Screening methods for antibody compositions

Abstract

The present invention provides a screening method for obtaining an antibody composition comprising at least two different monoclonal antibodies. The invention also relates to said antibody composition, and its use. Further, the invention provides a kit comprising said antibody composition. The invention also provides a method for determining a level of antibodies directed to an antigen in a sample.

Description

[0001] SCREENING METHODS FOR ANTIBODY COMPOSITIONS

[0002] Field of Invention

[0003] The present invention provides a screening method for obtaining an antibody composition comprising at least two different monoclonal antibodies, wherein the monoclonal antibodies are preferably non-chimeric. The invention also relates to said antibody composition, and its use. Further, the invention provides a kit comprising said antibody composition. The method further provides methods for determining a level of antibodies directed to an antigen in a sample.

[0004] Background

[0005] Reference standards are used to standardize procedures that are intended to quantify the amount of analyte in a sample. Standardization ensures correct unit assignment to the obtained raw signal of the assessment and lot-to-lot consistency, thereby enables high numeric reproducibility of a result. Result consistency is a prerequisite to refer to previously obtained results e.g. in case of analyte titre monitoring over time.

[0006] Additionally, application of the same reference standardization material to different assay methods allows the harmonization of otherwise hardly comparable results obtained by different methods. Scientific publications often also require a reference to unique and accredited reference material or performance controls to enable judgement of data quality, as well as relative comparison, of data from different studies using different methods.

[0007] Ideally, reference standards can be generated by definition of the contained amount of analyte in worldwide accepted units of the “systeme international (SI)” as established and maintained by the General Conference on Weights and Measures and based on the metric system. International quantification of a contained analyte is classically determined in mass per volume, corresponding SI unit would be kilogram per cubic metre (kg / m3) or subunits thereof (a widely accepted subunit of volume is defined in litres (I), with 1 L = 1 dm3). Equally, the amount of substance per volume can also be defined in mole per cubic metre (mol / m3) with mole defined by the amount of molecules per mass based on the Avogadro constant (NA). Molar definition is most relevant in cases where molar ratios of interaction partners are of primary interest. Reference standard material ideally contains an amount of analyte defined in SI units.

[0008] Certain analytes, however, are difficult to relate to SI units, e.g. in case the reactive subtraction cannot be separated from the non-reactive proportion of the same class of molecules. This is e.g. the case for immunoglobulins, i.e. antibodies, raised by a host in the course of an infection or vaccination. Whereas the total amount immunoglobulin in a human sample can be quantified in SI units, the amount of immunoglobulins specific to a defined target, for example towards the receptor binding domain (RBD) of the SARS-CoV-2 S1 protein, cannot. This is based on the minor differences between immunoglobulins which change its binding specificity, but do not change its mass.

[0009] Also, reactivity to a given target can be realized by a multitude of sequences of the complementarity-determining region (CDR) of an immunoglobulin. In a native (natural) humoral response, not only one monoclonal type of antibody is providing reactivity to the target, but a host always raises a polyclonal antibody response towards infection or vaccination. This does not only result from the typically broad variety of different pathogenic proteins providing a broad range of epitopes, but also is the case for comparably small targets like the RBD being also addressed with a polyclonal antibody response. Hence, antibody mediated reactivity is conveyed by this polyclonal mixture of target directed antibodies.

[0010] Still, quantification of the mounted antibody response is of medical interest and the prerequisite to monitor titer development over time or to compare individual test results and study data. This drives the need for suitable, well-defined, as well as, broadly and long-term available standard material which, at the same time, reflects sample characteristics to the best extent.

[0011] Dilution linearity is assigned to any standard material, as a proportional relation of the determined amount to the applied amount of standard material, is the characteristic of quantification and of functional standardization. However, linear dilution of polyclonal antibody mixture (such as the one found in a subject), and specific dilution characteristics are present in a polyclonal antibody mix. Of note, the dilution properties of a suitable standard material has to mirror the dilution properties of a native sample to allow useful quantification. Hence, a reference standard has to reflect the dilution dynamics of a polyclonal antibody mixture. Single monoclonal antibody based standards can reflect the dilution characteristics of polyclonal sample material, but only to a minor extent and are not considered suitable in the context of antibody quantification.

[0012] For this reason, standard material used so far is derived from pooled analyte positive human samples, hence contains a native polyclonal antibody mixture. By design, such standard material is of limited quantity, which depends on the initially acquired bulk mix volume. Follow-up standards can again be generated from new human samples, but have to be thoroughly aligned and validated to the preceding standard to ensure congruency of assigned units. However, any follow up material cannot be of identical antibody composition as the initial batch based on inevitable changes in source. This inevitable variation is due to the polyclonality generated within the individual immune response, which is highly dependent on coincidence (individual immune response), time (maturation of antibodies) and trigger (infection, reinfection, virus strain, vaccination).

[0013] Limited availability of any human sample based standard can lead to significant inconvenience and risks for continuity in diagnostic result generation. This was observed for the recently developed “First WHO International Standard for Anti-SARS-CoV-2 immunoglobulins, human” from NIBSC that faced high demand worldwide, and was therefore distributed in a limited amount, which unfortunately prohibited extensive use and generic application, but still became out of stock as early as 18 months after initial availability.

[0014] The present invention aims to ameliorate at least some of the shortcomings associated with the use of human sample based standards.

[0015] Summary of the invention

[0016] The present invention relates to the problem of generating standard material reflecting polyclonal antibody binding kinetics and associated competition of the contained antibodies for their respective binding site. This competition characteristically influences the dilution behavior of standard material of polyclonal origin. As shown in the Examples section of the present disclosure, certain concentrations show more pronounced competition than others giving rise to dilution properties reflecting these concentration dependent binding characteristics.

[0017] Strictly, monoclonal antibody dilution series do not show polyclonal antibody binding kinetics, as the contained antibodies are of the same type and bind to the same epitope. The present inventors believe that dilution behaviour is dependent on the affinity of the antibody and antibody / target ratio, but not on competition with antibodies e.g. binding close by and potentially affecting the accessibility of the epitopes of each other (in dependency of their individual concentrations).

[0018] The problem of generating standard material reflecting polyclonal antibody binding kinetics and associated competition of the contained antibodies for their respective binding site can be addressed by the present invention. The invention is based on the inventors’ surprising finding that certain combinations of monoclonal antibodies can replicate the dilution characteristics of patient obtained samples comprising polyclonal antibodies against a defined antigen. As shown in more detail in the Examples section, the inventors have tested a number of different combinations of two non-chimeric monoclonal antibodies against the SARS-CoV-2 Spike (S) protein RBD domain, SARS-CoV-2 Nucleocapsid (N) protein, or Dengue non- structural protein 1 (NS1). The inventors found that samples (derived for example from human plasma or serum) containing a single type of one of these monoclonal antibodies, or a combination of at least two different monoclonal antibodies that bind to the same epitope bin within an antigen do not reflect the dilution behaviour of human sample based standards.

[0019] However, surprisingly, the inventors’ found that when the two monoclonal antibodies each bind to different epitope bins of an antigen, the dilution behaviour of these antibodies reflects that of a real human sample based standard containing naturally produced, polyclonal antibodies.

[0020] Accordingly, in one aspect, the present invention provides a screening method for obtaining an antibody composition comprising at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, the method comprising the steps of:

[0021] • providing a first monoclonal antibody that specifically binds to a first epitope of an antigen and a second monoclonal antibody that specifically binds to a second epitope of the same antigen,

[0022] • combining the first and second monoclonal antibodies to obtain a composition of said monoclonal antibodies,

[0023] • generating a dilution curve of the composition of said monoclonal antibodies, and

[0024] • selecting the composition of said monoclonal antibodies, wherein the dilution curve has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

[0025] In an embodiment, the dilution curve may be generated by:

[0026] • preparing a dilution series of the composition of said monoclonal antibodies,

[0027] • measuring the reactivity signal of said dilution series of the composition, and

[0028] • plotting the reactivity signal over the sample concentration, thereby generating a dilution curve.

[0029] In an embodiment, the composition may comprise or consist of no more than five, no more than four, or no more than three different monoclonal antibodies, preferably non-chimeric monoclonal antibodies. Suitably, some or all of the epitopes of the monoclonal antibodies may be located within different epitope bins as measured by epitope binning. In an embodiment, the composition may comprise or consist of two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies. Suitably, the first and second epitopes may be located within different epitope bins as measured by epitope binning.

[0030] In an embodiment, the epitope binning may be measured by biolayer interferometry or surface plasmon resonance assay. Suitably, the epitope binning may be measured by surface plasmon resonance assay. Suitably, surface plasma resonance assay may be a Biacore™ immunoassay.

[0031] In an embodiment, the first monoclonal antibody may be an inhibitory antibody and the second monoclonal antibody may be a ternary complex specific antibody.

[0032] In an embodiment, the first and the second monoclonal antibodies may be ternary complex specific antibodies.

[0033] In an embodiment, the composition comprises or consists of an equal concentration of the no more than five, no more than four or no more than three monoclonal antibodies.

[0034] In an embodiment, the composition comprises or consists of an equal concentration of the first and second monoclonal antibodies.

[0035] In an embodiment, the concentration may be from 0.05nM to 50nM, from 0.05nM to 20nM, or from 0.15nM to 10nM.

[0036] In an embodiment, the dilution curve may comprise data points for at least three, at least four, or at least five different concentrations.

[0037] In an embodiment, the reactivity signal may be measured by an immunoassay, optionally wherein the immunoassay is selected from the group consisting of electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA).

[0038] In an embodiment, the antigen may be a virus or a fragment thereof, optionally wherein the virus may be selected from the group consisting of SARS-CoV-2 and Dengue virus.

[0039] In an embodiment, the antigen may be a fragment of SARS-CoV-2, optionally wherein the fragment may be a protein or a fragment of the protein, optionally wherein the protein may be selected from the group consisting of Spike (S) protein, Nucleocapsid (N) protein, Envelope (E) protein, membrane glycoprotein (G).

[0040] In an embodiment, the fragment of the S protein may be receptor binding domain (RBD). In an embodiment, the antigen may be a fragment of Dengue virus, optionally wherein the fragment may be a protein or a fragment of the protein, optionally wherein the protein may be selected from the group consisting of Nonstructural 1 (NS1) protein, envelope (E) protein, Premembrane (prM) protein, Nonstructural 3 (NS3) protein, Nonstructural 4A (NS4A) protein, and Nonstructural 5 (NS5) protein.

[0041] In another aspect, the present invention provides an antibody composition comprising at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first monoclonal antibody specifically binds to a first epitope of an antigen and the second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the composition exhibits a dilution curve according to the polynomial formula y=B2X2+Bix+Boand wherein the B2 parameter is positive.

[0042] In an embodiment, the antibody composition may comprise or consist of no more than five, no more than four, or no more than three different monoclonal antibodies, preferably non- chimeric monoclonal antibodies. Suitably, some or all of the epitopes of the monoclonal antibodies may be located within different epitope bins as measured by epitope binning.

[0043] In an embodiment, the antibody composition may comprise or consist of two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies. Suitably, the first and second epitopes may be located within different epitope bins as measured by epitope binning.

[0044] In an embodiment, epitope binning may be measured by biolayer interferometry or surface plasmon resonance assay. Suitably, epitope binning may be measured by surface plasmon resonance assay. Suitably, the surface plasma resonance may be measured by a Biacore™ immunoassay.

[0045] In an embodiment, the first monoclonal antibody may be an inhibitory antibody and the second monoclonal antibody may be a ternary complex specific antibody.

[0046] In an embodiment, the first and the second monoclonal antibodies may be ternary complex specific antibodies.

[0047] In an embodiment, the antibody composition may comprise or consist of an equal concentration of the no more than five, no more than four or no more than three monoclonal antibodies, preferably non-chimeric monoclonal antibodies. In an embodiment, the antibody composition may comprise or consist of an equal concentration of the first and second monoclonal antibodies, preferably non-chimeric monoclonal antibodies.

[0048] In an embodiment, the concentration may be from 0.05nM to 50nM, from 0.05nM to 20nM, or from 0.15nM to 10nM.

[0049] In an embodiment, the dilution curve may comprise data points for at least three, at least four, or at least five different concentrations.

[0050] In an embodiment, the antigen may be a virus or a fragment thereof, optionally wherein the virus may be selected from the group consisting of SARS-CoV-2 and Dengue virus.

[0051] In an embodiment, the antigen may be a fragment of SARS-CoV-2, optionally wherein the fragment may be a protein or a fragment of the protein, optionally wherein the protein may be selected from the group consisting of Spike (S) protein, Nucleocapsid (N) protein, Envelope (E) protein, membrane glycoprotein (G).

[0052] In an embodiment, the fragment of the S protein may be receptor binding domain (RBD).

[0053] In an embodiment, the antigen may be a fragment of Dengue virus, optionally wherein the fragment may be a protein or a fragment of the protein, optionally wherein the protein may be selected from the group consisting of NS1 protein, E protein, prM protein, NS3 protein, NS4A protein, and NS5 protein.

[0054] In an embodiment when the antigen is RBD, the at least two monoclonal antibodies may include a pair of antibodies selected from the group consisting of: 1 F12 and 4H10; 1 F12 and 2C11 ; 1 F12 and 1 H9; 1 F12 and 7G5; 1 F12 and 14F10; 2C11 and 7G5; 2C11 and 24F10; 1 H9 and 7G5; and 1 H9 and 14F10.

[0055] In an embodiment when the antigen is the N protein, the at least two monoclonal antibodies may include a pair of antibodies selected from the group consisting of: 1 B10 and 6C3; 6C3 and 3H7; and 6D10 and 6C3.

[0056] In an embodiment when the antigen is the NS1 protein, the at least two monoclonal antibodies may include a pair of antibodies selected from the group consisting of: 1.35.19 and 1.3.2; 1.35.19 and 2.75.23; and 2.75.23 and 2.67.94.

[0057] In a further aspect, provided herein is use of an antibody composition obtained by the screening method of the invention, as a reference sample and / or as a control in a diagnostic assay, preferably for determining the presence or absence, and / or concentration of antibodies in a patient sample which are directed against the same antigen as the antibodies in the antibody composition.

[0058] In an embodiment, the diagnostic assay may be an immunoassay, optionally wherein the immunoassay may be selected from the group consisting of electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA).

[0059] In an embodiment, the patient sample may be selected from the group consisting of blood sample, saliva sample, and urine sample, optionally wherein the blood sample may be selected from the group consisting of serum, plasma and whole blood.

[0060] In a further aspect, provided herein is use of an antibody composition according to the invention, as a reference sample and / or as a control in a diagnostic assay, preferably for determining the presence or absence, and / or concentration of antibodies in a patient sample which are directed against the same antigen as the antibodies in the antibody composition.

[0061] In an embodiment, the diagnostic assay may be an immunoassay, optionally wherein the immunoassay may be selected from the group consisting of electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA).

[0062] In an embodiment, the patient sample may be selected from the group consisting of blood, saliva, and urine, optionally wherein the blood may be selected from the group consisting of serum, plasma and whole blood.

[0063] In a further aspect, provided herein is a kit comprising the antibody composition obtained by the screening method of the invention.

[0064] In a further aspect, provided herein is a kit comprising the antibody composition of the invention.

[0065] In a further aspect, provided herein is a method for determining a level of antibodies directed to an antigen in a sample, the method comprising the steps of:

[0066] • obtaining a sample signal indicative of the level of antibodies directed to the antigen in the sample; and

[0067] • comparing the sample signal to a reference signal indicative of a known amount of a composition of at least two different monoclonal antibodies, wherein the first antibody specifically binds to a first epitope of the antigen and a second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the dilution curve of the composition of at least two different monoclonal antibodies has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

[0068] Except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to all aspects of the invention. Thus, by way of example, the considerations set out with reference to the method of the invention also apply to the kits and uses of the invention.

[0069] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0070] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0071] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0072] Various aspects of the invention are described in further detail below.

[0073] Brief description of the Figures

[0074] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0075] Figure 1 shows a comparison of dilution patterns of monoclonal antibodies, combinations of monoclonal antibodies and human serum. Only the combination of mAB 2C11 with mAB 7G5 reflects the dilution pattern of human reactive series. Neither user of monoclonal antibodies alone nor combinations of mABs from the same epitope bin reflect the dilution pattern of human reactive serum. The graph is based on data provided in Table 1.

[0076] Figure 2 is a graph showing dilution behaviour of representative native human samples. Native human samples show a characteristic dilution pattern resulting in a convex shape of the regression curve and a positive parameter “B2” in the associated quadratic equation. Raw signals for neat determination and individual dilutions of representative natively reactive serum and plasma samples are shown in relation to their relative concentration. Optimal polynomial regression resulted in the indicated quadratic equations. The equation factor “B2” is shown in bold.

[0077] Figure 3 is a graph showing dilution behaviour of single receptor binding domain (RBD) monoclonal antibodies. Monoclonal antibodies show dilution characteristics different from human polyclonal sample (shown in Figure 2) and cannot serve as reference standard material. Monoclonal antibodies were adjusted to a concentration of 10nM in negative human sample. A doubling dilution series in negative human sample was prepared from this 10nM starting level. Signals for 10nM concentrations and the generated dilutions are shown in relation to their absolute concentration. Optimal polynomial regression resulted in the indicated quadratic equations. The equation factor “B2” is shown in bold. Alphanumeric codes are the identifier of the investigated antibody clone.

[0078] Figure 4 shows the dilution behaviour of several combinations of monoclonal antibodies. Certain combinations of two monoclonal antibodies reflect the dilution characteristics human polyclonal samples have. Pairs of monoclonal antibodies as indicated were mixed in an equimolar ratio and adjusted to a concentration of 10nM in negative human sample. A doubling dilution series in negative human sample was prepared from this 10nM starting level. Signals for 10nM concentrations and the generated dilutions are shown in relation to their absolute concentration. Optimal polynomial regression resulted in the indicated quadratic equations. The equation factor “B2” is shown in bold. Alphanumeric codes are the identifier of the investigated antibody clone. Antibody mixes with a polyclonal dilution signature are shown in the first nine panels, and mixes that do not have a polyclonal dilution signature are shown in the last three panels of this figure.

[0079] Figure 5 shows that dilution series of native human samples positive for antibodies to the SARS CoV-2 Nucleocapsid protein show a convex dilution behavior resulting in a positive “B2” factor of the corresponding quadratic regression. A doubling dilution series of native human samples positive for antibodies to the SARS CoV-2 Nucleocapsid protein was prepared in negative human serum. Raw signals for neat determination and the respective dilutions of representative natively reactive serum samples are shown in relation to their relative concentration. Optimal polynomial regression resulted in the indicated best fit values for a quadratic equation. Arrows indicate the equation factor “B2” being positive for the regression analyses of both dilution series, c (rel), arbitrary sample concentration with neat sample set to 1 ; raw signal, direct analyzer signal (counts) obtained with the Elecsys SARS- CoV-2 (antiSpike RBD) assay. Figure 6 shows that certain mixes of monoclonal antibodies to the SARS-CoV-2 Nucleocapsid protein reflect the dilution characteristics human polyclonal samples have. Pairs of monoclonal antibodies as indicated were mixed in an equimolar ratio in negative human sample. A doubling dilution series in negative human sample was prepared from this starting level. These samples were assessed with the Elecsys SARS-CoV-2 (anti- Nucleocapsid) assay. Obtained signal are shown in relation to the sample concentration. Quadratic regression resulted in the associated factors shown in the tables. Results normalized to the individual starting point of each dilution (set to 1) are shown in the lower panel of plots to enable best comparison of the shape of the regression curves. A. Antibody pairs that mimic polyclonal binding behavior. Black arrows indicate the positive factor “B2” obtained exclusively for the combination of the indicated antibodies, whereas B2 is negative for the regressions on the dilution series of the single antibodies. B. Antibody pairs that bind independently and hence do not mimic polyclonal binding behavior. Grey arrows indicate the factor “B2” that does not change in prefix when dilution series of single antibodies or their combinations are assessed.

[0080] Figure 7 shows dilution series of native human samples positive for antibodies to the Dengue virus NS1 protein show an apparent linear dilution behavior that still follows a slightly convex regression curve resulting in a positive “B2” factor of the corresponding quadratic regression. A doubling dilution series of native human samples positive for antibodies to the Dengue virus NS1 protein was prepared in negative human serum. Raw signals for neat determination and the respective dilutions of representative natively reactive serum samples are shown in relation to their relative concentration. Optimal polynomial regression resulted in the indicated best fit values for a quadratic equation. Arrows indicate the equation factor “B2” being positive for the regression analyses of both dilution series, c (rel), arbitrary sample concentration with neat sample set to 1 ; raw signal, direct analyzer signal (counts) obtained with the Elecsys Dengue IgG (anti-Dengue NS1) assay.

[0081] Figure 8 shows that certain mixes of monoclonal antibodies to the Dengue NS1 protein reflect the dilution characteristics human polyclonal samples have. Pairs of monoclonal antibodies as indicated were mixed in an equimolar ratio in negative human sample. A doubling dilution series in negative human sample was prepared from this starting level. These samples were assessed with the Elecsys Dengue IgG (anti-Dengue NS1) assay. Obtained signal are shown in relation to the sample concentration. Quadratic regression resulted in the associated factors shown in the tables. Results normalized to the individual starting point of each dilution (set to 1) are shown in the lower panel of plots to enable best comparison of the shape of the regression curves. A. Antibody pairs that mimic polyclonal binding behavior. Black arrows indicate the positive factor “B2” obtained exclusively for the combination of the indicated antibodies, whereas B2 is negative for the regressions on the dilution series of the single antibodies. B. Antibody pairs that bind independently and hence do not mimic polyclonal binding behavior. Grey arrows indicate the factor “B2” that does not change in prefix when dilution series of single antibodies or their combinations are assessed.

[0082] Various aspects of the invention are described in further detail below.

[0083] Detailed Description

[0084] It is common practice to determine the concentration of a tested analyte (such as an antibody to a specific antigen) in a sample by using a “dilution curve” (also commonly referred to as a “calibration curve”, “standard curve” or “working curve”). Such a curve can be generated by plotting the interrelation between known amounts (for example concentrations) of an analyte (such as an antibody to the specific antigen) in a reference sample and values (also referred to herein as reactivity signals such as optical densities, florescence, etc.) corresponding to those known amounts. For the dilution curve to be informative, the dilution behaviour of the analyte in the reference sample must correspond to that of the tested analyte. This allows one to correlate the reactivity signals of the tested analyte to the reactivity signals on the dilution curve, thereby determine the amount of tested analyte.

[0085] As mentioned above, the present invention is based on the inventors’ finding that certain compositions comprising at least two different monoclonal antibodies each binding to a different epitope of an antigen, may be used as a reference sample, for example in diagnostic methods for determining the level of antibodies directed to an antigen in a sample, without the need for obtaining human sample based standards. As explained elsewhere in the present disclosure, such human sample based standards are associated with many drawbacks (such as limited quantities of material). Therefore, the present invention represents a significant improvement over the current status quo.

[0086] A screening method for obtaining an antibody composition

[0087] Accordingly, in the first aspect, the present invention provides a screening method for obtaining an antibody composition comprising at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, the method comprising the steps of: • providing a first monoclonal antibody that specifically binds to a first epitope of an antigen and a second monoclonal antibody that specifically binds to a second epitope of the same antigen,

[0088] • combining the first and second monoclonal antibodies to obtain a composition of said monoclonal antibodies,

[0089] • generating a dilution curve of the composition of said monoclonal antibodies, and

[0090] • selecting the composition of said monoclonal antibodies, wherein the dilution curve has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

[0091] The term “screening method” refers to a method of investigating whether an antibody composition comprising at least two different monoclonal antibodies has a desired dilution behaviour. In this context, a composition can be said to have a desired dilution behaviour, when a dilution curve of said composition has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive. An antibody composition that is selected on the basis of having this desired dilution behaviour may be suitable for use as a reference sample and / or control, for example in a diagnostic assay. Therefore, it can be said that the screening method is for obtaining a reference sample and / or control.

[0092] The antibody composition may comprise a sample obtained from a single reference subject or a group of reference subjects that is / are known or suspected of not having natural (native) antibodies against the antigen of interest. In other words the antibody composition may be obtained by combining the monoclonal antibodies with a sample obtained from a single reference subject or a group of reference subjects. Natural (native) antibodies against the antigen may develop in response to the reference subject being exposed to the antigen (for example as a result of an infection, vaccination, development of cancer cells, or coming into contact with an allergen). Accordingly, the reference subject is known or suspected of not being exposed to said antigen, and / or known or suspected of not being recently exposed to said antigen. By “recently” it is meant that the reference subject is known or suspected of not being exposed to the antigen at least 3 months, at least 6 months, at least 9 months, at least 12 months, or more prior, to acquiring the reference sample from the reference subject. Accordingly, the antibody composition is also known or suspected of not having natural (native) antibodies against the antigen of interest. In other words, it can be said that the antibody composition is substantially negative for (or substantially or completely devoid of) natural (native) antibodies against the antigen of interest. However, it will be understood that whilst the antibody composition is negative for natural (native) antibodies against the antigen of interest, the reference sample may comprise other antibodies against different antigens. Instead of containing natural (native) antibodies against the antigen, the antibody composition comprises monoclonal antibodies against the antigen, preferably non-chimeric monoclonal antibodies.

[0093] Suitably, the antibody composition comprises at least two different monoclonal antibodies, , preferably non-chimeric monoclonal antibodies wherein the first monoclonal antibody specifically binds to a first epitope of the antigen, and the second monoclonal antibody specifically binds to a second epitope of the same antigen.

[0094] Suitably, the antibody composition comprises no more than five, no more than four, or no more than three different monoclonal antibodies, preferably non-chimeric monoclonal antibodies. Suitably the antibody composition consists of no more than five, no more than four, or no more than three different monoclonal antibodies. In an embodiment where the antibody composition comprises more than two different monoclonal antibodies (for example three, four or five different monoclonal antibodies), at least two of those different monoclonal antibodies bind to a different epitope of the antigen (i.e. first of the at least two monoclonal antibodies binds a first epitope, and the second of the at least two monoclonal antibodies binds a second epitope).

[0095] Suitably, when the antibody composition comprises more than two monoclonal antibodies (for example three, four, five, or more), some or all of the monoclonal antibodies may bind to a different epitope of the antigen, providing that at least two of those different monoclonal antibodies bind to a different epitope of the antigen (i.e. first of the at least two monoclonal antibodies binds a first epitope, and the second of the at least two monoclonal antibodies binds a second epitope). It will be appreciated that in the context of the present disclosure, different epitopes refers to epitopes of an antigen that have a different amino acid sequence.

[0096] Suitably, the composition may consist of two different monoclonal antibodies, preferably non- chimeric monoclonal antibodies, wherein the first antibody specifically binds to a first epitope of the antigen, and the second monoclonal antibody specifically binds to a second epitope of the antigen.

[0097] Herein, when the antibody composition comprises (or consists) of at least two, at least three, etc. or two, three, etc. different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, it will be understood that this refers to the number of distinct monoclonal antibodies, rather than to the number of antibody molecules as such. By “different”, it is meant antibodies that have a different amino acid sequence. Different monoclonal antibodies may differ in their sequences by any number of amino acid residues (for example 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50 or more residues). By the same token, “different epitopes” refers to epitopes of an antigen that have a different amino acid sequence.

[0098] The epitope may differ by any number of amino acid residues (for example 1 , 2, 3, or more residues).

[0099] It will be appreciated that the monoclonal antibodies may be synthetically added to the antibody composition. By synthetically adding an amount of the monoclonal antibodies to the antibody composition, the amount (for example concentration) of the at least two different monoclonal antibodies in the antibody composition may be known. The term “amount” as used herein may refer to the concentration (for example nM). Suitably, only the total amount of the monoclonal antibodies in the antibody composition may be known. Suitably, the amount of each of the different monoclonal antibodies in the antibody composition may be known. The different types of monoclonal antibodies in the antibody composition may be present in equal amount (for example be equimolar) or be in different amounts (for example be non-equimolar).

[0100] As used herein, the term "antibody" refers to an immunoglobulin or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulphide bonds. The term "antibody" includes monoclonal antibodies, non-chimeric antibodies, recombinant antibodies, human antibodies, humanized antibodies and chimeric antibodies. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The terms "domain" and "domain" are used interchangeably herein. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs). Each VH and VL typically comprises 3 CDRs. In a suitable embodiment, the antibody may be selected from the group consisting of IgG, IgE, IgD, IgA and IgM, or an antigen binding fragment thereof. Suitably, the IgG may be selected from the group consisting of lgG1 , lgG2, lgG3, and lgG4. Suitably, the IgA may be selected from the group consisting of lgA1 and lgA2.

[0101] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different epitopes, each monoclonal antibody in said population is directed against a single epitope on the antigen. Monoclonal antibodies are typically produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same epitope on the antigen. The term “monoclonal” is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc. Suitably, the monoclonal antibody may be man-made. By man-made it is meant generated using standard laboratory techniques rather than by a natural immune response.

[0102] As used herein, the term “polyclonal antibodies” refers to a composition comprising a heterogenous mixture of antibodies capable of binding to different and / or the same epitopes within the antigen. For avoidance of doubt, the antibody composition of the invention is a heterogenous mixture of antibodies capable of binding to different epitopes within the antigen and therefore may be considered as comprising polyclonal antibodies to the antigen of interest. However, as mentioned elsewhere herein, the antibody composition is also known or suspected of not having natural (native) antibodies against the antigen of interest. In other words, it can be said that the antibody composition is substantially negative for (or substantially or completely devoid of) natural (native) antibodies against the antigen of interest. It will be understood that whilst the antibody composition is negative for natural (native) antibodies against the antigen of interest, the antibody composition may comprise other antibodies against different antigens.

[0103] The term "specifically binds," or "binds specifically to", or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x1 O'6M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In the context of the present disclosure the term “specifically bind” or “bind” may be used interchangeably, and have the same meaning. Furthemore, for avoidance of doubt, when reference is made to “antibodies directed to an antigen” herein, it shall be understood that such antibodies are antibodies which bind the antigen.

[0104] Suitably, in the context of the screening method of the invention, the first and second epitopes may be located on different epitope bins of the antigen. As explained elsewhere herein, the present inventors have found that certain combinations of at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first monoclonal antibody binds a first epitope of an antigen, and a second monoclonal antibody binds a second epitope of the same antigen, have a desired dilution behaviour. This desired dilution behaviour may be determined by generating a dilution curve of the composition comprising at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies. A composition with the desired dilution behaviour will have a dilution curve that has the polynomial formula y=B2X2+Bix+Bo, and wherein the B2 parameter is positive. Suitably, in context of the present disclosure, in order to determine whether the B2 parameter is positive, a polynomial dilution curve may be generated. The present inventors found that antibody combinations that have this desired dilution behaviour comprise at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first monoclonal antibody binds an epitope located within a first bin, and a second monoclonal antibody that binds an epitope located within a second bin.

[0105] The term “bin”, “bins” or “epitope bins” refer to areas of an antigen. Antibodies binding within the same epitope bin are likely to compete for the same or highly related epitope. Methods measuring (i.e. determining) epitope bins are known and widely used by those skilled in the art. Bins may be determined by a method known as “epitope binning” (also known as “epitope mapping”). In epitope binning, pairs of monoclonal antibodies specific to the same antigen may be tested to assess whether they block one another’s binding to a specific site of the antigen or not. The monoclonal antibodies that block binding to the same epitope are “binned” together, i.e. bind an epitope located in the same bin. Methods for epitope binning are known in the art. By way of example, epitope binning may be measured by biolayer interferometry or surface plasmon resonance assay. More suitably, epitope binning may be measured by surface plasmon resonance assay. “Biolayer interferometry” refers to an optical analytical technique that analyses the interference pattern of light reflected from a layer of immobilized protein on a biosensor tip and an internal reference layer. “Surface plasmon resonance” refers to the physical phenomenon in which incident light is converted strongly into electron currents at the metal surface for planar surfaces, and “localized surface plasmon resonance (LSPR)” can also be used for surface plasmon resonance of nanometer-sized metallic structures. The oscillating currents produce strong electric fields in the (non-conducting) ambient medium near the surface of the metal. The electric fields, in turn, induce electric polarization in the ambient medium. Electric polarization is well known to cause the emission of light at wavelengths characteristic of the medium, i.e., the “Raman wavelengths.” The Biacore™ immunoassay is an example of a surface plasmon resonance assay that can be used for epitope binning.

[0106] Exemplary methods for epitope binning are discussed for example in Nilvebrant, et al.

[0107] Y1 (Methods Mol Biol, 2018, 1785: 1-10) and Haynes, et al. (Communications biology, 2021 , 4, 1317), all of which are incorporated herein by reference. Epitope binning may be performed using a binning software such as Biacore™ Insight Epitope Binning Extension or the Carterra Epitope Software.

[0108] In an embodiment where the antibody composition comprises at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first monoclonal antibody specifically binds to a first epitope of the antigen, and the second monoclonal antibody specifically binds to a second epitope of the same antigen, the first and second epitopes may be located within different epitope bins.

[0109] In an embodiment where the antibody composition comprises more than two (for example no more than five, no more than four, or no more than three) different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, at least two of those different monoclonal antibodies bind different epitopes (i.e. the first of the at least two monoclonal antibodies binds a first epitope, and the second of the at least two monoclonal antibodies binds a second epitope), and the first and second epitopes may be located within different epitope bins.

[0110] By the same token, in an embodiment where the antibody composition consists of no more than five, no more than four, or no more than three different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, at least two of those different monoclonal antibodies bind different epitopes (i.e. the first of the at least two monoclonal antibodies binds a first epitope, and the second of the at least two monoclonal antibodies binds a second epitope), and the first and second epitopes may be located within different epitope bins.

[0111] Suitably, in the context of the screening method, the first monoclonal antibody may be an inhibitory antibody, and the second monoclonal antibody may be a ternary complex binding antibody.

[0112] Suitably, the first and second monoclonal antibodies may be both inhibitory antibodies.

[0113] Suitably, the first and second monoclonal antibodies may be both ternary complex binding antibodies.

[0114] The term “inhibitory antibody” as used herein means any antibody having inhibiting activity, which means that binding of the inhibitory antibody to an antigen, prevents binding to its physiological binding partner. As way of example, inhibitory antibodies against RBD of SARS- CoV-2 virus, inhibit binding of the RBD of the SARS-CoV-2 Spike protein to its binding partner ACE2 (present on the surface of host cells), thereby inhibiting fusion of the SARS-CoV-2 virus with the host cell, i.e. preventing infection of the host cell. Referring to the Examples section of the present disclosure, examples of inhibitory antibodies are 4H10, 7G5, 14F10, and 1 F12 (all of which are directed against SARS-CoV-2 RBD).

[0115] The term “ternary complex binding antibody” as used herein refers to an antibody that binds a complex comprising at least two peptides, as e.g. an antigen-antibody complex. At certain concentrations, such antibodies bridge two antigens to each other and this structure may be termed ternary complex. Referring to the Examples section of the present disclosure, examples of ternary complex binding antibodies are 1 H9, 3D11 , 2C11 , 6G12, and 13G2 (all of which are directed against SARS-CoV-2 RBD).

[0116] As used herein, the term “antigen” refers to any substance that is capable of inducing an adaptive immune response. Suitably, the antigen may be selected from the group consisting of a virus, bacterium, fungus, parasite, autoantigen, tumorantigen and allergen, ora fragment thereof (such as a protein, fragment of a protein, or a nucleic acid (for example RNA or DNA) or a fragment of a nucleic acid). The antigen may be natural or synthetic. A vaccine is an example of a synthetic antigen.

[0117] As exemplified by the Examples of the present disclosure, the antigen may be a virus, for example SARS-CoV-2, Dengue virus, or a fragment thereof.

[0118] Suitably the fragment may be of a protein expressed by SARS-CoV-2 or a fragment of a protein, and / or a nucleic acid (for example RNA) of SARS-CoV-2. Merely by way of example the protein may be selected from the group consisting of Spike (S) protein, Nucleocapsid (N) protein, Envelope (E) protein, membrane glycoprotein (G). A fragment of the S protein may be for example the receptor binding domain (RBD).

[0119] Suitably the fragment may be of a protein expressed by Dengue virus or a fragment of a protein, and / or a nucleic acid (for example RNA) of Dengue virus. Merely by way of example the protein may be selected from the group consisting of Nonstructural 1 (NS1) protein, envelope (E) protein, Premembrane (prM) protein, Nonstructural 3 (NS3) protein, Nonstructural 4A (NS4A) protein, and Nonstructural 5 (NS5) protein.

[0120] The screening method of the invention comprises the step of combining the first and second monoclonal antibodies, preferably non-chimeric monoclonal antibodies, to obtain a composition of said monoclonal antibodies. In this context, it will be appreciated that in an embodiment where the antibody composition comprises more than two different monoclonal antibodies, each of the different monoclonal antibodies may be combined. Therefore, in an embodiment where the composition comprises more than two different antibodies (for example, three, four, five, or more) all of the different monoclonal antibodies may be combined together to obtain a composition of said monoclonal antibodies.

[0121] The term “combining” as used herein means putting or adding together, so as to form a mixture of said monoclonal antibodies. The monoclonal antibodies may be combined in the presence of a liquid (such as a sample obtained from a reference subject, as mentioned elsewhere herein). Therefore, it will be appreciated that when it is said in the context of the present disclosure that the antibody composition “comprises no more than five”, etc. different monoclonal antibodies, the composition may comprise in addition to those monoclonal antibodies, other antibodies that may be natural in the liquid (such as a sample obtained from a reference subject) within which the may be combined. Therefore, in other words it can be said that the antibody composition may comprise a mixture of monoclonal antibodies, wherein the mixture consists of no more than five, no more than four, or no more than three different monoclonal antibodies. Suitably, the antibody composition may comprise a mixture of monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the mixture consists of a first monoclonal antibody and a second monoclonal antibody.

[0122] Upon combining the first and second monoclonal antibodies to obtain a composition of said monoclonal antibodies, the method involves the step of generating a dilution curve of the composition of said monoclonal antibodies. It will be appreciated that in an embodiment where the antibody composition comprises more than two different monoclonal antibodies (for example three, four, five, or more monoclonal antibodies), the method may involve the step of generating a dilution curve of the composition comprising all of said monoclonal antibodies.

[0123] The term “dilution curve” refers to the correlation between the amount (for example concentration) of the at least two different monoclonal antibodies in a composition of said monoclonal antibodies and a reactivity signal. The reactivity signal may be indicative of a known amount of the at least two different monoclonal antibodies in the composition of said monoclonal antibodies. The term “reactivity signal” as used herein refers to any detectable effect and / or change, caused and / or provided by an assay carried out on the composition of said monoclonal antibodies. The effect and / or change may be due to the presence of monoclonal antibodies directed to an antigen in the composition. Suitably, the effect and / or change may be proportional to the amount (for example concentration). The reactivity signal may be in the form of fluorescence, phosphorescence, chromogenicity, optical density, chemiluminescence, light-scattering, and / or Raman scattering. Various other forms of reactivity signals indicative of the amount of antibodies in a composition are well known to those skilled in the art.

[0124] It shall also be appreciated that methods of detecting fluorescence, phosphorescence, chromogenicity, optical density, chemiluminescence, light-scattering, and Raman scattering, and / or changes in fluorescence, phosphorescence, chromogenicity, optical density, chemiluminescence, light-scattering, and Raman scattering are also very well known to those skilled in the art. The reactivity signal may be assigned a value (such as a number). The units of the value may depend on the type of signal, for example the units may be relative fluorescence units (RFUs), optical density units (ODUs), or chemiluminescent units (CUs). The reactivity signal may be obtained by any suitable method known in the art.

[0125] In a suitable embodiment, the reactivity signal may be obtained by an assay selected from the group consisting of electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry, and radioimmunoassay. Different subtypes of these assays will be known to those skilled in the art. Merely by way of example, the ELISA may be selected from the group consisting of a double antigen sandwich ELISA, direct ELISA, indirect ELISA, and sandwich ELISA.

[0126] Suitably, the assay may be ECL. ECL provides a sensitive and precise measurement of the presence and concentration of an analyte of interest (such as a monoclonal antibody). Such technique uses labels or other reactants that can be induced to luminesce when electrochemically oxidized or reduced in an appropriate chemical environment. Such electrochemiluminescence is triggered by a voltage imposed on a working electrode at a particular time and in a particular manner. The light produced by the label is measured and indicates the presence or quantity of the analyte. For a fuller description of ECL techniques, reference is made to US Patent No. 5,221 ,605, US Patent No. 5,591 ,581 , US Patent No. 5,597,910, PCT published application W090 / 05296, PCT published application WO92 / 14139, PCT published application W090 / 05301 , PCT published application WO96 / 24690, PCT published application US95 / 03190, PCT application US97 / 16942, PCT published application US96 / 06763, PCT published application WO95 / 08644, PCT published application WO96 / 06946, PCT published application WO96 / 33411 , PCT published application W087 / 06706, PCT published application WO96 / 39534, PCT published application WO96 / 41175, PCT published application WO96 / 40978, PCT / US97 / 03653 and US patent application 08 / 437,348 (U.S. Patent No. 5,679,519). Reference is also made to a 1994 review of the analytical applications of ECL by Knight, et al. (Analyst, 1994, 119: 879- 890) and the references cited therein. Methods for generating a dilution curve will be well known to those skilled in the art. Merely by way of example, the method for generating a standard curve may comprise the steps of a) preparing a dilution series, b) measuring the reactivity signal of said dilution series, and c) plotting the reactivity signal over the sample concentration, thereby generating a dilution curve. Step a) of this exemplary method may comprise providing a single reference sample comprising a known amount of the analyte (i.e. composition of said monoclonal antibodies) and diluting it to provide multiple compositions of different, known, amounts of monoclonal antibodies. These multiple compositions of different, known, amounts of monoclonal antibodies may be referred to as “dilution series”. Suitably, the dilution curve may be generated by obtaining at least three, at least four, at least five, or more reactivity signals indicative of at least three, at least four, at least five, or more different known amounts (for example concentrations) of the at least two different monoclonal antibodies in the composition. In other words, the dilution curve may comprise data points for at least three, at least four, at least five, or more different concentrations of monoclonal antibodies. Merely by way of example, the concentration of monoclonal antibodies in the composition may be from about OnM to about 50nM, for example from about OnM to about 20nM, or for example from about OnM to about 10nM. Merely by way of example, in a dilution series, the concentration of monoclonal antibodies in the composition may be OnM, 0.25 nM, 0.5nM, 0.75nM, 1 ,25nM, 2.5nM, 5nM and 10nM. For avoidance of doubt, the term “measuring the reactivity signal of said dilution series” means obtaining a reactivity signal for each of the different multiple compositions of different, known, amounts of monoclonal antibodies. Upon measuring the reactivity signal of each of the samples (i.e. multiple compositions of different, known, amounts of monoclonal antibodies) in the dilution series, the reactivity signal of each of the samples within the dilution series may be plotted over the concentration of each of the samples within the dilution series.

[0127] Suitably, the composition of monoclonal antibodies may comprise an equal concentration of the monoclonal antibodies. In an embodiment, where the composition comprises more than two different monoclonal antibodies (for example, three, four, five, or more) some or all of the different monoclonal antibodies may be at an equal concentration. Suitably, the concentration may be from 0.05nM to 50nM, from 0.05nM to 20nM, or from 0.15nM to 10nM.

[0128] The screening method of the invention involves the step of selecting those compositions of monoclonal antibodies that produce a dilution curve that has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive. The B parameters are the polynomial coefficients and B2 is the guiding coefficient of this second order (i.e. quadratic) polynomial function. The sign of the guiding coefficient defines the orientation of the parabolic shape of a quadratic function. A positive guiding coefficient in a second order, i.e. quadratic, polynomial function (termed here: “B2” parameter) defines upward bending of the parabola described by this second order polynomial function. Upward bending of a parabola is also termed as “convex shape”. Those selected compositions of monoclonal antibodies may be referred to herein as “antibody compositions”. Said antibody compositions give rise to a further aspect of the invention as described herein. The selected antibody compositions may be used as reference samples and / or control. In other words it can be said that when the composition of monoclonal antibodies has a dilution curve that has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive, it is indicative of the composition of monoclonal antibodies being suitable for use as a reference sample and / or control.

[0129] The terms calibrator, validations, standardization or standard, verification may be used as synonym of the term “reference sample”

[0130] A “polynomial curve” is a curve that can be parametrized by polynomial functions of R[x], A polynomial curve may be obtained by polynomial curve fitting. Methods of polynomial curve fitting will be well known to those skilled in the art. As it will be appreciated by a person skilled in the art the formula y=B2X2+Bix+Bo is a quadratic function, wherein B2X2is the quadratic term, Bix is the linear term, and Bo is the constant term and refers to the y-axis intercept. It shall be also appreciated by a person skilled in the art that the terms “B2”, “Bi” and “Bo” are constants. A positive B2 value indicates that the polynomial dilution curve has a convex shape. For a polynomial dilution curve as described herein, y is the signal obtained, and x is the amount (for example concentration) of the at least two monoclonal antibodies.

[0131] The screening method of the invention may be high throughput (i.e. involve investigating a large number of monoclonal antibody combinations, for example 10, 20, 30 or more combinations). Alternatively, the screening method may involve investigating a small number (less than 10, for example 5, 4, 3, 2 or 1 ) of monoclonal antibody combination(s).

[0132] Antibody Compositions, Uses and Kits

[0133] In another aspect, the present invention provides an antibody composition comprising at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first monoclonal antibody specifically binds to a first epitope of an antigen and the second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the composition exhibits a dilution curve according to the polynomial formula y=B2X2+Bix+Boand wherein the B2 parameter is positive. The antibody composition may be obtainable by the screening method of the invention as described herein. The term “obtainable” as used herein also encompasses the term “obtained”. Thus, the antibody composition may be obtained by the screening method of the invention. Accordingly, the embodiments outlined above in relation to the method of screening apply equally to the antibody composition of the invention, as well as uses and kits of the invention, unless the context requires otherwise.

[0134] Suitably, the at least two monoclonal antibodies of the antibody composition may include a pair of antibodies selected from the group consisting of: 1 F12 and 4H10; 1 F12 and 2C11 ; 1 F12 and 1 H9; 1 F12 and 7G5; 1 F12 and 14F10; 2C11 and 7G5; 2C11 and 24F10; 1 H9 and 7G5; and 1 H9 and 14F10. As explained in the Examples section of the present disclosure, these antibodies bind the receptor binding domain (RBD) of the S protein of SARS-CoV-2. Accordingly, such an antibody composition may be particularly useful as a reference sample in a diagnostic assay for determining the levels of antibodies in a sample from a subject against the S protein RBD of SARS-CoV-2.

[0135] Suitably, the at least two monoclonal antibodies of the antibody composition may include a pair of antibodies selected from the group consisting of: 1 B10 and 6C3; 6C3 and 3H7; and 6D10 and 6C3. As explained in the Examples section of the present disclosure, these antibodies bind to the N protein of SARS-CoV-2. Accordingly, such an antibody composition may be particularly useful as a reference sample in a diagnostic assay for determining the levels of antibodies in a sample from a subject against the N protein of SARS-CoV-2.

[0136] Suitably, the at least two monoclonal antibodies of the antibody composition may include a pair of antibodies selected from the group consisting of: 1.35.19 and 1.3.2; 1.35.19 and 2.75.23; and 2.75.23 and 2.67.94. As explained in the Examples section of the present disclosure, these antibodies bind to the NS1 protein of Dengue virus. Accordingly, such an antibody composition may be particularly useful as a reference sample in a diagnostic assay for determining the levels of antibodies in a sample from a subject against the NS1 protein of Dengue.

[0137] In a further aspect, the present invention provides use of an antibody composition of the present invention as a reference sample and / or control in a diagnostic assay. Suitably, the diagnostic assay may be for determining the presence or absence, and / or concentration of antibodies in a patient sample, which are directed against the same antigen as the antibodies in the antibody compositions. Suitably, the diagnostic assay may be an immunoassay. Examples of immunoassays are provided elsewhere herein, and include electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA).

[0138] The diagnostic assay may involve the steps of: a) obtaining a reactivity signal indicative of the level of antibodies directed to the antigen in the sample; and b) comparing the reactivity signal to a signal indicative of a known amount of the antibody composition of the invention.

[0139] Herein, the reactivity signal obtained in a) may be referred to as “a sample signal”, and the reactivity signal obtained in b) may be referred to as “a reference signal”.

[0140] Suitably, the present invention provides a method for determining a level of antibodies directed to an antigen in a sample, the method comprising the steps of:

[0141] • obtaining a sample signal indicative of the level of antibodies directed to the antigen in the sample; and

[0142] • comparing the sample signal to a reference signal indicative of a known amount of a composition of at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first antibody specifically binds to a first epitope of the antigen and a second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the dilution curve of the composition of at least two different monoclonal antibodies has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

[0143] Optionally, the diagnostic assay and / or method for determining a level of antibodies may comprise the step of obtaining a reference signal. The reference signal may be obtained simultaneously or sequentially to the sample signal. As used herein, the term “sample signal” refers to the signal produced by the sample, i.e. signal indicative of the level of antibodies directed to the antigen in the sample. The term “simultaneously” as used herein does not necessarily mean that the sample signal and reference signal are obtained at the exact same time point. This term also includes signals that are obtained during the course of the same assay and / or the same assay run. The term “sequentially” as used herein means that the signals (sample signal and reference signal) are obtained in sequence, for example at an interval or intervals of minutes, hours, days or weeks. The step of obtaining the reference signal may be obtained before and or after the step of obtaining the signal. As used herein, the term “obtaining”, refers to measuring, receiving, or otherwise getting information regarding the level (for example concentration) of the antibodies in a sample directed to the antigen, and / or monoclonal antibodies in the antibody composition of the invention. The term “obtaining” shall be broadly understood to include passive obtaining (for example from a third party or storage element) and / or "active" acquiring (for example by performing an assay). Suitably, the information regarding the level of antibodies (for example in the sample and / or monoclonal antibodies in the antibody composition of the invention ) may be in the form of a reactivity signal. The strength of the reactivity signal obtained may depend upon the level of the analyte (such as antibodies directed to an antigen in the sample and / or monoclonal antibodies in the antibody composition of the invention).

[0144] It will be appreciated that the effect and / or change in reactivity signal may be relative to the levels of the substance (such as an antibody in the sample and / or monoclonal antibodies in the antibody composition of the invention), rendering the signal (or reference signal) indicative of the level of antibodies in the sample (or reference sample).

[0145] The term “comparing” as used herein refers to any suitable method of evaluating, calculating or processing data allowing one to determine the level of antibodies directed to an antigen in a sample by referring to the reference signal. It is to be understood that comparison generally refers to a comparison of corresponding parameters or values, such as comparing a fluorescence signal of a sample to a fluorescence signal of a reference sample, or a chemiluminescence signal of a sample to a chemiluminescence signal of a reference sample. The comparison may be performed manually or may be computer-assisted. Thus, the comparison may be made by a computing device. For computer-assisted comparison, the obtained signal may be compared with a suitable reference signal value stored by a computer program in a database. The computer program may further evaluate the result of the comparison, i.e. automatically provide the required evaluation in a suitable output format.

[0146] In the context of the present disclosure, the reference signal may be indicative of a known amount of at least two different monoclonal antibodies in the antibody composition upon generation of a dilution curve. Therefore, suitably, the diagnostic assay and / or method for determining a level of antibodies may comprise the step of generating the dilution curve.

[0147] The term “sample” as used herein refers to any biological matter in which antibodies are naturally found. Suitably, the sample may be selected from the group consisting of a blood sample, a saliva sample, and urine sample. Suitably the blood sample may be a plasma sample, a serum sample, or a whole blood sample. As used herein, the term “whole-blood” is a broad term and is used in its ordinary sense and refers, without limitation, to blood that has been withdrawn from a patient but that has not been otherwise processed, e.g., it has not been haemolysed, lyophilized, centrifuged, or separated in any other manner, after being removed from the patient. Whole-blood may contain amounts of other fluids, such as interstitial fluid or intracellular fluid, which may enter the sample during the withdrawal process or are naturally present in the blood. The term "serum", as used herein, refers to the whole blood component that results after blood clotting and removal of the resulting clot, the term “plasma” as used herein refers to the liquid fraction obtained by removing the blood cells contained in whole blood.

[0148] The sample may be obtained from the subject. Methods of obtaining samples from a subject are well known in the art. By way of example and not limitation the subject may be selected from the group consisting of a human, a monkey, a dog, a pig, a bovine, a rabbit, a guinea pig, and a rodent.

[0149] The term “reference sample” or “control” as used herein refers to an antibody composition of the invention that may be analysed in substantially the same manner as the patient’s sample (i.e. sample) and whose information may be compared to the information (such as signal) of the sample to obtain further information about the sample. Suitably, the reference sample may be the same type as the sample (for example the reference sample and sample may be both blood samples, for example plasma samples). As explained elsewhere herein in the context of an antibody composition, the reference sample may be obtained from a reference subject or group of reference subjects. Therefore, the reference sample is known or suspected of not having natural (native) antibodies against the antigen of interest. In other words, it can be said that the reference sample is substantially negative for (or substantially or completely devoid of) natural (native) antibodies against the antigen of interest. However, it will be understood that whilst the reference sample is negative for natural (native) antibodies against the antigen of interest, the reference sample may comprise other antibodies against different antigens. Instead, the reference sample may comprise monoclonal antibodies against the antigen of interest. Such monoclonal antibodies may be artificially added to provide the reference sample.

[0150] In another aspect, provided herein is a kit comprising the antibody composition of the invention. As mentioned elsewhere herein, the antibody composition may be obtainable by the screening method of the invention. Therefore, suitably, the antibody composition may be obtained by the screening method of the invention. Suitably, the kit may be for determining the levels of antibodies to an antigen in a sample. The term “antigen” and examples thereof are provided elsewhere herein.

[0151] In a suitable embodiment, the kit may be for determining the level of antibodies to SARS- CoV-2 in a sample. Suitably, the kit may be for determining the level of antibodies to the S protein of SARS-CoV-2, N protein of SARS-CoV-2, or a fragment thereof. Suitably, the fragment of the S protein may be RBD. Suitably, the kit may be for determining the level of antibodies to Dengue virus. Suitably, the kit may be for determining the level of antibodies to NS1.

[0152] Suitably, when the kit is for determining the level of antibodies to the S protein (or RBD of the S protein) of SARS-CoV-2, the kit may comprise an antibody composition, wherein the antibody composition comprises a pair of antibodies selected from the group consisting of: 1 F12 and 4H10; 1 F12 and 2C11 ; 1 F12 and 1 H9; 1 F12 and 7G5; 1 F12 and 14F10; 2C11 and 7G5; 2C11 and 24F10; 1 H9 and 7G5; and 1 H9 and 14F10. Suitably, the at least two antibodies are 1 F12 and 4H10. More suitably, the at least two monoclonal antibodies may be 1 F12 and 4H10. The details of these antibodies (i.e. 1 F12, 4H10; 2C11 ; 1 H9; 7G5; 14F10; and 2C11) such as their sequences are provided elsewhere herein.

[0153] Suitably, when the kit is for determining the level of antibodies to the N protein of SARS-CoV- 2, the kit may comprise an antibody composition, wherein the antibody composition comprises a pair of antibodies selected from the group consisting of: 1 B10 and 6C3; 6C3 and 3H7; and 6D10 and 6C3. The details of these antibodies (i.e. 1 B10, 6C3, 3H7, and 6D10), such as their sequences are provided elsewhere herein.

[0154] Suitably, when the kit is for determining the level of antibodies to the NS1 protein of Dengue virus, the kit may comprise an antibody composition, wherein the antibody composition comprises a pair of antibodies selected from the group consisting of: 1.35.19 and 1.3.2; 1.35.19 and 2.75.23; and 2.75.23 and 2.67.94. Exemplary details of these antibodies, such as their sequences are provided elsewhere herein.

[0155] The antibody composition may be provided in solution or lyophilized. As used herein, the term “in solution” means that the molecule (e.g. the monoclonal antibody, monoclonal antibodies, or antigen as discussed herein) is present in the liquid composition in its free form. Merely by way of example, the solution may be a buffer solution. Examples of the buffer solution include, but are not limited to, MES, HEPES, TES, ADA, ACES, bis-Tris, Tris, TES, CAPS, boric acid buffer solution, phosphate buffer solution and citrate buffer solution. Other suitable solutions for providing the monoclonal antibodies in solution will be known to those skilled in the art. The term “lyophilized” or “freeze-dried” includes a state of a substance that has been subjected to a drying procedure such as lyophilization, where at least 50% of moisture has been removed.

[0156] As mentioned, the kit may further comprise the antigen. The antigen may be immobilised to a solid support, lyophilized, or in solution. The terms “lyophilized” and “in solution” are described above. By “solid support” it is herein referred to a material which is insoluble, or can be made insoluble by a subsequent reaction. Numerous and varied solid supports are known to those in the art and include, without limitation, multi-well plates (such as 96-well plates), nitrocellulose, the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, membranes, and microparticles (such as latex particles).

[0157] Further examples of useful solid supports include: natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, crosslinked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulfates or carbonates of alkaline earth metals and magnesium, including barium sulfate, calcium sulfate, calcium carbonate, silicates of alkali and alkaline earth metals, aluminum and magnesium; and aluminum or silicon oxides or hydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials may be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initializing polymerization of synthetic polymers on a preexisting natural polymer. Further examples of useful solid supports include: a microfluidic chip, a silicon chip, a microscope slide, a microplate well, solid and semi-solid matrixes, resins, beads, biochips, multi-well plates, membranes, conducting and non-conducting metals, glass, magnetic supports, silica gels, polymeric membranes, particles, derivatized plastic films, derivatized glass, derivatized silica, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead; or Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

[0158] The antigen may be immobilised to the solid support directly or indirectly. By “directly” it is meant that the antigen is in contact with the solid support. By “indirectly” it is meant that the antigen is attached to the solid support through a linker. The term "linker," as used herein, refers to a chemical group or a molecule linking the antigen to the solid support. The linker may be, for example, a short amino acid sequence (for example between 3 and 100 amino acids in length, 10 and 75 amino acids in length, or 25 to 50 amino acids in length).

[0159] In some embodiments the kit further comprises a detecting reagent. “A detecting reagent” as used herein refers to a reagent for detecting an interaction between antibodies and the antigen. It will be appreciated that the reagent may be for detecting the interaction between the monoclonal antibodies provided in the kit and the antigen, and / or the antibodies in the sample the levels of which are to be determined. Many reagents for detecting an interaction between antibodies and the antigen are known in the art. Suitably, the reagent for detecting the interaction may be a secondary antibody. The term “secondary antibody” refers to an antibody that specifically binds to a primary antibody. In the context of the present disclosure, the “primary antibody” may be a monoclonal antibody provided in the kit (or the monoclonal antibody in the reference sample) and / or the antibody in the sample from the subject as described herein with reference to the method of the invention. Suitably, the secondary antibody will detect an interaction between the antibodies in the sample and / or the monoclonal antibodies in the reference sample by binding to antibodies in the sample and / or monoclonal antibodies in the reference sample.

[0160] A detecting reagent may comprise or consists of a detectable label. It will be appreciated that in some embodiments, the detectable label is what, upon binding of the antibodies (in the sample and / or reference sample), provides the signal indicative of the levels of antibodies as described in the context of the method of the invention. In some examples, the detectable label may be a fluorescent label, a chemiluminescent label, a bioluminescent label, a radioactive label, a paramagnetic label, biotin, or an enzyme. Examples of fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde or fluorescamine. Examples of chemiluminescent labels include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.

[0161] Examples of bioluminescent labels include luciferin, luciferase or aequorin.

[0162] Suitably, kit may comprise an instruction manual and / or may provide information regarding the use of its contents (for example instructions on how to generate a dilution curve for the antibody composition).

[0163] In one embodiment, the kit may comprise the monoclonal antibodies that together form the antibody composition of the invention.

[0164] Suitably, the kit may comprise or consist of at least two different monoclonal antibodies, preferably non-chimeric monoclonal antibodies, wherein the first monoclonal antibody specifically binds to a first epitope of the antigen and a second monoclonal antibody specifically binds to a second epitope of the same antigen, and wherein a dilution curve of a composition of said at least two monoclonal antibodies has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive. Suitably, the kit may comprise or consist of no more than five, no more than four, no more than three different monoclonal antibodies, wherein a dilution curve of a composition of said monoclonal antibodies has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

[0165] The monoclonal antibodies in the kit may be provided in a single container, or in separate containers. By way of example, the container may be a tube, vile, bottle, and the like.

[0166] In an embodiment, where the kit comprises more than two different monoclonal antibodies, for example no more than five, no more than four, or no more than three different monoclonal antibodies, some or all may be provided in a separate container. Alternatively, some or all may be provided in the same container.

[0167] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0168] EXAMPLES

[0169] Example 1 - Assessment of the dilution properties of polyclonal versus monoclonal antibody material

[0170] Using an immunoassay the inventors compared the different dilution properties for polyclonal versus monoclonal antibody material. The results are shown in Figure 1 and Table 1.

[0171] Table T. Relative signals obtained with dilution series of single monoclonal antibodies (mAB) and mAB combinations compared to a human serum dilution defining the reference dilution pattern. Indicated clones or equimolar mixes of the indicated clones of antibodies were diluted to the indicated concentrations (nM) in human negative serum. A dilution series of reactive human serum in the same negative serum was assessed in parallel as a reference for the required dilution pattern. Relative signals indicate the ratio in percent to the signal of the highest concentration of the individual series to enable comparison independent of obtained absolute signal. Epitope bins associated to the antibody used are indicated by capital letters.

[0172] Data in Table 1 show the dilution pattern discrepancy between polyclonal antibodies and monoclonal antibodies. The present invention addresses this discrepancy by mimicking polyclonal dilution characteristic through the use of two monoclonal antibodies that bind different epitope bins. Surprisingly, the use of two different monoclonal antibodies that do not bind different epitope bins does not reflect the dilution behaviour of true polyclonal sample material. Without wishing to be bound by this hypothesis, the inventors believe that the epitope bins should not be identical, at these antibody pairs might compete for the same or highly related epitope, which then is reflective of the less complex condition in a monoclonal antibody dilution. The inventors believe that only if the bins of the antibodies are different, but of sufficient spatial proximity that antibody binding affects binding efficacy of the other binder to a certain extent, then a polyclonal competition scenario is sufficiently mimicked.

[0173] This setup of reference standard material enables unlimited supply of identical quality and characteristics. The required monoclonal antibodies can be produced in large scale, are of defined sequence and generated from a stable cell line. Tedious pre-characterization of suitable human sample material, estimate of demand and generation of suitable amount of stock is not necessary, ethic approval is not required and the manufacturing is less resource intense.

[0174] Example 2 - Examples of specific antibody combinations

[0175] Materials and Methods

[0176] Reference dilution series of human serum

[0177] Individual human samples from donors convalescent from a native infection with SARS-CoV- 2 and hence positive for antibodies to the receptor binding domain (RBD) of the Spike 1 protein from SARS-CoV-2 were diluted with human serum that was negative for these antibodies. Native positive material contains a polyclonal mixture of antibodies resulting in a characteristic dilution pattern. All investigated human samples showed the same characteristic pattern. Here, we included two representative human sample dilution series as a reference.

[0178] Comparator dilution series of MAB and MAB mixes

[0179] Mass concentrations of the investigated MABs were determined by standard procedure (OD280 of purified MAB). The MABs were then adjusted to the indicated molar concentrations by spiking into human negative serum. For MAB mixes, the indicated concentrations refer to the total amount of MAB with each contained MAB contributing to this amount in equimolar ratio. Consecutive 1 :2 dilution series were prepared by further dilution in the same human negative serum.

[0180] Determination of reactivity

[0181] Reactivity of the samples used were determined with the Elecsys Anti-SARS-CoV-2 S assay according to IVD approved standard procedures as described in the method sheet (Mat#09289275190). In brief, this immunoassay specifically determines reactivity to the receptor binding domain (RBD) of the Spike 1 protein from SARS-CoV-2 by application of a double antigen sandwich format. It is a fully automated immunoassay running on cobas e analyzers.

[0182] Data read out and analysis

[0183] The data are shown as raw signals (“counts”) determined by the photomultiplier of the analyzer. Typically, raw signals are translated into units based on standardization and calibration. However, as this investigation seeks to determine most suitable material for standardization, comparison of raw signals was relevant for appropriate comparison of material characteristics. Raw signals can vary with run, analyzer and assay reagent used. To avoid this variation, all compared data were generated within the same run in batch enabling reliable comparison. Data analyses and calculations were performed with Microsoft Excel 2016. Graphs were generated with Microsoft Excel or Graphpad Prism 10.

[0184] Results

[0185] Dilution of human serum or plasma revealed a characteristic dilution pattern. Exemplary data sets are shown in Figure 2. The relation of the dilution level to the observed raw signal is described by polynomial regression leading to the indicated quadratic equations of the formula y=ax2+bx+c. The associated parameter “a” is positive, as the dilution pattern of reactive serum and plasma describes a convex curve. Despite individual absolute reactivity of neat human samples differs, of course, their dilution pattern follows the same mathematical characteristics which is driven by their polyclonal reactivity.

[0186] Candidate standard reference material was compared to the dilution pattern defined by native human sample with suitable material having to share the same mathematical characteristics, i.e. parameter a being positive.

[0187] The inventors investigated the dilution pattern of RBD-specific monoclonal antibodies for their relation of raw signal to concentration (Figure 3). The observed dilution pattern describes a concave curve and the determined parameter “a” is negative. The mathematical characteristics of the dilution pattern are discordant to that of native polyclonal samples. The observed dilution pattern was of the same characteristics for all monoclonal antibodies tested. These data show that suitable reference material cannot be composed of a single monoclonal antibody.

[0188] Whereas dilution behaviour of the native sample reference (polyclonal antibodies) might be influenced by steric hindrance and secondary binding effects, the dilution behaviour of monoclonal antibodies is believed by the inventors to be dependent on affinity and concentration of the monoclonal antibody only. This difference results in the significantly different dilution patterns.

[0189] The inventors assessed if the polyclonal dilution behaviours of a native sample can be mimicked by combination of a minimal set of monoclonal antibodies, anticipating that hindrance and epitope competition similar to that observed in a polyclonal antibody sample can occur.

[0190] In the examples provided below, different combinations of two different monoclonal antibodies (MAB), directed against the RBD of SARS-CoV-2 Spike 1 protein, were tested for their dilution pattern. Both antibodies were applied at equal molar ratio, indicated concentration indicate the final concentration of the mix (Figure 4).

[0191] Surprisingly, the inventors observed that certain combinations of as few as two MABs reflected the dilution characteristics of a native polyclonal sample (convex dilution curve, positive coefficient B2), whereas others did not (concave dilution curve, negative coefficient B2). Those combinations that reflected native sample dilution behaviours qualified as suitable candidates for a reference standard (native sample dilution behaviours, unlimited supply, absolute quantification in SI units possible).

[0192] Out of the identified candidates, the equimolar combination of MABs 1 F12 and 4H10 was selected to serve as the reference standard for the Elecsys Anti-SARS-CoV-2 S assay.

[0193] Epitope binning analyses

[0194] Despite the exact epitopes of the investigated antibodies are not known, the antibodies had already been assigned to epitope “bins” by SPR (as described in W02023 / 072904, which is incorporated here by reference). These bins are arbitrarily assigned, relative regions as determined by observation of relative binding competition. This means antibodies with the same epitope bin compete for binding to their specific epitope, antibodies with different bins can bind their epitope on the analyte without binding of one antibody affects binding of the other.

[0195] It became evident that MAB combinations from different bins resulted in the desired dilution behaviour, whereas the MAB combinations from the same bin did not. This finding suggests that a defined spatial relation of the epitopes is required to induce convex dilution characteristics. Of note, this spatial relation does not appear to be direct competition for the same or similar binding site, i.e. not antibodies of the same epitope bin. But a more distant influence of MABs on each other seems to be required to mimic the dilution characteristics of a native polyclonal sample.

[0196] Table 2: Relation of observed dilution patterns of MAB combinations to their assigned epitope bins. Matrix of tested MAB clones, their respective epitope bin and the observed dilution characteristics. Only combinations of MABs from different epitope bins resulted in the desired convex dilution pattern, MAB combinations from the same epitope bin showed concave dilution patterns similar to patterns of single MABs.

[0197] Example 3 - Binding characteristics to the SARS-CoV-2 Nucleocapsid protein

[0198] Binding characteristics to the SARS-CoV-2 Nucleocapsid protein were assessed with the Elecsys Anti-SARS-CoV-2 assay (which employs the Nucleocapsid protein as signal specifier. The dilution behavior of a native human serum sample positive for antibodies to SARS-CoV-2 Nucleocapsid protein is shown in Figure 5. Again, the observed dilution pattern describes a convex curve and, consequently, the “B2” factor of the associated equation of the fitted quadratic regression is positive.

[0199] In a next step, the inventors investigated the dilution pattern of available monoclonal antibodies towards the SARS-CoV-2 Nucleocapsid protein. The inventors identified antibody pairs that showed a similar dilution pattern to that of native serum (concave curve, positive “B2”), whereas the individual antibodies in separate dilution showed the opposing pattern (convex curve, negative “B2”). The inventors could also identify antibody patterns that do not change their regression pattern upon combination (“B2” of the dilution of the antibody combinations is not different from the single dilutions). Here, binding seems to be independent of the presence of a second antibody, no interference occurs and the dilution pattern remains unchanged. Results are shown in Figure 6.

[0200] Example 4 - Binding characteristics to the wing domain of the Dengue NS1 protein

[0201] Binding characteristics to the wing domain of the Dengue NS1 protein were assessed with the Elecsys Anti-Dengue IgG assay (which employs the wing domain of the Dengue NS1 protein as signal specifier). The dilution behavior of a native human serum sample positive for antibodies to Dengue NS1 protein is shown in Figure 7.

[0202] The binding pattern of dilution series of native polyclonal serum describes a convex curve, despite at a less prominent level which is visually difficult to determine. Still, the “B2” factor of the associated equation of the fitted quadratic regression is positive.

[0203] In analogy to the previous examples, the inventors investigated the dilution pattern of available monoclonal antibodies towards the Dengue NS1 protein. They could identify antibody pairs that showed a similar dilution pattern to that of native serum (concave curve, positive “B2”), whereas the individual antibodies in separate dilution showed the opposing pattern (convex curve, negative “B2”). The inventors also identified antibody patterns that do not change their regression pattern upon combination (“B2” of the dilution of the antibody combinations is not different from the single dilutions). Here, binding seems to be independent of the presence of a second antibody, no interference occurs and the dilution pattern remains unchanged.

[0204] Results are shown in Figure 8.

[0205] Summary

[0206] The inventors have shown that it is possible to apply suitable antibody pairs of monoclonal antibodies to mimic polyclonal binding behavior. This was experimentally confirmed in three independent setups.

[0207] The strength of the effect was different for each assay and antigen used, but the change of the generated dilution pattern towards the dilution characteristics of polyclonal human serum was observed in all three experiments.

[0208] The association of observed effects to the region of binding (“antibody bin”) was investigated for RBD. Specific spatial localization of the respective epitopes appears to be required for occurrence of the here described binding effects and explains presence of effects in certain, but not all of the additionally investigated antibody pairs on additionally investigated targets. These data further indicate that suitable reference standard material should not be composed of a single monoclonal antibody, as this typically does not match the dilution properties of polyclonal human sample.

[0209] ANTIBODY SEQUENCE INFORMATION

[0210] Table 3: sequences for antibody clone 2C11; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0211] Table 4: sequences for antibody clone 1H9; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0212] Table 5: sequences for antibody clone 7G5; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0213]

[0214] Table 6: sequences for antibody clone 1F12; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0215] Table 7: sequences for antibody clone 13G2; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0216]

[0217] Table 8 sequences for antibody clone 14F10; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0218] Table 9: sequences for antibody clone 3D11; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0219]

[0220] Table 10: sequences for antibody clone 4H10; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0221] Table 11: sequences for antibody clone 6G12; Antigen: SARS-CoV-2 Spike-RBD; Host: rabbit.

[0222]

[0223] Table 12: sequences for antibody clone 1B10; Antigen: SARS-CoV-2 Nucleocapsid; Host: rabbit

[0224] Table 13: sequences for antibody clone 6C3; Antigen: SARS-CoV-2 Nucleocapsid; Host: rabbit

[0225]

[0226] Table 14: sequences for antibody clone 3H7; Antigen: SARS-CoV-2 Nucleocapsid; Host: rabbit

[0227] Table 15: sequences for antibody clone 6D10; Antigen: SARS-CoV-2 Nucleocapsid; Host: rabbit

[0228]

[0229] Table 16: sequences for antibody clone 1F1; Antigen: SARS-CoV-2 Nucleocapsid; Host: rabbit

[0230] Table 17: sequences for antibody clone 1.3.2; Antigen: Dengue NS1; Host: mouse

[0231]

[0232] Table 18: sequences for antibody clone 2.75.23; Antigen: Dengue NS1; Host: mouse

[0233] Table 19: sequences for antibody clone 2.30.63; Antigen: Dengue NS1; Host: mouse

[0234]

[0235] Table 20-. sequences for antibody clone 1.37.3; Antigen: Dengue NS1; Host: mouse

[0236] EMBODIMENTS

[0237] 1 . A screening method for obtaining an antibody composition comprising at least two different monoclonal antibodies, the method comprising the steps of:

[0238] • providing a first monoclonal antibody that specifically binds to a first epitope of an antigen and a second monoclonal antibody that specifically binds to a second epitope of the same antigen,

[0239] • combining the first and second monoclonal antibodies to obtain a composition of said monoclonal antibodies,

[0240] • generating a dilution curve of the composition, and

[0241] • selecting the composition, wherein the dilution curve has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

[0242] 2. The according to embodiment 1 , wherein the dilution curve is generated by:

[0243] • preparing a dilution series of the composition of said monoclonal antibodies,

[0244] • measuring the reactivity signal of said dilution series of the composition, and

[0245] • plotting the reactivity signal over the sample concentration, thereby generating a dilution curve.

[0246] 3. The method according to embodiment 1 or 2, wherein the composition comprises or consists of no more than five, no more than four, or no more than three different monoclonal antibodies.

[0247] 4. The method according to embodiment 1 or 2, wherein the composition consists of two different monoclonal antibodies

[0248] 5. The method according to embodiment 3, wherein some or all of the epitopes of the monoclonal antibodies are located within different epitope bins as measured by epitope binning.

[0249] 6. The method according to embodiment 4, wherein the first and second epitopes are located within different epitope bins as measured by epitope binning.

[0250] 7. The method according to embodiment 5 or 6, wherein the epitope binning is measured by biolayer interferometry or surface plasmon resonance assay.

[0251] 8. The method according to embodiment 7, wherein the epitope binning is measured by surface plasmon resonance assay. The method according to embodiment 8, wherein the surface plasma resonance assay is a Biacore™ immunoassay. . The method according to any preceding embodiment, wherein the first monoclonal antibody is an inhibitory antibody and the second monoclonal antibody is a ternary complex specific antibody. . The method according to embodiment 3, or any one of embodiments 5 to 10, wherein the composition comprises or consists of an equal concentration of the no more than five, no more than four or no more than three monoclonal antibodies. . The method according to any one of embodiments 1 , 2 or 4 to 10, wherein the composition comprises or consists of an equal concentration of the first and second monoclonal antibodies. . The method according to embodiment 11 or 12, wherein the concentration is from 0.05nM to 50nM, from 0.05nM to 20nM, or from 0.15nM to 10nM. . The method according to any preceding embodiment, wherein the dilution curve comprises data points for at least three, at least four, or at least five different concentrations. . The method according to any preceding embodiment, wherein the reactivity signal is measured by an immunoassay, optionally wherein the immunoassay is selected from the group consisting electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA). . The method according to any preceding embodiment, wherein the antigen is a virus or a fragment thereof, optionally wherein the virus is selected from the group consisting of SARS-CoV-2 and Dengue virus. . The method according to embodiment 16, wherein the antigen is a fragment of SARS- CoV-2, optionally wherein the fragment is a protein or a fragment of the protein, optionally wherein the protein is selected from the group consisting of Spike (S) protein, Nucleocapsid (N) protein, Envelope (E) protein, membrane glycoprotein (G). . The method of embodiment 17, wherein the fragment of the S protein is receptor binding domain (RBD). . The method according to embodiment 16, wherein the antigen is a fragment of Dengue virus, optionally wherein the fragment is a protein or a fragment of the protein, optionally wherein the protein is selected from the group consisting of Nonstructural 1 (NS1) protein, envelope (E) protein, Premembrane (prM) protein, Nonstructural 3 (NS3) protein, Nonstructural 4A (NS4A) protein, and Nonstructural 5 (NS5) protein. . An antibody composition comprising at least two different monoclonal antibodies, wherein the first monoclonal antibody specifically binds to a first epitope of an antigen and the second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the composition exhibits a dilution curve according to the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive. . The antibody composition according to embodiment 20, wherein the composition comprises or consists of no more than five, no more than four, or no more than three different monoclonal antibodies. . The antibody composition according to embodiment 20 or 21 , wherein the composition consists of two different monoclonal antibodies. . The antibody composition according to claim 21 , wherein some or all the epitopes of the monoclonal antibodies are located within different epitope bins as measured by epitope binning. . The antibody composition according to claim 20 or 22, wherein the first and second epitopes are located within different epitope bins as measured by epitope binning. . The antibody composition according to embodiments 23 or 24, wherein epitope binning is measured by biolayer interferometry or surface plasmon resonance assay.. The antibody composition according to embodiment 25, wherein epitope binning is measured by surface plasmon resonance assay. . The antibody composition according to embodiment 26, wherein the surface plasma resonance is measured by a Biacore™ immunoassay. . The antibody composition according to any one of embodiments 20 to 27, wherein the first monoclonal antibody is an inhibitory antibody and the second monoclonal antibody is a ternary complex specific antibody. . The antibody composition according to embodiment 21 , or any one of embodiments 23 to 28, wherein the antibody composition comprises or consists of an equal concentration of the no more than five, no more than four or no more than three monoclonal antibodies. The antibody composition according to any one of embodiments 20, 22, or 24 to 28, wherein the antibody composition comprises or consists of an equal concentration of the first and second monoclonal antibodies. The antibody composition according to embodiment 28 or 29, wherein the concentration is from 0.05nM to 50nM, from 0.05nM to 20nM, or from 0.15nM to 10nM. The antibody composition according to any one of embodiments 20 to 30, wherein the dilution curve comprises data points for at least three, at least four, or at least five different concentrations. The antibody composition according to any one of embodiments 20 to 31 , wherein the antigen is a virus or a fragment thereof, optionally wherein the virus is selected from the group consisting of SARS-CoV-2 and Dengue virus. The antibody composition according to embodiment 32, wherein the antigen is a fragment of SARS-CoV-2, optionally wherein the fragment is a protein or a fragment of the protein, optionally wherein the protein is selected from the group consisting of Spike (S) protein, Nucleocapsid (N) protein, Envelope (E) protein, membrane glycoprotein (G). The antibody composition according to embodiment 33, wherein the fragment of the S protein is receptor binding domain (RBD). The antibody composition according to embodiment 32, wherein the antigen is a fragment of Dengue virus, optionally wherein the fragment is a protein or a fragment of the protein, optionally wherein the protein is selected from the group consisting of NS1 protein, E protein, prM protein, NS3 protein, NS4A protein, and NS5 protein. The antibody composition according to embodiment 34, wherein the at least two monoclonal antibodies include a pair of antibodies selected from the group consisting of: 1 F12 and 4H10; 1 F12 and 2C11 ; 1 F12 and 1 H9; 1 F12 and 7G5; 1 F12 and 14F10; 2C11 and 7G5; 2C11 and 24F10; 1 H9 and 7G5; and 1 H9 and 14F10. The antibody composition according to embodiment 33, wherein when the antigen is the N protein, the at least two monoclonal antibodies include a pair of antibodies selected from the group consisting of: 1 B10 and 6C3; 6C3 and 3H7; and 6D10 and 6C3. The antibody composition according to embodiment 35, wherein when the antigen is the

[0252] NS1 protein, the at least two monoclonal antibodies include a pair of antibodies selected from the group consisting of: 1.35.19 and 1.3.2; 1.35.19 and 2.75.23; and 2.75.23 and

[0253] 2.67.94. Use of an antibody composition obtained by the method according to any one of embodiments 1 to 19, as a reference sample and / or as a control in a diagnostic assay, preferably for determining the presence or absence, and / or concentration of antibodies in a patient sample which are directed against the same antigen as the antibodies in the antibody composition. The use according to embodiment 39, wherein the diagnostic assay is an immunoassay, optionally wherein the immunoassay is selected from the group consisting electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA). The use according to embodiment 39 or 40, wherein the patient sample is selected from the group consisting of blood sample, saliva sample, and urine sample, optionally wherein the blood sample is selected from the group consisting of serum, plasma and whole blood. Use of an antibody composition according to any one of embodiments 20 to 38, as a reference sample and / or as a control in a diagnostic assay, preferably for determining the presence or absence, and / or concentration of antibodies in a patient sample which are directed against the same antigen as the antibodies in the antibody composition. The use according to embodiment 42, wherein the diagnostic assay is an immunoassay, optionally wherein the immunoassay is selected from the group consisting electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA). The use according to embodiment 42 or 43 wherein the patient sample is selected from the group consisting of blood, saliva, and urine, optionally wherein the blood is selected from the group consisting of serum, plasma and whole blood. A kit comprising the antibody composition obtained by the method according to any one of embodiments 1 to 19. A kit comprising the antibody composition according to any one of embodiments 20 to 38. A method for determining a level of antibodies directed to an antigen in a sample, the method comprising the steps of: obtaining a sample signal indicative of the level of antibodies directed to the antigen in the sample; and

[0254] • comparing the sample signal to a reference signal indicative of a known amount of a composition of at least two different monoclonal antibodies, wherein the first antibody specifically binds to a first epitope of the antigen and a second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the dilution curve of the composition of at least two different monoclonal antibodies has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

Claims

CLAIMS1 . A screening method for obtaining an antibody composition comprising at least two different monoclonal antibodies, the method comprising the steps of:• providing a first monoclonal antibody that specifically binds to a first epitope of an antigen and a second monoclonal antibody that specifically binds to a second epitope of the same antigen,• combining the first and second monoclonal antibodies to obtain a composition of said monoclonal antibodies,• generating a dilution curve of the composition of said monoclonal antibodies, and• selecting the composition of said monoclonal antibodies, wherein the dilution curve has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

2. The method of claim 1 , wherein the dilution curve is generated by:• preparing a dilution series of the composition of said monoclonal antibodies,• measuring the reactivity signal of said dilution series of the composition, and• plotting the reactivity signal over the sample concentration, thereby generating a dilution curve.

3. An antibody composition comprising at least two different monoclonal antibodies, wherein the first monoclonal antibody specifically binds to a first epitope of an antigen and the second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the composition exhibits a dilution curve according to the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

4. The method according to claim 1 or 2, or antibody composition according to claim 3, wherein the antibody composition comprises or consists of no more than five, no more than four, or no more than three different monoclonal antibodies.

5. The method or antibody composition according to any preceding claim, wherein the first and second epitopes are located on different epitope bins as measured by epitope binning.

6. The method or antibody composition according to claim 5, wherein the epitope binning is measured by biolayer interferometry or surface plasmon resonance assay, optionally wherein the surface plasma resonance assay is a Biacore™ immunoassay.

7. The method or antibody composition according to any preceding claim, wherein the first monoclonal antibody is an inhibitory antibody and the second monoclonal antibody is a ternary complex specific antibody.

8. The method or antibody composition according to any preceding claim, wherein the antibody composition comprises or consists of an equal concentration of the at least two different monoclonal antibodies, optionally wherein the concentration is from 0.05nM to 50nM, from 0.05nM to 20nM, or from 0.15nM to 10nM.

9. The method or antibody composition according to any preceding claim, wherein the dilution curve comprises data points for at least three, at least four, or at least five different concentrations.

10. The method or antibody composition according to any preceding claim, wherein the antigen is a virus or a fragment thereof, optionally wherein the virus is selected from the group consisting of SARS-CoV-2 and Dengue virus.

11. Use of an antibody composition obtained by the method according to any one of claims 1 , 2, or 4 to 10, or use of the antibody composition according to any one of claims 3 to 10, as a reference sample and / or as a control in a diagnostic assay, preferably for determining the presence or absence, and / or concentration of antibodies in a patient sample which are directed against the same antigen as the antibodies in the antibody composition.

12. The use according to claim 11 , wherein the diagnostic assay is an immunoassay, optionally wherein the immunoassay is selected from the group consisting of electrochemiluminescence (ECL), ELISA, ELISPOT, flow cytometry and radioimmunoassay (RIA).

13. The use according to claim 11 or 12, wherein the patient sample is selected from the group consisting of blood sample, saliva sample, and urine sample, optionally wherein the blood sample is selected from the group consisting of serum, plasma and whole blood.

14. A kit comprising the antibody composition obtained by the method according to any one of claims 1 , 2, or 4 to 10, or a kit comprising the antibody composition according to any one of claims 3 to 10.

15. A method for determining a level of antibodies directed to an antigen in a sample, the method comprising the steps of:obtaining a sample signal indicative of the level of antibodies directed to the antigen in the sample; and comparing the sample signal to a reference signal indicative of a known amount of a composition of at least two different monoclonal antibodies, wherein the first antibody specifically binds to a first epitope of the antigen and a second monoclonal antibody specifically binds to a second epitope of the same antigen, wherein the dilution curve of the composition of at least two different monoclonal antibodies has the polynomial formula y=B2X2+Bix+Bo and wherein the B2 parameter is positive.

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