Compositions and methods for immobilising DFS70 on a substrate

Abstract

A composition for immobilising a biological material on a substrate for an assay, comprises: a biological material, wherein the biological material comprises or consists of a DFS70 protein; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound, such as GSH and / or DTT.

Description

[0001] COMPOSITIONS AND METHODS FOR IMMOBILISING DFS70 ON A SUBSTRATE

[0002] Field

[0003] The present disclosure relates to a composition for printing a biological material, for example DFS70, on a substrate for an assay such as a microarray.

[0004] A microarray is a two-dimensional array of a biological material, such as proteins, DNA, antigens or antibodies, lipids, or peptides, deposited and immobilised on a solid and typically flat substrate, typically made of functionalized glass or plastic. The substrate is generally selected to provide adequate binding with the biological material, but also to avoid any modification of the material, such as denaturation, cleavage or degradation in the case of a protein.

[0005] Typically, once the microarray of biological material has been deposited on the substrate, a blocking reagent is applied to the microarray surface to prevent unwanted materials binding to the surface of the substrate. The microarray is then reacted with a sample, and detection is carried out by suitable detection methods, such as the addition of one or more detection reagents, optionally followed by imaging techniques.. An example of such microarray-based technology used for blood grouping and donor disease screening is MosaiQ™ by AliveDx.

[0006] Deposition of the biological material is typically carried out by printing, for example using a system such as MosaiQ™ Manufacturing System (MMS), Arrayjet’s Inkjet Microarray Technology or Scienion’s sciFLEXARRAYER. However, printing requires the printed solution to have a viscosity within a workable range, typically a dynamic viscosity of at least 1 mPa.s, e.g. at least 2 mPa.s, and / or of about 1-20 mPa.s, e.g about 1-10 mPa.s, e.g. about 2-9 mPa.s, e.g. about 4-9 mPA.s, e.g. about 6-9 mPa.s. To that end, a viscosity modifier is typically included in the printing composition. It will be appreciated, however, that the optimum viscosity for a given printed solution may depend on the type of printing equipment used.

[0007] In order to ensure that the deposited biological material is immobilized on the substrate upon printing, it is desirable for the biological material to interact with, e.g. bind to, the substrate. Factors influencing binding of the biological material to the substrate include: the type of biological material; the type of substrate; - pH; ionic strength; viscosity.

[0008] The pH of the biological material composition is typically controlled using a buffering agent, such as a phosphate, a carbonate or an acetate buffer. The buffering agent may typically be provided at a concentration of about 5-300 mM in the composition.

[0009] The ionic strength of the biological material composition is typically controlled using a salt, such as NaCI. The salt may typically be provided at a concentration between about 0 and 5M. A typical salt concentration may be around 150 mM, as this may correspond to the osmolality of cell cytoplasm.

[0010] The viscosity of the biological material composition is typically controlled using a viscosity modifier, such as sucrose, trehalose, or glycerol. In particular, printing of proteinic molecules typically requires the presence of a viscosity modifier to avoid quick drying of the liquid material deposited on the substrate, which could have a negative impact on the protein integrity. The viscosity modifier may typically be provided at a concentration of about 0.5-60 % w / v or w / w, depending on the type of viscosifier.

[0011] Typical substrates have a coating that includes reactive groups which react, e.g. covalently, with the biological material, in order to promote binding. An example of such a substrate is a substrate, e.g. glass, coated with an epoxy-containing material such as an epoxysilane material. Such substrates as commonly referred to as “epoxy glass”. An advantage of these substrates is that the epoxy groups in the coating may react, typically covalently, with a number of chemical groups that are typically present in many biological materials, such as amino (secondary amines), hydroxyl, or thiol moieties, thereby enabling immobilization upon printing. For example, molecules of protein origin are capable of binding to epoxy groups via positively charged amino acids such as Arginine, Histidine or Lysine.

[0012] However, certain biological materials either do not include free reactive moieties capable of reacting with the reactive groups, e.g. epoxy, in the coating, or have reactive moieties whose reactivity is diminished or inhibited due to the conformation of the biological material. The dense fine speckled protein of 70 kD (DFS70), also known as lens epithelium derived growth factor protein of 75 kD (LEDGF / p75) and PC4 and SFRS1 Interacting protein 1 (PSIP1), is a multi-functional protein which plays important roles in the formation of transcription complexes, transcriptional activation, mRNA splicing regulation , DNA repair, and cell survival against stress. Due to its multiple functions, this protein has emerged as a key contributor to several human pathologies, including acquired immunodeficiency syndrome (AIDS), leukemia, cancer, ocular diseases, Rett syndrome as well as systemic autoimmune rheumatic diseases (SARD). Anti-DFS-70 antibodies in serum are considered as a “negative” biomarker for SARD, when they are detected as isolated (monospecific) autoantibodies and other SARD autoantibodies are not present. Further discussion may be found in the literature, for example: The significance of autoantibodies to DFS70 / LEDGFp75 in health and disease: integrating basic science with clinical understanding, Ochs et al., Clinical and Experimental Medicine, 2016, 16, 273; and Twenty years of research on the DFS70 / LEDGF autoantibody-autoantigen system: many lessons learned but still many questions, Ortiz- Hernandez et al., Autoimmunity Highlights, 2020, 11 , 3..

[0013] However, effective immobilization of the DFS70 protein on a microarray such as on MosaiQ® microarray, was found to be challenging, leading to relatively lower diagnostic sensitivity. In particular, it was observed that the discrimination between anti- DFS70 positive and anti-DFS70 negative samples was relatively poor when using conventional printing buffer compositions. Without wishing to be bound by theory, it is believed that in conventional printing conditions, not all epitopes of DFS70 are presented like in the unbound antigen. For example, some epitopes may not be accessible or may not have the native conformation, which may result in a low diagnostic sensitivity.

[0014] US 2004 / 063220 (Lebrun) discloses a device for immobilization of proteins which includes a hydrophobic polymeric layer attached to a rigid support.

[0015] WO 2009 / 095500 A1 (Debyser et al) discloses peptides having lentiviral replication inhibiting properties.

[0016] Journal of Molecular Biology, 2007, Volume 372, pages 407-421 , (BARTHOLOMEEUSEN et al) discloses differential interaction of HIV-1 integrase and JPO2 with the C-terminus of LEDGF / p75.

[0017] Journal of Molecular Biology, 2011 , Volume 410, pages 811-830, (MCNEELY et al) discloses in vitro DNA tethering of HIV-1 integrase by the transcriptional coactivator LEDGF / p75.

[0018] US 2010 / 261751 A1 (VAN LOOCK et al) discloses a method for determining one of the two Human Immunodeficiency Virus (HIV) integrase enzymatic activities in an in vitro assay.

[0019] It is an object of the present invention to obviate and / or mitigate the limitations and / or disadvantages associated with the prior art and / or with conventional methods and systems. It is an object of the present invention to provide a printing composition, e.g. a printing buffer, that promotes immobilisation of a biological material, e.g. of a DFS70 protein, on a substrate, e.g. functionalised glass, and / or that improves diagnostic sensitivity of a biological material, e.g. of a DFS70 protein, immobilised on a substrate, e.g. functionalised glass.

[0020] As explained above, the binding between a deposited biological material and a substrate depends on a number of factors, including for example the type of biological material, the type of substrate, pH, ionic strength, and viscosity.

[0021] The present invention is based on the surprising findings that the diagnostic sensitivity of a DFS70 protein and a substrate, e.g. a functionalised substrate such as epoxy glass, may be significantly improved by providing one or more additives in the printing composition. Without wishing to be bound by theory, it is believed that certain additives, such as dithiothreitol (DTT) or glutathione (GSH), may improve the presentation and / or accessibility of certain epitopes on a DFS70 protein, when immobilised on a substrate such as functionalised glass.

[0022] According to a first aspect there is provided a composition for printing a biological material on a substrate for an assay, the composition comprising: a biological material; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound.

[0023] The at least one additive may comprise or may be a thiol-containing C3-C12 compound, e.g. a thiol-containing C4-C10 compound.

[0024] The at least one additive may further comprise one or more moieties selected from the group consisting of: thiol, hydroxyl, amine, amide, and carboxyl.

[0025] The at least one additive may comprise or may be a C2-C20 compound comprising at least one thiol group, and one or more moiety selected from amine, amide and / or carboxyl. The at least one additive may comprise or may be a C4-C16 compound, e.g. a C8-C12 compound, comprising at least one thiol group, and one or more moiety selected from amine, amide and / or carboxyl. The at least one additive may comprise or may be a C4-C16 compound, e.g. a C8-C12 compound, e.g. a C10 compound, comprising at least one thiol group, and at least one amine group, amide group, and carboxyl group. In an embodiment, the at least one additive may comprise or may be glutathione (GSH).

[0026] The at least one additive may comprise or may be a C2-C20 compound comprising at least one thiol group and at least one hydroxyl group. The at least one additive may comprise or may be a C2-C12 compound, e.g. a C3-C8 compound, e.g. a 04 compound, comprising at least one thiol group and at least one hydroxyl group. The at least one additive may comprise or may be a C2-C12 compound, e.g. a C3-C8 compound, e.g. a 04 compound, comprising at least two thiol groups and at least two hydroxyl groups. In an embodiment, the at least one additive may comprise or may be dithiothreitol (DTT).

[0027] Thus, in an embodiment of the first aspect, there is provided a composition for printing a biological material on a substrate for an assay, the composition comprising: a biological material; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises dithiothreitol and / or glutathione.

[0028] The at least one additive may be present at a concentration of about 0.5-50 mM in the composition.

[0029] When the at least one additive comprises or consists of DTT, the at least one additive may be present at a concentration of about 0.5-10 mM, e.g. about 0.5-5 mM, e.g. about 1-5 mM, e.g. about 1mM, in the composition.

[0030] When the at least one additive comprises or consists of GSH, the at least one additive may be present at a concentration of about 1-50 mM, e.g. about 1-10 mM, e.g. about 2.5-5 mM, in the composition.

[0031] The at least one additive may comprise or may consist of dithiothreitol and glutathione. Advantageously, a synergistic effect may be observed by combining DTT and GSH.

[0032] Preferably, the printing buffer may be free of and / or may not comprise tris(hydroxymethyl)aminomethane (also known as ‘Tris’). Tris is a common additive in printing buffers. However, it is believed that, due to the presence amino groups in Tris, Tris competes with proteins such as DFS70 intended to be immobilised on epoxy glass. Therefore, in a preferred embodiment, the printing buffer comprising the at least one additive, is free of and / or does not comprise Tris. The printing buffer may comprise a viscosity modifier.

[0033] The concentration of the viscosity modifier may be in the range of about 30-60% w / v or w / w, depending on the type of viscosifier in the composition.

[0034] The viscosity modifier may comprise a carbohydrate, e.g. a monosaccharide, disaccharide, or oligosaccharide. Typically, the viscosity modifier may comprise a disaccharide, e.g. one or more compounds selected from the list consisting of sucrose, trehalose, lactose, maltose, cellobiose, and chitobiose. In an embodiment, the viscosity modifier may comprise or may consist of sucrose and / or trehalose.

[0035] The viscosity modifier may comprise a polyol such as glycerol.

[0036] The concentrations of the viscosifier may be expressed as %w / v or %w / w in the composition. For example, typically, when the viscosity modifier comprises a carbohydrate such as sucrose or trehalose, the concentration may be in %w / w. When the viscosity modifier comprises a polyol such as glycerol, the concentration may be in %w / v.

[0037] The composition may comprise a buffering agent, such as a phosphate, a carbonate or an acetate buffer. The buffering agent may typically be provided at a concentration of about 5-300 mM, e.g. about 10-250mM, e.g. about 20-200 mM in the composition.

[0038] The composition may comprise a salt, typically an inorganic salt. By such provision, the salt may affect the ionic strength of the composition, but may have limited or no reactivity with the biological material. Typically, the salt may comprise one or more compounds selected from the list consisting of sodium chloride (NaCI), calcium chloride (CaCh), magnesium chloride (MgCh), sodium bicarbonate (NaHCCh), potassium chloride (KCI), sodium sulphate (Na2SO4), calcium carbonate (CaCCh), and calcium phosphate (Ca3(PC>4)2. Conveniently, the last may comprise or may consist of NaCI.

[0039] The salt may typically be provided at a concentration between about 0 and 5M, e.g. between about 0.025M and 4M, e.g. about 150mM.

[0040] The composition may comprise a carrier or solvent, typically water such as distilled water, deionised water or the like.

[0041] The composition comprises a biological material. The term “biological material” will be herein understood as encompassing any biological material, whether naturally or synthetically made. Typically, the biological material may comprise a peptide, a protein (including recombinant proteins), an amino-acid, a nucleic acid (DNA and / or RNA), an oligonucleotide, a lipid, a carbohydrate, an enzyme, a metabolite, an antibody (including monoclonal and / or polyclonal antibodies and any (antigen binding) fragments thereof), an antigen, cells, red blood cells, plasma, serum or the like.

[0042] The biological material may comprise a protein and / or an antigen.

[0043] The biological material may comprise or may be a DFS70 protein. The DFS70 protein may be of animal origin, e.g. from a human or non-human mammal.

[0044] Typically, the substrate may be made of glass, silicon, or a polymer such as nitrocellulose.

[0045] The substrate may optionally be coated with a coating layer which may be selected so as to improve or alter properties of or interaction with the biological material, including adhesion, immobilisation, stabilisation, etc. The coating layer may comprise, may consist essentially of or may consist of a metal such as aluminium or gold, or a polymer such as hydrophilic polymers or hydrophilic polymer, e.g. polyacrylamide, epoxysilane, or the like.

[0046] Preferably, the substrate may comprise or may be functionalised glass, for example glass coated with a reactive electrophilic material such as an epoxy-containing material, e.g. an epoxysilane material. This type of substrate may be referred to as ‘epoxy glass’.

[0047] Advantageously, it was found that using one or more thiol-containing C2-C20 compounds in the printing buffer of a printing composition comprising a DSF70 protein, improved the positive / negative signal ratio during subsequent analysis when the composition was immobilised (e.g. printed) on epoxy glass.

[0048] The substrate may be referred to as a ‘chip’.

[0049] The assay may comprise or may be a microarray.

[0050] The assay and / or substrate may comprise a plurality of immobilised biomolecules, defining a microarray.

[0051] The plurality of immobilised biomolecules may each independently comprise a biomolecule selected from, or may each be independently selected from: dsDNA, TRIM21 ; SS-A 60; SS-B; Sm; Sm / RNP; U1 RNP; Scl-70; Jo-1 ; Chromatin; CENP-B; Ribosomal P proteins; RNA polymerase III; and CCP / ACPA. Typically, the size, e.g. diameter, of the discrete amounts, e.g. spots, of biological material immobilised to the surface of the substrate may be approximately 100pm - 300pm, e.g. about 150pm - 250pm, typically about 210pm + / -40 pm.

[0052] According to a second aspect there is provided a method of printing a biological material on a substrate for an assay, the method comprising: providing a composition comprising a biological material; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound; and printing the composition on the substrate.

[0053] The features relating to the composition described in the first aspect may equally apply here, and are not repeated, merely for brevity.

[0054] The method may comprise drying the composition.

[0055] The method may comprise printing the composition at one or more discrete locations on the substrate, e.g. on a surface thereof.

[0056] The method may comprise printing the composition at one or more discrete locations defining an array, e.g. a microarray.

[0057] The array may be or may define a one-dimensional, two-dimensional, or three dimensional array. Typically, the array may be or may define a two-dimensional array on a surface thereof.

[0058] Typically, a surface of the substrate, e.g. a surface on which the composition is printed and / or immobilized, is flat or planar or is substantially flat or planar. The terms “flat” and “planar” will be understood to refer to the shape of the surface at a macroscopic level, i.e., it be herein understood to mean that the surface does not include any wells or grooves that may be configured to receive a substance or composition. Rather, the substance or composition is printed and / or immobilised at different and / or discrete locations of the substrate on an otherwise substantially continuous surface. At least a portion of the substrate, e.g. a portion of the surface of the substrate configured to receive the composition or other substances, may be free of wells or recesses.

[0059] The method may further comprise blocking materials printed on the substrate, e.g. blocking the biological material of the composition, and / or any other substances printed and / or immobilised on the substrate. The method may comprise spraying a blocking composition onto at least a portion of the substrate, e.g. on the biological material.

[0060] The blocking composition may comprise, may consist of or may consist essentially of a blocking buffer.

[0061] According to a third aspect, there is provided an apparatus comprising: a substrate for an assay, and a composition printed on the substrate, the composition comprising: a biological material, wherein the biological material comprises or consists of a DFS70 protein; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound.

[0062] Preferably, the substrate may comprise or may consist of a functionalised glass material such as epoxy glass.

[0063] The composition may be a composition as described in relation to the first aspect, the features of which are not repeated here, merely for brevity.

[0064] For the avoidance of doubt, any feature described in respect of any aspect of the invention may be applied to any other aspect of the invention, in any appropriate combination. For example, method features may be applied to composition features and vice versa.

[0065] Brief Description of the Drawings

[0066] The present invention will now be further described in detail and with reference to the figures in which:

[0067] Figure 1 is a graph showing a comparison of positive-negative discrimination capability of DFS70 print formulations tested with 50 anti-DFS70 antibody negative samples (NEG) and 47 anti-DFS70 antibody positive samples (POS), with y-axis as signal intensity (SMN), showing the potential effect of concentrations of DFS70 (Figs laic), using a different viscosifier (trehalose - Fig 1 d), and using an additive (Figs 1e). Data were generated using the automated MosaiQ instrument, with microarrays printed by MMS;

[0068] Figure 2 is a graph showing a comparison of positive-negative discrimination capability of 100 pg / mL of DFS70 in 4 different print conditions tested with 49 anti- DFS70 antibody negative samples (NEG) and 48 anti-DFS70 antibody positive samples (POS) as in Figure 1 , showing the effect of the addition of DDT or GSH. The area under curve (AUG) for the ROC (receiver operating characteristics) curve is indicated for each condition, as well as its 95% confidence interval. Negative percent agreement (NPA) and positive percent agreement (PPA) for a potential cut-off are shown. In this example, the cut-off was applied to set NPA to 100%. Data were generated using the automated MosaiQ instrument, with microarrays printed by MMS;

[0069] Figure 3 is a graph showing a comparison of positive-negative discrimination capability of DFS70 for 3 different concentrations of DFS70 tested with 6 anti-DFS70 antibody negative samples (NEG) and 5 anti-DFS70 antibody positive samples (POS). Data were generated using the automated MosaiQ instrument, with microarrays printed by MMS;

[0070] Figure 4 is a graph showing an evaluation of the detection capability of 6 different DFS70 formulations containing as additive DTT only or both DTT and GSH. The x-axis represents the dilution of the positive sample from neat (d1) to dilution 1 / 512 (d512) for sample PS00004S; and to dilution 1 / 128 (d128) for sample TH00001S. Data were generated using the automated MosaiQ instrument, with microarrays printed by MMS;

[0071] Figure 5 is a graph showing a comparison of positive-negative discrimination capability of 100 pg / mL of DFS70 in 6 different print conditions tested with 31 anti-DFS70 antibody negative samples (NEG) and 45 anti-DFS70 antibody positive samples (POS), showing the effect of the addition of DTT or GSH. Differently from the other experiments, data were generated by manual immunoassay, with microarrays printed by sciFLEXARRAYER S3;

[0072] Detailed Description of the Drawings

[0073] In the present disclosure, reference is made to a number of terms, which have the meanings provided below, unless a context indicates to the contrary. The nomenclature used herein for defining compounds, in particular the compounds according to the invention, is in general based on the rules of the IIIPAC organisation for chemical compounds, specifically the “IIIPAC Compendium of Chemical Terminology (Gold Book)”. For the avoidance of doubt, if a rule of the IIIPAC organisation is in conflict with a definition provided herein, the definition herein is to prevail. Furthermore, if a compound structure is in conflict with the name provided for the structure, the structure is to prevail. The term “comprising” or variants thereof is to be understood herein to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0074] The term “consisting” or variants thereof is to be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, and the exclusion of any other element, integer or step or group of elements, integers or steps.

[0075] The term “about” herein, when qualifying a number or value, is used to refer to values that lie within ± 5% of the value specified. For example, if a temperature is specified to be about 5 to about 13 °C, temperatures of 4.75 to 13.65 °C are included.

[0076] Reference to physical states of matter (such as liquid or solid) refer to the matter’s state at 25 °C and atmospheric pressure unless the context dictates otherwise.

[0077] As explained above, certain thiol-containing additives, such as dithiothreitol (DTT) or glutathione (GSH), may improve the presentation and / or accessibility of certain epitopes on a DFS70 protein, when immobilised on a substrate such as functionalised glass. In particular, the present inventors have surprisingly discovered that the discrimination between positive and negative samples could be significantly improved by providing certain additives in the printing composition, e.g. in the printing buffer.

[0078] Examples

[0079] Experimental

[0080] Materials

[0081] Table 1 : Antigens full length DFS70 antigen

[0082] ** protein fragment containing immunodominant region

[0083] Substrate

[0084] Nexterion Epoxysilane coated glass (Schott Minifab) was used for peptide immobilisation and microarray manufacturing. Samples

[0085] PS00004S is a serum sample sourced from Plasma Services Group (Moorestown, NJ, USA). The measured value of anti-DFS70 antibodies was 616.1 U / mL using the EliA DFS70 assay (ThermoFisher). TH00001S is a serum sample sourced from Biosynex-Theradiag (Croissy-Beaubourg, France). The measured value of anti-DFS70 antibodies was 97.4 U / mL using the EliA DFS70 assay (ThermoFisher). The positivity cut-off of the EliA DFS70 method used to determine anti-DFS70 titres was >10 U / mL. Samples negative (n=50) for anti-DFS70 antibodies were sourced from Swiss Red Cross (Liebefeld, Switzerland). Samples positive for anti-DFS70 antibodies were sourced either from Rheumatology Institute Praha (Prague, Czech Republic) (n=48) or from Plasma Services Group (n=27). The anti-DFS70 serostatus of all these samples were confirmed by testing with the EliA DFS70 assay.

[0086] DFS70 buffer exchange

[0087] DFS70 antigen was buffer exchanged into carbonate buffer supplemented with 150 mM of NaCI and 1 % (w / v) of sucrose using the Zeba Spin Desalting columns from ThermoFisher® (catalog number 89877) and the supplier’s protocol was applied.

[0088] Methods

[0089] Microarray printing

[0090] Using sciFLEXARRAYER S3: The sciFLEXARRAYER S3 (Scienion, Berlin, Germany) is an automated piezo driven, non-contact dispensing system of ultra-low volumes specifically designed as an economical entry unit for academia and R&D labs. It is suitable for the production of DNA, protein and glycan arrays for research applications. Printing is performed on glass with dimensions similar to a microscope slide according to the manufacturer’s instructions. The spot size was about 150-200 micrometres in diameter, corresponding to a print volume of about 900-1000 pL. After deposition of spots onto glass, binding is carried out at 2-8 °C, in 50-60% humidity during one day.

[0091] Using MosaiQ manufacturing system: The MosaiQ instrument (AliveDx, Eysins, Switzerland) is a high-throughput and full traceability system, designed to provide multimodal, multiplexed testing with a single patient sample. The system uses microarrays that can harbour up to 132 spots, has 1 ,000 microarray loading capacity and uses QR codes and RFID tagging for easy management of the ready-to-use reagents. The MosaiQ system microarray was printed and assembled through the MosaiQ manufacturing system using piezo-electric, non-contact printing. The spot size was about 150-200 micrometres in diameter, corresponding to a print volume of about 900-1000 pL.

[0092] The DFS70 print formulations contained carbonate pH 9.5 as base buffer, a salt (NaCI or KCI) and sucrose as viscosity modifier unless specified otherwise. The print concentrations are in the 50-125 pg / ml range.

[0093] Immunoassay methods

[0094] Manual immunoassay: Slides printed using sciFLEXARRAYER S3 were blocked during 20 minutes with 2% BSA in phosphate buffer. Then, the blocking buffer was discarded and the sample to be tested was added and incubated for 20 min at 37 °C. After several washes in PBS-Tween 20 (0.05%), a secondary antibody conjugated HRP was added (monoclonal anti-human IgG antibody-HRP at 2 microgram / mL in the StabiIZyme HRP Conjugate stabilizer from Surmodics (ref SZ02)). The slides were left for incubation with this detection reagent for 30 min at 37 °C. After several washes in PBS-Tween 20 (0.05%), 3,3’,5,5’-tetramethylbenzidine (TMB) solution was added (SeramunBlau, ref S-710-#-TMB) and incubated 7 min at 37 °C. The colour development reaction was stopped by washing in milli RO water. Image acquisition was carried out using the Microblot Array Reader from Biovendor. Spot images were analysed using the reader software.

[0095] MosaiQ system: Single use microarrays were printed and assembled using the MosaiQ manufacturing system (AliveDx). The immunoassays were carried out using the microarrays on the fully automated MosaiQ 125 instrument (AliveDx). The MosaiQ system dilutes the patient sample with sample diluent solution and adds the patient sample to the microarray. An incubation period allows antibodies present in the sample to react with the immobilized peptides. After washing away unbound antibodies, HRP labelled antibodies against human IgG are dispensed to form conjugate complexes during an additional incubation step. Excess conjugate is eliminated during another wash cycle before TMB is added to the array and incubated. Following another wash cycle, images generated by the system for the array are analysed to determine the amount of antibodies bound to each spot. Results are obtained by reading the signal of the MosaiQ for each individual analyte. Wash buffer (ref 155041), MosaiQ Sample Diluent 2 (ref 155002), TMB Substrate (ref 155050) and IgG Conjugate (ref 155051) were supplied by Alive Dx. Results

[0096] Anti-DFS70 assay raw material selection

[0097] Initial tests investigated the most suitable type of DFS70 antigen for use in subsequent studies. Raw materials were selected from Diarect (full length DFS70 antigen) or Abbexa (protein fragment containing immunodominant region) suppliers.

[0098] Although both were considered suitable, DFS70 material from Abbexa was retained as the main candidate due to its superior detection capability and positivenegative discrimination

[0099] After resuspension, DFS70 antigen from Abbexa was in a Tris 20 mM pH8, 150mM NaCI buffer containing 0.01% sarcosyl and 5% trehalose for 250 pg / mL (batch specific information). Sarcosyl, also known as sodium lauroyl sarcosinate is an anionic surfactant and is a milder detergent than sodium dodecyl sulfate (SDS).Sacrosyl has an average micellar weight of 600 Da, therefore it should be possible to eliminate it through buffer exchange using 7 kDa cut-off Zeba columns. We compared the spot size and the signal intensity of the DFS70 antigen printed with and without buffer exchange of the raw material before print formulation preparation. Buffer exchanging DFS70 antigen into a mM carbonate pH 9.5, 150 mM NaCI buffer using two successive Zeba columns allowed good spot morphology.

[0100] DFS70 print formulation development

[0101] Based on preliminary screening results, carbonate pH 9.5, 150 mM NaCI, 30 or 40% sucrose was selected as the basis of the DFS70 print formulations printed on MMS and tested in MosaiQ.

[0102] A basic prototyping experiment was carried out, investigating the possible effects of varying the concentrations of DFS70 (Figs 1a-1c), using a different viscosifier (trehalose - Fig 1 d), and using an additive (Figs 1e). The results of a basic prototyping experiment are shown in Figures 1a-1e.

[0103] The following conclusions were drawn:

[0104] - Addition of certain additives such as BSA (7.5 pg / mL - Fig 1e) did not bring any improvement compared to the print formulation without any additives;

[0105] - No significant difference between sucrose or trehalose was observed. As such, sucrose was confirmed as the viscosity modifier for the print formulation.

[0106] - The signal intensity has increased as expected by increasing the DFS70 concentration in the formulation. Twice-buffer exchanged DFS70 was printed at 50, 75 and 100 pg / mL and as shown in Figure 1 , the median of signal intensity for the positives was 59, 72, and 81 units respectively. The 100 pg / mL print condition gave the best results in the experiment.

[0107] However, with an AUC of 0.80 [0.70-0.89] and a PPA of 57% at an NPA of 100%, its diagnostic accuracy was not considered satisfactory. In addition, it was observed that some samples were totally undetected, possibly hinting at an antigen presentation and / or epitope accessibility issue. Including additives that can influence protein conformation as well as testing even higher concentrations of DFS70 were identified as a desirable solution.

[0108] Another set of experiments was carried out, investigating the effects of:

[0109] (i) increasing the concentration of DFS70 in the print formulation from 100 to 250 pg / mL;

[0110] (ii) increasing the concentration of BSA in the print formulation from 7.5 to 52.8 pg / mL to locally increase DFS70 concentration and create “hot spots”; and

[0111] (iii) increasing the ionic strength to 2M NaCI in the print formulation to influence protein conformation;

[0112] It was observed that none of these variants resulted in improved performance:

[0113] Investigation of specific additives

[0114] The effect of two specific additives, namely Dithiothreitol (DTT) and Glutathione (GSH), was investigated.

[0115] Using the DFS70 antigen from Abbexa, formulations with DTT additive and with GSH were tested. Figure 2 shows the detection results when using:

[0116] • Figure 2a: 100 pg / mL DFS70, 100mM Carbonate pH 9.6, 150mM NaCI, 40% (w / v) sucrose, no additive;

[0117] • Figure 2b: 100 pg / mL DFS70, 100mM Carbonate pH 9.6, 150mM NaCI, 40% (w / v) sucrose, 1 mM DTT

[0118] • Figure 2c: 100 pg / mL DFS70, 100mM Carbonate pH 9.6, 150mM NaCI, 40% (w / v) sucrose, 10 mM GSH

[0119] • Figure 2d: 100 pg / mL DFS70, 100mM Carbonate pH 9.6, 150mM NaCI, 40% (w / v) sucrose, 50 mM GSH

[0120] As shown in Figure 2, it can be seen that the base formulation (Fig 2a) has a suboptimal differentiation between positive and negative samples. However, the addition of DDT (Fig 2b) or GSH (Fig 2c and 2d) significantly improved the discrimination between positive and negative samples. It can also be seen that a higher concentration of 50 mM of GSH resulted in higher signal intensity for positive samples (median 192) compared to a lower concentration of 10 mM (median 164).

[0121] Biomolecule concentration

[0122] Three different print conditions were assessed to evaluate the impact of capture agent concentration on assay performances. DFS70 was diluted in 100mM carbonate supplemented with 150mM of NaCI, 30mM of Glutathione and 40% (w / v) of sucrose, at a concentration of 75 (Fig 3a), 100 (Fig 3b) and 125pg / ml (Fig 3c), respectively. The DFS70 raw material used was two times buffer exchanged, as explained above.

[0123] Figure 3 illustrates the results. It can be seen that all three concentrations led to satisfactory Pos / Neg discrimination. .

[0124] Additive concentrations

[0125] Further experiments were carried out to investigate the effect of changing the type and the concentration of the additives in relation to discrimination of positive and patient populations, and in relation to the potential improvement of the detection of low positive samples. The additives tested were:

[0126] • DTT at different concentrations: 1mM, 2.5mM and 5mM

[0127] • A mixture of DTT and Glutathione at different concentrations: DTT at 1 mM with Glutathione at 5 or 10mM, and DTT at 2.5 with Glutathione at 5mM to assess potentializing effects between DTT and GSH

[0128] For all these examples, the DFS70 print concentration was set at 100 pg / ml and each spot was printed in a printing buffer comprising 100mM Carbonate pH 9.6, 150mM NaCI, 40% (w / v) sucrose.

[0129] The results carried out for these conditions, on two separate samples, are shown in Figures 4a, b and 4c, d respectively.

[0130] As shown in Figure 4a and 4c, higher concentrations of DTT (2.5 and 5 mM) result in higher signal compared to lower concentrations of DTT (1 mM). There is also a synergistic effect between GSH and DTT (Figure 4b, d). The combination of 1 mM DTT with 5 or 10 mM of GSH results in higher signal intensity compared to a formulation where the only additive is 1 mM DTT.

[0131] Other printing and assaying

[0132] All the experiments shown in Figures 1-4 were generated using microarrays printed with the MosaiQ Manufacturing System and assayed with the fully automated MosaiQ instrument. To illustrate that other printing and assaying systems can also be used, DFS70 formulations were printed with sciFLEXARRAYER S3 (Scienion, Berlin, Germany) and assayed manually as described in the Methods section. The results are shown in Figure 5. It can be seen that the base formulation (Fig 5a) has a suboptimal differentiation between positive and negative samples, with an AUG of 0.8573. However, the addition of DDT (Fig 5b, c) or GSH (Fig 5d to 5f) significantly improved the discrimination between positive and negative samples as well as the assay performance (AUG >0.99 for assays based on print formulations containing DTT and / or GSH, Table 2)

[0133] Table 2

Claims

CLAIMS:1 . A composition for immobilising a biological material on a substrate for an assay, the composition comprising: a biological material, wherein the biological material comprises or consists of a DFS70 protein; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound.

2. A composition according to claim 1 , wherein the at least one additive comprises or consists of a thiol-containing C3-C12 compound, optionally a thiol-containing C4-C10 compound.

3. A composition according to claim 1 or claim 2, wherein the at least one additive further comprises one or more moieties selected from the group consisting of: thiol, hydroxyl, amine, amide, and carboxyl.

4. A composition according to any one of the preceding claims, wherein the at least one additive is present at a concentration of about 0.5-50 mM in the composition.

5. A composition according to any one of claims 1 to 3, wherein the at least one additive comprises or consists of a C8-C12 compound comprising at least one thiol group, and one or more moiety selected from amine, amide and / or carboxyl.

6. A composition according to claim 5, wherein the at least one additive comprises or consists of glutathione (GSH).

7. A composition according to claim 5 or claim 6, wherein the at least one additive is present at a concentration of about 1-50 mM, optionally about 1-10 mM, optionally about 2.5-5 mM, in the composition.

8. A composition according to any one of claims 1 to 3, wherein the at least one additive comprises or consists of a C3-C8 compound comprising at least one thiol group and at least one hydroxyl group.

9. A composition according to claim 8, wherein the at least one additive comprises or consists of dithiothreitol (DTT).

10. A composition according to claim 8 or claim 9, wherein the at least one additive is present at a concentration of about 0.5-10 mM, optionally about 0.5-5 mM, optionally about 1-5 mM, optionally about 1mM, in the composition.

11. A composition according to any one of the preceding claims, wherein the at least one additive comprises dithiothreitol and glutathione.

12. A composition according to any one of the preceding claims, wherein the printing buffer comprises a viscosity modifier.

13. A composition according to claim 12, wherein the concentration of the viscosity modifier is in the range of about 30-60% w / v or w / w, in the composition.

14. A composition according to claim 10 or claim 11 , wherein the viscosity modifier comprises a disaccharide or a polyol.

15. A composition according to any one of the preceding claims, wherein the printing buffer comprises a buffering agent.

16. A composition according to any one of the preceding claims, wherein the printing buffer comprises a carrier or solvent.

17. A composition according to any one of the preceding claims, wherein the printing buffer is free of and / or does not comprise Tris.

18. A composition according to any one preceding claim, wherein the substrate comprises or consists of epoxy glass.

19. An apparatus comprising: a substrate for an assay, wherein the substrate comprises or consists of a functionalised glass material such as epoxy glass, and a composition printed on the substrate, the composition comprising:a biological material, wherein the biological material comprises or consists of a DFS70 protein; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound.

20. A method of immobilising a biological material on a substrate for an assay, the method comprising: providing a composition comprising: a biological material, wherein the biological material comprises or consists of a DFS70 protein; and a printing buffer, wherein the printing buffer comprises at least one additive, wherein the at least one additive comprises a thiol-containing C2-C20 compound; and printing the composition on the substrate.

21. A method according to claim 20, wherein the method comprises drying the composition.

22. A method according to claim 20or 21 , wherein the method comprises printing the composition at one or more discrete locations on a surface of the substrate.

23. A method according to any one of claims 20 to22, wherein the method further comprises blocking the composition printed on the substrate.

24. A method according to any one of claims 20 to 23, wherein the substrate comprises or consists of functionalised glass, optionally epoxy glass.

25. A method according to any one of claims 20 to 24, wherein the at least one additive comprises glutathione and / or dithiothreitol.

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