CN118759190A

CN118759190A - Chemiluminescent immunoassay CLIA kit for measuring TPS and method for measuring TPS

Info

Publication number: CN118759190A
Application number: CN202410961224.2A
Authority: CN
Inventors: 夏振伟; 刘振世; 薛玲; 基兰·贾加拉穆迪
Original assignee: Taizhou Zecen Biotechnology Co ltd
Current assignee: Taizhou Zecen Biotechnology Co ltd
Priority date: 2024-07-18
Filing date: 2024-07-18
Publication date: 2024-10-11

Abstract

The invention relates to the technical field of chemical determination, in particular to a chemiluminescent immunoassay CLIA kit for determining TPS, which comprises the following components: an anti-reagent A, the active ingredient of which is a first immune complex, the first immune complex is a first monoclonal antibody marked by alkaline phosphatase ALP, and the first monoclonal antibody is specifically combined with an M3 epitope of cytokeratin 18; the anti-reagent B comprises a second immune complex as an active ingredient, wherein the second immune complex is a second monoclonal antibody marked by fluorescein isothiocyanate FITC, and the second monoclonal antibody is specifically combined with an M21 epitope of cytokeratin 18; magnetic particle reagent, its active ingredient is magnetic bead as solid phase carrier; alkaline phosphatase substrates. The CLIA kit and the method for measuring TPS have high sensitivity and can detect TPS in a biological sample of a patient in a short time.

Description

Chemiluminescent immunoassay CLIA kit for measuring TPS and method for measuring TPS

Technical Field

The invention relates to the technical field of chemical determination, in particular to a chemiluminescent immunoassay CLIA kit for determining TPS and a method for determining TPS.

Background

Cancer is one of the major causes of death in the industrial world. In order to improve patient care, efforts are currently underway to find more information about prognosis, early signs of therapeutic response, and disease progression. Thus, tumor markers can be a meaningful class of substances to indicate tumors that may exist in vivo. The tumor marker can be a substance secreted by the tumor or a specific response of the human body to the presence of the tumor. Ideally, such biomarkers should be detected in a bodily fluid (e.g., urine, blood, or serum).

All eukaryotic cells have a cytoplasmic cytoskeletal structure, i.e., a filament composed of various intermediate filament proteins including cytokeratin. Cytoskeletal networks are responsible for the mechanical integrity of cells and are critical in cell processes such as cell division, motility and intercellular contact. It has now been found that more than 20 different cytokeratins, of which cytokeratins 8, 18 and 19 are the highest in simple epithelial cells. Cytokeratin has epithelial cell specificity and its pattern is generally preserved during the transformation of normal cells into malignant cells. When released from proliferating or apoptotic cells, cytokeratin can be used as a useful marker for epithelial malignancy, clearly reflecting the continued activity of the cells.

The clinical value of determining soluble cytokeratin fragments in body fluids is in the early discovery of recurrence of epithelial cell cancer and rapid assessment of the efficacy of the therapeutic response. The three most clinically used cytokeratin markers are the tissue polypeptide antigens TPA, tissue polypeptide specific antigens TPS and CYFRA21-1. Cytokeratin tumor markers can accurately predict disease states prior to traditional methods and provide a simple, noninvasive, inexpensive, and reliable tool for more effective management.

U.S. patent No. 10,386,363 discloses a method for detecting at least two cytokeratins 8, 18 and 19 in a sample. The specific method is that a sample is contacted with a solid carrier, and the solid carrier is combined with specific antibodies to cytokeratin 8, specific antibodies to cytokeratin 18 and specific antibodies to first epitope of cytokeratin 19, and cytokeratin in the sample is combined with the combined antibodies to form a complex. The complex is then contacted with a labeled antibody specific for dimers of cytokeratins 8 and 18 and a labeled antibody specific for a second epitope of cytokeratin 19, the labeled antibody is allowed to bind to the complex to form an immunocomplex of solid phase carrier-antibody-antigen-antibody-label, and the concentration of the immunocomplex is then detected. There is still a need for a rapid, sensitive and accurate detection method for measuring the useful biomarker TPS.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a chemiluminescent immunoassay CLIA kit for measuring TPS and a method for measuring TPS.

The technical scheme adopted for solving the technical problems is as follows: a chemiluminescent immunoassay CLIA kit for determining TPS comprising:

the active ingredient of the anti-reagent A is a first immune complex, the first immune complex is a first monoclonal antibody marked by alkaline phosphatase ALP, the first monoclonal antibody is specifically combined with an M3 epitope of cytokeratin 18, and the epitope consists of an amino acid sequence NSLREVEARYALQMEQLNG defined in SEQ ID NO. 1;

The active ingredient of the anti-reagent B is a second immune complex, the second immune complex is a second monoclonal antibody marked by fluorescein isothiocyanate FITC, the second monoclonal antibody is specifically combined with an M21 epitope of cytokeratin 18, and the epitope consists of an amino acid sequence VDGKVVSETNDTKVLR defined in SEQ ID NO. 2;

The magnetic particle reagent comprises sheep anti-FITC magnetic beads as active ingredients and is used as a solid phase carrier;

alkaline phosphatase substrates.

According to another embodiment of the invention, the second immunocomplex comprises a capture ligand conjugated to the other of the first monoclonal antibody and the second monoclonal antibody, wherein the solid support comprises an immobilized capture antibody that specifically binds to the capture ligand.

According to another embodiment of the invention, the second immunocomplex comprises Fluorescein Isothiocyanate (FITC) conjugated to the other of the first monoclonal antibody and the second monoclonal antibody, and the solid support comprises immobilized sheep anti-FITC antibody that specifically binds to FITC.

According to another embodiment of the invention, the solid support comprises magnetic beads.

According to another embodiment of the invention, the first immunocomplex comprises a first monoclonal antibody and the second immunocomplex comprises a second monoclonal antibody.

According to another embodiment of the present invention, further comprising, the first monoclonal antibody further comprises:

A VH domain complementarity determining region CDR1 of amino acid sequence NYTIH as defined in SEQ ID No. 3;

a VH domain complementarity determining region CDR2 of amino acid sequence YFNPSSGYNNYNQKFRD as defined in SEQ ID No. 4;

a VH domain complementarity determining region CDR3 of amino acid sequence LIPPFTY as defined in SEQ ID No. 5;

VL domain complementarity determining region CDR1 of amino acid sequence RASESVDNYGISFMN as defined in SEQ ID No. 6;

VL domain complementarity determining region CDR2 of amino acid sequence AASKEGS as defined in SEQ ID No. 7;

and the VL domain complementarity determining region CDR3 of amino acid sequence LQSKEVPFT as defined in SEQ ID NO. 8.

According to another embodiment of the invention, the first monoclonal antibody further comprises:

a VH domain of the amino acid sequence as defined in SEQ ID No. 9;

And/or the VL domain of the amino acid sequence as defined in SEQ ID NO. 10.

A heavy chain of an amino acid sequence as defined in SEQ ID NO. 11;

And/or the light chain of the amino acid sequence as defined in SEQ ID NO. 12.

According to another embodiment of the invention, the second monoclonal antibody further comprises:

A VH domain complementarity determining region CDR1 of amino acid sequence SFWMN as defined in SEQ ID No. 13;

A VH domain complementarity determining region CDR2 of amino acid sequence MLQPADNETKINQKLKD as defined in SEQ ID No. 14;

a VH domain complementarity determining region CDR3 of amino acid sequence GGVVTSYWYFDV as defined in SEQ ID No. 15;

VL domain complementarity determining region CDR1 of amino acid sequence KASQDVGTAVA as defined in SEQ ID No. 16;

VL domain complementarity determining region CDR2 of amino acid sequence WASTRHT as defined in SEQ ID No. 17;

And the VL domain complementarity determining region CDR3 of amino acid sequence QQFSRYPVT as defined in SEQ ID NO. 18.

a VH domain of the amino acid sequence as defined in SEQ ID No. 19;

And/or the VL domain of the amino acid sequence as defined in SEQ ID NO. 20.

According to another embodiment of the invention, the second monoclonal antibody comprises:

a heavy chain of an amino acid sequence as defined in SEQ ID NO. 21;

and/or the light chain of the amino acid sequence as defined in SEQ ID NO. 22.

According to another embodiment of the invention, further comprising, the alkaline phosphatase substrate is APS-5, AMPPD, PNPP, ABTS, o-phenylenediamine or tetramethylbenzidine, or AMPPD modified product CSPD, or a combination of BCIP and NBT.

According to another embodiment of the present invention, a substrate buffer comprising 0.1-1.0M Tris, 0.1-0.5% sodium sulfite, 0.5-2.0% sodium dodecyl sulfate and 0.15-0.5% bovine serum albumin having a pH of 9.0-10.0 is further included.

According to another embodiment of the invention, a calibrator comprising recombinant human cytokeratin 8 and recombinant human cytokeratin 18 in a molar ratio of 1:1 is also included.

According to another embodiment of the present invention, an antibody buffer solution comprising phosphate buffer, about 1% bovine serum albumin, about 1% bovine IgG and 0.05% preservative, which is ph7.0, is also included.

According to another embodiment of the invention, the TPS comprises dimers between cytokeratin 18 and cytokeratin 8.

A method for determining TPS using the CLIA kit described above, comprising the steps of:

s1, contacting a sample with a first immune complex, a second immune complex and a solid phase carrier;

s2, removing the supernatant from the solid phase carrier;

S3, adding alkaline phosphatase substrate to the solid-phase carrier;

S4, detecting chemiluminescence so as to detect the TPS content in the sample.

According to another embodiment of the invention, S1 comprises: reacting the sample with the first immune complex and the second immune complex of the CLIA kit at 35-45 ℃ to form a sandwich complex of the first immune complex-antigen (TPS) -second immune complex, adding the solid phase carrier of the CLIA kit into the sandwich complex, reacting at 35-45 ℃, and combining the sandwich complex on the solid phase carrier.

According to another embodiment of the invention, the solid support comprises magnetic beads; s2, exposing the combined magnetic beads to a magnetic field, adsorbing the magnetic beads by the magnetic field, and removing the supernatant.

One aspect of the invention relates to a chemiluminescent immunoassay CLIA kit for the determination of tissue polypeptide specific antigen TPS. The kit comprises an anti-reagent A, wherein the active ingredient of the anti-reagent A is alkaline phosphatase ALP labeled monoclonal antibody, the monoclonal antibody is specifically combined with an M3 epitope of cytokeratin 18, and the epitope consists of an amino acid sequence NSLREVEARYALQMEQLNG defined in SEQ ID NO. 1; the active ingredient of the anti-reagent B is fluorescein isothiocyanate FITC labeled second monoclonal antibody, the antibody is specifically combined with M21 epitope of cytokeratin 18, and the epitope consists of an amino acid sequence VDGKVVSETNDTKVLR defined in SEQ ID NO. 2. The magnetic particle reagent has sheep anti-FITC magnetic beads as the active components and is used as a solid phase carrier.

The invention relates to a method for detecting tissue polypeptide specific antigen TPS. The method comprises the steps of forming an immune complex of ALP-antibody-antigen-antibody-FITC by a sample, the ALP-labeled monoclonal antibody and the FTIC-labeled monoclonal antibody under certain conditions, and combining the immune complex with the goat anti-magnetic particle reagent of the CLIA kit to form a complex of ALP-antibody-antigen-antibody-FITC-goat anti-FITC-magnetic beads. The complex is adsorbed under the action of magnetic field to remove supernatant. The method further comprises adding alkaline phosphatase substrate of the CLIA kit to the complex and detecting chemiluminescence, thereby detecting the concentration of TPS.

The CLIA kit and the method for measuring TPS have the advantages that the sensitivity is high, and TPS in a biological sample of a patient can be detected in a short time.

Drawings

The invention will be further described with reference to the drawings and examples.

FIG. 1 is a schematic reaction diagram of the present invention;

FIG. 2 is a distribution of TPS in healthy individuals;

Fig. 3 is a statistical graph of the correlation between TPS CLIA and IDL TPS cube ELISA.

Detailed Description

TPS is a tumor biomarker and can be used for early diagnosis of various cancer diseases such as breast cancer, prostate cancer, ovarian cancer and gastrointestinal cancer, and can also be used for monitoring anti-cancer treatment and detecting cancer remission. As a tumor biomarker, TPS can also complement other biomarkers (such as cancer antigen 15-3 (CA 15-3), CA125, carcinoembryonic antigen CEA and prostate specific antigen PSA), improving the sensitivity of early detection of progressive cancer disease.

TPS can now be detected by enzyme-linked immunosorbent assay ELISA. Such TPS enzyme-linked immunosorbent kits are generally sensitive to early detection of various cancer diseases, but usually take several hours to complete the detection. The sensitivity of the method is equivalent to that of a TPS enzyme-linked immunosorbent kit, but the speed is obviously higher, the result can be obtained within one hour, and the result can be obtained within 30 minutes generally.

The CLIA kit and method of determining TPS of the invention are based on two monoclonal antibodies that specifically bind to different epitopes of cytokeratin 18. In more detail, one of the two monoclonal antibodies can specifically bind to the M3 epitope of cytokeratin 18, while the other monoclonal antibody can specifically bind to the M21 epitope of cytokeratin 18.

Cytokeratin 18, also known in the art as keratin 18, KRT18, CK18, CYK18 or K18, is a type I cytokeratin. It is probably the most common product of the intermediate filament protein family with cytokeratin 8. They are expressed in a single layer of epithelial tissue in humans. Cytokeratin 18 is often used together with cytokeratin 8, 19 to distinguish between epithelial cells and hematopoietic cells when detecting circulating tumor cells in the blood.

The M3 epitope corresponds to amino acid numbers 322 to 340 of cytokeratin 18 (SEQ ID NO: 23), while the M21 epitope is derived from the-helix 2B2 region of cytokeratin 18, corresponding to amino acid numbers 411 to 429 of cytokeratin 18.

This pair of monoclonal antibodies specifically bind to the M3 and M21 epitopes in cytokeratin 18, enabling the detection of tissue polypeptide specific antigen TPS comprising cytokeratin 18 in biological samples, including the detection of dimers between cytokeratin 18 and cytokeratin 8, and between cytokeratin 18 and cytokeratin 7.

Accordingly, one aspect of the invention relates to a chemiluminescent immunoassay kit for the determination of the tissue polypeptide-specific antigen TPS. The kit comprises a first conjugate comprising alkaline phosphatase ALP or acridinium ester AE or horseradish peroxidase HRP or other marker labeled with a first monoclonal antibody which specifically binds to the M3 epitope of cytokeratin 18 consisting of amino acid sequence NSLREVEARYALQMEQLNG defined in SEQ ID NO. 1. Or alkaline phosphatase ALP or acridinium ester AE or horseradish peroxidase HRP or other marker, which specifically binds to the M21 epitope of cytokeratin 18, which consists of the amino acid sequence VDGKVVSETNDTKVLR defined in SEQ ID NO. 2. The kit also comprises fluorescein isothiocyanate FITC or biotin BIO or other markers for labeling the first monoclonal antibody or the second monoclonal antibody. The kit also comprises anti-FITC magnetic beads or avidin SA magnetic beads or other magnetic bead forming magnetic particle reagents as solid phase carriers. Or the kit comprises alkaline phosphatase ALP or acridinium ester AE or horseradish peroxidase HRP or other markers for labeling the first monoclonal antibody or the second monoclonal antibody, and the other antibody is immobilized on magnetic beads to form a solid phase carrier. The kit further comprises substrates corresponding to the different labels.

The specificity of an antibody may be determined based on affinity and/or thermal sensitivity. Affinity, expressed by the equilibrium constant Kd for antigen-antibody dissociation, is a measure of the strength of binding between an epitope and an antibody binding site. The smaller the Kd value, the stronger the binding strength between the epitope and the antibody. In addition, affinity can also be expressed by an affinity constant Ka, i.e., 1/Kd. It will be clear to the person skilled in the art that the affinity can be determined in a manner known per se for specific antigens.

Titers are a measure of the strength of binding between an antibody and the antigen of interest. The potency is related to both the affinity between the epitope and the binding site of the antibody and the number of relevant binding sites present on the antibody.

Typically, the antibody binds to the antigen with an equilibrium constant Kd of 10-5 to 10-12M/liter or less, preferably 10-7 to 10-12M/liter or less, more preferably 10-8 to 10-12M/liter, i.e., an affinity constant Ka of 105 to 1012M-1 or more, preferably 107 to 1012M-1 or more, more preferably 108 to 1012M-1.

In general, any Kd value greater than 10-4M (or any Ka value less than 104M-1) is considered to be a non-specific binding. The antibody preferably binds to an M3 or M21 epitope with an affinity of less than 500nM, preferably less than 200nM, more preferably less than 10nM, such as less than 5nM or less, such as 1nM or less.

Specific binding of the antibody to the antigen or antigenic determinant may be determined by any suitable means known per se, including for example scatchard analysis and/or competitive binding assays, such as radioimmunoassay RIA, enzyme immunoassay EIA and sandwich competition assays, as well as different methods known in the art.

The antigen-binding fragment of an antibody as used herein may be selected from the group consisting of a single chain antibody, fv fragment, scFv fragment, fab fragment, F (ab ') 2 fragment, fab' fragment, fd fragment, single domain antibody (sdAb), scFv-Fc fragment, di-scFv fragment and multiple complementarity determining region CDRs.

In one embodiment, other monoclonal antibodies that do not bind to ALP may be immobilized on a solid support. In this case, the solid support preferably comprises a plurality of such immobilized monoclonal antibodies. For example, magnetic beads can be used as solid phase carriers for CLIA kits. In this case, the monoclonal antibody may be covalently bound to the magnetic beads. Such covalent attachment may use a variety of attachment variants, such as amino, carboxyl or thiol. For example, a carboxyl group hydrophilic magnetic bead binds to an amino group at a pH of 5 to 6 under room temperature (15 to 25 ℃) conditions, an amino group hydrophilic magnetic bead binds to an aldehyde group at neutral to high pH values under room temperature (15 to 25 ℃) conditions, a tosyl group hydrophobic magnetic bead binds to an amino group and a mercapto group at neutral pH values and 37 ℃, and an epoxy group hydrophilic magnetic bead binds to an amino group and a mercapto group at neutral pH values at room temperature (15 to 25 ℃) or even higher.

The above examples can also be used to immobilize monoclonal antibodies on other solid supports than magnetic beads, such as the surface of a multiwell plate.

In another embodiment, another monoclonal antibody that does not bind ALP is immobilized on a solid support. In this embodiment, the CLIA kit comprises an ALP-labeled first monoclonal antibody or second monoclonal antibody, and further comprises an immunocomplex consisting of a capture ligand that binds to the other of the first monoclonal antibody and the second monoclonal antibody, and then the solid support is labeled with a capture antibody that specifically binds to the capture ligand.

Thus, in this embodiment, the CLIA kit comprises two immune complexes, the first immune complex being one of the two monoclonal antibodies labeled with ALP and the second immune complex being the immune complex of the other monoclonal antibody with its ligand. The second immunocomplex may then be immobilized to the solid support by specific binding of the capture antibody on the solid support to the ligand moiety of the second immunocomplex.

Various ligands may be used in the second immunocomplex to immobilize the second immunocomplex on a solid support. A representative but non-limiting example of such a ligand is fluorescein isothiocyanate FITC. In such an example, the second immune complex comprises FITC-labeled first monoclonal antibody or second monoclonal antibody. The solid support bound anti-FITC antibodies that specifically bound to FITC.

FIG. 1 shows an example in which the CLIA kit includes a first immunocomplex represented by IDL2-ALP and a second immunocomplex represented by IDL 1-FITC. In this example, the solid support is a magnetic bead MB, comprising anti-FITC antibodies bound to the surface of the magnetic bead.

In another embodiment, the first monoclonal antibody and the second monoclonal antibody are bound to a solid support using another technique in addition to the ligand and the capture antibody. Another technique is to use a non-covalent bond between the monoclonal antibody and the solid support, such as a streptavidin-biotin bond, an avidin-biotin bond, or the use of protein A or G. For example, biotin may be used to label the first monoclonal antibody or the second monoclonal antibody as a second immunocomplex, and biotin may be attached to the solid support, and the second immunocomplex may be attached to the solid support via a streptavidin/avidin-biotin bond. Protein a or G conjugate magnetic beads can be used to capture IgG antibodies, but this approach cannot be used to specifically immobilize between the first and second monoclonal antibodies and the magnetic beads unless the monoclonal antibodies that bind to ALP belong to a class of antibodies that do not bind to protein a or G.

However, it may be preferable to use a second immunocomplex consisting of a ligand and to use a solid support consisting of an immobilized capture antibody instead of using streptavidin/avidin-biotin bonds to immobilize the monoclonal antibody, as this effectively avoids interference with endogenous biotin that may be present in the sample to be tested. This means that the assay sensitivity of the CLIA kit is generally higher if such streptavidin/biotin linkages can be avoided.

The solid support is preferably a magnetic particle, i.e., a magnetic bead, such as a carboxyl magnetic bead. In a particular embodiment, the magnetic particles have an average diameter between 0.8 and 1.2 μm.

In one embodiment, the first immune complex comprises a first monoclonal antibody and the second immune complex comprises a second monoclonal antibody. Thus, in this embodiment, the first immune complex is an ALP-labeled monoclonal antibody capable of specifically binding to the M3 epitope; the second immune complex is a ligand (e.g., FITC) labeled monoclonal antibody capable of specifically binding to the M21 epitope.

In one embodiment, the first monoclonal antibody that specifically binds to the M3 epitope has a variable heavy VH domain complementarity determining region 1CDR1 having the amino acid sequence of the VH domain complementarity determining region CDR2 of amino acid sequence YFNPSSGYNNYNQKFRD defined in SEQ ID NO. 3 and a VH domain complementarity determining region CDR3 of amino acid sequence LIPPFTY defined in SEQ ID NO. 5. The first monoclonal antibody further has a variable light VL domain complementarity determining region CDR1 of amino acid sequence RASESVDNYGISFMN defined in SEQ ID No. 6, a VL domain complementarity determining region CDR2 of amino acid sequence AASKEGS defined in SEQ ID No. 7, and a VL domain complementarity determining region CDR3 of amino acid sequence LQSKEVPFT defined in SEQ ID No. 8.

In one embodiment, the first monoclonal antibody has a VH domain with the amino acid sequence defined in SEQ ID No. 9. In another embodiment, the first monoclonal antibody has a VL domain with the amino acid sequence defined in SEQ ID NO. 10. In another embodiment, the first monoclonal antibody has a VH domain having the amino acid sequence defined in SEQ ID NO. 9 and a VL domain having the amino acid sequence defined in SEQ ID NO. 10.

In one embodiment, the first monoclonal antibody has a heavy chain with the amino acid sequence defined in SEQ ID NO. 11. In another embodiment, the first monoclonal antibody has a light chain with the amino acid sequence defined in SEQ ID NO. 12. In another embodiment, the first monoclonal antibody has a heavy chain with the amino acid sequence defined in SEQ ID NO. 11 and a light chain with the amino acid sequence defined in SEQ ID NO. 12.

A monoclonal antibody IDL2 having the CDR regions of SEQ ID NOS 3 to 8, the VH and VL domains of SEQ ID NOS 9 and 10 and the heavy and light chains of SEQ ID NOS 11 and 12 is disclosed in example 1.

In one embodiment, the second monoclonal antibody that specifically binds to the M21 epitope has a VH domain complementarity determining region CDR1 having amino acid sequence SFWMN defined in SEQ ID NO. 13, a VH domain complementarity determining region CDR2 having amino acid sequence MLQPADNETKINQKLKD defined in SEQ ID NO. 14, and a VH domain complementarity determining region CDR3 having amino acid sequence GGVVTSYWYFDV defined in SEQ ID NO. 15. The second monoclonal antibody has a VL domain complementarity determining region CDR1 having amino acid sequence KASQDVGTAVA defined in SEQ ID No. 16, a VL domain complementarity determining region CDR2 having amino acid sequence WASTRHT defined in SEQ ID No. 17, and a VL domain complementarity determining region CDR3 having amino acid sequence QQFSRYPVT defined in SEQ ID No. 18.

In one embodiment, the second monoclonal antibody has a VH domain with the amino acid sequence defined in SEQ ID NO. 19.

In another embodiment, the second monoclonal antibody has a VL domain with the amino acid sequence defined in SEQ ID NO. 20.

In another embodiment, the second monoclonal antibody has a VH domain with the amino acid sequence defined in SEQ ID NO. 19 and a VL domain with the amino acid sequence defined in SEQ ID NO. 20.

In one embodiment, the second monoclonal antibody has a heavy chain with the amino acid sequence defined in SEQ ID NO. 21.

In another embodiment, the second monoclonal antibody has a light chain with the amino acid sequence defined in SEQ ID NO. 22.

In another embodiment, the second monoclonal antibody has a heavy chain with the amino acid sequence defined in SEQ ID NO. 21 and a light chain with the amino acid sequence defined in SEQ ID NO. 22.

A monoclonal antibody IDL1 having the CDR regions of SEQ ID NOS 13 to 18, the VH and VL domains of SEQ ID NOS 19 and 20 and the heavy and light chains of SEQ ID NOS 21 and 22 is disclosed in example 1.

The CLIA kit comprises an alkaline phosphatase substrate.

The alkaline phosphatase substrate is APS-5, AMPPD, PNPP, ABTS, o-phenylenediamine or tetramethyl benzidine, or AMPPD modified product CSPD, or a combination of BCIP and NBT.

The APS-5 is [ (4-chlorobenzenesulfonyl) (10-methyl-9, 10-dihydroacridine methylene disodium phosphate ];

The AMPPD is [3- (2-spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxy cyclohexane disodium salt ];

The BCIP is p-toluidine blue;

the NBT is tetrazolium chloride.

The PNPP is p-nitrophenyl phosphate;

the ABTS is (2, 2' -biazo-bis-3-ethylbenzothiazoline-6-sulfonic acid).

This means that ALP (EC 3.1.3.1) is able to accelerate chemical reactions with substrates, resulting in luminescence values.

Examples of such substrates include dihydroxyacetone phosphate DHAP, which can be hydrolyzed by ALP to Dihydroxyacetone (DHA), which can be reacted with fluorescein to produce intense chemiluminescence (Kokado et al., Chemiluminescent assay of alkaline phosphatase using dihydroxyacetone phosphate as substrate detected with lucigenin, Luminescence (2002) 17(1): 5-10); amic acid adenine dinucleotide phosphate (NADP), which can be catalyzed by ALP to dihydroxyacetone, which can be reacted with fluorescein to produce intense chemiluminescence (Kitamura et al., A new highly sensitive chemiluminescent assay of alkaline phosphatase using lucigenin and its application to enzyme immunoassay, J Biolumin Chemilumin (1995) 10(1): 1-7);3-(2- spiral adamantane) -4-methoxy-4- (3-phosphoryl) -phenyl-1, 2-dioxa-ne disodium salt (AMPPD), which can be catalyzed by ALP to produce chemiluminescence (Bronstein et al., 1,2-Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays, Journal of Bioluminescence and Chemiluminescence (1989) 4(1): 99-111); p-nitrophenyl phosphate (pNPP), p-toluidine Blue (BCIP) and tetrazolium chloride blue (NBT) in combination, D-fluorescein phosphate, adamantane-1, 2-dioxacyclobutane Phenyl Phosphate (PPD) which can be catalyzed by ALP to (Girotti et al., Chemiluminescent Determination of Alkaline Phosphatase Activity in Serum, Analytical Letters (1994) 27(2): 323-335)、3-（4- methoxy spiro (1, 2-dioxacyclobutane-3, 2'- (5' -chloro) tricyclo [3.3.1.1 (3.7) ] decane) -4-yl) disodium phenyl phosphate (CSPD), and 1, 2-dioxacyclobutane is a U.S. patent: chemiluminescent substrates disclosed in patent nos. 6,461,876 and 7,422,908. Specific substrates that may be used for the liquid chemiluminescent substrate include APLS, lucigenin, tris, sodium sulfite, SDS and TWEEN 20. For example, the substrate may comprise about 120mg/L APLS, about 3.4mg/L lucigenin, about 1g/L SDS, about 10mg/L sodium sulfite, and about 0.31g/L TWEEN 20 in about 0.26mol/L Tris buffer (pH about 9.35). An example of such a chemiluminescent substrate is disclosed in chinese patent No. 103344633.

In one embodiment, the alkaline phosphatase substrate comprises APS-5 and luciferin nitrate.

Virtually any chemiluminescent substrate that can produce chemiluminescence by the ALP enzyme can be used in the CLIA kit.

In one embodiment, the CLIA kit comprises a substrate buffer comprising alkaline phosphatase substrate, the buffer consisting of 0.1-1.0M Tris, 0.1-0.5% sodium sulfite, 0.5-2.0% SDS, and 0.15-0.5% bovine serum albumin, at a pH of 9.0-10.0.

Preferably, 0.2-0.6% gloss extract is also included in the buffer.

In one embodiment, the CLIA kit comprises a set of calibrator consisting of TPS antigens at different concentrations, consisting of recombinant human cytokeratin 8 and recombinant human cytokeratin 18 in a molar ratio of 1:1.

In a particular embodiment, the calibrator is formulated from 4000-9000U/L TPS antigen diluted with a buffer containing about 1% bovine serum albumin at a pH of 7.5.+ -. 0.1.

In one embodiment, the CLIA kit comprises an antibody buffer consisting of about 1% bovine serum albumin, about 1% bovine IgG, and 0.05% preservative in phosphate buffer at pH 7.0±0.1.

Another aspect of the invention relates to a method of detecting TPS, which involves the use of a CLIA kit.

In one embodiment, the method comprises contacting the sample with a solid support of a CLIA kit according to the invention and the other of the first immunocomplex, the first monoclonal antibody and the second monoclonal antibody, see fig. 1. The method further comprises removing the supernatant from the solid support, adding alkaline phosphatase substrate of the CLIA kit to the solid support, and detecting the chemiluminescent value, thereby detecting the TPS content.

In one embodiment, the method comprises contacting the sample with a first immune complex, a second immune complex in a CLIA kit. In this embodiment, the method further comprises incubating the sample with the first immunocomplex, the second immunocomplex, and adding a solid phase carrier of the CLIA kit.

In one embodiment, the solid support comprises magnetic beads. In this embodiment, removing the supernatant includes exposing the sample to a magnetic field to adsorb the magnetic beads, and removing the supernatant from the adsorbed magnetic beads.

The sample is preferably a biological sample from a patient or subject.

In one 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 a preferred embodiment, as shown in FIG. 1, a sample (e.g., a serum sample) is contacted with a first immunocomplex (e.g., an ALP-labeled anti-M3 monoclonal antibody immunocomplex, preferably IDL 2-ALP) and a second immunocomplex (e.g., a ligand-labeled anti-M21 monoclonal antibody immunocomplex, preferably IDL-FITC). The sample is then incubated with the two immune complexes for a period of time, preferably 5 to 60 minutes, more preferably 5 to 30 minutes, more preferably 10 to 20 minutes, such as about 15 minutes.

In cytokeratin 18 monomers and complexes of cytokeratin 18 with other molecules (e.g., dimers between cytokeratin 18 and cytokeratin 8 and dimers between cytokeratin 18 and cytokeratin 7), these two immune complexes will specifically bind to M3 and M21 epitopes on cytokeratin 18. As shown in fig. 1, binding of both immune complexes to the M3 and M21 epitopes on the same cytokeratin 18 monomer or complex will form a sandwich complex during incubation.

The sample with the immunocomplexes is then contacted with a solid support, for example by adding anti-FITC magnetic beads to the sample or by adding the sample to anti-FITC magnetic beads. The sample is incubated in the presence of the solid support for a period of time, preferably at intervals of 1 to 30 minutes, more preferably 1 to 15 minutes, more preferably 2 to 10 minutes, such as about 5 minutes. As shown in FIG. 1, anti-FITC antibodies immobilized on a solid support (e.g., magnetic beads) will specifically bind to the FITC ligand in the second immunocomplex IDL 1-FITC.

The solid support will specifically bind to the sandwich complex described above, but will also specifically bind to the second immune complex and the second immune complex which binds to the cytokeratin 18 monomer or complex. Any non-specific binding to the solid support, such as to the first immunoconjugate, will be removed by washing the solid support in one or more washing steps. In these washing steps, the supernatant is preferably removed from the solid support and replaced with a washing liquid as disclosed in example 2 herein. If the solid support is in the form of magnetic beads, the supernatant can be easily removed from the solid support by exposing the sample to a magnetic field to collect the magnetic beads and remove the supernatant.

Alkaline phosphatase substrate is then added to the washed solid phase to produce a chemiluminescent reaction which is representative of the TPS-containing content of the original (biological) sample. Chemiluminescence may then be measured using a spectrometer or other luminescence detector or the like, wherein the measurement (e.g., in the form of relative light units RLUs) represents or indicates the level of TPS in the original sample.

In one embodiment, anti-FITC magnetic beads are present in Tris buffer consisting of methylcellulose and Bovine Serum Albumin (BSA). For example, the Tris buffer may be 0.1-1.0M Tris buffer, including 3-10% methylcellulose, 1-10% BSA, and 0.5-2.0mg/mL anti-FITC magnetic beads.

In one embodiment, the first immunocomplexes are present in Tris buffer containing BSA. For example, the Tris buffer may be a 0.05-0.15M Tris buffer at a pH of 7.0-8.5 containing 1-5% BSA and 1.0-5.0 [ mu ] g/mL (preferably 1.0-2.5 [ mu ] g/mL) of the first immunocomplex.

In one embodiment, the second immune complex is present in Tris buffer containing BSA. For example, the Tris buffer may be a 0.3-0.8M Tris buffer at a pH of 7.0-8.5 containing 1-5 mass% BSA and 0.5-2.0 μg/mL of the second immunocomplex.

In one embodiment, the Tris buffer of the first and second immunocomplexes may comprise a surfactant, such as Tween 20 and/or Triton X-100 (2- [4- (2, 4-trimethylpent-2-yl) phenoxy ] ethanol). In this embodiment, the surfactant is 0.01 to 0.5% by mass.

In one embodiment, the Tris buffer of the first and second immune agar may contain a preservative such as tetracycline, naN3, bronidox (5-bromo-5-nitro-1, 3-dioxane) and/or neomycin sulfate. In such embodiments, the preservative may be 0.01 to 0.1% by mass.

In one embodiment, the calibrator TPS antigen consists of recombinant human cytokeratin 8 and recombinant human cytokeratin 18 in a 1:1 ratio. The calibrator dilutions are preferably Tris buffers, e.g.pH 7.0-9.0, comprising 0.5-10% BSA and 0.5-3% Tween 20.

In one embodiment, the Tris buffer of the calibrator may comprise a preservative such as tetracycline, naN3, bronidox, and/or neomycin sulfate.

In one embodiment, the luminescent substrate is in the form of a Tris buffer, e.g., a 0.1-1.0M Tris buffer at a pH of 9.0-10.0, comprising AP substrate luminescent solution (APLS, see CN 103344633B), 0.1-0.5% sodium sulfite, 0.5-2.0% SDS, 0.2-0.6% fluorescein, 0.15-0.5% BSA. The Tris buffer preferably contains APLS luminescent substrate in a mass ratio of from 1:4 to 1:10.

In one embodiment, the wash solution is Tris-NaCl buffer, e.g., 0.1-1.0M Tris-NaCl buffer containing 1-4% Tween 20 and/or Triton X-100.

The method of detecting TPS in a sample using CLIA kit can yield a detection result within about 30 minutes.

The method and the CLIA kit can be used for early diagnosis of various cancers such as breast cancer, prostatic cancer, ovarian cancer, gastrointestinal cancer and the like, and can also be used for monitoring anticancer treatment and detecting cancer remission.

Examples

This example identifies two mouse monoclonal antibodies, referred to herein as IDL1 and IDL2, respectively, that bind to different epitopes of cytokeratin 18 (CK 18), as shown in SEQ ID NO:23 (GeneBank CAA 31375.1), below. IDL2 antibodies specifically bind to the M3 epitope of CK18, which corresponds to amino acid numbers 322 to 340 of CK18, as shown in the above figures. Another antibody IDL1 was prepared against epitope M21 of the-helix 2B2 region of CK18 (corresponding to amino acid numbers 411 to 429 of CK 18).

Materials:

hybridoma cells prepared from GenScript;

SMART Scribe ™ reverse transcriptase kit (Takara, trade name: 639537).

Zymogen Quick-RNA MINIPREP KIT (Zymogen, product number: R1051)

The research method comprises the following steps:

Total RNA was isolated from hybridoma cells according to the technical manual of Zymogen Quick-RNA MINIPREP KIT. The total RNA was then reverse transcribed into cDNA using isotype specific antisense primers or universal primers according to the technical manual of the SMART script ™ reverse transcriptase kit. Rapid amplification of cDNA Ends from GenScript the antibody fragments for VH and VL were SOP amplified following the RACE standard protocol. The amplified antibody fragments were cloned into standard cloning vectors, respectively. Colony polymerase chain reaction, colony PCR, was performed to screen clones with inserts of the correct size. The number of colonies with correct insert size sequenced for each fragment is no less than 5. The sequences of the different clones were aligned and consensus sequences of these clones were provided.

The isotype of IDL1 was determined to be mouse IgG1/kappa by sequence analysis of the constant region.

Examples

This example developed a chemiluminescent-tagged immunoassay CLIA using the two monoclonal antibodies IDL1 and IDL2 characterized in example 1.

Tables 1 to X below list the buffers used in this example 1. Unless otherwise indicated, the amounts of the reagents listed in the tables are expressed in mass percent.

TABLE 1 Tris buffer (pH 7.5)

TABLE 2 Tris buffer for calibrator

The reagents defined in Table 2 were added to the universal Tris buffer pH7.5 defined in Table 1.

TABLE 3 Tris buffer for anti-agents

The reagents defined in Table 3 were added to the universal Tris buffer pH7.5 defined in Table 1.

TABLE 4 Tris buffer for magnetic beads

TABLE 5 Tris buffer for luminescent substrates

TABLE 6 cleaning solution

Antibody IDL1 was labeled with FITC:

the antibody stock was desalted using a PD10 desalting column and purified by dialysis against carbonate buffer (0.1M Na2CO3, 0.1M Na2HCO 3) at pH8.0-10 at 2-8deg.C. The carbonate buffer was changed twice during desalting and the dialysate was left overnight. Desalted IDL1 antibody was collected, absorbance A280 at 280nm was detected using a spectrophotometer, and the concentration of IDL1 antibody was determined to be A280×0.74×dilution.

20-50. Mu.L of 5mg/mL fluorescein isothiocyanate FITC solution per mg of antibody was added to the desalted IDL antibody and incubated at room temperature (20 ℃ + -5 ℃) for 2 hours. Unbound FITC was removed using a carbonate buffer at ph8.0-10 and the solution was dialyzed overnight while the carbonate buffer was changed 2 or 3 times. FITC-labeled antibody IDL1 complex was diluted with carbonate buffer pH8.0-10 to give absorbance A280 < 2.0. The absorbance at A280 and A492 was measured, and the protein concentration (mg/mL) was calculated to be A280- (A492X 0.35)/1.4.

FITC-labeled antibody IDL1 was added to 0.3-0.8M Tris buffer containing 0.01-1% preservative (NaN 3, neomycin sulfate, tetracycline and/or brinetol) and 0.01-0.5% surfactant (Tween 20 or Triton X-100) to neutralize 1-5% bovine serum albumin BSA at a final concentration of 0.5-2.0 μg/mL.

Labeling antibody IDL2 with ALP:

The antibody stock was desalted using a PD10 desalting column and eluted with phosphate buffered saline PBS buffer, pH 7.5-8.0. Desalted IDL2 antibody was collected.

Alkaline phosphatase ALP was prepared in PBS buffer (pH 7.5-8.0) at a concentration of 1.0-5.0 mg/mL. The absorbance was measured at 280nm using a spectrophotometer and A280 should be 0.2-1.0.

Desalted antibody IDL2 was transferred to a brown glass bottle in the required amount to achieve a molar ratio of 1:2 to 1:10, and a calculated volume of AP solution was added, followed by mixing at room temperature for 4-5 hours. The AP-labeled antibody IDL2 is purified using a protein purifier chromatography column equilibrated with 0.1-0.5M bicarbonate buffer, pH 8.0-9.5. AP-labeled antibody IDL2 was eluted with 0.1-0.5M bicarbonate buffer at ph=8.0-9.5, collected and added to 0.05-0.15M Tris buffer containing 0.01-1% preservative (NaN 3, neomycin sulfate, tetracycline and/or bronidamine). ) And 0.01-0.5% surfactant (Tween 20 or Triton X-100) and 1-5% bovine serum albumin BSA, with a final concentration of 1.0-2.5 μg/mL.

Preparation of magnetic beads:

The carboxyl magnetic bead concentrate was thoroughly mixed and added to the reaction flask, which was placed in a magnetic field for 15 minutes. After all carboxyl beads settled, the supernatant was aspirated. 5 volumes of PBS buffer were added per 2 volumes of carboxyl beads and mixed for 20-30 minutes. The reaction flask was then placed in a magnetic field to allow all carboxyl beads to settle for 15 minutes, and the supernatant was aspirated. This washing step was repeated 3 times to sufficiently wash the carboxyl magnetic beads. Finally, the volume of the carboxyl magnetic bead solution is regulated to 10-50 mg/mL.

Sheep anti-FITC antibody was added to the carboxyl beads at a mass ratio of beads to anti-FITC antibody=100:1 while mixing at 2-8 ℃ for 18 hours. The beads were washed 3 times with PBS and diluted to 10mg/mL and stored at 2-8deg.C until use.

The preparation of the magnetic bead buffer is defined in table 4.

Preparation of a substrate solution:

AP luminescent substrates (luciferin, sigma) were prepared according to Table 5 in Tris buffer.

Preparation of a calibrator:

The calibrator was prepared using TPS standard material, whose TPS antigen was prepared from recombinant human cytokeratins CK8 and CK18 (PROGEN Biotechnik GmbH, germany) in a 1:1 ratio. Recombinant human cytokeratins CK8 and CK18 are expressed in E.coli and have purity of >95% as determined by SDS. The molecular weight of recombinant human cytokeratin CK8 is 52.5-53kDa, isoelectric point pi=6.1, while the molecular weight of recombinant human cytokeratin CK18 is 45-48kDa, isoelectric point=5.7. Briefly, according to Table 2, recombinant human cytokeratins CK8 and CK18 were mixed in Tris buffer at a ratio of 1:1. The mixture was incubated at 37 ℃ for 2 weeks, then again at 4 ℃ for 4 weeks to form CK8 and CK18 dimers. The concentration of calibrator antigen was 4000-9000U/L, diluted in Tris buffer according to Table 2 to give 10-1200U/L of calibrator.

CLIA measurement:

TPS in body fluid samples, serum samples were mixed with FITC-labeled antibody IDL1 (0.5 μg/mL), AP-labeled antibody IDL2 (0.8 μg/mL) and incubated at 37 ℃ for 15 minutes. TPS in the sample forms an AP-IDL2-TPS-IDL1-FITC sandwich complex with FITC-labeled antibody IDL1 and AP-labeled antibody IDL2, as shown in FIG. 1. Then, the sandwich complex was mixed with goat anti-FITC magnetic beads (2 mg/mL) and incubated at 37℃for 5 minutes to form an immune complex of AP-IDL2-TPS-IDL 1-FITC-goat anti-FITC magnetic beads, the complex was adsorbed in a magnetic field, the supernatant was removed, and the magnetic beads were washed with a washing liquid as defined in Table 6. 200 μl of the substrate solution was then added to the beads, suspended for 5 seconds, and the luminescence intensity was measured with respect to the light unit RLU.

Analytical sensitivity:

The blank LOB of CLIA was repeated 20 times with calibration buffer (calibrator a) and these measurements were repeated for 3 batches. The average RLU for 20 replicates per batch was calculated as the concentration based on the standard curve. The highest value of 6U/L in batch 3 is considered the LOB for CLIA.

The limit of detection LOD of CLIA was determined by serial dilution of a low calibrator (calibrator B) corresponding to 30U/L. Low calibrator was diluted 2-fold (B/2, B/4, B/8, B/16 and B/32), and analysis was repeated 10 times for each dilution. LOD is set to a coefficient of variation CV% below 20 corresponding concentration, i.e., 6U/L.

TABLE 7 luminescence intensity detection

Precision:

Precision of CLIA was determined by using one low value sample and one high value sample (136 and 490U/L). 10 replicates were performed for each sample in 3 different batches. CV was calculated, where the low value sample CV was 4.3% and the high value sample CV was 4.6%.

Accuracy/recovery:

The accuracy of CLIA was evaluated by calculating the recovery rate using a high calibrator (calibrator F) at 1200U/L added to the low value samples. The accuracy of CLIA is in the range of 85-115%.

TABLE 8 recovery rate

Dilution linearity:

the high calibrator was diluted four times (F, F/4, F/16, F/64 and F/256) to a concentration of about 5U/L and each dilution was analyzed in triplicate in three different batches. To evaluate the dilution linearity of CLIA. Each dilution was measured in triplicate and compared to the expected concentration. The dilution linearity showed a correlation of 99.9% with the expected value.

TABLE 9 dilution linearity

Serum samples of healthy subjects were added with standard TPS material up to 20000U/L at high concentration, each concentration was measured in triplicate. As high as 20000U/L no hook effect was observed.

TABLE 10 hook effect

Any interference, such as bilirubin, hemoglobin, triglycerides, rheumatoid factors, antinuclear antibodies ANA and human anti-mouse antibodies HAMA, is tested using common interferents. No significant interference was observed by CLIA.

Table 11-interfering substances: bilirubin; analyte: 5U/L

Table 12-interfering substances: bilirubin; analyte: 75U/L

Table 13-interfering substances: hemoglobin; analyte: 5U/L

Table 14-interfering substances: hemoglobin; analyte: 75U/L

Table 15-interfering substances: triglycerides; analyte: 5U/L

Table 16-interfering substances: triglycerides; analyte: 85U/L

Table 17-interfering substances: a rheumatoid factor; analyte: 5U/L

Table 18-interfering substances: a rheumatoid factor; analyte: 75U/L

Table 19-interfering substances: ANA; analyte: 5U/L

Table 20-interfering substances: ANA; analyte: 75U/L

Table 21-interfering substances: HAMA; analyte: 5U/L

Table 22-interfering substances: HAMA; analyte: 75 U/L

Cross-reactivity:

no cross-reaction with CK8 and CK19 was observed.

Stability:

The storage temperature of the CLIA kit is 2-8deg.C, and the shelf life is 12 months. Accelerated stability testing was performed at 37℃for 10 days. Accelerated stability testing verifies that the CLIA kit meets all requirements in terms of% CV.

Critical value:

The threshold was determined by measuring TPS levels in serum samples of 100 healthy human subjects. TPS values are in the range of 10-100U/L, with a critical value (95% CI) of 79U/L. The distribution frequency of TPS in healthy individuals is shown in figure 2.

Clinical contrast:

CLIA was compared to IDL TPS by ELISA using 100 serum samples from healthy subjects and 58 serum samples from different malignancies. Of 158 serum samples, 4 serum samples had TPS values higher than 1200U/L, which were excluded from the relevant analysis. Through regression analysis, the correlation between CLIA and idltps ELISA was 0.967 (P < 0.0001), as shown in fig. 3.

Furthermore, ROC curve analysis was performed with 100 serum samples of healthy subjects and 58 serum samples with different malignancy to compare the performance of CLIA and IDL TPS ELISA. The results show that both assays have similar sensitivities and positive and negative predictive values, as shown in table 23.

TABLE 23 characterization of clinical manifestations

AUC: area under curve

PPV: positive predictive value

NPV: negative predictive value

The above embodiments should be understood as several illustrative examples of the invention. It will be understood by those skilled in the art that various modifications, combinations and variations can be made to the embodiments without departing from the scope of the invention. In particular, where technically feasible, different partial solutions in different embodiments may be combined in other configurations.

Claims

1. A chemiluminescent immunoassay CLIA kit for the determination of TPS comprising:

magnetic particle reagent, its active ingredient is magnetic bead as solid phase carrier;

alkaline phosphatase substrates.

2. The chemiluminescent immunoassay CLIA kit for determining TPS according to claim 1, wherein said second immunocomplex comprises a capture ligand conjugated to the other of the first monoclonal antibody and the second monoclonal antibody, wherein said solid support comprises an immobilized capture antibody that specifically binds to capture ligand.

3. A chemiluminescent immunoassay CLIA kit for determining TPS according to claim 2 wherein said second immunocomplex comprises Fluorescein Isothiocyanate (FITC) conjugated to the other of the first monoclonal antibody and the second monoclonal antibody and said solid support comprises immobilized sheep anti-FITC antibody that specifically binds FITC.

4. The chemiluminescent immunoassay CLIA kit for determining TPS according to claim 1, wherein said magnetic beads are sheep anti-FITC magnetic beads.

5. The chemiluminescent immunoassay CLIA kit for determining TPS according to claim 1, wherein said first monoclonal antibody further comprises:

and a VL domain complementarity determining region CDR3 of amino acid sequence LQSKEVPFT as defined in SEQ ID No. 8;

Or (b)

A VH domain of the amino acid sequence as defined in SEQ ID No. 9;

And/or a VL domain of an amino acid sequence as defined in SEQ ID NO. 10;

Or (b)

A heavy chain of an amino acid sequence as defined in SEQ ID NO. 11;

And/or the light chain of the amino acid sequence as defined in SEQ ID NO. 12.

6. The chemiluminescent immunoassay CLIA kit for determining TPS according to claim 1, wherein said second monoclonal antibody further comprises:

And a VL domain complementarity determining region CDR3 of amino acid sequence QQFSRYPVT as defined in SEQ ID No. 18;

Or (b)

A VH domain of the amino acid sequence as defined in SEQ ID No. 19;

and/or a VL domain of an amino acid sequence as defined in SEQ ID NO. 20;

Or (b)

A heavy chain of an amino acid sequence as defined in SEQ ID NO. 21;

and/or the light chain of the amino acid sequence as defined in SEQ ID NO. 22.

7. A chemiluminescent immunoassay CLIA kit for determining TPS according to claim 1 wherein said alkaline phosphatase substrate is APS-5, AMPPD, PNPP, ABTS, o-phenylenediamine or tetramethylbenzidine, or AMPPD modified product CSPD, or a combination of BCIP and NBT.

8. The chemiluminescent immunoassay CLIA kit for determining TPS of claim 1 further comprising a substrate buffer comprising 0.1-1.0M Tris, 0.1-0.5% sodium sulfite, 0.5-2.0% sodium dodecyl sulfate and 0.15-0.5% bovine serum albumin at ph9.0-10.0.

9. The chemiluminescent immunoassay CLIA kit for determining TPS according to claim 1, further comprising a calibrator comprising a 1:1 molar ratio of recombinant human cytokeratin 8 to recombinant human cytokeratin 18.

10. The chemiluminescent immunoassay CLIA kit for determining TPS of claim 1 further comprising an antibody buffer solution comprising phosphate buffer, about 1% bovine serum albumin, about 1% bovine IgG and 0.05% preservative, ph7.0.

11. A method for determining TPS, characterized in that the CLIA kit according to any one of claims 1-10 is used and comprises the steps of:

s2, removing the supernatant from the solid phase carrier;

S3, adding alkaline phosphatase substrate to the solid-phase carrier;

S4, detecting chemiluminescence so as to detect the TPS content in the sample.

12. A method for determining TPS according to claim 11, characterized in that,

S1 comprises the following steps: reacting the sample with the first immune complex and the second immune complex of the CLIA kit at 35-45 ℃ to form a sandwich complex of the first immune complex-antigen (TPS) -second immune complex, adding the solid phase carrier of the CLIA kit into the sandwich complex, reacting at 35-45 ℃, and combining the sandwich complex on the solid phase carrier.

13. The method of measuring TPS according to claim 11, wherein S2 comprises exposing the bound beads to a magnetic field, adsorbing the beads by the magnetic field, and removing the supernatant.