CN114137222A

CN114137222A - Kit for detecting tissue metal protease inhibitor-1 in body fluid sample

Info

Publication number: CN114137222A
Application number: CN202111403439.5A
Authority: CN
Inventors: 万蒙; 王学忠
Original assignee: Jiangxi Lecheng Biological Medical Co ltd
Current assignee: Jiangxi Lecheng Biological Medical Co ltd
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-03-04

Abstract

The invention provides a kit for detecting tissue metal protease inhibitor-1 in a body fluid sample, which comprises A, B, C, D four compositions: the composition A at least contains a combination of a chaotropic ion, a surfactant, a dispersant and a coagulant; composition B is a composition containing at least a monoclonal antibody capable of binding to tissue metallo-protease inhibitor-1; composition C contains at least a polyclonal antibody capable of binding to tissue metal protease inhibitor-1, or one or more monoclonal antibodies, and is capable of binding to the polyclonal antibody of composition C when tissue metal protease inhibitor-1 in the sample binds to the monoclonal antibody of composition B; composition D is a standard sample containing a known concentration of tissue metallo-protease inhibitor-1. The kit can simply, conveniently and accurately carry out quantitative detection on TIMP-1 in a sample, is beneficial to determining and identifying cancer disease markers, and is further beneficial to the fields of diagnosis, medical treatment and the like.

Description

Kit for detecting tissue metal protease inhibitor-1 in body fluid sample

Technical Field

The invention relates to the technical field of biological detection, in particular to a kit for detecting tissue metal protease inhibitor-1 in a body fluid sample.

Background

Inhibitors of tissue metalloproteinases (TIMPs, abbreviated TIMPs) are specific inhibitors of matrix metalloproteinases (MMPs, abbreviated MMPs). TIMP not only binds to its specific MMP proenzyme in a 1:1 format to inhibit its activation, but also inactivates the active enzyme, thereby rendering MMPs non-degradable. The degree of balance between MMPs and TIMPs determines the net MMP activity, and is an important factor in determining extracellular matrix transformation. The metabolism of the hepatic extracellular matrix is mainly regulated by MMPs, which promote the degradation of the extracellular matrix, and TIMPs, which prevent the degradation of the extracellular matrix by inhibiting MMPs, thereby developing and promoting hepatic fibrosis.

Four types of TIMPs have been found, namely TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMP-1 and TIMP-2 inhibit the activity of all known MMPs and thus play a critical role in maintaining extracellular matrix deposition and degradation during physiologic or fibrotic processes, with TIMP-1 being more important than TIMP-2 in pathological liver processes. TIMP-1 is a 28.5KD glycoprotein that primarily inhibits the activity of interstitial collagenase, stromelysin and gelatinase B. Each TIMP has independent gene expression and functions, but the four amino acid sequences have partial homology and can combine with active MMPs to form a 1:1 complex.

Only TIMP-1 and TIMP-2 among the 4 TIMPs found so far were expressed in the liver, with TIMP-1 being the most significant. Numerous studies have shown that TIMP-1 and TIMP-2 are derived primarily from non-parenchymal cells rather than parenchymal cells. With liver injury or the development of liver cancer, hepatic stellate cell activation is transformed into myofibrillar cells, which are the major sources of type i collagen and TIMP-1, TIMP-2 in the fibrotic liver. Expression and secretion of active TIMP-1 is a feature of activated hepatic stellate cells that, once activated to express type I collagen, simultaneously prevent collagen degradation by expressing TIMP-1 and down-regulating interstitial collagenase activity. Therefore, the detection of TIMP-1 is of great significance for the early diagnosis of liver cancer.

TIMP-1 may also be used in a test to screen a large population for the occurrence of cancer. Furthermore, it relates to a related diagnostic method for monitoring the persistence of Minimal Residual Disease (MRD) or their recurrence of cancer disease in colorectal cancer patients. The method is based on measuring TIMP-1 in body fluids alone or in combination with other tumor markers, e.g. tumor markers.

These methods are based on measuring tissue inhibitor type 1 metalloprotease (TIMP-1) in various body fluids, including blood, plasma, serum, saliva, stool, cerebrospinal fluid, and urine. Studies have shown that individuals with a high probability of having cancer, such as colorectal cancer, are identified by elevated levels of TIMP-1 in body fluids, whereas individuals with low levels of TIMP-1 are less likely to have cancer. Also, individuals who have previously diagnosed cancer and are likely to later develop recurrent cancer with clinical manifestations, e.g., individuals with gastrointestinal cancer, colon cancer, may be identified by elevated levels of TIMP-1 in the body fluids after surgery, while individuals with low levels of TIMP-1 after surgery are less likely to have or later recur to cancer. Thus, detection of TIMP-1 in a body fluid sample can be used to identify individuals who are most likely to have early stage asymptomatic cancer, such as colon cancer or later develop recurrent colorectal cancer. In addition, the identified individual should be further examined and if cancer, recurrent cancer or minimal residual disease or recurrent cancer and minimal residual disease are found, the patient should be provided with surgery, radiation therapy, anti-tumor therapy, or any combination thereof, thereby increasing the chances of a cure and/or long-term survival of the individual.

The common TIMP-1 detection methods at present are ELISA and chemiluminescence methods, and have good reference values for judging the occurrence, development and recovery of hepatic fibrosis and screening and recurrence judgment of related cancers. However, both of these methods completely employ monoclonal antibodies and are therefore inaccurate for detecting bound TIMP-1 in a test sample, and neither method can be used for high throughput applications for screening large populations.

Disclosure of Invention

The purpose of the present invention is to provide a kit that can quantitatively detect TIMP-1 in a sample simply and accurately.

In order to achieve the above object, the technical solution provided by the present invention is as follows:

a kit for quantifying TIMP-1 in a sample comprising composition a, composition B, composition C, and composition D:

composition A: is a combination at least containing chaotropic ionic sodium bromide, a surfactant, a dispersant and a coagulant; the effects of composition a include: (i) eliminating the interference of high-concentration chyle and lipid; (ii) dissociating the bound TIMP-1, and dispersedly exposing the protein to be detected; step (iii) maintaining the stability of the antigen structure of TIMP-1; wherein the surfactant is selected from nonionic surfactant, anionic surfactant and zwitterionic surfactant, the dispersant is selected from sugar, salt and amino acid, and the coagulant is selected from polyethylene glycol;

the sample contains free form TIMP-1 and combined form TIMP-1 combined with metalloprotease, and long-term experiments show that the combined form TIMP-1 has no affinity or poor affinity with various monoclonal antibodies (monoclonal antibodies for short) or polyclonal antibodies, so that the subsequent quantification is inaccurate. The composition A containing the chaotropic sodium bromide, the surfactant, the dispersing agent and the coagulant can act on the combined TIMP-1 and dissociate and disperse, and the structure of the TIMP-1 is kept stable.

Composition B: the method comprises a step of immobilizing a monoclonal antibody on the surface of a latex microsphere by chemical covalent crosslinking, wherein the monoclonal antibody at least contains a monoclonal antibody capable of binding TIMP-1 and is used for capturing TIMP-1 antigen in a sample;

composition C: is a polyclonal antibody or more than one monoclonal antibody (hereinafter abbreviated as polyclonal antibody or polyclonal antibody) at least capable of binding TIMP-1; long-term research shows that due to the structural diversity of TIMP, the adoption of a monoclonal antibody is not enough to fully combine with the TIMP-1 to be measured, so that the measurement is inaccurate. Preferably, more than one monoclonal antibody is used, more preferably, a polyclonal antibody is used. A step for realizing turbidity measurement by immobilizing the polyclonal antibody on the surface of a latex microsphere through chemical covalent crosslinking, wherein when TIMP-1 in a sample is bound to the monoclonal antibody in the composition B, the polyclonal antibody can be bound to the polyclonal antibody in the composition C;

composition D: is a TIMP-1 standard sample containing known concentration; correlating the concentration of TIMP-1 with the resulting turbidity measurement to determine the TIMP-1 content of the sample at the time of measurement;

wherein a two-reagent mode can be formed in which the composition A is used as the first-step measurement reagent 1, and the composition B and the composition C are mixed as the second-step measurement reagent 2; a three-reagent model may be formed in which composition A is used as the first-step measurement reagent 1, composition B is used as the second-step measurement reagent 2, and composition C is used as the third-step measurement reagent 3;

wherein the reactant in the dissociation and dispersion combined TIMP-1 process is a combination of a surfactant and a dispersant, wherein the surfactant is selected from a nonionic surfactant, an anionic surfactant and a zwitterionic surfactant, and the dispersant is selected from a combination of sugar, salt and amino acid; the dissociation and dispersion agent is a combination containing sodium bromide;

wherein the binding protein reacting with TIMP-1 in the sample is a combination of a specific anti-TIMP-1 monoclonal antibody and a polyclonal antibody, and the combination ratio of the monoclonal antibody and the polyclonal antibody is not particularly limited as long as TIMP-1 in the sample can be captured and forms effective turbidity;

wherein: the monoclonal antibody and polyclonal antibody reacting with TIMP-1 in the sample are immobilized on the latex microsphere carrier in a manner not particularly limited, and can be physically adsorbed or chemically crosslinked.

According to the kit provided by the invention, the composition A at least contains the combination of chaotropic ionic sodium bromide, a surfactant, a dispersant and a coagulant, the composition A can eliminate the interference of high-concentration chyle and lipid, dissociate the combined TIMP-1, disperse and expose the tested protein, and maintaining the stability of the antigen structure of TIMP-1, wherein composition B comprises at least a monoclonal antibody capable of binding to TIMP-1, composition C comprises at least a polyclonal antibody capable of binding to TIMP-1, or one or more monoclonal antibodies, wherein when TIMP-1 in the test sample binds to the monoclonal antibody in composition B, can be combined with the polyclonal antibody of the composition C to realize full combination with the tested TIMP-1, therefore, the kit is used for realizing the procedure of measuring turbidity, so that the measuring result is more accurate, and the kit can be used for high-throughput application of screening a large number of people. The invention is useful for the determination and differential determination of cancer disease markers, and thus is useful in the fields of diagnosis, medical treatment, and the like.

Drawings

FIG. 1 is a graph showing the absorbance change and a standard curve for each concentration standard sample;

FIG. 2 is a comparison curve between the detection value of the kit of example 2 of the present invention and the detection value of the chemiluminescence method.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Several embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

The invention provides a kit for detecting tissue metal protease inhibitor-1 in a body fluid sample, which comprises the following A, B, C, D four compositions:

the composition A at least comprises a combination of a chaotropic ion, a surfactant, a dispersing agent and a coagulant, wherein the surfactant is selected from a nonionic surfactant, an anionic surfactant and a zwitterionic surfactant, the dispersing agent is selected from sugar, salt and amino acid, and the coagulant is selected from polyethylene glycol;

composition B is a composition containing at least a monoclonal antibody capable of binding to tissue metallo-protease inhibitor-1;

composition C contains at least a polyclonal antibody capable of binding to tissue metal protease inhibitor-1, or one or more monoclonal antibodies, and is capable of binding to the polyclonal antibody of composition C when tissue metal protease inhibitor-1 in the sample binds to the monoclonal antibody of composition B;

composition D is a standard sample containing a known concentration of tissue metallo-protease inhibitor-1.

The sample used in the measurement kit of the present invention may be whole blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, tissue homogenate, or the like, and is preferably plasma or serum.

As debonding and dispersing agents in the composition a of the present invention: is a combination of a surfactant containing sodium bromide and a dispersant; among these, optional nonionic surfactants are, for example: tween-20, Tween-80, Triton X-100 and Triton X-405; anionic surfactants such as sodium cholate, sodium deoxycholate, sodium taurocholate; zwitterionic surfactants such as CHAPS, CHAPSO; the dispersing agent is selected from sugars, salts, amino acids, such as: sucrose, sorbitol, mannose, sodium chloride, sodium bromide, phosphate, glycine, glycylglycine, potassium aspartate and sodium glutamate.

The sodium bromide is a Chaotropic ion (Chaotropic Ions) which acts to unfold or dissociate macromolecular structures without destroying the immunospecific activity. Chaotropic ions may be mentioned, for example, sodium bromide, sodium thiocyanate, perchlorate, iodide. Experimental research shows that the chaotropic ions applied to the kit of the invention are preferably sodium bromide and sodium thiocyanate, and more preferably sodium bromide.

The surfactant in composition A is an important composition which acts synergistically with the chaotropic ions, further promotes dissociation of the bound form TIMP-1 and helps to maintain the stability of the free form TIMP-1 protein structure. Preferred are anionic surfactants, nonionic surfactants; more preferably a nonionic surfactant, or a combination of an anionic surfactant and a nonionic surfactant; the concentration is preferably 0.01 to 1.0%, more preferably 0.05 to 0.5%.

The salts in the composition A have a synergistic dissociation effect and can be one or a combination of more than one; the concentration of sodium chloride is preferably 50 mM-500 mM, more preferably 150 mM-350 mM; the concentration of sodium bromide is preferably 50 mM-500 mM, more preferably 150 mM-350 mM.

The saccharide in the composition A can help to maintain and stabilize the dissociated TIMP-1 protein, and can be one or a combination of 2; wherein the concentration of the sucrose is preferably 20g/L to 250g/L, more preferably 50g/L to 200 g/L; the mannose concentration is preferably 20mM to 150mM, more preferably 60mM to 100 mM; the sorbitol concentration is preferably 50 mM-300 mM, more preferably 60 mM-200 mM.

The TIMP-1 protein in composition A, whose amino acids contribute to the maintenance and stabilization of dissociation, may be one or a combination of more; wherein the concentration of glycine is preferably 20 mM-150 mM, more preferably 50 mM-100 mM; the glycylglycine concentration is preferably 20 mM-150 mM, more preferably 50 mM-100 mM; the concentration of potassium aspartate is preferably 50 mM-500 mM, more preferably 100 mM-300 mM; the concentration of sodium glutamate is preferably 20mM to 80mM, more preferably 25mM to 50 mM.

The composition a may be an aqueous solution, preferably a buffer solution, the selection of the buffer is not particularly limited, and buffers such as: phosphate, Tris, MOPS, MOPSO, MES; the concentration of the buffer solution is preferably 10 mM-150 mM; more preferably 30 to 100 mM.

As additives for composition a, for example, preservatives such as sodium azide, ProClin 300, chelating agents such as EDTA; stabilizers such as bovine serum albumin, casein.

The compounds of composition A are commercially available products, such as Sigma-Aldrich, West Long chemical, national medicine Shanghai chemical company.

As the monoclonal antibody binding to TIMP-1 in the composition B, the monoclonal antibody is preferably immobilized on the surface of a latex microsphere through chemical covalent crosslinking, and is used for realizing a process of capturing TIMP-1 antigen in a sample. Any antibody or antibody fragment capable of specifically recognizing and binding TIMP-1, such as human or animal derived antibodies, recombinant antibodies, chimeric antibodies, is included. For example, commercially available mouse monoclonal antibodies from ThermoFisher, Abcam, and Italian Shenzhou Biotech.

The carrier used as the TIMP-1 monoclonal antibody comprises various micro-porous plates, test tubes, test strips, latex microspheres and magnetic particles. Immobilization may be by chemical covalent cross-linking or physical adsorption processes. Preferably, the coupling agent is fixed by chemical covalent crosslinking, and has the advantages of high coupling efficiency, high sensitivity and good stability.

The coupling method commonly used may employ a one-step method using carbodiimide (EDC) as a crosslinking agent, or a two-step method using carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as crosslinking agents. Taking latex microspheres as an example:

the one-step process comprises the following steps:

(1) uniformly mixing the monoclonal antibody and the carboxyl microspheres in a mass ratio of 1: 10-100 in a pH6.0025mmol/LMES coupling buffer solution; adding EDC according to the molar ratio of the EDC to carboxyl contained in the latex microspheres being 1: 1-10, mixing and stirring for 1-3 hours at 37 ℃;

(2) centrifuging, removing the supernatant, adding a Tris buffer solution with the pH of 8.00 and containing 0.1-10 percent BSA for resuspension, and centrifuging and cleaning for 2-3 times;

(3) adding 0.1-10% BSA-containing Tris buffer solution with the pH value of 8.00 to resuspend, so that the final concentration of the latex microsphere solution is 0.1-0.5% (W/V); and (5) standby.

Preferably, the one-step process further comprises the following steps:

(1) uniformly mixing the monoclonal antibody and the carboxyl microspheres in a mass ratio of 1: 20-50 in a pH6.0025mmol/LMES coupling buffer solution; adding EDC according to the molar ratio of the EDC to carboxyl contained in the latex microspheres being 1: 2-5, mixing and stirring for 2-3 hours at 37 ℃;

(2) centrifuging, removing the supernatant, adding a Tris buffer solution with the pH value of 8.00 and containing 0.1-5.0 percent BSA for resuspension, and centrifuging and cleaning for 2-3 times;

(3) adding 0.1-5.0% of BSA-containing Tris buffer solution with the pH value of 8.00 to resuspend, so that the final concentration of the latex microsphere solution is 0.1-0.5% (W/V); and (5) standby.

The two-step process comprises the following steps:

(1) suspending the carboxyl microspheres in 15-30 mM MES buffer solution with the pH value of 5.5-6.5;

(2) adding EDC into the solution in the step (1) according to the molar ratio of EDC to the surface carboxyl content of the latex microspheres of 1-10: 1; slowly adding NHS or Sulfo-NHS according to the molar ratio of NHS or Sulfo-NHS to EDC of 1-5: 1, and stirring for 10-30 minutes;

(3) centrifuging, removing the supernatant, adding 30-100 mM boric acid buffer solution with pH8.00 for resuspension, and centrifuging and cleaning for 1-3 times; adding 30-100 mM boric acid buffer solution with pH8.00 for resuspension;

(4) slowly adding the monoclonal antibody to be fixed into the solution obtained in the step (3) according to the final concentration of 100-1000 micrograms/ml, and stirring while adding; continuously stirring for 2-3 hours;

(5) centrifuging, discarding the supernatant, adding a diluent for resuspension, and repeatedly centrifuging and cleaning for 1-3 times in the way; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.1-0.5% (W/V); the diluent is 10-50 mM Tris-glycine buffer solution containing 0.1-0.5% Tween-20 and 0.1% NaN₃，pH7.20～8.50。

Preferably, the two-step process further comprises the following steps:

(1) suspending the carboxyl microspheres in 12-30 mM MES buffer solution with pH6.0-pH6.2;

(2) adding EDC into the solution in the step (1) according to the molar ratio of EDC to the surface carboxyl content of the latex microspheres of 1-5: 1; slowly adding NHS or sulfo-NHS according to the molar ratio of NHS or sulfo-NHS to EDC of 1-3: 1, and stirring for 20-30 minutes;

(3) centrifuging, removing the supernatant, adding 30-50 mM boric acid buffer solution with pH8.00 for resuspension, and centrifuging and cleaning for 1-3 times; adding 30-50 mM boric acid buffer solution with pH8.00 for resuspension;

(4) slowly adding the monoclonal antibody to be fixed into the solution obtained in the step (3) according to the final concentration of 100-500 micrograms/ml, and stirring while adding; continuously stirring for 2-3 hours;

(5) centrifuging, discarding the supernatant, adding a diluent for resuspension, and centrifuging and cleaning for 1-3 times; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.1-0.5% (W/V) for later use; the diluent is 10-50 mM Tris-glycine buffer solution containing 0.1-0.5% Tween-20 and 0.1% NaN₃，pH7.20～8.50。

The components of the reagent for diluting the composition B are not particularly limited, and various formulations can be used as long as they can suspend the latex microspheres therein and keep the antibody stable.

The latex microspheres are commercially available products with the diameter of 100-350 nm, such as Suzhou Kongo, JSR, Germany Merck.

As the polyclonal antibody binding TIMP-1 in the composition C, the polyclonal antibody is preferably immobilized on the surface of the latex microsphere through chemical covalent crosslinking, and when TIMP-1 in the sample is bound to the monoclonal antibody in the composition B, the polyclonal antibody can be bound with the polyclonal antibody of the composition C, so that the polyclonal antibody can be used for realizing the procedure of measuring turbidity. The polyclonal antibody includes any antibody or antibody fragment capable of specifically recognizing and binding TIMP-1, such as human or animal derived antibodies, recombinant antibodies, chimeric antibodies. For example, commercially available rabbit polyclonal antibody or goat polyclonal antibody from ThermoFisher, rabbit monoclonal antibody or goat polyclonal antibody from Abcam, and rabbit polyclonal antibody from Yinqiao Hill Biotech.

The carrier used as TIMP-1 polyclonal antibody comprises various micro-porous plates, test tubes, test strips, latex microspheres and magnetic particles. Immobilization may be by chemical covalent cross-linking or physical adsorption processes. Preferably, the coupling agent is fixed by chemical covalent crosslinking, and has the advantages of high coupling efficiency, high sensitivity and good stability.

As a fixation technique for TIMP-1 polyclonal antibody in composition C, it was not different from that of monoclonal antibody in composition B. Therefore, the description is omitted.

As a standard sample containing TIMP-1 at a known concentration in composition D, TIMP-1 may be derived from the serum of a patient or obtained by fermentation through genetic engineering expression. The standard sample preferably contains 3 to 6 TIMP-1 standard samples with different concentrations, and more preferably contains 5 to 6 TIMP-1 standard samples with different concentrations. The TIMP-1 standard is preferably in the form of a buffer solution, more preferably a lyophilized preparation, which is reconstituted with purified water prior to use. The buffer solution contains, but is not limited to: sodium phosphate, bovine serum albumin, sodium chloride, sucrose, tween-20, polyethylene glycol 6000, and an aqueous solution with a pH value of 7.20-7.40.

The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

Example 1

1. Reagent

Composition A as first agent (R1)

MOPSO buffer pH6.50

50mM sodium chloride

Sodium bromide 150mM

PEG 20000 5.0g/L

Tween-200.3%

BSA 0.06％

Composition B as a second agent (R2)

(1) Mixing the TIMP-1 monoclonal antibody and microspheres with the particle size of 280nm and the carboxyl content of 150ueq/g uniformly in an MES coupling buffer solution with the pH value of 6.0025mmol/L according to the mass ratio of 1: 25; adding EDC according to the molar ratio of the EDC to carboxyl contained in the latex microspheres of 1:2, and mixing and stirring for 2.5 hours at 37 ℃;

(2) centrifuging, removing the supernatant, adding a Tris buffer solution with the pH value of 8.10 and containing 3.0 percent BSA for resuspension, and centrifuging and cleaning for 2 times;

(3) adding 1.0% BSA-containing Tris buffer solution with pH8.03 to resuspend so that the final concentration of the latex microsphere solution is 0.3% (W/V) for later use.

Composition C as third agent (R3)

(1) Mixing TIMP-1 polyclonal antibody and 280nm microsphere with the particle size of 81ueq/g according to the mass ratio of 1:30, and uniformly mixing in MES coupling buffer solution with the pH value of 6.0025mmol/L; adding EDC according to the molar ratio of the EDC to carboxyl contained in the latex microspheres being 1:3, mixing and stirring for 3 hours at 37 ℃;

(2) centrifuging, removing supernatant, adding Tris buffer solution (pH8.05) containing 1.0% BSA, resuspending, centrifuging, and washing for 3 times;

(3) adding 0.5 percent BSA-containing Tris buffer solution with pH of 8.10 to resuspend the mixture so that the final concentration of the latex microsphere solution is 0.2 percent (W/V) for later use.

2. Test specimen

Composition D as a Standard sample

Sodium phosphate buffer 50mM pH7.4

Bovine serum albumin 5.0g/L

0.9 percent of sodium chloride

Sucrose 200g/L

Tween-200.2%

Polyethylene glycol 60002.0 g/L

Purified TIMP-1 was added to the above buffer solutions at 5 concentrations, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, and 800ng/mL, respectively, with pure water as 0.0 ng/mL.

3. Measuring parameters

A measurement device Hitachi 7180 biochemical analyzer;

analytical method two-point end-point method, measuring wavelength 700nm (without auxiliary wavelength);

the reagent sample ratio R1: S: R2: R3 is 200:10:20: 20.

As a result: the calibration curve is shown in fig. 1.

Example 2

1. Reagent

Composition A as first agent (R1)

Tris buffer pH8.00

50mM sodium chloride

50mM sodium thiocyanate

PEG 20000 10.0g/L

Triton X-100 0.2％

BSA 0.06％

Composition B

(1) Suspending the 280nm particle size microsphere with carboxyl content of 150ueq/g in 30mM MES buffer solution with pH of 6.0;

(2) adding EDC into the solution in the step (1) according to the molar ratio of 3:1 of the EDC to the carboxyl content on the surface of the latex microsphere; slowly adding NHS or sulfo-NHS according to the molar ratio of NHS or sulfo-NHS to EDC of 2:1, and stirring for 25 minutes;

(3) centrifuging, removing supernatant, adding 30mM boric acid buffer solution with pH8.00, resuspending, centrifuging, and cleaning for 1 time; adding 30mM boric acid buffer solution with pH8.00 for resuspension;

(4) slowly adding the monoclonal antibody to be fixed into the solution in the step (3) according to the final concentration of 300 micrograms/ml, and stirring while adding; and stirring was continued for 3 hours;

(5) centrifuging, discarding the supernatant, adding diluent for resuspension, centrifuging and cleaning for 2 times; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.3 percent (W/V) for later use; the diluent is 50mM Tris-glycine buffer solution containing 0.5% Tween-20 and 0.1% NaN₃，pH7.80。

Composition C

(1) Suspending the 280nm particle size microsphere with the carboxyl content of 150ueq/g in 25mM MES buffer solution with the pH value of 6.20;

(2) adding EDC into the solution in the step (1) according to the molar ratio of EDC to the surface carboxyl content of the latex microspheres being 1: 1; slowly adding NHS or sulfo-NHS according to the molar ratio of NHS or sulfo-NHS to EDC of 3:1, and stirring for 30 minutes;

(3) centrifuging, removing supernatant, adding 30mM pH8.02 boric acid buffer solution for resuspension, centrifuging and cleaning for 2 times; adding 50mM boric acid buffer solution with pH8.02 for resuspension;

(4) slowly adding the fixed polyclonal antibody to be required into the solution obtained in the step (3) according to the final concentration of 400 micrograms/ml, and stirring while adding; and stirring was continued for 3 hours;

(5) centrifuging, discarding the supernatant, adding diluent for resuspension, and centrifuging and cleaning for 1 time; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.2 percent (W/V) for later use; the diluent is 50mM Tris-glycine buffer solution containing 0.5% Tween-20 and 0.1% NaN₃，pH7.50。

Mixing the composition B and the composition C at a ratio of 1:1 to obtain a second reagent (R2)

2. Test specimen

13 parts of clinical serum sample with confirmed value by chemiluminescence method

3. Measuring parameters

Hitachi 7180 biochemical analyzer for measuring equipment

the ratio of reagent samples R1: S: R2 ═ 200:10: 40;

as a result: as shown in fig. 2, the kit of the present invention has significant correlation with comparative chemiluminescence methods.

Example 3

1. Reagent

The first reagent (R1) in example 1 and the second reagent (R2) in example 2 were used

2. Test specimen

10 normal human plasma specimens and 7 cancer patient plasma specimens

3. The parameters were measured as in example 2

As a result: as shown in Table 1, the kit can significantly distinguish the level of TIMP-1 in clinical plasma samples.

TABLE 1

In summary, according to the kit provided by the present invention, composition A is a composition containing at least chaotropic ionic sodium bromide, a surfactant, a dispersant and a coagulant, the composition A can eliminate the interference of high-concentration chyle and lipid, dissociate the combined TIMP-1, disperse and expose the tested protein, and maintaining the stability of the antigen structure of TIMP-1, wherein composition B comprises at least a monoclonal antibody capable of binding to TIMP-1, composition C comprises at least a polyclonal antibody capable of binding to TIMP-1, or one or more monoclonal antibodies, wherein when TIMP-1 in the test sample binds to the monoclonal antibody in composition B, can be combined with the polyclonal antibody of the composition C to realize full combination with the tested TIMP-1, therefore, the kit is used for realizing the procedure of measuring turbidity, so that the measuring result is more accurate, and the kit can be used for high-throughput application of screening a large number of people. The invention is useful for the determination and differential determination of cancer disease markers, and thus is useful in the fields of diagnosis, medical treatment, and the like.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A kit for detecting a tissue metallo-protease inhibitor-1 in a body fluid sample, comprising the following A, B, C, D four compositions:

2. The kit according to claim 1, wherein the cathepsin-1 monoclonal antibody and the cathepsin-1 polyclonal antibody are immobilized on latex particle carriers, respectively.

3. The kit of claim 1, wherein the chaotropic ion in composition a is sodium bromide.

4. The kit according to claim 1, wherein the binding protein that reacts with the tissue metal protease inhibitor-1 in the sample is a combination of a specific anti-tissue metal protease inhibitor-1 monoclonal antibody and a polyclonal antibody.

5. The kit according to claim 1, wherein the sample is any one of whole blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, and tissue homogenate.

6. The kit according to claim 1, wherein the kit is used to form a two-reagent mode in which composition A is used as the first-step measurement reagent 1, and composition B and composition C are mixed as the second-step measurement reagent 2.

7. The kit according to claim 1, wherein the kit is used to form a three-reagent model in which composition A is used as the first-step measurement reagent 1, composition B is used as the second-step measurement reagent 2, and composition C is used as the third-step measurement reagent 3.

8. The kit according to claim 1, wherein the kit is used for detecting the content of the tissue metal protease inhibitor-1 in the sample on a fully-automatic biochemical analyzer.