Chemiluminescence immunoassy

Abstract

A TPS CLIA kit comprises an immunoconjugate comprising an alkaline phosphatase conjugated to a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to one of the M3 and M21 epitopes of cytokeratin 18, a monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to other of the M3 and M21 epitopes, a substrate of alkaline phosphatase and a solid support. The monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to other of the M3 and M21 epitopes is immobilized or is intended to be immobilized to the solid support. The invention also relates to a method for detecting TPS in a sample using the TPS CLIA kit.

Description

[0001]CHEMILUMINESCENCE IMMUNOASSY TECHNICAL FIELD The present invention relates to cancer detection and in particular to kit and method that can be used in such cancer detection. BACKGROUND Cancer is one of the leading causes of death in the industrial world. In order to improve patient care, much effort is currently put into finding additional information related to the prognosis, early indications of treatment response, and disease progression. An interesting tool to this end is the use of cancer biomarkers, i.e., substances that are indicative of the presence of cancer in the body. A biomarker may be a molecule secreted by a tumor or a specific response of the body to the presence of cancer. Ideally, such biomarkers should be assayed in body fluids, such as urine, blood or serum.All eucaryotic cells have cytoplasmic cytoskeletal structures known as intermediate filaments composedof various intermediate filament proteins, including cytokeratins. The cytoskeletal network is responsiblefor the mechanical integrity of the cells and it is critical during cellular processes like cell division,motility and cell to cell contacts. At present more than 20 different cytokeratins have been identified, of which cytokeratin 8, 18 and 19 are the most abundant in simple epithelial cells. The cytokeratins are epithelial cell specific and the cytokeratin pattern is usually preserved during the transformation ofnormal cells into malignant cells. Upon release from proliferating or apoptotic cells, cytokeratins provideuseful markers for epithelial malignancies, distinctly reflecting ongoing cell activity. The clinical value of determining soluble cytokeratin protein fragments in body fluids lies in the early detection of recurrence and the fast assessment of the efficacy of therapy response in epithelial cell carcinomas. The three most applied cytokeratin markers used in the clinic are tissue polypeptide antigen (TPA), tissue polypeptide specific antigen (TPS), and CYFRA 21-1. Cytokeratin tumor markers can accurately predict disease status before conventional methods and offer a simple, non-invasive, cheap, and reliable tool for more efficient management (Barak et al., Clinical utility of cytokeratins as tumor markers, Clinical Biochemistry (2004) 37(7): 529-540).US 10,386,363 discloses a method for the detection of at least two of cytokeratins 8, 18 and 19 in asample. It is practiced by contacting the sample with a solid support having a first antibody withspecificity for cytokeratin 8, a second antibody with specificity for cytokeratin 18 and, optionally, a third antibody with specificity for a first epitope of cytokeratin 19 bound to it and allowing cytokeratins in the sample to bind to the bound antibodies to form complexes. The complexes are then contacted with a first labelled antibody with specificity for a dimer of cytokeratin 8 and 18 and optionally a second labelled antibody with specificity for a second epitope of cytokeratin 19 and allowing the labelled antibodies to bind to the complexes. The labelled antibodies bound to the complexes are then detected.There is still a need for a fast, sensitive and accurate assay that can be used to measure biomarkersuseful in cancer management. SUMMARY It is a general objective to provide a fast, sensitive and accurate assay that can be used to measure TPS. This and other objectives are met by embodiments as disclosed herein. The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.An aspect of the invention relates to a chemiluminescense immunoassay (CLIA) kit for determiningtissue polypeptide specific antigen (TPS) comprising cytokeratin 18. The kit comprises a firstimmunoconjugate comprising an alkaline phosphatase conjugated to one of a first monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to an M3 epitope of cytokeratin 18 consisting of amino acid sequence NSLREVEARYALQMEQLNG as defined in SEQ ID NO: 1 and a second monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to an M21 epitope of cytokeratin 18 consisting of amino acid sequence VDGKVVSETNDTKVLR as defined in SEQID NO: 2. The kit also comprises the other of the first monoclonal antibody, or the antigen-bindingfragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof. The kitfurther comprises a substrate of alkaline phosphatase and a solid support. The other of the firstmonoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, is immobilized or is intended to be immobilized to the solid support.Another aspect of the invention relates to a method for detecting TPS comprising cytokeratin 18. Themethod comprises contacting a sample with the first immunoconjugate, the other of the first monoclonalantibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, and the solid support of the CLIA kit according to above. The method alsocomprises removing a supernatant from the solid support. The method further comprises adding thesubstrate of alkaline phosphatase of the CLIA kit according to above to the solid support and detectingchemiluminescence to thereby detect the TPS comprising cytokeratin 18.The TPS CLIA kit and method of the invention has a high sensitivity capable of detecting TPS inbiological samples from patients in a short period of time. The TPS CLIA kit and method can thereby beused in cancer diagnosis, such as for early detection of various cancer diseases. BRIEF DESCRIPTION OF DRAWINGSThe embodiments, together with further objects and advantages thereof, may best be understood bymaking reference to the following description taken together with the accompanying drawings, in which:Fig.1 schematically illustrates operations of the TPS CLIA kit and method according to an embodiment; Fig.2 is a graph illustrating TPS levels in healthy subjects measured by the TPS CLIA kit according to an embodiment;Fig. 3 is a graph illustrating correlation between TPS® ELISA and the TPS CLIA kit according to anembodiment; andFig.4 is a graph comparing ROC curve analysis of TPS® ELISA and the TPS CLIA kit according to an embodiment. DETAILED DESCRIPTION The present invention relates to cancer detection and in particular to kit and method that can be used in such cancer detection. In particular, the invention relates to a chemiluminescence immunoassay (CLIA) kit and method for determining tissue polypeptide specific antigen (TPS). TPS is a tumor biomarker allowing not only for early diagnosis of various cancerous diseases, such as breast cancer, prostatecancer, ovarian cancer and gastrointestinal cancer, but can also be used for monitoring anti-cancertherapy and for detection of cancer remission. TPS as tumor biomarker also complements otherbiomarkers, such as cancer antigen 15-3 (CA 15-3), CA 125, carcinoma embryonic antigen (CEA), andprostate-specific antigen (PSA), to improve the sensitivity of early detection of progressive cancerdiseases.Today TPS can measured using enzyme-linked immunosorbent assays (ELISAs). Such TPS ELISA kitsare generally sensitive for early detection of various cancer diseases but typically require several hoursto run the assay. The TPS CLIA method of the invention has a sensitivity in pair with TPS ELISA kitsbut is significantly faster and can deliver results in less than one hour and typically already within 30 minutes. The TPS CLIA kit and method of the invention is based on the usage of two monoclonal antibodies, or antigen-binding fragments thereof, binding specifically to different epitopes of cytokeratin 18. In more detail, one of the two monoclonal antibodies, or antigen-binding fragments thereof, binds specifically to the so-called M3 epitope of cytokeratin 18, whereas the other monoclonal antibody, or the antigen- binding fragment thereof, binds specifically to the M21 epitope of cytokeratin 18.Cytokeratin 18, also referred to as keratin 18, KRT 18, CK-18, CYK18 or K18 in the art, is a type Icytokeratin. It is, together with its filament partner cytokeratin 8, perhaps the most commonly found products of the intermediate filament gene family. They are expressed in single layer epithelial tissuesof the body. Cytokeratin 18 is often used together with cytokeratin 8 and cytokeratin 19 to differentiatecells of epithelial origin from hematopoietic cells in tests that enumerate circulating tumor cells in blood. The M3 epitope corresponds to amino acid numbers 322 to 340 of cytokeratin 18 (SEQ ID NO: 23), whereas the M21 epitope is from the ^-helix 2B2 region of cytokeratin 18 corresponding amino acid numbers 411 to 429 of cytokeratin 18. The pair of monoclonal antibodies, or the antigen-binding fragments thereof, binding specifically to thiscombination of epitopes in cytokeratin 18 is able to detect tissue polypeptide specific antigen (TPS)comprising cytokeratin 18 in a biological sample, including detection of dimers between cytokeratin 18and cytokeratin 8 and also dimers between cytokeratin 18 and cytokeratin 7.An aspect of the invention therefore relates to a CLIA kit for determining TPS comprising cytokeratin 18. The kit comprises a first immunoconjugate comprising an alkaline phosphatase conjugated to one of a first monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to an M3 epitope of cytokeratin 18 consisting of amino acid sequence NSLREVEARYALQMEQLNG as defined in SEQ ID NO: 1 and a second monoclonal antibody, or an antigen-binding fragment thereof, binding specifically to an M21 epitope of cytokeratin 18 consisting of amino acid sequence VDGKVVSETNDTKVLR as defined in SEQ ID NO: 2. The kit also comprises the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof. The kit further comprises a substrate of alkaline phosphatase and a solid support. The other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, is immobilized or is intended to be immobilized to the solid support. The TPS CLIA kit, thus, comprises a first immunoconjugate between alkaline phosphatase (ALP) and the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M3 epitope or the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M21 epitope. The TPS CLIA kit also comprises other of the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M3 epitope or the monoclonal antibody, or the antigen- binding fragment thereof, binding specifically to the M21 epitope, which is immobilized to or intended to be immobilized to the solid support.Hence, in an embodiment, the TPS CLIA kit comprises the first immunoconjugate between ALP and themonoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M3 epitope, and the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M21 epitope immobilized to or intended to be immobilized to the solid support. In another embodiment, the TPS CLIA kit comprises the first immunoconjugate between ALP and the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M21 epitope, and the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M3 epitope immobilized to or intended to be immobilized to the solid support. The two monoclonal antibodies, or the antigen-binding fragments thereof, bind specifically to the M3 or M21 epitope of cytokeratin 18.The specificity of an antibody, or an antigen-binding fragment thereof, can be determined based onaffinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation of anantigen with the antibody, or the antigen-binding fragment thereof, (Kd), is a measure for the bindingstrength between an antigenic determinant and an antigen-binding site on the antibody, or the antigen- binding fragment thereof. The lesser the value of Kd, the stronger the binding strength between the antigenic determinant and the antibody, or the antigen-binding fragment thereof. Alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1 / Kd. As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.Avidity is the measure of the strength of binding between an antibody, or an antigen-binding fragmentthereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenicdeterminant and its antigen binding site on the antibody, or the antigen-binding fragment thereof, andthe number of pertinent binding sites present on the antibody, or the antigen-binding fragment thereof.Typically, antibodies, or antigen-binding fragments thereof, will bind to their antigen with a dissociationconstant (Kd) of 10-5to 10-12moles / liter (M) or less, and preferably 10-7to 10-12M or less and morepreferably 10-8 to 10-12 M, i.e., with an association constant (Ka) of 105 to 1012 M-1 or more, andpreferably 107to 1012M-1or more and more preferably 108to 1012M-1. Generally, any Kd value greater than 10-4M (or any Ka value lower than 104M-1) is generally consideredto indicate non-specific binding. Preferably, an antibody, or an antigen-binding fragment thereof, willbind to the M3 or M21 epitope with an affinity less than 500 nM, preferably less than 200 nM, morepreferably less than 10 nM, such as less than 5 nM or even lower, such as 1 nM or lower.Specific binding of an antibody, or an antigen-binding fragment thereof, to an antigen or antigenicdeterminant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich competition assays, and the different variants thereof known per se in the art.An antigen-binding fragment of an antibody as used herein can be selected from a group consisting ofa single chain antibody, a Fv fragment, a scFv fragment, a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, a di-scFv fragment andmultiple complementary determining region (CDR) regions.In an embodiment, the other monoclonal antibody, or the antigen-binding fragment thereof, that is not conjugated to ALP could be immobilized onto the solid support. In such a case, the solid support preferably comprises a plurality of such immobilized monoclonal antibodies, or the antigen-binding fragments thereof. As an example, magnetic beads could be used as solid support for the TPS CLIA kit. In such a case, the monoclonal antibody, or the antigen-binding fragment thereof, could be covalently bond to the magnetic beads. There are various attachment variants that could be used for such covalent binding, such as using amine groups, carboxyl groups or sulfhydryl groups. For instance, carboxylic acid-hydrophilic bead bind amine groups at pH 5-6 at room temperature, amine-hydrophilicbeads bind aldehydes at neutral to high pH at room temperature, tosyl groups-hydrophobic beads bindamine and sulfhydryl groups at neutral pH and 37^C and epoxy-hydrophilic beads bind amine andsulfhydryl groups at neutral pH at a range of temperatures. The above-described examples could also be used for immobilizing the monoclonal antibody, or the antigen-binding fragment thereof, to other solid supports than magnetic beads, such as surfaces of multi-well plates. In another embodiment, the other monoclonal antibody, or the antigen-binding fragment thereof, that is not conjugated to ALP is intended to be immobilized to the solid support. In such an embodiment, the TPS CLIA kit comprises a second immunoconjugate comprising a capture ligand conjugated to the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof. The solid support then comprises immobilized capture antibodies, or antigen-binding fragments thereof, binding specifically to the capture ligand. In this embodiment, the TPS CLIA kit therefore comprises two immunoconjugates one between ALP and one of the two monoclonal antibodies, or the antigen-binding fragments thereof, and the other between a ligand and the other of the two monoclonal antibodies, or the antigen-binding fragments thereof. The second immunoconjugate can then be immobilized to the solid support by a specificbinding between the immobilized capture antibodies and the ligand part of the secondimmunoconjugate. Various such ligands could be used in the second immunoconjugate to immobilize the second immunoconjugates onto the solid support. An illustrative, but non-limiting, example of such a ligand is fluorescein isothiocynate (FITC). In such an example, the second immunoconjugate comprises FITC conjugated to the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof. The solid support comprises immobilized anti-FITC antibodies, or antigen-binding fragments thereof, binding specifically to FITC. Such an example is shown in Fig.1, where the TPS CLIA kit comprises the first immunoconjugate represented by IDL2-ALP and the second immunoconjugate represented by IDL1-FITC. In this example, the solid support is magnetic beads (MB) comprising anti-FITC antibodies, or antigen-binding fragments thereof, immobilized to the surface of the magnetic beads. In another embodiment, another technology than ligand and capture antibodies is used to immobilize the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, onto the solid support. Such another technology could be using a non-covalent bond between the monoclonal antibody, or the antigen- binding fragment thereof, and the solid support represented by a streptavidin-biotin bond, an avidin- biotin bond or a using protein A or G. For example, streptavidin-conjugated or avidin-conjugated magnetic beads could be used together with a second immunoconjugate between the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, and biotin, or biotin-conjugated magnetic beads could be used together with a second immunoconjugate between the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, and streptavidin or avidin. Protein A or G conjugated magnetic beads could be used to capture IgG antibodies but such an approach does not allow specific immobilization between the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, and the magnetic beads unless the monoclonal antibody, other antigen-binding fragment thereof, conjugated to ALP is of an antibody class that does not bind to protein A or G.It may, though, be preferred to have a second immunoconjugate comprising a ligand and using a solidsupport comprising immobilized capture antibodies, or antigen-binding fragments thereof, rather than using streptavidin / avidin-biotin bond for immobilization of monoclonal antibodies, or antigen-bindingfragments thereof, as this effectively avoids any interference with endogenous biotin that may otherwisebe present in the sample to be tested. This means that the analytic sensitivity of the TPS CLIA will typically be higher if such streptavidin / avidin-biotin bonds can be avoided.The solid support is preferably in the form of beads and in particular magnetic beads, such as carboxylmagnetic beads. In a particular embodiment, magnetic particles have an average diameter within an interval of from 0.8 up to 1.2 µm. In an embodiment, the first immunoconjugate comprises the first monoclonal antibody, or the antigen- binding fragment thereof, and the second immunoconjugate comprises the second monoclonal antibody, or the antigen-binding fragment thereof. Hence, in this embodiment, the first immunoconjugate is an immunoconjugate between ALP and the monoclonal antibody, or the antigen- binding fragment thereof, binding specifically to the M3 epitope and the second immunoconjugate is an immunoconjugate between the ligand, such as FITC, and the monoclonal antibody, or the antigen- binding fragment thereof, binding specifically to the M21 epitope. In an embodiment, the first the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M3 epitope has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence NYTIH as defined in SEQ ID NO: 3, a VH domain CDR2 having amino acid sequence YFNPSSGYNNYNQKFRD as defined in SEQ ID NO: 4, and a VH domain CDR3 having amino acid sequence LIPPFTY as defined in SEQ ID NO: 5. The first monoclonal antibody, orthe antigen-binding fragment thereof, further has a variable light (VL) domain CDR1 having amino acidsequence RASESVDNYGISFMN as defined in SEQ ID NO: 6, a VL domain CDR2 having amino acidsequence AASKEGS as defined in SEQ ID NO: 7 and a VL domain CDR3 having amino acid sequenceLQSKEVPFT as defined in SEQ ID NO: 8.In an embodiment, the first the monoclonal antibody, or the antigen-binding fragment thereof, has a VHdomain having an amino acid sequence as defined in SEQ ID NO: 9. In another embodiment, first themonoclonal antibody, or the antigen-binding fragment thereof, has a VL domain having an amino acidsequence as defined in SEQ ID NO: 10. In a further embodiment, the first the monoclonal antibody, orthe antigen-binding fragment thereof, has a VH domain having an amino acid sequence as defined inSEQ ID NO: 9 and a VL domain having an amino acid sequence as defined in SEQ ID NO: 10.In an embodiment, the first the monoclonal antibody, or the antigen-binding fragment thereof, has aheavy chain having an amino acid sequence as defined in SEQ ID NO: 11. In another embodiment, thefirst the monoclonal antibody, or the antigen-binding fragment thereof, has a light chain having anamino acid sequence as defined in SEQ ID NO: 12. In a further embodiment, the first the monoclonalantibody, or the antigen-binding fragment thereof, has a heavy chain having an amino acid sequenceas defined in SEQ ID NO: 11 and a light chain having an amino acid sequence as defined in SEQ IDNO: 12.A monoclonal antibody having CDR regions according to SEQ ID NO: 3 to 8, VH and VL domainsaccording to SEQ ID NO: 9 and 10 and heavy and light chains according to SEQ ID NO: 11 and 12 is disclosed herein in Example 1 as IDL2. In an embodiment, the second the monoclonal antibody, or the antigen-binding fragment thereof, binding specifically to the M21 epitope has a VH domain CDR1 having amino acid sequence SFWMN as defined in SEQ ID NO: 13, a VH domain CDR2 having amino acid sequenceMLQPADNETKINQKLKD as defined in SEQ ID NO: 14 and a VH domain CDR3 having amino acidsequence GGVVTSYWYFDV as defined in SEQ ID NO: 15. The second the monoclonal antibody, orthe antigen-binding fragment thereof, has a VL domain CDR1 having amino acid sequenceKASQDVGTAVA as defined in SEQ ID NO: 16, a VL domain CDR2 having amino acid sequenceWASTRHT as defined in SEQ ID NO: 17 and a VL domain CDR3 having amino acid sequenceQQFSRYPVT as defined in SEQ ID NO: 18.In an embodiment, the second the monoclonal antibody, or the antigen-binding fragment thereof, has aVH domain having an amino acid sequence as defined in SEQ ID NO: 19. In another embodiment, thesecond the monoclonal antibody, or the antigen-binding fragment thereof, has a VL domain having anamino acid sequence as defined in SEQ ID NO: 20. In a further embodiment, the second themonoclonal antibody, or the antigen-binding fragment thereof, has a VH domain having an amino acidsequence as defined in SEQ ID NO: 19 and a VL domain having an amino acid sequence as defined inSEQ ID NO: 20.In an embodiment, the second the monoclonal antibody, or the antigen-binding fragment thereof, has aheavy chain having an amino acid sequence as defined in SEQ ID NO: 21. In another embodiment, thesecond the monoclonal antibody, or the antigen-binding fragment thereof, has a light chain having anamino acid sequence as defined in SEQ ID NO: 22. In a further embodiment, the second themonoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain having an amino acidsequence as defined in SEQ ID NO: 21 and a light chain having an amino acid sequence as defined inSEQ ID NO: 22. A monoclonal antibody having CDR regions according to SEQ ID NO: 13 to 18, VH and VL domains according to SEQ ID NO: 19 and 20 and heavy and light chains according to SEQ ID NO: 21 and 22 is disclosed herein in Example 1 as IDL1. The TPS CLIA kit comprises a substrate of ALP. This means that the ALP (EC 3.1.3.1) is capable of accelerating a chemical reaction using the substrate as starting material to generate a product upon generation of luminescence. Illustrate, but non-limiting, examples of such substrates include dihydroxyacetone phosphate (DHAP) and its ketal could be hydrolyzed by ALP into dihydroxyacetone (DHA), which reacts with lucigenin (10,10′-dimethyl[9,9′-biacridine]-10,10′-diium dinitrate) upon production of strong chemiluminescence (Kokado et al., Chemiluminescent assay of alkaline phosphatase using dihydroxyacetone phosphate assubstrate detected with lucigenin, Luminescence (2002) 17(1): 5-10), glycerol-3-phosphatase ornicotinamide-adenine-dinucleotide phosphate+ (NADP+) could be catalyzed into dihydroxyacetone by ALP, which reacts with lucigenin upon production of chemiluminescence (Kitamura et al., A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application toenzyme immunoassay, J Biolumin Chemilumin (1995) 10(1): 1-7), 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), which can be catalyzed by ALP upon generation of chemiluminescence (Bronstein et al., 1,2-Dioxetanes: Novel chemiluminescent enzyme substrates.Applications to immunoassays, Journal of Bioluminescence and Chemiluminescence (1989) 4(1): 99-111), para-nitrophenylphosphate (pNPP), a combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), D-luciferin phosphatase, adamantyl-1,2-dioxetane phenylphosphate (PPD) (Girotti et al., Chemiluminescent Determination of Alkaline Phosphatase Activity inSerum, Analytical Letters (1994) 27(2): 323-335), 3-(4-methoxyspiro [1,2-dioxetane-3,2´(5´-chloro)-tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate (CSPD), and chemiluminescent 1,2-dioxetanes asdisclosed in U.S. Patent Nos. 6,461,876 and 7,422,908. A particular substrate that could be used aliquid chemiluminescence substrate comprising 9-(4-chlorphenyl sulfobenzoyloyl methylene)-10-methyl-9, 10-dihydroacridine-disodium salt, N, N-dimethyl acridine nitrate, trihydroxymethyl amino methane,sodium sulfite, lauryl sodium sulfate and TWEEN® 20. For instance, the substrate could compriseabout 120 mg / L 9-(4-chlorphenyl sulfobenzoyloyl methylene)-10-methyl-9, 10-dihydroacridine-disodiumsalt, about 3.4 mg / L N, N-dimethyl acridine nitrate, about 1 g / L lauryl sodium sulfate, about 10 mg / Lsodium sulfite, about 0.31 g / L TWEEN® 20 and 0.26 mol / L of Tris buffer, pH 9.35. An example of sucha liquid chemiluminescence substrate is disclosed in Chinese Patent No.103344633. In an embodiment, the substrate of alkaline phosphatase comprises 9-(4-chlorphenyl sulfobenzoyloylmethylene)-10-methyl-9, 10-dihydroacridine-disodium salt and N, N-dimethyl acridine nitrate. In fact any chemiluminescent substrate that can be used to produce chemiluminescence by the enzyme ALP could be used in the TPS CLIA kit. In an embodiment, the TPS CLIA kit comprises a substrate buffer comprising the substrate of alkaline phosphatase in a buffer comprising 0.1-1.0 M Tris, 0.1-0.5 % sodium sulfite, 0.5-2.0 % sodium dodecyl sulfate, and 0.15-0.5 % bovine serum albumin, pH 9.0-10.0., preferably comprising 0.2-0.6 % lucigenin in the buffer.In an embodiment, the TPS CLIA kit comprises a calibrator standard comprising recombinant humancytokeratin 8 and recombinant human cytokeratin 18 at 1:1 molar ratio.In a particular embodiment, the calibrator standard comprises 4000-9000 U / L of the recombinanthuman cytokeratin 8 and the recombinant human cytokeratin 18 diluted in 1 % bovine serum albumin, pH 7.5.In an embodiment, the TPS CLIA kit comprises an antibody buffer solution comprising 1 % bovineserum albumin, 1 % bovine immunoglobulin G and 0.05 % preservative in phosphate-buffered saline, pH 7.0.Another aspect of the invention relates to a method for detecting TPS comprising cytokeratin 18 andwhere the method involves the use of the TPS CLIA kit.In an embodiment, the method comprises contacting a sample with the first immunoconjugate, theother of the first monoclonal antibody, or the antigen-binding fragment thereof, and the secondmonoclonal antibody, or the antigen-binding fragment thereof, and the solid support of the TPS CLIA kitaccording to the invention, see Fig.1. The method also comprises removing a supernatant from thesolid support, adding the substrate of ALP of the TPS CLIA kit to the solid support and detectingchemiluminescence to thereby detect the TPS comprising cytokeratin 18.In an embodiment, the method comprises contacting the sample with the first immunoconjugate, theother of the first monoclonal antibody, or the antigen-binding fragment thereof, and the secondmonoclonal antibody, or the antigen-binding fragment thereof of the TPS CLIA kit. In this embodiment,the method also comprises incubating the sample with the first immunoconjugate, the other of the firstmonoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, orthe antigen-binding fragment thereof and adding the solid support of the TPS CLIA kit.In an embodiment, the solid support comprises magnetic beads. In this embodiment, removing thesupernatant comprises exposing the sample to a magnetic field to capture the magnetic beads andremoving the supernatant from the captured magnetic beads. The sample is preferably a biological sample originating from a patient or subject. In an embodiment, the biological sample is selected from the group consisting of a blood sample, a plasma sample and a serum sample. In a preferred embodiment, the sample is a serum sample. In an a preferred embodiment, the sample, such as in the form of a serum sample, is contacted with the first immunoconjugate, such as the conjugate between ALP and the anti-M3 monoclonal antibody, or the antigen-binding fragment thereof, preferably IDL2-ALP, and the second immunoconjugate, such as the conjugate between the ligand and the anti-M21 monoclonal antibody, or the antigen-binding fragment thereof, preferably IDL-FITC, as shown in Fig.1. The sample is then incubated with the two immunoconjugates for a time period preferably selected within an interval of from 5 up 60 min,preferably within an interval of from 5 up 30 min, more preferably within an interval of from 10 up to 20min, such as about 15 min. The two immunoconjugates will bind specifically to M3 and M21 epitopes on cytokeratin 18 in cytokeratin 18 monomers and complexes between cytokeratin 18 and other molecules, such as dimers between cytokeratin 18 and cytokeratin 8 and cytokeratin 18 and cytokeratin 7. The two immunoconjugates bond to the M3 and M21 epitopes on the same cytokeratin 18 monomer or complex will form a sandwich complex during incubation as indicated in Fig.1. The sample with the immunoconjugates is then contacted with the solid support, such as by adding anti-FITC magnetic beads to the sample or by adding the sample to the anti-FITC magnetic beads. The sample is allowed to incubate in the presence of the solid support for a time period preferably selected within an interval of from 1 up to 30 min, preferably within an interval of from 1 up to 15 min, and more preferably within an interval of from 2 up to 10 min, such as about 5 min. The anti-FITC antibodies, or the antigen-binding fragments thereof, immobilized onto the solid support, such as magnetic beads, willthen bind specifically to the FITC ligand in the second immunoconjugate (IDL1-FITC) as shown in Fig.1.The solid support will bind specifically to the above-mentioned sandwich complexes but also to thesecond immunoconjugates and to the second immunoconjugate with bond cytokeratin 18 monomer or complex. Any non-specific bonding to the solid support, such as of the first immunoconjugate, will beremoved by washing the solid support at one or multiple wash steps. In such wash steps, thesupernatant is preferably removed from the solid support and replaced by wash buffers as disclosed in Example 2 herein. The supernatant can easily be removed from the solid support if the solid support is in the form of magnetic beads by exposing the sample to a magnetic field to collect the magnetic beads will removing the supernatant. An ALP substrate is then added to the washed solid phase to generate chemiluminescence that is representative of the amount of TPS comprising cytokeratin 18 present in the original (biological) sample. The chemiluminescence can then be measured, for instance, using a spectrometer or other luminescence detector, wherein the measured value, such as in the form of relative light units (RLUs), is representative or indicative of the amount of TPS comprising cytokeratin 18 in the original sample. In an embodiment, the anti-FITC magnetic beads are present in a Tris buffer comprisingmethylcellulose and bovine serum albumin (BSA). For instance, the Tris buffer could be a 0.1-1.0 MTris buffer comprises 3-10 mass percentage methylcellulose, 1-10 mass percentage BSA, and 0.5-2.0mg / mL anti-FITC magnetic beads.In an embodiment, the first immunoconjugate is present in a Tris buffer comprising BSA. For instance,the Tris buffer could be a 0.05-0.15 M Tris buffer, pH 7.0-8.5, comprising 1-5 mass percentage BSAand 1.0-5.0 µg / mL, preferably 1.0-2.5 µg / mL of the second immunoconjugate. In an embodiment, the second immunoconjugate is present in a Tris buffer comprising BSA. For instance, the Tris buffer could be a 0.3-0.8 M Tris buffer, pH 7.0-8.5, comprising 1-5 mass percentage BSA and 0.5-2.0 µg / mL of the first immunoconjugate. In an embodiment, the Tris buffers of the first and second immunoconjugates may comprise surfactants, such as Tween® 20 and / or TritonTMX-100 (2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol). In such an embodiment, the surfactant could, for instance, be present at 0.01-0.5 mass percentage. In an embodiment, the Tris buffers of the first and second immunoconjugates may comprise preservatives, such as tetracycline, NaN3, bronidox (5-bromo-5-nitro-1,3-dioxane) and / or neomycin sulfate. In such an embodiment, the preservatives could, for instance, be present at 0.01-0.1 mass percentage. In an embodiment, the calibration standard comprises a standard TPS product in the form ofrecombinant human cytokeratin 8 and recombinant human cytokeratin 18 at a 1:1 ratio. The calibrationstandard is preferably a Tris buffer, such as a Tris buffer of pH 7.0-9,0 comprising 0.5-10 masspercentage BSA and 0.5-3 mass percentage Tween® 20 (polysorbate 20, polyoxyethylene (20)sorbitan monolaurate). In an embodiment, the Tris buffers of the calibration standard may comprise preservatives, such as tetracycline, NaN3, bronidox and / or neomycin sulfate. In an embodiment, the luminescent substrate is in the form of a Tris buffer, such as a 0.1-1.0 M Trisbuffer, pH 9.0-10.0 comprising AP substrate luminescent liquid (APLS, see CN 103344633 B), 0.1-0.5mass percentage sodium sulfite, 0.5-2.0 mass percentage SDS, 0.2-0.6 mass percentage lucigenin, 0.15-0.5 mass percentage BSA. The Tris buffer preferably comprises the APLS luminescent substrate at a mass ratio of from 1:4 up to 1:10. In an embodiment, the wash buffer is a Tris-NaCl buffer, such as a 0.1-1.0 M Tris-NaCl buffer comprising 1-4 mass percentage Tween® 20 and / or TritonTMX-100. The method of detecting TPS in a sample using the TPS CLIA will present a detectable result already within about 30 minutes. The method and TPS CLIA kit of the invention can be used for early diagnosis of various cancerous diseases, such as breast cancer, prostate cancer, ovarian cancer and gastrointestinal cancer, and also for monitoring anti-cancer therapy and for detection of cancer remission. EXAMPLE EXAMPLE 1 This Example characterized two mouse monoclonal antibodies, denoted IDL1 and IDL2 herein, bindingto different epitopes of cytokeratin 18 (CK18) as shown in SEQ ID NO: 23 below (GeneBank CAA31375.1). The IDL2 antibody binds specifically to the M3 epitope of CK18 corresponding to amino acid numbers 322 to 340 of CK18 as shown above. The other antibody, IDL1, was raised against the epitope M21 from the ^-helix 2B2 region of CK18 corresponding amino acid numbers 411 to 429 of CK18. Human cytokeratin 18 (SEQ ID NO: 23) 10 20 30 40 50 MSFTTRSTFS TNYRSLGSVQ APSYGARPVS SAASVYAGAG GSGSRISVSR 60 70 80 90 100 STSFRGGMGS GGLATGIAGG LAGMGGIQNE KETMQSLNDR LASYLDRVRS 110 120 130 140 150 LETENRRLES KIREHLEKKG PQVRDWSHYF KIIEDLRAQI FANTVDNARI 160 170 180 190 200 VLQIDNARLA ADDFRVKYET ELAMRQSVEN DIHGLRKVID DTNITRLQLE 210 220 230 240 250 TEIEALKEEL LFMKKNHEEE VKGLQAQIAS SGLTVEVDAP KSQDLAKIMA 260 270 280 290 300 DIRAQYDELA RKNREELDKY WSQQIEESTT VVTTQSAEVG AAETTLTELR 310 320 330 340 350RTVQSLEIDL DSMRNLKASL ENSLREVEAR YALQMEQLNG ILLHLESELA360 370 380 390 400 QTRAEGQRQA QEYEALLNIK VKLEAEIATY RRLLEDGEDF NLGDALDSSN 410 420 430SMQTIQKTTT RRIVDGKVVS ETNDTKVLRHMaterials Hybridoma cells prepared by GenScript;SMARTScribe™ Reverse Transcriptase Kit (Takara, Cat. No.: 639537).Zymogen Quick-RNA Miniprep Kit (Zymogen, Cat No.: R1051) Methods Total RNA was isolated from the hybridoma cells following the technical manual of Zymogen Quick- RNA Miniprep Kit. Total RNA was then reverse transcribed into cDNA using isotype-specific anti-senseprimers or universal primers following the technical manual of SMARTScribe™ Reverse Transcriptase Kit. The antibody fragments of VH and VL were amplified according to the standard operating procedure (SOP) of rapid amplification of cDNA ends (RACE) of GenScript. Amplified antibody fragments were cloned into a standard cloning vector separately. Colony PCR was performed to screen for clones with inserts of correct sizes. No less than five colonies with inserts of correct sizes weresequenced for each fragment. The sequences of different clones were aligned and the consensussequence of these clones was provided. IDL1 heavy chain: DNA sequence (SEQ ID NO: 24) Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region-stop codon ATGGGATGGAGCTCTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGT CCAGCTGCTGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGAGTTTCAGTGAAGCTGTCCTGCA AGGCTTCTGGCTACTCCTTCAGCAGCTTCTGGATGAACTGGGTGAAGCAGAGGCCTGGACAA GGCCTTGAGTGGATTGGCATGCTTCAGCCCGCCGATAATGAAACTAAGATAAATCAGAAACT CAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCA GCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGGGGGGTAGTAACAAGTTAC TGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACC CCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGG GATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTG TCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTC AGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGG CCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATA TGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCAC CATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGG TCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAG GAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCT CAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAA CCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAG GAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGA CATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCA TCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGG GAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGA GAAGAGCCTCTCCCACTCTCCTGGTAAATGA IDL1 heavy chain: amino acid sequence (SEQ ID NO: 25) Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region MGWSSIILFLVATATGVHSQVQLLQPGAELVRPGVSVKLSCKASGYSFSSFWMNWVKQRPGQ GLEWIGMLQPADNETKINQKLKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCARGGVVTSY WYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSL SSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCI CTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPRE EQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNW EAGNTFTCSVLHEGLHNHHTEKSLSHSPGK ILD1 light chain: DNA sequence (SEQ ID NO: 26) Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region-stop codon ATGGAGACACATTCTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGAAGGAGA CATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAATAGGAGACAGGGTCAGCATCA CCTGCAAGGCCAGTCAGGATGTGGGTACTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAA TCTCCTAAACTACTGATTTACTGGGCATCCACCCGGCACACTGGAGTCCCTTATCGCTTCAC AGGCAGTGGATCTGGGACAGACTTCACTCTCACCATTAACAATGTGCAGTCTGAAGACTTGG CAGATTATTTCTGTCAGCAATTTAGCAGGTATCCAGTCACGTTCGGCTCGGGGACAAAGTTG GAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTT AACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATG TCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAG GACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGA ACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGA GCTTCAACAGGAATGAGTGTTAGILD1 light chain: amino acid sequence (SEQ ID NO: 27)Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region METHSQVFVYMLLWLSGVEGDIVMTQSHKFMSTSIGDRVSITCKASQDVGTAVAWYQQKPGQ SPKLLIYWASTRHTGVPYRFTGSGSGTDFTLTINNVQSEDLADYFCQQFSRYPVTFGSGTKL EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQ DSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC The isotype of IDL1 was determined to be mouse IgG1 / kappa as analyzed by the sequences of the constant region.ILD2 heavy chain: DNA sequence (SEQ ID NO: 28)Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region-stop codon ATGGAAAGGCACTGGGTCTTTCTACTCCTGTTGTCAGTAACTGCAGGTGTCCACTCCCAGGT CCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCA AGGCTTCTGGCTACACCTTTACTAACTACACGATACACTGGGTAAAACAGAGGCCTGGACAG GGTCTGGAATGGATTGGATACTTTAATCCTAGCAGTGGTTATAATAATTACAATCAGAAGTT CAGGGACAAGGCCACATTGACTGCAGACACATCCTCCACCACAGCCTACATGCGACTGAGCA GCCTGACATCTGAGGACTCTGCAGTCTATTTCTGTACAAGACTGATTCCACCGTTTACTTAC TGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCC ACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGG GCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCAC ACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTC CAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGG TGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAA GTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCC TAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGT TTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGC ACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTT CAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCA AAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAG GATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTG GCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATG GCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACT TTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCA CTCTCCTGGTAAATGAIDL2 heavy chain: amino acid sequence (SEQ ID NO: 29)Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region MERHWVFLLLLSVTAGVHSQVQLQQSGAELARPGASVKMSCKASGYTFTNYTIHWVKQRPGQ GLEWIGYFNPSSGYNNYNQKFRDKATLTADTSSTTAYMRLSSLTSEDSAVYFCTRLIPPFTY WGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH TFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPE VSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNS TFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAK DKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNT FTCSVLHEGLHNHHTEKSLSHSPGKILD2 light chain: DNA sequence (SEQ ID NO: 30)Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region-stop codon ATGGAGAAAGACACACTCCTGCTATGGGTCCTGCTTCTCTGGGTTCCAGGTTCCACAGGTGA CATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCT CCTGCAGAGCCAGCGAAAGTGTTGATAATTATGGCATTAGTTTTATGAACTGGTTCCAACAG AAACCAGGACAGCCACCCAAACTCCTCATCTTTGCTGCATCCAAGGAAGGATCCGGGGTCCC TGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTATGGAGG AGGATGATACCGCAATGTACTTCTGTCTGCAAAGTAAGGAGGTTCCATTCACGTTCGGCTCG GGGACGAGTTTGGAAACAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATC CAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCA AAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGT TGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAA GGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCAC CCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAGILD2 light chain: amino acid sequence (SEQ ID NO: 31)Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-constant region MEKDTLLLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQ KPGQPPKLLIFAASKEGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCLQSKEVPFTFGS GTSLETKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNS WTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC The isotype of IDL2 was determined to be mouse IgG1 / kappa as analyzed by the sequences of the constant region. EXAMPLE 2 This Example developed a chemiluminescense immunoassay (CLIA) utilizing the two monoclonal antibodies IDL1 and IDL2 characterized in Example 1. The following Tables 1 to X define buffers used in this Example 1. The contents of the reagents listed in these tables are in mass percentage unless specified otherwise. Table 1 - General Tris buffer pH 7.5Reagent name Manufacturer Concentration 1 L buffer dosage100 mM Tris0.1 M 12.12 g100 mM NaClSigma0.9 % 5.82 g4 M HCl pH 7.5 ~20 mLTable 2 – Tris buffer for calibratorsReagent name Manufacturer Concentration 1 L buffer dosageBSA Proliant 2 % 20 gTween® 201 % 10 mLTetracyclineSigma0.01 % 10 mgNeomycin sulfate 0.01 % 10 mgThe reagents defined in Table 2 are added to the general Tris buffer pH 7.5 defined in Table 1. Table 3 – Anti-reagent Tris bufferReagent name Manufacturer Concentration 1 L buffer dosageBSA Proliant 3 % 30 gMgCl20.41 g / L 0.41 gTetracyclineSigma0.01 % 10 mgNeomycin sulfate 0.01 % 10 mgThe reagents defined in Table 3 are added to the general Tris buffer pH 7.5 defined in Table 1. Table 4 – Tris buffer for magnetic beadsReagent name Manufacturer Concentration 1 L buffer dosageTris0.1 M 12.12 gSigmaNaCl 0.9 % 5.82 gBSA Proliant 1 % 10 g0.01 M ZnCl220 mL / L 20 mLSigmaMethylcellulose 5 % 50 gTable 5 – Tris buffer for luminescent substrateReagent name Manufacturer Concentration 1 L buffer dosageTris0.1 M 12.12 gNaCl 0.9 % 5.82 gSigmaLucigenin 0.3 % 30 mgSDS 1 % 10 gTable 6 – Wash bufferReagent name Manufacturer Concentration 1 L buffer dosageTris0.1 M 12.12 gNaCl 0.9 % 5.82 gSigmaTween® 20 1 % 10 mLTritonTM X-100 1 % 10 mLLabelling of antibody IDL1 with FITC The antibody stock solution was desalted using a PD10 desalting column, and dialyzed and purifiedwith a carbonate buffer (0.1 M Na2CO3, 0.1 M Na2HCO3) of pH 8.0-10 at 2-8°C. The carbonate bufferwas replaced twice during desalting, and the dialysate was left overnight. The desalted IDL1 antibodywas collected and detected at an absorbance of 280 nm (A280) using a spectrophotometer todetermine the IDL1 antibody concentration as A280×0.74×dilution factor.20 to 50 µl of a 5 mg / ml fluorescein isothiocyanate (FITC) solution per mg of antibody was added to thedesalted IDL antibody and incubated at room temperature (20^C±5^C) for 2 h. The carbonate bufferwith pH 8.0-10 was used to remove unbound FITC, and the solution was dialyzed overnight whilechanging the carbonate buffer 2 or 3 times. The FITC-labeled antibody IDL1 complexes were dilutedwith the carbonate buffer at pH 8.0-10 to obtain an absorbance A280<2.0. The absorbance wasdetected at A280 and A492, and the protein concentration (mg / ml) was calculated as A280-(A492×0.35) / 1.4.The FITC-labeled antibody IDL1 was added to a 0.3-0.8 M Tris buffer containing 0.01-1 %preservatives (NaN3, neomycin sulfate, tetracycline and / or bronidox) and 0.01-0.5 % surfactant(Tween® 20 or TritonTM X-100) and 1-5% bovine serum albumin (BSA) at a final concentration of 0.5-2.0 µg / mL.Labelling of antibody IDL2 with ALPThe antibody stock solution was desalted using a PD10 desalting column, and eluted with phosphate-buffered saline (PBS) buffer at pH 7.5-8.0. The desalted IDL2 antibody was collected.Alkaline phosphatase (ALP) was prepared in a PBS buffer (pH 7.5-8.0) at a concentration of 1.0-5.0mg / mL. The absorbance was measured by a spectrophotometer at 280 nm and A280 should be 0.2- 1.0. The desalted antibody IDL2 was transferred to a brown glass bottle in the required amount to achieve amolar ratio of 1:2 to 1:10 and the calculated volume of AP solution was added followed by mixing for 4-5 hours at room temperature. A protein purifier chromatographic separation column, equilibrated with0.1-0.5 M bicarbonate buffer with pH=8.0-9.5, was used for purification of the AP-labelled antibodyIDL2. The AP-labelled antibody IDL2 was eluted with 0.1-0.5 M bicarbonate buffer with pH=8.0-9.5,collected and added to a 0.05-0.15 M Tris buffer containing 0.01-1 % preservatives (NaN3, neomycinsulfate, tetracycline and / or bronidox) and 0.01-0.5 % surfactant (Tween® 20 or TritonTM X-100) and 1-5% bovine serum albumin (BSA) at a final concentration of 1.0-2.5 µg / mL. Preparation of magnetic beadsA carboxyl magnetic bead concentrate was mixed thoroughly and added to a reaction bottle that wasplaced in a magnetic field for 15 minutes. After all the carboxyl magnetic beads had settled, thesupernatant was aspirated. 5 volumes of PBS buffer were added per 2 volumes of carboxyl magneticbeads and mixed for 20-30 minutes. The reaction bottle was then placed in a magnetic field to allow allthe carboxyl magnetic beads to settle for 15 minutes, followed by aspiration of the supernatant. Thiscleaning step was repeated 3 times to fully clean the carboxyl magnetic beads. Finally, the volume of the carboxyl magnetic bead solution was adjusted to 10-50 mg / mL.Sheep anti-FITC antibodies were added to the carboxyl magnetic beads at a mass ratio of magneticbeads: anti-FITC antibody = 100:1, while mixing at 2-8°C for 18 hours. The magnetic beads werewashed 3 times with PBS and diluted to 10 mg / mL and stored at 2-8^C until use.A magnetic bead buffer was prepared as defined in Table 4. Preparation of substrate solutionThe AP luminescent substrate (lucigenin, Sigma) was prepared in a Tris buffer according to Table 5.Preparation of calibratorsThe calibrators were prepared by using TPS® standard material, which contains recombinant humanCK8 and CK18 proteins (PROGEN Biotechnik GmbH, Germany) at 1:1 ratio. Recombinant human CK8and CK18 were expressed in Escherichia coli at a purity of >95 % as determined by SDS. Recombinanthuman CK8 had a molecular weight of 52.5-53 kDa and an isoelectric point pI = 6.1, whereasrecombinant CK18 had a molecular weight of 45–48 kDa and pI = 5.7. In brief, recombinant humanCK8 and CK18 were mixed in 1:1 ratio in a Tris buffer according to Table 2. The mixture was incubatedat 37^C for 2 weeks and then it was incubated again for 4 weeks at 4^C so to form CK8 and CK18dimers. The calibrator material had a concentration of 4000-9000 U / L, which was diluted in the Trisbuffer according to Table 2 to obtain the calibrators ranging from 10 to 1200 U / L. CLIA measurementsBody fluid samples, serum samples – 30 µL, were mixed with FITC-labelled antibody IDL1 (0.5 µg / mL),the AP-labelled antibody IDL2 (0.8 µg / mL) and an antibody buffer solution (1% BSA, 1% bovine IgGand 0.05% Proxel in PBS pH of 7.0). Then the mixture was incubated at 37^C for 15 min. The FITC-labeled antibody IDL1 formed a sandwich complex with AP-labeled antibody IDL2 as shown in Fig. 1.Then, the sandwich complex was suspended with the anti-FITC conjugated magnetic particles (2mg / mL), placed in a magnetic field and allowed to set for 5 min in the magnetic field. The supernatantwas removed and the magnetic beads were washed with wash buffer as defined in Table 6. 200 µl ofthe substrate solution was added to the washed magnetic beads and suspended for 5 seconds followedby detection of luminescence intensity in relative light units (RLU) using a luminescence detector asshown in Fig. X. Analytical sensitivityThe limit of blank (LOB) of the CLIA was determined using the calibration buffer (calibrator A) repeatedwith 20 replicates and these measurements were repeated with 3 lots. The average RLUs from the 20replicates of each lot were calculated as concentration based on the standard curve. The highest value,6 U / L, out of the 3 lots was considered as LOB of the CLIA.The limit of detection (LOD) of the CLIA was determined by serial dilution of the low calibrator corresponding to 30 U / L (calibrator B). The low calibrator was diluted 2-fold (B / 2, B / 4, B / 8, B / 16 andB / 32) and each dilution was analyzed in 10 replicates. The LOD was set as the concentrationcorresponding to % coefficient of variation (CV) below 20, which was 6 U / L.Table 7 – Luminescence intensity detectionNo. Lot 1 Lot 2 Lot 31 107629 108560 1138262 107325 111752 1157273 110598 110064 1158994 107974 115975 1162305 105941 114172 1149316 108641 111762 1108447 107937 108711 1112288 105852 109561 1090869 113501 113610 11111410 112362 115795 10659911 106400 109797 11606512 114339 113740 11571113 107237 112728 11190714 109336 10S866 10832615 115926 112907 11552916 106794 108958 11513817 114346 113699 10841218 113488 114214 10713819 109604 108349 10905720 105649 115113 115706Average RLU 109544.03 111917 112424RLU SD 3293.54 2605.27 3408.14Average RLU+2×(RLU SD) 116131.099 117127.070 119239.982Result (≤6 U / L) 1.045 1.229 1.618PrecisionThe precision of the CLIA was determined by using one low control sample and one high controlsample (136 and 490 U / L) with assigned TPS® values. Each control sample was analyzed in 10replicates with 3 different lots. The %CV for the 136 U / L control was 4.3 and %CV for the 490 U / Lcontrol was 4.6.Table 8 - Reportable range (low limit), Experimental data results for first batchConcentration Concentration Concentration Concentration Concentration 1 (U / L) 2（U / L） 3（U / L） 4（U / L） 5（U / L）1 7.35 11.07 10.07 10.37 11.262 7.69 8.53 9.85 10.45 11.873 9.76 11.16 10.37 10.36 12.434 8.58 8.30 9.87 11.16 11.655 9.37 8.08 1052 10.82 12.166 9.38 9.09 10.36 10.92 12.147 8.83 7.23 9.82 11.08 12.228 6.63 10.22 10.35 10.30 11.529 10.47 9.23 9.42 11.41 11.6810 9.26 11.62 9.43 10.67 11.36AVE 8.732 9.453 10.006 10.754 11.827SD 1.1844 1.4916 0.3939 0.3876 0.3972CV% 1356% 15.78% 3.94% 3.60% 3.36%Table 9 - Reportable range (low limit), Experimental data results for second batchConcentration Concentration Concentration Concentration Concentration 1 (U / L) 2（U / L） 3（U / L） 4（U / L） 5（U / L）1 9.41 10.42 9.84 11.31 12.592 7.18 8.88 10.51 11.09 12.133 6.34 11.90 1013 11.13 12.214 9.80 10.84 10.99 11.68 12.345 7.04 9.80 10.84 11.61 12.796 10.93 7.56 10.01 11.60 12.967 1083 7.93 9.99 10.73 13.028 8.11 7.13 10.04 10.77 13.069 10.63 8.30 11.14 11.62 12.6910 6.33 9.62 9.90 11.55 12.43AVE 8.661 9.238 10.338 11.309 12.621SD 1.8711 1.5449 0.4882 0.3605 0.3381CV% 21.61% 16.72% 4.72% 3.19% 2.68%Table 10 - Reportable range (low limit), Experimental data results for third batchConcentration Concentration Concentration Concentration Concentration 1 (U / L) 2（U / L） 3（U / L） 4（U / L） 5（U / L）1 9.97 11.27 10.12 10.25 12.332 8.74 7.97 9.83 11.20 12.463 6.04 7.17 10.62 10.99 12.224 8.15 7.41 10.23 11.31 11.765 5.94 8.75 9.97 10.90 12.146 10.17 8.83 10.51 11.38 11.437 9.56 6.89 10.16 11.38 11.198 8.50 7.07 9.75 11.36 11.809 8.66 8.04 10.66 10.89 12.0910 6.83 11.72 10.14 11.01 12.21AVE 8.257 8.511 10.198 11.068 11.963SD 1.5301 1.7098 0.3158 0.3487 0.4108CV% 18.53% 20.09% 3.10% 3.15% 3.43%Table 11 – Reportable range (high limit)Reportable Experimental data Experimental data results: Experimental data results: range (highresults: first batch second batch third batchlimit) Con.1Con.2 Con.3Con.1Con.2 Con.3Con.1Con.2 Con.3(U / L) (U / L) (U / L) (U / L) (U / L) (U / L) (U / L) (U / L) (U / L)1 893.77 88212 1000.37 956.98 97442 1098.47 925.23 882.61 1227.622 872.92 92723 1474.26 878.03 965.33 125738 973.12 954.13 1118.123 919.04 907.47 1192.89 948.09 890.01 1166.30 992.30 962.42 1224.58AVE 895.24 905.61 122251 927.70 943.25 1174.05 963.55 933.05 190.11Dilution ratio 5 10 15 5 10 15 5 10 15Reduction4476.22 9056.07 18337 4638.50 9432.53 17610.75 4817.75 9330.53 17851.60concentration 60 Theoretical4500 9000 15000 4500 9000 15000 4500 9000 15000concentration Relative0.53% 0.62% 22.25% 3.08% 4.81% 17.41% 7.06% 3.679% 19.01%deviation (%) Accuracy / Recovery The accuracy of the CLIA was evaluated using the high calibrator with a concentration of 1200 U / L (calibrator F). The high calibrator was diluted fourfold (F, F / 4, F / 16, F / 64 and F / 256) up to the concentration of 5 U / L and each dilution was analyzed in triplicates with three different lots. The accuracy was calculated using measured concentration against the theoretical concentration. The accuracy of the CLIA was in the range of 85-115%. Table 12 - recoverylot Fitting conc.Fitting conc.Fitting conc.3 Concentration Recovery (85%-1 2 mean U / L 115%）20210101 148.07 143.82 136.57 142.82 96.52%20210102 145.81 149.67 144.06 146.51 99.59%20210103 140.95 143.80 137.35 140.70 94.75%Dilution linearity The dilution linearity of the CLIA was assessed by diluting the high calibrator of 1200 U / L into the measuring range of the CLIA (30-1200 U / L). Each dilution was measured in triplicates and comparedwith the expected concentration. Dilution linearity up to 5 U / L showed 99.9% correlation with theexpected values. Table 13 – dilution linearityDilution Factor Conc (average) Conc 1 Conc 2 Conc 31 1200 1186.0 1202.7 1196.51 / 4 300 266.1 239.2 246.21 / 16 75 74.8 77.3 80.71 / 64 18,8 16.9 20.1 10.21 / 128 9.4 9.5 9.6 9.81 / 256 47 4.1 4.3 4.4Correlation r 1.000 0.999 0.999Hook effect Serum samples from healthy subjects were spiked with very high concentrations of standard TPS® material up to 20000 U / L and each concentration was measured in triplicates. No hook effect wasobserved up to 20000 U / L.Table 14 – Hook effectFirst batch Second batch Third batchTheoretical Theoretical Theoretical concentration RLU concentration RLU concentration RLU U / L U / L U / L 900 4638101 900 4722172 900 46763731200 5911110 1200 5984008 1200 58247282300 8945386 2300 8803335 2300 80016254200 11889691 4200 11894037 4200 119940716000 12902646 6000 13031197 6000 1289024313000 14956762 13000 14531974 13000 1499574015000 12945123 15000 12777029 15000 1271674420000 11607232 20000 11637076 20000 11878169Interference Any interference was tested with common interferents, such as bilirubin, hemoglobin, triglycerides, Rheumatoid factor, anti-nuclear antibodies (ANA) and human anti-mouse antibodies (HAMA). No significant interference was observed for the CLIA. Table 15 - Interfering substances: bilirubin; Analyte: 5 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 15.13 5.14 5.11 5.11 5.10 5.032 5.03 5.10 5.13 5.11 5.14 5.033 5.03 5.07 5.08 5.13 5.06 5.05Mean value 5.06 5.10 5.11 5.12 5.10 5.04SD 0.06 0.04 0.03 0.01 0.04 0.01dobs -0.04 -0.01 0.06dc 0.040 0.013 0.01395% confidence-0.163 0.083 -0.051 0.031 0.023 0.104interval Interference No interference No interference No interferencecondition Table 16 - Interfering substances: bilirubin; Analyte: 75 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 171.85 75.61 78.08 74.31 75.47 74.172 75.62 72.27 72.32 72.46 76.69 73.503 79.03 75.84 71.69 78.27 76.29 73.85Mean value 75.50 74.57 74.03 75.01 76.15 73.84SD 3.59 2.00 3.52 2.97 0.62 0.34dobs 0.93 -0.98 2.31dc 2.261 3.359 037995% confidence-6.093 7.947 -11.412 9.445 1.133 3.487interval Interference No interference No interference No interferencecondition Table 17 - Interfering substances: hemoglobin; Analyte: 5 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 15.03 5.06 5.10 5.14 5.05 5.072 5.11 5.07 5.0s 5.11 5.09 5.123 5.05 5.13 5.06 5.04 5.03 5.10Mean value 5.06 5.09 5.0s 5.10 5.06 5.10SD 0.04 0.04 0.02 0.05 0.03 0.03dobs -0.02 -0.02 -0.04dc 0.043 0.058 0.02895% confidence-0.156 0.110 -0.197 0.164 -0.123 0.048interval Interference No interference No interference No interferencecondition Table 18 - Interfering substances: hemoglobin; Analyte: 75 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 171.60 72.06 72.85 76.84 76.74 77-202 76.66 74.26 72.42 76.27 77.40 70.283 70.74 71.65 78.10 74.16 75.29 75.54Mean value 73.00 72.66 74.46 75.76 76.48 74.34SD 320 1.40 3.16 1.41 1.08 3.61dobs 0.34 -1.30 2.14dc 1.588 1.598 4.08895% confidence-4.588 5.275 -6.260 3.660 -10.556 14.829interval Interference No interference No interference No interferencecondition Table 19 - Interfering substances: triglycerides; Analyte: 5 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 15.04 5.07 5.06 5.05 5.10 5.042 5.14 5.09 5.05 5.11 5.05 5.063 5.11 5.11 5.12 5.10 5.09 5.07Mean value 5.10 5.09 5.08 5.09 5.08 5.06SD 0.05 0.02 0.04 0.03 0.03 0.02dobs 0.01 -0.01 0.02dc 0.023 0.036 0.01795% confidence-0.064 0.077 -0.123 0.103 -0.030 0.077interval Interference No interference No interference No interferencecondition Table 20 - Interfering substances: triglycerides; Analyte: 85 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 174.89 76.75 78.16 77.65 74.80 78.362 76.47 74.07 71.34 71.33 75.57 75.183 73.60 71.53 72.93 76.81 73.86 77.28Mean value 74.99 74.12 74.14 75.26 74.74 76.94SD 1.44 2.61 3.57 3.43 0.86 1.62dobs 0.87 -1.12 -2.20dc 2.954 3.884 1.83095% confidence-8.301 10.041 -13.179 10.939 -7.878 3.485interval Interference No interference No interference No interferencecondition Table 21 - Interfering substances: rheumatoid factors; Analyte: 5 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 15.06 5.04 5.05 5.11 5.07 5.092 5.11 5.13 5.08 5.05 5.11 5.033 5.09 5.05 5.03 5.11 5.06 5.14Mean value 5.09 5.07 5.05 5.09 5.08 5.09SD 0.03 0.05 0.03 0.03 0.03 0.06dobs 0.01 -0.04 -0.01dc 0.056 0.039 0.06295% confidence-0.160 0.187 -0.158 0.085 -0.200 0.187interval Interference No interference No interference No interferencecondition Table 21 - Interfering substances: rheumatoid factors; Analyte: 75 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 176.95 76.11 72.36 70.42 75.54 71.842 74.59 70.55 74.86 70.47 76.97 77.543 74.65 74.86 75.01 74.22 69.88 77.71Mean value 75.40 73.84 74.08 71.70 74.13 75.70SD 1.35 2.92 1.49 2.18 3.75 3.34dobs 1.56 2.37 -1.57dc 3.301 2.466 3.78195% confidence-8.692 11.805 -5.285 10.031 -13.305 10.172interval Interference No interference No interference No interferencecondition Table 22 - Interfering substances: ANA; Analyte: 5 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 15.14 5.13 5.12 5.14 5.05 5.052 5.11 5.10 5.07 5.08 5.10 5.143 5.12 5.09 5.14 5.10 5.10 5.14Mean value 5.12 5.11 5.11 5.11 5.08 5.11SD 0.02 0.02 0.04 0.03 0.03 0.05dobs 0.02 0.00 -0.3dc 0.024 0.035 0.05995% confidence-0.056 0.090 -0.104 0.111 -0.209 0.156interval Interference No interference No interference No interferencecondition Table 23 - Interfering substances: ANA; Analyte: 75 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 172.81 73.48 70.23 72.70 69.56 68.532 76.85 76.43 70.15 71.33 7233 74.853 69.52 6942 68.78 74.98 70.32 73.78Mean value 73.06 73.11 69.72 73.00 70.74 72.39SD 3.67 3.52 0.82 1.84 1.43 3.38dobs -0.05 -3.28 -1.65dc 3083 2.086 3.82895% confidence-12.416 12.316 -9.761 3.195 -13.534 10.234interval Interference No interference No interference No interferencecondition Table 24 - Interfering substances: HAMA; Analyte: 5 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 15.09 5.13 5.04 5.07 5.07 5.032 5.10 5.14 5.13 5.06 5.14 5.113 5.11 5.07 5.11 5.04 5.04 5.14Mean value 5.10 5.11 5.09 5.06 5.08 5.09SD 0.01 0.04 0.05 0.02 0.05 0.06dobs -0.01 0.04 -0.01dc 0.043 0.017 0.06495% confidence-0.146 0.120 -0.017 0.090 -0.210 0.190interval Interference No interference No interference No interferencecondition Table 25 - Interfering substances: HAMA; Analyte: 75 U / LGroup A Group A Group B Group B Group C Group C experiment control experiment control experiment control 173.88 74.67 76.75 72.36 73.77 76.742 76.59 71.05 76.00 76.62 71.38 76.873 69.76 74.28 76.78 68.85 74.52 76.95Mean value 73.41 73.33 76.51 72.61 73.22 76.85SD 3.44 1.99 0.44 3.89 1.64 0.11dobs 0.08 3.90 -3.63dc 2.249 4.403 0.12095% confidence-6.904 7.058 -9.771 17.571 -4.002 -3.258interval Interference No interference No interference No interferencecondition Cross reactivity No cross reactivity was observed with CK8 and CK19. Stability The CLIA kit storage temperature is 2-8^C and it has a shelf life of 12 months. An accelerated stabilitytest was carried out at 37^C of 10 days. The accelerated stability test verified that the CLIA kit met allthe requirements in terms of %CV. Cut-off valueThe cut-off value was determined by measuring TPS levels in serum samples from 100 healthy humansubjects. The TPS values were in the range of 10-100 U / L with a cut-off (95% CI) value of 89 U / L. Thedistribution frequency of TPS in healthy individuals are shown in Fig.2.Assay comparisonThe CLIA was compared with IDL TPS® ELISA by using serum samples from 100 healthy subjects andserum samples from 58 with different malignancies. Out of 158 serum samples, 4 serum samples had TPS values above 1200 U / L and were excluded from the correlation analysis. By using Passing-Bablokregression analysis, the correlation between the CLIA and IDL TPS® ELISA was 0.94 (P<0.0001) asshown in Fig.3.Furthermore, receiver operating characteristic (ROC) curve analysis was performed to compare theperformance of the CLIA with IDL TPS® ELISA. The results showed that both assays had similarsensitivity as well as positive and negative predictive values as shown in Table 26 and Fig.4.Table 26 – Clinical performance characteristicsAssay Cut-off (U / L) AUC Sensitivity Specificity PPV NPVTPS CLIA 89 0.87 72.4 % 95 % 89.4 % 85.6 %TPS® ELISA 89 0.88 72.4 % 94 % 87.5 % 85.5 %AUC – area under curvePPV – positive predictive valueNPV – negative predictive valueThe embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible.

Claims

1. CLAIMS1. A chemiluminescense immunoassay (CLIA) kit for determining tissue polypeptide specificantigen (TPS) comprising cytokeratin 18, the kit comprises:a first immunoconjugate comprising an alkaline phosphatase conjugated to one of a firstmonoclonal antibody, or an antigen-binding fragment thereof, binding specifically to an M3 epitope ofcytokeratin 18 consisting of amino acid sequence NSLREVEARYALQMEQLNG as defined in SEQ IDNO: 1 and a second monoclonal antibody, or an antigen-binding fragment thereof, binding specificallyto an M21 epitope of cytokeratin 18 consisting of amino acid sequence VDGKVVSETNDTKVLR as defined in SEQ ID NO: 2; the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and thesecond monoclonal antibody, or the antigen-binding fragment thereof;a substrate of alkaline phosphatase; and a solid support, wherein the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, is immobilized or is intended to be immobilized to the solid support.

2. The CLIA kit according to claim 1, further comprising a second immunoconjugate comprising acapture ligand conjugated to the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof, wherein the solid support comprises immobilized capture antibodies, or antigen-binding fragments thereof, binding specifically to the capture ligand.

3. The CLIA kit according to claim 2, whereinthe second immunoconjugate comprises fluorescein isothiocyanate (FITC) conjugated to the other of the first monoclonal antibody, or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-binding fragment thereof; and the solid support comprises immobilized anti-FITC antibodies, or antigen-binding fragments thereof, binding specifically to FITC.

4. The CLIA kit according to any one of claims 1 to 3, wherein the solid support comprises magneticbeads.

5. The CLIA kit according any one of claims 1 to 4, whereinthe first immunoconjugate comprises the first monoclonal antibody, or the antigen-binding fragment thereof; and the second immunoconjugate comprises the second monoclonal antibody, or the antigen-binding fragment thereof.

6. The CLIA kit according any one of claims 1 to 5, wherein the first the monoclonal antibody, or theantigen-binding fragment thereof, has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence NYTIH as defined in SEQ ID NO: 3; a VH domain CDR2 having amino acid sequence YFNPSSGYNNYNQKFRD as defined in SEQ ID NO: 4; a VH domain CDR3 having amino acid sequence LIPPFTY as defined in SEQ ID NO: 5; a variable light (VL) domain CDR1 having amino acid sequence RASESVDNYGISFMN as defined in SEQ ID NO: 6; a VL domain CDR2 having amino acid sequence AASKEGS as defined in SEQ ID NO: 7; and a VL domain CDR3 having amino acid sequence LQSKEVPFT as defined in SEQ ID NO: 8.

7. The CLIA kit according to claim 6, wherein the first the monoclonal antibody, or the antigen-binding fragment thereof, has a VH domain having an amino acid sequence as defined in SEQ ID NO: 9; and / or a VL domain having an amino acid sequence as defined in SEQ ID NO: 10.

8. The CLIA kit according to claim 7, wherein the first the monoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain having an amino acid sequence as defined in SEQ ID NO: 11; and / or a light chain having an amino acid sequence as defined in SEQ ID NO: 12.

9. The CLIA kit according to any one of claims 1 to 8, wherein the second the monoclonal antibody,or the antigen-binding fragment thereof, has a variable heavy (VH) domain complementarity determining region 1 (CDR1) having amino acid sequence SFWMN as defined in SEQ ID NO: 13; a VH domain CDR2 having amino acid sequence MLQPADNETKINQKLKD as defined in SEQ ID NO: 14;a VH domain CDR3 having amino acid sequence GGVVTSYWYFDV as defined in SEQ ID NO: 15; a variable light (VL) domain CDR1 having amino acid sequence KASQDVGTAVA as defined in SEQ ID NO: 16; a VL domain CDR2 having amino acid sequence WASTRHT as defined in SEQ ID NO: 17; and aVL domain CDR3 having amino acid sequence QQFSRYPVT as defined in SEQ ID NO: 18.

10. The CLIA kit according to claim 9, wherein the second the monoclonal antibody, or the antigen-binding fragment thereof, has a VH domain having an amino acid sequence as defined in SEQ ID NO: 19; and / or a VL domain having an amino acid sequence as defined in SEQ ID NO: 20.

11. The CLIA kit according to claim 10, wherein the second the monoclonal antibody, or the antigen-binding fragment thereof, has a heavy chain having an amino acid sequence as defined in SEQ ID NO: 21; and / or a light chain having an amino acid sequence as defined in SEQ ID NO: 22.

12. The CLIA kit according to any one of claims 1 to 11, wherein the substrate of alkalinephosphatase is selected from the group consisting of 10,10′-Dimethyl[9,9′-biacridine]-10,10′-diiumdinitrate (lucigenin), 3-(4-methoxyspiro [1,2-dioxetane-3,2´(5´-chloro)-tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate (CSPD), 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), [(4-methoxy-4-(3-phosphoryloxy-4-chlorophenyl)] spiro [1,2-dioxetane-3, 2′-5-chloroadamantane] disodium salt, 9-(4-chlorphenyl sulfobenzoyloyl methylene)-10-methyl-9, 10-dihydroacridine-disodium salt and N, N-dimethyl acridine nitrate.

13. The CLIA kit according to any one of claims 1 to 12, further comprising a substrate buffercomprising the substrate of alkaline phosphatase in a buffer comprising 0.1-1.0 M Tris, 0.1-0.5 % sodium sulfite, 0.5-2.0 % sodium dodecyl sulfate, and 0.15-0.5 % bovine serum albumin, pH 9.0-10.0.,preferably comprising 0.2-0.6 % lucigenin in the buffer.

14. The CLIA kit according to any one of claims 1 to 13, further comprising a calibrator standardcomprising recombinant human cytokeratin 8 and recombinant human cytokeratin 18 at 1:1 molar ratio.

15. The CLIA kit according to any one of claims 1 to 15, further comprising an antibody buffersolution comprising 1 % bovine serum albumin, 1 % bovine immunoglobulin G and 0.05 % preservative in phosphate-buffered saline, pH 7.0.

16. The CLIA kit according to any one of claims 1 to 16, wherein the TPS comprises dimers betweencytokeratin 18 and cytokeratin 8 and optionally also dimers between cytokeratin 18 and cytokeratin 7.

17. A method for detecting tissue polypeptide specific antigen (TPS) comprising cytokeratin 18, themethod comprises: contacting a sample with the first immunoconjugate, the other of the first monoclonal antibody, orthe antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-bindingfragment thereof, and the solid support of the chemiluminescence immunoassay (CLIA) kit according toany one of claims 1 to 16;removing a supernatant from the solid support; adding the substrate of alkaline phosphatase of the CLIA kit according to anyone of claims 1 to16 to the solid support; anddetecting chemiluminescence to thereby detect the TPS comprising cytokeratin 18.

18. The method according to claim 17, wherein contacting the sample comprises:contacting the sample with the first immunoconjugate, the other of the first monoclonal antibody,or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-bindingfragment thereof of the CLIA kit according to any one of claims 1 to 16;incubating the sample with the first immunoconjugate, the other of the first monoclonal antibody,or the antigen-binding fragment thereof, and the second monoclonal antibody, or the antigen-bindingfragment thereof; and adding the solid support of the CLIA kit according to any one of claims 1 to 16 to the sample.

19. The method according to claim 17 or 18, whereinthe solid support comprises magnetic beads; and removing the supernatant comprises: exposing the sample to a magnetic field to capture the magnetic beads; and removing the supernatant from the captured magnetic beads.

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