Synthetic peptides for immunomolecular assays

Abstract

The invention relates to the field of immunoassays, in particular to the field of immunomolecular assays. The present invention provides an internal control for an immunomolecular assay, such as a dual recognition immunoassay or a proximity ligation assay. The internal control comprises a synthetic peptide having the following formula (I): R1-Z-R2 (I) wherein:-Z is a spacer consisting of 1 to 3 non-natural amino acids; -R1 and R2 are two different peptide moieties wherein the first peptide moiety of R1 or R2 comprises a Lys tag and the second peptide moiety comprises a His tag.

Description

Technical Field

[0001] The present invention relates to the field of immunoassays, and more particularly to the field of immuno-molecule assays. The present invention provides an internal control for immuno-molecule assays (such as dual-recognition immunoassays or proximity ligation assays). Background Art

[0002] There has been a continuous need to develop simple, minimally invasive tools for disease screening, diagnosis, and prevention. Proximity ligation assay (PLA) and the recently emerged proximity extension assay (PEA TM , Olink) may be suitable options to meet these needs. PEA TM and PLA belong to homogeneous dual-recognition immunoassays (DRIs), which have been shown to be highly sensitive, specific, and convenient to operate in detecting or quantifying one or more bioanalytes [1][2]. Proximity assays rely on the proximity probing principle, that is, the analyte is detected by the binding of at least two specific probes, and when these probes are close to each other after binding to the analyte, a nucleic acid-based signal can be generated, and this signal can be quantified [3]. Due to the characteristics of these assay methods, the native conformation of the antigen to be detected is retained.

[0003] In this specification, the two expressions "Dual-recognition immunoassay" and "Dual-Recognition Immunoassay" and their corresponding abbreviations "PEA" and "DRI" can be regarded as synonyms. One of the main limitations of the dual-recognition immunoassay embodiments described in the literature is the long assay time (excluding qPCR, more than 90 minutes), because multiple dilution and incubation steps are required in different buffers.

[0004] It has been previously demonstrated that it is possible to significantly shorten the result time of dual-recognition immunoassays to better meet the requirements of clinical diagnosis (unpublished results). The model analyte selected for this validation is procalcitonin, which is a biomarker, and shortening its assay time helps better manage patients. Procalcitonin is a small polypeptide, the blood level of which will increase, and it can be routinely and specifically measured early, especially in the case of bacterial infections. Procalcitonin helps to determine the severity of the infection, monitor the development of the infection and the response to treatment, and adjust the treatment plan. Generally, it is considered that when the value of procalcitonin is below 0.25 μg / L, bacterial infection can be excluded; a plasma dose higher than 0.5 μg / L indicates local infection, and a dose higher than 2.0 μg / L even indicates systemic bacterial infection; when the dose reaches 10 μg / L or higher, severe sepsis and / or septic shock can be diagnosed [4-7].

[0005] Compared to other more traditional heterogeneous immunoassay techniques (such as sandwich ELISA), a key advantage of a dual-recognition immunoassay like PEA TM is that it requires neither a solid phase nor washing, making the assay protocol simpler and easier to implement. However, dual-recognition immunoassays still involve multiple reagent addition, dilution, and incubation steps that are prone to human error (when performed manually) or system malfunctions (when automated). In particular, an inadvertent or accidental deviation from the correct protocol can result in false qPCR signals that may then be misinterpreted. To reduce this risk, a fail-safe internal control (IC) was developed. The role of this control is to indicate any failures that occur during the assay process (from sample addition to qPCR) so that the results can be flagged as invalid.

[0006] A fundamental requirement for such a control is that it should always produce the same level of signal (when the dual-recognition immunoassay protocol is correctly executed), regardless of the specific sample being assayed and the concentration of the analyte in the sample.

[0007] In the present disclosure, a synthetic peptide was generated that consists of two artificial non-human sequences separated by a neutral peptide spacer, and its concept as a fail-safe internal control in immuno-molecular assays was demonstrated by using this internal control in a dual-recognition immunoassay for detecting procalcitonin in human patient samples. Summary of the Invention

[0008] A first object of the present disclosure relates to a synthetic peptide having the following formula (I):

[0009] R1-Z-R2 (I)

[0010] Wherein:

[0011] Z is a spacer consisting of 1 to 3 unnatural amino acids;

[0012] R1 and R2 are different peptide moieties, where one of the peptide moieties in R1 or R2 contains a polyhistidine tag (or His tag), and the other peptide moiety contains a polylysine tag (or Lys tag).

[0013] Another object of the present disclosure relates to a composition comprising the synthetic peptide as defined herein, the synthetic peptide being dissolved in water, in the presence of a buffer, or in a biological fluid.

[0014] Another object of the present disclosure relates to a kit for use as an internal control or quantitative calibrator in immuno-molecular assays (such as dual-recognition immunoassays (e.g., PEA TM ), proximity extension or ligation assays, immuno-PCR), the kit comprising at least the following components:

[0015] (i) the synthetic peptides disclosed herein; and

[0016] (ii) at least two proximity probes, each proximity probe comprising an antigen-binding moiety conjugated to a nucleic acid domain,

[0017] wherein the first proximity probe comprises an anti-His tag antibody or an antigen-binding fragment thereof that specifically binds to the His tag, and the second proximity probe comprises an anti-Lys tag antibody or an antigen-binding fragment thereof that specifically binds to the Lys tag.

[0018] In a specific embodiment, the immunoassay is performed using a plasma or blood sample, such as a blood sample obtained from a human subject.

[0019] The present disclosure also relates to the use of the synthetic peptides disclosed herein or a kit comprising the synthetic peptides as an internal control or a quantitative calibrator in an immunoassay (preferably in a dual-recognition immunoassay).

[0020] Another object of the present disclosure is a method for detecting or quantifying an analyte by a dual-recognition immunoassay, the method comprising using the synthetic peptides disclosed herein as an internal control or a quantitative calibrator. DETAILED DESCRIPTION OF THE INVENTION

[0022] Synthetic peptide

[0023] The compounds of the present disclosure are synthetic peptides having the following formula (I):

[0024] R1-Z-R2 (I)

[0025] Wherein:

[0026] Z is a spacer consisting of 1 to 3 unnatural amino acids;

[0027] R1 and R2 are different peptide moieties, wherein one of the peptide moieties in R1 or R2 contains a Lys tag and the other peptide moiety contains a His tag.

[0028] Such synthetic peptides having both a Lys tag and a His tag are bispecific. As used herein, a bispecific compound refers to a compound comprising two different epitopes that can be specifically recognized by two different specific monoclonal antibodies. In the present disclosure, the synthetic peptides can be recognized by a monoclonal antibody that specifically binds to the Lys tag and a monoclonal antibody that specifically binds to the His tag. In particular, there is no substantial steric hindrance to prevent these two antibodies from binding simultaneously to their respective epitopes on the synthetic peptides.

[0029] The peptides of the present disclosure are synthetic peptides, i.e., peptides that do not exist in nature. The Lys tag and His tag are artificial sequences that do not exist in nature.

[0030] The synthetic peptides according to the present specification are conducive to long-term storage. In fact, peptides are usually stored at -20°C. However, it has been demonstrated that the synthetic peptides proposed in the present disclosure are stable for at least 12 weeks (86 days) at 4°C and at least 24 hours at room temperature. Therefore, these synthetic peptides can be used for a long time under common storage conditions and with a relatively low risk of significant degradation.

[0031] As used herein, the term "His tag" refers to a peptide sequence (polyhistidine peptide) containing 4 to 8 histidine residues, which can be specifically recognized by anti-His tag antibodies (such as 18D12

[10] ). A preferred embodiment of the His tag is the 6x histidine tag (MRGSHHHHHH) of SEQ ID NO:1, more specifically the 6x histidine tag (MRGSHHHHHHSVDES) of SEQ ID NO:2.

[0032] As used herein, the term "Lys tag" refers to a peptide sequence (polylysine peptide) containing 4 to 8 lysine residues, which can be specifically recognized by anti-Lys tag antibodies (such as 2G4A12 or 5F12E4

[10] ). A preferred embodiment of the Lys tag is the 6x lysine tag (GKKKKKKSV) of SEQ ID NO:3, more specifically the 6x lysine tag (GKKKKKKSVDESL) of SEQ ID NO:4. In the context of the present specification, the antibodies 2G4A12 and 5F12E4 may also be referred to as 2G4 or 5F12, respectively.

[0033] Spacer Z is used to provide an appropriate spacing between the two epitope tags. The inventors have indeed determined that a length of spacer Z between about 20 and about 30 angstroms is optimal for using the synthetic peptide as an internal control or quantitative calibrator in a dual-recognition immunoassay.

[0034] The spacer mainly consists of 1 to 3 unnatural amino acids. Preferably, the spacer is a dipeptide or tripeptide.

[0035] As used herein, the term "unnatural amino acid" refers to an amino acid that does not exist in natural polypeptide chains. Such unnatural amino acids may exist as secondary metabolites of bacteria, fungi, plants, or marine organisms, or can be obtained by chemical synthesis.

[0036] "Non-natural amino acid" also refers to an amino acid that does not belong to the 20 amino acids required to form all proteins found in humans and most other life forms. These 20 amino acids are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. It should be mentioned that selenocysteine and pyrrolysine are regarded as the 21st and 22nd amino acids respectively, and they are not non-natural amino acids either. After translation, these 22 amino acids can also be further modified by post-translational modification to increase the diversity of protein generation. In a preferred embodiment, the non-natural amino acid has the following formula:

[0037] -NH-(Z')-COO-, where Z' is a straight-chain saturated or unsaturated hydrocarbon chain optionally containing one or more heteroatoms, and the chain contains 6 to 20 non-hydrogen atoms.

[0038] In a preferred embodiment, Z' is a saturated or unsaturated hydrocarbon chain containing one or more heteroatoms, and the heteroatoms are selected from O, N, and S.

[0039] In a specific embodiment, the non-natural amino acid is 8-amino-2,4-dioxaoctanoic acid.

[0040] In a particularly preferred embodiment, Z is a dipeptide or tripeptide of 8-amino-2,4-dioxaoctanoic acid.

[0041] In another embodiment, Z is polyethylene glycol (PEG) of the formula H-(O-CH2-CH2) n -OH, where n is from 1 to 4, preferably n is equal to 2.

[0042] In another embodiment, Z is a peptide spacer having the formula (NH-(Z')-COO) n where n is from 1 to 3, Z' is a straight-chain saturated or unsaturated hydrocarbon chain optionally containing one or more heteroatoms, and the chain contains 6 to 20 non-hydrogen atoms; or is a structure of the formula H-(O-CH2-CH2) n -OH, where n is from 1 to 4, preferably n is equal to 2.

[0043] The N-terminus and C-terminus of the spacer are flanked by peptide moieties of R1 and R2 respectively.

[0044] In addition to the Lys tag or His tag, the peptide moieties of R1 and R2 may further contain 1, 2, 3, 4, or 5 additional amino acids at the N-terminus or C-terminus, provided that these additional amino acids do not respectively affect the simultaneous binding of the anti-His tag antibody and the anti-Lys tag antibody.

[0045] Thus, in one specific embodiment, R1 is a peptide having the following sequence:

[0046] X -10 X -9 X -8 X -7 (X -6 ) n X -5 X -4 X -3 X -2 X -1 , wherein:

[0047] X -10 is optional or is a nonpolar amino acid, preferably methionine;

[0048] X -9 is optional or is a basic amino acid, preferably arginine;

[0049] X -8 is optional or is a nonpolar amino acid, preferably glycine;

[0050] X -7 is optional or is a polar amino acid, preferably serine;

[0051] X -6 is histidine or lysine;

[0052] X -5 is optional or is a polar amino acid, preferably serine;

[0053] X -4 is optional or is a nonpolar amino acid, preferably valine;

[0054] X -3 is optional or is an acidic amino acid, preferably aspartic acid;

[0055] X -2 is optional or is an acidic amino acid, preferably glutamic acid;

[0056] X -1 is optional or is a polar amino acid, preferably serine; and

[0057] n is an integer between 4 and 8, preferably equal to 6.

[0058] In a more specific embodiment, R1 consists mainly of one of the following sequences: MRGSHHHHHH (SEQ ID NO:1) or MRGSHHHHHHSVDES (SEQ ID NO:2).

[0059] In other specific embodiments that can be combined with the foregoing embodiments, R2 is a peptide having the following sequence:

[0060] X +1 (X +2 ) p X +3 X +4 X +5 X +6 X +7 X +8 , wherein:

[0061] X +1 is optional or is a non-polar amino acid, preferably glycine;

[0062] X +2 is lysine or histidine;

[0063] X +3 is optional or is a polar amino acid, preferably serine;

[0064] X +4 is optional or is a non-polar amino acid, preferably valine;

[0065] X +5 is optional or is an acidic amino acid, preferably aspartic acid;

[0066] X +6 is optional or is an acidic amino acid, preferably glutamic acid;

[0067] X +7 is optional or is a polar amino acid, preferably serine;

[0068] X +8 is optional or is a non-polar amino acid, preferably leucine; and

[0069] p is an integer between 4 and 8, preferably equal to 6.

[0070] In a more specific embodiment, R2 consists mainly of one of the following sequences: GKKKKKKSV (SEQ ID NO:3) or GKKKKKKSVDESL (SEQ ID NO:4).

[0071] As used herein, the term "non-polar amino acid" refers to an amino acid in which the variable R group mainly comprises a hydrocarbon (for methionine, also contains a sulfur atom) and has no polarity. In specific embodiments of the R1 and R2 residues, the non-polar amino acids are selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0072] As used herein, the term "polar amino acid" refers to hydrophilic amino acids, including negatively charged, positively charged or uncharged amino acids. All polar amino acids (in an aqueous environment) have an OH or NH2 group and can thus form hydrogen bonds with other suitable groups. In a specific embodiment of the R1 and R2 residues, the polar amino acids are selected from serine, cysteine, threonine, tyrosine, asparagine and glutamine.

[0073] As used herein, the term "basic amino acid" refers to an amino acid having a basic side chain at neutral pH. In a specific embodiment of the R1 and R2 residues, the basic amino acids are selected from arginine, histidine and lysine.

[0074] As used herein, the term "acidic amino acid" refers to an amino acid having an acidic carboxylic acid group on its side chain, thereby rendering it acidic (a proton donor). In a specific embodiment of the R1 and R2 residues, the acidic amino acids are selected from aspartic acid and glutamic acid.

[0075] In a preferred embodiment, the synthetic peptide has the following sequence: MRGSHHHHHH-(N) m -GKKKKKKSV, where N is a non-natural amino acid having a side chain length of about 10 to 30 angstroms, and m is 1, 2 or 3. In a specific embodiment, N is a non-natural amino acid having the following formula:

[0076] -NH-(Z')-COO-, where Z' is a straight-chain saturated or unsaturated hydrocarbon chain optionally containing one or more heteroatoms, and the chain contains 6 to 20 non-hydrogen atoms.

[0077] In a more preferred embodiment, the synthetic peptide has the following sequence: MRGSHHHHHH-(N)m-GKKKKKKSV, where N is 8-amino-2,4-dioxaoctanoic acid, and m is 2 (SEQ ID NO:5) or 3 (SEQ ID NO:6).

[0078] In a preferred embodiment, the synthetic peptide has the following sequence: MRGSHHHHHHSVDES-(N)m-GKKKKKKSVDESL, where N is a non-natural amino acid having a side chain length of about 10 to 30 angstroms, and m is 1, 2 or 3. In a specific embodiment, N is a non-natural amino acid having the following formula:

[0079] -NH-(Z')-COO-, where Z' is a straight-chain saturated or unsaturated hydrocarbon chain optionally containing one or more heteroatoms, and the chain contains 6 to 20 non-hydrogen atoms.

[0080] In another preferred embodiment, the synthetic peptide has the following sequence: MRGSHHHHHHSVDES-(N)m-GKKKKKKSVDESL, where N is 8-amino-2,4-dioxaoctanoic acid and m is 2 (SEQ ID NO:7) or 3 (SEQ ID NO:8).

[0081] The compositions and kits of the present disclosure

[0082] The synthetic peptides of the present disclosure, particularly when used as an internal control or quantitative calibrator in a dual-recognition immunoassay, are included in an aqueous composition, which optionally further comprises a buffer.

[0083] In a preferred embodiment, the synthetic peptide is included in a sterile aqueous composition and is advantageously stored at -20 °C.

[0084] Buffers for use in compositions containing the synthetic peptides of the present disclosure are well known in the art and are, for example, selected from phosphate buffered saline (PBS), HEPES, and Tris-HCl.

[0085] In a specific embodiment, the composition is a concentrated solution for dilution in a reaction solution for performing a dual-recognition immunoassay. The content of the synthetic peptide in the concentrated solution can be two-fold, three-fold, four-fold, five-fold, ten-fold, twenty-fold or more than twenty-fold of its final concentration when used as an internal control in the dual-recognition immunoassay. Generally, the concentration of the synthetic peptide can be between 1 pg / mL and 1 mg / mL.

[0086] The composition may further comprise other compounds, including salts, proteins (such as bovine serum albumin), synthetic polymers (such as dextran or polyethylene glycol), or surfactants.

[0087] When used in conjunction with a pair of proximity probes comprising an anti-Lys tag antibody and an anti-His tag antibody, the composition is particularly useful as an internal control, as described in the following section.

[0088] Accordingly, the present disclosure also relates to a kit for use as an internal control or quantitative calibrator in a dual-recognition immunoassay, the kit comprising at least the following components:

[0089] - The synthetic peptide described in the previous section, preferably a synthetic peptide having the following sequence: MRGSHHHHHH-(N)m-GKKKKKKSV, or a synthetic peptide having the following sequence: MRGSHHHHHHSVDES(N)m-GKKKKKKSVDESL,

[0090] where N is 8-amino-2,4-dioxaoctanoic acid and m is 2 or 3, for example, a synthetic peptide of SEQ ID NO:6 or SEQ ID NO:8;

[0091] - At least two proximity probes, each proximity probe comprising an antigen-binding portion conjugated to a nucleic acid domain, wherein the first proximity probe comprises an anti-His tag antibody or an antigen-binding fragment thereof that specifically binds to the His tag, and the second proximity probe comprises an anti-Lys tag antibody or an antigen-binding fragment thereof that specifically binds to the Lys tag.

[0092] In certain embodiments, the synthetic peptide is selected from the synthetic peptides of SEQ ID NO:7 or SEQ ID NO:8.

[0093] Anti-His tag antibodies and anti-Lys tag antibodies are well known to those skilled in the art, and any commercially available related products are suitable for the kits described in this specification. In addition, those skilled in the art using hybridoma technology are able to prepare monoclonal antibodies that specifically bind to one of the sequences in the R1 and R2 peptide moieties.

[0094] As used herein, the term "proximity probe" refers to a probe that can be used in a dual-recognition immunoassay. A dual-recognition immunoassay refers to any assay method for detecting or quantifying an analyte in a sample, and which relies on the "proximity probing" principle, i.e., the analyte (or a synthetic peptide as an internal control) is detected by the binding of multiple (i.e., two or more, typically two or three) proximity probes, and when these proximity probes are brought close to each other by binding to the analyte, a signal is allowed to be generated.

[0095] Typically, each proximity probe comprises a nucleic acid domain (or portion, such as an oligonucleotide) linked to an antigen-binding domain (such as an antibody), and the generation of the signal involves the interaction between the nucleic acid moieties (such as the formation of a duplex by hybridization of complementary strands). In other words, the generation of the signal depends on the interaction between the proximity probes.

[0096] The term "hybridization" (hybridization or hybridizes) as used herein refers to the formation of a duplex between nucleotide sequences that have sufficient complementarity to form a duplex by Watson-Crick base pairing. Two nucleotide sequences are "complementary" to each other when they have base pair organizational homology. Complementary nucleotide sequences will specifically bind to each other under appropriate hybridization conditions to form a stable duplex.

[0097] For example, two sequences are complementary when a portion of the first sequence is capable of binding to a portion of the second sequence in an antiparallel manner, where the 3' end of each sequence binds to the 5' end of the other sequence, and each A, T(U), G, and C in one sequence aligns with T(U), A, C, and G, respectively, in the other sequence. The two sequences do not have to be completely homologous to be "complementary". Typically, two sequences have sufficient complementarity when at least about 85% (preferably at least about 90%, most preferably at least about 95%) of the nucleotides have homology in base pair organization over the defined length of the molecule or domain determined to be complementary. Thus, the nucleic acid domains of the first and second neighboring probes contain regions that are complementary to the nucleic acid domains of the other neighboring probe.

[0098] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain antigen-binding sites that immunospecifically bind an antigen. Thus, the term antibody includes not only intact antibody molecules but also antibody fragments and variants (including derivatives) of antibodies. Thus, an anti-His tag antibody is an antibody (or fragment thereof) that specifically binds to a polyhistidine tag. An anti-Lys tag antibody is an antibody (or fragment thereof) that specifically binds to a polylysine tag.

[0099] In native antibodies of rodents and primates, the two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, λ and κ. There are five major heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the recognition and specificity of binding to an antigen. The constant domain regions of the light chain (CL) and heavy chain (CH) confer important biological properties to the antibody, such as association of antibody chains, secretion, transplacental transport, complement binding, and binding to Fc receptors (FcR).

[0100] The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody depends on the structural complementarity between the antibody binding site and the epitope. The antibody binding site is mainly composed of residues from the hypervariable regions or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) can also participate in the antibody binding site or affect the structure of the entire domain and thus the binding site. The complementarity-determining region or CDR refers to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs, named L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, the antigen binding site typically consists of six CDRs, including the CDR sets of the V regions of the heavy and light chains. The framework region (FR) refers to the amino acid sequences located between the CDRs. Thus, the variable regions of the light and heavy chains typically contain 4 framework regions and 3 CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0101] Residues in the antibody variable domains are usually numbered according to the system designed by Kabat et al. This system is described in "Sequences of Proteins of Immunological Interest" published by Kabat et al. in 1987 (U.S. Department of Health and Human Services, National Institutes of Health, U.S.A.) (Kabat et al., 1992, hereinafter referred to as "Kabat et al."). This specification adopts this numbering system. The Kabat residue numbering does not always directly correspond to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions of structural components (whether framework regions or complementarity-determining regions (CDRs)) in the basic variable domain structure corresponding to the strict Kabat numbering. For a given antibody, the correct Kabat numbering of residues can be determined by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3). The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3).

[0102] In a specific embodiment, the antibodies provided herein are antibody fragments, and more specifically, any protein comprising the antigen-binding domain of the antibodies disclosed herein. Antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies.

[0103] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules consisting of a single molecule. A monoclonal antibody composition has a single binding specificity and affinity for a particular epitope.

[0104] The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds an antigen".

[0105] Specificity can also be demonstrated, for example, by the ratio of the affinity / avidity for binding to a specific antigen to the non-specific binding to other unrelated molecules, such as a ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or higher (in which case the specific antigen is a His tag or Lys tag peptide sequence).

[0106] As used herein, the term "avidity" refers to the strength of binding of an antibody to an epitope.

[0107] As used herein, an antibody that specifically binds an antigen (such as a His tag or Lys tag) refers to an antibody having a K D value of at least 100 nM, at least 10 nM, or at least 1 nM or lower for binding to the antigen, for example, as measured by surface plasmon resonance (SPR) assays (such as Biacore assays).

[0108] In a specific embodiment, the anti-His tag antibody against the 6x histidine tag (Ladavière et al; Bioconjug Chem. 1998; 9(6):655-61. Epub 1998 / 11 / 17. doi: 10.1021 / bc970208i. PubMed PMID: 9815157) comprises at least the heavy chain variable region (VH) and the light chain variable region (VL) of the following monoclonal antibody called 18D12. For example, such antibodies include the monoclonal antibody called "clone 4D11" provided by Merck (reference number 05-531), the monoclonal antibody called "6x-His Tag Monoclonal antibody (HIS.H8)" provided by ThermoFisher (reference number MA1-21315), or the antibody called "clone AD1.1.10" provided by R&S Systems (reference number MAB050).

[0109] In a specific embodiment, the anti-Lys tag antibody against the 6x lysine tag (Ladavière et al; Bioconjug Chem. 1998; 9(6): 655-61. Epub 1998 / 11 / 17. doi: 10.1021 / bc970208i. PubMed PMID: 9815157) comprises at least the heavy chain variable region (VH) and the light chain variable region (VL) of the following monoclonal antibodies called 2G4A12 or 5F12E4 (preferably 2G4A12).

[0110] In a preferred embodiment, the anti-His tag antibody for the kit is the 18D12 antibody.

[0111] In a preferred embodiment, the anti-Lys tag antibody for the kit is the 2G4A12 or 5F12E4 antibody, more preferably the 2G4A12 antibody.

[0112] In a more preferred embodiment of the kit, the anti-His tag antibody is 18D12, and the anti-Lys tag antibody is 2G4A12.

[0113] In a specific embodiment, the kit comprises at least:[[]]END

[0114] - A first proximity probe, which comprises an anti-His tag antibody (such as 18D12 or its antigen-binding region) conjugated to a first oligonucleotide;

[0115] - A second proximity probe, which comprises an anti-Lys tag antibody (such as 2G4A12 or 5F12E4 or its antigen-binding region) conjugated to a second oligonucleotide.

[0116] The oligonucleotide can be conjugated to the analyte-binding domain directly or through a linking group. The components can be covalently bound to each other through functional groups known in the art, which may be present on the components or introduced onto the components through one or more steps (such as oxidation reaction, reduction reaction, cleavage reaction, etc.). Functional groups that can be used to covalently bind the components to prepare the proximity probe include: hydroxyl group, sulfhydryl group, amino group, etc. The specific parts on different components that are modified to achieve covalent connection are selected such that they do not substantially and adversely affect the required binding affinity of the component to the target analyte. When necessary and / or desired, certain parts on the components can be protected using protecting groups known in the art, for example, see Green & Wuts, Protective Groups in Organic Synthesis (John Wley & Sons) (1991). Methods for preparing nucleic acid / antibody conjugates are well known to those skilled in the art. For example, see U.S. Patent No. 5,733,523, the disclosure of which is incorporated herein by reference.

[0117] In other embodiments, proximity probes can be prepared using in vitro protocols that generate nucleic acid-protein conjugates, i.e., molecules having a nucleic acid (such as a coding sequence) covalently bound to a protein, i.e., where the analyte-binding domain is generated in vitro by a vector encoding the proximity probe. Examples of such in vitro protocols of interest include: RepA-based protocols (such as see Fitzgerald, Drug Discov. Today (2000) 5:253-258 and WO 98 / 37186), ribosome display-based protocols (such as see Hanes et al., Proc. Natl Acad. Sci. USA (1997) 94:4937-42; Roberts, Curr Opin Chem Biol (1999) Jun; 3:268-73; Schaffitzel et al., J Immunol Methods (1999) Dec 10; 231:119-35; and WO 98 / 54312), etc.

[0118] In a specific embodiment, the first and second oligonucleotides conjugated to the anti-His tag antibody and the anti-Lys tag antibody, respectively, are designed to be able to hybridize when the antibodies bind to the synthetic peptides of the present disclosure (preferably the synthetic peptides of SEQ ID NO:6 or SEQ ID NO:8).

[0119] Typically, the first and second oligonucleotides have 29 to 56 bases. In one embodiment of the kit disclosed herein, one oligonucleotide has 29 bases and the other has 31 bases. In another embodiment of the kit disclosed herein, one oligonucleotide has 49 bases and the other has 56 bases.

[0120] In a specific embodiment, the length of the complementary region (i.e., the hybridization region) in the oligonucleotides for the kit can range from 4 to 30 bases, such as 6 to 20 bases, 6 to 18 bases, 7 to 15 bases, 8 to 12 bases, or 9 to 11 bases.

[0121] In a preferred embodiment, the pair of oligonucleotides have complementary sequences of GACGACTTC and GAAGTCGTC, respectively.

[0122] In a preferred embodiment, the kit of the present disclosure, in addition to the above synthetic peptide, at least further comprises:

[0123] (i) A first proximity probe comprising an anti-His tag antibody conjugated to the oligonucleotide of SEQ ID NO:9; and

[0124] (ii) A second proximity probe comprising an anti-Lys tag antibody conjugated to the oligonucleotide of SEQ ID NO:10. In a more preferred embodiment, the kit of the present disclosure, in addition to the above synthetic peptide, at least further comprises:

[0125] (i) A first proximity probe comprising the 8D12 anti-His tag antibody conjugated to the oligonucleotide of SEQ ID NO:9; and

[0126] (ii) A second proximity probe comprising the 2G4 anti-Lys tag antibody conjugated to the oligonucleotide of SEQ ID NO:10.

[0127] In an even more preferred embodiment, the kit of the present disclosure at least comprises:

[0128] (i) The synthetic peptide of SEQ ID NO:6 or SEQ ID NO:8;

[0129] (ii) A first proximity probe comprising the 8D12 anti-His tag antibody conjugated to the oligonucleotide of SEQ ID NO:9; and

[0130] (iii) A second proximity probe comprising the 2G4 anti-Lys tag antibody conjugated to the oligonucleotide of SEQ ID NO:10.

[0131] Method of using the synthetic peptide

[0132] The synthetic peptide, or a composition thereof, or a kit comprising the synthetic peptide, is advantageously used as an internal control or a quantitative calibrator in immunoassays, and preferably for immunoassays, such as dual recognition immunoassays, proximity ligation assays or immuno-PCR, to detect and / or quantify an analyte in a sample.

[0133] As used herein, the term "analyte" refers to any substance (e.g., molecule) or entity that is desired to be detected by an immunoassay (such as a dual recognition immunoassay). The analyte is the "target" of the dual recognition immunoassay. Thus, the analyte can be any biomolecule or chemical compound that is desired to be detected, such as a peptide or protein, a nucleic acid molecule or a small molecule (including organic and inorganic molecules). The analyte can be a cell, a microorganism (including a virus) or a fragment or product thereof. All that is required is that the analyte be able to bind at least two binding partners (more specifically, the analyte-binding domains of at least two proximity probes). In a preferred embodiment, the analyte is a protein or polypeptide and any molecule comprising a protein or polypeptide component. For example, the analyte is a protein that may be present in a blood sample (typically a blood sample).

[0134] As used herein, the term "internal control" more specifically refers to an indicator of any malfunction during the assay process so that the assay results can be marked as invalid. In a preferred embodiment, the internal control will produce substantially the same (expected) result regardless of the specific sample being assayed and the concentration of the analyte in the sample.

[0135] In this case, the internal control can also be used as a quantitative calibrator: the concentration of the internal control is known, and the signal obtained is also known. Thus, the various signals obtained when detecting the analyte (e.g., in a multiplex dual recognition immunoassay performed together with the internal control) can be adjusted by comparison with the signal obtained for the internal control.

[0136] As used herein, PEA TMIt is a dual-recognition immunoassay method in which two matching antibodies conjugated to unique nucleic acid domains (referred to herein as proximity probes) simultaneously bind an analyte in solution. This brings the two antibodies into proximity to each other, enabling their nucleic acid domains to hybridize and serve as a template for a DNA polymerase-dependent extension step. This generates a double-stranded DNA "barcode" that is unique for a specific antigen and is in quantitative proportion to the initial concentration of the target protein or analyte. PCR amplification is carried out immediately after hybridization and extension. The resulting DNA amplicons can then be detected and / or quantified. Methods using the dual-recognition immunoassay have been described in Lundberg et al. [1], Fredriksson et al. [2], and Greenwood et al. [3]. Other variants of proximity probe-based assays have also been described in the art. For example, the dual-recognition immunoassay is described in WO01 / 61037, U.S. Patent No. 6,511,809, WO03 / 055231, WO2005 / 123963, and WO2006 / 137932.

[0137] In a specific embodiment, an internal control is used in a method for detecting and / or quantifying an analyte in a sample, the method comprising at least the following steps:

[0138] (a) contacting the sample with at least one set of at least a first and a second proximity probe, each proximity probe comprising an analyte-binding domain (e.g., an antigen-binding domain) and a nucleic acid domain (e.g., an oligonucleotide) and being capable of binding the analyte simultaneously;

[0139] (b) after the proximity probes have bound to the analyte, causing the nucleic acid domains of the proximity probes to interact with each other, wherein the interaction comprises forming a duplex;

[0140] (c) detecting and / or quantifying the formation of the duplex, typically by extension and PCR amplification based on the duplex.

[0141] As used herein, the term "duplex" refers to the hybridization of two complementary nucleic acid domains of the proximity probes.

[0142] Step (c) may further comprise the following sub-steps:

[0143] (c1) extending the 3'-end of at least one nucleic acid domain in the duplex to generate an extension product;

[0144] (c2) amplifying and detecting the extension product.

[0145] As used herein, the term "amplification" or "amplified" generally refers to any method of increasing the copy number of an extension product or a portion thereof in an assay as a means of indicating the presence of a target analyte in a sample. For example, any amplification method known in the art can be used in the methods of the present disclosure, such as polymerase chain reaction (PCR), ligase chain reaction (LCR), and the like.

[0146] Obviously, in order to determine whether a sample contains a target analyte, it is not necessary to amplify the entire extension product. It is only necessary to amplify that portion of the extension product that was not present in the sample before the extension reaction occurred. For example, the extension product will actually contain two parts: a first "old" part (existing part) that contains the nucleotide sequence that makes up the nucleic acid domain of the adjacent probe, and a second "new" part (extension part) that contains the nucleotide sequence generated by the template extension reaction. In a specific embodiment, the detection of the second "new" or "extension" part can enable the detection of the target analyte, that is, if there is no analyte, no extension will occur and thus there will be no "new" or "extension" part.

[0147] Thus, in a preferred aspect of the present disclosure, the step of amplifying the extension product includes amplifying a portion of the extension part of the extension product. This portion of the extension part needs to be of sufficient length to be distinguishable from other sequences present in the sample. In fact, this portion of the extension part of the extension product serves as a unique identifier or signal corresponding to the presence of the target analyte. Therefore, if this portion contains nucleotide sequences that are not present in the sample, the amplification of this sequence is sufficient to indicate the presence and quantity of the target analyte in the sample.

[0148] Thus, this portion can contain at least 8 nucleotides, preferably at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides. The length of a portion of the extension part of the extension product can generally range from about 8 to about 90 nucleotides, about 12 to about 70 nucleotides, about 14 to about 50 nucleotides, about 16 to about 40 nucleotides.

[0149] Although it is conceivable to amplify the entire extension product, that is, the existing part and the extension part, it is sufficient that the amplification product contains at least a portion of the extension part of the extension product. In one aspect of the present disclosure, primers can be designed to flank either side of the extension part of the extension product and amplify this part (e.g., by PCR), and the amplification product (containing this portion of the extension product) can be detected as described below.

[0150] Examples of PCR amplification, DNA polymerase, and 3'-exonuclease for proximity extension assays are also described in WO2012104261 and WO2013113699 owned by Olink AB.

[0151] Generally, real-time PCR (RT-PCR) or quantitative PCR (qPCR) can be used.

[0152] In a particularly preferred embodiment, the extension product is amplified by PCR, where the PCR is quantitative PCR, and an intercalating dye is used to quantify the amplified nucleic acid molecules. In a preferred embodiment, the intercalating dye is selected from SYBR and EvaGreen TM .

[0153] Accordingly, the present disclosure also relates to a method for detecting and / or quantifying an analyte in a sample, typically a biological sample, the method comprising:

[0154] (i) at least one dual-recognition immunoassay, such as PEA TM , by incubating a test sample with at least a pair of proximity probes directed against the analyte to be detected;

[0155] (ii) at least one control test for the validity of the proximity assay, by incubating the test sample with a synthetic peptide disclosed herein (such as the synthetic peptide of SEQ ID NO:6) and a corresponding pair of proximity probes comprising an anti-His tag and an anti-Lys tag antibody provided in the kit of the present disclosure.

[0156] As used herein, the term "biological sample" refers to a fluid sample containing an analyte from a biological source (such as viruses and other microorganisms, plants, animals).

[0157] For example, the biological sample can be obtained from urine, blood (including but not limited to peripheral blood, plasma or serum), feces, sputum, saliva, bronchoalveolar lavage fluid, endotracheal aspirate, wound, cerebrospinal fluid, lymph node, exudate, and more generally, can be obtained from any human biopsy tissue or body fluid, tissue or material. In a more specific embodiment, the biological sample is a whole blood sample.

[0158] In a more specific embodiment, the analyte to be detected is procalcitonin.

[0159] The biological sample can be processed or not processed before being used for the dual-recognition immunoassay.

[0160] The dual-recognition immunoassay in step (i) and the control test in step (ii) can be performed simultaneously or sequentially, and can be performed on the same solid support or separate solid supports.

[0161] In a specific embodiment, they are performed in the same biological sample in a multiplex dual-recognition immunoassay.

[0162] Methods for performing multiplexed dual-recognition immunoassays are described, for example, by Assarsson et al.

[20] .

[0163] In addition to serving as an internal positive control, the synthetic peptides or kits of the present disclosure can also be used as quantitative calibrators in sample testing. Accordingly, the present disclosure relates to a method for quantitatively testing an analyte in a sample, the method comprising:

[0164] (i) at least one dual-recognition immunoassay, such as PEA TM , by incubating the test sample with at least one pair of proximity probes directed against the analyte to be assayed;

[0165] (ii) at least one control test for the validity of the proximity assay, by incubating the test sample with a synthetic peptide of the present disclosure (such as the synthetic peptide of SEQ ID NO:6) and a corresponding pair of proximity probes comprising an anti-His tag and an anti-Lys tag antibody provided in the kit of the present disclosure;

[0166] (iii) determining the amount of analyte in the test sample by comparing the signal obtained in step (i) with the signal obtained in step (ii).

[0167] Steps (i) and (ii) can be performed simultaneously in the same test sample using a multiplexed dual-recognition immunoassay.

[0168] To determine the amount of analyte in step (iii), the signal obtained with the proximity probes used for quantifying the internal control (i.e., the synthetic peptide of the present disclosure, such as the peptide of SEQ ID NO:6) in step (ii) is quantified to obtain a control value. At the same time, the signal obtained with the proximity probes used for detecting the analyte in step (i) is quantified to obtain a test value. Relative quantification is obtained by determining the ratio between the test value and the control value. If the absolute amount of the internal control in the test sample is known, absolute quantification can be obtained.

[0169] In a specific embodiment, to quantify the two signals, in a multiplexed dual-recognition immunoassay, the DNA amplicons representing the internal control and another DNA amplicon representing the analyte can be quantified (usually using qPCR or RT-PCR).

[0170] The present invention will now be further illustrated by the following figures and examples. However, these examples and figures should not be construed in any way as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1: The optical density (OD) at 405 nm of different tag antibodies when measuring different concentrations of Bitag 1 or Bitag 2 peptides (0, 1, and 5 μg / mL) by ELISA. 18D12 corresponds to the His-tag monoclonal antibody, and 2G4 and 5F12 correspond to the Lys-tag monoclonal antibodies. The results are the average of three replicate experiments performed under each condition.

[0172] Figure 2 : Evaluation of different peptides (a: Bitag 1; b: Bitag 2) using MML and MMC proximity probes. The results are expressed as Delta-Ct, corresponding to the difference between the Ct value for 0 ng / mL Bitag peptide and the Ct values for other concentrations (5 and 50 ng / mL respectively) of Bitag peptide. Ct corresponds to the cycle threshold, i.e., the minimum qPCR cycle number at which the fluorescence signal is above background. The results are the average of three replicate experiments.

[0173] Figure 3 : (a) Dual-recognition immunoassay in human plasma supplemented with different concentrations of Bitag 2 peptide. The results are expressed as Delta-Ct0-X (X is 0.5, 5, 50, and 500 ng / mL respectively), and correspond to the average of three replicate experiments. (b) Box plot of the dual-recognition immunoassay in human plasma supplemented with 0 (BT_0) or 50 ng / mL (BT_50) Bitag 2 peptide. The results are expressed as Ct values and obtained from 13 (BT_0) and 28 (BT_50) plasma samples. Each assay was performed in duplicate. (x): average.

[0174] Figure 4 : Procalcitonin dual-recognition immunoassay in PBS-BSA buffer supplemented with different concentrations of recombinant procalcitonin (rPCT) (0 and 0.1 ng / mL) and Bitag 2 peptide (0 to 200 ng / mL). The results are expressed as Ct values, corresponding to the average of three replicate experiments.

[0175] Figure 5 : Procalcitonin and internal control dual-recognition immunoassay in PBS-BSA buffer supplemented with different concentrations of rPCT (0 and 0.1 ng / mL) and Bitag 2 peptide (0 to 160 ng / mL). After the dual-recognition immunoassay (DRI), PCR amplification was performed using (a) specific PCR primers for the PCT DRI probe or (b) specific PCR primers for the internal control proximity probe respectively. The results are expressed as Ct values, corresponding to the average of three replicate experiments.

[0176] Figure 6: The procalcitonin double recognition immunoassay (red dots) and the internal control double recognition immunoassay (green triangles) were performed on three human plasma samples supplemented with different concentrations of rPCT (0.01 to 10 ng / mL) plus 50 ng / mL Bitag 2. The results are expressed as Ct values, corresponding to the mean of replicate experiments. Example

[0177] Example 1: Synthesis of Bitag-1 and Bitag-2

[0178] The synthesis of the synthetic peptides was carried out using an ABI433A synthesizer from Applied Biosystems (Foster City, CA, USA). Rink Amide MBHA resin (product number 855003, MerckMillipore, Molsheim, France) was used as the polymeric solid support. At the end of the chemical synthesis, the peptides were deprotected and cleaved from the polymer in the presence of a mixture of trifluoroacetic acid - ethanedithiol - triisopropylsilane - water (94 / 2.5 / 1 / 2.5, v / v) for about 2 hours. After removing the polymer by filtration, the peptides were precipitated and separated from diethyl ether at 0 °C. To improve purity, the peptides were purified by reverse-phase preparative high-performance liquid chromatography (HPLC) on a Vynac Denali TM C18, 10 μm column (Mandel Scientific Company Inc., Guelph, Ontario, Canada). Each peptide was eluted with a step gradient of acetonitrile in an aqueous solution containing 0.1% trifluoroacetic acid (acetonitrile concentration from 0 to 95%), and the percentage of acetonitrile in the gradient steps was chosen to optimize the separation of the corresponding target peptide peak. After this final step, two different analytical techniques were performed to verify and characterize the resulting peptides.

[0179] Example 2: Development of a dual-epitope peptide for double recognition immunoassay as a fail-safe internal control and its application in human plasma samples

[0180] Materials and Methods

[0181] Materials: Recombinant human procalcitonin (rPCT) was developed and produced by bioMérieux SA (Lyon, France) after expressing the protein in prokaryotic cells according to standard procedures [8]. rPCT was stored at -80 °C in PBS buffer containing 5% bovine serum albumin (BSA).

[0182] Monoclonal antibodies were generated and purified by bioMérieux SA according to standard procedures after immunization of mice with rPCT or peptides [9]. The rPCT antibodies were directed against the calcitonin domain of procalcitonin (8F12 monoclonal antibody) or the katacalcin domain of PCT (11E12 monoclonal antibody), and were non-commercial antibodies. Anti-tag antibodies were directed against the 6x histidine tag (MRGSHHHHHH) or the 6x lysine tag (GKKKKKKSV), and were designated 18D12 or 2G4A12 / 5F12E5, respectively

[10] . Peptides were synthesized in-house using Fmoc chemistry on an ABI433A peptide synthesizer from Applied Biosystems according to the manufacturer's instructions (Foster City, CA USA).

[0183] Whole blood samples were obtained from healthy donors of the French National Blood Center (EFS, Etablissement du Sang, Lyon, France). Samples were selected based on negative PCT testing. Fresh blood was centrifuged at 20,000 g for 5 minutes at 4 °C using a refrigerated microcentrifuge (Eppendorf 5424R, Montesson, France). After centrifugation, the supernatant (corresponding to plasma) was collected for the dual recognition immunoassay and stored at 4 °C before use. For all biological samples, informed consent was obtained prior to any experiments. All experiments were in accordance with relevant laws and institutional guidelines and followed the ethical standards of the Declaration of Helsinki.

[0184] Proximity probes: Proximity probes were prepared by covalently linking purified PCT monoclonal antibodies to HPLC-purified oligonucleotides. The oligonucleotides had a C6-amino modification at the 5' end and contained an 18-22 nucleotide sequence for PCR primer hybridization and a 9 nucleotide sequence for hybridization to a second proximity probe from the 5' to the 3' end. The synthesis, modification, and HPLC purification of the oligonucleotides were performed by Integrated DNA Technologies (Louvain, Belgium). Antibody-oligonucleotide conjugates were generated using a commercial Thunderlink kit (Abcam, Cambridge, UK) at an oligonucleotide to antibody ratio of 5:1 according to the manufacturer's instructions. The conjugation quality was checked by running the conjugate on a reducing 4-12% SDS-PAGE (Thermofisher, Waltham, MA, USA) followed by Coomassie blue staining (Thermofisher) according to the supplier's instructions (Abcam).

[0185] Two pairs of nucleotide sequences (one pair for PCT proximity probes and the other pair for internal control proximity probes) were generated randomly (https: / / molbiotools.com), while taking care to avoid homologous sequences and palindromic sequences. The melting temperature (Tm) of the PCR primer sequences was approximately 60 °C.

[0186] Enzyme-linked immunosorbent assay (ELISA): After overnight coating with the peptide diluted to 5 μg / mL in PBS buffer (pH 7.4, Euromedex, Souffelweyersheim, France), Nunc 96-well plates (Thermofisher) were blocked with PBS 1x-5% bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis, MI, USA) for 1 hour at room temperature (RT). After washing 4 times with PBS 1x-0.1% Tween 20, PBS 1x-1% BSA buffer containing the anti-tag antibody (1 μg / mL) was added and incubated at 37 °C for 1 hour. After incubation with alkaline phosphatase (AP)-labeled streptavidin (Jackson Immunoresearch, Ely, UK) diluted 1 / 50000 in PBS-BSA-0.5% at 37 °C for 30 minutes, PNPP substrate (Thermofisher) was used for color development. Absorbance levels (OD405) were read at 405 nm using an Infinite M Nano+ microplate reader (Tecan, Switzerland). All samples were tested in duplicate and three independent experiments were performed.

[0187] Proximity extension assay (PEA TM ): The proximity extension assay protocol and different buffers were those originally described by Lundberg et al. [1] and a more recently developed shorter protocol (unpublished results). The proximity extension assay was performed in a specific multi-well plate (Hard-Shell 96-well clear-shell PCR plate, BioRad, Hercule, CA, USA), with each sample run in triplicate. Briefly, 5 μL of the sample (PBS 1x-0.1% BSA buffer or human EDTA plasma with or without rPCT and / or IC peptide) was mixed with 5 μL of a mixture of two PEA TM conjugates (500 pM each) in probe incubation buffer and incubated at 37 °C for 60 minutes. After probe incubation, dilution buffer containing 40 mM of each dNTP was added. After incubation at 37 °C for 5 minutes, 96 μL of an extension mixture containing 26 U / mL of T4 DNA polymerase (Thermofisher) was added and incubated at 37 °C for an additional 3 minutes, followed by a heat inactivation step at 80 °C.

[0188] qPCR assay: For qPCR assay, 4 μL of the extension product was transferred into a qPCR plate and mixed with 36 μL of a qPCR mixture (SYBRGREEN mixture, Bio-Rad) containing 0.6 μM of each PCR primer. One-step qPCR was performed with an initial denaturation at 95 °C for 3 min, followed by denaturation at 95 °C for 1 s and annealing / extension at 63 °C for 10 s for 40 cycles. Finally, a melting step was performed by gradually increasing the temperature from 60 °C to 95 °C at 0.5 °C / s.

[0189] Statistical analysis: Statistical data analysis was performed using GraphPad Prism v4.03 software (GraphPad Software, San Diego, CA, USA). The Mann-Whitney U test, which can be used for unknown distributions of two small sample sets (n < 30), was used to compare continuous variables. Excel was used to plot histograms, and the confidence intervals (error bars) corresponded to 2 standard deviations.

[0190] Results

[0191] Internal control design selection: A key consideration in designing an internal control is the need to prevent interference from proteins naturally present in human blood products. After considering the use of non-human proteins as internal controls, synthetic peptides containing two non-human epitopes (more specifically, epitope tags) were ultimately selected for evaluation. His tags and Lys tags have previously been used in protein purification and functionalization of microplates in immunoassays [10,11].

[0192] As previously mentioned, the binding of a dual-recognition immunoassay probe to its target requires two epitopes. The binding of two neighboring probes (antibody conjugates) to these epitopes should not interfere with each other due to steric hindrance. Therefore, the two epitopes on the internal control peptide are separated by a neutral peptide bond, which acts as a spacer. The spacer consists of an Ado (aminodioxo-octanoic acid

[12] ) moiety, which is composed of a straight chain of three artificial amino acids with a total length of 10 Å. A tandem of two or three Ado spacers was inserted between the two epitope tags of the IC, resulting in distances of 20 Å and 30 Å, respectively. The peptide containing two Ado spacers is called Bitag 1, and the peptide containing 3 Ado spacers is called Bitag 2.

[0193] Selection and validation of Bitag peptides and proximity probes: First, to check whether the anti-tag antibodies indeed bind to their epitopes in the context of Bitag peptides, indirect ELISA experiments were performed by coating multi-well plates with two concentrations (1 μg / mL and 5 μg / mL) of Bitag 1 or Bitag 2 peptides. For both coated peptides, the 18D12 monoclonal antibody (anti-His tag) or 2G4 and 5F12 monoclonal antibodies (anti-Lys tag) were used, and detection was performed with an AP-conjugated anti-mouse conjugate.

[0194] Figure 1 It was shown that in the absence of peptides, a small signal was observed (OD405 < 0.126). This signal was 5.42-fold lower than the signal obtained in the presence of peptides (18D12 antibody, 1 μg of Bitag 1) and 9.17-fold lower (2G4 antibody, 5 μg of Bitag 1), confirming that the antibodies indeed specifically bind to the peptides. In addition, a small signal (between 0.124 and 0.138) was obtained in the absence of anti-tag antibodies, which reflects the weak non-specific binding of the AP conjugate. Overall, all antibodies bound to both Bitag peptides. The signal of 18D12 (anti-His) was observed to be lower than the signals obtained with the anti-Lys tag antibodies (2G4 and 5F12). For example, for 1 μg of Bitag 1 peptide, the OD405 of 18D12 was 0.617, while the OD405 of 2G4 was 0.993. For 5 μg of peptide, the OD405 of 18D12 increased significantly (+0.262), while the increases for 2G4 and 5F12 were smaller (+0.089 and +0.074, respectively). No significant difference was observed between the two anti-Lys tag antibodies 2G4 and 5F12. However, since 2G4 was systematically observed to have a slight advantage in terms of OD values, 2G4 was preferred over 5F12 in subsequent experiments.

[0195] Therefore, two monoclonal antibodies, 18D12 (anti-His tag) and 2G4 (anti-Lys tag), were used in the preparation of internal control proximity probes.

[0196] Second, to determine the optimal combination of spacer size and oligonucleotide length, proximity probes were sequentially tested on two Bitag 1 and Bitag 2 peptides. The first type of probe is called MML, which has a longer oligonucleotide version (49 and 56 bases), while the second type of probe is called MMC, which has a shorter oligonucleotide version (29 and 31 bases). For MML and MMC, the complementary sequence size at the 3'-end of each oligonucleotide is 9 nucleotides.

[0197] Figure 2Summarizes the results of the dual-recognition immunoassay obtained by adding three concentrations of Bitag peptides (0, 5, and 50 ng / mL) in PBS-BSA buffer. The results are expressed as Delta-Ct0-X, corresponding to the difference in Ct values between 0 and X ng / mL of the peptide. Figure 2 The graphs in Figure 2 show a significant increase in Delta-Ct observed for MMC compared to the MML probe. For example, for Bitag 2, at 5 ng / mL of the peptide, the gain of MMC relative to MML was 2.32, and at 50 ng / mL of the peptide it was 1.86. For MMC, Delta-Ct0-50 reached 7.08 (while for MML it was 5.22). Higher Delta-Ct values for MMC were also found for Bitag 1, but to a lesser extent.

[0198] Regardless of which peptide was tested, the MMC probe was most suitable for the dual-recognition immunoassay. The combination of the shorter MMC oligonucleotide with the peptide containing 3 Ado sequence spacers (Bitag 2) produced the highest Delta-Ct at the peptide concentrations tested.

[0199] To confirm these preliminary results obtained when diluting the peptide in PBS-BSA buffer, PEA was performed on pooled human plasma (from three plasma specimens) spiked with four concentrations (0 to 500 ng / mL) of Bitag 2 peptide using the MMC probe. TM The results are shown in Figure 3 as in a. Overall, although the assay presented similar dynamics to those obtained in PBS-BSA buffer, the Delta-Ct values seemed slightly lower compared to those obtained in PBS-BSA (a loss of 0.26 and 0.36 at 5 ng / mL and 50 ng / mL of the peptide, respectively). Additionally, at 500 ng / mL, a significant dynamic loss was observed (Delta-Ct50-500 was 0.9, while Delta-Ct5-50 was 2.72).

[0200] In summary, these results indicate that it is feasible to detect peptides with two epitope tags in a complex medium (pooled human plasma samples) using a rapid dual-recognition immunoassay protocol such as PEA tM .

[0201] Third, the concentration of Bitag 2 suitable for the internal control dual-recognition immunoassay must be selected. A concentration of 50 ng / mL was chosen because it provided a signal level (Ct value) significantly higher than the background but still within the dynamic range. To confirm the specificity of the results obtained using 50 ng / mL of Bitag 2 peptide, human plasma specimens were tested by the dual-recognition immunoassay after adding 50 ng / mL of the peptide (n = 25 samples) or without peptide supplement (n = 13 samples). The results obtained are summarized in Figure 3In b. According to the Mann-Whitney test, the difference between the two groups was significant (P < 0.001).

[0202] Interference between procalcitonin and the internal control: To examine whether the Bitag peptide would interfere with the detection of procalcitonin, a dual-recognition immunoassay protocol for detecting procalcitonin was performed in PBS-BSA buffer supplemented with two concentrations of rPCT (0 and 0.1 ng / mL) and six concentrations of Bitag 2 peptide (0 to 200 ng / mL). The results are as Figure 4 shown. For 0.1 ng / mL of rPCT, no significant difference was observed within the range of peptide concentrations added to the samples (mean = 34.38 ± 0.15). Similar results were obtained for 0 ng / mL of rPCT, except that a significant decrease in Ct value was observed at 160 ng / mL of peptide (35.9, while the mean for other peptide concentrations was 36.98).

[0203] To examine that the PCR primers used for qPCR amplification of the procalcitonin product would not interfere with the primers for the internal control adjacent probe, a dual-recognition immunoassay protocol for detecting procalcitonin was performed in PBS-BSA buffer containing two analytes, namely rPCT (0 and 0.1 ng / mL) and Bitag peptide (in the concentration range of 0 to 160 ng / mL). The dual-recognition immunoassay protocol was performed in parallel for the two analytes, but PCR amplification was carried out in the presence of specific primers for the procalcitonin DRI probe or specific primers for the internal control adjacent probe. As Figure 5 shown in a, when the PCT primer set was used, no significant difference in Ct values for different concentrations of Bitag peptide was observed. For 0.1 ng / mL of rPCT, the mean was 34.26 ± 0.31. For 0 ng / mL of PCT, a slight but significant increase in background noise was observed after adding 40 ng of peptide compared to the sample without added peptide. The Delta-Ct between these two conditions was 1.4. Interestingly, the Ct for 40 ng / mL of peptide was the same as that for 160 ng / mL of peptide.

[0204] When PCR amplification was carried out using the internal control PCR primer set (see Figure 6 b), no interference was observed in the presence of procalcitonin. Regardless of the presence or absence of procalcitonin (0.1 ng / mL), no significant difference in Ct values was obtained for each peptide concentration.

[0205] Therefore, both dual-recognition immunoassay protocols can be performed without significantly inhibiting their PCR amplification performance.

[0206] Application in human plasma specimens: Human plasma contains many endogenous potential interfering substances, such as antibodies with broad reactivity to murine antibodies, which may hinder the performance of dual-recognition immunoassays

[13] . To confirm the feasibility of simultaneously detecting procalcitonin and internal control in human samples, different concentrations of rPCT (0, 1, and 10 ng / mL) and 50 ng / mL of Bitag peptide were added to three plasma specimens collected from three different patients. As Figure 6 shown, rPCT at a concentration as low as 1 ng / mL could be detected in human plasma. As expected, as the procalcitonin concentration increased, the Ct decreased. The average Delta-Ct observed between 0 and 1 ng / mL of rPCT was 5.2. Between 1 and 10 ng / mL, the average Delta-Ct decreased to 2.8.

[0207] The Ct values of the internal control did not change significantly among the three rPCT concentrations and among different patients. For 0 ng / mL of PCT, the average Ct was 33.53 ± 0.11, and for 10 ng / mL of PCT, the average Ct was 34.23 ± 0.40.

[0208] All results obtained using Bitag 2 peptide were also replicated and confirmed on Bitag2' peptide (data not shown).

[0209] Discussion

[0210] In short, it has been shown that peptides composed of two epitope tags can serve as fail-safe internal controls in immunoassays (such as dual-recognition immunoassays, e.g., PEA TM ). The internal control (the synthetic peptide of the present disclosure) can be detected simultaneously with at least one diagnostically significant biomarker in human plasma specimens, with no cross-interference between the control and the analyte, and no evidence of significant interference with plasma proteins. The combination of shorter MMC oligonucleotides with peptides having a spacer containing 3 Ado sequences performed best when using a dual-recognition immunoassay protocol as a proof of concept.

[0211] In terms of the design and implementation of internal controls for immunoassays, first, it must be ensured that the internal control can achieve sufficient inter-batch reproducibility through a strictly controlled synthesis process. For this purpose, it is considered preferable to use recombinant proteins or synthetic peptides rather than naturally occurring molecules. Second, such a protein or peptide should not occur naturally in human samples because the signal associated with the internal control must always be the same regardless of the sample being tested. In addition, the internal control must have two different epitopes, and there must be two specific monoclonal antibodies (at least one antibody for each epitope) available as probes.

[0212] Ideally, each epitope should be mapped to understand its precise location on the three-dimensional structure of the protein analyte, if any. To facilitate production and avoid restricting the accessibility of epitopes on the protein surface, it is considered preferable to select a non-glycosylated protein. Finally, in terms of proximity and accessibility on the protein or peptide, the epitope must be suitable for proximity extension or ligation assay methods. In practice, if epitopes are buried within the protein or too far apart from each other, this can have a negative impact on the successful development of a dual-recognition immunoassay protocol. Taking these factors into account, it was decided to use synthetic peptides rather than non-human recombinant proteins (e.g., from plants, invertebrates) as ICS.

[0213] In a previous study, Arrarsson et al.

[14] chose to use additive controls consisting of a mixture of different recombinant proteins, including green fluorescent protein (GFP) and phycoerythrin. These controls were used to monitor the incubation steps of the proximity assay, identify possible outliers in the samples, and perform inter-plate normalization. Arrarsson et al. also used extension controls consisting of antibodies linked to oligonucleotides. In addition, the controls for qPCR consisted of double-stranded DNA templates. These controls have been shown to help improve the precision within assays (for multiplex assays) and between assays by compensating for technical differences.

[0214] Epitope tags are short peptides (3 - 14 amino acids) that are commonly used to facilitate protein purification or to monitor the localization, movement, modification, or interaction of proteins during physiological processes [15,16]. Epitope tags can be recognized and bound by tag-specific antibodies. A variety of epitope tags have been developed and used traditionally. They are selected such that they do not interfere with cellular functions or processing. They also have weak antigenicity so as not to bind to host antibodies. The tag can be placed at the N-terminus, C-terminus, or within the reading frame of the protein under study. They are typically derived from protein sequences of viruses and bacteriophages, such as the peptide tag of herpes simplex virus glycoprotein D or the peptide tag of bacteriophage T7 capsid protein [17,18].

[0215] Here, it was decided to evaluate peptides in which two epitope tags (His tag and Lys tag) are separated by a neutral spacer consisting of three tandem Ado bridges. In fact, the synthesis of these peptides is strictly controlled, and the distance between the two tags is determined by the number of Ado bridges. In addition, by nature, the peptides have poor structure and there is a low probability that the epitopes cannot be recognized by antibodies. Finally, there are several monoclonal antibodies available for each epitope tag of the peptide.

[0216] One of the common problems with using linear peptides is their insufficient stability [19,20]. For long-term storage, peptides are typically stored at -20 °C. However, it has been demonstrated that the peptides proposed in the present disclosure are stable for at least 12 weeks (86 days) at 4 °C and at least 24 hours at room temperature (data not shown). Thus, these peptides can be used for extended periods under common storage conditions with a low risk of significant degradation.

[0217] To determine the optimal balance between on the one hand peptide size and on the other hand the length of the oligonucleotide conjugated to the antibody, it has been demonstrated that on the scale of the peptide, the oligonucleotide must be as short as possible to bring about a significant improvement in assay sensitivity.

[0218] Under these conditions, the internal control can be detected by immunoassays (e.g., by proximity extension or ligation assays) within a concentration range corresponding to approximately three orders of magnitude of dynamic range. Additionally, the effect of peptide concentration on the performance of the procalcitonin dual recognition immunoassay is limited, and no interference between the two qPCR amplification systems was observed.

[0219] When tested in parallel with procalcitonin in several human samples, the Ct values of the internal control remained stable, while the Ct values of procalcitonin decreased as the procalcitonin concentration increased, as expected.

[0220] Furthermore, the internal control is mainly used as a failsafe control for the entire dual recognition immunoassay protocol, including qPCR. Thus, the internal control is designed to provide a qPCR signal (Ct value) that must fall within a predetermined acceptable range for the assay to be considered valid. However, the internal control can also be used as a calibrator, i.e., the signal obtained from the internal control is used to normalize the signal of the analyte.

[0221] In the present disclosure, the dual recognition immunoassay is performed in a microplate, and the internal control is added in the first step of the dual recognition immunoassay protocol. The internal control can also be applied to an automated version of the proximity extension / ligation assay protocol, most notably as part of a system used for diagnostic applications, e.g., in the context of a point-of-care solution.

[0222] Example 3: Description of the nucleotide sequences used in the present disclosure

[0223] SEQ ID NO: Sequence description 1 His-tag peptide 2 Long His-tag peptide 3 Lys-tag peptide 4 Long Lys-tag peptide 5 Bitag-1 6 Bitag-2 7 Bitag-1' 8 Bitag-2' 9 Complete oligonucleotide 1 sequence of Bitag 2MMC 10 Complete oligonucleotide 2 sequence of Bitag 2MMC

[0224]

[0225]

[0226] References

[0227] 1. Lundberg M, Eriksson A, Tran B, Assarsson E, Fredriksson S. Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. Nucleic Acids Res. 2011;39(15):e102. Epub 2011 / 06 / 08. doi:10.1093 / nar / gkr424. PubMed PMID: 21646338; PubMedCentral PMCID: PMCPMC3159481.

[0228] 2. Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gústafsdóttir SM, et al. Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol. 2002;20(5):473-7. Epub 2002 / 05 / 01. doi:10.1038 / nbt0502-473. PubMed PMID: 11981560.

[0229] 3. Greenwood C, Ruff D, Kirvell S, Johnson G, Dhillon HS, Bustin SA. Proximity assays for sensitive quantification of proteins. Biomol Detect Quantif. 2015;4:10-6. Epub 2016 / 04 / 15. doi:10.1016 / j.bdq.2015.04.002. PubMed PMID: 27077033; PubMed Central PMCID: PMCPMC4822221.

[0230] 4. Schuetz P, Albrich W, Mueller B. Procalcitonin for diagnosis of infection and guide to antibiotic decisions: past, present and future. BMC Med. 2011;9:107. Epub 2011 / 09 / 23. doi:10.1186 / 1741-7015-9-107. PubMed PMID: 21936959; PubMed Central PMCID: PMCPMC3186747.

[0231] 5. Wacker C, Prkno A, Brunkhorst FM, Schlattmann P. Procalcitonin as a diagnostic marker for sepsis: a systematic review and meta-analysis. Lancet Infect Dis. 2013;13(5):426-35. Epub 2013 / 02 / 05. doi:10.1016 / s1473-3099(12)70323-7. PubMed PMID: 23375419.

[0232] 6. Hamade B, Huang DT. Procalcitonin: Where Are We Now? Crit Care Clin. 2020;36(1):23-40. Epub 2019 / 11 / 18. doi:10.1016 / j.ccc.2019.08.003. PubMed PMID: 31733680; PubMed Central PMCID: PMCPMC6866676.

[0233] 7. Harbarth S, Holeckova K, Froidevaux C, Pittet D, Ricou B, Grau GE, et al. Diagnostic value of procalcitonin, interleukin-6, and interleukin-8 in critically ill patients admitted with suspected sepsis. Am J Respir Crit Care Med. 2001;164(3):396 - 402. Epub 2001 / 08 / 14. doi:10.1164 / ajrccm.164.3.2009052. PubMed PMID: 11500339.

[0234] 8. Schendel PF. Overview of protein expression in E. coli. Curr Protoc Mol Biol. 2001;Chapter 16:Unit16.1. Epub 2008 / 02 / 12. doi:10.1002 / 0471142727.mb1601s41. PubMed PMID: 18265122.

[0235] 9. Yokoyama WM. Monoclonal antibody supernatant and ascites fluid production. Curr Protoc Immunol. 2001;Chapter 2:Unit 2.6. Epub 2008 / 04 / 25. doi:10.1002 / 0471142735.im0206s40. PubMed PMID: 18432770.

[0236] 10. Ladavière C, Delair T, Domard A, Novelli-Rousseau A, Mandrand B, Mallet F. Covalent immobilization of proteins onto (Maleic anhydride-alt-methyl vinylether) copolymers: enhanced immobilization of recombinant proteins. Bioconjug Chem. 1998;9(6):655-61. Epub 1998 / 11 / 17. doi:10.1021 / bc970208i. PubMed PMID: 9815157.

[0237] 11. Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg HG. Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus. Proc Natl Acad Sci U S A. 1991;88(20):8972-6. Epub 1991 / 10 / 15. doi:10.1073 / pnas.88.20.8972. PubMed PMID: 1924358; PubMed Central PMCID: PMCPMC52633.

[0238] 12. Basak S, Mohottalage D, Basak A. Multibranch and pseudopeptide approach for design of novel inhibitors of subtilisin kexin isozyme-1. Protein Pept Lett. 2006;13(9):863-76. Epub 2006 / 11 / 15. doi:10.2174 / 092986606778256199. PubMed PMID: 17100641.

[0239] 13. Klee GG. Human anti-mouse antibodies. Arch Pathol Lab Med. 2000;124(6):921 - 3. Epub 2000 / 06 / 03. doi:10.5858 / 2000 - 124 - 0921 - hama. PubMed PMID: 10835540.

[0240] 14. Assarsson E, Lundberg M, Holmquist G, J, Thorsen SB, Ekman D, et al. Homogenous 96 - plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. PLoS One. 2014;9(4):e95192. Epub 2014 / 04 / 24. doi:10.1371 / journal.pone.0095192. PubMed PMID: 24755770; PubMed Central PMCID: PMCPMC3995906 commercializing the described method under the name Proseek Multiplex. Patent name: “Exonuclease enabled proximity extension assays” and number: WO2012104261A1. There are no new patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials.

[0241] 15. Kolodziej PA, Young RA. Epitope tagging and protein surveillance. Methods Enzymol. 1991; 194: 508 - 19. Epub 1991 / 01 / 01. doi: 10.1016 / 0076 - 6879(91)94038 - e. PubMed PMID: 1706460. Cravchik A, Matus A. A novel strategy for the immunological tagging of cDNA constructs. Gene. 1993; 137(1): 139 - 43. Epub 1993 / 12 / 27. doi: 10.1016 / 0378 - 1119(93)90262 - 2. PubMed PMID: 7506688.

[0242] 16. Hansen MK, Tams JW, Fahrenkrug J, Pedersen PA. Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae. Recept Channels. 1999; 6(4): 271 - 81. Epub 1999 / 07 / 21. PubMed PMID: 10412720.

[0243] 17. Humbert N, Schürmann P, Zocchi A, Neuhaus JM, Ward TR. High - yield production and purification of recombinant T7 - tag mature streptavidin in glucose - stressed E. coli. Methods Mol Biol. 2008; 418: 101 - 10. Epub 2008 / 02 / 22. doi: 10.1007 / 978 - 1 - 59745 - 579 - 4_9. PubMed PMID: 18287653.

[0244] 18. Wearley LL. Recent progress in protein and peptide delivery by noninvasive routes. Crit Rev Ther Drug Carrier Syst. 1991;8(4):331-94. Epub 1991 / 01 / 01. PubMed PMID: 1769066.

[0245] 19. Bogdanowich-Knipp SJ, Chakrabarti S, Williams TD, Dillman RK, Siahaan TJ. Solution stability of linear vs. cyclic RGD peptides. J Pept Res. 1999;53(5):530-41. Epub 1999 / 07 / 29. doi:10.1034 / j.1399-3011.1999.00052.x. PubMed PMID: 10424348.

[0246] 20. Assarsson E, Lundberg M, Holmquist G, Bjo¨rkesten J, Bucht Thorsen S, et al. (2014) Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability. PLoS ONE 9(4):e95192. doi:10.1371 / journal.pone.0095192.

Claims

1. A synthetic peptide having the following formula (I): R1-Z-R2 (I) Wherein: Z is a peptide spacer composed of 1 to 3 unnatural amino acids; R1 and R2 are two different peptide moieties, wherein one of the peptide moieties in R1 or R2 contains a polylysine tag, and the other peptide moiety contains a polyhistidine tag.

2. The synthetic peptide according to claim 1, wherein the length of the spacer Z is between about 10 and about 30 angstroms, preferably between about 20 and about 30 angstroms.

3. The synthetic peptide according to claim 1 or 2, wherein the spacer Z is a dipeptide or tripeptide of unnatural amino acids, and the length of each unnatural amino acid is about 10 angstroms.

4. The synthetic peptide according to any one of claims 1-3, wherein the unnatural amino acid is 8-amino-2,4-dioxaoctanoic acid.

5. The synthetic peptide according to any one of claims 1-4, wherein Z is a tripeptide of 8-amino-2,4-dioxaoctanoic acid.

6. The synthetic peptide according to any one of claims 1 - 3, wherein Z is a peptide spacer having the formula (NH-(Z')-COO) n wherein n is from 1 to 3, and Z' is a straight-chain saturated or unsaturated hydrocarbon chain optionally containing one or more heteroatoms, and the chain contains 6 to 20 non-hydrogen atoms; or Z is of the formula H-(O-CH2-CH2) n -OH, wherein n is from 1 to 4, and preferably n is equal to 2.

7. The synthetic peptide according to any one of claims 1-6, wherein R1 is a peptide having the following sequence: X -10 X -9 X -8 X -7 (X -6 ) n X -5 X -4 X -3 X -2 X -1 , wherein: -X -10 is optional or a non-polar amino acid, preferably methionine; -X -9 is optional or is a basic amino acid, preferably arginine; -X -8 is optional or a non-polar amino acid, preferably glycine; -X -7 is optional or is a polar amino acid, preferably serine; -X -6 is histidine or lysine; -X -5 is optional or a polar amino acid, preferably serine; -X -4 is optional or a non-polar amino acid, preferably valine; -X -3 is optional or a polar acidic amino acid, preferably aspartic acid; -X -2 is optional or is a polar acidic amino acid, preferably glutamic acid; -X -1 is optional or is a polar amino acid, preferably serine; And -n is an integer between 4 and 8, preferably equal to 6.

8. The synthetic peptide according to any one of claims 1-7, wherein R2 is a peptide having the following sequence: X +1 (X +2 ) p X +3 X +4 X +5 X +6 X +7 X +8 , where: -X +1 is optional or a non-polar amino acid, preferably glycine; -X +2 is lysine or histidine; -X +3 is optional or is a polar amino acid, preferably serine; -X +4 is optional or is a non-polar amino acid, preferably valine; -X +5 is optional or is a polar acidic amino acid, preferably aspartic acid; -X +6 is optional or is a polar acidic amino acid, preferably glutamic acid; -X +7 is optional or is a polar amino acid, preferably serine; -X +8 is optional or is a non-polar amino acid, preferably leucine; and -p is an integer between 4 and 8, preferably equal to 6.

9. The synthetic peptide according to any one of claims 1-8, which has the following formula (II): X -10 X -9 X -8 X -7 (X -6 ) n X -5 X -4 X -3 X -2 X -1 (N) m X +1 (X +2 ) p X +3 X +4 X +5 X +6 X +7 X +8 (II) Wherein: -X -10 is a non-polar amino acid, preferably methionine; -X -9 is a basic amino acid, preferably arginine; -X -8 is a non-polar amino acid, preferably glycine; -X -7 is a polar amino acid, preferably serine; -X -6 is histidine; -N is 8-amino-2,4-dioxaoctanoic acid; -X +1 is a non-polar amino acid, preferably glycine; -X +2 is lysine; -X +3 is a polar amino acid, preferably serine; -X +4 is a non-polar amino acid, preferably valine; -X -5 、X -4 、X -3 、X -2 、X -1 、X +5 、X +6 、X +7 and X +8 do not exist; -m is 2 to 4, preferably equal to 3; And -n and p are 4 to 8, preferably equal to 6.

10. The synthetic peptide according to claim 9, wherein: -X -10 is a nonpolar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X -9 is arginine; -X -8 is a nonpolar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X -7 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine and glutamine; -X -6 is histidine; -X +1 is a non-polar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X +2 is lysine; -X +3 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine and glutamine; -X +4 is a non-polar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -m is equal to 2 or 3, preferably 3; and -n and p are equal to 5 or 6.

11. The synthetic peptide according to claim 9 or 10, which has the following sequence: MRGSHHHHHH-(N) m -GKKKKKKSV (SEQ ID NO:6), where N is 8-amino-2,4-dioxaoctanoic acid, and m is 3.

12. The synthetic peptide according to any one of claims 1-8, which has the following formula (III): X -10 X -9 X -8 X -7 (X -6 ) n X -5 X -4 X -3 X -2 X -1 (N) m X +1 (X +2 ) p X +3 X +4 X +5 X +6 X +7 X +8 (III) Wherein: -X -10 is a non-polar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X -9 is a basic amino acid; -X -8 is a non-polar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X -7 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine, and glutamine; -X -6 is histidine; -X -5 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine, and glutamine; -X -4 is a nonpolar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X -3 is an acidic amino acid selected from aspartic acid and glutamic acid; -X -2 is an acidic amino acid selected from aspartic acid and glutamic acid; -X -1 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine, and glutamine; -N is 8-amino-2,4-dioxaoctanoic acid; -X +1 is a nonpolar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X +2 is lysine; -X +3 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine, and glutamine; -X +4 is a non-polar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -X +5 is an acidic amino acid selected from aspartic acid and glutamic acid; -X +6 is an acidic amino acid selected from aspartic acid and glutamic acid; -X +7 is a polar amino acid selected from serine, cysteine, threonine, tyrosine, asparagine, and glutamine; -X +8 is a nonpolar amino acid selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; -m is 2 to 4, preferably equal to 3; and -n and p are 4 to 8, preferably equal to 6.

13. The synthetic peptide according to claim 12, which has the following sequence: MRGSHHHHHHSVDES-(N)m-GKKKKKKSVDESL (SEQ ID NO:8), wherein N is 8-amino-2,4-dioxaoctanoic acid, and m is 3.

14. A kit, comprising at least the following components: - The synthetic peptide according to any one of claims 1-13; and - At least two proximity probes, each proximity probe comprising an antigen-binding portion conjugated to a nucleic acid domain, wherein the first proximity probe comprises an anti-His tag antibody or an antigen-binding fragment thereof that specifically binds to the His tag, and the second proximity probe comprises an anti-Lys tag antibody or an antigen-binding fragment thereof that specifically binds to the Lys tag.

15. Use of the synthetic peptide according to any one of claims 1 to 13 or the kit according to claim 14 as an internal control or quantitative calibrator in an immunoassay, preferably as an internal control or quantitative calibrator in an immunomolecular assay.

16. The use according to claim 15, wherein the immunoassay is a dual recognition immunoassay, proximity ligation assay or immuno-PCR, preferably a dual recognition immunoassay, such as proximity extension assay or proximity ligation assay, and for example a multiplex dual recognition immunoassay.

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