CN114414802A

CN114414802A - Early diagnosis kit based on four combined detections of liver cancer and application

Info

Publication number: CN114414802A
Application number: CN202111115480.2A
Authority: CN
Inventors: 曹哲; 祝跃球
Original assignee: Hunan Laituofu Biotechnology Co ltd
Current assignee: Hunan Laituofu Biotechnology Co ltd
Priority date: 2021-09-23
Filing date: 2021-09-23
Publication date: 2022-04-29

Abstract

The invention belongs to the technical field of immunodetection, and discloses a liver cancer tumor marker based on four liver cancer tumor markers: a combined detection kit of aldo-keto reductase 1B10(AKR1B10), abnormal prothrombin (DCP), Alpha Fetoprotein (AFP) and alpha fetoprotein heteroplasmon (AFP-L3). The kit comprises: streptavidin magnetic beads; biotin labeling of AKR1B10, DCP, AFP antibodies; acridinium ester labels AKR1B10, DCP, and AFP antibodies; LCA magnetic beads, a cleaning solution and an eluent; AKR1B10, DCP and AFP calibrator and quality control product. And analyzing and judging the risk of the potential development of the subject to the liver cancer by simultaneously detecting the contents of the four tumor markers in the serum of the subject. The invention adopts the combined detection of four tumor markers to improve the sensitivity and specificity of the early diagnosis of the liver cancer and provides more reliable basis for the clinical diagnosis of the liver cancer.

Description

Early diagnosis kit based on four combined detections of liver cancer and application

Technical Field

The invention belongs to the technical field of immunodetection, and particularly relates to a liver cancer specific marker aldone reductase 1B10(AKR1B10), Alpha Fetoprotein (AFP), alpha fetoprotein heteroplasmons (AFP-L3) and abnormal prothrombin (DCP) combined determination kit, which can be used for early screening, diagnosis, curative effect judgment, prognosis evaluation or recurrence monitoring of liver cancer or high risk groups.

Background

Primary Hepatocellular Carcinoma (HCC) is one of the clinically common malignant tumors, the main causes of the HCC are cirrhosis, hepatitis, alcoholic and non-alcoholic fatty liver, and these patients are collectively called high risk group of liver cancer. According to the statistics of the world health organization, in 2020, liver cancer has 91 ten thousand new cases and 83 ten thousand death cases all over the world. Liver cancer is not evident in its early stage and, once diagnosed, usually has progressed to a late stage, losing the best clinical treatment period. At present, the diagnosis of the liver cancer in clinic mainly adopts imaging examination and serum marker detection, and the imaging detection has low sensitivity and high price, so the method is not suitable for large-scale screening of high-risk groups. The clinical common liver cancer serum markers comprise alpha-fetoprotein, alpha-fetoprotein heteroplasmon and abnormal prothrombin, although the clinical common liver cancer serum markers have certain diagnostic value, the diagnosis of a single item still has limitations, if the clinical common liver cancer serum markers are combined to detect simultaneously, the sensitivity and specificity of the early diagnosis of the liver cancer can be greatly improved, and the phenomena of missed detection and false detection of serum detection can be greatly improved.

Alpha-fetoprotein (α FP or AFP) is a carcinoembryonic glycoprotein with a relative molecular mass of about 68kDa, synthesized by fetal liver and yolk sac. In adults, AFP is produced by hepatocytes, and its content in serum is very small, but when hepatocytes become cancerous, the AFP-producing function is restored, and the AFP content in serum increases sharply as the condition worsens. The detection of the AFP level in serum has extremely high clinical value for early diagnosis of the primary liver cancer, so that the long-term increase of AFP is regarded as a specific index of the primary liver cancer. However, not all liver cells of liver cancer patients secrete AFP, and about 30-40% of AFP of patients is negative or low in concentration, so that the method has obvious limitation.

The alpha-fetoprotein heteroplasmon is a single-chain glycoprotein, the sugar content is about 4%, the sugar chain structure of AFP synthesized in different tissue cells is slightly different, and the AFP with different sugar chain structure is collectively called AFP heteroplasmon. Based on the different binding capacity of AFP to lentil Lectin (LCA), it is classified into AFP-L1, AFP-L2 and AFP-L3. AFP-L1 is mainly expressed in benign liver diseases and cannot bind to LCA; AFP-L2 is mainly present in pregnant women, with minor binding to LCA; AFP-L3 is unique to hepatoma cells and specifically binds to LCA. When the ratio of AFP-L3 in the total AFP exceeds 10%, the incidence rate of liver cancer is more than 95%.

Abnormal prothrombin (DCP) is a Vitamin K-deficient or Antagonist-II Induced Protein (Protein Induced by Vitamin K Absense or Antagonist-II, PIVKA-II) that can be present in the serum of patients with Vitamin K deficiency or hepatocellular carcinoma (HCC). The study shows that the serum DCP level of the patients with liver diseases except HCC is slightly increased, but the serum DCP level of the patients with HCC is obviously increased, and the DCP is increased in 90 percent of the patients with HCC, and the mean value is as high as 900 ug/L.

The aldoketoreductase 1B10(Aldo-keto Reductase 1B10, AKR1B10) gene is located on chromosome 7q33, encodes a protein consisting of 316 amino acids, namely AKR1B10 protein, and has a molecular weight of 36.02 kDa. The AKR1B10 protein can be secreted from tumor cells into the blood circulation by a lysosome-mediated, non-classical secretion pathway. The function of the cell protective agent is mainly to protect cells from carboxyl toxicity damage; and stabilizing acetyl-coa carboxylase alpha (ACC α) against ACC α degradation, thereby modulating fatty acid and lipid synthesis and promoting growth, proliferation and migration of tumor cells. Relevant studies show that AKR1B10 is highly expressed in human primary liver cancer tissues and is a novel liver cancer serum marker.

However, the accuracy and reliability of the individual detection of the serum markers of liver cancer have more or less problems.

Disclosure of Invention

In view of the above problems, the present inventors have developed a combined assay kit for detecting liver cancer specific markers aldone reductase 1B10(AKR1B10), alpha-fetoprotein (AFP), alpha-fetoprotein heteroplasmon (AFP-L3) and abnormal prothrombin (DCP). The kit can be used for detecting the concentration of AKR1B10, AFP-L3 and DCP protein in serum, urine, milk, intestinal fluid, stool or tissue samples of a subject. The kit can be used for simultaneously detecting the concentrations of four tumor markers, and greatly improving the sensitivity and specificity of early diagnosis of liver cancer. Can be clinically used for early screening, diagnosis, curative effect judgment, prognosis evaluation or relapse monitoring of liver cancer or high risk groups.

Specifically, the invention relates to a kit for determining aldehyde ketone reductase 1B10(AKR1B10) protein which is a liver cancer specific marker. An aldoketoreductase 1B10(AKR1B10), Alpha Fetoprotein (AFP), alpha fetoprotein heteroplasmon (AFP-L3) and abnormal prothrombin (DCP) combined determination kit and application thereof.

The kit can be used for early screening, diagnosis, curative effect judgment, prognosis evaluation or relapse monitoring of liver cancer or high risk groups.

A first reagent, a reagent component required for detection of AKR1B 10;

a second reagent for detecting a component of the reagent required for AFP;

a third agent, which isolates the required reagent components for AFP-L3;

and a fourth reagent for detecting the components of the reagent required by the DCP.

And respectively containing calibration products and quality control products of AKR1B10, AFP and DCP antigens with different concentrations.

Wherein the content of the first and second substances,

a) the first reagent comprises: a biotin-labeled rabbit anti-AKR 1B10 polyclonal antibody, an acridinium ester-labeled mouse anti-AKR 1B10 monoclonal antibody and streptavidin magnetic beads. The buffer was 0.1M PBS (pH7.0. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300.

b) The second reagent comprises: a biotin-labeled mouse anti-AFP monoclonal antibody, an acridinium ester-labeled mouse anti-AFP monoclonal antibody and streptavidin magnetic beads. The buffer was 0.1M PBS (pH7.0. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300.

c) The third reagent comprises: LCA magnetic beads, a cleaning solution and an eluent. The LCA magnetic beads are prepared by coupling agglutinin (LCA) and NHS magnetic beads. The buffer was 0.1M PBS (pH7.0. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300. The cleaning solution is prepared from Tris-HCl, PBS or NaHCO3 with certain concentration, and the preferable preparation method of the cleaning solution is as follows: 50mM Tris-HCl (pH 7.5. + -. 0.1); the eluent is prepared from Tris-HCl, PBS, sucrose, glucan or D-mannoside with certain concentration, and the preferable preparation method of the eluent is as follows: 0.2M PBS (pH7.0. + -. 0.1), 5M D-mannoside.

d) The fourth reagent comprises: a mouse anti-DCP monoclonal antibody marked by biotin, a mouse anti-DCP monoclonal antibody marked by acridinium ester and streptavidin magnetic beads. The buffer was 0.1M PBS (pH7.0. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300.

Preferably, the kit further comprises:

the calibration and quality control products of AKR1B10, AFP and DCP antigens with different concentrations are prepared by diluting natural antigens (or recombinant antigens) of AKR1B10, AFP and DCP to specified concentrations, and the buffer solution is 0.1M PBS (pH7.0 +/-0.1), 1% BSA, 0.05% Tween20 and 0.05% Proclin 300. The method comprises the following steps: AKR1B10 calibrator CAL 1300 pg/mL, CAL 26000 pg/mL; AKR1B10 quality control QC 1330 pg/mL, QC 21800 pg/mL. AFP calibrator CAL 115 ng/mL, CAL 2180 ng/mL; AFP quality control QC 110 ng/mL, QC 2100 ng/mL. The DCP calibrator CAL 1200 mAU/mL and CAL 28000 mAU/mL; DCP quality control QC 1150 mAU/mL, QC 2450 mAU/mL.

The reagent components (such as some necessary buffers) not described in detail in the kit of the present invention, the external package of the kit, and the independent packaging containers for each reagent component can be performed according to the conventional operations in the field, and the requirements are met.

The subject to which the kit of the invention can be applied is selected from serum, urine, milk, intestinal fluid, stool or a tissue sample. The detection application is early screening, diagnosis, curative effect judgment, prognosis evaluation or relapse monitoring of liver cancer or high risk groups.

The liver cancer four-item combined determination kit provided by the invention has the following advantages:

a) the content of four proteins of AKR1B10, AFP-L3 and DCP can be measured simultaneously, the sensitivity and specificity of early diagnosis of liver cancer are greatly improved, the detection time and cost can be greatly reduced, and no products of the same type exist clinically at present;

b) the biotin-labeled capture antibody and the acridinium ester-labeled detection antibody are used for labeling, separating and purifying through chemical reaction, so that the detection sensitivity is greatly improved. Meanwhile, reagents of all components are independently packaged, so that cross reaction among different antigens or antibodies is avoided.

c) Furthermore, the invention adopts a magnetic particle chemiluminescence analysis method, so that immunoreactions are easier to mix and combine uniformly, the background value is low, the detection of signals is facilitated, and the final sensitivity and specificity of the kit are improved;

d) in addition, the kit has good stability and the effective period can reach more than one year.

Drawings

FIG. 1 shows that the kit of the invention detects the content of AKR1B10, AFP-L3 and DCP in the serum of normal people and high risk people.

FIG. 2 shows that the kit of the invention can detect the content of AKR1B10, AFP-L3 and DCP in the serum of normal human and liver cancer patients.

Detailed description of the invention

The invention provides a liver cancer four-item combined assay kit (magnetic particle chemiluminescence method), and specific examples are only used for more clearly illustrating the invention, and the invention is further described by referring to the following examples. The examples are for illustration only and do not limit the invention in any way.

Some of the reagent materials used in the examples are commercially available (the mouse anti-AKR 1B10 monoclonal antibody is a product of the present company, patent No. ZL 20141057977.0, and the grant publication No. CN 104650234B), the equipment used is also the existing equipment in the field, and the experimental methods without specific conditions are all conventional methods and conditions well known in the field.

Example 1 preparation of a kit for the four-item Co-assay of liver cancer.

Firstly, a biotin labeled antibody preparation method comprises the following steps:

the rabbit anti-AKR 1B10 polyclonal antibody labeled by biotin is taken as an example:

(1) the rabbit anti-AKR 1B10 polyclonal antibody to be labeled was replaced with 1x PBS buffer using a desalting column.

(2) Mixing the antibody in the step (1) with biotin with the concentration of 10mM, and reacting for 2 hours at room temperature (18-25 ℃) by gentle shaking with the molecular ratio of biotin to antibody being 20: 1.

(3) And replacing the labeled antibody solution into 1 XPBS solution by using a desalting column, removing free biotin, and storing at-80 ℃ to prepare the required biotin-labeled antibody.

The preparation of other biotin-labeled antibodies can be carried out according to the above-described method.

The preparation method of the acridinium ester labeled antibody comprises the following steps:

for example, acridinium ester-labeled murine anti-AKR 1B10 monoclonal antibody:

(1) the murine anti-AKR 1B10 monoclonal antibody to be labeled was replaced with desalting column into 1 × PBS solution as buffer.

(2) Mixing the antibody in the step (1) with acridinium ester with the concentration of 1mg/ml, wherein the molecular ratio of the acridinium ester to the antibody is 5: 1, the reaction was carried out at room temperature (18-25 ℃) for 30 minutes with gentle shaking.

(3) And replacing the labeled antibody solution into 1 XPBS solution by using a desalting column, removing free acridinium ester, and storing at-80 ℃ to prepare the required acridinium ester labeled antibody.

Preparation of other acridinium ester labeled antibodies can be performed according to the methods described above.

Thirdly, the preparation method of the LCA magnetic beads comprises the following steps:

lectin (LCA) and NHS magnetic beads are commercial raw materials, can be purchased by raw and auxiliary material suppliers, and are coupled to prepare the LCA magnetic beads.

(1) The NHS beads were separated with a magnetic stand, washed with an appropriate amount of coupling buffer and the residual solution removed.

(2) And (3) dissolving an appropriate amount of LCA in the coupling buffer solution to prepare an LCA solution with the concentration of 1 mg/mL.

(3) Mixing the NHS magnetic beads treated in the step (1) with an LCA solution, wherein the mixing ratio of the NHS magnetic beads (g) to the LCA (mg) is 1: 1-1: 5, mix well for 2 hours at room temperature.

(4) 0.1M PBS (pH7.0. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin300 was used. And repeatedly cleaning the connected LCA magnetic beads, and removing the LCA which is not coupled to obtain a solution, namely the LCA magnetic beads.

Fourthly, the preparation method of the calibration material and the quality control material comprises the following steps:

taking AKR1B10 calibrator and quality control as examples:

and dissolving AKR1B10 antigen in 0.1M PBS solution to prepare AKR1B10 calibrator, wherein the concentrations of the AKR1B10 calibrator are respectively as follows: CAL 1300 pg/mL, CAL 26000 pg/mL; wherein the 0.1M PBS solution has a pH of 7.0 + -0.1 and contains 1% BSA, 00.05% Tween20, and 0.05% Proclin 300.

Dissolving AKR1B10 antigen in 0.1M PBS solution to prepare AKR1B10 quality control product, wherein the AKR1B10 quality control product has the following concentrations: QC 1330 pg/mL, QC 21800 pg/mL; wherein the 0.1M PBS solution has a pH of 7.0 + -0.1 and contains 1% BSA, 00.05% Tween20, and 0.05% Proclin 300.

The preparation of other tumor marker calibrators and quality controls can be carried out in accordance with the methods described above.

Example 2 detection procedure of the liver cancer four-item combined assay kit.

The detection steps of the first reagent, the second reagent and the fourth reagent are as follows:

(1) adding a proper volume of each sample into 3 reaction cups, and adding corresponding volumes of AKR1B10, AFP and DCP calibrator and quality control materials into the reaction cups.

(2) Biotin antibodies and acridine ester antibodies to AKR1B10, AFP and DCP were added to the corresponding reaction cups in amounts of 100uL, respectively.

(3) The reaction cups were incubated at 37 ℃ for 10 minutes, then 30uL of streptavidin magnetic beads were added to each reaction cup, and incubation was continued at 37 ℃ for 20 minutes.

(4) The magnetic beads were adsorbed under the action of magnetic force, the liquid in the reaction cup was aspirated, and washing was repeated 3 times.

(5) Under the action of the pre-excitation liquid and the excitation liquid, the acridinium ester is excited to generate photons, and the photon intensity is positively correlated with the concentration of the substance to be detected in the reaction cup.

(6) And fitting a curve according to the concentration values and the luminescence values of the AKR1B10, the AFP and the DCP calibrators, and respectively calculating the concentrations of the AKR1B10, the AFP and the DCP samples.

The second and third reagent detection steps are as follows:

(1) 100uL of each sample was added to the reaction cuvette, and 30uL of LCA magnetic beads was added.

(2) And (3) placing the reaction cup at 37 ℃ for incubation for 10 minutes, adsorbing magnetic beads under the action of magnetic force, sucking liquid in the reaction cup, and adding 300uL of cleaning solution to repeatedly wash for 3 times.

(3) Adding 100uL of eluent, incubating for 2 minutes at 37 ℃, adsorbing magnetic beads under the action of magnetic force, and absorbing a proper amount of eluent to transfer into a new reaction cup.

(4) And respectively adding the biotin antibody and the acridinium ester antibody in the second reagent into the reaction cups, wherein the addition amount is 100 uL.

(5) The reaction cups were incubated at 37 ℃ for 10 minutes, then 30uL of streptavidin magnetic beads were added to each reaction cup, and incubation was continued at 37 ℃ for 20 minutes.

(6) The magnetic beads were adsorbed under the action of magnetic force, the liquid in the reaction cup was aspirated, and washing was repeated 3 times.

(7) Under the action of the pre-excitation liquid and the excitation liquid, the acridinium ester is excited to generate photons, and the photon intensity is positively correlated with the concentration of the substance to be detected in the reaction cup.

(8) And fitting a curve according to the concentration value and the luminous value of the AFP calibrator to calculate the concentration of the AFP-L3 sample.

Example 3 liver cancer four items combined determination kit normal reference value.

The same test method as that in example 2 was used to select 228 healthy normal human samples, and the normal reference value (P) was determined in 95% percentile₉₅(228+1) × 95% ═ 217). According to the arrangement order of the measured concentrations of the samples, in 228 serum samples, the concentration of AKR1B10 with the number corresponding to P95-217 is 373.5pg/mL, the concentration of DCP is less than or equal to 161.45mAU/mL, the concentration of AFP is 8.7ng/mL, the ratio of AFP-L3% is less than or equal to 10%, and the detailed results are shown in Table 1, (AFP-L3% is the concentration ratio of AFP-L3 and AFP).

TABLE 1.228 measurement of healthy normal human serum samples

Therefore, it was determined that the normal reference value for AKR1B10 was ≦ 350pg/mL, the normal reference value for AFP was ≦ 8.7ng/mL, the normal reference ratio for AFP-L3/AFP was ≦ 10%, and the normal reference value for DCP was ≦ 40mAU/mL (see Table 2).

TABLE 2 liver cancer four normal reference values

Example 4 liver cancer four-item combined assay kit for detecting serum samples of normal people and high risk people.

By adopting the same detection method as that in the example 2, serum samples of 40 healthy normal persons and 75 high risk persons are selected for detection, the concentrations of AKR1B10, AFP-L3 and DCP in the serum samples of the subjects are respectively calculated according to the concentration value and the luminous value of the calibrator and a fitting curve, and the detailed results are shown in Table 3. Results were analyzed using Graphpad 4.0 statistical software. The concentration of AKR1B10 in healthy normal people and high risk people is 208.7 +/-15.76 pg/mL and 303.8 +/-27.79 pg/mL respectively; the concentration of AFP is 4.002 + -0.3133 ng/mL and 33.9 + -4.735 ng/mL respectively; the concentration of AFP-L3 was 3.189 + -0.3032 ng/mL and 4.458 + -1.191 ng/mL, respectively; the concentration of DCP was 80.08. + -. 5.495mAU/mL and 178.3. + -. 19.99mAU mL, respectively (see FIG. 1).

TABLE 3.75 serum sample measurements for high risk group

In high risk population, the negative coincidence rate 77.33% (58/75), AFP negative coincidence rate 41.33% (31/75), AFP-L3 negative coincidence rate 85.33% (64/75) and DCP negative coincidence rate 61.33% (46/75) of AKR1B10 are analyzed separately; the total negative coincidence rate of the combined liver cancer tumor marker analysis is 96.00 percent (72/75). The results suggest that in the early screening of liver cancer high risk population, the detection sensitivity of the high risk population can be greatly improved by comparing the four-item combined detection of liver cancer with single analysis, and the method has important guiding significance for the early screening of liver cancer (see table 4).

TABLE 4.75 negative match rates for high risk group

Example 5 liver cancer four-item combined assay kit for detecting serum samples of normal human and liver cancer patients.

By adopting the same detection method as that of the example 2, serum samples of 40 healthy normal persons and 68 liver cancer patients are selected for detection, the concentration values of AKR1B10, AFP-L3 and DCP in the serum samples of the subjects are respectively calculated according to the concentration values and the luminous values of the calibrator and a fitting curve, and detailed results are shown in Table 5. Results were analyzed using Graphpad 4.0 statistical software. The concentration of AKR1B10 in healthy normal people and liver cancer patients is 208.7 +/-15.76 pg/mL and 1319 +/-180.1 pg/mL respectively; the concentration of AFP is 4.002 + -0.3133 ng/mL and 265.8 + -48.46 ng/mL respectively; the concentration of AFP-L3 was 3.189 + -0.3032 ng/mL and 67.02 + -13.52 ng/mL, respectively; the concentration of DCP was 80.08. + -. 5.495mAU/mL and 1981. + -. 379.3mAU/mL, respectively (see FIG. 2).

TABLE 5.68 serum samples of liver cancer

In liver cancer patients, the positive coincidence rate of AKR1B10 is analyzed separately, namely 73.53% (50/68), 66.18% (45/68) of AFP, 83.82% (57/68) of AFP-L3 and 70.58% (48/68) of DCP; the total positive coincidence rate of the four tumor marker analyses of the combined liver cancer is 95.59% (65/68), and the combined liver cancer shows particular advantages. The results are shown in Table 6.

TABLE 6.68 positive match rate of liver cancer samples

As can be seen from Table 6, in the diagnosis of patients with confirmed liver cancer, the positive detection rate of liver cancer patients can be significantly improved by comparing the four-item combined liver cancer detection with the single-item analysis.

Claims

1. A combined detection kit for liver cancer diagnosis is characterized by comprising the following components aiming at the tumor markers: (ii) reagents for detecting the concentration of aldoketoreductase 1B10, alpha fetoprotein heteroplasmons, and abnormal prothrombin in a sample from a subject.

2. The kit of claim 1, wherein the kit is used for early screening, diagnosis, treatment judgment, prognosis evaluation or recurrence monitoring of liver cancer or high risk group.

3. The kit of claim 1 or 2, comprising separately packaged first reagents that are components of reagents required for detecting AKR1B 10; the second agent is a component of the agent required to detect AFP; the third agent is a component of the agent required for the isolation of AFP-L3; and the fourth reagent is a reagent component required for detecting DCP;

and a calibrator and a quality control product which respectively contain AKR1B10, AFP and DCP antigens with different concentrations.

4. The kit of claim 3, wherein:

the first reagent is a biotin labeled AKR1B10 antibody and an acridinium ester labeled AKR1B10 antibody; the second reagent is biotin-labeled AFP antibody and acridinium ester-labeled AFP antibody; the third reagent is LCA magnetic beads, a cleaning solution and an eluent which are used for separating AFP-L3 protein; the fourth reagent is a biotin-labeled DCP antibody and an acridinium ester-labeled DCP antibody.

5. The kit of claim 3, wherein: the first reagent is a biotin-labeled rabbit anti-AKR 1B10 polyclonal antibody and an acridinium ester-labeled mouse anti-AKR 1B10 monoclonal antibody; the second reagent is a biotin-labeled rabbit anti-AFP polyclonal antibody and an acridinium ester-labeled mouse anti-AFP monoclonal antibody; the fourth reagent is biotin-labeled rabbit anti-DCP polyclonal antibody and acridinium ester-labeled mouse anti-DCP monoclonal antibody.

6. The kit of claim 4, wherein: the biotin-labeled AKR1B10, AFP and DCP antibodies are prepared by coupling a mouse anti-AKR 1B10 monoclonal antibody, a mouse anti-AFP monoclonal antibody and a mouse anti-CDP monoclonal antibody with biotin respectively; the acridine ester labeled AKR1B10, AFP and DCP antibodies are prepared by coupling a mouse anti-AKR 1B10 monoclonal antibody, a mouse anti-AFP monoclonal antibody and a mouse anti-CDP monoclonal antibody with acridine ester respectively; the LCA magnetic bead for separating AFP-L3 protein is prepared by coupling NHS magnetic bead with agglutinin.

7. The kit of claim 4, wherein: the cleaning solution is Tris-HCl, PBS or NaHCO₃The solution prepared, the preferred cleaning solution is: 50mM Tris.HCl (pH 7.5. + -. 0.1); the eluent is solution prepared from Tris-HCl, PBS, sucrose, glucan or D-mannosidePreferred eluents are: 0.2M PBS, pH 7.0. + -. 0.1, 5M D-mannoside.

8. The kit of claim 7, wherein: the cleaning solution is: 50mM Tris.HCl, pH7.5 + -0.1; the eluent is: 0.2M PBS, pH 7.0. + -. 0.1, 5M D-mannoside.

9. The kit of claim 3, wherein: the calibration material and the quality control material of the AKR1B10, AFP and DCP antigens with different concentrations comprise: AKR1B10 calibrator CAL 1300 pg/mL, CAL 26000 pg/mL; the quality control products QC 1330 pg/mL and QC 21800 pg/mL of AKR1B 10;

AFP calibrator CAL 115 ng/mL, CAL 2180 ng/mL; AFP quality control QC 110 ng/mL, QC 2100 ng/mL;

the DCP calibrator CAL 1200 mAU/mL and CAL 28000 mAU/mL; QC 1150 mAU/mL and QC 2450 mAU/mL of the DCP quality control;

the buffer was 0.1M PBS (pH7.0. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300.

10. A kit according to any one of claims 1 to 9, wherein the subject sample is selected from serum, urine, milk, intestinal fluid, stool or a tissue sample.