FIELD OF THE INVENTION

[0001] The disclosure relates to a method of bioconjugation where two or more biomolecules are conjugated via metal coordination complexes. More particularly, the disclosure relates to the use of certain metal coordination complexes to directly conjugate two or more biomolecules, including peptides, polypeptides, proteins and the like.

BACKGROUND OF THE INVENTION

[0002] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

[0003] Cross-linking agents are used in a wide variety of life sciences applications. In polymer chemistry cross-linking describes the formation of a covalent bond between two polymer chains and, depending on the type of cross-linker and its cross-linking density, different material properties can be created from the same starting polymers. Where proteins and other biomolecules are cross-linked the technique may be referred to as bioconjugation. Commonly, three different types or cross-linkers are described: homobifunctional, heterobifunctional and photoreactive. These cross-linking agents have been used for protein interaction studies, histochemistry, sterilisation, conjugate formation and many other applications. In some applications, it is important to maintain the functionality of the protein after cross-linking. One example is to maintain antibody - antigen recognition in challenging areas such as targeted nanomedicine. There are many different proposed solutions but more sophisticated strategies that increase the likelihood of maintaining protein functionality are generally more synthetically complex leading to challenges such as high manufacturing costs as well as achieving consistency and reproducibility of product.

[0004] Homobifunctional cross-linking agents are those that contain two or more identical reactive ends capable of coupling to specific functional groups such as carboxylic acids, primary amines, sulfhydryls, etc. , on proteins or other molecules of interest. One such cross-linker is glutaraldehyde, a linear dialdehyde, but there are many others commercially available. These cross-linkers randomly cross-link like functional groups and, while useful in limited situations, can easily create a broad range of poorly defined conjugates including large, polymerised protein aggregates.

[0005] Cross-linking agents that may respond to inherent stresses or changes in the system are generally classed under reversible cross-linking chemistries and are often applied to self-healing materials. Metal ions have been included as reversible crosslinking agents with different binding strengths depending on the metal ion and the polymer being cross-linked. One such example, used to maintain antibody functionality, is immobilization of the antibodies onto nanomaterials coated with metal - phenolic networks such as Fe ¹¹¹ , Co", Cu", Ni" or Zn" with tannic acids (Nano Lett. 2020, 20, 4, 2660-2666). It was found that such networks (and especially with Co") preferentially immobilized antibodies via the Fc region and improved association with antigen. However, this approach simply binds antibodies to nanomaterials and is not a solution phase method of cross-linking two or more proteins. For solution phase cross-linking, it would be necessary to synthesize such a metal-phenolic network onto one protein, a tedious synthetic process even in the unlikely event that the protein so modified would maintain its functional structure.

[0006] Another approach employing metal ions is described in Immobilised Metal ion Affinity Chromatography (IMAC) (lleda, E.K.M., Gout, P.W., and Morganti, L. J. Chromatography A, 988 (2003) 1-23). In IMAC, the metal ions are immobilised through metal chelating groups covalently attached to some solid support with free coordination sites to which protein can bind through the poly-histidine tag. Subsequently, the bound protein can be released by competition with imidazole and other such chelating agents. As with metal - phenolic networks, the technology is an immobilisation of a protein to a solid support and not a method of cross-linking proteins in solution. Even if one can incorporate a metal ion into one protein in order to bind a different protein having a polyhistidine tag, this interaction is an intrinsically low affinity interaction and would most likely simply dissociate under conditions commonly encountered in immunoassays.

[0007] Another approach that uses metal ions to form coordination complexes between proteins and synthetic materials without the need for prior modification of the protein (such as the addition of poly-histidine tags) is described in Use of Metal Complexes (International publication no. WO 2006/002472) and Conjugating Molecules to Particles (International publication no. WO 2015/021509), both by the present applicant. But as with other approaches described, the method involves binding of a protein onto some form of solid support, such as particles.

[0008] The methods as described in the PCT publications mentioned above, Use of Metal Complexes and Conjugating Molecules to Particles, in the name of the present applicant mitigated the weaknesses of some of the approaches of the prior art by allowing quantitative coupling of antigen without agglutination, without the need for tight control over ratios and concentrations of reagents, and provided formation of a stable metal coordinated particle for later use. These benefits, however, absolutely required the presence of some solid support, such as a particle. Metal complex activated particles were understood to provide for rapid kinetics of binding of target molecules such that if two different target molecules were added as a mixture in a certain ratio then the rate of incorporation onto such an activated particle would largely reflect this ratio, thereby providing for simple batch production and improved reproducibility. The approach could not employ metal complexes for cross-linking proteins or other biomolecules in solution as the reaction kinetics were simply too rapid to allow any control over such solution phase cross-linking of proteins or other biomolecules.

SUMMARY OF THE INVENTION

[0009] It would be desirable to provide a simple homobifunctional cross-linking agent that allowed the formation of uniform bioconjugates or discrete biomolecule clusters in solution. Such an approach may also be useful for the formation of gels and resins incorporating a relatively uniform level of cross-linking and still including functional biomolecules, such as proteins, within its network.

[0010] Further, there is a need for a simple homobifunctional cross-linker that provides appropriate control over its reactivity to allow for formation of relatively uniform bioconjugates or discrete protein clusters comprising one or more types of different proteins including but not limited to IgM mimics, antibody-enzyme or antibody- streptavidin conjugates, and the like. Additionally, it would be useful if such a crosslinking agent had the ability to minimise any likely protein functional damage.

[0011] The present disclosure addresses one or more of these needs or provides a solution or a useful alternative to one or more of the problems or approaches of the prior art. The present disclosure provides simple, one step cross-linking agents which are suitable for cross-linking biomolecules, such as proteins, in the solution phase to form uniform bioconjugates, protein clusters and/or protein gels of advantageously uniform cross-linking density while still maintaining protein function. The approach employs suitable metal complexes with a modified or tailored reactivity to provide for appropriate stable cross-linking while minimising any functional/conformational damage and, optionally, employs suitable buffer conditions to assist on modification of reactivity.

[0012] In a first aspect of the disclosure, although not necessarily the broadest aspect, there is provided a biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex.

[0013] In a second aspect of the disclosure, there is provided a method of forming a biomolecule conjugate, the biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex, the method including the steps of:

(a) providing a liquid formulation comprising two or more biomolecules;

(b) contacting the liquid formulation comprising the two or more biomolecules with a metal coordination complex, to thereby form the biomolecule conjugate.

[0014] In embodiments, the liquid formulation comprising two or more biomolecules may be a liquid formulation comprising a population of biomolecules.

[0015] The population of biomolecules may be population of biomolecules which are all of a single type or the population may be a mixed population of two or more populations of different biomolecules.

[0016] In embodiments the two or more biomolecules or population of biomolecules are in the solution phase in the liquid formulation at the time of forming the biomolecule conjugate.

[0017] In a third aspect of the disclosure, there is provided a functional substrate comprising at least one biomolecule conjugate, the biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex, and the at least one biomolecule conjugate associated with a substrate material.

[0018] In a fourth aspect of the disclosure, there is provided a method of forming a functional substrate comprising at least one biomolecule conjugate including the steps of:

(a) providing a biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex;

(b) contacting the biomolecule conjugate with a substrate preformulation, the substrate preformulation adapted to form a substrate; and

(c) allowing the biomolecule conjugate to be incorporated into the substrate as it forms, to thereby form the functional substrate.

[0019] In embodiments of the first to fourth aspects, the two or more biomolecules may be the same or different. [0020] In embodiments of the first to fourth aspects, the two or more biomolecules may be selected from the group consisting of proteins, polypeptides, oligopeptides and peptides.

[0021] In embodiments of the first to fourth aspects, the metal coordination complex is an oligomeric metal coordination complex.

[0022] The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis. Consequently, features specified in one section may be combined with features specified in other sections as appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] FIG 6 shows the absorbance readings of lgM:biotinylated Goat anti-rat antibody cross-linked with Solution 4 compared to an antibody mixture without the cross-linker in a lateral flow half strip model. The Streptavidin-RPE reporter can only detect IgM if the IgM is conjugated to the biotinylated antibody. The wicking of the antibody complex was slower than its individual components but was not too big («1 micron) so as to prevent efficient flow through the membrane.

[0029] FIG 7 shows the absorbance readings of IgM: biotinylated anti-HCG antibody cross-linked with Solution 4 compared to an antibody mixture without the cross-linker in a lateral flow half strip model. The Streptavidin-RPE reporter can only detect IgM if the IgM is conjugated to the biotinylated antibody. The wicking of the antibody complex was slower than its individual components but was not too big («1 micron) so as to prevent efficient flow through the membrane.

[0030] FIG 8 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.

[0031] FIG 9 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.

[0032] FIG 10 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.

[0033] FIG 11 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 1mg/ml SARS-CoV Ab + 1 mg/ml BSA. Three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.

[0034] FIG 12 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 150|jg Ab per mg of Eu particles on a test line formed with 0.2mg/ml SARS-CoV Ab + 1mg/ml BSA. Three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared.

[0035] FIG 13 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.

[0036] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present disclosure is predicated, at least in part, on the understanding that certain metal coordination complexes, particularly certain oligomeric metal coordination complexes, can act as a solution phase homobifunctional cross-linking agents that can form stable bioconjugates, biomolecule conjugate clusters and/or gels of varying crosslinking densities, with a range of biomolecules.

[0038] The oligomeric metal coordination complexes employed for this purpose must be modified. The applicant has previously demonstrated the rapid binding of target molecules to unmodified complexes in, for example, Conjugating Molecules to Particles (International publication no. WO 2015/021509). This document demonstrated the extremely rapid kinetics of binding of proteins and the like to the unmodified metal complexes. For this reason, in that approach, it was essential to allow the metal coordination complexes to bind to a surface of a particle and subsequently wash the particle to ensure all unbound complexes were removed. The positive charge of the metal complex helps maintain good dispersion of particles and it was only at this point that the metal coordination complex activated particle could be exposed to a solution comprising the target molecules which had to be in the desired ratio. The rapid kinetics of binding then allowed those target molecules to bind to the particle surface via the metal coordination complexes in generally the same ratio as they were present in the solution, thereby joining the target molecules via the metal complex activated particle which was effectively acting as a linker. As is demonstrated in the examples herein, if target biomolecules are exposed purely in the solution phase to such unmodified metal coordination complexes, which are not bound to a particle or other substrate, then the rapid binding kinetics work unfavourably to form large biomolecule aggregates (well above 1 micron in size) which are polydisperse and which, due to uncontrolled and extremely rapid binding, do not maintain the desired functionality of the original biomolecules. It was therefore understood that metal coordination complexes were not an appropriate cross-linking approach to solution ligation of biomolecules without some form of solid support or substrate being present and to which the metal complexes must be first bound.

[0039] The present disclosure presents for the first time the knowledge that metal coordination complexes can have their reactivity modified or tuned down to thereby provide for a much better controlled solution phase bioconjugation approach. This can result in the formation of biomolecule conjugates, without the need for any substrate or support to be present during the conjugation process, which maintain an appropriate level of functionality of the original unlinked biomolecules.

[0040] It will be appreciated that the biomolecule conjugates described herein will not, in embodiments, likely simply be two biomolecules joined by a single strand of metal coordination complex. Rather, the metal coordination complexes will generally be in oligomeric form and will bond with multiple different biomolecules thereby providing for a biomolecule network which is formed from multiple biomolecules interconnected with metal coordination complexes.

[0041] It has been further found that control of not just the nature of the modified metal coordination complex but also the concentration of the reactants and, in embodiments, control of the temperature and buffer conditions can alter the reactivity of the metal coordination complexes and lead to the formation of relatively uniform biomolecule conjugates, clusters or networks thereof showing not only desired polydispersity levels but also maintaining functionality of the bound biomolecules. [0042] As used herein, the terms “biomolecule” or “biomolecules” refer to any compound isolated from a living organism, as well as analogs (including engineered and/or synthetic analogs), derivatives, mutants or variants and/or biologically active fragments of the same. For example, the biomolecule can be a protein (e.g., enzyme), nucleic acid, nucleotide, carbohydrate or lipid. In some embodiments, the biomolecule can be an engineered or synthetic analog of a compound isolated from a living cell that is structurally different from the compound but retains a biological activity characteristic of that compound. In embodiments, the “biomolecule” or “biomolecules” refer to such molecules which are made up, at least in part, of amino acids. In embodiments, the terms refer to biological molecules which are peptides or proteins or fragments of either.

[0043] In a first aspect of the disclosure, although not necessarily the broadest aspect, there is provided a biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex.

[0044] In embodiments, the two or more biomolecules may be the same or different.

[0045] In embodiments, at least one of the biomolecules, comprises amino acids.

[0046] Preferably, each biomolecule comprises amino acids.

[0047] In embodiments, the two or more biomolecules may each comprise at least one peptide or a fragment thereof.

[0048] In embodiments wherein the two or more biomolecules may each comprise at least one peptide or a fragment thereof, the at least one peptide or fragment thereof may be associated with one or more further peptides, ligands, coenzymes, cofactors and/or saccharides.

[0049] In embodiments, the two or more biomolecules may be independently selected from the group consisting of proteins, polypeptides, oligopeptides, peptides, glycoconjugates, globulins, steroid-binding proteins, antibodies, antigens, haptens, enzymes, or fragments thereof. [0050] In certain embodiments, the two or more biomolecules comprise a peptide or polypeptide or protein secondary structure.

[0051] In certain embodiments, the two or more biomolecules comprise a polypeptide or protein tertiary structure.

[0052] In certain embodiments, the two or more biomolecules comprise a polypeptide or protein quarternary structure.

[0053] In embodiments in which the two or more biomolecules are proteins, or fragments thereof, they may be the same or different. If the biomolecule conjugates are formed in the presence of a range of biomolecules then a bioconjugate population may be formed in which biomolecules may be joined in all possible combinations and/or in which a bioconjugate network is formed interconnecting a number of different biomolecules.

[0054] In certain embodiments disclosed herein, the two or more biomolecules may be independently selected from the group consisting of an antibody, 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.

[0055] In any of the embodiments disclosed herein, the two or more biomolecules may be independently selected from the group consisting of an antigen, an epitope of an antigen, an antibody, and an antigenically reactive fragment of an antibody.

[0056] In some embodiments, the two or more biomolecules 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. For example, the two or more biomolecules 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 nucleocapsid, or an antigen fragment thereof.

[0057] The biomolecules of the disclosure are directly conjugated, one to the other. In embodiments the biomolecules are conjugated directly by the metal coordination complex to form an interconnected network of biomolecule conjugates.

[0058] By “directly conjugated” it is intended that the biomolecules are not conjugated by the metal coordination complexes through a separate support or substrate or the like. In other words, the metal coordination complex is bonded only to the biomolecules and additional metal coordination complex at the point of the bioconjugates being formed. That is, the metal coordination complexes are not bonded to a substrate or support at the point of exposure to the two or more biomolecules.

[0059] In embodiments, the biomolecule conjugate is a solution-phase bioconjugate. That is, the biomolecule conjugate was formed with each biomolecule and the metal coordination complex purely in the solution phase.

[0060] In embodiments, the two or more biomolecules may be bonded to any region of the metal coordination complex. This is so because the metal coordination complex was not bonded to a surface, substrate or particle and so all ‘surfaces’ or regions not bound to other metal coordination complexes are available for bonding to the biomolecules.

[0061] In embodiments, the biomolecule conjugate is not bound to a physical support or substrate or particle at formation.

[0062] In one embodiment, the biomolecule conjugate is not bound to a polymer that is not a metal coordination complex bonding the two or more biomolecules.

[0063] In embodiments, the biomolecule conjugate substantially comprises only biomolecules and metal coordination complex.

[0064] In one embodiment, the biomolecule conjugate consists or consists essentially of the two or more biomolecules and metal coordination complex. [0065] The two or more biomolecules are conjugated by the metal coordination complex through non-covalent bonds. Bioconjugation is often described as involving covalent bonding between cross-linking agent and biomolecule but, as discussed further herein, the present disclosure advantageously relies on multiple dative or coordinate bonds between the metal coordinate complex and the biomolecules.

[0066] In embodiments, the biomolecule conjugate may be viewed as a biomolecule conjugate network or cluster or polymer, which terms may be used interchangeably herein.

[0067] In embodiments, the biomolecule conjugate network or cluster or polymer is a discrete biomolecule conjugate network or cluster or polymer.

[0068] In embodiments, the biomolecule conjugate network or cluster or polymer only comprises bonds between biomolecules and metal coordination complex and between metal coordination complex and metal coordination complex.

[0069] In embodiments, the biomolecule conjugate network or cluster or polymer does not comprise a solid support, substrate or particle.

[0070] In embodiments, the biomolecule conjugate network or cluster or polymer has an average diameter of between 10 nm to less than 1000 nm, 10 nm to less than 900 nm, 10 nm to less than 800 nm, 10 nm to less than 700 nm, 10 nm to less than 600 nm, 10 nm to less than 500 nm, 10 nm to less than 400 nm, 10 nm to less than 300 nm, 10 nm to less than 200 nm, 10 nm to less than 175 nm, between 20 nm to less than 1000 nm, 20 nm to less than 900 nm, 20 nm to less than 800 nm, 20 nm to less than 700 nm, 20 nm to less than 600 nm, 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 1000 nm, 30 nm to less than 900 nm, 30 nm to less than 800 nm, 30 nm to less than 700 nm, 30 nm to less than 600 nm, 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 1000 nm, 40 nm to less than 900 nm, 40 nm to less than 800 nm, 40 nm to less than 700 nm, 40 nm to less than 600 nm, 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 1000 nm, 50 nm to less than 900 nm, 50 nm to less than 800 nm, 50 nm to less than 700 nm, 50 nm to less than 600 nm, 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 nm, between 75 nm to less than 1000 nm, 75 nm to less than 900 nm, 75 nm to less than 800 nm, 75 nm to less than 700 nm, 75 nm to less than 600 nm, 75 nm to less than 500 nm, 75 nm to less than 400 nm, 75 nm to less than 300 nm, 75 nm to less than 200 nm, 75 nm to less than 175 nm, between 90 nm to less than 1000 nm, 90 nm to less than 900 nm, 90 nm to less than 800 nm, 90 nm to less than 700 nm, 90 nm to less than 600 nm, 90 nm to less than 500 nm, 90 nm to less than 400 nm, 90 nm to less than 300 nm, 90 nm to less than 200 nm, 90 nm to less than 175 nm, between 100 nm to less than 1000 nm, 100 nm to less than 900 nm, 100 nm to less than 800 nm, 100 nm to less than 700 nm, 100 nm to less than 600 nm, 100 nm to less than 500 nm, 100 nm to less than 400 nm, between 150 nm to less than 800 nm, 150 nm to less than 600 nm, 150 nm to less than 500 nm, or between 175 nm to less than 700 nm, 175 nm to less than 600 nm, 175 nm to less than 500 nm, such as between about 200 nm to about 400 nm.

[0071] In any embodiment described herein, the biomolecule conjugate network or cluster or polymer has an average diameter of between 20 nm to 500 nm.

[0072] References herein to diameter or average diameter of the biomolecule conjugate network, clusters or polymers is reference to the Z-average size which can be measured by standard approaches known in the field and including dynamic light scattering (DLS).

[0073] It is a particular advantage of the present disclosure that nanosized clusters or networks can be consistently formed with peptides and proteins. The applicant has 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, for example modified with carboxylate ligands, provides for consistent outcomes which are further enhanced in terms of PDI with the use of elevated temperatures and appropriate buffer conditions as described herein. [0074] In embodiments, the biomolecule conjugate network or cluster or polymer may comprise a water-soluble polymer. The water-soluble polymer may be included in the formation of the biomolecule conjugate network or cluster or polymer to add an additional functional or structural property to the biomolecule conjugate network or cluster or polymer. For example, the water-soluble polymer may, in one embodiment, be polyacrylic acid (PAA) but a range of water-soluble polymers are known in the art which may be incorporated to provide a variety of functionalities.

[0075] The biomolecule conjugate network or cluster or polymer is, as described, a solution phase ligation approach and so only water-soluble polymers may be included when the biomolecule conjugate network or cluster or polymer is forming.

[0076] In embodiments, the metal ion of the metal coordination complex is selected from the group consisting of chromium, ruthenium, iron, cobalt, titanium, aluminium, zirconium, and combinations thereof. In embodiments, the metal ion of the oligomeric metal coordination complex is selected from the group consisting of chromium, ruthenium, titanium, iron, cobalt, aluminium, zirconium, rhodium and combinations thereof.

[0077] In any of the embodiments or aspects described herein, the metal ion of the metal coordination complex is chromium.

[0078] The metal ion of the metal coordination complex may be present in any applicable oxidation state. For example, 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.

[0079] In an embodiment in which the metal ion is a chromium ion, it is preferred that the chromium has an oxidation state of III.

[0080] The metal ion may be associated with any suitable counter-ions such as are well-known in metal-ligand coordination chemistry. [0081] In certain embodiments, mixtures of different metal ions may be used, for example, to form a plurality of different oligomeric metal coordination complexes. In such cases, it is preferred that at least one metal ion is chromium.

[0082] 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.

[0083] In embodiments, the metal coordination complex is a chromium oligomeric metal coordination complex such as a chromium (III) oligomeric metal coordination complex. In embodiments, the 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.

[0084] Exemplary oxo-bridged chromium structures are provided below, albeit without indication of any appropriate modification of reactivity for biomolecule conjugate formation: [0085] On contact with some substrate particle, such as described in Conjugating to Particles (PCT/AU2014/050181), at least one of the water or hydroxyl groups (or whatever ligands may be present) on the oligomeric metal coordination complexes is replaced by a dative bond with the particle surface. This is illustrated below wherein “X” represents the dative bond to the particle surface.

[0086] It will also be appreciated, in the situation described, that multiple water or hydroxyl or other ligands present on the oligomeric metal coordination complex may be replaced by a dative bond with the particle surface but when such oligomeric metal complexes are added in excess there is no cross-linking between particles. When the excess is washed off the substrate particles, what remains is an oligomeric metal complex activated substrate which has several useful properties. Due to its size and oligomeric nature, binding to the particle surface is stable, and there still remains coordination potential to bind biomolecules, 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. This results in a binding of affinity agents representative of the ratio of such agents in any solution to which the metal complex-coated particles are exposed to. As previously described, it is critical to first add such oligomeric metal complexes in excess to some substrate such as a particle, and then wash off the excess to leave only an oligomeric metal complex activated substrate/particle.

[0087] Given the above discussion, any mixing of such unmodified oligomeric metal complexes with biomolecules in solution phase will lead to a rapid, uncontrolled reaction resulting in an insoluble aggregate of cross-linked biomolecules.

[0088] Instead, if at least one or more of the water or hydroxyl groups (or whatever ligands may be present) on the oligomeric metal coordination complex are replaced with more strongly coordinating ligands, which may be referred to herein as capping agents or coordination agents, interchangeably, the oligomeric metal coordination complexes to which the two or more biomolecules 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 oligomeric metal coordination complexes and two or more biomolecules in solution is generated, then the multiplicity of different coordination ligands naturally presenting on the two or more biomolecules may or may not be able to compete with the capping or coordinating agents of the modified oligomeric metal coordination complexes depending on the coordination strength of the capping or coordinating agents and their excess. Unlike synthetic polymers which have repeating ligands with the same metal coordination potential, biological polymers such as proteins include various ligands with different strengths of coordination to metal complexes. The capping agents and its excess set some threshold by which only a limited number of more strongly coordinating ligands on the biomolecule can coordinate to the metal complexes. In such a situation, uncontrolled coordination of metal complexes to any ligand in the biomolecule is minimised to encourage intermolecular cross-linking of biomolecules by the oligomeric metal complexes. While stability is maintained by a multiplicity of a small set of coordinate bonds, each individual coordinate bond compared to covalent coupling is relatively weak and reversible allowing for better maintenance of biomolecule functional structure. The capping agents may be part of the oligomeric metal complex or may be present in the solvent/buffer that contains the oligomeric metal complexes and/or the biomolecules before they are combined. Assuming some set amount of oligomeric metal complexes, the presence of capping agents in too high an excess may totally prevent the formation of biomolecule - metal complex networks. At the other extreme, the lack of sufficiently strong capping agents can lead to a multiplicity of metal ion - ligand coordination resulting in the formation of insoluble biomolecule aggregates. The control of reactivity also allows time for uniform mixing and thus for networks or relatively uniform clusters to form rather than highly polydisperse aggregates. This is as opposed to ‘standard’ or ‘unmodified’ oligomeric complexes which are highly reactive and would simply react completely almost immediately after contact with the two or more biomolecules to form non- or minimally functional aggregates.

[0089] It will be appreciated that there may be at least about 30% more intermolecular binding of the biomolecules with the oligomeric metal coordination complex described herein to form an interconnected network of biomolecule conjugates 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 the biomolecules with the oligomeric metal coordination complex described herein to form an interconnected network biomolecule conjugates than the same oligomeric metal coordination complex which has not been so modified. [0090] It will be appreciated by a person of skill in the art that the exchange of capping agents from the oligomeric metal coordination complex will be affected by the nature of other coordinating agents also present in the solution and so can be influenced by the use of, for example, a buffer solution comprising buffer salts such as phosphates which can coordinate with the oligomeric metal coordination complexes. Similarly, it will be appreciated that such competition and exchanges will be influenced by the concentration of such competing coordination ligands and by the pH of the solution.

[0091] While the discussion in relation to this first aspect of the disclosure relates to a metal coordination complex, preferably an oligomeric metal coordination complex, directly and non-covalently bonded to the at least two biomolecules, it will be appreciated that the 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 biomolecule conjugate has formed. This is because the bonding with the biomolecules will result in multiple previously separate oligomeric metal coordination complexes being bound to each biomolecule. In solution, some of these individual coordinate bonds will break and reform with the same or different biomolecules and so what has been referred to above as a biomolecule conjugate network or cluster or polymer is formed with the formerly oligomeric metal coordination complexes essentially forming the strands or connections of the network between separate biomolecules as hubs. Based upon the interconnected network formed, the biomolecule conjugate network or cluster or polymer may be viewed as polymeric, or at least ‘extended’ in nature even though it has been formed, in part, from metal coordination complexes, such as oligomeric metal coordination complexes.

[0092] It will be appreciated that the interconnected biomolecule conjugate network or cluster or polymer formed by crosslinking of the biomolecules and the metal coordination complexes, is a dynamic system due to the nature of the association between the components. Without wishing to be bound by theory, it is postulated that the oligomeric metal coordination complexes are directly bonded with the biomolecules through avidity or multi-component bonding and so each biomolecule is directly bonded to what becomes a metal coordination complex network through multiple coordinate bond interactions the accumulated strength of which results in anchoring of the biomolecules to the oligomeric metal coordination complex as if they were bonded via standard covalent bonding. As discussed, this may be viewed as forming an interconnected biomolecule conjugate network or cluster or polymer. However, any individual coordination bond between the metal ion in the oligomeric metal coordination complexes and the two or more biomolecules is relatively weak (in comparison to covalent) and can break as a result of a local stressor such as the biomolecules preference to maintain its preferred conformational structure. This also allows for desirable freedom of movement or orientation allowing the biomolecules to be advantageously functionally available.

[0093] In embodiments of the first aspect of the disclosure, there is provided a biomolecule conjugate network comprising two or more peptide, polypeptide or protein biomolecules directly conjugated, one to the other, and interconnected through non- covalent bonds by an oligomeric chromium metal coordination complex. In such embodiments, the biomolecule conjugate network may have been formed in the absence of a solid support, substrate or particle. That is, all components may have been in the solution phase when the biomolecule conjugate network formed.

[0094] In such embodiments, the biomolecule conjugate network is not bonded through a metal coordination complex to a solid support, substrate or particle.

[0095] In such embodiments, the biomolecule conjugate of the network, the biomolecules, and the metal coordination complexes may be of a nature and interact in manners as described in any other embodiment of the first aspect described.

[0096] In a second aspect of the disclosure, there is provided a method of forming a biomolecule conjugate, the biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex, the method including the steps of:

(a) providing a liquid formulation comprising a population of two or more biomolecules; (b) contacting the liquid formulation comprising the population of two or more biomolecules with a metal coordination complex, to thereby form the biomolecule conjugate.

[0097] The two or more biomolecules and oligomeric metal coordination complex and biomolecule conjugate may be as described for any embodiment, or combination of embodiments, of the first aspect.

[0098] As discussed, the advantages of the present disclosure relate, at least in part, to the ability to have the metal coordination complexes linking the two or more biomolecules to form those bonds in solution in a controlled fashion by reducing the reaction kinetics and adjusting the competition rates between the ligands on the biomolecule with those not on the biomolecule. This approach is demonstrated within the examples to provide for improvements in functional availability and/or mobility of the biomolecules versus bonding with unmodified metal coordination complexes.

[0099] The solution presented herein is to employ modified oligomeric metal coordination complexes, being relatively unreactive, or reduced reactivity, metal complexes which will form bonds to the two or more biomolecules at an appropriate rate to allow sufficient time for uniform mixing, leading to controlled size and conjugate uniformity of the interconnected biomolecule conjugate network. Without wishing to be bound by theory, the inventors believe that by controlling the kinetics of coordination of the modified oligomeric metal coordination complex to the two or more biomolecules, it is possible to form a stable interconnected biomolecule conjugate network useful for subsequent applications.

[00100] The degree of modification of the oligomeric metal coordination complexes, for example the extent or excess of capping or coordinating agents resulting in the modified oligomeric metal coordination complex, the presence of other competing ligands in solution such as those provided by buffer salts, the concentration of all of these components and the pH of the reaction can be controlled in tandem to modify the speed of formation of the biomolecule conjugate network. As shown in the experimental section, adjustment of these parameters, alone or in concert, can have a direct effect on the biomolecule functionality in the final biomolecule conjugate network.

[00101] In embodiments, the modified oligomeric metal coordination complex may be defined as a reduced reactivity oligomeric metal coordination complex, especially relative to the same metal ion which is fully hydrated (for example a hexahydrate).

[00102] In embodiments, the modified 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 metal coordination complex but in a fully hydrated state (for example in the form of a hexahydrate). In one embodiment, the unmodified metal coordination complex has non- or weakly coordination anions as ligands.

[00103] In embodiments, the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity as compared with an unmodified 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.

[00104] In embodiments, the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity due to the presence of stronger coordinating capping agents at appropriate concentration and strength compared with an unmodified metal complex, for example an unmodified oxo- bridged chromium (III) complex having only weakly or non-coordinating anions or ligands.

[00105] In embodiments, the unmodified oxo-bridged chromium (III) complex used for comparison purposes may be that as formed in Solution 1 of Example 1 in the examples section.

[00106] In embodiments, the modified oligomeric metal coordination complex is modified such that its reactivity to, or speed to bond with, one of at least one of the biomolecules is reduced as compared with the same oligomeric metal coordination complex which has not been so modified.

[00107] In embodiments, the biomolecule 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.

[00108] In embodiments, the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity with monoclonal antibodies to virus antigens such as SARS-CoV2, Flu A/B viruses 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).

[00109] In embodiments, the reduced reactivity of the modified oligomeric metal coordination complex may be defined as a reduced level of reactivity with monoclonal antibodies to virus antigens such as SARS-CoV2, Flu A/B viruses or polyclonal antibodies such as goat anti-mouse antibodies as compared with that of an oxo-bridged chromium (III) complex. In embodiments, 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.

[00110] In embodiments of any of the aspects described herein, the at least one modified 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.

[00111] In embodiments, the modified oligomeric metal coordination complex has been modified to display capping agent groups coordinately bound to the metal of the oligomeric metal coordination complex. The capping agents will alter the reaction kinetics of the now modified oligomeric metal coordination complex with ligands on two or more biomolecules 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 metal coordination complexes will therefore react more slowly and a more limited selection of ligands on the biomolecules depending on the type and concentration of capping agent to form an appropriate biomolecule conjugate network.

[00112] In embodiments, the method may further include the step of selecting or controlling the relative extent of the total coordination capacity of the 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 oligomeric metal coordination taken up by capping agents (as measured by that remaining following formation of the oligomeric metal coordination complex itself - as a coordination interaction is reversible, this percentage is the starting percentage taken up by the capping agents). For example, 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 100%, 200%, 400% or 600%. Where 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. In this situation, the degree of excess also changes the reaction kinetics of the now modified oligomeric metal coordination complex with the biomolecules as there are more capping agents in competition.

[00113] In embodiments, competing coordinating ligands may be supplied as part of the buffer solution in which the biomolecule conjugation is performed. 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 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.

[00114] Appropriate capping agents will therefore be those which slow down coordination of the modified oligomeric metal coordination complexes with the biomolecules but do not prevent it. With increasing amount of oligomeric metal complex in proportion to the biomolecules, there is an increasing tendency for uncontrolled biomolecule aggregation and with increasing amount of capping agents there is a decreasing tendency to coordinate and form biomolecule clusters. In combination, 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 biomolecules using standard unmodified oligomeric metal coordination complexes, the metal complexes will simply form tightly bound aggregates with the biomolecules 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 metal coordination complexes modified with different capping agents and exposed to the same biomolecules. Similarly, the level of functionality of the biomolecules can be tested by running parallel reactions with different ratios of metal complex and capping groups relative to the amount of biomolecule that was used.

[00115] In embodiments, 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.

[00116] In embodiments, 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.

[00117] In embodiments, the capping or coordinating agent may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, maleate, sulphate, phosphate, and hydroxyacetate. In embodiments, the capping group 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.

[00118] In embodiments, 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. In embodiments, 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. In embodiments, the capping group may be selected from the group consisting of oxalate, malonate, succinate, glutarate, adipate, maleate, citrate, and aconitate. In embodiments, the capping group may be selected from the group consisting of formate, acetate, propionate, oxalate, malonate, succinate, glutarate, maleate, citrate, and aconitate. For example, the capping group may be selected from the group consisting of acetate, oxalate, malonate, succinate, and citrate. In one example, the capping group may be selected from the group consisting of acetate, oxalate, phosphate and succinate. In a preferred example, the capping group may be selected from the group consisting of acetate, oxalate, and succinate. The capping group may be oxalate. The capping group may be succinate.

[00119] In embodiments, the capping or coordinating agent may be carboxylate or phosphate, preferably carboxylate. For example, the carboxylate may be a dicarboxylate or a tricarboxylate, preferably a dicarboxylate.

[00120] In embodiments, 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.

[00121] In embodiments, 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.

[00122] In embodiments, the capping group 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. [00123] In embodiments, the capping or coordinating agent is not simply a counterion of the oligomeric metal coordination complex or a group donated by a base. For example, in forming oligomeric metal coordination complexes it is common to expose the metal complex to a base, such as ethylene diamine, which simply encourage formation of the desired complexes. While the amine nitrogen may be, to a small degree, incorporated into the formed oligomeric metal coordination complex it does not have a significant enough effect on the subsequent reactivity of the oligomeric metal coordination complex to be considered a capping agent. Therefore, in one embodiment, the capping agent is not one donated by a base, including ethylene diamine.

[00124] The step of forming the modified 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.

[00125] 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.

[00126] In embodiments, the step of contacting the liquid formulation comprising the population of two or more biomolecules with a metal coordination complex may also include contacting the liquid formulation comprising the population of two or more biomolecules with a water-soluble polymer. The water-soluble polymer may be selected from any available in the prior art and which would have coordinating ligands to bond to the metal coordination complex and so become a component of the forming biomolecule conjugate network or cluster or polymer.

[00127] In embodiments, the step of adjusting the pH may include adjusting the pH of the solution in which the oligomeric metal coordination complexes themselves are forming (prior to exposure to the biomolecules) to ensure the desired degree of modification. This may comprise allowing the pH to become more acidic due to the release of hydrogen ions by the metal salts employed or it may comprise the addition of a base, such as ethylene diamine or a metal hydroxide, to mop up some of the released hydrogen ions to prevent the solution becoming too acidic. If a base is added then the amount will be such that the solution is still acidic, as defined above.

[00128] In embodiments, the modified metal coordination complex can be formed via the direct reduction of chromium (VI) oxide in the presence of suitable capping groups, such as carboxylate groups including acetate and oxalate groups from the corresponding acids. Once the complex is synthesised, the pH can be adjusted as required.

[00129] It will also be appreciated that, in embodiments, the modified oligomeric metal coordination complex may include various capping groups having on/off rates which are appreciably slower than pre-existing water and other ligand groups, and hence will affect coordination with an additional component of the formulation.

[00130] The oligomeric metal coordination complex will be discussed below, in terms of available variations in the synthetic approach and the potential for differences thereby achieved in the final product.

[00131] The oligomeric metal coordination complexes can be formed by providing conditions for forming electron donating groups for bridging or otherwise linking or bonding two or more metal ions. When not already commercially available, this can be done by providing a pH above pH 1 , and preferably between about 1 to 5, or about 2 to 5 to the solution when forming the complexes. Clearly, the chosen pH will depend on the approach by which modification of the oligomeric metal coordination complex is to be achieved. That is, while a pH above 3.8 may be appropriate for forming the oligomeric metal coordination complex when they are to be modified by use of capping groups, a pH below 3.8 is highly desirable for the oligomeric metal coordination complexes formed in aqueous solutions.

[00132] Various chromium salts such as chromium chloride, chromium nitrate, chromium sulphate, chromium acetate, chromium perchlorates, may be used to form a chromium-based oligomeric metal coordination complex. Unless pre-existing in some oligomeric form and used ‘as is’, these salts are mixed with an alkaline solution, such as sodium hydroxide, potassium hydroxide, lithium hydroxide, sodium bicarbonate, sodium sulphite and ammonium hydroxide to form different metal coordination complexes. Organic reagents that can act as bases such as ethylene diamine, bis(3- aminopropyl)diethylamine, pyridine, imidazoles, can also be used. The size and structure of the oligomeric metal coordination complex can vary with pH, temperature, choice of solvent and other conditions.

[00133] In embodiments, a liquid carrier or solvent forming the liquid formulation comprising the two or more biomolecules may be an aqueous or organic solvent, or mixture thereof. In embodiments, the liquid carrier has at least some aqueous component. The liquid carrier may preferably be an aqueous solution. The liquid carrier may be water or an alcohol or a mixture thereof. The alcohol may be methanol, ethanol, propanol, isopropanol or butanol. In one embodiment, the liquid carrier is water or isopropanol. In one embodiment, the liquid carrier is water.

[00134] Preferably, the liquid carrier is an aqueous carrier.

[00135] In embodiments, the liquid carrier is or comprises a buffer salt.

[00136] In embodiments, the liquid carrier is or comprises a buffer solution.

[00137] Buffer salts and solutions comprising same are well-known in the art and may comprise, for example, phosphate salts.

[00138] In one embodiment, the method further includes the step of warming the liquid formulation during or following bioconjugate formation.

[00139] In embodiments, the method may include the step of contacting the liquid formulation comprising the population of two or more biomolecules with the metal coordination complex at a temperature above 20 °C, above 25 °C, or above 26 °C, or above 27 °C, or above 28 °C, or above 29 °C or above 30 °C, or above 31 °C, or above 32 °C, or above 33 °C or above 34 °C, or above 35 °C all of which are considered to form the lower end of a temperature range with an upper limit being less than 45°C, less than 42 °C or less than 40 °C. For example, the conjugation between metal coordination complex and the two or more biomolecules may occur at a temperature between 20 to 45 °C or between 30 to 40 °C. [00140] The experiments described herein demonstrate that an advantageous level of polydispersity or uniformity can be achieved, in combination with the use of modified metal complexes, if the conjugation is carried out at these temperatures which are elevated above room temperature (23 °C) but below temperatures which are likely to damage the biomolecule functionality or structure.

[00141] In embodiments of the second aspect, there is provided a method of forming a biomolecule conjugate, the biomolecule conjugate comprising two or more peptide, polypeptide or protein biomolecules directly conjugated, one to the other, and interconnected through non-covalent bonds by an oligomeric chromium metal coordination complex, the method including the steps of:

(a) providing a liquid formulation comprising two or more peptide, polypeptide or protein biomolecules;

(b) contacting the liquid formulation comprising the two or more peptide, polypeptide or protein biomolecules with a modified oligomeric chromium metal coordination complex, to thereby form the biomolecule conjugate.

[00142] The two or more peptide, polypeptide or protein biomolecules may be populations of such species which may be the same or different.

[00143] In embodiments, the biomolecule conjugate is a biomolecule conjugate network.

[00144] In embodiments, the modified 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 biomolecules.

[00145] In embodiments, the liquid formulation comprising the population of two or more peptide, polypeptide or protein biomolecules 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 30 °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.

[00146] In a third aspect of the disclosure, there is provided a functional substrate comprising at least one biomolecule conjugate, the at least one biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non- covalent bonds by a metal coordination complex, and the at least one biomolecule conjugate associated with a substrate material.

[00147] The biomolecule conjugate, two or more biomolecules, and metal coordination complex may be as described in any embodiment, or combination of embodiments, of the first aspect.

[00148] In a fourth aspect of the disclosure, there is provided a method of forming a functional substrate comprising at least one biomolecule conjugate including the steps of:

(a) providing a biomolecule conjugate comprising two or more biomolecules directly conjugated, one to the other, through non-covalent bonds by a metal coordination complex;

(b) contacting the biomolecule conjugate with a substrate preformulation, the substrate preformulation adapted to form a substrate; and

(c) allowing the biomolecule conjugate to be incorporated into the substrate as it forms, to thereby form the functional substrate.

[00149] The biomolecule conjugate, two or more biomolecules, and metal coordination complex may be as described for any embodiment, or combination of embodiments, of the first aspect.

[00150] In embodiments of the third and/or fourth aspect, the substrate may be a polymeric substrate.

[00151] In embodiments of the third and/or fourth aspect, the substrate is a gel or resin. EXAMPLES

Example 1 : Preparation of metal coordination complex solutions.

[00152] Examples of 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.

Solution 1

[00153] In this example, chromium perchlorate hexahydrate (45.9 g) was dissolved into 480 mL of purified water and mixed thoroughly until all solid dissolved. Similarly, 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.

Solution 2

[00154] Similar to Solution 1 , different ratios of 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. As an example, 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.

Solution 3

[00155] In this example, 500 ml 100mM acetate buffer at pH 3.6 is added dropwise to 500 ml of Solution 2 with stirring, and then left to equilibrate to a pH of approximately 3.0 to form Solution 3A. Depending on the pH of the respective solutions and concentration of acetate buffer, different versions of Solution 3 can be formed. One example (Solution 3B) is when double the amount of acetate is added. Even the method of synthesis (to form hydroxo and oxo bridging) can form different versions of Solution 3 having different acetate ligands coordination strengths to the metal ion of the complex. It is not necessary for the acetate ligand to be all coordinated to the metal complex and it will be appreciated that more than one type of ligand comprising the buffer can also be used.

Solution 4

[00156] In this example, 500 ml 100mM oxalic acid buffer at pH 3.5 is added dropwise to 500 ml of Solution 2 with stirring, and then left to equilibrate to a pH of 3.0 to 3.5. Depending on the pH of the respective solutions and concentration of oxalate acid buffer, different versions of Solution 4 can be formed. This can be considered another representative modified oligomeric metal coordination complex.

Solution 5

[00157] Similar to Solution 4, alternative excesses of oxalic acid can be used to generate solutions having a far larger excess of oxalic acid to the metal complex of Solution 2. As an example, using 200mM of oxalic acid buffer will produce a metal ion to oxalic acid ratio of 1 :2 (Solution 5A), and by using more dilute mixtures, even higher oxalic acid excesses can be added to give metal ion to oxalic acid ratios of 1 :4 (Solution 5B). Metal complex solutions with many other different metal ions to ligand ratios can be formed.

Solution 6

[00158] In this example, 500 ml 100mM succinic acid buffer at pH 3.5 is added dropwise to 500 ml of Solution 2 with stirring, and then left to equilibrate to a pH of 3.0 to 3.5. This can be considered another representative modified oligomeric metal coordination complex. Similar to Solution 4 and 5, different ratios of succinic acid to Solution 2 can be used to generate solutions having a far larger excess of succinic acid to the metal complex of Solution 2. Such examples are metal ion to succinic acid ratio of 1 :1 (Solution 6A), ratio of 1 :2 (Solution 6B) and ratio of 1 :4 (Solution 6C). Many different ligands, combinations and ratios can be formed. Example 2: Clustering Size, Rate and Type with Oligomeric Metal Complexes.

Example 2a

[00159] In this example, 8 mg/ml of Bovine Serum Albumin (BSA) was dissolved in 25mM MES buffer at pH 6.0. To 500 .L BSA (final cone of 4 mg/ml) samples, 500 .L of Solution 1 having a final concentration of 4 mM were mixed. Samples were transferred into a zeta potential cuvette for measurement on a Malvern Nano ZS Zetasizer over a 60 min time period. As shown Figure 1 , Solution 1 complex (unmodified) was highly reactive resulting in rapid protein aggregation. Within 30 mins, large insoluble (cloudy suspension) aggregates ranging in size between 2 to over 8 microns (average of approximately 4 microns) were formed. Diluting the Solution 1 complex to 2mM still gave cloudy solutions with similar size aggregates as before. However, smaller clusters in the 200 to 700 nm range also remained (data not shown). Decreasing the amount of unmodified metal complexes led to a mixture of heterogenous sized clusters of which the majority existed as insoluble aggregates.

Example 2b

[00160] Using the same procedure as in Example 2a, the characteristics of Solution 3A and 3B (modified by acetate capping ligands) at 2, 4 and 8mM concentrations were assessed. As shown Figure 2a, Solution 3A complex still formed large aggregates using the higher concentrations. But at 2mM, the reaction has slowed to give cluster sizes of around 110nm after 7 hrs. With Solution 3B (Figure 2b) there was an opposite trend where the higher concentrations did not form any clusters. The lowest concentration of Solution 3b gave clusters of around 50 nm after 7 hours. A large excess of capping groups has the potential to stop cluster formation completely.

Example 2c

[00161] Using the same procedure as before, the effect of 4mM Solution 4 (modified via oxalic acid capping ligand) was tracked over 60 mins at room temperature. As shown in Figure 3, there is little change indicating that this capping group at this concentration prevented coordination of metal complexes to form protein clusters. However, if the same reaction is performed at 37°C over 120 min (Figure 4), small uniform clusters of BSA are being formed. By controlling the protein: metal complex ratios, buffer and temperature conditions, it is possible to form very uniform protein clusters.

Example 2d

[00162] The characteristics of the oxalate modified metal complexes at different concentrations and ratios were further assessed for forming controlled protein clusters. As shown in the example, metal complexes having three different oxalic acid capping ratios were compared, ie, metal complex to oxalic acid 1 :1 (Solution 4), 1 :2 (Solution 5A) and 1 :4 (Solution 5B). The different BSA/metal complex mixtures were compared with BSA/water mixture (Control) over a 7-hour period. Figure 5a shows the rate of BSA clusters (in nanometres) being formed with different dilutions of Solution 4. The higher concentration of Solution 4 led to uncontrolled aggregation of BSA molecules which crashed out of solution within 60 minutes while the low (2mM) concentration of Solution 4 did not lead to much cluster formation. The latter was comparable to the Control (having no metal complex). However, at 4mM Solution 4, there was a steady increase in BSA cluster size stabilising around 140nm after 7 hrs. Figure 5b shows the result of a similar experiment using a metal complex to oxalic acid ratio of 1 :2 (Solution 5A) and Figure 5c shows the rate using a metal complex to oxalic acid ratio of 1 :4 (Solution 5B). The trend to forming larger clusters with Solution 5A and 5B is the opposite of using Solution 4. The lowest concentration (2mM) of Solution 5A gave the largest clusters stabilising around 800nm after 7 hrs. Solution 5B at the lowest concentration also gave the largest clusters, which was stabilizing around 100nm after 7 hrs.

Example 2e

[00163] Using the same procedure as above, the effect of different capping agents are compared are similar conditions. 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). As shown, 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.

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.

[00164] 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. While the underlying mechanism is not fully determined, the selection of capping group and excess not only allows fine control over protein cluster formation, it was thought that improved antibody functionality could be achieved with such clusters compared to non-clusters when immobilized on substrates. The use of these methods to form protein clusters and improve lateral immunoassays was investigated in the following.

Example 3: Test functionality of IgM Biotinylated Ab Cluster

[00165] A conventional lateral flow half strip test was used to exemplify the present invention. An anti-IgM test line and a biotinylated antibody control lines were stripped onto nitrocellulose membranes. If an IgM antibody was conjugated to a biotinylated antibody, such a protein cluster would be captured on the test line and can be reported using Streptavidin-RPE (Code:016-110-084, Jackson Immunoresearch) with a fluorescence and absorbance reader (Axxin). If the lgM:biotinylated Ab cluster was not formed, the IgM captured on the test line would not have any biotinylated Ab to report with Streptavidin-RPE. Two different conjugates, a., an IgM: biotinylated anti-HCG Ab and b., lgM:biotinylated goat anti-rat Ab complexes were formed using Solution 4.

[00166] In brief, 0.1 mg/ml of Biotin-Goat anti-rat IgG (Control Line) and 1 mg/ml of Goat anti-human IgM (Test Line) in carbonate buffer, pH8.5 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.

[00167] Two IgM-Biotinylated IgG complexes were formed using modified oligomeric metal complex, Solution 4. In this example, one complex was 0.2 mg/ml of Human IgM (No. 18260, Sigma Aldrich) mixed with 0.2mg/ml of Biotinylated Goat anti-rat IgG (No. 112-065-003, Jackson ImmunoReseach), and cross-linked with 2mM Solution 4. The other complex was a 0.2 mg/ml of Human IgM mixed with 0.2mg/ml of Biotinylated anti- HCG (Cat#2H8B, HyTest), cross-linked with 2mM Solution 4. The antibody cross-linked with Solution 4 was compared to the antibody mixture which was not cross-linked with Solution 4 using a half strip model. As shown in Fig 6 and 7, IgM cross-linked with biotinylated IgG was reported on the test line when streptavidin-RPE was added. In contrast, the mixture of IgM and biotinylated IgG without Solution 4 would have captured IgM but would not have been observable without the cross-linked biotinylated IgG. These cross-linked proteins wicked easily through the porous membrane confirming that insoluble aggregates were not forming but the flow rate was slower when compared to non-cross-linked mixtures.

Example 4: Binding Antibody to Eu Particles (Control Conjugate).

Preparation of an unmodified metal coordination complex-activated Europium Particle

[00168] 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.

Antibody binding to unmodified metal coordination complex-activated Europium Particle [00169] Pre-activated europium particles (50pL, 10mg/mL), as described above, were added into 200pl of DI water and centrifuged for 5 minutes. The supernatant was removed, and an equal volume of DI water was added. After resuspending the pellet using bath sonicator and vortexing, it was centrifuged and the supernatant removed, and suspended in 25mM MES buffer, pH6.0 (120pL).

[00170] Stock COVID-19 mAb (Cat#40143-MM08, Sino Biological) was diluted to 600pg/mL in MES buffer. The activated europium particle suspension was mixed with an equal volume of 600pg/mL COVID-19 mAb was sonicated and vortexed to fully disperse the particles. The conjugate particle was left on a tube rotator for 1 hour and then 25pL of blocking buffer (10% BSA in MES buffer, Bovine Serum Albumin, A7030, Sigma) was added. After fully dispersing, the blocked conjugate particle was left for another hour on a tube rotator. 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 5: Binding Antibody Clusters to Eu Particles (Cluster Conjugates).

Preparation of metal coordination complex-activated Europium Particle

[00171] The metal coordination complex-activated Europium particles were prepared using the general approach of Example 3.

Preparation of Antibody Clusters using modified metal coordination complexes

[00172] As an example, 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. An equal volume of 2mM Solution 4 and 1.2mg/mL COVID-19 mAb solution was mixed to give a final concentration of 600pg/mL of antibody and 1mM of Solution 4 in a total volume of 125pL.

[00173] This mixture was left for 20 to 25 minutes before mixing with pre-activated particles and is equivalent to 150pg of mAb with 0.25mM of Solution 4 per mg of particle. By adjusting the volumes and ratios of antibodies with different metal complex solutions, many combinations can be formed and compared.

Antibody Cluster binding to activated Europium Particle [00174] The Antibody Cluster formed above conjugated to unmodified metal coordination complex-activated Europium particles in the manner as described in Example 3.

Example 6: Comparison on Cluster Conjugate vs Control Conjugate (I)

[00175] A lateral flow half test strip was used to compare any differences between the Control (Example 4) and antibody cluster (Example 5) conjugates. In brief, 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.

[00176] The Control, COVID-19 detection mAb (150pg per mg of Eu particles) and three different clusters formed with 150pg COVID-19 detection mAb and 0.5 mM, 0.25mM and 0.125mM of Solution 4 per mg of Eu particles were compared. The samples of conjugate particles were assessed using laser diffraction by a zetasizer (Malvern: Model nanoseries Z). The zeta size, polydispersity index (Pdl) and potential of the conjugate particles are shown in Table 3. Essentially there was a small increase in size between the plain antibody conjugate (Control) and the three antibody cluster conjugates. All particles show excellent uniformity from the Pdl with the zeta potential shifting to be negative in the case of cluster conjugation. Table 3 - Zeta Size and Potential of mAb conjugated Particles comparing the 3 different mAb clusters formed with Solution 4.

[00177] 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 8, the antibody cluster conjugates, formed with Solution 4, had different outcomes indicating that the functional activity of antibodies in the clusters were different. The clusters formed with 150pg COVID-19 detection mAb with 0.25mM Solution 4 per mg of Eu particles gave slightly better specific binding signals than Covid-19 mAb only particles in this experiment (Sn/SO of 23.0 vs 29.8). The clear difference in performance between the clusters indicate that the functional activity can be manipulated by the amount and type of modified metal coordination complexes. | _ 400 | _ 23.0 | _ 22.2 | _ 29.8 | _ 22.7 |

Table 4: Lateral flow results comparing use of antibody conjugates (Clusters vs Control).

Example 7: Titration of Conjugated Particles; Comparison of Antibody Cluster vs Control (II)

[00178] Using the lateral flow half test strip described in Example 6, conjugate particle concentrations were compared at 2pg particles per strip, 0.1 pg particles per strip, 0.05pg particles per strip and 0.025pg particles per strip for the Control (150pg COVID- 19 detection mAb) and a cluster formed with 150pg COVID-19 Ab and 0.25mM Solution 4 per mg of Eu particles. As shown in Figure 9, the antibody cluster conjugate clearly gave better results compared to the Control.

Example 8: Using Less Antibody per Conjugate Particle: Comparison of Antibody Clusters vs Control (III)

[00179] Using the lateral flow half test strip described in Example 6, conjugates formed with only 50pg COVID-19 detection mAb) were compared, ie, Control vs Cluster Conjugate formed with 50pg COVID-19 Ab and 0.25mM Solution 4 per mg of Eu particles. As shown in Figure 10, the antibody cluster conjugate clearly gave better results compared to the Control indicating that with less antibody, there was more functional antibody in the cluster compared to a non-cluster.

Example 9: Comparison of Antibody Cluster vs Control (IV): The Effect of different Ligand Concentrations to Metal Coordination Complex.

[00180] 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. In brief, conjugates (Control and Solution 5A mediated antibody clusters) were assessed at 50pg Ab per mg of Eu particles on a test line formed with 1 mg/ml SARS-CoV Ab + 1mg/ml BSA and as well, 150|jg Ab per mg of Eu particles were also assessed on a test line formed with 0.2mg/ml SARS-CoV Ab + 1mg/ml BSA. For each set, three different Solution 5 concentrations, 0.125mM, 0.0625mM and 0.03125mM were compared. As shown in Figure 11 , there was little difference in outcome compared to the Control when 50pg Ab per mg of Eu particles were used but as shown in Figure 12, the differences were significant when 150pg Ab per mg of Eu particles were used. There are large assay improvements using 0.125mM concentration of Solution 5A and worse outcomes with the other concentrations when compared to Control. The different capping groups, their concentration with respect to available antibody forms clusters with different characteristics including improving and/or decreasing the functionality of antibodies.

Example 10: Binding Antibody Clusters to Gold Nanoparticles.

[00181] 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 ALI40 nanoparticles. In brief, antibody clusters formed from, A., 10pL of 1mM 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.

[00182] mAb on gold colloids were assessed on a test line formed with 0.5mg/ml SARS-CoV Ab + 1 mg/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 13. Antibody clusters formed with both Solution 4 and 5A at the relevant concentrations passively bound to gold nanoparticles and gave improved assay performance over the Control.