WO2019207115A1 - Improved immunoassays - Google Patents

Abstract

The present invention relates to improved immunoassays with the use of a heat-treated preparation of Immunoglobulins to reduce false positives. The invention furthermore relates to the use of one or more Fc fragments to reduce false positives in immunoassays.

Description

Improved immunoassays

Technical field

The present invention relates to an immunoassay comprising at least one antibody capable of binding an antigen of interest in a sample, and a heat-treated preparation of Immunoglobulins.

Background

One of the most used immunoassays is the sandwich (capture) immunoassay (e.g. ELISA), where one monoclonal antibody (MAb) is used to capture an antigen (Ag) or other analyte of interest, and another (labelled) MAb is used to quantify the Ag/analyte (Holm et al. 2015).

Rheumatoid factors (RFs), present in 50-90 % of patients with autoimmune connective tissue diseases and 5-10 % of persons with infections and approximately 5 % of apparently healthy persons, interfere with sandwich immunoassays due to their ability to (cross)react with the capture and detecting MAbs (Holm et al. 2015) thereby giving erroneous and false positive results. RFs are immunoglobulins (Igs) recognizing the Fc part of other Igs. They are mainly found in blood samples from patients with rheumatoid diseases (e.g. rheumatoid arthritis (RA)) and occur in different forms, but the most prominent are IgM and IgA antibodies (Abs) (RFs) binding to the Fc part of IgG (Holm et al. 2015, Mageed et al. 1997, Dorner et al. 2014, Newkirk et al. 2002). The epitopes of RFs are characterised in a few cases and reside in the Fc part, often in the

CH2/CH3 groove or the CH3/CH3 groove (Corper et al. 1997, Duquerroy et al. 2007). An apparent paradox is how RFs can circulate in blood together with IgG in large amounts in patients with rheumatoid diseases. RFs can bind to the capture and/or detector antibodies to yield a signal even in the absence of analytes in immunoassays such as nephelometry and ELISA. Some heterophilic antibodies (HAbs) also interfere with sandwich immunoassays, depending on the species of origin of the Abs employed (Holm et al. 2015).

The involvement of RFs in false positive results has been recognized for a long time and many attempts have been made to eliminate RF interference with sandwich immunoassays. Currently, several reagents/products are available for

avoiding/minimizing interference of HAbs/RFs with sandwich immunoassays, notably Heteroblock (Thermo-Fisher/Omega) and Heterophilic Blocking Reagent (HBR) (Scantibodies Laboratory). Especially HBR is useful for minimizing false positive results in sandwich immunoassays. The composition and manufacture of these reagents are not known, however, both Heteroblock and HBR have been characterized in some detail (Holm et al. 2015). Disadvantages of these reagents are that they have a heterogeneous composition and that their modes of production are unknown.

The present invention relates to a method of overcoming interference with the results of immunoassays by non-specific binding of heterophilic antibodies in the sample to the antibodies present in the immunoassay.

Summary

The inventors have discovered two conformations of IgG; an open and a closed native conformation. The open conformation has exposed RFs binding sites in contrast to the closed native conformation. As shown in the examples provided herein, the closed conformation of IgG can transition to an open conformation of IgG upon binding to a surface, upon binding to an antigen (Ag) and upon heat treatment. The binding site for the majority of RFs resides in the same site as the binding site for C1q, which becomes exposed upon Ag binding. RF epitopes that are not exposed in the closed native conformation can therefore be regarded as cryptic epitopes. This resolves the apparent paradox of how C1q and RFs can circulate in high concentrations together with IgG in healthy persons and patients with rheumatic diseases, since the binding sites on IgG for C1q and RFs are exposed upon Ag binding.

In order to prevent false positive results to occur in diverse immunoassays, a preparation of Immunoglobulins can be heat-treated to allow RFs binding to the Fc part of said preparation that is now exposed upon heat treatment. This method can thus remove RFs and prevent them from interfering in the immunoassay.

The invention relates to an immunoassay comprising: at least one antibody capable of binding an antigen of interest in a sample, and a heat-treated preparation of

Immunoglobulins. The inventors have found that the immunoassay of the present invention exhibit a significant improvement in avoiding substantial distortions in measured concentrations or presence of the analyte due to HAMA and/or heterophilic antibodies such as rheumatoid factors present in some human samples. The immunoassay is improved by using a heat-treated preparation of immunoglobulins.

In one aspect, the present invention concerns a method of performing an

immunoassay, the method comprising: a. loading a sample comprising an antigen of interest to an immunoassay, wherein the immunoassay comprises at least one antibody capable of binding the antigen of interest,

b. contacting the sample with a heat-treated preparation of

Immunoglobulins comprising IgG, and

c. detecting the complex between the capture antibody and antigen of interest using a secondary antibody or turbidometry.

In one aspect, the present invention relates to the use of a heat-treated preparation of Immunoglobulins to reduce false positives in immunoassays.

In one aspect, the present invention relates to the use of one or more Fc fragments to reduce false positives in immunoassays.

In one aspect, the present invention relates to the use of one or more recombinant Fc fragments to reduce false positives in immunoassays.

Description of Drawings

Figure 1 . Immunoglobulin models.

A. Schematic structure of an immunoglobulin G (IgG) molecule consisting of two heavy chains (HC) of y type and two light chains (LC) of either k or l type linked by disulphide bridges. The variable part of a HC (VH) together with the variable part of a LC (VL) forms the antigen binding site. A LC together with VH and CH1 domains form a fragment antigen-binding (Fab) part (“arm”) and the CH2 and CH3 domains constitute the fragment constant or crystallisable (Fc) part with effector functions.

B. The structure of IgD, IgA, IgE and IgM. The nomenclature is similar to that of an IgG molecule, except that the HCs are named d, a, e, m respectively. IgM and IgE have an extra domain in the HCs. Figure 2. Immunoassay formats used in this work.

A. Simple antigen (Ag) antibody (capture) ELISA, B. Bead-based fluorescent capture sandwich immunoassay, biotin-streptavidin-PE (bS-PE), C. Rheumatoid factor (RF) ELISA, D. RF sandwich/bridging assay.

Figure 3. RF reactivity with IgG. RFs do not react in a bead-based fluorescent sandwich/bridging immunoassay with covalently immobilized IgG and biotinylated IgG but does so after exposure of the immobilized and biotinylated IgG to elevated temperature (57 ^°C). A-C. Native IgG (A) or IgG incubated at 57 ^°C for 4 h (B) or 24 h (on) (C) was covalently immobilized on fluorescent beads and incubated with RF- positive or -negative serum samples in the presence of biotinylated IgG, which was either non-heated or had been incubated at 57 ^°C for 4 h or 24 h. The positive control was rabbit antibodies to human IgG (RaHIgG). For each panel (A) to (C), B-IVIG represents IgG in solution, B-IVIG (57 dg, 4h) represents solution IgG that has been heat treated for 4h and B-IVIG (57 dg. on) represents solution IgG that has been heat treated for 24h. D. Reactivity of RF-positive and -negative sera to IgG, IgM or IgA immobilized on fluorescent beads (positive control: RaHIgG, control beads had no immobilized Ig). E. RFs react with heat-treated immobilized IgG but not with heat- treated IgA or IgM or non-heated IgG, IgA or IgM. F. Temperature dependence of RF reactivity. IgG incubated at the indicated temperatures before immobilization was tested for reaction with RF-positive or -negative sera in a bridging assay with biotin- IgG in solution. Final detection was with phycoerythrin (PE)-streptavidin.

Figure 4. Infliximab (IFX, Remicade) reacts with RFs after heat-treatment. A. Infliximab incubated at 57 ^°C and then immobilized covalently on beads reacts with RFs but shows no reaction with RF-negative sera. B. Immobilized IFX incubated first at 37 ^°C - 57 ^°C reacts with RaHIgG and can be bridged to native IFX in solution by TNF but not by RFs. No reaction is seen with IFX incubated at 62 ^°C or 67 ^°C due to precipitation of the IFX.

Figure 5. IgG reactivity with RF. Reaction of different IgG forms with Rheumatoid factors in inhibition assays. IgG (A) or Infliximab (Remicade) (B) was coated on the surface of polystyrene ELISA plates and incubated with RF-containing serum or control serum (healthy donor serum) in the absence or presence of the indicated

concentrations of inhibitor (native or heat-treated IgG). Native (N), heat-treated (denatured) (D), intravenous immunoglobulin (IVIG), pooled human sera with no RFs (donor pool, DP).

Figure 6. IgG conformational change upon heating allowing interaction with C1q. C1q was immobilized on the surface of ELISA wells and incubated with biotin-labelled IgG, which had been pre-incubated at the indicated temperatures. The interaction was maximal, when IgG had been subjected to heating at 57 ^°C.

Figure 7. IgG conformational change upon antigen binding allowing interaction with RFs. Infliximab was immobilized on beads and incubated with RF-positive or -negative sera in the presence or absence of tumor necrosis factor (TNF).

Figure 8.A Model of the native resting (closed) Infliximab IgG structure.

Definitions

As used herein, the term“antibody” (Abs) refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and encompasses polyclonal antibodies, monoclonal antibodies, and fragments thereof, as well as molecules engineered from

immunoglobulin gene sequences. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as m, d, a, g, e, which in turn define the immunoglobulin classes, IgM, IgD, IgA, IgG, and IgE, respectively. Antibody and immunoglobulin (Igs) can be used interchangeably. All Igs/Abs share the same basic unit design of two identical heavy chains (HCs) with a N- terminal variable domain (VH) and three or four constant domains (CH 1 -CH4) and two identical light chains (LCs) with an N-terminal variable domain (VL) and a C-terminal constant domain (CL) all linked by disulphide bonds. The variable N-terminal domains of the light and heavy chains together form two antigen binding sites and together with the CH1 and CL domains, these form the parts (arms) known as fragment antigen binding (Fab). The four (six in IgM and IgE) constant domains together form a part designated fragment crystallisable (or constant) (Fc). The fragment crystallisable region (Fc region) is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.

A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation.

As used herein, the term“sample” generally refers to a biological material being tested for and/or suspected of containing an analyte of interest. The biological material may be derived from any biological source. Examples of biological materials include, but are not limited to, stool, whole blood, serum, plasma, red blood cells, platelets, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, ascites fluid, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, soil, etc. The test sample may be used directly as obtained from the biological source or following a pre-treatment to modify the character of the sample. For example, such pre-treatment may include preparing plasma from blood, diluting viscous fluids and so forth. Methods of pre-treatment may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, etc.

Heterophilic antibodies are a group of antibodies exhibiting multispecificity that react with heterogeneous antigens.

Rheumatoid factors are autoantibodies that bind to antigenic determinants on the Fc portion of IgG (or more rarely on other antibodies, such as for example IgM or IgA)..

Serum is defined as blood plasma with its clotting factors removed.

In the context of the present invention the term "antigen" is used to refer to an immuno specific reagent which complexes with antibodies present in the test sample. An antigen is a substance comprising at least one antigenic determinant or epitope capable of interacting specifically with the target antibody it is desired to detect, or any capture agent interacting specifically with the variable region or complementarity determining regions of said antibody. The antigen will typically be a naturally occurring or synthetic biological macromolecule such as for example a protein or peptide, a polysaccharide or a nucleic acid and can include antibodies or fragments thereof such as anti-idiotype antibodies.

A cryptotope is an antigenic site or epitope hidden in a protein or virion by surface subunits. Cryptotopes are antigenically active after their exposure following for example a conformational change or a dissociation of protein. A cryptotope can also be referred to as a cryptic epitope.

An epitope is a discrete site to which an antibody binds.

Detailed description

The present invention provides an improved immunoassay comprising at least one antibody capable of binding an antigen of interest in a sample, and a heat-treated preparation of Immunoglobulins.

In one embodiment the antibody capable of binding an antigen of interest in a sample is a capture antibody bound to a solid surface. The capture antibody is specific to the target antigen.

Immunoassays

In one embodiment the immunoassay is selected from the group consisting of lateral flow immunochromatographic assays, sandwich assays, bead-based immunoassays, ELISA assays such as sandwich ELISA and nephelometry/turbidometry assays, enzyme immunoassays, fluoroimmunoassay, chemiluminescenceimmunoassay and radioimmunoassay.

The immunoassay described herein is a biochemical test that measures the presence or concentration of an antigen of interest in a sample through the use of an antibody or immunoglobulin. Typically, an antibody specific to the antigen of interest interacts with the antigen in an immunoassay. The antibody can be labelled, directly or indirectly such that those bound to the antigen could release a detectable signal. Presence or concentration of the antigen of interest can be determined based on the level of the detectable signal. The immunoassay described herein can involve the use of different types of labels, including enzymes, radioactive isotopes, DNA reporters, fluorogenic reporters, electrochemiluminescent tags, oligonucleotides, nanoparticles,

chemiluminophores, fluorophores, fluorescence quenchers, chemiluminescence quenchers, or biotin, all of which are well known in the art. In some instances, the immunoassay can involve a catalyst such as an enzyme to amplify the signal.

Alternatively, the immunoassay described herein can be performed without the use of a label, e.g. nephelometry/turbidometry assays.

ELISAs are generally well known in the art. The ELISA assay used in the detection immunoassay described herein can be in any format known in the art, including direct ELISA, Sandwich ELISA, competitive ELISA, and multiple and ready-to-use ELISA. In a typical“direct” or“indirect” ELISA, an antibody having specificity for the antigen of interest is immobilized on a solid surface (e.g., the wells of a standard microtiter assay plate, or the surface of a microbead or a microarray) and a sample comprising, e.g., bodily fluid or substances extracted from bodily fluids, to be tested for the presence of the antigen of interest is brought into contact with the immobilized antibody. Any antigen of interest in the sample will bind to the immobilized antibody. The bound antibody/antigen complexes may then be detected using any suitable method.

In one embodiment, a detection antibody, which specifically recognizes an epitope of the antigen, which may be different from the epitope recognized by the immobilized antibody, is used to detect the antibody/antigen complexes. The detection antibody is usually labelled with a detectable marker (directly or indirectly).

In one embodiment the immunoassay further comprises a detection antibody bound directly or indirectly to a detection moiety. The one-step procedure, direct detection, relies upon a single antibody which has been covalently joined to a detection moiety and directed against the target of interest. The two-step procedure, indirect detection uses two antibodies or an enzyme conjugate comprising an enzyme conjugated with the other member of the receptor/ligand pair, e.g., streptavidin, can be brought into contact with the second antibody. A substrate of the enzyme is then added to produce a product that releases a detectable signal. The primary antibody is unconjugated and a secondary antibody bound to a detection moiety directed against the primary antibody is used for detection.

In some examples, the detection moiety can be an enzyme such as peroxidase, alkaline phosphatase, or galactosidase, allowing quantitative detection by the addition of a substrate for the enzyme which generates a detectable product, for example a coloured, chemiluminescent or fluorescent product. Other types of detectable labels known in the art may be used with equivalent effect. In other examples, the second antibody may be labelled with a member of a receptor/ligand pair, for example, biotin.

The immunoassay described herein can be nephelometry which is a technique used in immunology to determine the levels of several blood plasma proteins. It is performed by measuring the turbidity in a water sample by passing light through the sample being measured. In nephelometry the measurement is made by measuring the light passed through a sample at an angle. Antibody and the antigen are mixed in concentrations such that only small aggregates are formed that do not quickly settle to the bottom. The amount of light scattering is measured and compared to the amount of scatter from known mixtures. The amount of the unknown is determined from a standard curve. Nephelometry can be used to detect either antigen or antibody, but it is usually run with antibody as the reagent and the patient antigen as the unknown. Rheumatoid factors can interfere in nephelometry resulting in false positive results due to the binding of RFs with the antibody detecting the antigen of interest.

A radioimmunoassay (RIA) is an immunoassay that uses radiolabelled molecules in a stepwise formation of immune complexes. A RIA is a very sensitive in vitro assay technique used to measure concentrations of substances, usually measuring antigen concentrations (for example, hormone levels in blood) by use of antibodies.

Lateral flow immunochromatographic assays, are simple devices intended to detect the presence (or absence) of a target analyte in a sample (matrix). Typically, these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. The technology is based on a series of capillary beds, such as pieces of porous paper, microstructured polymer, or sintered polymer. Each of these components has the capacity to transport fluid (e.g., urine) spontaneously.

The first component (the sample pad) acts as an adsorbent pad onto which the test sample is applied and holds an excess of sample fluid. Once soaked, the fluid migrates to the second component (conjugate or reagent pad) that contains detection antibodies conjugated to coloured particles (colloidal gold, nanoparticles, or latex microspheres) and specific to the analyte of interest in sample. The analyte of interest binds to these detection antibodies while migrating further through the third capillary bed. This material has one or more areas (often called stripes), where a capture antibody has been immobilized on the surface. By the time the sample-conjugate mix reaches these stripes, analyte has been bound to the detection antibody and the capture antibody binds the complex.

After a while, when more and more fluid has passed the stripes, antibody-antigen complexes accumulate and the stripe-area changes colour. Typically, there are at least two stripes: one (the control) that captures any antibodies and thereby shows that reaction conditions and technology worked fine, the second contains a specific capture antibody and only captures those antibody-antigen complexes proving the result of the immunoassay. After passing these reaction zones the fluid enters the final porous material, a waste container.

RFs can interfere in lateral flow immunochromatographic assays resulting in false positive results due to the binding of RFs to the Fc part of the antibody detecting the antigen of interest.

In one embodiment the antibody capable of binding an antigen of interest and/or detection antibody comprises an Fc region of an IgG. Heterophilic antibodies such as rheumatoid factors bind the Fc region of antibodies.

In one embodiment the capture antibody is bound to a surface, such as a bead for example the surface of a microbead or a filter, or a plate such as a microtiter plate.

In a particular embodiment the antibody capable of binding an antigen of interest in a sample and/or detection antibody is an IgG, such as human lgG1 , lgG2, lgG3, or lgG4, or mouse IgG, such as lgG1 , lgG2a, lgG2b, lgG2c, lgG3, or rabbit IgG. The four human subclasses, lgG1 , lgG2, lgG3, and lgG4, which are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. These regions are involved in binding to both IgG-Fc receptors (FcyR) and C1q and can thus be bound by RFs.

In another embodiment the antibody capable of binding an antigen of interest in a sample and/or detection antibody is a human immunoglobulin such as IgG, IgA, IgM, IgD, IgE, or a mouse immunoglobulin such as IgG, IgA, IgM, IgD, IgE or a rabbit immunoglobulin such as IgG, IgA, IgM, IgE. There are five immunoglobulin classes (isotypes) of antibody molecules found in serum: IgG, IgM, IgA, IgE and IgD. They are distinguished by the type of heavy chain they contain. IgG molecules possess heavy chains known as g-chains; IgMs have m-chains; IgAs have a-chains; IgEs have e- chains; and IgDs have d-chains. The variation in heavy chain polypeptides allows each immunoglobulin class to function in a different type of immune response or during a different stage of the body’s defence. The amino acid sequences that confer these functional differences are located mainly within the Fc domain. RFs can bind to different antibody isotypes.

Preparation of Immunoglobulins

According to the invention a preparation of heat-treated Immunoglobulins is added to prevent RF interference in the immunoassay.

In one embodiment the preparation of Immunoglobulins has been heat treated at a temperature of at least 42^qC, such as at least 50‘G, preferably at least 53 G, such as at least 55 °C, for example at least 57 ^qC. The purpose of the heat treatment is to allow exposure of cryptic epitopes thereby enabling heterophilic antibodies (such as rheumatoid factors) to bind to the Fc region of the heated immunoglobulin instead of binding to the antibodies of the assay.

In one embodiment, the heat-treated preparation of Immunoglobulins exposes at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 100%, of the cryptic epitopes. The percent of exposed cryptic epitopes recognised by rheumatoid factors depend on time of heat treatment of the preparation of Immunoglobulins. The higher the amount of exposed cryptic epitopes, the higher percentage of bound rheumatoid factors to the heat-treated preparation of

Immunoglobulins, the lower the percentage of false positive results.

In one embodiment the preparation of Immunoglobulins has been heated for at least 30 min, such as at least 1 hour, such as at least 1 day, such as at least 1 week, such as at least 1 month. The longer the preparation of Immunoglobulins is heated the more cryptic epitopes are exposed.

In one embodiment the heat-treated preparation of Immunoglobulins comprises IgG as heterophilic antibodies such as rheumatoid factors preferably binds to the Fc region of IgG. In another embodiment the heat-treated preparation of Immunoglobulins comprises at least the Fc region of IgG where the rheumatoid factor’s epitope is located.

In one embodiment the immunoassay comprises at least 1 ug/mL such as at least 10ug/ml_, such as at least 100ug/ml_, such as at least 1 mg/ml_, such as at least 10mg/ml_ of the heat-treated preparation of Immunoglobulins. In another embodiment, the immunoassay comprises between 1 pg/mL and 10mg/ml_, 1 pg/mL and 10pg /ml_, 10pg/mL and 100pg /mL, 100pg/mL and 1 mg/ml_, 1 mg/ml_ and 10mg/ml_ of the heat- treated preparation of Immunoglobulins.

In one embodiment the heat-treated preparation of Immunoglobulins is present in an amount at least 2-fold, such as at least 10-fold, such as at least 20-fold, such as at least 100-fold, such as at least 1000-fold compared to RFs. The purpose of a higher amount of heat-treated preparation of Immunoglobulins compared to RFs is to certify that all the rheumatoid factors present in the sample bind to the preparation of

Immunoglobulins thus preventing false positive results.

In one embodiment the heat-treated preparation of Immunoglobulins is a turkey or a chicken immunoglobulin.

In one embodiment the heat treated immunoglobulin preparation is mammalian.

In one embodiment the mammal is selected form the group consisting of a mouse, a goat, a donkey, a camel, a hamster, an Armenian hamster, a rat, a rabbit, a guinea pig, a horse, a sheep, a cow and a human. Heterophilic antibodies and rheumatoid factors can bind to Immunoglobulins from different species.

In one embodiment the heat-treated preparation of Immunoglobulin is derived from serum. Immunoglobulins are mainly found in serum.

In one embodiment the heat-treated preparation of Immunoglobulins has been purified to remove non-immunoglobulin proteins. The purpose of the purification is to prevent any unspecific bindings and only target heterophilic antibodies such as rheumatoid factors. In one embodiment the heat-treated preparation of Immunoglobulins has been purified by protein A chromatography, physicochemical fractionation, class-specific affinity, antigen-specific affinity, or immunoprecipitation. Antibody purification involves selective enrichment or specific isolation of antibodies from serum (polyclonal antibodies), ascites fluid or cell culture supernatant of a hybridoma cell line (monoclonal antibodies). Purification methods range from very crude to highly specific and can be classified as follows:

Physicochemical fractionation is a purification method based on differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples. This isolates a subset of sample proteins that includes the immunoglobulins. Class-specific affinity is a method based on solid-phase binding of particular antibody classes (e.g., IgG) by immobilized biological ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins. This purifies all antibodies of the target class without regard to antigen specificity. Antigen-specific affinity is a method based on affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains. This purifies all antibodies that bind the antigen without regard to antibody class or isotype. In one embodiment the heat-treated preparation of Immunoglobulins comprises one or more recombinant IgGs.

An aspect of the present disclosure is a method of performing an immunoassay which comprises: a. loading a sample comprising an antigen of interest to an immunoassay, wherein the immunoassay comprises at least one antibody capable of binding the antigen of interest,

b. contacting the sample with a heat-treated preparation of

Immunoglobulins comprising IgG, and

c. detecting the complex between the capture antibody and antigen of interest using a secondary antibody or turbidometry.

In one embodiment the sample is incubated with the heat-treated preparation of Immunoglobulins prior to loading. The purpose of this step is to remove the heterophilic antibodies such as rheumatoid factors from the sample before the antigen of interest is in contact with an antibody capable of binding said antigen. The rheumatoid factors will thus not be in contact with capture and/or detection antibodies since they will be bound to the heat-treated preparation of Immunoglobulins.

In one embodiment the heat-treated preparation of Immunoglobulins is present in the immunoassay.

An embodiment of the present invention is the use of a heat-treated preparation of Immunoglobulins to reduce false positives in immunoassays.

Fab and Fc fragments

As an alternative to heat-treated Immunoglobulins, the preparation comprises one or more Fc fragments. The C1 q epitope in the Fc fragments is not cryptic and is thus available for binding to RFs.

An embodiment of the present invention is the use of one or more recombinant Fc fragments to reduce false positives in immunoassays. Fc fragments have exposed cryptic epitopes recognised by heterophilic antibodies such as rheumatoid factors. These Fc fragments can thus hijack the rheumatoid factors present in the sample to be analysed in the immunoassay and prevent false positive results.

In on embodiment, the Fc fragment comprises epitopes recognised by heterophilic antibodies, preferably recognised by rheumatoid factors.

In one embodiment the Fc fragment is a pFc’ fragment. Antibodies can be cleaved into different fragments usually into fab fragments and Fc fragments. The enzymes papain and IdeS digest antibodies into two F(ab) fragments and one Fc fragment while the enzyme pepsin digests antibodies into a F(ab’)2 fragment containing two antigen binding F(ab) portions linked together by disulphide bonds and a very small pFc’ fragment. F (ab) and F (ab’) 2 fragment antibodies still bind to antigens while Fc fragment antibodies do not. In one embodiment RFs binds to pFc’.

In one embodiment the Fc fragment is a turkey or a chicken Fc fragment. In another embodiment the Fc fragments are mammalian. In one embodiment the mammal is selected from the group consisting of a mouse, a goat, a donkey, a camel, a hamster, an Armenian hamster, a rat, a rabbit, a guinea pig, a horse, a sheep, a cow and a human.

In one embodiment the Fc fragment is a recombinant Fc fragment.

Items

The invention is in the following described as numbered items.

1. An immunoassay comprising:

a) at least one antibody capable of binding an antigen of interest in a sample, and

b) a heat-treated preparation of Immunoglobulins. 2. The immunoassay according to item 1 , wherein the antibody capable of binding an antigen of interest in a sample is a capture antibody.

3. The immunoassay according to item 1 , wherein the immunoassay is selected from the group consisting of lateral flow immunochromatographic assays, sandwich assays, bead-based immunoassays, ELISA assays such as sandwich ELISA and nephelometry/turbidometry assays, enzyme immunoassays, fluoroimmunoassays, chemiluminescenceimmunoassays and

radioimmunoassays. 4. The immunoassay of any of the preceding items, further comprising a detection antibody bound directly or indirectly to a detection moiety.

5. The immunoassay of item 2, wherein the detection moiety is bound to the

detection antibody via a specific binding pair, such as streptavidin and biotin.

6. The immunoassay of item 2 or 3, wherein the detection moiety is selected from the group consisting of a fluorochrome or an enzyme.

7. The immunoassay according to any of the preceding items, wherein the

antibody capable of binding an antigen of interest in a sample and/or detection antibody is a human immunoglobulin such as IgG, IgA, IgM, IgD, IgE, or a mouse immunoglobulin such as IgG, IgA, IgM, IgD, IgE or a rabbit

immunoglobulin such as IgG, IgA, IgM, IgE. The immunoassay according to any of the preceding items, wherein the antibody capable of binding an antigen of interest in a sample and/or detection antibody is an IgG, such as human lgG1 , lgG2, lgG3, or lgG4, or mouse IgG, such as lgG1 , lgG2a, lgG2b, lgG2c, lgG3, or rabbit IgG. The immunoassay according to any of the preceding items, wherein the antibody capable of binding an antigen of interest in a sample and/or detection antibody comprises an Fc region of an IgG. The immunoassay according to any of the preceding items, wherein the capture antibody is bound to a surface, such as a bead or a filter, or a plate such as a microtiter plate. The immunoassay according to any of the preceding items, wherein the preparation of Immunoglobulins has been heat treated at a temperature of at least 42 °C, such as at least 45 °C, such as at least 50‘G, preferably at least 53 °C, such as at least 55 °C, for example at least 57 ^qC. The immunoassay according to any of the preceding items, wherein the preparation of Immunoglobulins has been heat treated to expose cryptic epitopes recognised by heterophilic antibodies, preferably recognised by rheumatoid factor. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins exposes at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least

100%, of the cryptic epitopes. The immunoassay according to any of the preceding items, wherein the preparation of Immunoglobulins has been heated for at least 30 min, such as at least 1 hour, such as at least 1 day, such as at least 1 week, such as at least 1 month. 15. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins comprises IgG.

16. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins comprises at least the Fc region of IgG.

17. The immunoassay according to any of the preceding items, wherein the

immunoassay comprises between 1 pg/mL and 10mg/ml_, 1 pg/mL and 10pg /ml_, 10pg/mL and 100pg /ml_, 100pg/mL and 1 mg/ml_, 1 mg/ml_ and 10mg/ml_ of the heat-treated preparation of Immunoglobulins.

18. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins is present in an amount at least 2-fold, such as at least 10-fold, such as at least 20-fold, such as at least 100-fold, such as at least 1000-fold compared to the amount of RFs or HAbs (heterophilic antibodies) in the sample.

19. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins is a turkey or a chicken

immunoglobulin.

20. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins is mammalian.

21. The immunoassay according to item 20, wherein the mammal is selected form the group consisting of a mouse, a goat, a donkey, a camel, a hamster, an Armenian hamster, a rat, a rabbit, a guinea pig, a horse, a sheep, a cow and a human.

22. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins is derived from serum.

23. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins has been purified to remove non immunoglobulin proteins.

24. The immunoassay according to item 23, wherein the heat-treated preparation of Immunoglobulins has been purified by protein A chromatography, physicochemical fractionation, class-specific affinity, antigen-specific affinity, or immunoprecipitation.

25. The immunoassay according to any of the preceding items, wherein the heat- treated preparation of Immunoglobulins comprises one or more recombinant

IgGs.

26. A method of performing an immunoassay, the method comprising: a. loading a sample comprising an antigen of interest to an immunoassay, wherein the immunoassay comprises at least one antibody capable of binding the antigen of interest,

b. contacting the sample with a heat-treated preparation of

Immunoglobulins comprising IgG, and

c. detecting the complex between the capture antibody and antigen of interest using a secondary antibody or turbidometry.

27. The method of item 26, wherein the sample is incubated with the heat-treated preparation of Immunoglobulins prior to loading.

28. The method of item 26, wherein the heat-treated preparation of

Immunoglobulins is present in the immunoassay.

29. The method of any of the items 26-28, wherein the immunoassay is an

immunoassay according to any of the preceding items 1 -25.

30. Use of a heat-treated preparation of Immunoglobulins to reduce false positives in immunoassays. 31. The use of item 30, wherein the preparation of Immunoglobulins has been heat treated at a temperature of at least 42^qC, such as at least 45^qC, such as at least 50 °C, preferably at least 53 °C, such as at least 55 °C, for example at least 57 ^qC.

32. The use of item 30 or 31 , wherein the preparation of Immunoglobulins has been heat-treated for at least 30 min, such as at least 1 hour, such as at least 1 day, such as at least 1 week, such as at least 1 month. 33. The use of any of the preceding items 30 to 32, wherein the preparation of Immunoglobulins has been heat treated to expose cryptic epitopes recognised by heterophilic antibodies, preferably recognised by rheumatoid factor.

34. The use of any of the preceding items 30 to 33, wherein the heat-treated

preparation of Immunoglobulins is a turkey or a chicken immunoglobulin.

35. The use of any of the preceding items 30 to 33, wherein the heat-treated

preparation of Immunoglobulins is mammalian.

36. The use of item 35, wherein the mammal is selected form the group consisting of a mouse, a goat, a donkey, a camel, a hamster, an Armenian hamster, a rat, a rabbit, a guinea pig, a horse, a sheep, a cow, and a human.

37. The use of any of the preceding items 30 to 36, wherein the heat-treated

preparation of Immunoglobulins is derived from serum.

38. The use of any of the preceding items 30 to 37, wherein the heat-treated

preparation of Immunoglobulins has been purified to remove non

immunoglobulin proteins.

39. The use of item 38, wherein the heat-treated preparation of Immunoglobulins has been purified by protein A chromatography, physicochemical fractionation, class-specific affinity, antigen-specific affinity, or immunoprecipitation.

40. The use of any of the preceding items 30 to 39, wherein the heat-treated

preparation of Immunoglobulins comprises one or more recombinant IgGs.

41. The use of any of the preceding items 30 to 40, wherein the heat-treated

preparation of Immunoglobulins comprises one or more Fc fragments.

42. Use of one or more Fc fragments to reduce false positives in immunoassays.

43. The use of item 42, wherein the Fc fragment is recombinant.

44. The use of item 42 or 43, wherein the Fc fragment comprises epitopes

recognised by heterophilic antibodies, preferably recognised by rheumatoid factors. 45. The use of any of the preceding items 42 to 44, wherein the Fc fragment is a pFc’ fragment.

46. The use of any of the preceding items 42 to 45, wherein the Fc fragment is a turkey or a chicken Fc fragment.

47. The use of any of the preceding items 42 to 46, wherein the Fc fragments are mammalian.

48. The use of item 47, wherein the mammal is selected from the group consisting of a mouse, a goat, a donkey, a camel, a hamster, an Armenian hamster, a rat, a rabbit, a guinea pig, a horse, a sheep, a cow and a human.

Examples

Materials and Methods

Materials

Human IgG (intravenous immunoglobulin (IVIG) containing 60, 33, 3 and 2% of lgG1 , lgG2, lgG3 and lgG4, respectively (20)) was from (Statens Serum Institut (SSI), Copenhagen, Denmark). Horseradish peroxidase-conjugated rabbit IgG to human IgM or IgA was from DAKO (Copenhagen, Denmark) ortho-phenylenediamine (oPD) substrate was from (Kem-En-Tek, Copenhagen, Denmark).

Phenolred, Na₂C0₃, H₂S0 , 2-(N-morpholino)-ethanesulfonic acid (MES), Biotin-N- hydroxysuccinimidester, bovine serum albumin (BSA), citric acid and urea were from Sigma (St. Louis, MO, USA). Ethanolamine, H₂0₂, KCI, KH₂P0₄, MgCI₂, NaCI, Na₂HP0₄, NaOH, NaN₃ and Tween 20 were from Merck (Darmstadt, Germany). Triton X-100 was from Applichem GmBH (Gatersleben, Germany). Luminex beads were from Luminex Corporation (Millipore, Billerica, MA, USA). Phycoerythrin (PE)-conjugated streptavidin was from Lifetechnologies (Grand Island, NY, USA). Human lgG1 , lgG2, lgG3, lgG4 were from The Binding Site (Birmingham, UK). Remicade® (Infliximab) was from Janssen Biotech, Horsham, PA, USA. N-hydroxysulfosuccinimide (Sulfo-NHS) and the 1 -ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) crosslinker was from Thermo Scientific (Rockford, IL, USA). The bis-(sulfosuccinimidyl) suberate (BS3) crosslinker was from ProteoChem (Hurricane, UT, USA). Bolt™ 4-12% Bis-Tris Plus gel was from Invitrogen (Carlsbad, CA, USA). PageRuler™ Prestained Protein Ladder was from Thermo Scientific (Rockford, IL, USA). RunBlue LDS Sample Buffer 4X and InstantBlue™ Coomassie staining solution was from Expedeon

(Cambridgeshire, United Kingdom). Bolt™ Sample Reducing Agent and Bolt™ MES DS Buffer were from Fischer Scientific (Carlsbad, CA, USA). Phosphate buffered saline (PBS), NH4HCO3, Dithiothreitol (DTT), lodacetamide (IAA), Acetonitrile (ACN),

Trifluoroacetic acid (TFA) and Formic Acid (FA) were from (Sigma Aldrich, Germany). Poros R2 resin (20pm) was from Applied Biosystems (Foster City, CA, USA). EMpore C18 lining was from 3M Company (Eagan, MN, USA). GLS-L10 pipette tips were from Rainin (Mettler Toledo, Germany). SafeSeal Low Binding tubes were from Sorenson Bioscience Inc. (Murray, UT, USA). Trypsin was from Promega (Southampton, United Kingdom). Ultra-High Quality Milli-Q water (UHQ) was produced using Elga LabWater instruments (18.2MW) (Lane End, United Kingdom)

Patient sera

RF-positive sera and healthy control sera were obtained from the biobank at Statens Serum Institut (SSI). Dependent on the experiment conducted, varying numbers of patient sera were used, as noted in the figure legends. The authors did not have direct contact with any patients or donors and were neither involved in drawing/collection of samples. Sera were used anonymously and therefore no written consent was required.

Eva Green fluorescence assay was carried out as described by Duus et al. (2013).

Immunoassays

The different immunoassays used in this work are illustrated in Fig. 2 and described below.

Rheumatoid factor enzyme-Hnked immunosorbent assays

IgM RF and IgA RF levels were determined by ELISA as described by Hoier-Madsen et al. (1986). Briefly, human IgG (10 pg/ml in 50 mM sodium carbonate, pH 9.6) was coated with 100 mI/well in Maxisorp polystyrene plates at 5 °C overnight (ON). After blocking at 5 °C ON with 200 mI/well of buffer A (0.5 M NaCI, 1.5 mM KH2PO4, 2.7 mM KCI, 8.1 mM Na2HP04, 1 % BSA, 0.1 % Triton X-100, 0.001 % phenol red, pH 7.2) and washing (3 x 1 min) with buffer B (0.5 M NaCI, 2.7 mM KCI, 8.1 mM Na2HP04, 0.1 % Tween 20, pH 7.2) plates incubated 1 hour at room-temperature (RT) with standards, controls and sera diluted 1 :100 in buffer A. Plates were washed (3 x 1 min) with buffer B and incubated 1 hour at RT with horseradish peroxidase-conjugated rabbit IgG to human IgM or IgA diluted 1 :9000 or 1 :5000, respectively, in buffer B. After 3 x 1 min washing with buffer B wells were incubated for 15 min with 50 mI/well 0.03 % (w:w) H₂O₂, 0.7 mg/ml ortho-phenylenediamine substrate in buffer C (67 mM Na2HP04, 35 mM citric acid, pH 5.0) and the reaction was then stopped by addition of 150 mI/well 1 M H₂SO₄. The absorbance was measured at 492 nm with background subtraction at 690 nm. Similarly, Capture sandwich assays were carried out by coating with native or heat-treated IgG (0.1 -10 pg/ml) in carbonate buffer followed by incubation with sera in incubation buffer or TTN buffer (50 mM Tris pH 7.5, 1 % Tween, 0.15 M NaCI) with or without native or heat-treated IgG, followed by detection with alkaline phosphatase (AP)-conjugated rabbit or goat immunoglobulins against human IgG, IgA or IgM in TTN buffer (1 :1000 or 1 :2000 dilution) and quantification with para-nitrophenylphosphate (pNPP) in AP buffer (1 M ethanolamine, 0.5 mM MgCI₂, pH 9.8) and absorbance reading at 450 nm with background subtraction at 690 nm.

The IgM RF concentration is given as international units (IU)/ml and IgA RF

concentration is given as U/ml. The cut-off value of the RF analyses (20 lU/ml for IgM RF, 20 U/ml for IgA RF) is set relatively high to compensate for the fact that many apparently healthy persons (especially with older age) contain RFs (values between 15 and 20 lU/ml are considered as a twilight-zone).

Bead-based fluorescent capture immunoassays

Briefly, human IgG was immobilised to fluorescent microsphere beads (Luminex) following the manufacturer’s instructions. IgG was coupled to 6.25x10⁵ pre-activated carboxylated microsphere beads using MES (50 mM, pH 5.0) with mixing for 2 hours at RT. Following peptide coupling, the beads were washed and stored in storage buffer (PBS, 0.1 % BSA, 0.02% Tween-20, 0.05% NaN₃, pH 7.4) at 4°C. Protein-Ab interactions were measured by incubating approximately 5000 beads with human sera (1 :100 dilution) with or without addition of native or heat-treated immunoglobulins in various concentrations or with TNF or other control reagents for 45 min at RT.

Following incubation, the microsphere beads were washed with assay buffer (PBS, 1 % BSA, pH 7.4) (3x1 min). Next, PE-conjugated goat anti-human IgG was added to the microsphere beads and incubated for 35 min at RT and washed with assay buffer. Finally, approximately 50 beads of each sample were measured on a Bioplex reader (Biosource, Camarillo, CA, USA) and protein-antibody interactions determined as described by Holm et al. (2015) and Skogstrand et al. (2005). Chemical crosslinking

Infliximab samples were diluted to a concentration of 1 pg/pL in 100mM PBS buffer.

The samples were incubated at 37°C, 47°C and 57°C, respectively, for 2h. An additional sample was thawed immediately prior to crosslinking. Bis- sulfosuccinimidylsuberate (BS3) crosslinker was incubated at room temperature (RT) before use. BS3 was dissolved in ultra high quality water (UHQ) for a final

concentration of 22mM BS3. BS3 was added to the Ab samples to a final concentration of 2.2mM BS3. The samples were incubated at RT for 45min. Crosslinking was quenched using 10OmM NH₄HCO₃ for 15min. The samples were lyophilized prior to SDS-PAGE.

Ethyl (dimethylaminopropyl) carbodiimide (EDC) crosslinking

Infliximab samples were diluted to a concentration of 1 pg/pL in 100mM PBS buffer.

The samples were incubated at RT, 37°C and 50°C, respectively for 2h. An additional sample was thawed immediately prior to crosslinking. EDC and Sulfo-NHS was incubated at RT before use and dissolved in 100mM PBS. Sulfo-NHS and EDC was added for a final ratio of 1 :1 (w: w).The samples were incubated at RT for 90min. Crosslinking was quenched using 100mM NH4HCO3 for 15min. The samples were lyophilized prior to SDS-PAGE.

SDS-PAGE

The samples were dissolved in lithium dodecylsulfate (LDS) sample buffer, Reducing agent and UHQ water and incubated at 70°C for 10min. The samples were loaded onto a 4-12% Bis-Tris gel. A 10-180kDa protein ladder was loaded for mass reference. Electrophoresis was carried out for 30-45min at 200V and 400mA. The proteins in the gels were stained using a Coomassie Brilliant Blue-based protein stain for 15min. The gels were de-stained in UHQ water for 30min, two times.

Digestion

Relevant gel bands were excised from the gel into separate low-binding tubes. Excess Coomassie stain was removed by adding 50% acetonitrile (can) and incubating the samples on a shaker for 15min, two times. The gel bands were dried using 100% acetonitrile (can), 20pL 10mM Dithiothreitol (DTT) was added and the samples were incubated for 20min at 57°C for reduction. The samples were cooled to RT and excess liquid was removed and immediately replaced with 20pL 50mM iodoacetamide (IAA). The samples were incubated for 30min at RT in the dark for alkylation. The gel bands were washed using 100mM NH₄HC0₃. The gel bands were dehydrated using 100% ACN. Six ng/L trypsin solution was added to the dried gel bands and the samples were incubated on ice for 45min. Excess liquid was removed and an additional volume of 50mM NH₄HCO₃ was added. The samples were incubated overnight (ON) at 37°C. The liquid from the gel band samples was transferred to new low-binding tubes. The gel bands were dehydrated using 100% ACN and the excess liquid was also transferred to the new tubes. The samples were lyophilized before micropurification.

Micropurification

Micropurification columns were created in house by inserting a C18 matrix membrane into a 200 pipette tip. R2 resins were suspended in 100% ACN and added on top of the membrane creating an approximately 1cm column. The column was equilibrated using 0.1% trifluoroacetic acid TFA. The lyophilized samples were re-dissolved in 0.1% TFA and added to the column. The column was washed using 0.1% (TFA). The sample was eluted using 60% ACN/0.1% TFA. The samples were lyophilized prior to LC-MS/MS analysis.

LC-MS/MS

The digested peptides were dissolved in 0.1% TFA and approximately 1 pg peptides were analysed using an EASY-nanoLC 1000 system (Thermo Scientific, Germany) coupled to either the Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer or the G- Exactive™ HF mass spectrometer. The peptides were loaded onto a 2.5cm in-house packed Reprosil-Pur 120 C18-AG (5pm: Dr. Maisch GmbH, Germany) precolumn with an internal diameter of 100pm and eluted directly onto a 19cm in-house packed Reprosil-Pur C18-AG column (3 pm; Dr. Maisch GmbH, Germany) with an internal diameter of 75pm. A 104min HPLC gradient with a flowrate of 250 nL/min was used, which used an increasing concentration of Solvent B (95% ACN, 0.1% formic acid (FA) in the following increments: 1 -3% for 3min, 3-25% for 80min, 25-45% for 10min, 45- 100% for 3min and 100% for the final 8 min. Solvent A was 0.1% FA.

Both mass spectrometers were operated in data dependent acquisition mode and each analysis begins with an MS1 scan performed/detected in the Orbitrap (resolution:

120.000, scan range: 300-2000 m/z, AGC target: 400.000, maximum injection time: 100ms, RF lens: 30%). For each MS1 spectrum, the 15 most intense peaks were selected for MS2. Monoisotopic Peak determination is set to Peptide and dynamic exclusion was switched on (Exclude after 1 time, Exclusion duration: 15s, Mass tolerance: ±10ppm, Exclude isotopes). The selected MS2 precursors were isolated in the quadrupole at 1 .0 Th (atomic mass units) (m/z) and fragmented using high collision dissociation (HCD) at collision energy of 30%. MS2 fragments were detected in the Orbitrap (resolution: 30.000, first mass: 1 10 m/z, AGO target: 200.000).

Results

Rheumatoid factors do not react with covalently immobilized IgG or IgG in solution but reacts with IgG adsorbed on a hydrophobic surface

Initial experiments, aimed at developing a RF assay based on fluorescent (Luminex) multiplex bead technology, showed that sera having either IgM RF, IgA RF or both did not react in a sandwich/bridging assay (Fig. 2) with ion exchange (lEX)-purified human IgG covalently immobilized on beads through amide bonds with lysine side chains (Fig. 3A). Also, no reaction of RFs was seen with covalently immobilised human IgM or IgA (Fig. 3). These results were obtained with sera, which had been shown to contain RFs by routine ELISA with protein A-purified IgG, and it was confirmed that they also reacted with the IEX-purified IgG in the routine ELISA, where proteins were passively adsorbed (coated) on a hydrophobic polystyrene surface (results not shown).

In the bead-based assay, human IgG was immobilized on fluorescent beads and the beads were then incubated with human sera and biotinylated human IgG together (bridging assay) followed by detection of bead-bound biotin-lgG with PE-labelled streptavidin. In principle, any RF present would be capable of cross-linking bead-bound IgG and biotin-labelled IgG in solution, provided that the immobilized IgG adopted an RF-binding conformation (Fig. 2D). As a positive control, rabbit Igs against human IgG gave a strong reaction, verifying the viability of the assay (Fig. 3). Furthermore, dilution series and experiments with sequential incubation of IgG-beads first with sera, then with biotin-labelled IgG and finally with PE-streptavidin (sequential bridging assay, Fig. 2D) showed no reaction (results not shown), demonstrating that the lack of RF reaction was not a consequence of suboptimal reagent/serum ratios, but rather was due to the covalently immobilised native IgG and native IgG in solution having compact closed conformations not reacting with RFs.

A conformational change in IgG allows for RF interaction From the experiments described above, we hypothesized that the RF epitopes could be hidden in IgG (cryptic epitopes) and only become exposed by conformational changes, such as may occur upon antigen binding in vivo or in vitro, upon

immobilization on hydrophobic surfaces (e.g. the polystyrene surface used in routine ELISA for RF) or by certain physico-chemical conditions (e.g. elevated temperature). This prediction was verified by subjecting the immobilised IgG and the biotinylated IgG to elevated temperatures (Fig. 3B, C). These experiments showed that the covalently immobilised heat-treated IgG was capable of RF binding, whereas the soluble

(biotinylated) IgG was incapable of binding RF but acquired the ability to do so after incubation at 57 ^qC. Temperatures below 47/48 'Ό did not induce the conformational change necessary for RF binding and temperatures above 58/59 °C resulted in diminished/abolished interaction (Fig. 3D, results not shown). This was due to more extensive structural changes and/or precipitation of the IgG (unfolding of the IgG (as measured by Eva Green fluorescence assay (Duus et al. 2013)) showed a broad peak corresponding to Tm values of 70-80 'Ό (results not shown)). Exposure of the IgG to pH 3-1 1 , 1 % SDS or Tween 20, 0.5 M NaCL or 2-8 M urea did not induce the conformational change leading to RF binding (results not shown).

Importantly, as described above, the IEC-purified IgG adsorbed on the polystyrene surface at room temperature acquired the ability to bind RFs (as performed in routine ELISA RF assays with protein A-purified IgG), showing that the physical interaction with the hydrophobic surface induced a conformational change allowing RF binding (as opposed to the covalently immobilised IgG and IFX on beads) (data not shown). In agreement with this, heat-treated IgG adsorbed directly on the polystyrene surface exhibited similar RF binding as the open heated IgG (data not shown) in the“simple” RF assay (Fig. 2A) and the heat-treated IgG retained the ability to bind protein G (results not shown). In the bridging ELISA assay with native IgG immobilized at room temperature, heat-treated (biotin-labelled) IgG (captured by the bridging RFs) showed binding at 47-57 °C (data not shown) as also observed in the bead-based assay. When immobilized on streptavidin coated on the polystyrene surface of the ELISA plate, the biotin-labelled IgG only showed binding after heat treatment (results not shown). When RF-containing sera were coated on the polystyrene surface of ELISA plates, they only bound (biotin-labelled) IgG, if it had been incubated at 57 ^°C first (results not shown). Also, another indication of a conformational change in the IgG was that heat-treated IgG showed somewhat diminished reaction with rabbit antibodies to human IgG (RaHIgG) (results not shown). Reactivity of lgG1, IgG 2, lgG3, lgG4, and monoclonal IgGs with RFs

The experiments described above were conducted using a natural mixture of human IgGs (i.e. IVIG), primarily containing lgG1 (60 %, 33 %, 3 % and 2 % of lgG1 , lgG2, lgG3 and lgG4, respectively) (Laursen et al. 2014). To investigate whether this picture only relates to lgG1 or to all IgGs, purified lgG1 , lgG2, lgG3 and lgG4 were heat- treated, coupled to microspheric beads and analysed for RF reactivity. Independent of the antibody subtype/isotype, reactivity with RFs was found when the IgGs had been pre-incubated at 57 ^°C (data not shown).

Experiments with Infliximab showed identical behaviour as observed with IgG (IVIG), except that the monoclonal Infliximab changed conformation at a somewhat lower temperature (Fig. 4B and data not shown).

Etanercept also reacted with RFs when coated in ELISA wells, whereas in solution no reaction was seen, and Nivolumab, Ipilimumab and other monoclonal antibodies also reacted with RFs when coated on ELISA plates (results not shown).

Binding, inhibition and elution experiments

Inhibition experiments (Fig. 5) verified the conclusions reached by the solid phase assays, as heat-treated IgG was capable of inhibiting the RF binding to immobilized IgG and Infliximab, whereas native IgG showed no inhibition.

Elution experiments confirmed the reaction of RFs with both“natural” IgG (IVIG) and Infliximab, since RFs eluted from IVIG reacted with Infliximab and vice versa (data not shown).

Heat-treated IgG was still capable of binding various antigens (tetanus toxoid, diphtheria toxoid, Epstein Barr virus nuclear antigen 1 ) and heat-treated Infliximab retained the ability to bind tumor necrosis factor (results not shown). Also, heat-treated IgG retained the ability to bind immobilized protein A, and immobilized protein A bound to immobilized heat-treated IgG with a strength comparable to non-treated IgG (results not shown).

When IgG was immobilized on protein G, the heat-treated IgG showed increased binding of ConA (data not shown) which is another indication of a conformational change. Conversely, When Con A was immobilized and incubated with IgG or Infliximab, interaction was only seen after treatment at 57 ^°C, indicating that the structural carbohydrate became accessible upon“opening” of the IgG (results not shown). A conformational change in IgG allows interaction with C1q

When C1q was coated on the surface of ELISA plates, it did not show interaction with IgG in solution unless the IgG had been subjected first to elevated temperature, an effect which was maximal at 57 ^°C (Fig. 6). Similarly, when the heat-treated IgG (biotinylated) was immobilized on streptavidin-coated ELISA well surfaces, it was only heat-treated IgG that showed binding to C1q (results not shown).

Ag binding exposes (“cryptic”) RF epitopes

When infliximab was immobilized on beads and incubated with (biotin-labelled) Infliximab in the presence of RFs, no binding was seen unless the Infliximab

(immobilized and in solution) had been heat-treated (Fig. 4). However, when this experiment was performed in the presence of TNF (which itself can crosslink Infliximab molecules due to its trivalency), strongly increased signals were obtained, indicating that TNF binding to Infliximab induced an RF-binding conformation (Fig. 7). Binding of IgG to immobilized (ELISA format) tetanus toxoid (TT), diphtheria toxoid (DT) or Epstein Barr virus nuclear antigen 1 (EBNA1 ) also allowed RF binding to the Fc part and binding of Infliximab to immobilized TNF also induced an RF-binding conformation (results not shown).

Accessibility of the IgG hinge region

We generated Fab and Fc fragments of the human IgG by cleavage with IdeS protease as described by von Pawel-Rammingen et al. (2002). No difference in cleavage pattern was seen for IgG and heat-treated IgG, showing that the hinge region is accessible on both native“closed” and on“open” IgG (results not shown).

Chemical cross-linking and molecular modelling

The full three-dimensional structure of Infliximab is not readily available. However, crystal structures of elements of the antibody are available in the Protein Data Bank (https://www.rcsb.org/). The entries 4G3Y and 5VH5 were used to construct the full structure, and the structure of the hinge region was obtained from entry T3D3514 of the Toxin and Toxin Target Database (http://www.t3db.ca/). This constructed sequence was applied to construct a full 3D model of the intact antibody, using the 3D structure automated modelling system SWISS-MODEL (Biasini, M et al 2014). The modelling returned three unique 3D structures. The highest scoring figure showed a traditional IgG structure.

Infliximab samples were incubated for 2 hours at 37°C, 47°C and 57°C, respectively, prior to chemical crosslinking. As previously shown, IgGs undergoes a conformational change allowing RF to bind to the Ab following an incubation at 57°C for 1 h. It follows, that the crosslinking pattern of the samples incubated at 37°C and 57°C should be different. The chemical crosslinker BS3, which has a spacer-arm length of 1 1 .4A, targets primary amines, covalently linking amine groups of lysine residues or the N- terminus located within the appropriate distance of each other (25A for Ca-Ca. Each sample was prepared in triplicate. After crosslinking, the samples were separated by SDS-PAGE. The gel bands corresponding to the mass of a monomeric antibody were excised from the gel and digested using trypsin (data not shown). The resulting peptides were examined using liquid chromatography tandem mass spectrometry (LC- MS) using a Q-Exactive HF mass spectrometer. Data analysis was performed using the software MassAI.

Only MS/MS spectra representing cross-links with a score higher than 12 were accepted and all were manually verified. Data analysis was approached by considering the open IgG (Y-shaped) structure and dividing the crosslinks obtained into either a ‘supporting’ category or an‘overlength’ category. The supporting category contains crosslinks located within crosslinking distance (Ca-Ca 25A), measured between residues in the same domain. The overlength category contains crosslinks between residues not expected to be within crosslinking distance according to Figure 8. The overlength crosslinks were subsequently used to model the Ab in closed formation.

Table 1. Crosslinks found from supporting and overlength categories.

Table 1 shows a list of the crosslinks found, both from the validating and overlength categories. Three significant overlength crosslinks were found to support a closed conformation. Mainly the crosslink between the LC N-terminal and the HC Lys417 residue was significant, as these residues, were located at opposite ends of the open conformation, too far apart for a crosslink to occur between them. As the samples originated from a gel-band corresponding to a monomer, with a mass between 130- 180kDa, this crosslink did not occur between two separate antibodies. Interestingly, the same crosslink was found in the samples incubated at 57°C, suggesting that the two hours incubation still left an amount of Abs remaining in the closed formation. This conclusion was supported by experiments showing that maximal“opening” of the IgG was only obtained after 7 days of incubation at 57 ^°C, where the ability of the heat- treated IgG to bind RFs was maximal (results not shown). Experiments with combinations of sequential biotinylation and/or acetylation of native and heat-treated IgG showed that while confirming the“closed” structure of native IgG, the chemical cross-linking reduced the ability of IgG to bind tetanus toxoid and RF in a concentration-dependent manner (results not shown). Both IgG and infliximab subjected to cross-linking with BS3 (or glutaric aldehyde) lost the ability to bind RFs when coated on polystyrene surfaces of ELISA wells, indicating a“locked” conformation (results not shown). Biotinylation of native IgG did not abolish the ability to bind RF upon immobilization on a hydrophobic surface, while biotinylation of heat-treated IgG (57 ^°C) reduced RF binding by approximately 50 %. Acetylation of native and heat-treated IgG abolished interaction with RFs in accordance with the smaller size of the acetylation reagent (acetic anhydride) (results not shown).

References

Biasini, M. et al. SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014; 42:252-258

Corper AL , Sohi MK , Bonagura VR , Steinitz M , Jefferis R , Feinstein A , Beale D , Taussig MJ , Sutton BJ . Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction. Nat Struct Biol 1997; 4:374 - 81.

Duquerroy S, Stura EA, Bressanelli S, Fabiane SM, Vaney MC, Beale D, Hamon M, Casali P, Rey FA, Sutton BJ, Taussig MJ. Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and lgG1 Fc reveals a novel epitope and evidence for affinity maturation. J Mol Biol 2007; 368: 1321 - 31.

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Holm BE, Sandhu N, Tronstrom J, Lydolph M, Trier NH, Houen G. Species cross reactivity of rheumatoid factors and implications for immunoassays. Scand J Clin Lab Invest. 2015; 75:51 -63.

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Laursen IA, Blou L, Sullivan JS, Bang P, Balstrup F, Houen G. Development, manufacturing and characterization of a highly purified, liquid immunoglobulin G preparation from human plasma. Transfus Med Hemother. 2014; 41 :205-12. Mageed RA, Borretzen M, Moyes SP, Thompson KM, Natvig JB. Rheumatoid factor autoantibodies in health and disease. Ann NY Acad Sci 1997; 815:296-31 1.

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Claims

Claims

1. An immunoassay comprising:

a) at least one antibody capable of binding an antigen of interest in a sample, and

b) a heat-treated preparation of Immunoglobulins.

2. The immunoassay according to claim 1 , wherein the antibody capable of

binding an antigen of interest in a sample is a capture antibody, and further comprising a detection antibody bound directly or indirectly to a detection moiety.

3. The immunoassay according to any of the preceding claims, wherein the

antibody capable of binding an antigen of interest in a sample and/or detection antibody comprises an Fc region of an IgG.

4. The immunoassay according to any of the preceding claims, wherein the

preparation of Immunoglobulins has been heat treated at a temperature of at least 42 °C, such as at least 45 °C, such as at least 50‘G, preferably at least 53 °C, such as at least 55 °C, for example at least 57 ^qC.

5. The immunoassay according to any of the preceding claims, wherein the

preparation of Immunoglobulins has been heated for at least 30 min, such as at least 1 hour, such as at least 1 day, such as at least 1 week, such as at least 1 month.

6. The immunoassay according to any of the preceding claims, wherein the heat- treated preparation of Immunoglobulins comprises at least the Fc region of IgG.

7. The immunoassay according to any of the preceding claims, wherein the heat- treated preparation of Immunoglobulins is mammalian.

8. A method of performing an immunoassay, the method comprising: a. loading a sample comprising an antigen of interest to an immunoassay, wherein the immunoassay comprises at least one antibody capable of binding the antigen of interest, b. contacting the sample with a heat-treated preparation of

Immunoglobulins comprising IgG, and

c. detecting the complex between the capture antibody and antigen of interest using a secondary antibody or turbidometry.

9. The method of claim 8, wherein the sample is incubated with the heat-treated preparation of Immunoglobulins prior to loading, or wherein the heat-treated preparation of Immunoglobulins is present in the immunoassay.

10. Use of a heat-treated preparation of Immunoglobulins to reduce false positives in immunoassays, wherein the preparation of Immunoglobulins has been heat treated at a temperature of at least 42^qC, such as at least 45^qC, such as at least 50 °C, preferably at least 53 °C, such as at least 55‘O, for example at least 57 ^qC.

1 1. The use of claim 10, wherein the preparation of Immunoglobulins has been heat-treated for at least 30 min, such as at least 1 hour, such as at least 1 day, such as at least 1 week, such as at least 1 month.

12. The use of claim 10 or 1 1 , wherein the preparation of Immunoglobulins has been heat treated to expose cryptic epitopes recognised by heterophilic antibodies, preferably recognised by rheumatoid factor.

13. The use of any of the preceding claims 10 to 12, wherein the heat-treated

preparation of Immunoglobulins is derived from serum.

14. The use of any of the preceding claims 10 to 13, wherein the heat-treated

preparation of Immunoglobulins has been purified to remove non

immunoglobulin proteins.

15. Use of one or more Fc fragments to reduce false positives in immunoassays, wherein the Fc fragment comprises epitopes recognised by heterophilic antibodies, preferably recognised by rheumatoid factors.