intensity (or prominence) of the peak is measured as a proxy for number of ThT molecules bound, and the diffusion time (residence time in the focal volume) is measured as a proxy for physical size of the fibril. The comparison of scatter plots of Intensity vs diffusion times, before and after incubation in monomeric synuclein, shows that sonicated fibrils can propagate as they increase in intensity or physical size.Example 3: Labelling a-synuclein fibrils

[0100] Fibril fragments of recombinant WT human a-syn were labelled with Alexa488 dyes at the lysine positions (a-syn-Alexa488) by suspending in 100mM phosphate buffer pH 8.3, incubating with NHS ester Alexa488 in 5x molar excess, then washing 10x by spinning and resuspending the pellet in PBS.

[0101] Fibril fragments of modified human a-syn were labelled at cysteine positions 9 and 121 with Alexa647 and tetramethylrhodamine (TMR) (a-syn-TMR-Alexa647) by suspending in 50mM Tris buffer pH 7.25, incubating with maleimide dyes (TMR and Alexa647) in 5x molar excess, then washing 10x by spinning and resuspending the pellet in PBS.Example 4: Loading of mesh with fluorescent-labelled a-synuclein fibrils

[0102] Stainless steel mesh (wire diameter 0.03mm and opening size 0.025mm, Edcon Steel) was cleaned extensively before use. The cleaning consisted of 3x successive washing in propan-1-ol in a sonicator bath (3x 5min), followed by 1 rinse with pure water. The mesh was then transferred into a 2% SDS solution, and sonicated for 15 min. The mesh was washed with pure water 10 times and placed in a clean petri dish to dry for 15 min at 70°C.

[0103] Cleaned mesh was incubated in fibril fragments (a-syn-Alexa488 or a-syn-TMR- Alexa647) for 10min under gentle shaking (300rpm). After 10min, the contaminated meshwas transferred to a clean tube, and washed 5x with pure water. The mesh was spun at 100rpm to remove any water and left to dry in the Eppendorf tube for 10min at 37°C.Example 5: Decontamination experiments with fluorescent-labelled a-synuclein fibrils

[0104] 4 control samples (cleaned and non-contaminated), 4 samples contaminated with a-syn-Alexa488 and a-syn-TMR-Alexa647 and 4 samples contaminated with a-syn-TMR- Alexa647 were imaged on a Biorad multi-fluorescence reader. The spectrum and intensity of fluorescence were collected with different exposure times (0.1s, 1s, 10s and 100s). For each exposure, three configurations were acquired, with blue excitation, green and red excitation, and acquisition in the channels corresponding to Alexa488, TMR and Alexa647. As shown in Figure 3, prion contamination on the mesh was measured by quantifying Alexa488 fluorescence intensity.

[0105] After imaging, the contaminated mesh and controls were treated with Autozyme PR Prion Deactivator (Ecolab), following the dilution recommended by the manufacturer.

[0106] Two samples contaminated with a-syn-Alexa488, 2 samples contaminated with a-syn-TMR-Alexa647 and 2 control samples were placed in 1.5 mL Eppendorf tubes with 1mL Autozyme solution. The tubes were incubated at 60°C using an Eppendorf Thermomixer for 10min.

[0107] Two samples contaminated with a-syn-Alexa488, 2 samples contaminated with a-syn-TMR-Alexa647 and 2 control samples were placed in a tunnel washer (Miele) with Autozyme solution. The rapid washing cycle was started at 60°C and interrupted after 10min of washing.

[0108] After treatment, the samples were collected and dried in clean Eppendorf tubes for 15min at 60°C and imaged on the Biorad multi-fluorescence reader using the same settings as before. As shown in Figure 4, samples contaminated with a-syn-Alexa488 that were incubated at 60°C using an Eppendorf Thermomixer in Autozyme solution appear entirely black, with no detectable residual fluorescence, shown in the plot profile across the surface.

[0109] The experiments show that Autozyme successfully removed a-syn-Alexa488 and a-syn-TMR-Alexa647 (Figure 5). The decontaminated mesh shows even slightly lower background than the controls, which had been thoroughly washed in propan-1 -ol, SDS and water. The efficacy of the Autozyme in the washer (red bars) is comparable, if not better, than the efficiency found in static conditions. This may be due to constant agitation in the washers renewing the enzymes at the surface of the mesh. In all cases, no residual prions on the mesh were detected after treatment.Example 6: Development of a prion surrogate

[0110] The protocols used to make a-syn fibrils in Example 2 were used to create whey protein fibrils. Fibrils were generated in different buffer conditions, at low temperatures (37°C) and the fibrils produced characterized.

[0111] To determine the viability of whey protein isolate (WPI) for growing fibrils, a 12% Bis-Tris gel was conducted with the sample to determine purity and confirm that p- lactoglobulin (16kDA) was present. The gel confirmed that the protein was present at both 1mg / ml and 5mg / ml concentrations of WPI dissolved in 40mM of Dithiothreitol (DTT) w / 0.04% NaN3(Figure 6). 5mg / ml WPI was selected for further experiments based on the abundance of protein revealed from the gel results.

[0112] 5 buffers were selected to determine which would be optimal for WPI fibril growth. The buffers used and their respective compositions are as follows; Ultra-High KCI (100mM Tris pH 7.5 + 1M KCI), PBS 2x (5.36mM KCI + 2.94mM KH2PO4+ 274mM NaCI + 16.2mM Na2HPO4), Artificial Cerebral Spinal Fluid (aCSF) (120mM NaCI + 0.01% NaN3+2.5mM KCI + 1mM NaH2PO4+ 10mM Glucose + 1.3mM MgCI2+2.7mM NaHCO3), Caughey (80mM NaPi + 340mM NaCI + 0.003% SDS + 0.04% NaN3, and a Low Salt pH 2.0 buffer (10mM Tris + 0.02% NaN3).

[0113] The samples were shaken at 1000rpm at 37°C for 7 days before being sonicated in a water bath for further growth results and snap frozen after ~10 days of incubation (3 days post-sonication) in -80°C. To achieve the correct concentration of fibrils, 5mg of WPI was weighed and dissolved in 500|JL of DTT w / 0.04% NaN3. From this solution, 500|JL of each respective buffer was added making a final volume of 1ml with a final WPI concentration of 5mg / ml. A 10mg / ml solution was made in a similar fashion however it was determined that 5mg / ml would allow for the best fibril growth results after data analysis of the monitored fibrils. The fibrils were then periodically monitored via single molecule fluorescent spectroscopy (Zeiss Axio Inverted microscope equipped with single photon counters from Micro Photon Devices Sri) throughout their 10-day growth period.

[0114] In comparison, the 5mg / ml WPI fibrils in aCSF buffer strikingly mimicked the recombinant WT human a-Synuclein (aSyn fibrils) after 7 days of growth in the same conditions. To monitor this growth in both the WPI and aSyn fibrils, 10pM of Thioflavin T (ThT) was used as a dye to fluoresce the fibrils giving the response observed from the spectroscopy measurements. There is an observable “clumping” effect in the acidic low salt condition of the WPI fibrils whereas the aCSF produce independent, single peak numbers (Figure 7) that mimic the aSyn fibrils (Figure 8).Example 7: Labelling WPI fibrils

[0115] WPI fibrils grown in aCSF at 5mg / ml were labelled with Cyanine 5.5 (Cy5.5) dye. To make the fluorescent-labelled fibrils, 2mg of Cy5.5 was dissolved in 200|JL of DMSO. From here, the Cy5.5 / DMSO composition was combined with 2ml of 2x PBS and 2ml of H2O. The Cy 5.5 dye was then purified via centrifugation (~4000rpm, 10 minutes per cycle) until flow through was clear. 50|JM Tris no salt pH 8.0 was used as the wash buffer.

[0116] Experiments were conducted with 90% of 5mg / ml unlabelled WPI fibrils and 10% of 5mg / ml WPI fibrils labelled with the Cy 5.5 dye. To achieve the appropriate concentration, a stock solution of 800|JL of 12.5mg / ml of Cy 5.5 (50mg / 4ml) was added to 200|JL 40mM DTT w / 0.04% NaN3to achieve the correct 10mg / ml for the initial step. For the unlabelled portion, 10mg of WPI was dissolved in 1ml of 40mM DTT w / 0.04% NaN3. To then achieve the “90:10” unlabelled to labelled ratio, 450|JL of unlabelled WPI was combined with 50|JL of fluorescent-labelled WPI, followed by then adding 500|JL of aCSF buffer (5mg / ml). Fibrils were shaken at 1000rpm and incubated at 37°C for 6 days when fibril patterns were deemed appropriately sized for storage. Growth patterns were recorded, and samples were stored at -80°C after 6 days of growth. The overall peak number and intensities of the fluorescent- labelled fibrils increased as incubation progresses. Noticeable peak growth is observed from the raw single fluorescent spectroscopy traces with similar peak profiles as the previously discussed recombinant aSyn from patient sample (Figure 9).Example 8: Urea resistance of fluorescent-labelled WPI fibrils

[0117] Urea resistance testing was conducted on fluorescent-labelled WPI fibrils and recombinant WT human aSyn fibrils. With single fluorescent spectroscopy of fibrils, total peak intensity should be considered conjunctively with the peak number. While there were differences between the fibril types (i.e. , fluorescent-labelled WPI fibrils vs aSyn fibrils) at low urea concentrations, the peak number graphs aligned closely at higher concentrations of urea (Figure 10). This shows that the fibril types have similar urea resistance (i.e., resistance to chemical denaturation) and shows that the structural stability of the fibril core is similar between WPI fibrils and a-Syn fibrils. Accordingly, fluorescent-labelled WPI fibrils are suitable for use as prion surrogates.Example 9: Decontamination experiments with fluorescent-labelled WPI fibrils

[0118] Various concentrations of the fluorescent-labelled 5mg / ml WPI fibrils were loaded onto surgical grade mesh of different opening sizes (wire diameter 0.03mm and opening size 0.034 mm, wire diameter 0.03mm and opening size 0.043mm, wire diameter 0.04mm and opening size 0.062 mm, Edcon Steel). Fibril concentrations were determined in a logarithmicfashion (5mg / ml, 1.66mg / ml, 0.555mg / ml, 0.185mg / ml, 0.0617mg / ml) and a 20pL drop of the fibril solution was placed on a 17mm x 9mm rectangular section of the meshes. Alternatively, the meshes were dipped in the fibril solution. The meshes were left to dry at room temperature (~23°C) for around 2 hours before being imaged on the BioRad Chemidoc with Cy 5.5 exposure settings. Meshes with opening sizes ranging from 0.034 mm to 0.062 mm gave similar results (Figure 11). Preferred meshes have an opening size between 0.025 mm and 0.1 mm, with an optimal size of about 0.05 mm.

[0119] Larger meshes (wire diameter up to 0.3mm and opening size up to 0.6mm) were also tested by dipping them in a solution of fluorescent-labelled 5mg / ml WPI fibrils in aCSF. Imaging of the mesh was done by exciting with a blue light (450-490nm) and collecting with a Cy5.5 filter (to image the WPI fibrils). Fluorescent images of a mesh with a wire diameter of 0.19mm and an opening size of 0.318mm is shown in Figure 12 and demonstrates that good signal to noise ratios were produced for the fluorescent-labelled WPI fibrils.

[0120] To determine the best starting concentration, either "drop" or "dip" methods were performed on mesh with 0.043 mm size. The drop method consists of depositing the fluorescent of Cy5.5-labelled WPI sample in the middle of the cut mesh, before drying. The drip method consists of immersing part of the mesh in the tube containing the fluorescent Cy5.5-labelled WPI sample, then drying the mesh after removing the excess of solution. The meshes were then imaged on the BioRad system using the Cy5 setting, and constant exposure for all samples.

[0121] Figure 13 shows that a minimal concentration of 0.25mg / mL is required to obtain sufficient fluorescent signal. To maximise sensitivity of the system, a concentration 10x above this value was selected, i.e., deposit at 2.5mg / mL (2.5g per Liter).

[0122] This minimal value of 0.25mg / mL was confirmed with further quantitative analysis showing that the profile of fluorescence reveals inhomogeneities below 0.1mg / mL (Figure 14).Example 10: Decontamination experiments with fluorescent-labelled WPI fibrils

[0123] Cy5.5-labelled WPI fibrils coated mesh (drop or dip method) was submerged in 1ml of a 1 in 100 dilution of Autozyme PR in individual tubes and incubated at 60°C for 30 minutes shaking at 900rpm. Untreated control mesh and Autozyme treated mesh were then imaged together on the ChemiDoc Cy 5 setting (Figure 15).

[0124] Cy5.5-labelled WPI fibrils coated mesh (drop method, size 0.043 mm) was submerged in 1 ml of a 1 in 100 dilution of Autozyme PR in individual tubes and incubated at 60°C for 30 minutes shaking at 900rpm. Untreated control mesh and Autozyme treated meshwere then imaged together on the ChemiDoc Cy 5 setting. The fluorescence across the mesh is measured, and the profile demonstrates the presence or absence of residual signal. In all cases, even for the highest loading of the mesh, the fluorescence is completely removed by Autozyme treatment (Figure 16).

Claims

CLAIMS1. A contamination indicator comprising mesh coated with fluorescence-labelled protein fibrils.

2. The contamination indicator according to claim 1, wherein the mesh is wire mesh.

3. The contamination indicator according to claim 2, wherein the wire mesh has an opening size of 0.025 mm to 0.1 mm.

4. The contamination indicator according to claim 2 or claim 3, wherein the wire mesh has a wire diameter of 0.02 mm to 0.05 mm.

5. The contamination indicator according to any one of claims 2 to 4, wherein the wire mesh is formed from stainless steel.

6. The contamination indicator according to claim 5, wherein the stainless steel is surgical steel.

7. The contamination indicator according to any one of claims 1 to 6, wherein the protein fibrils are prion fibrils.

8. The contamination indicator according to claim 7, wherein the prion fibrils are alpha synuclein fibrils.

9. The contamination indicator according to claim 8, wherein the alpha synuclein fibrils are human alpha synuclein fibrils.

10. The contamination indicator according to claim 8, wherein the alpha synuclein fibrils are mouse alpha synuclein fibrils.

11. The contamination indicator according to claim 8, wherein the alpha synuclein has the amino acid sequence of any one of SEQ ID Nos 1 to 3.

12. The contamination indicator according to any one of claims 1 to 6, wherein the protein fibrils are a non-pathogenic protein fibrils.

13. The contamination indicator according to any one of claims 1 to 6 or 12, wherein the protein fibrils are whey protein fibrils.

14. The contamination indicator according to claim 13, wherein the whey protein fibrils are formed in artificial cerebrospinal fluid.

15. The contamination indicator according to any one of claims 1 to 14, wherein the fluorescent label is selected from the group consisting of Alexa Fluor 488, Alexa Fluor 647, tetramethylrhodamine and Cyanine 5.5.

16. The contamination indicator according to any one of claims 1 to 15, wherein the indicator is a protein contamination indicator.

17. A method for monitoring decontamination of an object, comprising positioning the contamination indicator according to any one of claims 1 to 16 near the object during decontamination and then measuring the amount of fluorescence on the indicator.

18. The method according to claim 17, wherein the object is a surgical or dental instrument.

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