WO2023056528A1 - Dosage à écoulement latéral - Google Patents

Dosage à écoulement latéral Download PDF

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Publication number
WO2023056528A1
WO2023056528A1 PCT/AU2022/051210 AU2022051210W WO2023056528A1 WO 2023056528 A1 WO2023056528 A1 WO 2023056528A1 AU 2022051210 W AU2022051210 W AU 2022051210W WO 2023056528 A1 WO2023056528 A1 WO 2023056528A1
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Prior art keywords
metal coordination
affinity
oligomeric
lateral flow
agents
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PCT/AU2022/051210
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English (en)
Inventor
Nobuyoshi Joe Maeji
Chang-Yi Huang
Manuel Christoph Wieser
Kai-Anders HANSEN
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Anteo Technologies Pty Ltd
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Priority claimed from AU2021903235A external-priority patent/AU2021903235A0/en
Application filed by Anteo Technologies Pty Ltd filed Critical Anteo Technologies Pty Ltd
Publication of WO2023056528A1 publication Critical patent/WO2023056528A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/0014—Use of organic additives
    • C08J9/0052—Organo-metallic compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2207/00—Foams characterised by their intended use
    • C08J2207/10—Medical applications, e.g. biocompatible scaffolds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2301/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives
    • C08J2301/08—Cellulose derivatives
    • C08J2301/16—Esters of inorganic acids
    • C08J2301/18—Cellulose nitrate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N2021/752—Devices comprising reaction zones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7756—Sensor type
    • G01N2021/7759—Dipstick; Test strip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present disclosure relates to an approach and method to improve the detection accuracy and performance of rapid analyte detection devices, such as lateral flow immunoassays.
  • the present disclosure relates to an improved lateral flow device and method of detecting an analyte using same.
  • Point of Care (PoC) tests such as lateral flow devices
  • PoC tests have evolved from qualitative assessment of a single analyte to semi- and fully quantitative devices including the potential for multiplexing.
  • Semi-quantitative is commonly described in terms of detecting a target analyte to some defined thresholds, such a zero, low, medium or high, while fully quantitative usually means the translation of a test line, via a dedicated reader, to a numerical readout that can be compared on a calibration curve.
  • readers, software tools, high sensitivity labels and other components of a lateral flow device to digitise the output of a test line and remove the subjectivity inherent in qualitative methods.
  • the quantitative output of rapid lateral flow devices still cannot match those achieved using automated equipment in modern pathology labs, let alone molecular diagnostic devices.
  • the comparison can be considered an unfair one as laboratory-based tests use specialist equipment (automated mixing, washing, temperature regulation, etc.) to maximise reproducibility, sensitivity and specificity.
  • enzyme-based signal amplification is commonly used in immunoassays and reverse transcription polymerase chain reaction (RT- PCR), isothermal amplification technologies (IAT) and related technologies are a testament to the power of amplification for achieving high sensitivity and specificity in molecular tests.
  • IAT isothermal amplification technologies
  • reaction kinetics are related to the rate constant, concentration, and reaction time of the analyte with its capture agent. Assuming the same capture agents are used, maximising reaction rate without appreciably increasing time depends on the reactant concentration. To achieve high analytical sensitivity, the choices are pre-concentration of the analyte of interest (which adds to time and cost) or somehow increasing the number of effective binding sites. There are many different strategies which have been attempted but simply maximising the density of affinity agents per unit surface area does not necessarily improve performance.
  • the present disclosure provides methods for presenting affinity agents in what may be considered, in a functional and conformational sense, a quasi-quaternary form.
  • a protein s structure is organised around its amino acid sequence (Primary), which affects its local spatial arrangement of the polypeptide backbone (Secondary), which affects its entire three-dimensional structure (Tertiary).
  • Quaternary structure describes the three- dimensional arrangement of protein subunits in the case of multi-protein structures.
  • IgM antibodies form pentamers to allow multipoint, i.e. avidity binding which compensates for their often low individual affinity for a binding partner.
  • a lateral flow device comprising:
  • conjugate pad including mobile conjugate particles comprising binding agents associated with the conjugate particles
  • a membrane comprising immobilised capture agents associated with the membrane, wherein the binding agents are associated with the conjugate particles and/or the capture agents are associated with the membrane, by oligomeric metal coordination complexes.
  • a region of the membrane is a membrane test line upon which the capture agents are immobilised.
  • the first aspect may extend to a method of detecting an analyte in a sample including the step of applying the sample to the high sensitivity lateral flow device.
  • a method for the detection of an analyte in a sample including the steps of:
  • a method of forming a lateral flow device including the step of: incorporating
  • conjugate pad including mobile conjugate particles comprising binding agents associated with the conjugate particles
  • a membrane comprising immobilised capture agents associated with the membrane, wherein the binding agents are associated with the conjugate particles and/or the capture agents are associated with the membrane, by oligomeric metal coordination complexes, into the device to thereby form the lateral flow device.
  • the affinity agents may be binding agents or capture agents.
  • the substrate may be a conjugate particle or a membrane substrate.
  • the substrate when the affinity agent is a binding agent then the substrate is a conjugate particle and when the affinity agent is a capture agent then the substrate is a membrane substrate.
  • the first oligomeric metal coordination complex and the second oligomeric coordination complex may be the same or different.
  • the first oligomeric metal coordination complex is a chromium oligomeric metal coordination complex.
  • the substrate may be associated directly or indirectly with the second oligomeric metal coordination complex.
  • FIG 1 shows the zeta size of Bovine Serum Albumin (BSA) aggregates formed when unmodified oligomeric metal complexes (Solution 1) is added after (a) 1 minute, (b) 30 minutes.
  • BSA Bovine Serum Albumin
  • FIG 2 shows the zeta size of Bovine Serum Albumin (BSA) clusters after the addition of different concentrations modified oligomeric metal complexes, (a) Solution 3A and (b) Solution 3B over 7 hours.
  • BSA Bovine Serum Albumin
  • FIG 3 shows the zeta size of Bovine Serum Albumin (BSA) clusters formed when modified oligomeric metal complexes (Solution 4) is added at room temperature after (a) 1 minute, (b) 10 minutes, (c) 20 minutes and (d) 60 minutes.
  • BSA Bovine Serum Albumin
  • FIG 4 shows the zeta size of Bovine Serum Albumin (BSA) clusters formed when modified oligomeric metal complexes (Solution 4) is added at 37 °C after (a) 1 minute, (b) 10 minutes, (c) 60 minutes, and (d) 120 minutes.
  • BSA Bovine Serum Albumin
  • FIG 5 shows the zeta size of the Bovine Serum Albumin (BSA) clusters formed using different concentrations of modified oligomeric metal complexes, (a) Solution 4, (b) Solution 5A and (c) Solution 5B over 7 hours
  • BSA Bovine Serum Albumin
  • FIG 6 compares the performance differences in a COVID-19 antigen test of antibody clusters formed using three different concentrations of modified oligomeric metal complex (Solution 4) compared to Control.
  • the clusters and control were immobilised onto Eu particles activated with unmodified oligomeric metal coordination complexes.
  • FIG 7 compares the performance of a COVID-19 antigen test for one example of an antibody cluster compared to a Control when using different amounts of conjugate particles; 2pg particles per strip, 0.1 pg particles per strip, 0.05pg particles per strip and 0.025pg particles per strip.
  • FIG 8 compares the performance of a COVID-19 antigen test for one example of an antibody cluster compared to a Control when using 50pg COVID-19 detection mAb per mg of Eu particles.
  • FIG 9 compares the performance of a COVID- 19 antigen test for antibody clusters formed with different excesses of capping groups (Solution 5A) compared to a Control when using 50pg COVID-19 detection mAb per mg of Eu particles on a test line formed with 1 mg/ml SARS-CoV Ab + 1 mg/ml BSA. Three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.
  • FIG 10 compares the performance of a COVID-19 antigen test for antibody clusters formed with different excesses of capping groups (Solution 5A) compared to a Control when using 150pg Ab per mg of Eu particles on a test line formed with 0.2mg/ml SARS-CoV Ab + 1 mg/ml BSA. Three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.
  • FIG 11 compares the performance differences in a COVID-19 antigen test of antibody clusters formed on gold nanoparticles using modified oligomeric metal complex under two conditions compared to Control.
  • FIG 12 shows the signal intensity in RFU of the capture line of a lateral flow test strip on which mouse IgG antibody conjugated to a nanoparticle (detection moiety) is captured by the goat anti-mouse antibody, passively immobilised, at three different pH values (pH 5.2, 6.0 and 8.5) to nitrocellulose membranes.
  • the fluorescence intensity is an indicator of the number of Europium nanoparticles (detection moiety) captured on the capture line.
  • FIG 13 shows the signal intensity in RFU of the capture line of a test strip where the mouse IgG antibody conjugated to a nanoparticle (detection moiety) is captured by the goat anti-mouse antibody cross-linked by an oligomeric metal complex (Solution 4) at two concentrations (0.1 mM and 0.5mM) and at three different pH values (pH 5.2, 6.0 and 8.5) striped onto nitrocellulose membrane.
  • the fluorescence intensity is an indicator of the number of Europium nanoparticles captured on the capture line.
  • the signal obtained via passive binding at pH 8.5 (Control) is shown on the far right.
  • FIG 14 shows the signal intensity in RFU of the capture line where the mouse IgG antibody Conjugate is captured by the cluster formed with goat anti-mouse antibody and an oligomeric metal complex (Solution 4) on the nitrocellulose membrane.
  • the membrane was pre-blocked with BSA prior to striping of antibody clusters.
  • FIG 15 shows the signal intensity in RFU of the capture line where the mouse IgG antibody Conjugate is captured by the cluster formed with goat anti-mouse antibody and an oligomeric metal complex (Solution 6C) in carbonate buffer (pH 8.5).
  • FIG 16 shows the relative signal intensity (Sn/SO) of FluA antigen in a lateral flow sandwich assay with capture by clusters formed with anti-FluA antibody and an oligomeric metal complex (Solution 6C).
  • maximising sensitivity/specificity in a lateral flow assay involves optimisation of the reaction kinetics of the whole system and not just optimisation of a test line on a membrane or improved functionality of binding agent on a conjugate particle. While each method may improve sensitivity, to a degree, within some antigen-specific assay, it is not a general solution to maximising specific signal while minimising nonspecific signal.
  • Capture agents which are usually antibodies, needs to be “localised” on a substrate which can negatively affect diffusion rates. Some approaches try to increase the capture agent density but this can lead to steric crowding and poor accessibility.
  • the present disclosure provides for the binding of affinity agents, such as antibodies, to components of a lateral flow device by the use of modified oligomeric metal coordination complexes.
  • the oligomeric metal coordination complexes are modified in the sense that their reactivity to, or rate of forming associations with, the oligomeric metal coordination complexes is reduced compared with the same oligomeric metal coordination complexes which have not been so modified.
  • the modified oligomeric metal coordination complex preferentially mediates inter-molecular binding of affinity agents to form affinity agent clusters of controllable size and enhances its binding to its binding partner or target molecule.
  • the same oligomeric metal coordination complex which has not been so modified leads to rapid uncontrolled aggregation and severe damage to its functional conformation in part due to the unmodified metal complex also forming many intra-molecular interactions within an individual affinity agent.
  • the use of such modified oligomeric metal coordination complex at the appropriate concentration to the affinity agent may provide a simple generic solution to form more reactive affinity agents that can easily bind to particles, membrane or any other substrate material useful in lateral flow immunoassays.
  • the additive used in binding an affinity agent to a substrate, as described herein is a mixture of affinity agent and modified oligomeric metal coordination complex.
  • the concentration, ratio, time and temperature used to prepare the mixture can be modified.
  • the mixture may be left to provide an affinity cluster that can bind to particles and/or membranes by passive binding.
  • excess modified metal complexes can be included to also provide additional metal complex coordination between the substrate and the affinity agent cluster. It will be appreciated that the characteristics of the modified oligomeric coordination complex advantageously allows less metal complex to be used as it preferentially promotes inter- molecular coordination of affinity agent clusters allowing greater rotational freedom or otherwise improve the functional availability thereof.
  • the present disclosure further provides for the use of a similar approach in the formation of small antibody, or other binding/capture agent, clusters by the use of metal coordination complexes and the binding of such clusters to particles or membranes using metal coordination complexes having, in certain embodiments, modified or tailored, preferably reduced, reactivity.
  • the simplicity of the approach also allows for the integration of appropriate antibodies to remove heterophile antibodies and other interfering substances that lead to non-specific binding and a decrease in assay specificity.
  • affinity agent or “affinity cluster” is broad enough to include within its scope both typical assay capture agents, such as antibodies as may be used in a sandwich assay, and analytes, as may be used in a competitive assay, which are associated with the metal coordination complexes described herein. So long as the affinity agent associated with the oligomeric metal coordination complexes can participate in a binding event having appropriate specificity with a target molecule (which equally may be an antibody or analyte) in a sample of interest then it may, for the purposes of the present disclosure, be considered an affinity agent and, when multiple such agents are bound by metal coordination complexes, an affinity cluster.
  • An affinity cluster requires two or more such affinity agents bound by oligomeric metal coordination complexes which have been modified but, in preferred embodiments, results in a plurality or multiplicity of such binding agents interconnected by oligomeric metal coordination complexes to form an interconnected affinity network cluster.
  • the terms “specificity” indicates that the affinity agent binds preferentially to the target molecule, antibody or analyte of interest or binds with greater affinity to the target (analyte) than to other molecules within a sample.
  • an antibody will selectively bind to the antigen against which it was raised.
  • a DNA molecule will bind to a substantially complementary sequence and not to unrelated sequences under stringent conditions.
  • Specific binding can refer to a binding reaction that is determinative of the presence of a target in a heterogeneous population of molecules (e.g., proteins and other biologies). Thus, under designated conditions (e.g.
  • the specific ligand or antibody binds to its particular “target” molecule and does not bind in a significant amount to other molecules present in the sample.
  • a lateral flow device comprising:
  • conjugate pad including mobile conjugate particles comprising binding agents associated with the conjugate particles
  • the lateral flow device has a 50% or greater increase in analytical sensitivity for a given analyte compared with a similar assay which does not use the oligomeric metal coordination binding approach described herein. In further embodiments, the lateral flow device has a 50% or greater increase in analytical sensitivity for a given analyte compared with a similar assay that uses affinity agents that are not bound in the form of affinity clusters.
  • the binding agents are associated with the conjugate particles and/or the capture agents are associated with the membrane, through avidity bonding, including dative bonds, with the oligomeric metal coordination complexes.
  • the binding agents are associated with the conjugate particles and the capture agents are associated with the membrane, through oligomeric metal coordination complexes.
  • the coordinating ligands which are not binding agent and/or capture agent associations may be capping groups or small coordinate ligands present in the assay following addition of a sample.
  • the small coordinate ligands may be groups from the buffer or small molecules presenting electron-donating groups.
  • the oligomeric metal coordination complexes have been modified, as defined herein.
  • the oligomeric metal coordination complexes of the first aspect may be the same as the first oligomeric metal coordination complex as defined herein.
  • a plurality of the binding agents and/or capture agents are interconnected by oligomeric metal coordination complexes to form a first affinity cluster and/or a second affinity cluster.
  • the first affinity cluster is associated with the conjugate particle and the second affinity cluster is associated with the membrane.
  • the first affinity cluster and/or the second affinity cluster are an interconnected network of affinity agents associated with and connected by the first oligomeric metal coordination complex.
  • the first affinity cluster is a binding agent cluster associated with conjugate particles by a second oligomeric metal coordination complex.
  • the second affinity cluster is a capture agent cluster associated with the membrane by a second oligomeric metal coordination.
  • oligomeric metal coordination complex can provide for the formation of:
  • affinity clusters formed by oligomeric metal coordination complex that binds to a substrate by a second metal complex activated substrate and/or
  • affinity clusters in situ where the oligomeric metal coordination complex can form affinity clusters as well as binding to substrate.
  • a lateral flow device comprising:
  • affinity agents with the oligomeric metal coordination complex described herein there may be at least about 30% more inter-molecular binding of affinity agents with the oligomeric metal coordination complex described herein to form affinity agent clusters of controllable size and enhanced binding to its binding partner or target molecule than the same oligomeric metal coordination complex which has not been so modified. In some embodiments, there may be at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% more inter-molecular binding of affinity agents with the oligomeric metal coordination complex described herein to form affinity agent clusters than the same oligomeric metal coordination complex which has not been so modified.
  • an LF device typically comprises a sample pad, a conjugate pad, a membrane, typically made of nitrocellulose, that contains test and control lines, and a wicking pad. Each component will typically overlap to encourage appropriate capillary flow of the test sample.
  • a liquid sample such as blood, serum, plasma, urine, saliva, or solubilised solids, is added directly to the sample pad and from there is wicked through the LF device.
  • the sample pad neutralises the sample and filters unwanted particulates allowing the sample to flow unimpeded to the conjugate pad which contains, for example, strongly coloured or fluorescent conjugate nanoparticles that are provided with an antibody on their surface.
  • the nanoparticles may be colloidal gold which is common in commercial LF devices.
  • these dried nanoparticles are reconstituted and mix with the sample. If there are any target analytes in the sample that the antibody recognises, these will bind to the antibody.
  • the analyte-bound nanoparticles then flow through the nitrocellulose membrane and across one or more test lines and a control line.
  • the test line is the primary read-out of the diagnostic and consists of immobilised capture agents that can bind the nanoparticle to generate a signal that is correlated to the presence of the target molecule/analyte in the sample.
  • the fluid continues to flow across the strip until it reaches the control line.
  • the control line contains capture ligands that will bind the nanoparticle conjugate with or without the target molecule/analyte present in solution to confirm that the assay is working properly.
  • the fluid flows into the wicking pad which may be needed to absorb all of the sample liquid to ensure that there is consistent flow across the test and control lines. Once all the sample has passed across the test and control lines, the assay is complete and the user can read the results.
  • the LF device comprises at least a sample pad; a conjugate pad; and a membrane.
  • a lateral flow device comprising:
  • an affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex
  • the affinity agents may be binding agents or capture agents.
  • the substrate may be a conjugate particle or a membrane substrate.
  • the substrate when the affinity agent is a binding agent then the substrate is a conjugate particle and when the affinity agent is a capture agent then the substrate is a membrane substrate.
  • the lateral flow device may comprise:
  • an affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex; and (e) a substrate associated with a second oligomeric metal coordination complex, and the affinity cluster associated with the substrate by the second oligomeric coordination complex, wherein the affinity cluster and associated substrate are located within the conjugate pad and the membrane test line.
  • the lateral flow device may comprise:
  • a conjugate pad comprising a first affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex, and the first affinity cluster is associated with a conjugate particle and a second oligomeric metal coordination complex;
  • a membrane comprising a test line comprising a second affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex, and the second affinity cluster associated with the membrane by a second oligomeric coordination complex.
  • the lateral flow device may comprise:
  • a substrate associated with a second oligomeric metal coordination complex wherein the first affinity cluster associated with the substrate by the second oligomeric coordination complex is an affinity cluster associated with a conjugate particle and the second oligomeric metal coordination complex, and located within the conjugate pad, and wherein the second affinity cluster associated with the substrate by the second oligomeric coordination complex is an affinity cluster associated with and located within the membrane test line.
  • the LF device may further comprise a wicking pad and other components which are standard in the art.
  • the sample pad may be made of natural and/or synthetic porous, microporous, mesoporous, or macroporous materials capable of receiving a sample fluid and laterally conducting the sample fluid by capillary action.
  • the sample pad may be made from a range of suitable materials well-known in the art and including, without limitation, cellulose, nitrocellulose, paper, silica, cotton, glass (e.g., glass fiber), or synthetic material (e.g., polyester, polyethylene, polymers, rayon, nylon, etc.).
  • the sample pad may be treated by a buffer (e.g., an organic compound such as tris or tris(hydroxymethyl)aminomethane) to mitigate sample variabilities (pH, protein concentration, viscosity, salt concentration, etc.).
  • a buffer e.g., an organic compound such as tris or tris(hydroxymethyl)aminomethane
  • the buffer compound may be coated, impregnated, or otherwise applied or deposited on the sample pad and then dried.
  • the conjugate pad may be made of natural and/or synthetic porous, microporous, mesoporous, or macroporous materials capable of receiving the sample fluid from the sample pad.
  • the conjugate pad may be made from a range of suitable materials well-known in the art and including, without limitation, glass (e.g., glass fibre), cellulose, nitrocellulose, paper, silica, cotton, or synthetic material (e.g., polyester, polyethylene, polymers, rayon, nylon, etc.).
  • the sample pad and conjugate pad may be a single continuous pad.
  • the conjugate pad may contain affinity clusters comprising multiple binding agents that are capable of binding the target analyte in the sample fluid.
  • the affinity clusters may be coupled, via the second metal coordination complex, to a label or reporter component which is referred to herein as a conjugate particle and which is preferably a nanoparticle. In its natural state the conjugate particle may be readily visible either to the naked eye, or with the aid of an optical filter.
  • the binding agent of the affinity clusters may be an antibody, an antigen, a protein, a nucleic acid, etc., as described above, that is capable of binding to the target analyte.
  • the conjugate particle may be selected from the group consisting of nanoparticles such as, without limitation, metallic sols (e.g., colloidal gold or gold sol), dye sols, fluorescent particles including europium particles and chelates thereof, coloured latex particles, carbon, and the like.
  • metallic sols e.g., colloidal gold or gold sol
  • dye sols e.g., colloidal gold or gold sol
  • fluorescent particles including europium particles and chelates thereof, coloured latex particles, carbon, and the like.
  • the conjugate particle will, in this embodiment, be associated with the second oligomeric metal coordination complex as is described in further detail herein.
  • the conjugate particle is a europium or gold particle or nanoparticle then it will have second metal coordination complexes associated with its surface, either directly or indirectly.
  • the sample fluid may solubilise the conjugate particle and associated affinity clusters. If the sample fluid contains the target analyte, the target analyte may bind with the binding agents of the affinity clusters and form an immunocomplex.
  • the conjugate pad is therefore one component of the LF device of the present disclosure in which the affinity clusters may be located and associated with a substrate, in this embodiment the conjugate particle, by the second metal coordination complex.
  • the membrane of the LF device may be considered, in part, a capture zone which includes a test line that may be embedded in the membrane.
  • the capture zone may optionally include a control line that may also be embedded in the membrane.
  • the membrane may be made of materials which are well-known in the art including, without limitation, cellulose, nitrocellulose, paper, silica, cotton, glass (e.g., glass fibre), or synthetic material (e.g., polyester, polyethylene, polymers, rayon, nylon, etc.) that allow the sample to flow downstream from the conjugate pad into the membrane and, optionally, towards a wicking pad by capillary action.
  • the test line may be made of a porous material such as those specified for the membrane.
  • the test line in a sandwich assay format, may contain a capture agent that is immobilised on the test line, optionally via the second metal coordination complex and does not flow downstream when the porous material of the test line is wetted by the sample.
  • the capture agent immobilised on the test line may be the same or different to the binding agent associated with the conjugate particle on the conjugate pad.
  • the capture agent contained on the test line may be an immobilised antibody cluster that is capable of binding to the immunocomplex that is formed from binding of the analyte with the conjugate particle and associated affinity cluster on the conjugate pad.
  • the immunocomplex moves over the test line it binds with the immobilised antibody cluster on the test line resulting in a second immunocomplex that thereby colours the test line.
  • the intensity of the coloured test line is correlated with the density of the analyte in the sample fluid.
  • the second immunocomplex includes the analyte that is bound with the binding agent cluster and conjugate particle at one site and is bound with the immobilised capture agent cluster at the test line.
  • the test line may contain the immobilised analyte molecule (or a protein-analyte complex), which is considered to now be the capture agent bound within an affinity cluster with the first metal coordination complex. If the sample liquid does not contain the analyte, the conjugate particle-binding agent antibody that is solubilized by the sample liquid may flow from the conjugate pad into the test line and may bind to the analyte at the test line, resulting in a coloured test line that indicates the lack of the target analyte in the sample liquid.
  • the conjugate particle-binding agent antibody that is solubilized by the sample liquid may flow from the conjugate pad into the test line and may bind to the analyte at the test line, resulting in a coloured test line that indicates the lack of the target analyte in the sample liquid.
  • the analyte may bind to the conjugate particle-binding agent antibody on the conjugate pad and may therefore prevent the conjugate particle-binding agent antibody from binding the analyte at the test line.
  • the test line may not change colour/fluoresce, indicating the presence of the analyte in the sample fluid.
  • the membrane or capture zone may optionally include a control line as is known in the art.
  • the control line may contain an immobilised antibody that binds to the free conjugate particle-binding agent resulting in a coloured control line, which confirms that the test has operated correctly regardless of whether or not the target analyte has been present in the sample.
  • the control line may contain an immobilised analyte molecule (or a protein-analyte complex) that binds to the free conjugate particle-binding agent antibody resulting in a coloured control line, which confirms that the test has operated correctly regardless of whether or not the target analyte has been present in the sample.
  • the LF device may be a multiplex assay device and therefore, in one embodiment, comprise multiples of the conjugate particles and multiple test and even control lines on the one device. These will, as appropriate, have different capture and binding agents to be binding partners of the further analytes being tested for. In each instance, at least one of the binding agents associated with one of the classes of conjugate particle or the capture agents associated with one of the test lines will be so associated by through oligomeric metal coordination complexes, as described herein. It will also be appreciated that the multiplex approach may take the form of an array of individual strips, each comprising a sample pad, conjugate pad, membrane with test line etc.
  • the LF device may employ (i) affinity clusters (of binding agents) associated with the mobile conjugate particles in the conjugate pad via the second metal coordination complexes; or (ii) affinity clusters (of capture agents) associated with and immobilised on the test line of the membrane via second metal coordination complexes; or (iii) both (i) and (ii) are present.
  • the LF device may additionally comprise a heterophile capture zone which may be included to capture and retain heterophile antibodies which can otherwise interfere with the assay sensitivity and specificity of desired analyte binding.
  • the heterophilic capture zone may, similarly to the test line described herein, have appropriate antibodies bound to a region of the membrane via oligomeric metal coordination complexes with the bound antibodies being anti- to the relevant heterophilic antibodies.
  • This heterophile capture zone may be located anywhere on the assay device prior to the test line and so may, for example, be located in the sample pad, the conjugate pad or the region of the membrane prior (in the direction of sample flow) to the test line.
  • the affinity cluster is directly associated with the second oligomeric coordination complex. [0083] The affinity cluster is directly associated with the second oligomeric coordination complex through coordinate bonds which are non-covalent bonds.
  • the affinity cluster is an interconnected network of affinity agents associated with and connected by the first oligomeric metal coordination complex.
  • the affinity cluster may be a polymeric interconnected affinity cluster particle comprising the plurality of affinity agents coordinately bonded with, and interconnected by, the first oligomeric metal coordination complex.
  • the affinity cluster has an average diameter of between 20 nm to less than 500 nm, 20 nm to less than 400 nm, 20 nm to less than 300 nm, 20 nm to less than 200 nm, 20 nm to less than 175 nm, between 30 nm to less than 500 nm, 30 nm to less than 400 nm, 30 nm to less than 300 nm, 30 nm to less than 200 nm, 30 nm to less than 175 nm, between 40 nm to less than 500 nm, 40 nm to less than 400 nm, 40 nm to less than 300 nm, 40 nm to less than 200 nm, 40 nm to less than 175 nm, between 50 nm to less than 500 nm, 50 nm to less than 400 nm, 50 nm to less than 300 nm, 50 nm to less than 200 nm, 50 nm to less than 175 n
  • the affinity cluster or polymer has an average diameter of between 20 nm to 250 nm or between 30 nm to 250 nm or between 40 nm to 250 nm.
  • nanosized affinity clusters can be consistently formed with affinity agents such as antibodies.
  • affinity agents such as antibodies.
  • the applicant has previously demonstrated the ability to form such nanosized clusters with synthetic polymers, such as PAA and CMC, but doing so with peptides and, particularly, large proteins is much more challenging.
  • the approach disclosed herein employing modified oligomeric metal coordination complexes provides for consistent outcomes in the affinity clusters which are further enhanced in terms of PDI with the use of elevated temperatures and appropriate buffer conditions as described herein.
  • the affinity agents may be selected from biomolecules specific for a target molecule. That is, the affinity agent biomolecule and the target molecule or analyte may be binding partners.
  • the plurality of affinity agents may be an at least one antigen agent.
  • the plurality of affinity agents may be a protein and/or nucleic acid-based affinity agent.
  • the plurality of affinity agents may be independently selected from the group consisting of an antibody, an antigenically reactive fragment of an antibody, an antigen, an epitope of an antigen, a monoclonal antibody, a polyclonal antibody, an antibody fragment, an antibody peptide, an antibody mimetic, an antibody fusion protein, a phage display, a nucleic acid aptamer, a fibronectin display, a peptide-nucleic acid aptamer, and a non-antibody protein scaffold.
  • the affinity agents may be independently selected from antigen binding proteins, such as polyclonal antibodies, monoclonal antibodies and antigen binding fragments thereof, that bind specifically to one or more of: SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis, malaria, respiratory syncytial virus (RSV), Ebola virus (EBOV), human cytomegalovirus (HCMV) and influenza.
  • antigen binding proteins such as polyclonal antibodies, monoclonal antibodies and antigen binding fragments thereof, that bind specifically to one or more of: SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis, malaria, respiratory syncytial virus (RSV), Ebola virus (EBOV), human cytomegalovirus (HCMV) and influenza.
  • the affinity agents may be independently selected from antigen binding proteins, such as antibodies and antigen binding fragments thereof, that specifically bind to CoV spike or nucleocapsid protein, influenza hemagglutinin or nucle
  • the affinity agents may be termed either binding agents or capture agents.
  • binding agents and capture agents may take the same form of biomolecules/affinity agents as described above, it may be convenient to refer to an affinity agent which is associated with a conjugate particle as a ‘binding agent’, based on its binding of the target analyte; and to refer to an affinity agent which is immobilised on a test line of the membrane as a ‘capture agent’ based on its specificity of binding for, and so capture of, the conjugate particle and bound analyte complex.
  • the substrate may be a conjugate particle or a membrane substrate.
  • the substrate when the affinity agent is a binding agent then the substrate is a conjugate particle and when the affinity agent is a capture agent then the substrate is a membrane substrate and preferably a test line thereof.
  • the polymer-like affinity cluster formed by cross-linking of the plurality of affinity agents and the first oligomeric metal coordination complex is a dynamic system due to the nature of the association between the two components.
  • the first oligomeric metal coordination complex is associated with the affinity agents through avidity or multi-component bonding and so the affinity agents are directly bonded to the first oligomeric metal coordination complex through multiple coordinate bond interactions the accumulated strength of which results in anchoring of the affinity agents to the first oligomeric metal coordination complex as if they were bonded via standard covalent bonding. This may be viewed as forming an interconnected affinity network in the form of a small cluster.
  • any individual coordination bond between the metal ion in the first oligomeric metal coordination complex and the affinity agents is relatively weak and can break as a result of a local stressor which allows for rotational freedom of movement or orientation allowing a significant portion of the affinity agents to be optimally functionally available.
  • PCT/AU2016/051132 Method of Controlled Competitive Exchange
  • the first agent capped metal complex is added in excess to the membrane so that there is residual coordination potential after the metal complex is coordinated to the substrate. This requires that the excess metal complex be washed off before competition with competing agent. If the metal complex was not added in excess to the available coordination sites of the substrate, the metal complex will exhaust all coordinating sites in binding the substrate and no competition occurs. In the case of porous membranes, it also leads to blockages of the pores. Controlling excess is especially difficult when only a part of a membranes needs to be treated such as in stripping membranes with affinity agent.
  • Example 3 and 4 disclosed in Method of Controlled Competitive Exchange exemplifies acetate (first agent) capped metal complexes bound to a nitrocellulose membrane (i.e. substrate) and exchanged with streptavidin (Example 3) and an antibody (Example 4).
  • a mixture of metal complex and affinity agent which may form soluble, functional affinity agent clusters of affinity agent cross-linked via metal complexes with other affinity agents.
  • Such a cross-linked network of affinity agents through metal complexes are not described in Method of Controlled Competitive Exchange.
  • the affinity agent is the first agent on the substrate there is no competition by a competing agent.
  • a metal complex - affinity agent complex i.e. an affinity agent cluster
  • this invention describes the formation of affinity agent clusters in solution by mixing an affinity agent with suitable modified metal complexes.
  • the polymer-like affinity cluster may be formed by cross-linking of the plurality of affinity agents and the first oligomeric metal coordination complex while maintaining functionality of original unlinked affinity agents.
  • the first oligomeric metal coordination complex and the second oligomeric coordination complex may be the same or different.
  • the metal ion of the first and/or second oligomeric metal coordination complex is selected from the group consisting of chromium, ruthenium, iron, cobalt, titanium, aluminium, zirconium, and combinations thereof.
  • the metal ion of the first and/or second oligomeric metal coordination complex is selected from the group consisting of chromium, ruthenium, titanium, iron, cobalt, aluminium, zirconium, rhodium and combinations thereof.
  • the metal ion of the first and/or second oligomeric metal coordination complex may be chromium.
  • the metal ion of the first and/or second oligomeric metal coordination complex may be present in any applicable oxidation state.
  • the metal ion may have an oxidation state selected from the group consisting of I, II, III, IV, V, or VI, as appropriate and obtainable under standard conditions for each individual metal. The person of skill in the art would be aware of which oxidation states are appropriate for each available metal.
  • the first oligomeric metal coordination complex is a chromium oligomeric metal coordination complex.
  • the second oligomeric metal coordination complex is a chromium oligomeric metal coordination complex.
  • the metal ion is a chromium ion
  • it is preferred that the chromium has an oxidation state of III.
  • the metal ion may be associated with any suitable counter-ions such as are well- known in metal-ligand coordination chemistry.
  • mixtures of different metal ions may be used, for example, to form a plurality of different oligomeric metal coordination complexes.
  • at least one metal ion is chromium.
  • the substrate may be associated directly or indirectly with the second oligomeric metal coordination complex.
  • the affinity cluster is associated with the first metal coordination complex-coated conjugate particle it may be that the conjugate particle has been coated, partially or fully, with a third metal coordination complex and a protein or other polymer has been bonded to that third metal coordination complex.
  • the second metal coordination complex may then be associated with that protein or polymer layer.
  • affinity clusters can be formed first, and then added to oligomeric metal coordination complex activated substrates.
  • Modified oligomeric metal coordination complex can be used to activate the substrate or non-modified oligomeric metal coordination complex may be first used to activate the substrate and these bound complexes subsequently modified via capping groups or other coordinating ligands to adjust the subsequent strength of association with the affinity cluster.
  • the affinity agent, substrate and modified oligomeric metal complex can be reacted simultaneously (one-pot approach) or varied by changing the order of addition to form small or larger clusters with stronger or weaker association to the substrate.
  • affinity clusters are associated, via second metal coordination complexes, with both the conjugate particle and the test line then the nature of the second metal coordination complex associated with the conjugate particle and the membrane of the test line may be the same or different.
  • the lateral flow (LF) device and the components thereof, may be as described in any one or more embodiments of the first aspect.
  • the lateral flow device comprises a sample pad; a conjugate pad; and a membrane.
  • the affinity agents may be binding agents or capture agents.
  • the substrate may be a conjugate particle or a membrane substrate.
  • the substrate when the affinity agent is a binding agent then the substrate is a conjugate particle and when the affinity agent is a capture agent then the substrate is a membrane substrate.
  • the first oligomeric metal coordination complex and the second oligomeric coordination complex may be the same or different.
  • the first oligomeric metal coordination complex is a chromium oligomeric metal coordination complex.
  • the substrate may be associated directly or indirectly with the second oligomeric metal coordination complex.
  • the contacting of any LF device described herein with the sample may be the contacting of the sample pad of the LF device with a liquid test sample.
  • the sample may be human or animal bodily fluid, such as one or more of urine, blood, serum, plasma, saliva, sweat, milk, mucous, semen, vaginal or urethral secretions, and the like.
  • the sample may also be a fluid taken from sources other than a human or an animal.
  • the sample may naturally be a liquid, may be a liquid diluted with another liquid, such as water, or may have originally been in a solid form (e.g., a tissue sample) and is treated to be in liquid form for the application to the LFA device.
  • the target analytes may be substances such as, without limitation, proteins, haptens, enzymes, hormones, infectious disease agents, immunoglobulins, polynucleotides, steroids, drugs, nucleic acids, markers for gene mutations, etc.
  • the method of the second aspect may further include the step of reading the outcome of the assay by observing the test line after a sufficient period of time. Whether stronger colour or fluorescence intensity or another readout is provided, increased analytical sensitivity is achieved as compared with assays using non-clusters and/or traditional covalent or passive binding of affinity agent to substrate.
  • the increased density of functional affinity agents increases the on-rate of the analyte to bind on the test line, and for the conjugate particle with binding agent to bind the analyte.
  • Specific binding (SB) is increased while non-specific binding (NSB) is reduced or minimised by the improved functionality of affinity agents, and optionally by removal of heterophilic antibodies by placement of other capture lines for removal of contaminants by the same method.
  • a method of forming a lateral flow device including the step of: incorporating (i) an affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex and (ii) a substrate associated with a second oligomeric metal coordination complex wherein the affinity cluster is associated with the substrate through the second oligomeric coordination complex, into a component of the device to thereby form the lateral flow device.
  • a method of forming a lateral flow device including the step of: incorporating
  • conjugate pad including mobile conjugate particles comprising binding agents associated with the conjugate particles
  • a membrane comprising immobilised capture agents associated with the membrane, wherein the binding agents are associated with the conjugate particles and/or the capture agents are associated with the membrane, by oligomeric metal coordination complexes , into the device to thereby form the lateral flow device.
  • a plurality of the binding agents and/or capture agents are interconnected by oligomeric metal coordination complexes to form a first affinity cluster and/or a second affinity cluster.
  • the first affinity cluster is associated with the conjugate particle and the second affinity cluster is associated with the membrane.
  • the first affinity cluster and/or the second affinity cluster are an interconnected network of affinity agents associated with and connected by the first oligomeric metal coordination complex.
  • the first affinity cluster is a binding agent cluster associated with conjugate particles by a second oligomeric metal coordination complex.
  • the second affinity cluster is a capture agent cluster associated with the membrane by a second oligomeric metal coordination.
  • the method of forming the lateral flow device may include the step of: incorporating:
  • an affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex
  • the method of forming the lateral flow device may include the step of: incorporating:
  • an affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex
  • the method of forming the lateral flow device may include the step of: incorporating:
  • a conjugate pad comprising a first affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex, and the first affinity cluster is associated with a conjugate particle and a second oligomeric metal coordination complex;
  • a membrane comprising a test line comprising a second affinity cluster comprising a plurality of affinity agents interconnected by a first oligomeric metal coordination complex, and the second affinity cluster associated with the membrane by a second oligomeric coordination complex, into the device to thereby form the lateral flow device.
  • the method of forming the lateral flow device may include the step of: incorporating:
  • a substrate associated with a second oligomeric metal coordination complex wherein the first affinity cluster associated with the substrate by the second oligomeric coordination complex is an affinity cluster associated with a conjugate particle and the second oligomeric metal coordination complex, and located within the conjugate pad, and wherein the second affinity cluster associated with the substrate by the second oligomeric coordination complex is an affinity cluster associated with and located within the membrane test line, into the device to thereby form the lateral flow device
  • the LF device is as described in any one or more embodiments of the first or second aspects.
  • the method of the third aspect may therefore include the step of forming the affinity cluster by providing a liquid formulation comprising a plurality of affinity agents and contacting this with a modified first oligomeric metal coordination complex.
  • Metals are known to form a range of oligomeric metal coordination complexes.
  • Preferred ligands for forming the oligomeric metal coordination complex are those that include nitrogen, oxygen, or sulfur as dative bond forming groups. More preferably, the dative bond forming groups are oxygen or nitrogen. Even more preferably, the dative bond forming group is an oxygen-containing group which assist in olation to form the oligomeric complexes. In embodiments, the oxygen-containing group is selected from the group consisting of oxides, hydroxides, water, sulphates, phosphates, or carboxylates.
  • the first and/or second oligomeric metal coordination complex is a chromium (III) oligomeric metal coordination complex.
  • the first and/or second oligomeric metal coordination complex is an oxo-bridged chromium (III) oligomeric coordination complex.
  • This complex may optionally be further oligomerised with one or more bridging couplings such as carboxylic acids, sulphates, phosphates and other multi-dentate ligands.
  • oligomeric metal coordination complex may be replaced by a dative bond with the substrate surface but when such oligomeric metal complexes are added in excess, and then the excess washed off the substrate, what remains is an oligomeric metal complex activated substrate which has several useful properties. Due to its size and oligomeric nature, on binding to a substrate there still remains coordination potential to bind affinity agents or other polymers or materials. The binding to affinity agents and the like is highly reactive due to the multiplicity of coordination sites which gives multi-component or avidity binding characteristics driven by multiple charge-charge and coordination interactions. As previously described, it is critical to first add such oligomeric metal complexes in excess to some substrate, and then wash off the excess to form an oligomeric metal complex activated substrate.
  • the oligomeric metal coordination complexes to which the affinity agents are exposed are modified in terms of a ‘tuning down’ of their reactivity. Without wishing to be bound by theory, it is believed this means that when a liquid formulation of the modified first oligomeric metal coordination complexes and the affinity agents in solution is generated, then the multiplicity of potential coordinating ligands naturally presenting on the affinity agents will have changed.
  • biological polymers such as, proteins include different types of ligands with different strengths of coordination to metal complexes.
  • capping agents sets some threshold by which only a limited number of ligands presenting on the affinity agent can coordinate. In such a situation, uncontrolled coordination of metal complexes to any ligand in the affinity agent is minimised, as only a limited number of ligands can coordinate per individual affinity agent which encourages intermolecular cross-linking of affinity agents by the oligomeric metal complexes.
  • first oligomeric metal coordination complexes may no longer exist as discrete oligomeric complexes or may not be able to be truly identified as such once the affinity cluster has formed. This is because the bonding with the affinity agents will result in multiple previously separate first oligomeric metal coordination complexes being bound to each affinity agent.
  • affinity cluster or polymer is formed with the formerly oligomeric metal coordination complexes essentially forming the strands or connections of the network between separate affinity agents as hubs.
  • the affinity or cluster or polymer may be viewed as polymeric, or at least ‘extended’ in nature even though it has been formed, in part, from oligomeric metal coordination complexes.
  • the degree of modification of any particular oligomeric metal coordination complex can be achieved, for example the extent or excess of smaller coordinating agents (described as capping groups), to form the corresponding modified oligomeric metal coordination complex.
  • coordinating agents described as capping groups
  • a larger molecule such as an affinity agent, having multiple coordination potential will, over time, compete off a small coordinating agent.
  • the stability of such coordinating agents to exchange is dependent on the pH, the basic coordination strength, multi-valency, method of synthesis and as well any extra coordinating agents (same or different) are present in the liquid formulation.
  • the use of such modifications can delay coordination of such modified oligomeric metal complexes to affinity agent from minutes, to hours or even days.
  • the modified first and/or second oligomeric metal coordination complex may be defined as a reduced reactivity oligomeric metal coordination complex, especially relative to the same oligomeric metal complex which is fully hydrated (for example a complex formed with non-coordinating counter-ions), such as described as Solution 1 in the Examples.
  • the modified first and/or second oligomeric metal coordination complex is modified such that its reactivity is reduced as compared with the same oligomeric metal coordination complex which has not been so modified, for example the same oligomeric metal coordination complex but with non-coordinating counter-ions in a fully hydrated state (for example in the form of a hexahydrate).
  • the unmodified metal coordination complex has non- or weakly coordination anions as ligands.
  • the reduced reactivity of the modified first and/or second oligomeric metal coordination complex may be defined as a reduced level of reactivity as compared with an otherwise corresponding unmodified oligomeric metal complex, for example an unmodified oxo-bridged chromium (III) complex.
  • the unmodified metal complex may be a fully hydrated metal complex.
  • the oxo-bridged chromium (III) complex may be a fully hydrated oxo-bridged chromium (III) complex.
  • the unmodified oxo-bridged chromium (III) complex used for comparison purposes may be that as formed in ‘Solution T of Example 1 in the examples section.
  • the modified first and/or second oligomeric metal coordination complex is modified such that its reactivity to, or speed to bond with, the affinity agents is reduced as compared with the same oligomeric metal coordination complex which has not been so modified.
  • the affinity agent used to assess the reduced reactivity by comparison to that with an unmodified oligomeric metal coordination complex is an antibody such as monoclonal antibodies to virus antigens such as SARS-CoV2, Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies.
  • the reduced reactivity of the modified first and/or second oligomeric metal coordination complex may be defined as a reduced level of reactivity with monoclonal antibodies to virus antigens, such as SARS-CoV-2 virus, or polyclonal antibodies, such as goat anti-mouse antibodies, as compared with that of a corresponding unmodified metal complex, especially a corresponding fully hydrated metal complex (such a complex has non- or weakly coordination anions as ligands).
  • virus antigens such as SARS-CoV-2 virus
  • polyclonal antibodies such as goat anti-mouse antibodies
  • the reduced reactivity of the modified first and/or second oligomeric metal coordination complex may be defined as a reduced level of reactivity with monoclonal antibodies to virus antigens such as SARS-CoV-2 virus or polyclonal antibodies such as goat anti-mouse antibodies as compared with that of an oxo-bridged chromium (III) complex.
  • virus antigens such as SARS-CoV-2 virus or polyclonal antibodies such as goat anti-mouse antibodies
  • the oxo-bridged chromium (III) complex used for comparison purposes may be that as formed in Solution 1 of Example 1 in the examples section.
  • the at least one modified first and/or second metal coordination complex is a capped metal coordination complex, which may otherwise be referred to as one having stronger coordinating ligands as capping agents and/or having stronger competing coordinating ligands provided in solution.
  • the modified first and/or second oligomeric metal coordination complex has been modified to display capping agent groups coordinately bound to the metal of the first and/or second oligomeric metal coordination complex.
  • the capping agents will alter the reaction kinetics of the now modified first and/or second oligomeric metal coordination complex with the ligands on the affinity agents as they will be more resistant to being displaced (due to their greater relative coordinating potential) than, for example, simple counterions.
  • the moieties of the first and/or second metal coordination complexes will therefore react more slowly and with a more limited selection of ligands on the affinity agent depending on the type and concentration of capping agent to form an appropriate affinity cluster.
  • the method may further include the step of selecting or controlling the relative extent of the total coordination capacity of the first and/or second oligomeric metal coordination complex which is taken up by the capping agent groups, such as carboxylate or phosphate capping or coordinating groups. That is, there may be benefits in choosing or modifying the % of the total coordination capacity of the metal ions of the first and/or second oligomeric metal coordination taken up by capping agents (as measured by that remaining following formation of the first and/or second oligomeric metal coordination complex itself - as a coordination interaction is reversible, this percentage is the starting percentage taken up by the capping agents).
  • the % of the total coordination capacity taken up by capping or coordinating agents may be greater than 10%, or 20% or 30% or 40% or 50% any of which values may be combined to form a range with a maximum value of less than 600%, 400%, 200% or 100.
  • the capping or coordination agents is in excess of the available coordination potential of the oligomeric metal complex, this excess leads to greater competition for coordination to the available oligomeric metal complex.
  • the degree of excess also changes the reaction kinetics of the now modified first and/or second oligomeric metal coordination complex with the affinity agent as there are more capping agents in competition.
  • competing coordinating ligands may be supplied as part of the buffer solution in which the affinity cluster is being formed.
  • Components comprising different buffer salts or other additives can also function as capping or coordinating agents when they exchange with the original ligand on the first metal coordination complex and become bound and can replace or augment capping agents on the oligomeric metal complex to further tune the reaction kinetics of the now modified oligomeric metal coordination complex.
  • Appropriate capping agents will therefore be those, which slow down coordination of the modified first and/or second oligomeric metal coordination complexes with the affinity agent but do not prevent it.
  • affinity agent clusters of any desired size can be formed with some appropriate level of intermolecular coordination to maintain a stable cluster. Without this control, such as in the approach of binding affinity agents using standard unmodified oligomeric metal coordination complexes, the metal complexes will simply form tightly bound aggregates with the affinity agents and will not provide for appropriate functionality of said biomolecules.
  • the displacement of the capping agents should occur over an appropriate commercial timeframe, which can be easily tested by running parallel reactions of oligomeric first and/or second metal coordination complexes modified with different capping agents and exposed to the same biomolecules.
  • the level of functionality of the affinity agent clusters can be tested by running parallel reactions with different ratios of metal complex and capping groups relative to the amount of affinity agent.
  • useful capping or coordinating agents may be those that include nitrogen, oxygen, or sulphur as dative bond forming groups. More preferably, the dative bond forming groups of the capping agent are oxygen or nitrogen. Even more preferably, the capping or coordinating group is one comprising a dative bond forming group which is an oxygen containing group.
  • the oxygen containing group of the capping or coordinating agent is selected from the group consisting of sulphates, phosphates, carboxylates, sulphonic acids and phosphonic acids.
  • the capping or coordinating agent may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, sulphate, phosphate, and hydroxy acetate.
  • the capping group may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, citrate, sulphate, phosphate, an amino acid, and hydroxyacetate.
  • the capping or coordinating agent may be selected from the group consisting of formate, propionate, oxalate, malonate, succinate, glutarate, maleate, citrate, aconitate, sulphate, phosphate, and hydroxyacetate.
  • the capping or coordinating agent may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, citrate, sulphate, phosphate, an amino acid, naphthalene acetate, and hydroxyacetate.
  • the capping or coordinating agent may be selected from the group consisting of oxalate, malonate, succinate, glutarate, adipate, maleate, citrate, and aconitate.
  • the capping or coordinating agent may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, glutarate, maleate, citrate, and aconitate.
  • the capping or coordinating agent may be selected from the group consisting of acetate, oxalate, malonate, succinate, and citrate.
  • the capping group may be selected from the group consisting of acetate, oxalate, phosphate and succinate.
  • the capping group may be selected from the group consisting of acetate, oxalate, and succinate.
  • the capping or coordinating agent may be oxalate.
  • the capping or coordinating agent may be succinate.
  • the capping or coordinating agent may be carboxylate or phosphate or a combination thereof, preferably carboxylate.
  • the carboxylate may be a dicarboxylate or a tricarboxylate, preferably a dicarboxylate.
  • the capping or coordinating agent is a monodentate, bidentate or multidentate capping agent. In embodiments, the capping agent is a monodentate or bidentate capping agent.
  • the capping or coordinating agent has a molecular mass of less than 1000 Daltons, or less than 500 Daltons, or less than 400 Daltons, or less than 300 Daltons. Any of these values may be combined with a lower value of 10, 30 or 50 Daltons to form a range of molecular mass values for the capping agent such as 10 to 1000, 10 to 500, 10 to 400 or 10 to 300 Daltons.
  • the capping or coordinating agent may be a dicarboxylate or tricarboxylate having a molecular mass of less than 1000 Daltons, or less than 500 Daltons, or less than 400 Daltons, or less than 300 Daltons. Any of these values may be combined with a lower value of 10, 30 or 50 Daltons to form a range of molecular mass values for the capping agent such as 10 to 1000, 10 to 500, 10 to 400 or 10 to 300 Daltons.
  • the capping or coordinating agent is not simply a counterion of the oligomeric metal coordination complex or a group donated by a base.
  • a base such as ethylene diamine
  • the capping agent is not one donated by a base, including ethylene diamine.
  • the step of forming the modified first and/or second oligomeric metal coordination complex may include contacting the oligomeric metal coordination complex with a solution comprising a capping agent, such as a carboxylate or phosphate ligand-containing solution.
  • a capping agent such as a carboxylate or phosphate ligand-containing solution.
  • the method may further include the step of adjusting the pH of a liquid solution comprising the modified metal coordination complex and/or controlling the temperature of the liquid solution to be between 15 to 45 °C or 15 to 40 °C or 15 to 38 °C or 15 to 30 °C.
  • the modified first and/or second oligomeric chromium metal coordination complex is an oligomeric chromium metal coordination complex comprising a degree of carboxylate or phosphate capping agents at the time of contacting the affinity agents to form the affinity cluster.
  • the liquid formulation comprising the affinity agents is contacted with the modified oligomeric chromium metal coordination complex at a temperature of greater than 15 °C and less than 45 °C, or greater than 20 °C and less than 45 °C, or greater than 25 °C and less than 42 °C, or greater than 25 °C and less than 40 °C, or greater than 33 °C and less than 45 °C, or greater than 33 °C and less than 42 °C, or greater than 33 °C and less than 40 °C, or greater than 35 °C and less than 45 °C, or greater than 35 °C and less than 42 °C, or greater than 35 °C and less than 40 °C.
  • the liquid formulation comprising the affinity agents is contacted with the modified oligomeric chromium metal coordination complex at a temperature of about 37 °C.
  • Lower temperatures may be appropriate for storage of the liquid formulation and the higher end of these ranges may be appropriate for cluster formation.
  • the affinity cluster is a binding agent cluster associated with conjugate particles, via the second oligomeric metal coordination complex.
  • the formation of the affinity cluster itself requires the use of modified first oligomeric metal coordination complexes but in such embodiments it may be also preferred that the second oligomeric metal coordination complex is a modified oligomeric metal coordination complex as outlined above. This is because the affinity clusters may bind less strongly to these second modified oligomeric metal coordination complexes in such a way that more of the binding agents are functionally available for binding with the analyte, thereby effectively increasing the density of available binding agents.
  • the binding events with the affinity clusters may be much more rapid and strong and would likely result in more binding agents being involved in bonding with the metal ions of the second oligomeric metal coordination complexes and so not available for analyte binding.
  • the capping group is part of the buffer in which the affinity agent cluster is bound to the conjugate particles.
  • the affinity cluster is immobilised on the test line of the LFA device membrane.
  • the second oligomeric metal coordination complexes may be modified or unmodified. The considerations are the same and it may be preferred that the affinity clusters are associated with second modified oligomeric metal coordination complexes on the membrane test line.
  • that binding may be binding through oligomeric metal coordination complexes available from the affinity cluster itself and/or binding through excess oligomeric metal coordination complexes, which we present in the liquid formulation. This approach may be preferred as it may be practically challenging to accurately coat this region of the membrane and then directly coat again the same region with the affinity cluster.
  • antibodies may be located at any other region or zone of the LFA device. As discussed above, it may be desirable to include agents to capture any heterophile antibodies or other interference agents which may be present in the sample. Further, the control line will be provided with suitable antibodies as previously described. At any of these regions or zones or lines the antibodies may be associated with the relevant component of the LFA device via third, fourth or further oligomeric metal coordination complexes. In some embodiments these complexes may be applied to the LFA device as unmodified oligomeric metal coordination complexes. In alternative embodiments, some or all of the oligomeric metal coordination complexes may be modified oligomeric metal coordination complexes. The choice may depend simply upon convenience or upon the need for sensitivity at that particular region or zone.
  • a liquid carrier is used for applying the second oligomeric metal coordination complexes to the relevant substrate.
  • this liquid carrier may be an aqueous carrier.
  • the step of contacting the membrane with the liquid carrier may be a step of striping the membrane.
  • the contacting will only occur on portions of the membrane, as described, to form test and/or control and/or interference removal regions for the assay.
  • Striping of the test and control lines onto a membrane is well-known in the art with a number of commercially available dispensing instruments available.
  • Non-contact dispensing such as spray or jetting typically requires less volume to stripe, but can result in greater run-to-run variability.
  • Contact dispensing systems on the other hand have relatively low run-to-run variability, but require additional volume in order to stripe the same amount of material.
  • the choice of the striping approach and the specifics of the liquid formulation to allow for effective striping are well understood in the art and are easily tailored to provide the appropriate width of striped area, reagent density and the like.
  • the affinity clusters and conjugate particles may be incorporated by standard approaches for loading a conjugate pad with binding reagents and will include a step of removal of any liquid carrier.
  • the affinity clusters and second oligomeric metal coordination complex may be applied in the same liquid formulation to the LFA device.
  • the second oligomeric metal coordination complex may be coated on the conjugate particle or be applied to the test line for binding there.
  • the metal ion of the first and second oligomeric metal coordination complexes may be the same and may preferably be chromium as previously described. If the second oligomeric metal coordination complexes are applied to the LFA device separately to the affinity clusters then it may be possible that the metal ion of the first and second oligomeric metal coordination complexes are different although chromium is generally preferred.
  • Example 1 Preparation of metal coordination complex solutions.
  • oligomeric metal coordination complexes are described. Depending on the metal ion, salt, the base, the final pH and other ligands used, the metal coordination complex solutions exhibit different binding properties.
  • chromium perchlorate hexahydrate 45.9 g was dissolved into 480 mL of purified water and mixed thoroughly until all solid dissolved.
  • 8 mL of ethylene diamine (EDA) solution was added to 490 mL of purified water. The solutions were combined by the dropwise addition of the EDA solution into the chromium salt solution and stirred overnight at room temperature, and then left to equilibrate to a pH of approximately 4.5.
  • chromium perchlorate and ethylenediamine solution can be used to generate solutions having a different pH such as pH 3.0, 4.0, pH 5.0 or some other pH.
  • chromium perchlorate hexahydrate (103.5 g) was dissolved into 1000 mL of purified water and mixed thoroughly until all solid dissolved. 8 mL of ethylene diamine solution was added to 1000 mL of purified water. The solutions were combined by the dropwise addition of the EDA solution into the chromium salt solution, and stirred overnight at room temperature, and then left to equilibrate to the desired pH.
  • BSA Bovine Serum Albumin
  • Table 1 shows the trends with metal complex to oxalic acid ratio of 1 :4 (Solution 5B) and Table 2 shows the trends with metal complex to succinic acid ratio of 1 :4 (Solution 6C).
  • the rate of BSA cluster formation is different and changes with not only the capping agent but also the ratio between the protein and the modified oligomeric metal complex.
  • Table 1- the zeta size in nanometres of the Bovine Serum Albumin (BSA) clusters formed using oxalic acid capped (Solution 5B) at different ratios to BSA.
  • BSA Bovine Serum Albumin
  • Table 2 the zeta size in nanometres of the Bovine Serum Albumin (BSA) clusters formed using succinic acid capped (Solution 6C) at different ratios to BSA.
  • BSA Bovine Serum Albumin
  • the capping groups allow fine control over the rate of protein cluster formation and its final size. At some set concentration of oligomeric metal complex, a lower excess of suitable capping groups increases the availability of ligands on the protein that can potentially coordinate with the metal complex. The trend would be towards more intramolecular interactions within a protein which can lead to functional damage. At the extreme, it results in insoluble protein aggregates. Alternatively, larger excess of capping groups will restrict coordination only to the most reactive of ligands on the protein. In this case, the trend is towards more intermolecular cross-linking between proteins.
  • Example 3 Binding Antibody to metal-coordination complex activated particles (Control).
  • a suspension of Europium-chelate latex particles (Merck ref# F1-Eu-030) was sonicated and then the particles separated from the supernatant by centrifugation at 12000 g for 10 minutes. After removing the supernatant, the particles were redispersed in an equal volume of Solution 1. After constant mixing on a rotary mixer for 3 hours, the particles were then separated from Solution 1 by centrifugation and washed twice with DI water. Particles were then checked for monodispersity and size using laser diffraction using a zetasizer (Malvern: Model nanoseries Z). Activation was demonstrated by a change in charge from negative to positive. Their concentration was evaluated against a known standard using the fluorescent readout of a spectrophotometer (Tecan model Infinite M200 Pro). The final concentration was adjusted to 10mg/mL.
  • the suspension was again centrifuged to remove supernatant and resuspended in an equal volume of 50mM TRIS buffer, pH8.0. After repeating this wash step, the conjugated particle was left in TRIS buffer at a concentration of 10mg/mL.
  • Example 4 Binding Antibody Clusters formed by modified metal coordination complexes to unmodified metal coordination - activated particles.
  • Stock Solution 4 (oxalic modified oligomeric metal coordination complexes) was diluted to 2mM with DI water and stock COVID-19 mAb was diluted to 1.2mg/mL in 25mM MES buffer, pH6.0.
  • a lateral flow half test strip was used to compare any differences between the Control (Example 3) and antibody cluster (Example 4) conjugates.
  • goat anti mouse IgG was diluted to 0.2 mg/mL in carbonate buffer (pH8,5).
  • COVID-19 mAb capture was diluted to 1 mg/mL in carbonate buffer and then mixed with 1 L of 100mg/mL BSA to give a BSA concentration of 1 mg/mL BSA.
  • the COVID-19 mAb and GAM solutions were striped onto nitrocellulose membrane on a plastic support (HF090 card HF090MC100, Millipore) using the Linomat V (CAMAG). After striping, the ligand membrane was dried for 2 hours at 37°C in a fan forced incubator. The membrane was then stored in a sealed foil pouch with desiccant until use.
  • the COVID-19 antigen was used at 5 different concentrations; 25, 50, 100, 200 and 400 pg/ml, and Blank was also included.
  • the stock 10mg/mL of conjugate particles were diluted to 2pg/mL in TRIS buffer, pH 8.0 with 1% TWEEN + 0.25% BSA. 20pL was used for each strip.
  • Antigen capture was detected using a fluorescent reader (Axxin). As shown in Table 4 and Figure 6, the antibody cluster conjugates, formed with Solution 4, had different outcomes indicating that the functional activity of antibodies in the clusters were different.
  • Example 8 Comparison of Antibody Cluster vs Non-cluster (IV): The Effect of different Ligand Concentrations to Metal Coordination Complex.
  • a lateral flow half test strip was used to compare any differences in outcome with Solution 5A (in comparison to Solution 4) in the formation of antibody clusters.
  • conjugates Control and Solution 5A mediated antibody clusters
  • three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.
  • Example 9 Comparison of Antibody Cluster vs Non-cluster (V): Use of Gold Nanoparticles.
  • a lateral flow half test was used to compare any differences between antibodies, clusters vs non-cluster, when the detection antibody was passively immobilized on AU40 nanoparticles.
  • antibody clusters formed from, A., 10pL of 1 mM Solution 4 with 10pL of 1200pg/ml COVID-19 mAb, and B., 10pL of 0.5mM Solution 5A with 10pL of 1200pg/ml COVID-19 mAb, mixed for 30 mins and then diluted to a final concentration of 3.2pg/ml. These two clusters were compared to the Control (mAb at 3.2pg/ml without any metal complex).
  • Gold colloids (1mL at OD1) were added to 3 tubes and centrifuged to form pellets. After removing 500 pL of supernatant, the pellet was vortexed to resuspend and disperse the colloids. After adding a 500 pL of mAb solution (clusters and Control), the tubes were vortexed to fully disperse the colloids and left on a tube rotator at 25rpm for 1 hr at room temperature. 10%BSA (50pL) blocking buffer was then added and the tubes vortexed and left on a rotator for another 1 hr. The tubes were placed in the centrifuge and the samples were spun down to remove supernatant. The pellet was resuspended in 1000 pL 2mM Boric acid, 0.05% sodium azide buffer, pH 9.0. As before, the size and zeta potential were measured using a Zeta Sizer and shown in Table 5.
  • Table 5 Zeta Size and Potential of mAb conjugated Gold nanoparticles comparing clusters formed with Solution 4 and 5A.
  • mAb on gold colloids were assessed on a test line formed with 0.5mg/ml SARS- CoV Ab + 1mg/ml BSA and a Control Line formed with 0.2mg/ml goat anti-mouse antibody. The outcome of these half strip tests is shown in Figure 11.
  • Example 10 Comparison of Antibody Cluster vs Non-cluster on Membranes (Control).
  • a mouse monoclonal antibody for FluA (Cat#CO1736, Meridian) was conjugated to a nanoparticle (detection moiety) and captured with the ligand, goat anti-mouse antibody (Lampire), striped on a nitrocellulose membrane on a plastic support (HF090 card HF090MC100, Millipore).
  • the analyte/detection moiety mouse IgG antibody
  • Europium nanoparticles Merck Ref#F1-Eu-030
  • Solution 1 The analyte/detection moiety
  • Europium-chelate latex particles were separated from solution by centrifugation and then the particles were redispersed in Solution 1 to a concentration of 2mg/mL. Particles were then separated from Solution 1 by centrifugation and washed twice with DI water. 0.04 pg of mouse IgG conjugate was used for each strip.
  • Analyte bound to the capture ligand was detected using a fluorescent reader (Axxin).
  • the goat anti-mouse antibody was immobilised onto the membrane in the presence of oxalate capped metal coordination complexes (Solution 4).
  • 25 pl of goat anti-mouse antibody 0.5 mg/ml
  • 3 different striping buffers MES buffer, pH 5.2 and pH 6.0, and Carbonate buffer, pH 8.5
  • MES buffer, pH 5.2 and pH 6.0, and Carbonate buffer, pH 8.5 were vortexed and mixed with 25 pl of both 0.2 and 1.0 mM of Solution 4 for 20 mins to form a final 0.25 mg/ml antibody in 0.1- and 0.5-mM Solution 3.
  • the controlled application was executed using the Linomat V (CAMAG) at a volume of 1 pL/cm.
  • CAMAG Linomat V
  • the ligand membrane was dried for 2 hours at 37°C in a fan forced incubator. The membrane was then stored in a sealed foil pouch with desiccant until use. [00215]
  • the Control for this example is passive immobilization of ligand onto nitrocellulose membrane.
  • the GAM antibody in Carbonate buffer, pH 8.5 gave the best results so this was used as the benchmark for comparison purposes.
  • Example 11 Comparison of Antibody Cluster vs Non-cluster: Striping using higher concentration.
  • the Antibody and Solution 4 were mixed and striped onto the membrane between 10 and 20 mins.
  • 25 pl of goat anti-mouse antibody (1.0 mg/ml) in MES buffer at pH 6.0 was mixed with 25 pl of both 2.0 and 4.0 mM of Solution 3 to form a final 0.5 mg/ml antibody in 1.0- and 2.0 mM Solution 4.
  • two conjugate loadings of 0.04 pg (Control) and 0.08 pg were also compared. All other conditions were the same as in the previous Example.
  • This example shows a clear increase (approx. 50%) in signal compared to passive (Figure 13).
  • Example 12 Striping Antibody/Metal Complex Clusters to Blocked Membranes.
  • the Antibody and Solution 4 were mixed and striped onto the membrane (10 to 20 mins).
  • the procedure is as described in Example 11 but, in this example, the membrane was ‘pre-blocked’ using 5 mg/ml BSA (A3070, Sigma) in either MES pH 6.0 or carbonate pH 8.5 (for the Control) buffer.
  • BSA BSA
  • the signal for the Control was depressed compared to using non-blocked membranes but there was still partial binding.
  • certain antibody/metal complex mixtures gave almost 3x increase in signal when compared to the Control ( Figure 14). While passive binding could not be eliminated, there seems to be enhanced binding in the case of antibody clusters when compared to using non-blocked membranes shown in Figure 13.
  • Example 13 Striping Antibody/Metal Complex Clusters formed using Alternative Capping Agent.
  • the Antibody and Solution 6C were mixed and striped onto the membrane (10 to 20 mins).
  • the procedure is as described in Example 4 except a different buffer and metal complex was used.
  • 25 l of goat anti-mouse antibody (1.0 mg/ml) in carbonate buffer at pH 8.5. was mixed with 25 pl of both 1.0, 2.0 and 4.0 mM of Solution 6C to form a final 0.5 mg/ml antibody in 0.5-, 1.0- and 2.0 mM Solution 6C.
  • the clusters formed in carbonate buffer gave even better results in the case of 2mM Solution 6C ( Figure 15).
  • Example 14 Comparison of Antibody/Metal Complex Clusters in a Sandwich Assay.
  • a mouse monoclonal antibody for FluA (Cat#10-l50H, Fitzgerald) was conjugated to Europium nanoparticles (Merck Ref#F1-Eu-030) as the detection moiety and captured on two test lines comprising an anti-FluA antibody (Cat# 7304, Medix) and an antiFluB antibody (Cat#9906, Medix) diluted to 0.5mg/mL in carbonate buffer striped onto nitrocellulose membrane on a plastic support (HF090 card HF090MC100, Millipore) using the Linomat V (CAMAG).
  • the membrane was blocked with 5mg/ml BSA in carbonate buffer prior to striping the test lines. After striping, the ligand membrane was dried for 2 hours at 37°C in a fan forced incubator. The membrane was then stored in a sealed foil pouch with desiccant until use.
  • the Flu A antigen was used at 3 different concentrations; 2.75, 5.5 and 11 ng/ml in PBS, 1 %BSA, pH7.4, and Blank was also included.
  • the stock 10mg/mL of conjugate particles were diluted to 2pg/mL in Lysis buffer (Tris buffer pH8, containing N-lauroyl sarcosine sodium salt, L-aspartic acid, Tergitol, sodium dodecyl sulfate sodium deoxycholate and sodium azide) and 25pL was used for each strip.
  • Antigen capture was detected using a fluorescent reader (Axxin).
  • the antibody cluster formed with Solution 6C had different outcomes indicating that the functional activity of antibodies in the clusters were different.
  • the clusters gave low background signals and low cross reactivity to the Flu B antigen compared to the Control.
  • the cluster formed with 2mM Solution 6C there was significant improvement in Sn/SO.
  • the clear difference in performance between the clusters and Control indicate that the functional activity can be manipulated by the amount and type of modified metal coordination complexes.

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Abstract

La présente invention concerne de manière générale une approche et un procédé pour améliorer la précision de détection et les performances de dispositifs de détection d'analytes rapides, tels que des dosages immunologiques à écoulement latéral. En particulier, la présente invention concerne de manière générale un dispositif à écoulement latéral amélioré et un procédé de détection d'un analyte l'utilisant.
PCT/AU2022/051210 2021-10-08 2022-10-07 Dosage à écoulement latéral WO2023056528A1 (fr)

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