CN114778823A

CN114778823A - Determination reagent, kit and quantitative method for human asialoglycoprotein receptor

Info

Publication number: CN114778823A
Application number: CN202210437843.2A
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: 2022-04-25
Filing date: 2022-04-25
Publication date: 2022-07-22

Abstract

The invention provides a determination reagent for a human asialoglycoprotein receptor, which comprises the following components: the first reagent comprises buffer solution, an anti-interference agent A, a dispersing agent and a coagulant; a second agent comprising a polyclonal antibody linked to an insoluble carrier, a monoclonal antibody directed against the specific domain of sH2a EGHRG pentapeptide. The buffer solution, the anti-interference agent A, the dispersing agent and the coagulant are used in a combined mode, and then the buffer solution, the anti-interference agent A, the dispersing agent and the coagulant are matched with the polyclonal antibody connected with the insoluble carrier and the monoclonal antibody of the anti-sH 2a specific structural domain EGHRG pentapeptide to achieve rapid detection of the human asialoglycoprotein receptor in the sample, and meanwhile the accuracy of the detection result is guaranteed.

Description

Determination reagent, kit and quantitative method for human asialoglycoprotein receptor

Technical Field

The invention relates to the technical field of biological detection, in particular to a determination reagent, a kit and a quantitative method for a human asialoglycoprotein receptor.

Background

The human asialoglycoprotein receptor (ASGPR) is a membrane heteromeric substance that is expressed only in hepatocytes. The soluble secreted form of sH2a is not produced by shedding on the cell surface, but rather by intracellular cleavage of its membrane-bound precursor, encoded by an alternatively spliced form of the receptor H2 subunit.

ASGR2(asialoglycoprotein receiver 2) is heterogeneousOne of the two subunits of oligomeric ASGPR, which is specifically expressed on hepatocytes. It is also known as H2 and has high homology to another subunit known as H1. ASGPR is present on the plasma membrane of hepatocytes, toward the sinusoidal side, and undergoes endocytic cycling. The receptor is a type II membrane protein, therefore, ASGR2 has an N-terminus facing the cytoplasm, a transmembrane region, a stem region and a Ca²⁺Dependent CRD (carbohydrate recognition domain). The molecular weight of this subunit is 50 kDa.

The alternative splice variant of H2a of the H2 subunit of the human asialoglycoprotein receptor differs from the H2b variant in that an additional pentapeptide EGHRG is present in the outer domain alongside the membrane span. This difference results in the retention and degradation of H2a in the endoplasmic reticulum when no H1 subunit is expressed in the cell. In contrast, a large fraction of the mono-expressed H2b was processed by golgi apparatus and reached the cell surface. Using a novel specific anti-H2 a antibody, researchers found that in HepG2 cells, H2a was rapidly cleaved into a 35-kDa fragment that contained the entire outer domain, most of which was secreted into the culture medium. The cleavage site of the secretory fragment is located at the luminal end of the membrane span. No membrane bound H2a left the ER, indicating that the pentapeptide is a signal for ER retention and degradation of the membrane form, but does not prevent secretion of the cleaved soluble form. H2a does not form a membrane receptor complex with H1l as does H2 b. Thus, H2a is not a subunit of the receptor, but is a precursor to secreted forms of the protein; signal peptidases may be responsible for the cleavage of soluble fragments. Thus, the membrane-proximal sequence functions as a signal anchor sequence (H2b) or as a signal sequence for cleavage, regulating the function of the transmembrane domain of type ii membrane proteins, which produces a secreted product (H2 a).

An alternatively spliced form of the ASGR2 subunit, known as sH2a, is found in the serum of healthy individuals. This form of serum levels varies in liver fibrosis and defines the status of hepatocyte function. Thus, it has potential as a non-invasive marker of liver function and fibrosis.

The incidence of liver cancer (HCC) in our country is on the 4 th of all tumor incidence, and liver cancer diagnosis at present mainly depends on means such as imaging examination, serum markers, needle biopsy and the like. Among them, serum markers are mainly represented by a recognized and clinically applied alpha-fetoprotein, an alpha-fetoprotein heteroplasmon, abnormal prothrombin and the like, and with the development of protein and molecular biology technologies, new markers are continuously discovered and gradually applied to the clinic, such as microRNA, glypican, N-sugar markers and the like, but the clinical requirements on early diagnosis of liver cancer and disease hierarchical management are still far from being met.

The current common sH2a detection method is only ELISA and has good reference value for judging the occurrence, development and recovery of hepatic fibrosis and screening and relapse judgment of related cancers. However, the above-mentioned methods and kits are all solid-phase methods using monoclonal antibodies, and require labeling of detection antibodies, which are cumbersome to produce and prepare, and not suitable for application in high-throughput full-automatic biochemical analyzers.

Because the sH2a protein has a simple structure, is strong in homology with H1 and H2b, has a similar structure, and is not easy to obtain an effective antibody suitable for an immunoturbidimetric platform, a homogeneous detection kit applied to a full-automatic biochemical analyzer is not found at present.

Disclosure of Invention

Based on this, the object of the present invention is to provide a reagent, a kit and a method for determining the amount of human asialoglycoprotein receptor, which at least solve the above-mentioned deficiencies of the related art.

The invention provides a determination reagent for a human asialoglycoprotein receptor, which comprises the following components:

the first reagent comprises buffer solution, an anti-interference agent A, a dispersing agent and a coagulant;

a second agent comprising a polyclonal antibody linked to an insoluble carrier, a monoclonal antibody directed against the specific domain of sH2a EGHRG pentapeptide.

The invention has the beneficial effects that: the buffer solution, the anti-interference agent A, the dispersing agent and the coagulant are used in a combined mode, and then the buffer solution, the anti-interference agent A, the dispersing agent and the coagulant are matched with the polyclonal antibody connected with the insoluble carrier and the monoclonal antibody of the anti-sH 2a specific domain EGHRG pentapeptide to achieve rapid detection of the human asialoglycoprotein receptor in the sample, and meanwhile accuracy of a determination result is guaranteed.

In addition, the reagent for measuring a human asialoglycoprotein receptor provided by the present invention may have the following technical features:

the concentration of the buffer solution is 10 mmol/L-300 mmol/L, and the buffer solution is at least one of Tris buffer solution, MOPS buffer solution, MES buffer solution, phosphate buffer solution and glycylglycine buffer solution.

Further, the anti-interference agent A comprises a surfactant, a chaotropic ion and an amino acid;

wherein the concentration of the surfactant is 0.01-1.0%, and the surfactant is at least one of nonionic surfactant, anionic surfactant and zwitterionic surfactant;

the chaotropic ions are at least one of sodium bromide, sodium thiocyanate and potassium iodide;

the amino acid is at least one of glycine, glycylglycine, potassium aspartate, sodium glutamate and glutamic acid.

Further, the dispersant is at least one of organic acid, saccharide and salt;

wherein the concentration of the organic acid is 10 mmol/L-200 mmol/L, and the organic acid is at least one of citric acid, tartaric acid, malic acid and succinic acid;

the saccharide is at least one of sucrose, sorbitol, mannose and trehalose, the concentration of the sucrose is 50-300 g/L, the concentration of the sorbitol is 50-300 mmol/L, the concentration of the mannose is 20-150 mmol/L, and the concentration of the trehalose is 10-100 mmol/L;

the salt is at least one of sodium chloride, sodium sulfate and phosphate, the concentration of the sodium chloride is 50 mmol/L-500 mmol/L, the concentration of the sodium sulfate is 10 mmol/L-100 mmol/L, and the concentration of the phosphate is 20 mmol/L-200 mmol/L.

Further, the concentration of the coagulant is 0.1% -6.0%, and the coagulant is at least one of polyethylene glycol 6000 and polyethylene glycol 20000.

Further, the anti-human asialoglycoprotein receptor antibody is immobilized on an insoluble support comprising latex particles, magnetic particles, silica particles.

Further, the first reagent further comprises a preservative, a chelating agent and a stabilizing agent.

Another objective of the invention is to provide a kit for detecting a human asialoglycoprotein receptor, which comprises the above-mentioned detection reagent, and the kit further comprises a standard sample containing a known concentration of sH2 a.

Further, the standard sample is a water and/or serum mixed solution containing buffer, sodium chloride, bovine serum albumin, sucrose, mannitol, tween, EDTA, and a preservative.

Another object of the present invention is to provide a method for quantifying a human asialoglycoprotein receptor, which is applied to the above-mentioned assay reagent, for the purpose of diagnosis and treatment of non-diseases, the method comprising the steps of:

(1) mixing a sample with the first reagent for reaction to obtain a first reaction solution, wherein the first reagent is used for eliminating interference of high-concentration chyle and lipid in the sample and dispersedly exposing the detected protein in the sample;

(2) mixing the first reaction solution with the second reagent for reaction to obtain a second reaction solution;

(3) and reading the absorbance value of the second reaction solution at the corresponding wavelength, and calculating the content of the human asialoglycoprotein receptor.

In the invention, the buffer solution, the anti-interference agent A, the dispersing agent and the coagulant are used for eliminating the interference of high-concentration chyle and lipid, dispersing and exposing the detected protein, keeping the stability of the sH2a antigen structure, enhancing the strength of antigen-antibody binding turbidity and further ensuring that the detection result is more accurate.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a graph showing the absorbance change and the standard curve of each concentration standard sample in example two of the present invention;

FIG. 2 is a graph showing the comparison between the reagent kit detection value and the ELISA method detection value in the third embodiment of the present invention.

Detailed Description

So that the manner in which the features and aspects of the invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. The experimental procedures described in the following examples are only for demonstrating the feasibility of the present invention, and the application of the present invention is not limited thereto. The experimental procedures mentioned in the examples are, unless otherwise specified, all the procedures in the conventional experiments; the reagent consumables mentioned are conventional reagent consumables, unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Example one

A method for quantifying the human asialoglycoprotein receptor for non-disease diagnostic and therapeutic purposes, comprising the steps of:

(1) mixing a sample with a first reagent containing a buffer solution, an anti-interference agent A, a dispersing agent and a coagulant for reaction to obtain a first reaction solution, wherein the first reagent is used for eliminating interference of high-concentration chyle and lipid in the sample and dispersing and exposing detected protein in the sample;

(2) mixing the first reaction solution with a second reagent containing a polyclonal antibody connected with an insoluble carrier and a monoclonal antibody of an anti-sH 2a specific structural domain EGHRG pentapeptide for reaction to obtain a second reaction solution;

(3) and reading the absorbance value of the second reaction solution at the wavelength of 600nm, and calculating the content of the human asialoglycoprotein receptor.

It should be noted that the apparatus used in the above quantitative method is a conventional apparatus in the art, and may be a Hitachi 7180 full-automatic biochemical analyzer.

In the quantitative method in the embodiment, the buffer solution, the anti-interference agent A, the dispersing agent and the coagulant are used for eliminating the interference of high-concentration chyle and lipid, dispersing and exposing the detected protein, keeping the stability of the sH2a antigen structure, and enhancing the strength of antigen-antibody binding turbidity, so that the determination result is more accurate.

The reagent adopted in the method is a reagent for measuring the human asialoglycoprotein receptor, and comprises the following components:

and a second reagent comprising a polyclonal antibody linked to an insoluble carrier, a monoclonal antibody against the specific domain of sH2a, EGHRG pentapeptide.

In the application, the first reagent is a combination containing buffer solution, anti-interference agent A, dispersing agent and coagulant, wherein the buffer solution is selected from one of Tris buffer solution, MOPS buffer solution, MES buffer solution, phosphate buffer solution and glycylglycine buffer solution;

the anti-interference agent A is a combination containing a surfactant, chaotropic ions and amino acids, wherein the surfactant is selected from one of a nonionic surfactant, an anionic surfactant and a zwitterionic surfactant; the chaotropic ion is selected from one of sodium bromide, sodium thiocyanate and potassium iodide; the amino acid is selected from aspartic acid or glutamic acid;

the dispersant is selected from organic acid, saccharide, and salt, and the coagulant is polyethylene glycol.

The first reagent can have the following functions: (1) eliminating the interference of high-concentration chyle and lipid; (2) dispersedly exposing the tested protein, and keeping the stability of the sH2a antigen structure; (3) the strength of antigen-antibody binding turbidity is enhanced.

The buffer solution in the first reagent of the present invention is not particularly limited, and the purpose of the buffer solution is to provide a pH range necessary for the detection solution, and in this example, the buffer solution is Tris buffer solution, and one or more of Tris buffer solution, MOPS buffer solution, MES buffer solution, phosphate buffer solution, and glycylglycine buffer solution may be used, and the buffer solution concentration is 10mmol/L to 300mmol/L, and the pH range is 5.0 to 10.0.

In the present application, the anti-interference agent a is a combination comprising a surfactant, a chaotropic ion, and an amino acid, wherein, in the present embodiment, the surfactant is a nonionic surfactant: tween-20 at a concentration of 0.01% to 1.0%, although in other embodiments the surfactant is one or more of a nonionic surfactant, an anionic surfactant, and a zwitterionic surfactant, and optional nonionic surfactants include: tween-20, Tween-80, Triton X-100, Triton X-405, NP-40, Brij-35; optional anionic surfactants include: sodium cholate, sodium deoxycholate, sodium taurocholate; alternative zwitterionic surfactants include: CHAPS and CHAPSO.

Specifically, the chaotropic ions are at least one of sodium bromide, sodium thiocyanate and potassium iodide, the chaotropic ions are used for unfolding or dissociating the combination of the antigen and the blood protein without destroying the specific activity of the antigen, and the concentration of the chaotropic ions is 10 mmol/L-300 mmol/L.

In this embodiment, preferably, sodium bromide is used as the chaotropic ion.

Furthermore, the amino acid in the anti-interference agent A is helpful for maintaining and stabilizing the sH2a antigen protein in the sample, and can be one or a combination of glycine, glycylglycine, potassium aspartate, sodium glutamate and glutamic acid, and the concentration range of the selected glycine is 20 mmol/L-150 mmol/L; selecting the concentration range of glycylglycine to be 20 mmol/L-150 mmol/L; the concentration range of the potassium aspartate is 50 mmol/L-500 mmol/L; the concentration range of the sodium glutamate is 20 mmol/L-800 mmol/L.

In this embodiment, the amino acid in the anti-interference agent a is glycine, and the concentration of the glycine is 50mmol/L to 100 mmol/L.

The dispersant in the first reagent of the present invention is a combination of an organic acid, a saccharide and a salt, and the organic acid is one or more selected from citric acid, tartaric acid, malic acid and succinic acid. The organic acid can also be selected from its salt form, such as sodium citrate, potassium sodium tartrate, sodium malate, and sodium succinate. The concentration of the organic acid is 10 mmol/L-200 mmol/L.

In this embodiment, the organic acid is citric acid with a concentration of 20mmol/L to 100 mmol/L.

Specifically, the saccharide is helpful for maintaining and stabilizing the sH2a antigen protein in the sample, and can be one or a combination of more of sucrose, sorbitol, mannose and trehalose, and the concentration range of the sucrose is selected to be 50 g/L-300 g/L; selecting mannose with the concentration range of 20 mmol/L-150 mmol/L; the concentration range of the sorbitol is 50 mmol/L-300 mmol/L; the concentration range of the trehalose is 10 mmol/L-100 mmol/L.

In the embodiment, the sugar is sucrose, and the concentration is 100 g/L-250 g/L.

Furthermore, the salts have the functions of resisting interference and enhancing antigen-antibody reaction, can be one or a combination of more of sodium chloride, sodium sulfate and phosphate, and the concentration range of the sodium chloride is 50 mmol/L-500 mmol/L; selecting the concentration range of sodium sulfate to be 10 mmol/L-100 mmol/L; the concentration range of the selected phosphate is 20 mmol/L-200 mmol/L.

In this embodiment, the salt is sodium chloride with a concentration range of 100mmol/L to 350 mmol/L.

In the present application, the coagulant in the first reagent of the present invention is selected from the group consisting of polyethylene glycol 6000 and polyethylene glycol 20000, and the concentration of the coagulant is in the range of 0.1 to 6.0%.

In this embodiment, the coagulant is polyethylene glycol 6000 with a concentration of 0.2-2.0%.

In addition, as an additive of the first reagent in the present application, sodium azide is used as a preservative, and in some alternative embodiments, Proclin300 and gentamicin sulfate can also be used as the preservative; EDTA is selected as the chelating agent; the stabilizer is bovine serum albumin, and in some alternative embodiments, the stabilizer is casein.

The compounds in the first reagent of this example were all commercially available.

In the present application, the second reagent is a combination of a polyclonal antibody and a monoclonal antibody, each of which is capable of binding to a characteristic domain of sH2a, and the antibodies are immobilized on the surface of latex microspheres by chemical covalent crosslinking, and used for binding to sH2a antigen in a sample; the polyclonal antibody in the second reagent can be quantitatively measured on a full-automatic biochemical analyzer without distinguishing the capture antibody from the detection antibody and marking the antibodies with fluorescein, enzyme and the like.

In this example, the polyclonal antibody binding to sH2a antigen in the second agent of the present invention is ASGR2 antibody, and the monoclonal antibody is an antibody against the characteristic antigenic domain of sH2a, wherein the characteristic antigenic domain of sH2a is the protein domain formed by the specific domain EGHRG pentapeptide.

As a connecting process of an antibody and an insoluble carrier, the antibody is firstly combined with Bovine Serum Albumin (BSA) through chemical covalent crosslinking to form an anti-sH 2a antibody-BSA conjugate, and then the antibody is fixed on the insoluble carrier through a physical adsorption or chemical crosslinking mode, so that the antibody is fully combined with a tested sH2a antigen, the turbidity determination is enhanced, and the determination result is more accurate. The procedure for binding sH2a antigen in a sample is realized by fixing the sH2a antigen on the surface of a latex microsphere through chemical covalent crosslinking, and the procedure comprises any antibody or antibody fragment capable of specifically recognizing and binding sH2a, such as human or animal antibodies, recombinant antibodies and chimeric antibodies. For example, commercially available rabbit polyclonal antibody and mouse monoclonal antibody from ThermoFisher, rabbit polyclonal antibody and mouse monoclonal antibody from Abcam, rabbit polyclonal antibody and rabbit monoclonal antibody from Yinqiao Hitachi Biotech, and rabbit polyclonal antibody from Huamei Biotech.

As insoluble carriers of sH2a antibody, various latex microspheres, magnetic particles, silica particles were included. 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.

Conventional coupling methods may employ carbodiimide (EDC) and/or N-hydroxysuccinimide (NHS) as cross-linking agents. Taking latex microspheres as an example:

(1) anti-sH 2a antibody-BSA coupling process steps:

(1.1) uniformly mixing the anti-sH 2a antibody, a polyclonal antibody with the concentration of 5.0mg/mL, or a monoclonal antibody, or a mixed antibody of the polyclonal antibody and the monoclonal antibody, and BSA (bovine serum albumin) according to the mass ratio of 1:3 in 25mmol/L MES buffer solution with the pH value of 6.20;

(1.2) adding EDC dissolved in the same MES buffer solution, and stirring at room temperature for 30 minutes, wherein the final concentration of EDC is 50% of the BSA concentration;

(1.3) standing at 6 ℃ for later use.

In the anti-sH 2a antibody-BSA coupling process, the concentration range of the anti-sH 2a antibody is more than or equal to 1.0mg/mL, the mass ratio is 1: 1-1: 5, the stirring action time at room temperature is 10-120 minutes, the final concentration of EDC is 20-80% of the BSA concentration, and the standing temperature is 2-8 ℃.

(2) Process steps for immobilization of anti-sH 2a antibody-BSA conjugate on latex microspheres:

(2.1) suspending the carboxyl microspheres in30 mmol/L MES buffer solution with pH of 6.0;

(2.2) adding EDC into the solution in the step (2.1) according to the molar ratio of 5:1 of EDC to the carboxyl content on the surface of the latex microspheres; slowly adding NHS or Sulfo-NHS into the mixture according to the molar ratio of NHS or Sulfo-NHS to EDC of 3:1, and stirring for 20 minutes;

(2.3) centrifuging, removing the supernatant, adding 50mmol/L boric acid buffer solution with the pH value of 8.00 for resuspension, and centrifuging and cleaning for 2 times; then adding 50mmol/L boric acid buffer solution with the pH value of 8.00 for resuspension;

(2.4) slowly adding the required immobilized anti-sH 2a antibody-BSA conjugate to the solution of step (2.3) at a final concentration of 500. mu.g/ml, and stirring while adding; and stirring is continued for 2 hours;

(2.5) centrifuging, removing the supernatant, adding diluent for resuspension, and repeatedly centrifuging and cleaning for 2 times; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.6% (W/V); the diluent is 100mmol/L Tris-glycylglycine buffer solution, which contains 4.0% Tween-20, 1.0% NaN3 and has a pH value of 8.00.

In the process of fixing the anti-sH 2a antibody-BSA conjugate on the latex microspheres, the concentration of the buffer solution in the step (2.1) is 15-30 mmol/L, pH is 5.5-6.5; in the step (2.2), the molar ratio of the content of carboxyl on the surfaces of the EDC and the latex microspheres is 2-10: 1, the molar ratio of NHS or sulfo-NHS to EDC is 3-5: 1, and the stirring time is 10-30 minutes; in the step (2.3), the resuspension concentration of the boric acid buffer solution is 30-100 mmol/L, and the solution is centrifugally cleaned for 1-3 times; in the step (2.4), slowly adding the anti-sH 2a antibody-BSA conjugate to be fixed according to the final concentration of 10-1000 micrograms/ml, and stirring while adding; continuously stirring for 2-3 hours; and (3) repeatedly carrying out centrifugal cleaning for 1-3 times in the step (2.5), wherein the final concentration of the latex microsphere solution is 0.1% -0.8% (W/V), and the diluent is 10-200 mmol/L Tris-glycylglycine buffer solution which contains 0.1-10.0% of Tween-20 and 1.0% of NaN3 and has the pH value of 6.50-8.50.

The reagent component of the second reagent is not particularly limited as long as it can suspend the latex microspheres therein to keep the antibody stable, and various formulations are possible.

In the application, the diameter range of the latex microspheres is 100-350 nm, and the latex microspheres in the embodiment are commercial products with the diameter of 150 nm.

Another objective of the invention is to provide a kit for detecting the human asialoglycoprotein receptor, which comprises the detection reagent and a standard sample containing a known sH2a concentration.

Wherein the standard pattern contains sH2a with known concentration, and the sH2a can be derived from serum of patients or obtained by genetic engineering expression fermentation. The standard samples contain 1-6 sH2a standard samples with different concentrations, and in this embodiment, the standard samples contain 1-5 sH2a standard samples with different concentrations. The standard sample of sH2a is in the form of a buffer solution or a lyophilized preparation, in this example, which is reconstituted with purified water before use. The buffer solution contains, but is not limited to: sodium hydrogen phosphate, bovine serum albumin, Tween-20, sodium chloride, sucrose, mannitol, EDTA, polyethylene glycol 6000, mixed water with pH of 6.00-8.00 and/or bovine serum solution.

The buffer solution of the standard sample is water and/or bovine serum solution containing the following components and concentrations: the concentration of the sodium hydrogen phosphate is 20 mmol/L-300 mmol/L, in this embodiment 150 mmol/L; the concentration of bovine serum albumin is 0.1% -6.0%, in this embodiment 3.0%; the concentration of the sodium chloride is 50 mmol/L-500 mmol/L, in this embodiment 150 mmol/L; the concentration of the sucrose is 20 g/L-500 g/L, in the embodiment, 200 g/L; the concentration of mannitol is 20 mmol/L-150 mmol/L, in this example 50 mmol/L; the concentration of the Tween-20 is 0.1 to 1.0 percent, and is 0.5 percent in the embodiment; the concentration of EDTA is 1.0 mmol/L-15 mmol/L, in this example 5 mmol/L; the concentration of the polyethylene glycol 6000 is 2 g/L-60 g/L, and in the embodiment, the concentration is 50 g/L.

The buffer solution of the standard sample further contains a protective agent selected from sodium azide, gentamicin, imidazolidinyl urea, and 2-chloroacetamide, and in this embodiment, sodium azide is selected.

The standard sample can be prepared into a liquid solution or a freeze-dried product.

The kit is implemented on a full-automatic biochemical analyzer for detecting the content of the human asialoglycoprotein receptor in a sample.

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

Example two

1. Standard sample

A standard sample is prepared from the following buffer solution components to serve as a calibration solution, one of the same 3 parts is placed in a closed manner at 2-8 ℃ to serve as a control, the other two parts are respectively placed in an opened bottle at 2-10 ℃ and a closed manner at 37 ℃, and the results are shown in table 1, and the stability of the calibration solution meets the daily detection requirements.

The calibration solution comprises the following components:

50mmol/L glycylglycine buffer solution with pH 7.4;

50.0g/L bovine serum albumin;

35mmol/L glutamic acid;

5.0mmol/L EDTA.2Na；

0.9% sodium chloride;

200g/L of sucrose;

10.0g/L polyethylene glycol 6000;

1.0L of pure water;

0.1% sodium azide;

pure sH2a was added to the above buffer.

TABLE 1

Sealing at 2-8 deg.C	Opening the bottle for 1 day at 2-10 deg.C	Sealing at 37 deg.C for 7 days	Sealing at 37 deg.C for 15 days
				57ng/mL	55ng/mL	60ng/mL	62ng/mL

5 concentrations of sH2a standard samples were prepared using the above calibration buffer: 15.6ng/mL, 31.25ng/mL, 62.5ng/mL, 125ng/mL, 250ng/mL, pure water as 0.0 ng/mL.

2. Reagent

Reagent 1 as the first reagent (R1)

50mmol/L pH6.20MES buffer solution;

30mmol/L citric acid;

100mmol/L sodium bromide;

50mmol/L aspartic acid;

11.0g/L PEG 20000；

0.2% tween-20;

0.05％BSA；

0.05% sodium azide.

Reagent 2 as a second reagent (R2)

(1) anti-sH 2a antibody-BSA conjugation process steps:

(1.1) mixing an anti-sH 2a polyclonal antibody and a monoclonal antibody 1:1, mixing the antibody concentration of 1.0mg/mL and BSA according to the mass ratio of 1:2 in 25mmol/L MES buffer solution with the pH value of 6.20;

(1.2) adding EDC dissolved in the same MES buffer solution, and stirring at room temperature for 20 minutes, wherein the final concentration of EDC is 50% of the BSA concentration;

(1.3) standing for later use at 2-8 ℃.

(2.1) suspending the latex microspheres with the particle size of 280nm and the surface carboxyl content of 150ueq/g in 20mmol/L MES buffer solution with the pH value of 6.10;

(2.2) adding EDC into the solution in the step (2.1) according to the molar ratio of EDC to the carboxyl content on the surface of the latex microspheres of 9: 1; slowly adding NHS or Sulfo-NHS into the mixture according to the molar ratio of NHS or Sulfo-NHS to EDC of 3:1, and stirring for 20 minutes;

(2.3) centrifuging, removing the supernatant, adding 50mmol/L boric acid buffer solution with the pH value of 8.00 for resuspension, and centrifuging and cleaning for 3 times; then adding 50mmol/L boric acid buffer solution with the pH value of 8.00 for resuspension;

(2.4) slowly adding the required immobilized sH2a-BSA conjugate into the solution in the step (2.3) according to the final concentration of 100 micrograms/ml, and stirring while adding; and stirring is continued for 2 hours;

(2.5) centrifuging, removing the supernatant, adding diluent for resuspension, and repeatedly centrifuging and cleaning for 3 times; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.15% (W/V); the diluent is 50mmol/L Tris-glycylglycine buffer solution, and contains 0.5% Tween-20, 0.1% NaN3, and pH7.80.

3. Measuring parameters

A measurement device Hitachi 7180 biochemical analyzer;

two-point end point method of analytical method, measuring wavelength 600nm (without secondary wavelength); the reagent sample ratio R1: S: R2 is 200:10: 50.

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

EXAMPLE III

1. Reagent

Reagent 1 as the first reagent (R1)

70mmol/L, pH7.50 Tris buffer solution;

30mmol/L succinic acid;

100mmol/L sodium bromide;

35mmol/L sodium glutamate;

15.0g/L PEG 20000；

0.3% tween-20;

0.02％BSA；

0.03% sodium azide.

Reagent 2 as a second reagent (R2)

(1) anti-sH 2a antibody-BSA coupling process steps:

(1.1) mixing an anti-sH 2a polyclonal antibody with BSA according to the mass ratio of 2:3, wherein the antibody concentration is 6.0mg/mL, and the mixture is mixed in MES buffer solution with the concentration of 20mmol/L and the pH value of 6.10;

(1.3) mixing an anti-sH 2a monoclonal antibody with the antibody concentration of 1.0mg/mL and BSA according to the mass ratio of 2:3 in 20mmol/L MES buffer solution with the pH value of 6.10;

(1.4) adding EDC dissolved in the same MES buffer, and stirring at room temperature for 20 minutes until the final concentration of EDC is 50% of the BSA concentration;

(1.5) placing the polyclonal antibody and monoclonal antibody sH2a-BSA conjugate at 2-8 ℃ for later use.

(2) Respectively fixing the polyclonal anti-sH 2a antibody-BSA conjugate and the monoclonal anti-sH 2a antibody-BSA conjugate on the latex microspheres according to the following steps:

(2.1) suspending the latex microspheres with the particle size of 300nm and the surface carboxyl content of 89ueq/g in 25mmol/L MES buffer solution with the pH value of 6.00;

(2.2) adding EDC into the solution in the step (2.1) according to the molar ratio of EDC to the carboxyl content on the surface of the latex microspheres of 5: 1; slowly adding NHS or Sulfo-NHS into the mixture according to the molar ratio of NHS or Sulfo-NHS to EDC of 4:1, and stirring for 15 minutes;

(2.3) centrifuging, removing the supernatant, adding 80mmol/L boric acid buffer solution with the pH value of 8.00 for resuspension, and centrifuging and cleaning for 3 times; adding 80mM boric acid buffer solution with the pH value of 8.00 for resuspension;

(2.4) slowly adding the required immobilized anti-sH 2a antibody-BSA conjugate to the solution of step (2.3) at a final concentration of 200. mu.g/ml, and stirring while adding; and stirring is continued for 2 hours;

(2.5) centrifuging, removing the supernatant, adding diluent for resuspension, and repeatedly centrifuging and cleaning for 3 times; then adding diluent for resuspension to ensure that the final concentration of the latex microsphere solution is 0.20% (W/V); the diluent is 50mmol/L Tris-glycylglycine buffer solution, and contains 0.6% Tween-20, 0.2% NaN3, and pH7.50.

(2.6) mixing the multi-anti-sH 2a antibody-BSA conjugate solidified microspheres and the monoclonal antibody sH2a antibody-BSA conjugate solidified microspheres according to the ratio of 1:1 for later use.

2. Measuring parameters

A measuring device Hitachi 7180 biochemical analyzer;

analytical two-point end-point method, measuring the wavelength of 546nm (without auxiliary wavelength); the reagent sample ratio R1: S: R2: 200:12: 50.

3. Test specimen

19 clinical serum samples which have been confirmed by ELISA;

as a result: as shown in FIG. 2, the kit of the present invention has significant correlation with the comparative ELISA method.

Example four

1. Reagent

The first reagent (R1) in example two and the second reagent (R2) in example three were used

2. Test specimen

15 parts of normal human plasma specimen and 13 parts of hepatic fibrosis patient plasma specimen;

3. measurement of parameters in the same manner as in example III

As a result: as shown in table 2 below, the present kit was able to significantly differentiate the levels of CHI3L1 in clinical plasma specimens.

TABLE 2

EXAMPLE five

Stability of reagents

The results of taking 3 parts of each of the reagents R1 and R2 of example 2 and example 3, placing one part of the reagents in a sealed state at 2-8 ℃ as a control, and placing the other two parts of the reagents in an open bottle at 2-10 ℃ and 37 ℃ respectively are shown in Table 3, and the stability of the reagents meets the daily detection requirements.

TABLE 3

In conclusion, according to the kit provided by the invention, the first reagent consists of a buffer solution, an anti-interference agent A, a dispersing agent and a coagulant, and can eliminate the interference of high-concentration chyle and lipid, disperse and expose the detected protein, maintain the stability of the sH2a antigen structure and enhance the strength of antigen-antibody binding turbidity; in addition, the second reagent consists of a polyclonal antibody connected with an insoluble carrier and a monoclonal antibody of an anti-sH 2a specific EGHRG pentapeptide related domain, and can be fully combined with sH2a in a test sample, so that the turbidity measuring process is realized, the measuring result is more accurate, the kit can be used for screening high-throughput application of a large number of people, and the invention is beneficial to determining and identifying hepatic fibrosis diseases.

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

Claims

1. An assay reagent for a human asialoglycoprotein receptor comprising:

the first reagent comprises a buffer solution, an anti-interference agent A, a dispersing agent and a coagulant;

2. The reagent for measuring a human asialoglycoprotein receptor according to claim 1, wherein the buffer has a concentration of 10 to 300mmol/L, and the buffer is at least one of Tris buffer, MOPS buffer, MES buffer, phosphate buffer, and glycylglycine buffer.

3. The reagent for determining the human asialoglycoprotein receptor according to claim 1, wherein the anti-interference agent a comprises a surfactant, a chaotropic ion, and an amino acid;

4. The reagent for measuring a human asialoglycoprotein receptor according to claim 1, wherein the dispersant is at least one of an organic acid, a saccharide, and a salt;

5. The reagent for detecting the receptor for human asialoglycoprotein according to claim 1, wherein the concentration of the coagulant is 0.1-6.0%, and the coagulant is at least one of polyethylene glycol 6000 and polyethylene glycol 20000.

6. The reagent for determining the human asialoglycoprotein receptor according to claim 1, wherein said anti-human asialoglycoprotein receptor antibody is immobilized on an insoluble support, and said insoluble support comprises latex particles, magnetic particles, and silica particles.

7. The reagent for determining the human asialoglycoprotein receptor according to claim 1, wherein the first reagent further comprises a preservative, a chelating agent, and a stabilizer.

8. A kit for a human asialoglycoprotein receptor comprising the assay reagent of any one of claims 1 to 7, wherein said kit further comprises a standard sample containing a known concentration of sH2 a.

9. The kit for human asialoglycoprotein receptor according to claim 8, wherein said standard sample is a water and/or serum mixed solution containing buffer, sodium chloride, bovine serum albumin, sucrose, mannitol, tween, EDTA, preservative.

10. A method for quantifying a human asialoglycoprotein receptor, for non-disease diagnostic and therapeutic purposes, said method for quantifying a human asialoglycoprotein receptor being applied to the assay reagent of any one of claims 1 to 7, wherein said method for quantifying a human asialoglycoprotein receptor comprises the steps of: