Functionalised Nanoparticle

Abstract

A functionalised nanoparticle that is at least in part coated by a polymer, wherein the polymer comprises charged and uncharged groups at a ratio ranging from 4:1 to 1:4 and the functionalised nanoparticle is conjugatable or can be functionalised to conjugate with a biomolecule.

Claims

1. A functionalised nanoparticle that is at least in part coated by a polymer, wherein the polymer comprises charged and uncharged groups at a ratio ranging from 4:1 to 1:4 and the functionalised nanoparticle is conjugatable or can be functionalised to conjugate with a biomolecule.

2. The functionalised nanoparticle according to claim 1, wherein the charged groups are negatively charged.

3. The functionalised nanoparticle according to claim 1, wherein at least one of the charged groups is a maleate group.

4. The functionalised nanoparticle according to claim 1, wherein at least one of the uncharged groups is a sulfonate group.

5. The functionalised nanoparticle according to claim 1, wherein the nanoparticle is a quantum dot.

6. The functionalised nanoparticle according to claim 1, wherein the nanoparticle is bonded to a linker that is conjugatable with a biomolecule.

7. A method of producing a functionalised nanoparticle comprising: providing a nanoparticle; providing a polymer comprising charged and uncharged groups at a ratio ranging from 4:1 to 1:4; and mixing the nanoparticle and the polymer to form the functionalised nanoparticle that is at least in part coated by the polymer and that is conjugatable or can be functionalised to conjugate to a biomolecule.

8. The method according to claim 7, including reacting the nanoparticle with the polymer to form the functionalised nanoparticle.

9. The method according to claim 7, including functionalising the polymer to be conjugatable with a biomolecule.

10. The method according to claim 7, including reacting the functionalised nanoparticle with a linker to form a nanoparticle-linker conjugate that is connectable to a biomolecule.

11. The method according to claim 7, including reacting the polymer with a linker to form a polymer-linker conjugate that is connectable to a biomolecule.

12. A nanoparticle-biomolecule conjugate comprising a functionalised nanoparticle according to claim 1 that is conjugated to a biomolecule, wherein the biomolecule can be bonded to a target analyte.

13. A method of producing a nanoparticle-biomolecule conjugate comprises: providing a nanoparticle; providing a polymer comprising charged and uncharged groups at a ratio ranging from 4:1 to 1:4; mixing the nanoparticle and the polymer to form a functionalised nanoparticle which is at least in part coated by the polymer; and reacting the functionalised nanoparticle with a biomolecule to form the nanoparticle-biomolecule conjugate, wherein the nanoparticle-biomolecule conjugate can be bonded to a target analyte.

14. The method according to claim 13, including reacting the nanoparticle with the polymer to form the functionalised nanoparticle.

15. The method according to claim 13, including reacting the functionalised nanoparticle with a linker to form a nanoparticle-linker conjugate.

16. The method according to claim 15, including reacting the nanoparticle-linker conjugate with a biomolecule to form the polymer-biomolecule conjugate.

17. The method according to claim 13, including reacting a biomolecule with a linker to form a biomolecule-linker conjugate.

18. The method according to claim 17, including reacting the biomolecule-linker conjugate with the functionalised nanoparticle to form the polymer-biomolecule conjugate.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0086] FIG. 1 illustrates a nanoparticle-biomolecule conjugate according to an embodiment of the present invention.

[0087] FIG. 2 illustrates the complexation of the nanoparticle-biomolecule conjugate of FIG. 1 with an antigen.

[0088] FIG. 3 is a flow diagram illustrating methods of producing a coated nanoparticle and a nanoparticle-biomolecule conjugate according to the present invention.

DETAILED DESCRIPTION

[0089] The present invention relates to a nanoparticle-biomolecule conjugate that can be used to detect a disease, and a method of synthesising the conjugate.

[0090] One of the motivations of the present invention is the realization that fluorescence detection is considered to be more sensitive than colourimetric detection because lower concentrations of fluorescent materials can be detected compared to coloured material. In some instances, it may be possible with the right instrument to detect a single QD or dye.

[0091] However, to achieve such sensitive detection, background signals should be minimized. The present invention achieves this by providing the polymeric coating, which minimises non-specific adsorption of the nanoparticles (e.g., QDs).

[0092] This enables the present invention to detect a disease with a combination of fluorescence detection and low non-specific adsorption.

[0093] QDs fluoresce brighter than other fluorescent molecules such as dyes.

[0094] Brightness is defined as the product of the molar absorption coefficient multiplied by fluorescence quantum yield.

[0095] Molar absorption coefficient for QDs ranges from 100,000-1,000,000 M.sup.−1 cm.sup.−1, while the coefficient for dyes ranges from 25,000-250,000 M.sup.−1 cm.sup.−1.

[0096] Quantum yield for dyes can range from 1% to over 90%, depending on the dye. Quantum yield for QDs can be up to 100%, but typically drops to 20% when the particles are dispersed into water.

[0097] One advantage of the present invention is that the quantum yield of the QD can be retained at about 80% in water.

[0098] Use of a QD in the functionalized nanoparticle may provide up to 4 times the brightness of dyes.

[0099] The high signal to analyte ratio allows detection of low concentrations of a target analyte such as a disease-specific antigen. As a result, the use of QDs as the nanoparticle can facilitate early diagnosis and treatment.

[0100] However, the use of QDs is not straightforward. One challenge faced by the applicant is the conjugation between QDs and antibodies, for example in their application with LFA, enzyme-linked immunosorbent assay (ELISA) or other methods. This is because different reaction conditions often have to be established for each antibody, resulting in increased costs. Often, these reaction conditions involve high temperatures and/or pressures, and extreme pH conditions.

[0101] The present invention provides a functionalized nanoparticle that can be conjugated with a range of antibodies using mild reaction conditions to form a nanoparticle-biomolecule conjugate.

[0102] Reaction temperatures may range from 4-30° C. Suitably, the reaction temperature ranges from 15-25° C.

[0103] Reaction pressures may range from 1-1.5 atm. Suitably, the reaction pressure is at 1 atm.

[0104] Reaction pH may range from 7 to 9. Suitably, the reaction pH ranges from 7 to 8. More suitably, the reaction pH ranges from 7 to 7.5.

[0105] The biomolecule concentration may range from 100 μM-1 nM.

[0106] The nanoparticle-biomolecule conjugate comprises a nanoparticle that has an outer surface that is functionalized with a biomolecule, suitably an antibody, that can complex with a target analyte such as an antigen. However, it can be appreciated that the biomolecule can be any other type of protein.

[0107] With reference to FIG. 1, the nanoparticle-biomolecule conjugate 100 comprises a nanoparticle 12 in the form of a QD, suitably a CdSe/ZnS core shell QD.

[0108] It is believed that the QD is bonded to the polymer 14 via a combination of interaction between the —SH functional group on the polymer with the surface of the QD, and interaction between the aromatic ring and other portions of the polymer with the surface of the QD.

[0109] The polymer 14 that has a 1:1 ratio of negatively charged sulfonate groups (Group 4 molecules in the polymer examples below) and uncharged maleate groups to form a functionalised nanoparticle 10. Examples of suitable polymers (P1 and P2) are illustrated below:

##STR00002##

[0110] The polymer 14 has pendant thiol and azide groups (Group 1 molecules in the examples above). In some embodiments, the polymer may have pendant carboxyl and/or hydroxyl groups. These functional groups enable conjugation with a biomolecule.

[0111] Each polymer 14 also includes pendant polyethyleneglycol (PEG) chains having M.sub.ws of 600 and 750 for P1 and 600 for P2 (Group 6 molecules in the examples).

[0112] The pendant azide group on the polymer-coated nanoparticle can react with alkyne groups on a di-functional linker 16 to form a functionalized nanoparticle that can conjugate with an antibody 18.

[0113] The synthesized polymer 1 stabilizes the QD in water, provides the QD with a high quantum yield, low non-specific adsorption and allows the functionalised QD to be modified with a variety of functional groups for conjugation with a biomolecule.

[0114] The linker 16 may react with the polymer to form a polymer-linker conjugate that can react with an antibody 18 to form the nanoparticle-antibody conjugate 10.

[0115] Alternatively, the linker 16 is reacted with an antibody 18 to form a linker-antibody conjugate that can react with a polymer-coated nanoparticle to form the nanoparticle-antibody conjugate 10.

[0116] This provides an antibody-covered QD 10 can complex with a suitable antigen 20 which enables detection of a relevant pathogen (see FIG. 2).

[0117] In the example illustrated in FIG. 3, a nanoparticle-biomolecule conjugate 10 is formed by taking the following steps: [0118] 1. Separately synthesising/obtaining a nanoparticle 12 (e.g., a CdSe/ZnS core shell QD) and a polymer 14. [0119] 2. Reacting the nanoparticle 12 and polymer 14 to form a functionalised nanoparticle 10 in which the polymer 14 is bonded to the nanoparticle 12 and coats the outer surface of the nanoparticle 12. [0120] 3. The functionalised nanoparticle 10 is added to a buffer solution, preferably a BBS solution. [0121] 4. The functionalised nanoparticle 10 in solution can be stored for later use or processed to form the nanoparticle-biomolecule conjugate 100. [0122] 5. To form the nanoparticle-biomolecule conjugate 100, the functionalised nanoparticle 10 is purified by spin filtering. Thereafter, there are three synthetic pathways that can be taken to form the nanoparticle-biomolecule conjugate 100. [0123] 6. In pathway 1, the functionalised nanoparticle 10 is reacted with a linker 16 to form a nanoparticle-linker conjugate. The nanoparticle-linker conjugate is then reacted with a biomolecule 18, which in this example is an antibody, to form the nanoparticle-biomolecule conjugate 100. [0124] 7. In pathway 2, a biomolecule 18 such as an antibody is reacted with a linker 16 to form a biomolecule-linker conjugate. The biomolecule-linker conjugate is then reacted with the functionalised nanoparticle 10 to form the nanoparticle-biomolecule conjugate 100. [0125] 8. In pathway 3, the functionalised nanoparticle 10 is reacted directly with a biomolecule 18 to form the nanoparticle-biomolecule conjugate 100. [0126] 9. The nanoparticle-biomolecule conjugate 100 is stored in a buffer solution for future use.

[0127] In order to detect a target analyte, a solution of anti-ECM (MM01) antibody in PBS is spotted onto a test strip and dried.

[0128] A solution of ECM in QDs-anti-ECM-antibody in a washing buffer is then prepared. The ECM solution is applied to the antibody-loaded strip and dried. The dried strip is washed with wash buffer and dried again before being checked for fluorescence under UV light.

EXAMPLE

Polymer Synthesis

1. α,ω-Dichloro-PEG600

[0129] Poly(ethylene glycol) (M.sub.w 600, Sigma-Aldrich, 60.0 g) was dried by heating under vacuum for 1 h in an oil bath at 80-90° C. with constant stirring, then cooled to ambient temperature. A solution of thionyl chloride (22 mL, 36 g, 300 mmol) in diethyl ether (38 mL) was added using a dropping funnel over 50 min. The mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure with heating at 70° C., and the residue was diluted with CH.sub.2Cl.sub.2 (40 mL) and re-evaporated under reduced pressure with heating at 70° C. Nuclear magnetic resonance (NMR) was used to characterise the polymer with parameters as follows: 1H NMR δ 3.74-3.77 (4H, t, CH.sub.2Cl), 3.62-3.70 (45H, m, CH.sub.2O), 2.62 (0.24H, m, OH). IR ν (cm.sup.−1) 2866 (C—H), 1097 (ether), 743 (C—Cl); no discernible —OH above 3100.

2. α,ω-bisazido-PEG600

[0130] Crude α,ω-dichloro-PEG600 (41.5 g, 65 mmol) was dissolved in DMF (Dimethylformamide) (330 g). KHCO.sub.3 (0.88 g) and NaN.sub.3 (12.7 g, 196 mmol) were added. The mixture was stirred and heated at 80-90° C. under nitrogen for 20 h. DMF was evaporated under vacuum and dichloromethane (275 mL) was added. Inorganic solids were removed by filtration and the solvent evaporated under reduced pressure at 50° C., yielding a yellow liquid (47.3 g, 87% pure as assessed by 1H NMR). Nuclear magnetic resonance (NMR) was used to characterise the polymer with parameters as follows: 1H NMR δ 3.37-3.40 (4H, t, CH.sub.2N.sub.3), 3.65-3.69 (49H, m, CH.sub.2O). Sample contains 1.3 mol of DMF per mol of bisazide (0.15 g/g).

3. α-Amino-ω-azido-PEG600(H2N-PEG-N3)

[0131] Crude α,ω-bisazido-PEG600 (47.2 g, 87% pure) was dissolved in 1 M HCl (184 mL) in a 1 L flask and cooled to 0° C. Toluene (380 mL) was added and the mixture stirred vigorously under a stream of nitrogen. A solution of triphenylphosphine (18.2 g, 69.6 mmol) in toluene (200 mL) was added using a dropping funnel over 2 h. The mixture was allowed to warm to room temperature and was stirred under nitrogen overnight. The aqueous layer was separated, washed with toluene (3×100 mL) and then chilled on ice. Solid KOH (90 g) was added to the aqueous phase, which was then extracted with CH.sub.2Cl.sub.2 (3×75 mL). The extract was dried (MgSO.sub.4), filtered and the solvent evaporated, yielding 37 g of a light yellow oil. Nuclear magnetic resonance (NMR) was used to characterise the polymer with parameters as follows: 1H NMR δ 3.37-3.41 (2H, t, CH.sub.2N.sub.3), 3.62-3.69 (45H, m, CH.sub.2O), 3.52-3.55 (2H, t, CH.sub.2CH.sub.2NH.sub.2), 2.86-2.89 (2H, t, CH.sub.2NH.sub.2), 2.17-2.21 (2H, m, NH.sub.2). IR ν (cm-1) 3366 (NH.sub.2, weak), 2100 (N.sub.3), 1591 (amine, weak).

4. Polymer Backbone

[0132] ##STR00003##

[0133] DoPAT (2-(Dodecylthiocarbonothioylthio)propionic acid) (0.35 g, 1 mmol) and AIBN (80 mg, 0.5 mmol) and maleic anhydride (3.675 g, 37.5 mmol) was dissolved in 1,4 dioxane (10 mL). A second solution of styrenesulfonate (2.575 g, 12.5 mmol) in water (10 mL) was prepared. The two solutions were mixed together and degassed three times. The reaction was heated at 70° C. for overnight. The solution was precipitated from acetone and centrifuged 3 times.

5. 10th Generation (10th G) Polymer

[0134] ##STR00004##

[0135] Polymer backbone of item 4 (180 mg, 0.92 mmol), cysteamine (17.9 mg, 0.148 mol (1/6)), CH.sub.3-PEG-OH (M.sub.w 750 g/mol, 666.1 mg, 0.888 mmol) and NH.sub.2-PEG-N.sub.3 (M.sub.w 600 g/mol, 88.8 mg, 0.148 mmol) were added into water (3 mL). Another solution was prepared by dissolving EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, 568.4 mg, 2.96 mmol) and NHS (hydroxysuccinimide, 340.46 mg, 2.96 mmol) in water (10 mL). The above described two solutions were mixed together overnight. The resulting polymer was purified by spin filter three times (50K size, 6000 g, 10 mins).

6. 11th-Generation (11th G) Polymer

##STR00005##

[0136] Polymer backbone of item 4 (180 mg, 0.92 mmol), cysteamine (17.9 mg, 0.148 mol (1/6)), O-(2-Aminopropyl)-O′-(2-methoxyethyl)polypropylene glycol (M.sub.w 600 g/mol, 640.8 mg, 0.888 mmol) and NH.sub.2-PEG-N.sub.3 (M.sub.w 600 g/mol, 88.8 mg, 0.148 mmol) were added into water (3 mL). Another solution was prepared by dissolving EDC (1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, 568.4 mg, 2.96 mmol) and NHS (hydroxysuccinimide, 340.46 mg, 2.96 mmol) in water (10 mL). Two solutions were mixed together overnight. The resulting polymer was purified by spin filter three times (50K size, 6000 g, 10 mins).

QDs MUA (11-Mercaptoundecanoic Acid) Phase Transfer

[0137] The phase transfer protocol described below was applied to different type of QDs solutions. [0138] 1. A QDs solution (in chloroform 3 mL) was precipitated with methanol, centrifuged and redispersed in hexane (9 mL). [0139] 2. A second solution was prepared by dissolving KOH (0.2 g) and MUA (2.78 mmol, 0.78 g) in methanol (9 mL). [0140] 3. Both solutions were mixed in a centrifuged tube and shaken overnight. (two ways, rotating vertically @ 30 rmp; or rotating horizontally @ 50 rmp). [0141] 4. After the phase transfer, the colourless hexane phase was separated from methanol phase. [0142] 5. To separate the QDs, the methanol phase was centrifuged. The precipitated QDs were redispersed in KOH solution (3 mL, 0.1 M). This solution was washed with chloroform.

[0143] Ligand Exchange with 10th G or 11th G Polymer [0144] 1. A QDs solution (in MUA, 400 uL) was added into polymer solution (1 mL, 50 mg/mL). [0145] 2. The solution was mixed overnight (two ways, rotating vertically @30 rpm; or rotating horizontally @50 rpm). [0146] 3. The QDs solution was run a pre-packed desalting column to remove the MUA ligands. Using BBS buffer solution as the eluent. [0147] 4. The QDs solution in BBS buffer was further purified by spin filtering (three times, 6000 rcf, 10 min).

Conjugation Protocol

[0148] The conjugation protocol was carried out over two days as described below.

[0149] DAY 1: Linker+Antibody [0150] 1. Anti-biotin antibody (20 uL, 2 mg/ml, 0.04 mg, 2.67e-10 mol) and linker (2*2.35=4.7e-10 mol, 0.1 mM, 4.71 μL) in BBS buffer (40 uL) was mixed in a 200 μL tube for 18 hours at RT on an orbital shaker (750 rpm). For modification of thiol groups and -amino groups, which occurs selectively at physiological pH (7.0-7.5), phosphate buffers are ideally suited. More strongly basic lysine amines require more alkaline pH, in the range of 8.0-9.5, where phosphate solutions do not buffer well. For these reactions, carbonate/bicarbonate (pH of 100 mM bicarbonate is 9.2) or borate buffers are quite satisfactory. No additional buffer was added. [0151] 2. Quench with 50 mM glycine. [0152] 3. Purify by spin filter (50K size) three times (6000 RCF, 8 min) using BBS. [0153] 4. Collect the concentrated protein in a 200 μL tube, washing the filter once with 2-3 drops of BBS. The volume should be below ˜130 μL, to accommodate the QDs. [0154] 5. Characterise the modification via LC-ESI-TOF.  Note: The reaction can be refrigerated for 2 days instead of 18 hrs at RT.

[0155] DAY 2: Linker-Antibody Conjugate+QDs [0156] 1. The antibody-linker (2.35e-10 mol) from the previous step was added to the QD (2.35 e-10 mol, 0.5 μM, 4.7e-4 L, 470 μL) in BBS buffer, and mixed for 18 hours at RT on an orbital shaker (750 rpm). The initial protocol used 5.88e-11 mol QDs (the ratio antibody:QD was 4:1). [0157] 2. Purify by spin filter (300K size) three times using PBS-T (Invitrogen conditions: 1500 RCF, 30 min; quick: 4000 RCF, 5 min). Gently resuspend the QD after each run of spinfiltration using a 100 μL tip. [0158] 3. Collect the concentrated QDs in a 1.5 mL tube, washing the filter twice with 2 drops of BBS. [0159] 4. Filter the QDs using a small filter, like a 4 mm 0.22 μm Millex syringe filter. A 0.45 μm filter can be used if the solution has a large amount of aggregates. [0160] 5. Store the QDs in the fridge until needed. [0161] 6. Characterise the conjugation via DLS. [0162] 7. Dilute QDs to the desired concentration using the antibody diluent buffer before use.  Note 1: the reaction can be put in the fridge for 2 days instead of 18 hrs at RT.  Note 2: The molar ratio Ab:QD used here is 4:1.

LFA Protocol

[0163] DAY 2: Spot the Capture Antibody [0164] 1. Spot 1 μL of a solution 1 mg/ml of the anti-ECM (MM01) antibody in PBS. Spot near the adsorption pad away from the antibody line. [0165] 2. Immediately after spotting, put the strips in an oven at 37° C. for 2 hours. [0166] 3. Leave the strips to cool down, and close the strips in a tube with some desiccant.

[0167] DAY 3: Virus Detection in Washing Buffer [0168] 1. Filter the QDs to remove aggregates to reduce background noise. [0169] 2. Prepare solutions of ECM in QDs-anti-ECM-antibody (R030) with various concentration from 5 μM to 5 nM (in washing buffer: BBS buffer, Tween20 5%, BSA 5%). [0170] 3. Flow 35 μL of the ECM solution to the strip and wait until dry (10 minutes). [0171] 4. Wash with 50 μL of wash buffer, waiting 10 minutes to dry before each washing. [0172] 5. Check the fluorescence under UV light.

EXPERIMENTAL

[0173] Materials [0174] Linker stock solution=0.1 mM in anhydrous DMSO, divided in single doses of 5 μL each (in 200 μL tubes). Store at −20° C. [0175] QDs solution=QD-azide; 3.sup.rd generation, concentration=0.9 μM. Store at 4° C. [0176] Anti-biotin antibody=polyclonal antibody raised in goat, product #B3640 from Sigma-Aldrich. Store at −20° C. [0177] BSA=bovine serum albumin for immunoassay (protease free, fatty acid free, essentially globulin free), product #A7030 from Sigma-Aldrich. Store at 4° C. [0178] Biotin-NHS=NHS-dPEG®4-biotin, product #QBD10200 from Sigma-Aldrich, dissolved in anhydrous DMSO to a concentration of 1 mM and divided into single doses of 1 μL each in 200 μL tubes. Store at −20° C. [0179] Anti-Hendra polyclonal antibodies=raised in rabbit, about 20 mg/ml, divided into single doses of 1 μL each (in 200 μL tubes). Store at −20° C. [0180] Anti-Hendra monoclonal antibodies=human antibodies, 9.2 mg/ml, divided into single doses of 5 μL each (in 200 μL tubes). Store at −20° C. [0181] Human serum=product #S-2145 from Sigma-Aldrich. Store at −20° C. [0182] Hendra virus=concentrated and gamma-irradiated, stored at −80° C.

Buffers

[0183] Both Commercial and home-made buffers were used as listed below.

Note: “borate buffered”=no NaCl. “Borate buffered saline”=with NaCl. “Normal saline” is solution isotonic to blood that contains 9 g/L NaCl in water (0.154 M). However, in the buffer recipes the NaCl is usually 8 g/L to get the same ionic strength.

[0184] Commercial Buffers [0185] PBS 1×=PBS 10 mM made up by dissolving capsules from ThermoScientific in MilliQ water, as required by manufacturer (https://www.thermofisher.com/order/catalog/product/18912014).

[0186] Home-Made Buffers [0187] Dulbecco PBS (1×)=10 mM, pH-7.4, 8 g/L NaCl [0188] Recipe: 1.15 g/L Na.sub.2HPO.sub.4+0.2 g/L KH.sub.2PO.sub.4+8 g/L NaCl+0.2 g/L KCl. [0189] PBS-T=PBS+0.05% v/v Tween20. Use 0.5% v/v for a stronger washing. [0190] Note: density PBS 1×=1.01 g/ml; density Tween20=1.1 g/ml.fwdarw.0.05% v/v=0.055 w/v=0.055 w/w. [0191] Antibody dilution buffer=PBS-T+5% w/v BSA and 30 mM glycine. For 1 ml of buffer, 50 mg BSA and 22.5 uL of glycine stock solution, brought to 1 ml with PBS-T is required. [0192] Glycine stock solution 1% w/w=100 mg glycine in 1 g of PBS. [0193] MOPSr=MOPS 25 mM, pH 7.6, 0.8 g/L NaCl (r=reduced NaCl) [0194] Recipe: [0195] Tris buffer saline (aka Trizma)=10 mM, pH-8.3, 8 g/L NaCl. [0196] Recipe: [0197] 1.2 g/L Tris+−8 mL of HCl 1M (check the pH)+8 g/L NaCl. [0198] Bring to 1 L with milliQ. [0199] OR [0200] 1.6 g/L Tris-HCl+8 g/L NaCl (a solution of Tris-HCl should have pH-8.3). Bring to 1 L with milliQ. [0201] Borate buffer saline=10 mM, pH-9.4, 8 g/L NaCl. [0202] Recipe: 0.62 g/L boric acid+0.26 g/L NaOH+8 g/L NaCl. [0203] Bring to 1 L with milliQ water. Check the pH. [0204] Note: use 5 mL of a freshly made 1 M solution of KOH if too difficult to weigh the KOH pellets.

[0205] Equipment [0206] 200 μL polypropylene tubes. [0207] “Nanosep” spinfilters from Pall Corporation, 300 kDa Omega membrane (orange). [0208] “Amicon” spinfilters from Merk, 50 kDa membrane, 0.5 ml. This filter reduces the volume from 500 μL to 50 uL. This means that 3 rounds of purification reduce the concentration of unwanted molecules by 1,000-fold. [0209] 4 mm syringe filters “Millex”, 0.22 μm, PVDF (http://www.merckmillipore.com/AU/en/product/Millex-Syringe-Filter-Durapore-PVDF-Non-sterile,MM_NF-SLGVRO4NL). [0210] Lateral flow strips with one control line. The control line is biotinylated mouse IgG1 from eBiosciences (CD4 PSD 038, Cat number: 13-4714-85, https://www.thermofisher.com/antibody/product/Mouse-IgG1-kappa-clone-P3-6-2-8-1-Isotype-Control/13-4714-85).

Methods

Standard Procedures

[0211] The aliquots of proteins were prepared inside a laminar flow cabinet using sterile tips and tubes to avoid bacterial contamination.

Characterisation

[0212] A Biacore assay was used to check that the antibodies (polyclonal, biotinylated monoclonal) are active.

[0213] It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.

[0214] It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

[0215] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.