Transglutaminase substrates for labeling

Abstract

Disclosed are fusion polypeptides that are substrates for white Kunitz's transglutaminase. The fusion polypeptides comprise one or more FKBP chaperone proteins and a target polypeptide. Each of these elements is separated from adjacent elements by a linker amino acid sequence. It has been found that it is advantageous to insert a glutamic acid-containing transglutaminase recognition motif into the linker amino acid chain. The subsequent labeling reaction catalyzed by the transglutaminase surprisingly provides a labeled fusion polypeptide with superior properties compared to chemically randomly labeled fusion polypeptides of similar design. Assays and kits for detecting a target antibody in a sample in vitro are provided.

Description

Background Technology

[0001] This disclosure relates to an immunoassay for detecting antibodies against specific antigens in a sample. Antibody isotypes found in blood, such as, but not limited to, IgG, IgE, IgD, and monomeric IgA, are bivalent, meaning each has two antigen-binding sites. Each binding site can bind to the antibody's target antigen. Typically, it is necessary to detect antigen-specific antibodies in a sample. For example, antibodies specific to antigens derived from pathogens can indicate exposure to that pathogen. In vitro detection of such antibodies in samples obtained from patients can provide specific medical value in diagnosing diseases caused by that pathogen.

[0002] Immunoassays that detect antibodies utilize this characteristic. In a general sense, such assays provide an antigen that is contacted with a sample suspected of containing an antibody specific to and capable of binding to that antigen. If the antibody is present in the sample, it reacts with the antigen to form a complex, i.e., an immunoreaction product. In this complex, one of the antibody's antigen-binding sites attaches to the antigen through physical interaction. Immunoassays detect these immunoreaction products, i.e., the complex.

[0003] Detection can be performed using a label. The antigen can be labeled, and the immunoreaction product containing the labeled antigen is detected. In a specific embodiment, the immunoreaction product containing the labeled antigen is separated from unbound labeled antigen and sample material that did not participate in the immune reaction. If the antigen is already present in the sample, the label is separated together with the immunoreaction product. In this case, the detection of the label indicates the presence of the immunoreaction product, thereby indicating the presence of the target antibody in the sample.

[0004] An important prerequisite for an immune response is that in vitro antibody detection requires an antigen that is stable in the sample to present the antibody. The desired stable antigen retains its conformation and solubility, thereby ensuring its ability to bind to the target antibody in a reproducible manner (and specifically under the conditions of the in vitro test setup).

[0005] Therefore, several technical challenges exist, particularly considering antigens derived from peptides or polypeptides. Specifically, the production of purified formulations of antigens used in immunoassays may be hampered by solubility issues, as some antigens tend to aggregate and eventually precipitate. In such cases, the required amount of antigen for the immunoassay may not be sufficient. Furthermore, due to conformational changes, in vitro purified antigens may not adequately reflect the original antigens in vivo. In this situation, the ability to bind target antibodies in vitro is impaired.

[0006] Further challenges exist in labeling antigens as peptides or polypeptide chains (i.e., amino acid sequences). Label attachment typically involves chemical reactions. A typical example is amine-reactive crosslinking chemistry, which couples a label to the primary amine group of the antigen, often using N-hydroxysuccinimide esters. Primary amines are present at the N-terminus of each polypeptide chain and in the side chain of lysine (K) amino acid residues. By its very chemical principle, such reactions have virtually no site specificity. Chemically reacting the amine groups of the antigen and attaching the label to these groups can mask key portions of the antigen, preventing antibody binding sites from binding. Titration experiments are necessary to determine the appropriate concentrations of label, crosslinking agent, and antigen when excessive crosslinking can, in extreme cases, lead to complete functional inactivation of the antigen (i.e., antibody binding can no longer occur). Therefore, the label density on the antigen is not always optimal, and after the crosslinking reaction, the antigen is always present in a distribution of unlabeled, overlabeled, high-density labeled, or low-density labeled antigens. Depending on the severity of the undesirable labeling effect, the detection process in immunoassays can be compromised and requires laborious and time-consuming optimization efforts.

[0007] FK506-binding proteins (FKBPs) have been identified in many eukaryotes, from yeast to humans, and function as protein folding chaperones for proline-containing proteins. One example of a prokaryotic FKBP-type polypeptide is SlyD. The bacterial slyD gene (exemplarily from *Escherichia coli*) encodes an FKBP-type peptidyl-prolyl cis-trans isomerase (PPIase). SlyD is a bacterial two-domain protein that functions as a molecular chaperone, prolyl cis / trans isomerase, and nickel-binding protein. Chaperone functions located in one domain of SlyD involve diarginine translocation and increase the catalytic efficiency of the prolyl cis-trans isomerase domain in protein folding by two orders of magnitude. Non-limiting examples of such FKBP-type chaperone proteins are presented herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. Further examples are documented in the literature, such as Zoldák G. & Schmid FX (2011) J Mol Biol 406, 176-194 and Scholz C. et al. (2006) Biochemistry 45, 20-33, and other literature.

[0008] The problems concerning antigen solubility and conformation have been previously resolved. As reported in WO2003000877A2, many members of the peptidyl prolyl isomerase (PPI) class, particularly those from the FKBP-type protein family, not only exhibit catalytic activity but also have a desired effect on the solubility of polypeptides that would otherwise tend to aggregate. They achieve this by forming soluble complexes with polypeptides that readily aggregate in their unassisted and dissociated forms. Such polypeptides, which would otherwise be difficult or insoluble under physiological conditions, become soluble under mild physiological conditions (i.e., without the need for solubilizers such as detergents or dissociators) once they are bound to a suitable PPI chaperone protein in the complex.

[0009] WO2007077008A1 reports a chimeric fusion polypeptide comprising a polypeptide sequence containing a polypeptide-binding region of a non-human chaperone protein, a polypeptide sequence fused to the N-terminus of the non-human chaperone polypeptide sequence with an FKBP polypeptide or an FKBP-like domain, and a polypeptide sequence fused to the C-terminus of the non-human chaperone polypeptide sequence with an FKBP polypeptide or an FKBP-like domain.

[0010] EP1780282A1 discloses the following finding: by linking an antigen to an FKBP chaperone protein in a fusion polypeptide, enhanced solubility and reduced aggregation of the antigen can be advantageously achieved without solubilization, instead of forming a complex with the FKBP chaperone protein. This document reports a fusion polypeptide containing an amino acid sequence of a specific rubella virus E1 antigen fused at the N-terminus to an FKBP chaperone protein amino acid sequence. EP2127679A1 reports a recombinant-derived fusion polypeptide containing at least one amino acid sequence corresponding to the SlpA chaperone protein and at least one amino acid sequence corresponding to the target polypeptide.

[0011] As is clear, the technical problems of undesirable aggregation and precipitation of purified antigens can be mitigated by recombining the antigen as the amino acid sequence of the target peptide in a fusion peptide, wherein one or more copies of the FKBP chaperone protein amino acid sequence are present or attached to the N-terminal portion of the target. In practice, the target peptide and each FKBP chaperone protein domain are linked by coupled amino acid sequences that function as linkers between any of these elements and adjacent elements in the fusion peptide. However, technical challenges remain regarding the attachment of labels to such fusion peptides via chemical reactions. Again, in the case of fusion peptides, chemical cross-linking can mask key portions of the antigen. Furthermore, chemical cross-linking can affect the FKBP chaperone protein portion. As a result, the desired function of the chaperone protein may be impaired, thereby compromising the stability of the antigenic portion of the fusion peptide. Moreover, the problem of random distribution of labels remains. Conventional chemical strategies for peptide modification are difficult to control and produce heterogeneous populations of labeled peptides with variable stoichiometry, where each member of the population possesses its own in vitro properties. Furthermore, the amount of target peptides or labels in a chemical reaction is uncontrollable and therefore uncertain, and typically follows a Poisson distribution, which can cause problems when quantitative reactions are desired.

[0012] For at least these and other reasons, there is a need in the art to provide better alternatives to stabilized target peptides (such as antigens) for immunoassays.

[0013] The discovery of novel transglutaminases (Kutzneria albida transglutaminase; KalbTGase) and the identification of their corresponding peptide substrates have been described (Steffen et al., (2017) J Biol Chem292, 15622-15635). KalbTGase catalyzes the formation of heteropeptide bonds between donor acyl groups (particularly donor acyl groups on the glutamine (Gln, Q) side chain) and alkylamine donor groups (e.g., alkylamine donor groups on the lysine (Lys, K) side chain). Motifs YRYRQ (SEQ ID NO: 20) and RVRQR (SEQ ID NO: 21) are particularly suitable glutamine-containing motifs (Qtag) for KalbTGase. Steffen et al. (2017), ibid., reported RYESK (SEQ ID NO: 73) as a lysine-containing acceptor motif (Ktag) for KalbTGase. However, in addition to the lysine side chain, KalbTGase can process a variety of other amine donor groups as Ktags. One example is biotin-dPEG(23)-NH2 (Steffen et al. (2017), ibid.).

[0014] The introduction of KalbTGase-mediated artificial bioorthogonal groups for site-specific and stoichiometric peptide modification may offer a solution to the problems discussed above. However, unless significant effort is invested in determining the permissible sites for inserting Qtag amino acid sequences, the necessary insertions into target peptides (such as antigenic amino acid sequences) are expected to alter their conformation. Similarly, inserting Qtag motifs into the amino acid sequence of FKBP chaperone proteins raises similar concerns. Summary of the Invention

[0015] The inventors have discovered that, for fusion peptides, inserting a Qtag into the coupling amino acid sequence between two adjacent FKBP chaperone proteins is highly suitable for providing attachment sites for KalbTGase-mediated Ktag coupling. Furthermore, the inventors have found that inserting a Qtag into the coupling amino acid sequence between an FKBP chaperone protein and an adjacent target peptide is also highly suitable for providing attachment sites for KalbTGase-mediated Ktag coupling. Such Qtag insertions not only provide bioorthogonal addition of the label in a site-specific and stoichiometric manner, but also surprisingly demonstrate a significant improvement in the signal-to-noise ratio for specific fusion peptides labeled with this enzyme, as a KalbTGase substrate, and in label detection. Moreover, such labeled substrates exhibit surprisingly improved temperature stability.

[0016] In the first aspect, this report provides a recombinant KalbTGase substrate comprising a fusion peptide of formula I.

[0017]

[0018] in

[0019] The hyphen "-" represents a peptide bond;

[0020] n is an integer from 1 to 6;

[0021] A pair of square brackets “[” and “]” delimits the N-terminal and C-terminal boundaries of an amino acid sequence that appears n times;

[0022] A n For FKBP chaperone protein amino acid sequences, and in the case of n > 1, each A n The amino acid sequence of the FKBP chaperone protein is selected independently;

[0023] L n For coupled amino acid sequences, and in the case of n > 1, each L nThe amino acid sequences are selected independently;

[0024] B represents the amino acid sequence of the target polypeptide;

[0025] And among them

[0026] Each L n Having formula IIa or formula IIb

[0027]

[0028] in

[0029] m is an integer from 1 to 10;

[0030] A pair of square brackets “[” and “]” delimits the N-terminal and C-terminal boundaries of an amino acid sequence that appears m times;

[0031] T represents a flexible or rigid linker amino acid sequence containing 5 to 500 amino acids;

[0032] R m For flexible or rigid linker amino acid sequences containing 5 to 500 amino acids, and in the case of m > 1, each R m For independently selected flexible or rigid linker amino acid sequences containing 5 to 500 amino acids;

[0033] Qtag m The motif is an amino acid sequence containing an acyl donor glutamine residue for KalbTGase transglutaminase activity, and in the case of m > 1, each Qtag m The amino acid sequence motif is independently selected and contains an acyl donor glutamine residue for KalbTGase transglutaminase activity;

[0034] The condition is that SEQ ID NO: 18 and SEQ ID NO: 19 are excluded from Formula I.

[0035] In a second aspect, this report provides a method for forming a target polypeptide with a covalently attached marker, the method comprising the following steps:

[0036] a. Providing a KalbTGase substrate according to the first aspect and all embodiments thereof, said KalbTGase substrate comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity;

[0037] b. Providing a marker conjugate, wherein in the marker conjugate, the marker is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase;

[0038] c. Under conditions that allow transglutaminase activity to occur, the fusion peptide of (a) and the labeled conjugate of (b) are contacted with KalbTGase;

[0039] This allows the acyl donor glutamine residue to react with the primary amine group to form a covalent bond.

[0040] This forms the target polypeptide with the covalently attached marker.

[0041] In a third aspect, this report provides a method for forming a target polypeptide with covalently attached trapping groups, the method comprising the following steps:

[0042] a. Providing a KalbTGase substrate according to the first aspect and all embodiments thereof, said KalbTGase substrate comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity;

[0043] b. Providing a capture group conjugate, wherein in the capture group conjugate, the capture group is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase;

[0044] c. Under conditions that allow transglutaminase activity to occur, contact the fusion peptide of (a) and the capture group conjugate of (b) with KalbTGase;

[0045] This allows the acyl donor glutamine residue to react with the primary amine group to form a covalent bond.

[0046] This forms the target polypeptide with covalently attached trapping groups.

[0047] In the fourth aspect, this report provides a labeled target polypeptide that is obtained or available by means of the methods described in the second aspect and in all embodiments herein.

[0048] In the fifth aspect, this report provides a target polypeptide having a covalently attached trapping group, which is obtained or obtainable by means of the methods described in the third aspect and all embodiments herein.

[0049] In a sixth aspect, this report provides a composition suitable for detecting a target antibody specific for an antigenic amino acid sequence in an isolated sample, wherein the composition comprises a labeled target polypeptide according to the fourth aspect and all embodiments herein, wherein the antigenic amino acid sequence is contained in the labeled target polypeptide.

[0050] In the seventh aspect, this report provides a method for detecting a target antibody (=X) specific for an antigen amino acid sequence (=Y) in an isolated sample, the method comprising:

[0051] a. An immune reaction mixture is formed by mixing a body fluid sample suspected of containing X with a labeled target polypeptide according to the fourth aspect and all embodiments herein, wherein Y is contained in the labeled target polypeptide.

[0052] b. Maintaining the immunoreaction blend of step (a) for a period of time sufficient to allow X present in the body fluid sample to react immunoreact with Y contained in the labeled target polypeptide to form an immunoreaction product (=X:Y) in the blend; and

[0053] c. Detect the presence and / or concentration of X:Y formed in step (b);

[0054] This allows for the detection of target antibodies specific to the amino acid sequence of the antigen in the separated sample.

[0055] In the eighth aspect, this report provides (i) the KalbTGase substrate according to the first aspect and all embodiments herein, (ii) the label conjugate and (iii) the use of KalbTGase for the production of labeled target peptides, wherein in the label conjugate, the label is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase.

[0056] In the ninth aspect, this report provides the use of labeled target peptides obtained or available by the methods described in the fourth aspect and all embodiments herein for the detection of target antibodies specific to the amino acid sequence of an antigen in isolated samples.

[0057] In the tenth aspect, this report provides the use of labeled target peptides according to the fourth aspect and all embodiments herein, and individual target peptides with covalently attached capture groups according to the fifth aspect and all embodiments herein, for detecting target antibodies specific to the amino acid sequence of an antigen in isolated samples.

[0058] In the eleventh aspect, this report provides a component kit for detecting a target antibody specific to an antigenic amino acid sequence in an isolated sample, the kit comprising a labeled target polypeptide according to the fourth aspect and all embodiments herein, the labeled target polypeptide comprising the antigenic amino acid sequence.

[0059] In the twelfth aspect, this report provides a DNA encoding a fusion polypeptide comprising a target polypeptide and one or more acyl donor glutamine residues for KarbTGase transglutaminase activity, wherein a codon for an N-terminal methionine is appended to a nucleotide sequence encoding an amino acid sequence of a KarbTGase substrate according to the first aspect and all embodiments herein.

[0060] In the thirteenth aspect, this report provides an expression vector for recombinant expression in a transformed organism, the expression vector comprising DNA as described in the twelfth aspect and all embodiments herein.

[0061] In the fourteenth aspect, this report provides a prokaryotic host organism stably transformed with an expression vector according to the thirteenth aspect and all embodiments herein, wherein the transformed host organism is capable of expressing the encoded KarbTGase substrate from the expression vector.

[0062] In aspect fifteen, this report provides a method for producing recombinant KalbTGase substrates, the method comprising the following steps:

[0063] (a) Providing and culturing the transformed host organism according to aspect fourteen and all embodiments herein,

[0064] (b) Expressing the recombinant KalbTGase substrate in the transformed host organism described in (a), and

[0065] (c) Purify the expressed recombinant KalbTGase substrate from the transformed host organism.

[0066] This produces the recombinant KalbTGase substrate. Attached Figure Description

[0067] Figure 1 depicts a Ktag with Ru markers.

[0068] Figure 2 depicts a Ktag with a biotin group.

[0069] Figure 3 depicts a size exclusion chromatography chromatogram, which is performed after a Ru labeling reaction in the presence of KalbTGase to separate the products.

[0070] Figure 4 shows a Superdex® 200 chromatogram of the purified fusion peptide with a biotin-capturing group attached using KalbTGase in A.

[0071] A Superdex® 200 chromatogram of the purified fusion peptide with a ruthenium-labeled marker, which was attached using KalbTGase, is depicted in B. Detailed Implementation

[0072] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0073] Elements of the invention will now be described. These elements are listed along with specific embodiments; however, it should be understood that they can be combined in any manner and in any number to create other embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention solely to the explicitly described embodiments. This description should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context otherwise requires, any arrangement and combination of the elements described herein should be considered as disclosed in the specification of this application.

[0074] Unless the context clearly indicates otherwise, the terms “a,” “an,” and “the” generally include a plural reference. As used herein, “a plurality of” is understood to mean more than one. For example, a plurality means at least two, three, four, five, or more. Unless specifically stated or obvious from the context, as used herein, the term “or” is understood to be inclusive.

[0075] "Direct repetition" refers to repetition without further interruption or any further intercalation of amino acid sequences.

[0076] "Functional variant" refers to a polypeptide that has a similar function to the unmodified reference polypeptide. In one embodiment, a functional variant of the FKBP chaperone protein amino acid sequence retains the chaperone protein function. "Functional variant" also includes additional peptide sequences, such as histidine tags or N-terminal or C-terminal sequence changes, which may be in the case of cloning artifacts.

[0077] "N-terminus and C-terminus" refer to the corresponding ends of elements present in the fusion polypeptide, or the corresponding ends of the fusion polypeptide as a whole.

[0078] The term "target polypeptide" refers to any polypeptide of interest provided that the polypeptide contains one or more antigenic determinants.

[0079] "Linker amino acid sequence" refers to flexible and rigid linker amino acid sequences as disclosed and discussed in Chen X. et al. (2013) Adv Drug Deliv Rev. 65, 1357–1369.

[0080] The term "recombinant" refers to an amino acid or nucleotide sequence that has been intentionally modified by recombination methods. The term "recombinant nucleic acid" as used herein refers to nucleic acids initially formed in vitro in a form not normally found in nature, typically through the manipulation of nucleic acids by endonucleases. Therefore, for the purposes of this invention, isolated linear mutant DNA polymerase nucleic acids or expression vectors formed in vitro by linking normally non-conjugated DNA molecules are considered recombinant. It should be understood that once a recombinant nucleic acid is prepared and reintroduced into a host cell, it will replicate non-recombinantly using the host cell's in vivo cellular mechanisms rather than through in vitro manipulation; however, once such nucleic acid is recombinantly produced, it is considered recombinant for the purposes of this invention, even though it subsequently replicates non-recombinantly. "Recombinant polypeptide" or "recombinant-generated polypeptide" is a polypeptide prepared using recombination technology, i.e., through the expression of a recombinant nucleic acid as explained above.

[0081] The term "vector" refers to a segment of DNA, typically double-stranded, into which a segment of foreign DNA may have been inserted. Vectors can be, for example, plasmid-derived. Vectors contain a "replicon" polynucleotide sequence that facilitates autonomous replication of the vector within the host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally present in the host cell; said heterologous DNA may be, for example, a replication vector molecule, encoding a selectable or screenable marker, or encoding a transgene. Vectors are used to transport foreign or heterologous DNA into a suitable host cell. Once inside the host cell, the vector can replicate independently of or integrate with the host chromosomal DNA, and multiple copies of the vector and its inserted DNA can be generated. Additionally, the vector may contain essential elements that allow the inserted DNA to be transcribed into mRNA molecules or otherwise cause the inserted DNA to replicate into multiple copies of RNA. Some expression vectors also additionally contain sequence elements adjacent to the inserted DNA that increase the half-life of the expressed mRNA and / or allow the mRNA to be translated into protein molecules. Therefore, numerous mRNA and polypeptide molecules encoded by the inserted DNA can be rapidly synthesized.

[0082] The term "fusion polypeptide" refers to a polypeptide consisting of a linear sequence of two or more structural units, each of which is a peptide or polypeptide, and wherein two adjacent structural units are linked by peptide bonds. Fusion polypeptides can be produced in vivo or in vitro as a continuous translation product. Alternatively, fusion polypeptides can be provided using in vitro chemical synthesis. Their use as folding accessory proteins for target polypeptides is disclosed, specifically as additives to immunoassay mixtures. "Target polypeptide" refers to any polypeptide of interest, provided that the polypeptide contains one or more antigenic determinants.

[0083] The term "integer" is a positive natural number, which in a specific embodiment is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any subset thereof.

[0084] The term "sample" refers to a portion or section of a tissue, organ, or individual, typically smaller than such tissue, organ, or individual, intended to represent the whole tissue, organ, or individual. At the time of analysis, the sample provides information about the state of the tissue or the health or disease status of the organ or individual. Examples of samples include, but are not limited to, liquid samples such as blood, serum, plasma, synovial fluid, urine, saliva, and lymph; or solid samples such as tissue extracts, cartilage, bone, synovium, and connective tissue. Analysis of a sample can be performed on a visual or chemical basis. Visual analysis includes, but is not limited to, microscopic imaging or radiographic scanning of a tissue, organ, or individual to allow for morphological evaluation of the sample. Chemical analysis includes, but is not limited to, detecting the presence or absence of a specific indicator or changes in the amount, concentration, or level of a specific indicator. A sample is an in vitro sample isolated from the body, which will be analyzed in vitro and will not be transferred back into the body.

[0085] The terms “measurement,” “measuring,” “detecting,” or “detection” preferably include qualitative, semi-quantitative, or quantitative measurements. The term “detection of presence” is a descriptive measurement that indicates the presence or absence of a quantity without making any declaration (e.g., yes or no declaration). The term “detection amount” refers to a quantitative measurement in which an absolute quantity (ng) is detected. The term “detection concentration” refers to a quantitative measurement in which the quantity is determined relative to a given volume (e.g., ng / ml).

[0086] The term "antigen" is a molecule or molecular structure that binds to an antigen-specific antibody (Ab) or a B-cell antigen receptor (BCR). The presence of an antigen in the body typically triggers an immune response. In vivo, each antibody is specifically produced to match the antigen after the cells of the immune system come into contact with it; this allows for precise identification or matching of the antigen and initiation of a specific response. In most cases, an antibody reacts and binds to only one specific antigen; however, in some cases, antibodies may cross-react and bind to more than one antigen. Antigens are typically proteins, peptides (linker amino acid sequences), and polysaccharides (monosaccharides / simple glycans) or combinations thereof. For the purposes of this invention, the antigen is used as a specific component in an immunoassay, specifically binding to antibodies present in the analytical sample and bound to the antigen.

[0087] The term "chaperone protein" refers to a protein folding aid that assists in the folding of other proteins and maintains their structural integrity. Along with cyclin, FKBP belongs to the immunophilin family. Fifteen proteins in the human genome encode sequences that share significant homology with 12 kDa proteins that are targets of potent immunosuppressive macrolides such as FK506 or rapamycin. The 12 kDa prototype of FK506-binding protein (FKBP), called FKBP-12, is an abundant intracellular protein. FKBP12 functions as a PPIase, catalyzing the interconversion between prolyl cis / trans conformations. FKBPs are involved in a variety of cellular functions, including protein folding, cell signaling, apoptosis, and transcription. They function by directly binding to and altering the conformation of their target proteins, thus acting as molecular switches. Examples of folding aids are described in detail in WO2003000877. For example, chaperone proteins of the peptidyl prolyl isomerase class, such as those of the FKBP family, can be used for fusion with antigen variants. Examples of suitable FKBP chaperone proteins as fusion couplers are FkpA (aa 26-270, UniProt ID P45523), SlyD (1-165, UniProt ID P0A9K9), and SlpA (2-149, UniProt ID P0AEM0). Another suitable chaperone protein as a fusion coupler is Skp (21-161, UniProt ID P0AEU7), a trimer chaperone protein from the periplasm of *E. coli*, which does not belong to the FKBP family. The complete sequence of the chaperone protein is not always required. Functional fragments of the chaperone protein that still possess the desired capabilities and functions (so-called binding-capable modules) can also be used (see WO199813496). "FKBP chaperone protein" specifically refers to FKBP-type peptidyl prolyl isomerases. Its specific and advantageous uses as part of a fusion polypeptide have been reported in WO2003000878, EP1780282, WO2007077008, EP2127679, WO2012150320, WO2014072305, and WO2014072306. Specific, but not limiting, examples of FKBP chaperone proteins are FkpA, SlyD, SlyD-like proteins, triggering factors, and their functionally active variants, including truncated, amino acid-deleted, inserted, or substituted versions, as well as their affinity-tagged (e.g., His-tagged) versions.

[0088] The term "peptide" refers to a chain of at least two and fewer than ten amino acids linked together by peptide bonds.

[0089] "Amino acid" refers to any monomeric unit that can be combined to form a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following twenty naturally or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0090] A peptide bond is an amide bond between the α-carboxyl group of the first amino acid and the α-amino group of the second amino acid. A peptide bond is different from the covalent bond generated by transglutaminase activity, which is a heteropeptide bond.

[0091] The term "coupled amino acid sequence" refers to a segment of amino acids located between two FKBP chaperone protein amino acid sequences or between an FKBP chaperone protein amino acid sequence and a target polypeptide. In this disclosure, the coupled amino acid sequence contains one or more Qtags. An "element" refers to a structural unit or group of two or more structural units that are part of a fusion polypeptide, including connecting bonds between adjacent structural units. Structural units may be identical or different. For the purposes of this disclosure, elements include "T", "R", etc. m “L” n “A” n “B”, “Qtag” m ".

[0092] "KalbTGase" refers to the transglutaminase of *Kuznerella whiteum* as described by Steffen W. et al. (2017) J Biol Chem 292, 15622-15635. In one embodiment, KalbTGase is the polypeptide of SEQ ID NO: 65.

[0093] "KalbTGase substrate" refers to a polypeptide capable of undergoing a transglutaminase reaction catalyzed by KalbTGase, wherein an isopeptide bond is formed between a donor acyl group (particularly a donor acyl group on the glutamine (Gln, Q) side chain) and an alkylamine donor group. This effect can be clearly seen in immunoassays designed to detect antibodies bound to antigens contained in fusion polypeptides, wherein the fusion polypeptide contains one or more Qtags, which are covalently attached to a label via a Ktag and KalbTGase catalytic activity.

[0094] The inventors have discovered that, for fusion peptides, inserting a Qtag into the coupling amino acid sequence located between two adjacent FKBP chaperone proteins is highly suitable for providing an attachment site for KalbTGase-mediated Ktag coupling. Furthermore, the inventors have discovered that inserting a Qtag into the coupling amino acid sequence located between an FKBP chaperone protein and an adjacent target peptide is also highly suitable for providing an attachment site for KalbTGase-mediated Ktag coupling. Therefore, in a first aspect, this report provides a recombinant KalbTGase substrate comprising a fusion peptide of formula I.

[0095]

[0096] in

[0097] The hyphen "-" represents a peptide bond;

[0098] n is an integer from 1 to 6;

[0099] A pair of square brackets “[” and “]” delimits the N-terminal and C-terminal boundaries of an amino acid sequence that appears n times;

[0100] A n For FKBP chaperone protein amino acid sequences, and in the case of n > 1, each A n The amino acid sequence of the FKBP chaperone protein is selected independently;

[0101] L n For coupled amino acid sequences, and in the case of n > 1, each L n The amino acid sequences are selected independently;

[0102] B represents the amino acid sequence of the target polypeptide;

[0103] And among them

[0104] Each L n Having formula IIa or formula IIb

[0105]

[0106] in

[0107] m is an integer from 1 to 10;

[0108] A pair of square brackets “[” and “]” delimits the N-terminal and C-terminal boundaries of an amino acid sequence that appears m times;

[0109] T represents a flexible or rigid linker amino acid sequence containing 5 to 500 amino acids;

[0110] R m For flexible or rigid linker amino acid sequences containing 5 to 500 amino acids, and in the case of m > 1, each R m For independently selected flexible or rigid linker amino acid sequences containing 5 to 500 amino acids; and

[0111] Qtag m The motif is an amino acid sequence containing an acyl donor glutamine residue for KalbTGase transglutaminase activity, and in the case of m > 1, each Qtag m The amino acid sequence motif is independently selected and contains glutamine residues, which are acyl donors for KalbTGase transglutaminase activity.

[0112] In a specific embodiment of this aspect, SEQ ID NO: 18 and SEQ ID NO: 19 are excluded from Formula I.

[0113] In one embodiment of the recombinant KalbTGase substrate, the first aspect A n The group consisting of functional variants of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or those with 85% or higher sequence identity. In a more specific embodiment of the recombinant KalbTGase substrate, the sequence identity is any one of 90%, 95%, and 99%. In another embodiment of the recombinant KalbTGase substrate, all A n Derived from a single member of the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and said A n The amino acid sequences are at least 90% identical. In another embodiment of the recombinant KalbTGase substrate, each A nThe amino acid sequence functionally retains the FKBP chaperone protein activity in the fusion polypeptide. In another embodiment of the recombinant KalbTGase substrate, the integer n is 2 to 4.

[0114] In one embodiment of the recombinant KalbTGase substrate, B is the antigenic amino acid sequence. In a specific embodiment of the recombinant KalbTGase substrate, the antigenic amino acid sequence is derived from a peptide or polypeptide originating from a pathogen selected from the group consisting of: mammalian pathogenic viruses, mammalian pathogenic bacteria, mammalian pathogenic single-celled or multi-celled parasites, mammalian cancer cells, and prions. In a more specific embodiment of the recombinant KalbTGase substrate, the antigen amino acid sequence is derived from a peptide or polypeptide selected from a member of the group consisting of: human immunodeficiency virus, vaccinia virus, rubella virus, poliovirus, adenovirus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, dengue virus, Japanese encephalitis virus, varicella-zoster virus, cytomegalovirus, herpes simplex virus, genital herpes virus, Epstein-Barr virus, rotavirus, chikungunya virus, West Nile virus, tick-borne encephalitis virus, Zika virus, yellow fever virus, Marburg virus, Ebola virus, measles virus, mumps virus, rabies virus, MERS coronavirus, SARS coronavirus, and SARS coronavirus-2. In yet another more specific embodiment of the recombinant KalbTGase substrate, B comprises the amino acid sequence of SEQ ID NO: 76 or SEQ ID NO: 77. In another, more specific embodiment of the recombinant KalbTGase substrate, the antigenic amino acid sequence is derived from a peptide or polypeptide selected from a member of the group consisting of: Vibrio cholerae, Salmonella typhimurium, Salmonella typhi, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Helicobacter pylori, Bordetella pertussis, Streptococcus pyogenes, Streptococcus pneumoniae, Haemophilus influenzae, Clostridium tetani, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium leprae, Rickettsia rickettsia, Rickettsia kosherii, Rickettsia japonica, Rickettsia kosherii, Rickettsia japonica, Rickettsia kespermiae, Treponema pallidum, Neisseria gonorrhoeae, Neisseria meningitidis, Coccidioides immitis, Toxoplasma gondii, Borrelia auriculi, Borrelia gallini, Borrelia burgdorferi, Entamoeba histolytica, Plasmodium falciparum, Plasmodium spp., and Trypanosoma cruzi.

[0115] In one embodiment of the recombinant KalbTGase substrate, Qtag mSelected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30. SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64. In one embodiment of the recombinant KalbTGase substrate, m is 2 to 10, and each Qtag mIndependently selected from the group consisting of: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30. SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43. SEQ ID NO: 44. SEQ ID NO: 45. SEQ ID NO: 46. SEQ ID NO: 47. SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO: 64. In a more specific embodiment of the recombinant KalbTGase substrate, Qtag m Selected from YRYRQ (SEQ ID NO: 20) and RVRQR (SEQ ID NO: 21), or, in the case where m > 1, each Qtag m Independently selected from the group consisting of YRYRQ (SEQ ID NO: 20) and RVRQR (SEQ ID NO: 21). In yet another more specific embodiment of the recombinant KalbTGase substrate, all Qtags are selected. m They are the same.

[0116] In one embodiment of the recombinant KalbTGase substrate, R m T is selected from:

[0117] (i) A flexible linker amino acid sequence consisting of glycine (G) and optionally serine (S),

[0118] as well as

[0119] (ii) A rigid linker amino acid sequence consisting of glutamic acid (E), alanine (A) and lysine (K).

[0120] In one embodiment of the recombinant KalbTGase substrate, T and / or R m At least one flexible linker amino acid sequence is an independently selected amino acid sequence having the structure shown in Formula III.

[0121]

[0122] in

[0123] A pair of square brackets “[” and “]” delimits the N-terminal and C-terminal boundaries of an amino acid sequence that has been p or q times;

[0124] t is an integer from 1 to 5, representing the number of consecutive glycines when t is any one of 2 to 5;

[0125] r is zero or an integer from 1 to 3, representing the number of consecutive serines when r is 2 or 3;

[0126] q is an integer from 1 to 5, and

[0127] p is an integer from 1 to 10.

[0128] In one specific embodiment of the recombinant KalbTGase substrate, t is 2 or 3. In another specific embodiment of the recombinant KalbTGase substrate, r is 1. In yet another specific embodiment of the recombinant KalbTGase substrate, p is any one of 2 to 5, and in yet another specific embodiment p is 3 or 2.

[0129] In one embodiment of the recombinant KalbTGase substrate, T and each R m The amino acid sequence is a flexible linker. In one specific embodiment of the recombinant KalbTGase substrate, T and / or R... m At least one flexible linker amino acid sequence is selected from the group consisting of the following amino acid sequences.

[0130]

[0131] In one embodiment of the recombinant KalbTGase substrate, at least one L n Choose the group consisting of the following: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

[0132] In one embodiment of the recombinant KalbTGase substrate, T and / or any R m At least one rigid linker amino acid sequence is an independently selected amino acid sequence of formula IV.

[0133]

[0134] in

[0135] v is an integer from 2 to 5, and

[0136] w is an integer from 1 to 6.

[0137] In one specific embodiment of the recombinant KalbTGase substrate, v is 3. In another specific embodiment of the recombinant KalbTGase substrate, w is 2 to 5, and in yet another specific embodiment w is 3. In still another specific embodiment of the recombinant KalbTGase substrate, T or any R m At least one rigid linker amino acid sequence is an amino acid sequence of formula IVa.

[0138]

[0139] In one specific embodiment of the recombinant KalbTGase substrate, at least one L n Choose the group consisting of the following: SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76.

[0140] In a second aspect, this report provides a method for forming a target polypeptide with a covalently attached marker, the method comprising the following steps:

[0141] a. Providing a KalbTGase substrate according to the first aspect and all embodiments thereof, said KalbTGase substrate comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity;

[0142] b. Providing a marker conjugate, wherein in the marker conjugate, the marker is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase;

[0143] c. Under conditions that allow transglutaminase activity to occur, the fusion peptide of (a) and the labeled conjugate of (b) are contacted with KalbTGase;

[0144] This allows the acyl donor glutamine residue to react with a primary amine group to form a covalent link, thereby forming the target polypeptide with a covalently attached label.

[0145] In a third aspect, this report provides a method for forming a target polypeptide with covalently attached trapping groups, the method comprising the following steps:

[0146] a. Providing a KalbTGase substrate according to the first aspect and all embodiments thereof, said KalbTGase substrate comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity;

[0147] b. Providing a capture group conjugate, wherein in the capture group conjugate, the capture group is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase;

[0148] c. Under conditions that allow transglutaminase activity to occur, contact the fusion peptide of (a) and the capture group conjugate of (b) with KalbTGase;

[0149] This allows the acyl donor glutamine residue to react with a primary amine group to form a covalent link, thereby forming the target polypeptide with a covalently attached trapping group.

[0150] In one embodiment of the method for forming a target polypeptide with a covalently attached marker or trapping group, the KalbTGase is the polypeptide of SEQ ID NO: 65.

[0151] In the fourth aspect, this report provides a labeled target polypeptide that is obtained or available by means of the methods described in the second aspect and in all embodiments herein.

[0152] In the fifth aspect, this report provides a target polypeptide having a covalently attached trapping group, which is obtained or obtainable by means of the methods described in the third aspect and all embodiments herein.

[0153] In a sixth aspect, this report provides a composition suitable for detecting a target antibody specific for an antigenic amino acid sequence in an isolated sample, wherein the composition comprises a labeled target polypeptide according to the fourth aspect and all embodiments herein, wherein the antigenic amino acid sequence is contained in the labeled target polypeptide. In one embodiment, the label is selected from the group consisting of fluorescent dyes, chemiluminescent labels, iridium-containing electrochemiluminescent labels, ruthenium-containing electrochemiluminescent labels, single-chain oligonucleotides or analogs thereof, and radiolabels. In a more specific embodiment, the single-chain oligonucleotide analog consists of L-LNA monomers known in the art, such as those from WO2019243391 and WO2020245377. In yet another more specific embodiment, the label comprises tris(2,2'-bipyridine)ruthenium(II) ions. Other examples of labels suitable for the purposes of this report are disclosed in WO2017153574.

[0154] In one embodiment of a composition suitable for detecting a target antibody specific to an antigenic amino acid sequence in a separated sample, the composition further comprises a separate molecule containing an unlabeled antigenic amino acid sequence or a molecular mimic thereof, the separate molecule being attached to a capture group. In one specific embodiment, the separate molecule is a target polypeptide having a covalently attached capture group according to the fifth aspect of this report. In yet another specific embodiment, the capture group is selected from the group consisting of haptens, digoxigenin, and biotin.

[0155] In the seventh aspect, this report provides a method for detecting a target antibody (=X) specific for an antigen amino acid sequence (=Y) in an isolated sample, the method comprising:

[0156] a. An immune reaction mixture is formed by mixing a body fluid sample suspected of containing X with a labeled target polypeptide according to the fourth aspect and all embodiments herein, wherein Y is contained in the labeled target polypeptide.

[0157] b. Maintaining the immunoreaction blend of step (a) for a period of time sufficient to allow X present in the body fluid sample to react immunoreact with Y contained in the labeled target polypeptide to form an immunoreaction product (=X:Y) in the blend; and

[0158] c. Detect the presence and / or concentration of X:Y formed in step (b);

[0159] This allows for the detection of target antibodies specific to the amino acid sequence of the antigen in the separated sample.

[0160] In one embodiment of the method for detecting a target antibody (= X) that is specific to an antigen amino acid sequence (= Y) in a separated sample, step (c) includes the following steps:

[0161] (a) Contact the immunoreaction blend of step (a) or the blend having the immunoreaction product of step (b) with a single molecule (= Z), wherein Z contains a capture group and an unlabeled antigen amino acid sequence or a molecular mimic thereof;

[0162] (b) The blend of step (i) is maintained for a period of time sufficient to allow the formation of an immunoreaction product in which X simultaneously binds Y and Z (= Z:X:Y);

[0163] (c) Capture Z:X:Y and separate Z:X:Y from the blend described in step (ii); and

[0164] (d) Detect the markers present in the captured Z:X:Y.

[0165] In one embodiment of a method for detecting a target antibody (= X) specific for an antigenic amino acid sequence (= Y) in a separated sample, the molecular mimic of the antigenic amino acid sequence is an anti-idiotype antibody or aptamer capable of forming an immunoreaction with the target antibody, wherein the antigen is capable of competing with the aptamer or anti-idiotype antibody for binding to the target antibody. In one specific embodiment, the molecular mimic is an antibody-binding fragment of the anti-idiotype antibody.

[0166] In another embodiment of the method for detecting a target antibody (= X) that is specific to an antigen amino acid sequence (= Y) in a separated sample, Z is a target polypeptide having a covalently attached capture group as described in the fifth aspect of this report.

[0167] In yet another embodiment, step (iii) includes capturing Z:X:Y on a solid phase and separating the solid phase having the captured Z:X:Y from the blend of step (ii). In one specific embodiment, the solid phase is any of the magnetic, paramagnetic, or superparamagnetic beads known in the art. In yet another more specific embodiment, the surface of the solid phase comprises streptavidin, and Z comprises biotin as a capturing group.

[0168] In the eighth aspect, this report provides (i) the KalbTGase substrate according to the first aspect and all embodiments herein, (ii) the label conjugate and (iii) the use of KalbTGase for the production of labeled target peptides, wherein in the label conjugate, the label is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase.

[0169] In the ninth aspect, this report provides the use of labeled target peptides obtained or available by the methods described in the fourth aspect and all embodiments herein for the detection of target antibodies specific to the amino acid sequence of an antigen in isolated samples.

[0170] In the tenth aspect, this report provides the use of labeled target peptides according to the fourth aspect and all embodiments herein, and individual target peptides with covalently attached capture groups according to the fifth aspect and all embodiments herein, for detecting target antibodies specific to the amino acid sequence of an antigen in isolated samples.

[0171] In the eleventh aspect, this report provides a component kit for detecting a target antibody specific to an antigenic amino acid sequence in an isolated sample, the kit comprising a labeled target polypeptide according to the fourth aspect and all embodiments herein, the labeled target polypeptide comprising the antigenic amino acid sequence.

[0172] In one embodiment of a component kit for detecting a target antibody specific for an antigenic amino acid sequence in a separated sample, the kit further comprises individual molecules containing a capture group and an unlabeled antigenic amino acid sequence or a molecular mimic of it. In one embodiment, the molecular mimic of the antigenic amino acid sequence is an anti-idiotype antibody or aptamer capable of forming an immunoreaction with the target antibody, wherein the antigen is capable of competing with the aptamer or anti-idiotype antibody for binding to the target antibody. In a specific embodiment, the molecular mimic is an antibody-binding fragment of the anti-idiotype antibody. In another embodiment, the individual molecule is a target polypeptide having a covalently attached capture group as described in the fifth aspect herein.

[0173] In another embodiment of a component kit for detecting target antibodies specific to the antigen amino acid sequence in a separated sample, the kit further comprises a solid phase capable of capturing the capturing group. In a more specific embodiment, the solid phase is any of magnetic, paramagnetic, or superparamagnetic beads known in the art. In yet another more specific embodiment, the surface of the solid phase comprises streptavidin, and the capturing group is biotin.

[0174] In the twelfth aspect, this report provides a DNA encoding a fusion polypeptide comprising a target polypeptide and one or more acyl donor glutamine residues for KarbTGase transglutaminase activity, wherein a codon for an N-terminal methionine is appended to a nucleotide sequence encoding an amino acid sequence of a KarbTGase substrate according to the first aspect and all embodiments herein.

[0175] In the thirteenth aspect, this report provides an expression vector for recombinant expression in a transformed organism, the expression vector comprising DNA as described in the twelfth aspect and all embodiments herein.

[0176] In the fourteenth aspect, this report provides a prokaryotic host organism stably transformed with an expression vector according to the thirteenth aspect and all embodiments herein, wherein the transformed host organism is capable of expressing the encoded KarbTGase substrate from the expression vector.

[0177] In aspect fifteen, this report provides a method for producing recombinant KalbTGase substrates, the method comprising the following steps:

[0178] (a) Providing and culturing the transformed host organism according to aspect fourteen and all embodiments herein,

[0179] (b) Expressing the recombinant KalbTGase substrate in the transformed host organism described in (a), and

[0180] (c) Purify the expressed recombinant KalbTGase substrate from the transformed host organism.

[0181] This produces the recombinant KalbTGase substrate.

[0182] Example

[0183] Example 1: Expression and purification of recombinant HIV1 gp41 and 6hel antigens

[0184] Small-scale preparation of recombinant 6hel antigen for high-throughput screening

[0185] Twenty-two plasmids containing gp41-6hel genes with different point mutations and C-terminal hexahistine tags were synthesized in Twist Bioscience and cloned into pET29a via NdeI (5' end) and XhoI (3' end) restriction sites.

[0186] Small-scale expression of recombinant gp41-6hel protein

[0187] After diluting the plasmid to 5 to 10 ng / µl in 10 mM Tris-HCl buffer (pH 8.5), add 1 µl of DNA to HT96 BL21 (DE3) competent cell plates (Novagen). Transform according to the manufacturer's protocol, and plate 20 µL of the transformation reaction product onto 48-well LB-kanamycin (50 µg / ml) agar plates (Teknova).

[0188] All variants were cultured and expressed in 96-well plates. For pre-culture, one colony of each mutant was selected from each well in a 96-well flat-bottom microtiter plate containing 200 µl of 4x yeast-kanamycin (50 µg / ml) medium. Additionally, each plate contained at least one wild-type gp41 antigen as a reference. Cells were grown overnight at 37°C without shaking. For long-term storage, 50 µl of 50% (v / v) glycerol was added before freezing. Expression was performed in 96-well deep-well plates, each well containing 1000 µl of 4x yeast-kanamycin (50 µg / ml) medium containing 0.1 mM IPTG. Mutants were expressed at 30°C and 800 rpm (Microplate Shaker TiMix; Edmund Bühler GmbH) and harvested after 16 hours by centrifugation at 4700 rpm for 10 minutes.

[0189] Small-scale purification of recombinant gp41-6hel protein

[0190] For small-scale purification of the 6hel antigen, bacterial cell pellet from a 1 ml *E. coli* culture was lysed using 125 µl of 100% BugBuster® (Merck Millipore) according to the manufacturer's protocol. After adding 125 µl of 2x equilibration buffer (0.1 M NaH₂PO₄ pH 8.0; 1% (v / v) Tween-20; 1 M NaCl; 40 mM imidazole) and removing the cell lysate by centrifugation (4700 rpm, 10 min), the lysate was transferred to a 96-well V plate (Corning) using a Biomek robotic pipette tip. Small-scale purification was performed using a PhyTips (PhyNexus) robotic pipette tip pre-loaded with Ni-NTA resin. First, equilibrate the phytips with equilibration buffer (0.05 M NaH₂PO₄ pH 8.0; 0.5% (v / v) Tween-20; 0.5 M NaCl; 20 mM imidazole). Then transfer them to the sample for protein binding. To remove non-specifically bound proteins, wash the phytips twice with wash buffer 1 (0.05 M NaH₂PO₄ pH 8.0; 0.5% (v / v) Tween-20; 0.5 M NaCl; 20 mM imidazole), followed by two washes with wash buffer 2 (0.05 M NaH₂PO₄ pH 8.0; 0.5% (v / v) Tween-20; 0.15 M NaCl; 20 mM imidazole). Finally, the 6hel antigen was eluted in 100 µl of elution buffer (0.05 M NaH2PO4 pH 8.0; 0.5% (v / v) Tween-20; 0.15 M NaCl; 200 mM imidazole). Protein samples were analyzed by SDS-PAGE gel chromatography. After Ni-NTA purification, the buffer was exchanged to conjugation buffer (0.15 M KH2PO4 pH 8.0; 0.1 M KCl; 0.5 mM EDTA) using a Pierce™ 96-well microdialysis plate according to the Pierce Biotechnology instructions.

[0191] Small-scale ruthenium acylation and biotinylation of recombinant gp41-6hel protein

[0192] Conjugation was performed using NHS Chemistry in black 96-well half-zone plates (Corning). Prior to conjugation, protein concentrations in each well were determined in a microtiter plate using a Pierce™ BCA protein assay kit (ThermoFisher) via BCA assay. 180 µl of BCA solution was added to 20 µl of purified antigen per well and measured at 562 nm using a Tecansunrise™ microplate reader.

[0193] The antigen (approximately 1 mg / ml) and label were rapidly mixed to a final antigen-to-label ratio of 1:4 (ruthenium conjugation) and 1:5 (biotin conjugation), with a DMSO concentration of 10% (v / v). The plate was incubated at 600 rpm for 30 minutes at room temperature. The labeling reaction was terminated by adding L-lysine to a final concentration of 10 µM. For small-scale preparations, unbound ruthenium label was not removed, but unbound biotin label was removed using a PD MultiTrap™ G-25 96-well plate (GE Healthcare). The concentrations of ruthenium-acylated and biotinylated antigens were determined using a BCA assay as described above. The ruthenium-acylated and biotinylated gp41-6hel mutant was stored at 4°C until evaluation using an Elecsys assay system.

[0194] In summary, 171 (71%) of the 242 6hel mutations were successfully purified, labeled, and further evaluated by immunoassay. The remaining 71 variants either failed to be expressed due to DNA synthesis failure or the yield of purified protein was too low for labeling.

[0195] Large-scale preparation of recombinant HIV1 gp41 and 6hel antigens for thorough screening

[0196] Plasmids containing recombinant HIV1 gp41 (aa536-681) and 6hel genes (with different point mutations and C-terminal hexahistine tags) were synthesized at Eurofins Genomics GmbH and cloned into pET24a(+) via NdeI (5' end) and XhoI (3' end) restriction sites.

[0197] In addition, recombinant gp41 (aa536-681) was fused at the N-terminus with two SlyD chaperone proteins from Escherichia coli via a glycine-serine-rich linker (Scholz, C. et al., J. Mol. Biol. (2005) 345, 1229-1241) to produce the EcSlyD-EcSlyD-gp41 fusion protein, which will be referred to as gp41 in the following text.

[0198] gp41 and the 6hel construct were expressed in BLR(DE3) E. coli cells using standard LB medium and IPTG induction at 37°C for three hours. Cells were harvested by centrifugation (20 min, 5000 g) and stored at -20°C after further processing.

[0199] Large-scale purification of recombinant HIV1 gp41 and 6hel antigens

[0200] Recombinant HIV1 gp41 and 6hel antigens were purified under denaturing conditions and then subjected to on-column renaturation. Specifically, the bacterial pellet from 700 ml of *E. coli* culture was resuspended in lysis buffer (50 mM sodium phosphate, pH 8.0; 4 M guanidine chloride; 5 mM imidazole) and stirred at room temperature for 90 min. For purification, the cell lysates were centrifuged and filtered (5 / 0.8 / 0.2 µm). The clear supernatant was added to a Roche cOmpleteHis-tag purification column equilibrated with lysis buffer. Non-specifically bound proteins were removed from the column by thoroughly washing to baseline with lysis buffer. Antigen refolding was performed by on-column renaturation using refolding buffer (50 mM sodium phosphate, pH 8.0; 100 mM NaCl). The refolded target proteins were eluted from the column using elution buffer containing imidazole (50 mM sodium phosphate, pH 8.0; 50 mM imidazole; 100 mM NaCl). For buffer exchange and purification, the protein was applied to a Superdex 200 column, which was equilibrated with SEC buffer 1 (50 mM Tris-HCl pH 8.0; 150 mM KCl) for site-specific labeling or with SEC buffer 2 (150 mM potassium phosphate pH 8.9; 100 mM KCl; 0.5 mM EDTA) for labeling using NHS chemistry. Gp41 eluted in three peaks, with one main peak representing the oligomeric arrangement. The oligomeric fractions were concentrated and biotinylated and ruthenylated. 6hel eluted in one peak, concentrated, and biotinylated and ruthenylated.

[0201] Example 2: Ruthenium acylation and biotinylation of recombinant gp41 protein

[0202] Large-scale ruthenylation and biotinylation of recombinant HIV1 gp41 and 6hel antigens using NHS chemistry.

[0203] For antigen conjugation with biotin or ruthenium, the protein concentration in SEC buffer 2 should ideally be 10 mg / ml. Conjugation was performed using NSH Chemistry at a 1:4 molar ratio of antigen to label and a 5% (v / v) concentration of DMSO. The label and antigen were rapidly mixed and stirred at room temperature for 30 minutes. The labeling reaction was terminated by adding L-lysine to a final concentration of 10 mM. For large-scale preparation, free unbound label was removed from the reaction by size exclusion chromatography using a Superdex 200 Increase (GE Healthcare) column equilibrated with storage buffer (50 mM sodium phosphate pH 7.5; 100 mM KCl; 0.5 mM EDTA). The concentration of ruthenium-acylated antigen was determined by BCA assay, and the concentration of biotinylated antigen was determined by absorbance measurement at 280 nm.

[0204] Large-scale ruthenylation and biotinylation of recombinant HIV1 gp41 and 6hel using transglutaminase

[0205] Recombinant transglutaminase from *KalbTG* can be used for site-specific labeling of antigens by forming Gln-Lys isopeptide bonds between Q-tag-containing antigens and corresponding K-tag-containing markers (Steffen, W. et al., J. Mol. Biol. (2017) 292, 15622-1563). For conjugation of HIV1 antigens with biotin or ruthenium, the protein concentration in SEC buffer 2 should ideally be 10 mg / ml. Conjugation was performed at a Qtag to marker molar ratio of 1:5 and an enzyme to antigen deficiency ratio of 1:300. The antigen, marker, and activated enzyme were mixed and incubated at 37°C for 20 hours with gentle mixing. After 20 hours of incubation, the reaction was terminated by adding 10 mM ammonium sulfate. Finally, free unbound label and KalbTG were removed from the labeled antigen by size exclusion chromatography using a Superdex 200 Increase (GE Healthcare) column equilibrated with storage buffer (50 mM sodium phosphate pH 7.5; 100 mM KCl; 0.5 mM EDTA). The concentration of ruthenium-acylated antigen was determined by BCA assay, and the concentration of biotinylated antigen was determined by absorbance measurement at 280 nm.

[0206] Large-scale biotinylation of recombinant HIV1 gp41 and 6hel using sorting enzymes

[0207] Recombinant sorting enzymes can be used to site-specifically label antigens by forming a peptide bond between a threonine residue at the C-terminal sorting enzyme recognition site (LPETG) and a glycine residue in the corresponding label. For conjugation of HIV1 antigen to biotin via sorting enzyme, the ideal protein concentration in phosphate-free SEC buffer 1 should be 10 mg / ml. Conjugation is performed in the presence of 10 mM calcium chloride at an antigen-to-label ratio of 1:50 and an enzyme input of 50 U / µmol antigen. The antigen, label, and activated enzyme are mixed and incubated at 37°C for 1 hour with gentle mixing. After 1 hour of incubation, the reaction mixture is loaded onto Roche cOmplete His-labeled resin to remove the sorting enzyme and unlabeled antigen from the reaction mixture. Finally, free, unbound label was removed by size exclusion chromatography using a Superdex 200 Increase (GE Healthcare) column equilibrated with storage buffer (50 mM sodium phosphate, pH 7.5; 100 mM KCl; 0.5 mM EDTA). The concentration of biotinylated antigen was determined by absorbance measurement at 280 nm.

[0208] Example 3: Biochemical analysis of recombinant HIV1 gp41 and 6hel antigens

[0209] Spectroscopic measurements of recombinant HIV1 gp41 and 6hel antigens

[0210] Protein concentrations were measured using a NanoDrop One® microUV / visible spectrophotometer (Thermo Scientific). The molar extinction coefficient (ε280 nm) of the antigen was calculated using the equation reported by Pace et al. (Protein Sci. 1995 Nov;4(11):2411-23).

[0211] Table 1: Protein parameters of five optimal recombinant HIV1 gp41 and 6hel antigens

[0212]

[0213] Circular dichroism (CD) spectrum of recombinant HIV1 6hel antigen

[0214] The far-UV CD spectra (190–250 nm) of the 6hel antigen were recorded using a Jasco-720 spectropolarimeter and ultimately converted to the mean residue ellipticity (λmrw). All samples were diluted to 0.21 mg / mL in 50 mM potassium phosphate (pH 7.5), 100 mM KCl, and 0.5 mM EDTA. The spectrometer was adjusted during measurements as follows: 0.2 cm path length, scan range 190–330 nm, scan rate 20 nm / min, bandwidth 2.0 nm, resolution 0.5 nm, and response time 1 second. All spectra were measured nine times and averaged.

[0215] In the far UV range, CD spectroscopy allows for the analysis of secondary structure proteins because absorption in this UV range is primarily caused by peptide bonds. Therefore, the far UV CD spectroscopy of the full-helical 6hel antigen provides reliable insights into antigen structure and the impact of point mutations on protein folding, compared to mutant variants.

[0216] HPLC analysis of recombinant HIV1 6hel antigen

[0217] To analyze the purity and aggregation tendency of the mutant antigen and to estimate the molecular weight of the purified 6hel antigen, HPLC analysis was performed. Therefore, at least 25 µg of recombinant protein was loaded onto a Superdex 200 column using 50 mM potassium phosphate (pH 7.5), 100 mM KCl, and 0.5 mM EDTA as the mobile phase. Internal HPLC standards were also analyzed for reference. HPLC analysis allowed for the assessment of the aggregation behavior of the mutant 6hel antigen compared to the wild-type construct.

[0218] Example 4: Immunoreactivity of different recombinant HIV1 gp41 and 6hel antigens in anti-HIV immunoassay

[0219] Immunoreactivity (antigenicity) of HIV1 gp41 and 6hel variants was assessed in an automated Elecsys® cobas analyzer (Roche Diagnostics GmbH) using a dual-antigen sandwich (DAGS) format. Signal detection in the automated Elecsys® cobas analyzer is based on electrochemiluminescence. In the DAGS assay format, biotinylated capture antigens are immobilized on the surface of streptavidin-coated magnetic beads, while the same detection antigen is conjugated with a ruthenium complex. Upon activation, the ruthenium complex switches between redox states 2+ and 3+, generating a light signal. In the presence of a specific immunoglobulin (in this case, anti-HIV IgG antibodies in human serum), the ruthenium complex bridges to the solid phase, triggering light emission at 620 nm at the electrode by the addition of tripropylamine.

[0220] This study investigated all 171 mutant variants of recombinant 6hel expressed and labeled on a small scale (Figure 3) to assess their binding potential with anti-HIV1 IgG antibodies.

[0221] Immunoreactivity of different recombinant HIV1 gp41 and 6hel antigens in DAGS assay settings

[0222] To conduct a more thorough analysis, approximately 20 optimal mutations in the 6hel antigen were expressed and labeled on a large scale and fully analyzed in a DAGS assay setting. These mutations were identified as having improved immunospecificity during initial screening. Furthermore, the same selected mutations were also transferred to the HIV1 gp41 antigen (WO03 / 000877) and their specificity was evaluated. In addition to these constructs, 6hel and gp41 antigens containing the most promising combinations of mutations were generated and evaluated.

[0223] Specifically, different gp41-biotin or 6hel-biotin and gp41-ruthenium or 6hel-ruthenium antigens were used in reagent buffer 1 (R1) and R2, respectively. Labeled recombinant gp41 antigens were used in R1 and R2 at concentrations ranging from 30 ng / ml to 300 ng / ml. The concentrations of various labeled 6hel antigens in R1 and R2 ranged from 2 ng / ml to 130 ng / ml, depending on the mutation.

[0224] To avoid immune cross-reaction via the chaperone protein fusion unit of the recombinant HIV1 gp41 antigen, a large excess (5 to 30 µg / ml) of unlabeled EcSkp-EcSlyD-EcSlyD (EP2893021(B1)) or chemically polymerized EcSlyD-EcSlyD was added to the reaction buffer as an anti-interference agent.

[0225] To assess the specificity and sensitivity of different recombinant HIV1 gp41 and 6hel antigens, Elecsys measurements were analyzed using HIV-negative, positive, and seroconverted samples.

[0226] The results for the three optimal antigens (SEQ ID No. 1, 2, and 3) are shown in Figure 6. Specifically, A) Cutoff index (COI) of 10 highly positive HIV samples from patients infected with different HIV-1 subtypes. As with the standard AHIVI module, all samples tested with the modified anti-HIV module (AHIVII) were positive. A COI value < 1 was indicated as non-reactive, while samples with a COI > 1 indicated the presence of anti-HIV antibodies. B) Performance comparison of the standard Elecsys HIV Duo assay (black HIV Duo I) with the optimized Elecsys HIV Duo II assay (gray HIV Duo II), and separate comparisons of the gray and black anti-HIV modules AHIV I and AHIV II, respectively. This comparison was performed on five commercially available seroconversion plates (1–5), with blood drawn sequentially. The optimized anti-HIV II module showed higher sensitivity compared to the AHIV I module. The higher sensitivity of the AHIV II module was particularly evident in serological conversion plates 2 and 3. In both plates, nine or five blood draws (highlighted in gray) were negative in the AHIV I module, but significantly positive in the optimized AHIV II module. This higher sensitivity reduced the risk of a second window period post-infection and significantly reduced the risk of false-negative HIV results. The C) graph and D) figures represent the specificity of the current and optimized anti-HIV modules, respectively, in black and gray. The specificity of the two modules was determined using 6046 routine HIV-negative samples from different vendors. With the HIV-positive threshold set to COI >1 (highlighted in bold), the standard AHIV I module showed four false-positive samples, resulting in a specificity of 99.92. The optimized AHIV II module, however, showed no signal for COI >1, resulting in a specificity of 100. In conclusion, the experiments conducted here demonstrate that the AHIV II module significantly improves sensitivity and specificity compared to the AHIV module.

[0227] Example 5: Data from external specificity studies

[0228] To thoroughly assess the specificity of the mutated and thus optimized gp41 and 6hel antigens, 15,242 routine blood samples were analyzed in an external study (Figure 7a). This assessment was performed by an independent laboratory using the Elecsys HIV Duo AHIV module and the optimized Elecsys HIV Duo II assay (containing SEQ ID NO 79, 80, and 81).

[0229] In this study, 44 samples produced false positive signals in the Elecsys HIV Duo I assay (99.71% specificity), while only 7 false positive samples were detected using the optimized HIV Duo II assay (99.95% specificity) (Figure 7A). The 21 false positive samples in the Elecsys HIV Duo I assay and the 4 false positive samples in the HIV Duo II assay were caused by gp41 and 6hel antigens within the anti-HIV module of the HIV Duo Elecsys assay (Figure 7b). Therefore, the significant improvement in specificity of the mutated and optimized gp41 and 6hel antigens can also be seen in independent external studies, and the interference potential of the gp41 antigen was reduced to <20% after optimization.

[0230] Example 6: Improving the sensitivity and specificity of HIV immunoassays through a combination of mutation and optimized HIV gp41 antigen.

[0231] Compared to HIV immunoassays containing the HIV gp41 antigen included in SEQ ID NO. 10, not only did the combination of SEQ ID NO. 79, 80, and 81 show significantly improved antigenicity, but combinations of two optimized HIV gp41 antigens (SEQ ID NO. 79 and 80 or SEQ ID NO. 79 and 81) also showed significantly improved immunoreactivity (Figure 8). The specificity of different antigen combinations was assessed by analyzing 103 HIV-negative samples (Figure 8a). When using the unoptimized gp41 antigen, only 91 of the 103 negative samples (88.35%) showed a COI between 0.02 and 0.05, while when using different combinations of mutated gp41 and 6hel antigens, at least 98% of the negative samples were within this COI range (Figure 8a, columns 2-3). Therefore, sample scattering was significantly reduced when using optimized antigens. In addition, not only were specificity and scattering improved, but the sensitivity of different combinations of gp41 and 6hel antigens optimized was also significantly better (Figure 8b). For example, sample Sero01 clearly showed much higher sensitivity in all combinations using mutant antigens, with SEQ ID NO. 79, 80 and 81 being the optimal combinations.

[0232] Example 7: Rutheniumization of a fusion peptide containing four YRYRQ Qtags

[0233] Recombinant fusion peptides containing Qtag were labeled using a ruthenium-labeled conjugate, provided as a conjugate with a Ktag peptide (GRYESKG, SEQ ID NO: 95) containing an alkylamine donor group for a lysine residue of KalbTGase. In a separate labeling reaction, the Qtag-containing fusion peptide was mixed with a Ru conjugate containing Ktag. KalbTGase was added and incubated. Conditions were as previously described by Steffen et al. (2017) J BiolChem 292, 15622-15635. Unreacted labeled Ktag conjugates were separated from the labeled fusion peptides by size exclusion chromatography. Elution was monitored by measuring optical density at 280 nm and 455 nm.

[0234] In the same manner, any fusion polypeptide having a Qtag is labeled. An exemplary Ru label is disclosed in Figure 1. Suitable compounds for biotin-Ktag attachment are disclosed in Figure 2. Biotin-Ktag attachment using KalbTGase is performed similarly to the above, replacing the Ru label with biotin-Ktag.

[0235] Example 8: Applications in immunoassays

[0236] In an immunoassay for detecting a target antibody (=X) specific to an antigen amino acid sequence (=Y) in a sample, using a ruthenium-labeled fusion polypeptide of SEQ ID NO: 96 as described in Example 7, the method comprises...

[0237] (a) An immune reaction mixture is formed by mixing a serum or plasma sample, with or without X, with a labeled target peptide, wherein the labeled target peptide contains dengue virus NS1 antigen.

[0238] (b) Maintaining the immunoreaction blend of step (a) for a period of time sufficient to allow X present in the body fluid sample to react immunoreact with Y contained in the labeled target polypeptide to form an immunoreaction product (=X:Y) in the blend; and

[0239] (c) Perform the following steps:

[0240] (d) Contact the immunoreaction blend of step (a) or the blend having the immunoreaction product of step (b) with a single molecule (= Z), wherein Z contains biotin as a capture group and an unlabeled antigen amino acid sequence;

[0241] i. Maintain the blend from step (d) for a period of time sufficient to allow the formation of an immunoreaction product in which X simultaneously binds Y and Z (= Z:X:Y);

[0242] ii. Z:X:Y is captured on magnetic beads coated with streptavidin, and Z:X:Y is separated from the blend of step (ii) by magnetic force; and

[0243] iii. Detect the markers present in the captured Z:X:Y region fixed on the magnetic bead.

[0244] Detection was performed on an ELECSYS® analyzer capable of detecting immobilized Ru labels by electrochemiluminescence (ESL). Parallel experiments were conducted using conventionally prepared Ru-labeled fusion peptides without Qtags. These molecules were labeled using a cross-linking reaction. Samples were provided that were positive or negative for antibodies known to be specific to the dengue virus NS1 antigen. Table 2 summarizes the data.

[0245] Based on the double-antigen sandwich principle, the typical duration of measurement is 18 minutes.

[0246] First incubation: A defined volume of sample, biotinylated recombinant antigen, and the same recombinant antigen labeled with a ruthenium complex are mixed to form a sandwich complex.

[0247] Second incubation: After the addition of streptavidin-coated microparticles, the complex binds to solids via the interaction of biotin and streptavidin.

[0248] The reaction mixture is drawn into a measuring cell, where particles are magnetically trapped onto an electrode surface. Unbound material is then washed away with a specific buffer solution that simultaneously provides the luminescent reagent.

[0249] A voltage is then applied to the electrodes to induce chemiluminescence emission, which is then measured using a photomultiplier tube.

[0250] Table 2: ESL Photon Count

[0251]

[0252] As can be clearly seen from the data in Table 2, in the case of KalbTGase-labeled fusion peptides, the baseline signal generated by the negative samples is much lower than the baseline signal in the experiments of chemically labeled fusion peptides.

[0253] Example 9: Applications in immunoassays, different temperature protocols

[0254] This effect could be reproduced when Example 8 was repeated after incubating the labeled fusion peptide at 4°C and 35°C for one week. This indicates that the stability of the KarbTGase-labeled fusion peptide is increased.

[0255] Table 3a: Comparison of ESL photodetector reagents after storage at 4°C and 35°C for 1 week.

[0256]

[0257] Table 3b: Relative light counts after incubation at 35℃ compared to incubation at 4℃ (100%)

[0258]

[0259] The data show that when Qtag, KtagRu, and KalbTGase labels are used to label the fusion peptides, not only do the photocounts of the negative control samples remain very stable, but the photocounts of the positive control samples also remain very stable.

[0260] Example 10: Applications in immunoassays

[0261] Example 8 was repeated in a modified form, using a different antigen, specifically HIV gp41 instead of the dengue virus NS1 antigen. The fusion polypeptide was the fusion polypeptide of SEQ ID NO: 86. Using the ruthenium-labeled fusion polypeptide of SEQ ID NO: 86 as described in Example 7, in an immunoassay for detecting a target antibody (=X) specific to the antigen amino acid sequence (=Y) in a sample, the method included...

[0262] (a) An immune reaction mixture is formed by mixing a serum or plasma sample containing or without X with a labeled target peptide, wherein the labeled target peptide contains the HIV gp41 antigen.

[0263] (b) Maintaining the immunoreaction blend of step (a) for a period of time sufficient to allow X present in the body fluid sample to react immunoreact with Y contained in the labeled target polypeptide to form an immunoreaction product (=X:Y) in the blend; and

[0264] (c) Perform the following steps:

[0265] (d) Contact the immunoreaction blend of step (a) or the blend having the immunoreaction product of step (b) with a single molecule (= Z), wherein Z contains biotin as a capture group and an unlabeled antigen amino acid sequence;

[0266] i. Maintain the blend from step (d) for a period of time sufficient to allow the formation of an immunoreaction product in which X simultaneously binds Y and Z (= Z:X:Y);

[0267] ii. Z:X:Y is captured on magnetic beads coated with streptavidin, and Z:X:Y is separated from the blend of step (ii) by magnetic force; and

[0268] iii. Detect the markers present in the captured Z:X:Y region fixed on the magnetic bead.

[0269] Further variations were introduced by attaching biotin to Z via chemical coupling or by KalbTGase-mediated labeling.

[0270] The assay was performed on an ELECSYS® analyzer capable of detecting immobilized Ru markers via electrochemiluminescence (ESL).

[0271] Table 4: Comparison of ruthenium labeling and biotin labeling using chemical or KalbTGase labeling, with the fusion peptide consistently at 70 ng / ml.

[0272]

[0273] As can be clearly seen from the table, the KarbTGase-based labeling yields the best results not only for attaching ruthenium labels but also for attaching biotin labels to fusion peptides with Qtags.

[0274] Example 10: Applications in immunoassays

[0275] Fusion peptides of SEQ ID NO: 91, 92, 93, 94, and 96 were labeled with ruthenium using KalbTGase. The fusion peptides with the trapping group were biotinylated chemically. The ruthenium-labeled fusion peptides differed in their linker amino acid sequences and the Qtags used. The signals generated by all labeled peptides in immunoassays for antibody detection were compared. Z:X:Y sandwich complexes were immobilized, and ELECSYS® photocounts were measured.

[0276] Table 5: Signal yield of fusion peptides using SEQ ID NO: 91, 92, 93, 94 and 96

[0277] The biotin-labeled density in Z was the same for every experiment.

[0278]

[0279] Example 11: Quantification of Ru-labeled incorporation

[0280] The product obtained from KalbTGase incubation as described in Example 7 was subjected to size exclusion chromatography using a Superdex® 200 column. Elution was monitored by measuring the absorbance at 455 nm and 280 nm wavelengths. An exemplary elution profile is shown in Figure 3. Fractions under the asterisk-labeled peaks were combined. Protein concentration was determined using the BCA assay. The molar ratio of Ru-labeled peptides to each labeled fusion peptide was calculated using the extinction coefficient of the individual Ru-labeled peptides as a reference. The ratio was typically found to be between .5 and 4, depending on the availability of the Qtag to the labeling reaction in the presence of KalbTGase. An exemplary chromatogram of the purified Ru-labeled fusion peptide is shown in Figure 4, along with another chromatogram of the fusion peptide with biotin attached to the Qtag.

Claims

1. A recombinant KalbTGase substrate comprising a fusion polypeptide of formula I, in The hyphen "-" represents a peptide bond; n is an integer from 1 to 6; A pair of square brackets "[" and "]" defines the N-terminal and C-terminal boundaries of an amino acid sequence that appears n times; A n For FKBP chaperone protein amino acid sequences, and in the case of n > 1, each A n The amino acid sequence of the FKBP chaperone protein is selected independently; L n For coupled amino acid sequences, and in the case of n > 1, each L n The amino acid sequences are selected independently; B represents the amino acid sequence of the target polypeptide; And among them Each L n Having formula IIa or formula IIb in m is an integer from 1 to 10; A pair of square brackets "[" and "]" defines the N-terminal and C-terminal boundaries of an amino acid sequence that appears m times; T represents a flexible or rigid linker amino acid sequence containing 5 to 500 amino acids; R m For flexible or rigid linker amino acid sequences containing 5 to 500 amino acids, and in the case of m > 1, each R m For independently selected flexible or rigid linker amino acid sequences containing 5 to 500 amino acids; Qtag m The motif is an amino acid sequence containing an acyl donor glutamine residue for KalbTGase transglutaminase activity, and in the case of m > 1, each Qtag m The amino acid sequence motif is independently selected and contains an acyl donor glutamine residue for KalbTGase transglutaminase activity; The condition is that SEQ ID NO: 18 and SEQ ID NO: 19 are excluded from Formula I.

2. The recombinant KalbTGase substrate according to claim 1, wherein A n Selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or their functional variants having 85% or higher sequence identity.

3. The recombinant KalbTGase substrate according to claim 1 or 2, wherein n is 2 to 6.

4. The recombinant KalbTGase substrate according to claim 3, wherein each A n The group consisting independently of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or their functional variants having 85% or higher sequence identity.

5. The recombinant KalbTGase substrate according to claim 3 or 4, wherein all A n Derived from a single member of the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, and said A n The amino acid sequences are at least 90% identical.

6. The recombinant KalbTGase substrate according to any one of claims 3 to 5, wherein each A n The amino acid sequence in the fusion polypeptide functionally retains the FKBP chaperone protein activity.

7. The recombinant KalbTGase substrate according to any one of claims 1 to 6, wherein B is the antigenic amino acid sequence.

8. The recombinant KalbTGase substrate of claim 7, wherein B comprises the amino acid sequence of SEQ ID NO: 76 or SEQ ID NO:

77.

9. The recombinant KalbTGase substrate according to any one of claims 1 to 8, wherein Qtag m Selected from YRYRQ (SEQ ID NO: 20) and RVRQR (SEQ ID NO: 21), or, in the case where m > 1, each Qtag m The group consisting of YRYRQ (SEQ ID NO: 20) and RVRQR (SEQ ID NO: 21) is selected independently.

10. The recombinant KalbTGase substrate according to any one of claims 1 to 17, wherein R m T is selected from: (i) A flexible linker amino acid sequence consisting of glycine (G) and optionally serine (S), as well as (ii) A rigid linker amino acid sequence consisting of glutamic acid (E), alanine (A) and lysine (K).

11. The recombinant KalbTGase substrate according to any one of claims 1 to 10, wherein at least one L n The group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 is selected.

12. The recombinant KalbTGase substrate of claim 10, wherein T and / or any R m At least one rigid linker amino acid sequence is an independently selected amino acid sequence of formula IV. in v is an integer from 2 to 5, and w is an integer from 1 to 6.

13. The recombinant KalbTGase substrate according to any one of claims 1 to 10, wherein at least one L n The group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76 is selected.

14. A method for forming a target polypeptide having a covalently attached marker, the method comprising the steps of: (a) Providing a KalbTGase substrate according to any one of claims 1 to 13, the KalbTGase substrate comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity; (b) Providing a marker conjugate, wherein in the marker conjugate, the marker is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase. (c) Contact the fusion peptide of (a) and the labeled conjugate of (b) with KalbTGase under conditions that allow transglutaminase activity to occur; This allows the acyl donor glutamine residue to react with the primary amine group to form a covalent bond. This forms the target polypeptide with the covalently attached marker.

15. A method for forming a target polypeptide having a covalently attached trapping group, the method comprising the steps of: (a) Providing a KalbTGase substrate according to any one of claims 1 to 13, the KalbTGase substrate comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity; (b) Providing a capture group conjugate, wherein in the capture group conjugate, the capture group is covalently attached to a Ktag, wherein the Ktag is a lysine-containing receptor motif of KalbTG or a functional analog thereof, wherein the Ktag contains a primary amine group capable of reacting with an acyl donor glutamine residue for KalbTGase transglutaminase activity in the presence of KalbTGase. (c) Contact the fusion peptide of (a) and the capture group conjugate of (b) with KalbTGase under conditions that allow transglutaminase activity to occur; This allows the acyl donor glutamine residue to react with the primary amine group to form a covalent bond. This forms the target polypeptide with covalently attached trapping groups.

16. A labeled target polypeptide, which is obtained or can be obtained by the method according to claim 14.

17. A target polypeptide having a covalently attached trapping group, said target polypeptide being obtained or obtainable by the method according to claim 15.

18. A DNA encoding a fusion polypeptide comprising a target polypeptide and one or more acyl donor glutamine residues for KalbTGase transglutaminase activity, wherein a codon for an N-terminal methionine is appended to a nucleotide sequence encoding an amino acid sequence of a KalbTGase substrate according to any one of claims 1 to 13.

19. An expression vector for recombinant expression in a transformed organism, the expression vector comprising the DNA according to claim 18.

20. A prokaryotic host organism stably transformed using the expression vector according to claim 19, wherein the transformed host organism is capable of expressing the encoded KarbTGase substrate from the expression vector.

21. A method for producing recombinant KalbTGase substrate, the method comprising the following steps: (a) Providing and culturing the transformed host organism according to claim 20, (b) Expressing the recombinant KalbTGase substrate in the transformed host organism described in (a), and (c) Purify the expressed recombinant KalbTGase substrate from the transformed host organism. This produces the recombinant KalbTGase substrate.

22. A composition suitable for detecting a target antibody specific to an antigenic amino acid sequence in an isolated sample, wherein the composition comprises a labeled target polypeptide according to claim 16, wherein the antigenic amino acid sequence is contained in the labeled target polypeptide.

23. The composition of claim 22, wherein the marker is selected from the group consisting of fluorescent dyes, chemiluminescent markers, iridium-containing electrochemiluminescent markers, ruthenium-containing electrochemiluminescent markers, single-stranded oligonucleotides or analogs thereof, and radiolabelers.

24. The composition of claim 22 or 23, wherein the composition further comprises individual molecules comprising an unlabeled antigenic amino acid sequence or a molecular mimic thereof attached to a capture group.

25. The composition of claim 24, wherein the individual molecule is the target polypeptide of claim 17 having a covalently attached trapping group.

26. A method for detecting a target antibody (=X) specific for an antigen amino acid sequence (=Y) in an isolated sample, the method comprising: (a) An immune reaction mixture is formed by mixing a body fluid sample suspected of containing X with the labeled target polypeptide according to claim 16, wherein Y is contained in the labeled target polypeptide. (b) Maintaining the immunoreaction blend of step (a) for a period of time sufficient to allow X present in the body fluid sample to react immunoreact with Y contained in the labeled target polypeptide to form an immunoreaction product (= X:Y) in the blend; and (c) Detect the presence and / or concentration of X:Y formed in step (b); This allows for the detection of target antibodies specific to the amino acid sequence of the antigen in the separated sample.

27. The method of claim 26, wherein step (c) comprises the following steps: (i) Contact the immunoreaction blend of step (a) or the blend having the immunoreaction product of step (b) with a single molecule (= Z), wherein Z contains a capture group and an unlabeled antigen amino acid sequence or a molecular mimic thereof; (ii) The blend of step (i) is maintained for a period of time sufficient to allow the formation of an immunoreaction product in which X simultaneously binds Y and Z (= Z:X:Y); (iii) Capturing Z:X:Y and separating Z:X:Y from the blend described in step (ii); and (iv) Detect the markers present in the captured Z:X:Y.

28. The method of claim 27, wherein Z is the target polypeptide having a covalently attached trapping group as described in claim 17.

29. The method according to claim 27 or 28, wherein step (iii) comprises capturing Z:X:Y on a solid phase and separating the solid phase having the captured Z:X:Y from the blend of step (ii).

30. Use of labeled target peptides obtained or available by the method of claim 16 for detecting target antibodies specific to the amino acid sequence of an antigen in an isolated sample.

31. The use of the labeled target peptide of claim 16 and the target peptide of claim 17 having a covalently attached capture group for detecting a target antibody specific to an antigen amino acid sequence in an isolated sample.

32. A component kit for detecting a target antibody specific to an antigenic amino acid sequence in an isolated sample, the kit comprising a labeled target polypeptide according to claim 16, the labeled target polypeptide comprising the antigenic amino acid sequence.

Images