Method and kit for elimination of rheumatoid factor interference in immunoassays

Abstract

Method and kit for elimination of rheumatoid factor interference in immunoassays The invention relates to an in vitro method of reducing and / or eliminating unwanted detection of an IgG antibody in an immunoassay on a biological sample containing rheumatoid factor (RF), the method comprising contacting said biological sample with a RF interference-reducing protein comprising an amino acid sequence of SEQ ID NO. 1, 2 or 3, and capable of binding to the Fc domain of IgG antibodies. Also disclosed is an in vitro method for detecting antibodies specific to an antigen in a biological sample that belong to one or more immunoglobulin classes other than IgG, which makes use of the aforementioned RF interference-reducing protein. There are further described a kit for carrying out the method of the invention and the use in said method of a fusion protein comprising said RF interference-reducing protein linked to a fusion partner.

Description

[0001] Method and kit for elimination of rheumatoid factor interference in immunoassays

[0002] Technical field

[0003] The present invention relates to a method of reducing and / or eliminating unwanted detection of IgG antibodies in an immunoassay performed on a biological fluid sample containing rheumatoid factor (RF). The present invention also relates to a method and kit for the detection of antigen- specific antibodies of one or more immunoglobulin classes other than IgG in a biological fluid sample containing both interfering IgG antibodies specific to the same antigen and interfering rheumatoid factor (RF).

[0004] Background art

[0005] Rheumatoid factor (RF) is one of the most common sources of interference in immunoassays, particularly in IgM testing. RFs are autoimmune antibodies, usually of the IgM class, but existing also as IgG, IgA and IgE, directed against the Fc portion of an individual’s IgG (Van Boekel et al., Arthritis Res. 2001 Nov 6;4(2):87-93). RFs are found in the blood of up to 25% of older individuals, primarily in patients suffering from rheumatoid arthritis, a multifactorial disease whose etiology still needs to be fully resolved. Naturally occurring RFs are also detectable in healthy individuals, most likely arising as part of the immune response against certain bacterial or viral antigens, e.g. to lipopolysaccharide or Epstein-Barr virus (Westwood et al. Rheumatology 2006; 45:379-385). RF circulate in blood alongside with their target antigens (IgGs) without reacting with them, as it has been demonstrated that IgG epitopes become accessible only upon antigen binding or following certain physico-chemical treatments since the native state of IgG is a compact (closed) form, thus preventing RF binding. Upon binding of the antibody to the respective antigen, a conformational change is induced, resulting in the exposure of the Fc domain recognized by RF (Maibom-Thomsen et al. PLoS One, 2019; 14(6): 1-28).

[0006] Interference from RF when measuring antibody levels is essentially due to the ability of rheumatoid factor to bridge between the capture antigen and the detection reagent in the immunoassay setup, even in the absence of the target antibody, thereby generating a false signal. Immunoassays particularly susceptible to interference from rheumatoid factor are indirect IgM assays, in which a solid phase material, coated with an antigen, is used to capture antigen- specific IgM antibodies, and the IgM antibody-antigen complex thus formed is then detected employing an anti-IgM conjugate (e.g. an enzyme conjugate). Typically, in these assays binding of antigen- specific IgG antibodies present in serum to the capture phase also occurs, and upon binding, the binding sites on IgG for RFs are exposed. IgM RFs bound to the Fc constant domain of captured IgGs are then recognized by the anti-IgM conjugate used for antibody detection, thus generating a false-positive IgM reaction. As rheumatoid factor can be also of IgA or IgE isotype, although more rarely, the aforementioned mechanism may also occur in indirect immunoassays for the detection of IgA or IgE antibodies, where secondary antibodies that recognize these classes of immunoglobulins are employed.

[0007] RF has been identified as an interferent in several assays, including EBV IgM (Ho et al., J. Clin. Microbiol. 1989, 27, 5, 952), thyroid hormones (Despres et al., Clin. Chem., 1998, 44, 440), HSV 1+2 IgM (Pan et al., Lab Medicine, 2018, 49, 369), HBsAg (Xu et al., Clin. Biochem., 2013, 46, 9, 799).

[0008] In view of the above, the interference of RF in antibody immunoassays represents a critical issue in laboratory medicine due to their widespread employment, thus potentially causing harmful consequences for the patient if unreliable results are produced.

[0009] Methods to eliminate RF interference in immunoassays may involve the removal of IgG antibodies from serum. Several techniques for the separation of IgG from IgM to improve testing performance have been reported (Martins et al., Clin. Diagn. Lab. Immunol., 1995, Vol 2, No 1, 98). Among these techniques, several make use of particular bacterial proteins, such as staphylococcal Protein A and G, that are known to bind with high specificity and affinity to the Fc-domain of IgGs, thus resulting particularly effective in clearing much of the IgG present in serum. Alternative methods for removing IgGs prior to IgM testing employ anion-exchange chromatography.

[0010] However, the above-described methods have been shown not to be particularly suitable for routine and high-volume serology testing as they are labour-intensive, may result in unintended removal of the IgM analyte (i.e. partial analyte stripping), and are not specific for all IgG subclasses. Moreover, the addition of recombinant staphylococcal Protein A or Protein G directly to the assay reagents may cause assay interference per se, due to the crossrecognition of these bacterial proteins towards the mouse antibodies that are typically used in immunoassays, either for analyte capture and / or detection. Furthermore, since staphylococcal Protein A or Protein G contain multiple IgG-binding sites per molecule, a bridge between interfering IgGs and detection reagents used in the assay may form and generate unwanted signals, similarly to RF interference (Rispens and Vidarsson, Antibody Fc, 2014, Chapter 9, 159-177).

[0011] US 5,698,393 discloses an approach aiming at removing RF interference in immunoassays based on the addition of neutralization buffers to the reaction mix, in order to adjust the pH to an optimal range at which the RF interference is minimized.

[0012] Most modern immunoassays are designed with specific protective measures against interference from RF. Commercial blockers are available, consisting of highly concentrated blends of anti-RF antibodies which can be spiked into the immunoassay reagents to a target concentration. Another method widely adopted to avoid RF interference relies on the use of goat (or other animal-derived) anti-human IgG hyperimmune serum which acts as IgG- stripping agent when added to the assay reagents to a target dilution. However, this method suffers from several drawbacks, such as a reduced lot-to-lot consistency of goat sera, the potential interference due to carry over of the serum when used above certain percentages, and a low assay throughput due to the required pre-dilution step of the test sample with a solution containing the goat serum.

[0013] The group A streptococcus, Streptococcus pyogenes, is an important human pathogen that is estimated to be the ninth leading cause of deaths due to microbial infections worldwide. Members of the S. pyogenes M protein family are key virulence factors that contribute to the pathogenesis of bacterial infections and their binding of blood proteins, such as complement regulatory proteins, plasminogen, albumin, fibrinogen, and immunoglobulins, is thought to contribute to pathogenesis. The M protein family is composed of M protein (Emm), M- related protein (Mrp), and an M-like protein (Enn), which are part of the Mga regulon. The components of the Mga regulon can vary depending upon the serotype. Some serotypes express only Emm, whereas other serotypes express Emm, Mrp and / or Enn. Infections caused by S. pyogenes are almost entirely restricted to humans, but the molecular basis for this host preference is poorly understood. Plasminogen binding has been linked to host specificity of group A streptococcal infections, and the ability of S. pyogenes to selectively bind immunoglobulins from certain species is thought to contribute to this host specificity and to virulence (Cedervall et al., Biochemistry, 1997, 36, 16, 4987).

[0014] M-related proteins (Mrps) are dimeric a-helical coiled-coil cell membrane-bound surface proteins. During infection, Mrps recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring bacteria enhanced resistance to phagocytosis and increased growth in human blood. However, Mrps show a high degree of sequence diversity, but diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranges from 3.7 to 11.1 nM for mixed IgG. Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Dimeric Mrps bind to IgG with a 1: 1 stoichiometry (Proctor et al., J. Biol. Chem., 2024, 300, 2, 105623).

[0015] Mrp4 (M-related protein 4) is a key virulence factor that contributes to resistance of Streptococcus pyogenes to phagocytosis by binding fibrinogen and human IgG at two separate and distinct binding domains. Mrp4 selectively binds to the Fc domain of human IgG, but not IgA or IgM or IgE, and it preferentially binds subclasses IgGl>IgG4>IgG2>IgG3; Mrp4 doesn’t bind goose, guinea pig, rat, chicken, cow, mouse and goat IgGs. The IgG-binding domain of Mrp is localized in the A-repeat region and does not have any significant degree of similarity to the IgG-binding domains of M proteins and protein H: Mrp4 contains 3 A repeats. Both a recombinant Mrp4 which contains a single A repeat and a recombinant Mrp which contains three A-repeats bind human IgGs but the latter is ~40-fold more effective inhibitor than the former (Courtney et al., PLoS ONE, 2013, 8, 10, e78719).

[0016] Summary of the invention In the light of the foregoing, there is therefore a strong need of providing effective approaches aiming at reducing and / or eliminating interference caused by rheumatoid factor (RF) in antibody immunoassays, which overcome the drawbacks and technical limitations of the prior art and enable achieving reliable assay results, which is crucial in medical diagnostics.

[0017] These and other needs are addressed by the present invention, which in a first aspect provides an in vitro method of reducing and / or eliminating unwanted detection of an IgG antibody in an immunoassay performed on a biological fluid sample containing rheumatoid factor (RF), the method comprising contacting said biological fluid sample with a RF interferencereducing protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2 and 3, said RF interference-reducing protein being capable of binding to the Fc domain of IgG antibodies.

[0018] According to the invention, the amino acid sequences of SEQ ID NO. 1, 2 and 3 consist of an IgG-binding domain of a Mrp4 protein from Streptococcus pyogenes.

[0019] In one embodiment, the immunoassay is an antibody immunoassay, preferably an immunoassay for the detection of antigen- specific antibodies of one or more immunoglobulin classes other than IgG, more preferably an immunoassay for the detection of antigen- specific antibodies of an IgM, IgA and / or IgE immunoglobulin class.

[0020] In another aspect of the present invention is provided an in vitro method for the detection of antibodies specific to an antigen in a biological fluid sample, said antibodies being of one or more immunoglobulin classes other than IgG, said biological fluid sample containing interfering elements comprising both IgG antibodies specific to the same antigen as the antibodies to be detected and rheumatoid factor (RF), the method comprising the steps of: i) contacting the biological fluid sample with a capture moiety comprising said antigen, whereby immunocomplexes between the capture moiety and the antigen- specific antibodies present in the biological fluid sample are formed, ii) contacting said immunocomplexes with a RF interference-reducing protein as above defined and allowing binding of the RF interference-reducing protein to IgG immunocomplexes, thereby blocking the binding of rheumatoid factor to said IgG immunocomplexes, and iii) detecting the immunocomplexes formed by the antibodies of the one or more immunoglobulin classes other than IgG.

[0021] In a further aspect of the present invention is provided a kit for the detection of antibodies specific to an antigen in a biological fluid sample, said antibodies being of one or more immunoglobulin classes other than IgG, said biological fluid sample containing interfering elements comprising both IgG antibodies specific to the same antigen as the antibodies to be detected and rheumatoid factor (RF), the kit comprising:

[0022] - a capture moiety comprising said antigen specific to the antibodies to be detected,

[0023] - a RF interference-reducing protein as above defined, and

[0024] - means for detecting the antigen- specific antibodies of one or more immunoglobulin classes other than IgG.

[0025] Also provided herein is the use of a fusion protein in any of the methods of the invention, wherein the fusion protein comprises a RF interference-reducing protein as above defined and a fusion partner, the RF interference-reducing protein being linked to the fusion partner, optionally through a peptide linker.

[0026] Other features and advantages of the present invention are defined in the appended claims which form an integral part of the description.

[0027] Description of the invention

[0028] A key feature of the present invention in all its aspects is the use of a Mrp4 protein from Streptococcus pyogenes, or a portion thereof, as an effective inhibitor of rheumatoid factor interference in an immunological method. As it will be illustrated in detail in the experimental section that follows, the present inventors surprisingly observed that false positive results caused by RF interference in an immunoassay for the detection of non-IgG antibodies, such as an IgM immunoassay, performed on serum samples also containing interfering IgGs, were abolished by incorporating in this assay an incubation step in the presence of S. pyogenes Mrp4, or a portion thereof comprising at least one IgG-binding domain.

[0029] Remarkably, it was also observed that the use of Mrp4, or an IgG-binding portion thereof, as RF interference-reducing agent advantageously increased assay sensitivity for IgM detection in serum samples from patients undergoing seroconversion during the natural course of a viral infection (Figure 7).

[0030] The above observations are totally unexpected as further experiments conducted by the present inventors using a monoclonal antibody in the antibody immunoassay having same specificity as Mrp4 towards the IgG Fc domain, did not achieve suppression of rheumatoid factor interference (Table 3). These surprising results were further confirmed by employing an anti-human IgG polyclonal antibody (Table 4).

[0031] Without limiting the invention to specific theories, the results obtained by the present inventors indicate that said Mrp4 protein, or an IgG-binding portion thereof, acts by blocking RF binding sites on IgG antibodies, thereby preventing rheumatoid factor from binding to these antibodies. Consequently, in an immunoassay employing said protein or portion thereof, for example an immunoassay directed to antigen- specific antibodies of an immunoglobulin class other than IgG (such as IgM, IgA and / or IgE antibodies), rheumatoid factors can no longer interact with interfering IgG antibodies present in the sample, i.e. IgGs having the same antigen specificity as the antibodies target of the assay, and the undesired detection of said IgGs via RF resulting in false positivity does no longer occur.

[0032] To the best of the inventors' knowledge, the use of S. pyogenes Mrp4, or an IgG-binding portion thereof, in immunological assays is not disclosed in the prior art, particularly not as a solution to the problem of reducing or eliminating rheumatoid factor (RF) interference in an antibody immunoassay.

[0033] Therefore, the present invention relates to an in vitro method of reducing and / or eliminating unwanted detection of an IgG antibody in an immunoassay as above defined. Further, the present invention relates to an in vitro method as above defined, for the detection of antibodies specific to an antigen in a biological fluid sample, said antibodies being of one or more immunoglobulin classes other than IgG, said biological fluid sample containing interfering elements comprising both IgG antibodies specific to the same antigen as the antibodies to be detected and rheumatoid factor (RF).

[0034] As used herein, the term "interference” refers to the presence of a substance or factor in a biological sample, which can cause erroneous or misleading results by affecting the assay's ability to accurately detect or measure the target analyte. In immunoassays, interference can occur when non-target molecules, such as rheumatoid factor (RF), bind to assay components, leading to false-positive or false-negative results.

[0035] As used herein, the term "interfering substance” refers to a substance that may alter the determination of a test antigen or antibody in an immunoassay, for example by interacting with one or more reagent antibodies. Interfering substances may lead to falsely elevated or falsely low analyte measures, thereby altering the correct value of the immunoassay result.

[0036] As used herein, the term “antigen” is used to refer to any substance which exhibits specific immunological reactivity with a target antibody present in the test sample.

[0037] The term "antigen- specific" as used herein, means the ability of an antibody to recognize an antigen specifically as a unique molecular entity and distinguish it from another with very high precision.

[0038] “Detection” refers to a qualitative or quantitative determination of an analyte in a sample, such as an antibody specific against an antigen. More specifically, “detecting” may include detecting the presence of an analyte, and / or quantifying said analyte in the sample.

[0039] In all aspects of the present invention, the RF interference-reducing protein is characterized in that it comprises or consists essentially or consists of an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2 and 3, and is capable of binding to the Fc domain of IgG antibodies. In the context of the present description, the amino acid sequence of Mrp4 protein from S. pyogenes is set forth as SEQ ID NO. 4 as available from the NCBI database under accession number P30141 (https: / / www.ncbi.nlm.nih.gov / search / all / ?term=P30141). The nucleotide sequence encoding S. pyogenes Mrp4 is herein provided as SEQ ID NO. 6.

[0040] According to the invention, the amino acid sequence of SEQ ID NO. 1 corresponds to residues 179 to 283 of SEQ ID NO. 4, and the amino acid sequence of SEQ ID NO. 2 corresponds to residues 191 to 296 of SEQ ID NO. 4. Such sequences have been disclosed in the art as overlapping, IgG-binding domains of Mrp4 protein (Proctor EJ el al, Journal of Biological Chemistry, Volume 300, Issue 2, 2024, 105623; Courtney HS, and Li Y. PLoS One. 2013 Oct 25;8(10):e78719). Still according to the invention, the amino acid sequence of SEQ ID NO. 3 corresponds to residues 179 to 296 of the amino acid sequence of SEQ ID NO. 4. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 3 is herein provided as SEQ ID NO. 7.

[0041] In a preferred embodiment of all aspects of the invention, the RF interference-reducing protein comprises or consists essentially or consists of the amino acid sequence of SEQ ID. 3. According to this embodiment, the RF interference-reducing protein of SEQ ID. 3 is optionally conjugated with a histidine tail at the amino-terminal (N-terminal) or carboxyterminal (C-terminal) end.

[0042] In the context of the present invention, the term “conjugated” refers to the presence of a covalent bond between the amino acid at the N-terminal end of the RF interference-reducing protein of the invention and the amino acid at the C-terminal end of the histidine tail or, vice versa, the presence of a covalent bond between the amino acid at the C-terminal end of the peptide of the invention and the amino acid at the N-terminal end of the histidine tail, whether or not preceded by a linker.

[0043] An exemplary amino acid sequence of the RF interference-reducing protein according to the aforementioned embodiment is as set forth in SEQ ID NO. 5 wherein amino acids 1 to 24 correspond to the His-tag plus a linker and amino acids 25 to 142 correspond to SEQ ID NO. 3.

[0044] In another preferred embodiment of all aspects of the invention, the RF interference-reducing protein comprises or consists essentially or consists of the amino acid sequence of SEQ ID NO. 4.

[0045] In still another preferred embodiment of all aspects of the invention, the RF interferencereducing protein is provided in the form of a fusion protein, wherein said RF interferencereducing protein is linked to a fusion partner, optionally through a peptide linker.

[0046] Hereinafter the expression “fusion partner” is intended to mean a protein, or a portion thereof, or a peptide, which is linked via a covalent bond to the RF interference-reducing protein.

[0047] According to this embodiment, it is envisaged that the RF interference-reducing protein may be linked either to the C-terminal or the N-terminal of a fusion partner as above defined, optionally, via a peptide linker. Preferably, the linker may be a peptide comprising 3 to 20 amino acid residues or consisting of 3 to 20 amino acid residues.

[0048] A fusion partner according to the invention may be any protein able to improve expression levels, solubility, stability, folding of the RF interference-reducing protein, or any protein suitable to be used for affinity purification purposes. Preferably, the fusion partner is a protein selected from the group consisting of FkpA, SlyD, trigger factor, maltose binding protein (MBP), thioredoxin, glutathione-S -transferase, Fc-portion of IgG. Fusion partners are routinely employed in protein sciences and their use within the context of the present invention is therefore well within the knowledge of the person skilled in the art.

[0049] According to the present invention, the RF interference-reducing protein or a fusion protein comprising said protein may be a recombinant protein, wherein the term "recombinant", as used herein, refers to a protein produced using genetic engineering approaches at any stage of the production process, for example by fusing a nucleic acid encoding the protein to a strong promoter for overexpression in cells or tissues or by engineering the sequence of the protein itself. The person skilled in the art is familiar with methods for engineering nucleic acids and proteins encoded (for example, described in Sambrook et al (1989), Molecular Cloning, CSH or in Brown T. A. (1986), Gene Cloning - an introduction, Chapman & Hall) and for producing and purifying native or recombinant proteins (for example Handbooks "Strategies for Protein Purification", "Antibody Purification", published by GE Healthcare Life Sciences , and in Burgess, R. R., Deutscher, M. P. (2009): Guide to Protein Purification).

[0050] Alternatively, the RF interference-reducing protein may be an isolated protein, wherein the term "isolated" means that the protein has been enriched compared to its state upon production using a biotechnological or synthetic approach and is preferably pure, i.e. at least 60, 70, 80, 90, 95 or 99 percent of the protein in the respective liquid consists of said protein as judged by SDS polyacrylamide gel electrophoresis followed by Coomassie blue staining and visual inspection.

[0051] Thanks to the unique mechanism underlying the present invention, which provides an indirect block of RF interference by selectively occupying RF binding sites on IgG antibodies, the methods of the invention advantageously achieve suppression of RF activity regardless of the specific RF isotype present in the test sample. Accordingly, the interfering rheumatoid factor (RF) present in the biological fluid sample according to the invention may be of any antibody isotype, for example an IgM, IgA, and / or IgE RF isotype.

[0052] The term “immunoassay” as used herein, refers to a highly selective bioanalytical method that enables to determine the presence or concentration of analytes in a solution using an antibody or an antigen as a biorecognition agent.

[0053] In certain embodiments of the invention, the immunoassay may be an indirect assay in which an antigen having specificity for the antibodies under test, optionally immobilized on a solid surface, is used to bind / capture said antibodies, and the bound / captured antibody / antigen complexes are then detected, preferably by means of a secondary conjugated antibody capable of recognizing the antigen-specific antibodies.

[0054] Obviously, the use of any type of immunoassay format, the selection of which falls within the skills of the ordinary person of skill in the art, is within the scope of the present invention.

[0055] As aforesaid, the present invention also provides an in vitro method for the detection of antigen- specific antibodies of one or more immunoglobulin classes other than IgG in a biological fluid sample, wherein said sample comprises interfering elements comprising both IgG antibodies specific to the same antigen as the antibodies to be detected and rheumatoid factor (RF).

[0056] Preferably, said antigen-specific antibodies to be detected are IgM, IgA and / or IgE antibodies.

[0057] In a first step of the method of the invention, the biological fluid sample to be tested is brought into contact with a capture moiety comprising the antigen having specificity for the antibodies under test, whereby immunocomplexes between the capture moiety and the antigen- specific antibodies present in the biological fluid sample are formed. In other words, the antigen- specific antibodies present in the biological sample are captured by the capture moiety.

[0058] The term "capture moiety" as used herein, refers to a moiety which is capable to bind selectively to an antibody, thus enabling said antibody to be captured and isolated from a biological sample.

[0059] Any non-IgG antibody of the desired specificity present in the biological fluid sample will bind to said capture moiety. When the sample contains interfering IgG antibodies specific for the same antigen recognized by the non-IgG antibodies under test, said interfering IgGs will also be captured by the capture moiety. Accordingly, in the first step of the method of the invention immune complexes are formed by the binding of both non-IgG antibodies and interfering IgGs to the capture moiety.

[0060] According to the invention, the immunocomplexes thus formed (i.e. the antigen- specific antibodies bound to the the capture moiety) are then brought into contact with a RF interference-reducing protein as above defined to allow binding of said protein to the IgG immunocomplexes (i.e. the interfering IgGs bound to the capture moiety), so as to block the access of rheumatoid factor present in the sample to said IgGs. Advantageously, the use of the RF interference-reducing protein according to the invention enables to suppress the ability of RF to bridge between interfering IgGs and detection reagents, thereby preventing the undesired IgG detection leading to false positive signals in antibody immunoassays.

[0061] The immunocomplexes formed by the antigen- specific antibodies of the one or more immunoglobulin classes other than IgG (i.e. captured non-IgG antigen- specific antibodies) may then be detected using any suitable detection means, typically a secondary antibody that specifically recognizes a specific class of immunoglobulins that is different from IgG.

[0062] In a preferred embodiment of the method of the invention, the capture moiety is immobilized on a solid support. Non-limiting examples of suitable solid supports are the wells of a microtiter plate, the surface of a microparticle such as a latex, polystyrene, silica, chelating sepharose or magnetic beads, membranes, strips or chips.

[0063] Preferably, the solid support is a bead, more preferably a paramagnetic micro-particle (PMP). The capture moiety may be covalently bound to the solid surface, or non-covalently attached through nonspecific bonding. For example, the capture moiety may be in a biotinylated form and immobilization may be performed by using a solid phase carrying (strept)avidin.

[0064] Suitable antigens according to the invention may include, but are not limited to, naturally occurring proteins, recombinant or synthetic proteins or polypeptides, synthetic peptides, peptide mimetics, polysaccharides and nucleic acids. Preferably, in the method of the invention the antibodies under test of one or more immunoglobulin classes other than IgG, have specificity for an antigen from a microorganism, more preferably from a bacterial or viral microorganism, even more preferably from a microorganism selected from the group consisting of Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Mumps virus (MuV), Measles virus (MV), Mycoplasma pneumoniae, and any combination thereof. Figure 2 illustrates by way of example a preferred embodiment of the invention, wherein an IgM immunoassay is conducted in the presence of a RF interference-reducing protein according to the invention. Where the biological fluid sample contains interfering IgG antibodies with same specificity as the test IgM antibodies, these IgGs bind to the capture moiety employed in the immunoassay for capturing the IgM, optionally immobilized onto a solid support. Then, the interfering rheumatoid factors present in the sample interact with the interfering IgG antibodies bound to the capture moiety and the immunocomplexes thus formed are then recognized by labeled anti-IgM antibodies used as assay detection means, whereby a false positive signal is generated (Figure 2A). In the presence of a RF interferencereducing protein according to the invention, the interaction of interfering rheumatoid factors with captured IgG antibodies is prevented by the binding of this protein to the Fc domain of IgGs, and advantageously only the antigen- specific IgM antibodies are detected (Figure 2B).

[0065] Since a biological fluid sample may contain various RF isotypes, it is envisaged that the assay scheme illustrated in Figure 2 can be easily applied also to a method for the detection of antigen- specific IgA and / or IgE antibodies. Accordingly, the scope of the present invention is not limited to a method for the detection of IgM antibodies only, but it includes also the detection of antigen- specific IgA and / or IgE antibodies.

[0066] The immunoassay formats that can be used for practicing the method of the present invention and that can be incorporated in a kit form are many, and include, for example, enzyme-linked immunosorbent assays (ELISA) also referred to as enzyme immunoassays (EIA), chemiluminescent immunoassays (CLIA), fluorescence immunoassays, enzyme-linked immunoassays (ELISA), luminescence immunosorbent assays (LISA), radioimmunoassays (RIA), luminex-based bead arrays. The use of any type of immunoassay format, the selection of which falls within the skills of the person skilled in the art, is within the scope of the present invention.

[0067] In the method of the invention, specific immunological binding of the antibodies of one or more immunoglobulin classes other than IgG to the capture moiety can be detected by means of a detection agent conjugated to one or more detectable labels. The detectable label may be any substance capable of producing a signal that is detectable by visual or instrumental means. Suitable labels for use in the present invention include for example fluorescent compounds, chemiluminescent compounds, radioactive compounds, enzymes, enzyme substrates, molecules suitable for colorimetric detection, binding proteins, and epitopes. In practice, any signal molecule or label known in the art may be incorporated in embodiments of the method and kit of the present invention.

[0068] In a preferred embodiment, the immunocomplexes formed by the antibodies of the one or more immunoglobulin classes other than IgG may be detected using a secondary antibody. Typically, the secondary antibody is directed towards an epitope common to one or more classes of human immunoglobulins such as e.g. IgM, IgA or IgE antibodies, and is generated in an animal species that is different from the animal from whom the biological fluid sample was taken.

[0069] Preferably, the secondary antibody used in the method of the invention is an anti- IgM, antiIgA and / or anti-IgE antibody, more preferably is an anti-IgM, anti-IgA and / or anti-IgE antibody from a non-human animal labeled with a detectable label.

[0070] In any aspects of the present invention, the biological fluid sample is preferably selected from the group consisting of whole blood, serum, plasma, and urine. The biological fluid sample may optionally include further components, such as for example: diluents, preservatives, stabilizing agents and / or buffers. If needed, dilutions of the biological fluid sample are prepared using any suitable diluent buffer known in the art.

[0071] A preferred sample according to the invention is a biological fluid sample of human origin.

[0072] The preferred embodiments of the method according to the invention described above can be combined with each other as required, and the implementation of these combinations falls within the skills of the person skilled in the art.

[0073] Thanks to the above-illustrated features, the method according to the invention achieves suppression of RF interference by providing a selective blocking of RF binding sites on the Fc domain of interfering IgG antibodies possibly present in a test biological fluid sample. Consequently, the present invention advantageously avoids the effects of partial stripping, i.e. unintended removal of the analyte by pre-analytical sample treatment, or analyte blocking that may occur when prior art agents having non-specific binding activity, such as hyperimmune sera, are employed in antibody immunoassays for the purpose of reducing RF interference.

[0074] The binding selectivity of the RF interference-reducing protein of the present invention is also particularly advantageous over the use of Staphylococcal Protein A or Protein G, which, by binding to the Fc region of immunoglobulins from various species, may subtract from the immunoassay the animal-derived antibodies typically used as detection reagents. More specifically, cross -reactivity of Protein G towards mouse IgGs may cause significant interference with the mouse monoclonal antibodies typically used as reagents in immunoassays. In particular, in the case of indirect assays for detection of IgM antibodies, the use of Protein G may lead to false negative results by binding the mouse anti-human IgM antibody used for detection and preventing the recognition of the IgM analyte captured on solid phase.

[0075] In addition to safeguarding assay sensitivity, the method of the present invention allows also to overcome other relevant limitations associated with the use of hyperimmune serum as RF interference blocker. As well-known in the art, the production of such serum is a poorly controllable process and the resulting product is usually highly heterogeneous and poorly characterizable from a biochemical standpoint. Accordingly, it is often observed that there can be considerable variation across different lots of hyperimmune sera, including e.g. the specific IgG titer and the polyclonality grade, ultimately resulting in a significantly different functional performance. Advantageously, the present invention provides for the use of a protein reagent that can be prepared in highly purified and homogeneous form through highly reproducible processes, which enable an easy characterization of the biochemical properties of the resulting product.

[0076] Still a further advantage of the method of the invention over prior art methods utilizing hyperimmune serum for reducing RF interfering activity is the possibility to adopt high- throughput assay protocols, without the requirement of laborious and time-consuming sample pretreatment procedures.

[0077] Also, the methods of the invention advantageously provide a remedy against the inherent risk of sample carry-over associated with the use of hyperimmune serum or highly concentrated matrices as the RF interference-reducing protein according to the invention may be easily washed away from tips, capillaries, needles, cuvettes and other parts of an instrument.

[0078] The means suitable for performing the in vitro method of the invention are assembled into a kit in order to facilitate the use thereof.

[0079] Therefore, a further aspect of the invention is a kit for the detection of antibodies specific to an antigen in a biological fluid sample, said antibodies being of one or more immunoglobulin classes other than IgG, said biological fluid sample containing interfering elements comprising both IgG antibodies specific to the same antigen as the antibodies to be detected and rheumatoid factor (RF), the kit comprising: (i) a capture moiety comprising said antigen specific to the antibodies to be detected, (ii) a RF interference-reducing protein as above defined, and (iii) means for detecting the antigen- specific antibodies of one or more immunoglobulin classes other than IgG.

[0080] Suitable capture moieties and detection means to be used in the kit of the invention are as described above in connection with the method of the invention.

[0081] The kit of the invention may further comprise a solid support such as, without limitation, latex, polystyrene, silica, chelating sepharose, magnetic beads, membranes, strips, chips, microparticles, nanoparticles, super paramagnetic particles, a microtiter plate, a cuvette, a lateral flow device, a flow cell, or any surface to which the capture moiety can be passively or covalently bound.

[0082] It will be understood that any embodiment of the method as described above may be incorporated in the kit of the present invention. A further aspect of the invention is the use of a kit as above defined for carrying-out any of the in vitro methods according to the invention.

[0083] As discussed above, a fusion protein that comprises a RF interference-reducing protein according to the invention and a fusion partner is particularly suitable to be used in any of the methods of the invention. Accordingly, the use of a fusion protein in any of the methods according to the invention falls within the scope of the invention, said fusion protein being characterized by comprising a RF interference-reducing protein as defined above and a fusion partner, wherein said RF interference-reducing protein is linked to the fusion partner, optionally through a peptide linker.

[0084] Preferred embodiments of the fusion protein for the use according to the invention are as described above in connection with the methods of the invention.

[0085] A preferred fusion partner is selected from a bacterial chaperone FkpA and a bacterial maltose binding protein (MBP). More preferably, the bacterial chaperone FkpA comprises or consists essentially or consists of the amino acid sequence of SEQ ID NO. 8 and / or the bacterial MBP comprises or consists essentially or consists of the amino acid sequence of SEQ ID NO. 9. In one embodiment, there are also provided isolated nucleic acid molecules comprising or consisting of a nucleotide sequence encoding a bacterial chaperone FkpA or a bacterial MBP, hereinafter designated as SEQ ID NO. 10 and SEQ ID NO. 11, respectively.

[0086] In certain embodiments of the invention, the fusion protein comprising the RF interferencereducing protein may further comprise additional components such as an affinity tag for purification or a signal peptide. Among the most common affinity tags, polyhistidine tags (“His-tag”) attached at the C-terminal or N-terminal of the protein of interest are routinely employed in protein sciences and their use within the context of the present invention is therefore well within the knowledge of the person skilled in the art. However, other affinity tags such as, for example, Arg5, Strep-tag II, FLAG, fluorescein (FITC), Poly(A), Poly(dT) and biotin may be employed. Techniques for the production of epitope-tagged recombinant proteins are generally known in the art. In a preferred embodiment, the fusion protein according to the invention comprises or consists essentially or consists of the amino acid sequence of SEQ ID NO. 12 (corresponding to SEQ ID NO. 8 linked to SEQ ID NO. 3 through a linker, and comprising an N-terminal His-tag).

[0087] In another preferred embodiment, the fusion protein according to the invention comprises or consists essentially or consists of the amino acid sequence of SEQ ID NO. 13 (corresponding to SEQ ID NO. 9 linked to SEQ ID NO. 3 through a linker comprising a His-tag).

[0088] An exemplary amino acid sequence of a fusion protein according to the invention is set forth as SEQ ID NO. 12, wherein amino acids 1 to 18 correspond to the His-tag followed by a linker, amino acids 19 to 263 correspond to a FkpA fusion partner (SEQ ID No. 8), amino acids 264 to 274 correspond to a linker, and amino acids 275 to 392 correspond to SEQ ID No. 3 of the RF interference-reducing protein of the invention.

[0089] Yet another exemplary amino acid sequence of a fusion protein according to the invention is set forth as SEQ ID NO. 13, wherein amino acids 1 to 368 correspond to a MBP fusion partner (SEQ ID No. 9), amino acids 369 to 390 correspond to the His-tag followed by a linker, and amino acids 391 to 508 correspond to SEQ ID No. 3 of the RF interferencereducing protein of the invention.

[0090] The fusion protein according to the invention can be prepared artificially through a recombinant DNA technology. For example, the genes or nucleic acid molecules encoding the RF interference-reducing protein and the fusion partner may be linked with each other to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion protein. Accordingly, the present invention provides an isolated nucleic acid comprising or consisting essentially or consisting of a nucleotide sequence encoding a fusion protein as above defined.

[0091] A preferred isolated nucleic acid according to the invention comprises or consists essentially or consists of a nucleotide sequence selected from SEQ ID NO. 14 (encoding the fusion protein of SEQ ID NO. 12) and SEQ ID NO. 15 (encoding the fusion protein of SEQ ID NO. 13).

[0092] Further, the present invention provides an expression vector comprising the isolated nucleic acid as defined above, and optionally further comprising a promoter sequence and a polyadenylation signal sequence, as well as a host cell comprising the above expression vector.

[0093] Recombinant expression vectors for use in the manufacture of proteins or peptides are known and described in the state of the art, therefore the selection and use thereof are within the skills of those of ordinary skill in the art. Such vectors can be prokaryotic or eukaryotic vectors. Preferably, the cell system used for the expression of the expression vector of the invention is selected from prokaryotic systems, for example Escherichia coli host cells.

[0094] The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section, reference is made to the appended drawings, wherein

[0095] - Figure 1 shows the binding curves of a RF interference-reducing protein of the invention to human IgG, IgM, IgA and IgE antibodies, expressed in nanometers per second (nm / s);

[0096] - Figure 2 illustrates a schematic representation of an embodiment of the method of the invention. (A) Unwanted detection of an interfering antigen- specific IgG antibody, bound to a capture moiety, by a labelled secondary antibody recognizing an interfering RF in an indirect IgM immunoassay. (B) Elimination of unwanted detection of the interfering antigenspecific IgG antibody, bound to a capture moiety, by using of a RF interference-reducing protein of the invention, capable of binding to the Fc domain of said IgG antibody;

[0097] - Figure 3 shows luminescence signals detected for anti-EBV IgM antibodies in 10 RF- positive and IgM-negative serum samples containing increasing RF concentrations. Comparison between an anti-EBV IgM immunoassay employing no agent to eliminate RF interference (grey dots) and the method of the invention, employing a RF interferencereducing protein for said purpose (black dots), demonstrates the generation of false positive signals only in the first condition. Concentration values are expressed in International Units over ml (lU / ml), and signal values are expressed in relative light units (RLU);

[0098] - Figure 4 shows the correlation between anti-EBV IgM detection signals obtained for IgM- negative (graph on the left) and IgM-positive (graph on the right) serum samples using MBP- Mrp4-BD vs FkpA-Mrp4-BD in the method of the invention for the elimination of RF interference. The values are expressed in relative light units (RLU).

[0099] - Figure 5 shows the average signal values of EBV IgM-positive (gray bars) and -negative serum samples (black bars) tested with an immunoassay using goat serum to eliminate RF interference vs the method of the invention, employing a RF interference-reducing protein of the invention for said purpose. The values are expressed in relative light units (RLU);

[0100] - Figure 6 illustrates the dynamics of anti-EBV antibody responses over time during primary infection, convalescence, virus carriership and reactivation (Middeldorp, Curr. Top. Microbiol. Immunol., 2015, 391:289-323);

[0101] - Figure 7 shows two graphs reporting the signal values obtained for IgM detection in two seroconversion sera panels taken at different time points during EBV primary infection, using an immunoassay employing goat serum to eliminate RF interference (dotted line) vs the method of the invention, employing a RF interference-reducing protein for said purpose (solid line). Time points are expressed in days, and signal values are expressed in relative light units (RLU).

[0102] Declaration under Art 170 bis, paragraphs 2, 3 and 4, of the Italian Industrial Property Code

[0103] The present invention has been attained in accordance with the provisions established by Article 170-bis, paragraphs 2 and 4 of the Italian Industrial Property Code.

[0104] EXAMPLES

[0105] 1. Cloning and expression of a RF interference-reducing protein according to the invention

[0106] The present inventors employed standard cloning techniques for preparing the vector for the expression of a RF interference-reducing protein according to the invention, comprising the region spanning amino acid residues from 179 to 296 of the Streptococcus pyogenes Mrp4 protein (SEQ ID NO. 3) linked at the N-terminal to a His-tag, hereinafter referred to as Mrp4- BD (SEQ ID NO. 5). Briefly, the synthetic gene encoding for Mrp4-BD, optimized for expression in E. coli, was commissioned to GeneArt (Invitrogen - Thermo Fisher Scientific, Carlsbad, CA) and inserted in pET24 (Merck KGaA, Darmstadt, Germany), between Ndel and Xhol restriction sites. New England Biolabs (NEB, Beverly, Massachusetts) was the supplier for the restriction enzymes used in the cloning steps.

[0107] The expression of the RF interference-reducing protein was obtained by transformation of E. coli strain BL21(DE3) (Merck KGaA, Darmstadt, Germany) with the above defined expression construct. Selected transformants were inoculated from glycerol stock into 50 mL LB broth containing the kanamycin and grown overnight at 37°C. Overnight cultures were then diluted 1:50 in LB medium containing kanamycin, and cells were grown at 37°C. When OD600 reached 0,6, protein expression was induced by adding 1 mM IPTG and cells were harvested by centrifugation 3 hours after induction.

[0108] 2. Cloning and expression of fusion proteins according to the invention

[0109] For their experiments, the present inventors made use of fusion proteins according to the invention, comprising (i) a RF interference-reducing protein comprising the region spanning amino acid residues from 179 to 296 of S. pyogenes Mrp4 protein (SEQ ID NO. 3) linked at the N-terminal to E. coli FkpA protein (SEQ ID NO. 8) through a linker, and comprising an N-terminal His-tag, hereinafter referred to as FkpA-Mrp4-BD, or (ii) the region of S. pyogenes Mrp4 protein as above defined linked at the N-terminal to E. coli maltose binding protein (MBP) (SEQ ID NO. 9) through a linker comprising a His-tag, hereinafter referred to as MBP-Mrp4-BD. In particular, the fusion protein FkpA-Mrp4-BD consists of the amino acid sequence of SEQ ID NO. 12, and the fusion protein MBP-Mrp4-BD consists of the amino acid sequence of SEQ ID NO. 13. Standard cloning techniques were used for preparing the vectors for the expression of the Mrp4 fusion proteins. Briefly, the synthetic genes encoding for FkpA-Mrp4-BD and MBP-Mrp4-BD, optimized for expression in E. coli, were commissioned to GeneArt (Invitrogen - Thermo Fisher Scientific, Carlsbad, CA). In particular, the synthetic gene encoding for FkpA-Mrp4-BD (SEQ ID NO. 14) was cloned between Ndel and Xhol restriction sites in pET24 (Merck KGaA, Darmstadt, Germany). Similarly, the mutant C-terminal region of the E. coli MBP gene (E385A, K388A, D389A, R393N; SEQ ID NO. 11) described by Center et al. (Center, R.J. et al, 1998. Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein. Protein Sci. 7, 1612-1619), flanked by Notl and Xhol restriction sites, was cloned between Ncol and PstI restriction sites in pMAL-c2X, replacing the wild type C-terminal of MBP and the poly linker. In this vector, the S. pyogenes Mrp4 synthetic gene (SEQ ID NO. 7) was cloned between Notl and Xhol restriction sites to obtain the MBP-Mrp4-BD encoding construct (SEQ ID NO. 15).

[0110] New England Biolabs (NEB, Beverly, Massachusetts) was the supplier for the restriction enzymes used in the cloning steps.

[0111] The expression of the fusion proteins of the invention was obtained by transformation of E. coli strain BL21(DE3) (Merck KGaA, Darmstadt, Germany) with the above defined expression constructs. Selected transformants were inoculated from glycerol stock into 50 mL LB broth containing the appropriate selective antibiotic, and grown overnight at 37°C. Overnight cultures were then diluted 1:50 in LB medium containing the selective antibiotic and grown at 37°C. When OD600 reached 0.6, protein expression was induced by adding 1 mM IPTG and cells were harvested by centrifugation 3 hours after induction.

[0112] 3. Purification of the proteins of the invention

[0113] Cell paste containing the FkpA-Mrp4-BP fusion protein was sonicated and centrifuged, and the protein was purified by Immobilized-Metal Affinity Chromatography (IMAC) using a HisTrap excel column (Cytiva, 17371205). Unbound proteins were eliminated by flowing phosphate buffer through the IMAC column. Bound FkpA-Mrp4-BP fusion protein was eluted by flowing an imidazole solution through the IMAC column (phosphate 50 mM, NaCl 500 mM, Imidazole 500 mM).

[0114] The chromatographic fractions were analyzed by SDS-PAGE and the most concentrated eluted fractions were pooled together. The resulting IMAC pool was then subjected to a gel filtration chromatographic step (GFC) by means of a HiLoad 26 / 600 Superdex 200 prep grade column (Cytiva, 28989336) that was previously equilibrated in the storage buffer (phosphate 50 mM, NaCl 150 mM, pH 7.5). The eluted fractions were analyzed by SDS- PAGE and those containing the FkpA-Mrp4-BP fusion protein were pooled together. The concentration of the purified FkpA-Mrp4-BP fusion protein was determined spectrophotometrically.

[0115] The same procedure was performed to purify the Mrp4-BD and MBP-Mrp4-BD proteins of the invention.

[0116] 4. Assessment of the antibody binding selectivity of a RF interference-reducing protein of the invention

[0117] The present inventors carried out in vitro tests aimed at the evaluation of the binding selectivity of a RF interference-reducing protein towards different classes of immunoglobulins. Specifically, Bio-Layer Interferometry (BLI, Octet® - Sartorius) was employed using the biotinylated fusion protein FkpA-Mrp4-BD, immobilized on streptavidin-coated sensors, and purified human immunoglobulins of the G, M, A and E classes. In particular, IgGs were purchased from Merck (cod 15154), IgAs from Rockland (cod. 009-0106), IgEs from Invitrogen (cod. DIA HE1-0), and IgMs were recombinantly produced in-house from CHO cell line.

[0118] Immunoglobulins were assayed in Octet 10X Kinetic Buffer (Sartorius) at a concentration of 5 nM, with the only exception of IgMs which were tested at 2.5 nM. Association and dissociation were monitored for 10 minutes. Between measurements of different classes of antibodies, the biosensor surfaces were regenerated by exposure to 10 mM Glycine at pH 3 for 30 seconds, followed by Neutralization Buffer for 5 seconds, for a total of 5 regeneration cycles.

[0119] All measurements were corrected for baseline drift by subtracting a control well containing Running Buffer only. Data were analyzed using a 1: 1 interaction model on the Octet Analysis Studio 13.0 Software. Curves showing no response (< 0.1 nm) were not fitted with the software. Figure 1 shows the binding curves of the RF interference-reducing protein of the invention towards human immunoglobulins of the G, A, E ad M classes, expressed in distance over time (nm / second). Of particular note, said protein of the invention was able to efficiently and selectively bind only to IgG antibodies, as indicated by the kinetic and affinity constants described in Table 1 below, demonstrating a comparable binding dynamic to a purified mouse monoclonal antibody against the Fc domain of human IgGs (B68). Signal peak was registered at 600 s and equalled 0.201 nm, suggesting a pronounced interaction between the RF interference-reducing protein according to the invention and IgGs. The above data collectively support the use of Mrp4 or a portion thereof for reducing / eliminating RF interference in an immunoassay context for the detection of IgM, IgA or IgE antibodies.

[0120] Table 1 Kinetic binding constants and affinity constants of RF interference-reducing protein FkpA-Mrp4-BD, or anti-human IgG monoclonal antibody B68, towards human immunoglobulins of the G, A, E and M classes.

[0121] 5. Evaluation of the IgG-blocking effects of a RF interference-reducing protein of the invention

[0122] The present inventors carried out in vitro experiments in an immunoassay context, aimed at the assessment of the ability of a RF interference-reducing protein of the invention to act as an IgG-blocking agent. Particularly, an immunoassay for anti-EBV IgGs was employed to evaluate the capability of the protein of the invention to bind IgGs and inhibit the detection of these antibodies in a EBV IgG-positive serum sample. In these experiments, the assayed sera did not contain any interfering RF. Briefly, the inventors set up a chemiluminescent immunoassay using a RF interference-reducing protein according to the invention, based on an indirect high-throughput format suitable to be performed on the LIAISON® XL platform, a fully automated chemiluminescence analyzer.

[0123] In these experiments, the inventors employed both the Mrp4-BD protein and the FkpA- Mrp4-BD fusion protein as described in Paragraphs 1 and 2 of the Example section, respectively. For comparison, the purified anti-human IgG mouse monoclonal antibody B68, having the same antigen specificity of the proteins of the invention, was used. The sample was initially incubated with the assay diluent, containing said proteins or monoclonal antibody and magnetic particles coated with EBV viral capsid antigen (capture moiety), in which anti-EBV IgG antibodies present in the biological fluid sample bound to the capture moiety, resulting in the formation of immune complexes on magnetic particles. Subsequently, Mrp4-BD, FkpA-Mrp4-BD, or the monoclonal antibody, bound to the Fc domain of captured IgG antibodies. Unbound reagents were then removed with a wash cycle. In a second incubation step, the detection antibody was added, followed by removal of unbound material by means of a wash cycle. Afterwards, the starter reagents were added and a flash chemiluminescence reaction was thus induced. The light signal was measured by a photomultiplier as relative light units (RLU) and is indicative of IgG antibodies present in the sample that were not bound by the RF interference-reducing protein according to the invention.

[0124] Assay protocol

[0125] A total of 5 pl of sample were incubated for 13 minutes with 475 pl of assay diluent and 20 pl of magnetic particles coated with EBV viral capsid antigen (1stincubation time). Unbound reagents were removed with a wash cycle. Afterwards, 200 pl of detection antibody were added and incubated for 10 minutes (2ndincubation time). Unbound material was then removed with a wash cycle. Finally, the starter reagents were added, and the emitted light was measured.

[0126] Reagents and formulations A. Solid phase

[0127] Magnetic particles coated with VC A pl8 synthetic peptide (Van Grunsven et al. J. Infect. Dis. 1994, 170(1): 13-19) were diluted at a final concentration of 0.375% (w / v) in 8 g / L NaCl, 0.2 g / L KC1, 1.44 g / L Na2HPO4*H2O, 0.24 g / L KH2PO4, 1 g / L BSA, 1 g / L NaN3 pH 7.4.

[0128] B. Assay diluent

[0129] Assay diluent was composed as follows: fusion protein FkpA-Mrp4-BD (SEQ ID NO. 12), or anti-human IgG mouse monoclonal antibody B68, dissolved at 240 pg / ml in 1.6 g / L Na2HPO4, 1.2 g / L KH2PO4, 6.4 g / L NaCl, 0.2 KC1, 0.9 g / L EDTANa2 + 32 g / L BSA, 2 g / L Tween 20, 2 ml / L Proclin® 300, pH 7. Protein Mrp4-BD (SEQ ID NO. 5) was dissolved in the same solution at 80 pg / ml, thus being equimolar to FkpA-Mrp4-BD.

[0130] C. Detection antibodies

[0131] For detection of IgG antibodies, a mouse monoclonal antibody to human IgG conjugated to an isoluminol derivative, in phosphate buffer pH 7.4, was employed.

[0132] Assay results

[0133] Serial dilutions of an EBV IgG-positive serum sample were tested using the high throughput assay protocol and the related conditions described above. Surprisingly, both RF interference-reducing proteins Mrp4-BD and FkpA-Mrp4-BD provided a strong inhibition of IgG detection, comparable to that exhibited by the anti-human IgG monoclonal antibody, achieving > 90% inhibition at 240 pg / ml, compared to absence of IgG blocking (Table 2 below).

[0134] Table 2 Inhibition of EBV IgG detection by the RF interference-reducing proteins Mrp4-BD and FkpA-Mrp4-BD, and monoclonal antibody B68 in serial dilutions of an EBV IgG positive sample.

[0135] 6. Evaluation of the RF interference-reducing effects

[0136] The present inventors carried out a set of experiments using an in vitro method, aimed at the evaluation of the RF interference-reducing effects of a RF interference-reducing protein according to the invention, as exemplified in Figure 2. Particularly, the inventors employed the indirect chemiluminescence immunoassay as defined above, for the detection of IgM antibodies to EBV in human serum samples, in a high-throughput format suitable to be performed on the LIAISON® XL platform. In the following, experiments relating to different embodiments of the method of the invention are described.

[0137] 6.1 First Embodiment of the method of the invention

[0138] In a first embodiment, the method of the invention employed the RF interference-reducing protein FkpA-Mrp4-BD in a chemiluminescence immunoassay for the detection of IgM antibodies against EBV. Specifically, the assayed samples contained antigen- specific IgMs, interfering IgGs specific to the same antigen and RF. Particularly, three experiments were carried out in parallel: in the presence of either the RF interference-reducing protein FkpA- Mrp4-BD, the anti-human IgG monoclonal antibody B68, or a commercial anti-human IgG goat polyclonal antibody in the sample diluent. Following sample incubation with said assay diluent and magnetic particles coated with the EBV viral capsid antigen (capture moiety), both anti-EBV IgM and anti-EBV IgG antibodies present in the biological fluid sample were bound to the capture moiety, resulting in the formation of antigen- specific IgM and IgG immune complexes on magnetic particles. In the presence of FkpA-Mrp4-BD, said protein bound to the Fc domain of captured IgG antibodies, thereby blocking RF binding to said IgG immune complexes. Same mechanism can be hypothesized in the presence of the anti-human IgG monoclonal or polyclonal antibodies. Unbound reagents were then removed with a wash cycle. In a second incubation step, the detection antibody was added, followed by removal of unbound material by means of a wash cycle. Afterwards, the starter reagents were added and a flash chemiluminescence reaction was thus induced. The light signal was measured by a photomultiplier as relative light units (RFU). These RFU values are indicative of the presence of IgM antibodies in the sample or antigen- specific IgG antibodies bound by RF and detected by the detection antibody, causing a false positive result.

[0139] Assay protocol

[0140] A total of 5 pl of sample were incubated for 13 minutes with 475 pl of assay diluent and 20 pl of magnetic particles coated with EBV viral capsid antigen (1stincubation time). Unbound reagents were removed with a wash cycle. Afterwards, 200 pl of detection antibody were added and incubated for 10 minutes (2ndincubation time). Unbound material was then removed with a wash cycle. Finally, the starter reagents were added, and the emitted light was measured.

[0141] Reagents and formulations

[0142] A. Solid phase

[0143] Magnetic particles coated with VC A pl8 synthetic peptide (Van Grunsven et al. J. Infect. Dis. 1994, 170(1): 13-19) were diluted at a final concentration of 0.375% (w / v) in 8 g / E NaCl, 0.2 g / E KC1, 1.44 g / L Na2HPO4*H2O, 0.24 g / L KH2PO4, 1 g / L BSA, 1 g / L NaN3 pH 7.4.

[0144] B. Assay diluent

[0145] Assay diluent for the EBV IgM immunoassay was composed as follows: FkpA-Mrp4-BD fusion protein, dissolved at 240 pg / ml in 1.6 g / L Na2HPO4, 1.2 g / L KH2PO4, 6.4 g / L NaCl, 0.2 KC1, 0.9 g / L EDTANa2 + 32 g / L BSA, 2 g / L Tween 20, 2 ml / L Proclin® 300, pH 7.

[0146] For comparative purposes, an anti-human IgG mouse monoclonal antibody (B68) or an antihuman IgG goat polyclonal antibody (Jackson ImmunoResearch Laboratories Inc., Cat. No. 109-005-088) was employed in the assay diluent in place of the reference RF interferencereducing protein of the invention.

[0147] C. Detection antibodies

[0148] For detection of IgM antibodies, a mouse monoclonal antibody to human IgM conjugated to an isoluminol derivative, in phosphate buffer pH 7.4, was employed.

[0149] Assay results

[0150] Comparative analysis with an anti-human IgG monoclonal antibody

[0151] A panel of 233 serum samples, classified according to a commercial EBV IgM assay employing goat serum as IgG blocking agent (LIAISON® EBV IgM - Diasorin 310500), was tested in an immunoassay for the detection of IgM antibodies against EBV according to the protocol above described. Samples were purchased from the following suppliers: Etablissement Francais du Sang (EFS) Centre-Atlantique, collection August 2017 (blood donors) and Biomex. Based on data collected from negative and positive samples, elaborated through ROC (Receiver Operating Characteristics) analysis to achieve the highest overall agreement (positive / negative classification) with the commercial EBV IgM immunoassay, cut off values were set as follows: 110,000 RLU for testing in the presence of the FkpA- Mrp4-BD fusion protein of the invention and 166,915 RLU for testing using an anti-human IgG mouse monoclonal antibody.

[0152] As shown in Table 3 below, among samples classified as negative with the commercial anti- EBV IgMs immunoassay, six were identified as false positive when assessed using the antihuman IgG mouse monoclonal antibody as IgG blocking agent and RF interference suppressor. Further assessments using the LIAISON® EBV IgG assay (Diasorin, 310510) and the Abcam’s Anti-Rheumatoid Factor IgM ELISA kit (Cat nr. abl78653) demonstrated the presence of antigen- specific IgGs and RF in said samples, indicative of strong interfering effects in IgM detection. Remarkably, use of the RF interference-reducing protein FkpA- Mrp4-BD of the invention allowed to correctly classify said samples as negative for EBV IgMs, thereby exhibiting a surprisingly effective inhibition of RF interference by said fusion protein.

[0153] Table 3 EBV IgM detection in six false positive samples, tested using an anti-human IgG mouse monoclonal antibody (B68) or the RF interference-reducing protein FkpA-Mrp4-BD of the invention, and related EBV IgG and RF concentrations.

[0154] Comparative analysis with an anti-human IgG polyclonal antibody

[0155] The present inventors further compared the performance of FkpA-Mrp4-BD as an RF interference-reducing protein with that of a commercial anti-human IgG goat polyclonal antibody, in an EBV IgM immunoassay using the samples and the protocol as above described. As a result of the test, a significant reduction of false-positive IgM signal was observed by using the FkpA-Mrp4-BD fusion protein averaging over 90% across all tested samples (measured in Relative Light Units, RLU). In contrast, the use of the anti-human IgG polyclonal antibody as RF interference-reducing agent resulted in a substantially lower reduction in the false-positive IgM signal, specifically less than 60%.

[0156] These data, presented in Table 4, confirm the superior capability of the FkpA-Mrp4-BD fusion protein to substantially reduce RF interference, thereby achieving enhanced immunoassay specificity and reliability.

[0157] Of note, these findings are totally unexpected. Given the ability of a polyclonal antibody to recognize multiple epitopes on the target antigen (i.e., human IgGs), such reagent was expected to exhibit a RF interference-reducing effect comparable to, if not greater than, that of the FkpA-Mrp4-BD fusion protein, by efficiently blocking RF binding to the interfering IgGs present in the samples.

[0158] Table 4 EBV IgM detection in four false-positive samples tested using the following RF interference-reducing agents: an anti-human IgG mouse monoclonal antibody (B68), an antihuman IgG goat polyclonal antibody (Jackson ImmunoResearch Laboratories Inc., Cat. No. 109-005-088) and the RF interference-reducing protein FkpA-Mrp4-BD of the invention. Average value

[0159] Elimination ofRF interference

[0160] A set of 10 serum samples, positive for RF and anti-EBV IgGs at different concentrations and negative for anti-EBV IgMs, according to the reference commercial assay (Diasorin - 310500), was assayed for the presence of anti-EBV IgM antibodies using the method of the invention employing FkpA-Mrp4-BD as the RF interference-reducing agent. In parallel, said samples were tested in the absence of an IgG-blocking agent for reduction of RF interference. Samples, purchased from Biomex, were also assessed for RF titer using Abcam’s Anti-Rheumatoid Factor IgM ELISA kit (Cat nr. abl78653), and for EBV IgG antibody concentration employing the commercial LIAISON® VCA IgG assay (310510 - Diasorin), as shown in Table 5 below.

[0161] Table 5 RF and EBV IgG concentrations of 10 serum samples, assessed with Abeam’ s kit (Cat. Nr. Abl78653) and Diasorin’s IgG assay (310510), respectively.

[0162] As shown in Figure 3, detection signals for IgM antibodies measured in the aforementioned serum samples in absence of an IgG-blocking agent were found to increase in correlation with RF concentrations, expressed in lU / ml, thereby resulting in false positivity. Surprisingly, employment of the RF interference-reducing protein of the invention completely abolished said signal increase. Overall, these data demonstrate a marked RF interference-reducing effect of the method of the invention by blocking undesired antigenspecific IgG detection.

[0163] 6.2 Second Embodiment of the method of the invention

[0164] The present inventors carried out further experiments using the RF interference-reducing protein MBP-Mrp4-BD (designated as SEQ ID NO. 13), in a chemiluminescence immunoassay for the detection of IgM antibodies against EBV. Briefly, the same set of 233 serum samples, purchased from Etablissement Francais du Sang (EFS) Centre-Atlantique, collection August 2017 (blood donors) and Biomex, was assayed using the protocol and reagents described in Paragraph 6.1 of the Example section, in the presence of the MBP- Mrp4-BD fusion protein dissolved at 240 pg / ml in assay diluent. The light signal was measured by a photomultiplier as relative light units (RLU) and is indicative of IgM antibodies present in the sample or antigen-specific IgG antibodies bound by RF and detected by the detection antibody, causing a false positive result.

[0165] Assay results

[0166] Luminescence signals obtained from these experiments making use of the MBP-Mrp4-BD fusion protein, were compared to the results illustrated in Example 6.1 above, relating to the FkpA-Mrp4-BD protein. Of note, Figure 4 shows a strong linear correlation for both positive and negative serum samples between antibody detection signals obtained using MBP-Mrp4- BD and FkpA-Mrp4-BD, suggesting that assay performance of the RF interference-reducing proteins herewith disclosed is independent of the selected fusion partner.

[0167] 6.3 Third Embodiment of the method of the invention

[0168] In a third embodiment, the method of the invention employed the RF interference-reducing protein Mrp4-BD (SEQ ID NO. 5) in a chemiluminescence immunoassay for the detection of IgM antibodies against EBV. The experiments were conducted according to the protocol described above in Paragraph 6.1 of the Example section. Briefly, an EBV IgM negative sample containing both RF and interfering IgGs was assayed in the presence of the Mrp4- BD protein dissolved at 80 pg / ml in assay diluent. The light signal was measured by a 5 photomultiplier as relative light units (RLU). In this experiment, an assay signal is indicative of the binding of RF to antigen-specific interfering IgG antibodies leading to a false positive result.

[0169] As shown in Table 6 below, the use of Mrp4-BD in the method of the invention enabled the 0 correct classification of the assayed serum sample (EBM-01-I02) as EBV IgM-negative, thereby preventing the RF-mediated undesired detection of interfering IgGs present in the sample. Moreover, it is noted that a similar result was obtained when the FkpA-Mrp4-BD fusion protein was employed on the same sample (see Table 3 above). Accordingly, these findings demonstrate that suppression of RF interference by the proteins of the invention 5 relies solely on the unexpected properties of Mrp4, irrespective of the presence or identity of a fusion partner.

[0170] Table 6 EBV IgM detection in an IgM negative sample, tested using the following RF interference-reducing agents: an anti-human IgG mouse monoclonal antibody (B68) or the 0 FkpA-Mrp4-BD and Mrp4-BD proteins of the invention. The concentrations of EBV IgG antibodies and RF are also reported.

[0171] Assay results 5 Comparative analysis with goat serum and RF blockers

[0172] The present inventors further conducted a comparative evaluation of the performance of the Mrp4 proteins of the invention versus various RF interference-reducing agents known in the

[0173] 5 art. Specifically, additional immunoassay experiments were carried out on a panel of contrived and human serum samples that exhibit different combinations of EBV IgM positivity, interfering IgGs positivity and RF presence. The assay was carried out by employing the following RF interference-reducing agents: goat serum (LIAISON® EBV IgM - Diasorin 310500), two commercially available RF blockers dissolved at 200 pg / ml in assay diluent (Molecular Depot, Cat. A2010009, and Meridian, Cat. BN1200) and the Mrp4- BD protein dissolved at 80 pg / ml in assay diluent. The light signal was measured as described above, and it correlates either to the detection of EBV IgM antibodies present in the sample or to the RF-mediated detection of interfering IgGs, causing a false positive result.

[0174] 15

[0175] As a first experiment, the effects of the aforementioned RF interference-reducing agents on the signal intensity of detected EBV IgMs was assessed on a panel of six EBV IgM-positive contrived serum samples known to be RF-negative. As illustrated in Table 7 below, the light signal intensity associated with true EBV IgM positivity was maintained almost unaltered

[0176] 20 by employing Mrp4-BD in comparison with a control assay carried out without the addition of any IgG blocking. Conversely, a significant decrease in EBV IgM signal intensity was observed in the presence of goat serum (up to 69%), RF blocker (up to 93%) and K-block (up to 99%).

[0177] 25 Table 7 EBV IgM detection in EBV IgM positive and RF negative contrived serum samples, tested in the presence of the following RF interference-reducing agents: goat serum, two commercial RF suppressors (RF blocker and K-block), or the Mrp4-BD protein of the invention. Also shown are the concentrations of EBV IgM antibodies.

[0178] Overall, these results show that, differently from Mrp4-BD, all the RF suppressors employed in the assay caused a substantial decrease in the antibody detection signal, thereby leading to a dramatic reduction of the assay sensitivity.

[0179] To further validate the above results, the intensity of the detection signal for EBV IgM antibodies was assessed in the EBM-01-I02 serum sample. As aforesaid, this sample is independently known as EBV IgM negative, EBV IgG positive and RF positive. Remarkably, the use of RF suppressors other than Mrp4-BD in the immunoassay resulted in a detection signal intensity that is close to, or even higher than, that observed for confirmed IgM positive samples (Table 8 below). These data demonstrate that, unlike the Mrp4-BD protein of the invention, the commercially available agents for suppression of RF interference in immunoassays do not allow for a reliable discrimination between true IgM- positive and IgM-negative detection signals in serum samples.

[0180] Table 8 EBV IgM detection in the EBM-01-I02 sample, tested using the following RF interference-reducing agents: goat serum, two commercial RF suppressors (RF blocker and K-block) or the Mrp4-BD protein of the invention. The difference with the lowest IgM- positive RLU value as shown in Table 7 is also reported.

[0181] 7. Evaluation of assay performance using a RF interference-reducing protein of the invention

[0182] The present inventors carried out further experiments in an immunoassay context aimed at the evaluation of assay performance in the presence of a RF interference-reducing protein of the invention. Specifically, the indirect chemiluminescence immunoassay protocol described in Paragraph 6.1 of the Example section was employed for the detection of IgM antibodies to EBV in human serum samples, in a high-throughput format suitable to be performed on the LIAISON® XL platform. In the following, different experiments using a RF interference-reducing protein of the invention are described.

[0183] 7.1 Comparative analysis with goat serum

[0184] To evaluate assay performance, the inventors analysed luminescence signals obtained for anti-EBV IgMs in the aforementioned panel of 233 serum samples using the method of the invention outlined above, in the presence of the RF interference-reducing protein FkpA- Mrp4-BD. Remarkably, employment of said protein provided a difference between average RLU values obtained for anti-EBV IgM positive and negative samples equal to 790,343. The difference as measured was significantly increased compared to the difference obtained with the commercial immunoassay using goat serum as IgG-blocking agent (275,725 RLU, shown in Table 9 below). Collectively, these data show an improved assay performance with the method of the invention because of the enhanced discrimination between positive and negative results thereby achieved (Figure 5).

[0185] Table 9 Comparison of average RLU values for positive and negative samples assayed for the presence of anti-EBV IgM with an immunoassay using goat serum as IgG blocking agent (LIAISON® EBV IgM assay, 310500) or the protocol employing the RF interference- reducing protein FkpA-Mrp4-BD of the invention.

[0186] 7.2 Detection ofanti-EBV IgM antibodies in seroconversion panels

[0187] Further experiments were carried out to assess anti-EBV IgM detection using the method of the invention, as above outlined, in two seroconversion panels purchased from Biomex (Catalog No: SCP-EBV-001 and SCP-EBV-002), to investigate assay performance on specimens from patients undergoing antibody isotype conversion during the natural course of EBV infection. Specifically, as shown in Figure 6, while the acute stage of infection is mainly characterized by elevated titers of IgM and IgG antibodies against the viral capsid antigen (VCA), which are widely adopted as biomarkers for the primary EBV infection, the later convalescence phase is defined by a significant increase in IgGs towards the EBV- associated nuclear antigen (EBNA). For comparative purposes, the anti-human IgG mouse monoclonal antibody B68 was employed in the assay diluent in place of the RF interferencereducing protein FkpA-Mrp4-BD. Based on data collected from negative and positive samples, elaborated through ROC (Receiver Operating Characteristics) analysis to achieve the highest overall agreement (positive / negative classification) with the commercial EBV IgM immunoassay, cut off values were set as follows: 110,000 RLU for testing in the presence of the FkpA-Mrp4-BD fusion protein of the invention and 166,915 RLU for testing using an anti-human IgG mouse monoclonal antibody. Of particular note, Figure 7 shows that the method of the invention resulted in significantly higher antibody detection signals for every seroconversion time point, expressed in days, compared to the immunoassay using goat serum. Samples from the first seroconversion panel corresponding to acute primary infection time points, as indicated by the absence of EBNA IgGs, were unclassified for IgM positivity by the assay employing goat serum, and mistakenly classified as IgM negative using the monoclonal antibody.

[0188] Advantageously, these samples were unequivocally and correctly classified as IgM positive using the method of the invention, suggesting enhanced IgM sensitivity by employing the RF interference-reducing protein FkpA-Mrp4-BD. Furthermore, correct classification of IgM-negative samples from the second seroconversion panel corresponding to time points of the convalescence infection phase, as indicated by EBNA IgG positivity, was obtained with the method of the invention, thereby outperforming the assay using goat serum, which resulted in indeterminate outcomes. In addition, the protocol employing the anti-human IgG monoclonal antibody further generated two false positive samples. Collectively, these results indicate a significantly increased assay sensitivity achieved by using the RF interferencereducing protein of the invention (Table 10 below).

[0189] Table 10 Detection of anti-EBV IgM antibodies in seroconversion panels, tested using an immunoassay employing goat serum (Diasorin, 310500), an anti-human IgG monoclonal antibody or the method of the invention employing the RF interference-reducing protein FkpA-Mrp4-BD. Also shown is EBNA IgG positivity, according to the LIAISON® EBNA IgG kit (310520).

Claims

CLAIMS1. An in vitro method of reducing and / or eliminating unwanted detection of an IgG antibody in an immunoassay performed on a biological fluid sample containing rheumatoid factor (RF), the method comprising contacting said biological fluid sample with a RF interference-reducing protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2 and 3, said RF interference-reducing protein being capable of binding to the Fc domain of IgG antibodies.

2. An in vitro method for the detection of antibodies specific to an antigen in a biological fluid sample, said antibodies being of one or more immunoglobulin classes other than IgG, said biological fluid sample containing interfering elements comprising both IgG antibodies specific to the same antigen as the antibodies to be detected and rheumatoid factor (RF), the method comprising the steps of: i) contacting the biological fluid sample with a capture moiety comprising said antigen, whereby immunocomplexes between the capture moiety and the antigen- specific antibodies present in the biological fluid sample are formed, ii) contacting said immunocomplexes with a RF interference-reducing protein as defined in claim 1 and allowing binding of the RF interference-reducing protein to IgG immunocomplexes, thereby blocking the binding of rheumatoid factor to said IgG immunocomplexes, and iii) detecting the immunocomplexes formed by the antibodies of the one or more immunoglobulin classes other than IgG.

3. The in vitro method according to claim 2, wherein the antibodies of the one or more immunoglobulin classes other than IgG are selected from the group consisting of IgM, IgA, IgE, and any combination thereof4. The in vitro method according to any of claims 1 to 3, wherein the RF interferencereducing protein comprises or consists of the amino acid sequence of SEQ ID NO. 4.

5. The in vitro method according to any of claims 1 to 4, wherein the RF interference-reducing protein is linked to a fusion partner, optionally through a peptide linker, the fusion partner being preferably selected from the group consisting of FkpA, SlyD, trigger factor, maltose binding protein (MBP), thioredoxin, glutathione-S-transferase, Fc-portion of IgG, and any combination thereof.

6. The in vitro method according to any of claims 1 to 5, wherein the interfering rheumatoid factor (RF) in the biological fluid sample is a IgM, IgA, or IgE RF isotype.

7. The in vitro method according to any of claims 1 to 6, wherein the biological fluid sample is selected from the group consisting of whole blood, plasma, serum or urine.

8. The in vitro method according to any of claims 2 to 7, wherein the capture moiety is immobilized on a solid support.

9. The in vitro method according to any of claims 2 to 8, wherein step (iii) is carried out by means of a detection agent conjugated to one or more detectable labels, the one or more detectable labels being preferably selected from the group consisting of enzymatic labels, isotopic labels, chemiluminescent labels, fluorescent labels, dyes, alkaline phosphatase (AP) labels, biotin labels, and any combination thereof.

10. The in vitro method according to claim 9, wherein the detection agent comprises an anti-IgM, anti-IgA or anti-IgE antibody.

11. The in vitro method according to any of claims 1 to 10, wherein the specific antigen is from a microorganism, said microorganism being preferably selected from the group consisting of Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Mumps virus (MuV), Measles virus (MV), Mycoplasma pneumoniae, and any combination thereof.

12. A kit for the detection of antibodies specific to an antigen in a biological fluid sample, said antibodies being of one or more immunoglobulin classes other than IgG, said biological fluid sample containing interfering elements comprising both IgG antibodies specific to thesame antigen as the antibodies to be detected and rheumatoid factor (RF), the kit comprising:- a capture moiety comprising said antigen specific to the antibodies to be detected,- a RF interference-reducing protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2 and 3, said RF interference-reducing protein being capable of binding to the Fc domain of IgG antibodies, and- means for detecting the antigen- specific antibodies of one or more immunoglobulin classes other than IgG.

13. The kit according to claim 12, wherein the antibodies of the one or more immunoglobulin classes other than IgG are selected from the group consisting of IgM, IgA, IgE, and any combination thereof.

14. The kit according to claim 12 or 13, wherein the RF interference-reducing protein comprises or consists of the amino acid sequence of SEQ ID NO. 4.

15. The kit according to any of claims 12 to 14, wherein the capture moiety is immobilized on a solid support, the solid support being preferably selected from the group consisting of latex, polystyrene, silica, chelating sepharose, magnetic beads, membranes, strips and chips.

16. The kit according to any of claims 12 to 15, wherein the detection means comprises an anti-IgM, anti-IgA or anti-IgE antibody.

17. Use of a kit as defined in any of claims 12 to 16 for carrying out an in vitro method according to any of claims 1 to 11.

18. Use of a fusion protein in an in vitro method according to any of claims 1 to 11, wherein the fusion protein comprises a RF interference-reducing protein linked to a fusion partner, said RF interference-reducing protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 1, 2 and 3, and being capable of binding to the Fc domain of IgG antibodies.

19. The use according to claim 18, wherein the RF interference-reducing protein is linked to the fusion partner through a peptide linker.

20. The use according to claim 18 or 19, wherein the fusion partner is selected from the group consisting of FkpA, SlyD, trigger factor, maltose binding protein (MBP), thioredoxin, glutathione-S-transferase, Fc-portion of IgG, and any combination thereof.

Images