Composition for measuring alpha-fetoprotein isoforms and its application

Abstract

The present invention relates to in vitro diagnostic reagents, and in particular to a composition for determining alpha-fetoprotein heterogeneities and its application. The composition comprises a first reagent group for capturing alpha-fetoprotein heterogeneities and a second reagent group for separating and determining alpha-fetoprotein heterogeneities in the same reaction system; the first reagent group comprises a lectin magnetic bead reagent and a sample diluent; the second reagent group comprises an AFP capture magnetic bead reagent, a signal-labeled AFP detection antibody reagent, and an eluent; or the second reagent group comprises a streptavidin magnetic bead reagent, a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent, and an eluent; or, the first reagent group comprises a lectin magnetic bead reagent and a streptavidin magnetic bead reagent; the second reagent group comprises a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent, and an eluent. The composition has strong specificity, high sensitivity, and strong instrument versatility, and can greatly improve the detection efficiency of AFP-L3.

Description

Technical Field

[0001] The present invention relates to an in vitro diagnostic reagent, in particular to a composition for measuring alpha-fetoprotein heterogeneities and an application thereof. Background Art

[0002] Alpha-fetoprotein (AFP) is generally considered a relatively specific tumor marker for HCC (hepatocellular carcinoma), and persistently elevated AFP is a risk factor for HCC. Currently, some European and American researchers believe that AFP's sensitivity and specificity are low, and the 2010 American Association for the Study of Liver Diseases (AASLD) guidelines no longer include AFP as a screening indicator. However, HCC in my country is mostly associated with HBV (hepatitis B virus) infection, which differs from the pathogenic factors of HCC in Western countries (mostly HCV, alcohol, and metabolic factors). Therefore, based on the results of randomized trials (RCTs) in China and current practice, AFP will continue to be included in routine HCC surveillance and screening.

[0003] Serum AFP and its variants are important indicators for diagnosing liver cancer and the most specific tumor markers. They are commonly used in China for liver cancer screening, early diagnosis, postoperative monitoring, and follow-up. AFP levels ≥400 μg / L for more than one month, or ≥200 μg / L for two months, can be excluded if other causes of AFP elevation are excluded, including pregnancy, germline embryonic tumors, active liver disease, and secondary liver cancer. However, 30%-40% of liver cancer patients test negative for AFP, including those with ICC, well-differentiated and poorly differentiated HCC, or HCC that has undergone necrosis and liquefaction, in whom AFP may not be elevated. Therefore, AFP alone cannot diagnose all liver cancers. The positive rate of AFP for liver cancer diagnosis is generally 60%-70%, sometimes with significant variation.

[0004] AFP is a single-chain glycoprotein. The sugar chain structure of serum AFP varies among patients with different liver diseases. Based on their affinity for lentil agglutinin, AFP can be divided into three types: AFP-L1, secreted by benign liver cells; AFP-L2, produced by pregnant women; and AFP-L3, commonly known as an alpha-fetoprotein variant, produced by malignant cancer cells. AFP-L3 is a highly specific marker for liver cancer diagnosis and is considered a new-generation liver cancer marker. The FDA approved this marker for liver cancer early warning in 2005, setting a positive cutoff value of 10% for AFP-L3 in the diagnosis of liver cancer. At the Fourth National Cancer Conference in 1999, AFP-L3 was listed as one of the liver cancer markers in the clinical diagnostic criteria for primary liver cancer. AFP-L3 is recognized as a more specific marker for primary liver cancer than AFP alone.

[0005] Differentiating liver cancer from benign liver diseases. AFP levels are often elevated in patients with primary liver cancer, but many benign liver diseases can also have elevated AFP levels. It is sometimes difficult to distinguish benign from malignant lesions based solely on AFP results. In these cases, AFP variant detection is clinically valuable, especially for patients with AFP levels between 30 and 400 ng / mL. Yozhiaki conducted a prospective study of 361 patients with cirrhosis. Among 53 patients with AFP levels of 30 μg or higher, 21 developed liver cancer two years later. At the time of liver cancer diagnosis, 39% of patients had AFP levels below 400 μg / L. Comparison of AFP values ​​at the beginning of the study between hepatocellular carcinoma (HCC) and non-HCC groups revealed no significant differences. However, the study revealed that the types of AFP variants in lesions differed. For HCC, the positive rate for LCA was 87.12%, with a false-positive rate of 21.5%. ConA was also positive at an 89.17%, with a false-positive rate of 17.15%. Current research considers an AFP-L3 level greater than 10% as a positive indicator for liver cancer.

[0006] Postoperative monitoring for liver cancer. After liver cancer resection, serum AFP levels decrease. The rate of decrease depends on the amount of AFP remaining in the body and its half-life. It generally turns negative within two months, with the disappearance of AFP variants. If AFP levels drop significantly but do not turn negative, and the variants do not change significantly, this suggests incomplete surgery and the presence of residual margins, vascular cancer, satellite nodules, or metastases. If the variants drop below 25%, while AFP and variant concentrations remain relatively constant, this may indicate hepatitis or cirrhosis.

[0007] Abnormal embryonic development and fetal congenital disorders. During normal pregnancy, the AFP content in maternal serum and the AFP content in the embryo are in equilibrium. However, if the fetus is malformed or the placental barrier is abnormal, fetal serum may leak into the amniotic fluid or amniotic fluid into maternal serum, causing a sharp increase in maternal AFP in the amniotic fluid or serum. However, measuring only the total amount of AFP has certain limitations. Experiments have shown that AFP and / or AFP variants may be positive in neural tube defects, anencephaly or spina bifida, childhood hepatoblastoma, biliary atresia, gonadal tumors, and malignant teratomas.

[0008] AFP-L3 is the only protein produced by cancer cells in a patient's liver. This test was studied in a multicenter, prospective, double-blind, long-term clinical trial in Canada and the United States. Results showed that patients with elevated AFP-L3 (above 15%) had a seven-fold increased risk of developing hepatocellular carcinoma over the following 21 months. According to established hepatocellular carcinoma oncology practice guidelines, these patients have an extremely high risk of developing hepatocellular carcinoma.

[0009] Currently, the AFP-L3 detection methods include plant lectin affinity immunoelectrophoresis technology, i30 detection system technology, sugar capture centrifugal column pre-treatment technology and other detection methods (such as lectin detection). Among them, plant lectin affinity immunoelectrophoresis technology and The i30 detection system has high technical requirements, cumbersome operation, and expensive reagents, limiting its clinical application. The sugar capture spin column (CN100588942C) requires numerous manual procedures, complex steps, and a large number of supporting equipment, which is time-consuming and cannot be automated, making it impossible to achieve high-throughput sample testing. Furthermore, this method requires the corresponding AFP detection reagents after AFP-L3 separation to obtain complete test results. Furthermore, Shanghai Liangrun Biopharmaceutical Technology Co., Ltd. disclosed in CN105785043A3 a kit for the quantitative detection of AFP-L3%. However, due to the low affinity of lectins for sugar chains, the high concentration of lectins used in the test significantly affects the separation and detection of AFP-L3, resulting in a small sensitivity and linear range for AFP-L3. Beijing Rejing Biotechnology Co., Ltd. disclosed in CN108627653B a composition and kit for separating and detecting alpha-fetoprotein heterogeneities. The composition and kit contain a separation reagent and a detection reagent. The separation reagent contains AFP-L3 separation magnetic beads, and the detection reagent contains AFP-L3 detection magnetic beads. The separation and detection of alpha-fetoprotein heterogeneities require multiple steps, and the instrument measurement efficiency is low.

[0010] Immunomagnetic bead separation technology is to covalently link the affinity ligands of the target molecule, such as antibodies and proteins, to the surface of magnetic beads to obtain active magnetic beads. When the active magnetic beads are mixed with a solution containing the target molecule, a magnetic bead-ligand-target molecule complex is formed due to the affinity binding of the target molecule and its ligand. The magnetic bead-ligand-target molecule complex is then separated by the magnetism between the magnetic bead separator (magnetic stand or magnetic rod) and the magnetic beads. After washing with a washing solution to remove non-specific binding impurities, the target molecule is finally separated from the magnetic beads with an eluent, thereby achieving separation and purification of the target molecule. Immunomagnetic bead separation technology has the unique advantages of solid-phase reagents and the high specificity of immunological reactions, and has fast separation speed, high efficiency and good repeatability. Based on magnetic separation technology, AFP-L3 can be separated using LCA (lentil agglutinin), and then the concentration of AFP-L3 is measured using an AFP assay kit to further obtain the proportion of AFP-L3 in AFP. The AFP-L3 measurement process includes lectin capture of AFP-L3, washing (removal of unbound matter), elution (separation of AFP-L3 from the lectin), and AFP-L3 concentration measurement. There are two approaches to fully automated AFP-L3 measurement (extraction and detection). In the first approach, the instrument requires independent modules: an extraction module and a detection module. The extraction module performs the first three steps, while the detection module performs AFP-L3 concentration measurement. In the second approach, the instrument incorporates at least three incubation steps: lectin capture of AFP-L3 (incubation 1), elution (separation of AFP-L3 from the lectin) (incubation 2), and AFP-L3 concentration measurement (incubation 3). Both approaches place high demands on the instrument, resulting in high design and manufacturing costs and low measurement efficiency.

[0011] Typically, immune reactions can be completed in one or two steps, so most fully automated chemiluminescence immunoassays support one- and two-step reactions. However, due to the limited availability of three-step reactions, high instrument design costs, and low test efficiency, few instruments support the three-step method. Therefore, there is a need to develop a composition and method for the determination of AFP variants that simplifies the detection steps and enhances instrument versatility, thereby improving detection efficiency. Summary of the Invention

[0012] To address the problems and deficiencies in the aforementioned field, the present invention provides a reagent composition for measuring AFP-L3 using a two-step method. This composition boasts strong specificity, high sensitivity, and broad instrument versatility, significantly improving the efficiency of AFP-L3 detection and possessing significant application value for the early diagnosis and treatment of liver cancer.

[0013] The present invention provides a composition for measuring alpha-fetoprotein isoforms, which comprises a first reagent set for capturing alpha-fetoprotein isoforms and a second reagent set for separating and measuring alpha-fetoprotein isoforms in the same reaction system;

[0014] The first reagent group includes a lectin magnetic bead reagent and a sample diluent; the second reagent group includes an AFP capture magnetic bead reagent, a signal-labeled AFP detection antibody reagent, and an eluent, wherein the AFP capture magnetic beads are formed by coupling an AFP capture antibody to a magnetic bead; or the second reagent group includes a streptavidin magnetic bead reagent, a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent, and an eluent;

[0015] Alternatively, the first reagent group includes a lectin magnetic bead reagent and a streptavidin magnetic bead reagent; the second reagent group includes a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent, and an eluent;

[0016] The AFP capture antibody and the AFP detection antibody target different AFP antigen epitopes.

[0017] In the second reagent set, the eluent is a separate reagent, or is contained in at least one reagent constituting the second reagent set.

[0018] In some embodiments of the present invention, the first reagent set includes a lectin magnetic bead reagent and a sample diluent; the lectin magnetic bead reagent contains lectin magnetic beads with a volume fraction of 5% to 50%; and the sample diluent contains 1 mM to 100 mM tris(hydroxymethyl)aminomethane.

[0019] In some embodiments of the present invention, the second reagent group includes an AFP capture magnetic bead reagent, a signal-labeled AFP detection antibody reagent and an eluent; the AFP capture magnetic bead reagent contains AFP capture magnetic beads at a concentration of 0.1 to 1.0 mg / mL; the signal-labeled AFP detection antibody reagent contains a signal-labeled AFP detection antibody at a concentration of 0.01 μg / mL to 1.0 μg / mL.

[0020] In some embodiments of the present invention, the second reagent group includes a streptavidin magnetic bead reagent, a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent and an eluent; the streptavidin magnetic bead reagent contains streptavidin magnetic beads at a concentration of 0.1 to 5 mg / mL; the biotin-labeled AFP capture antibody reagent contains a biotin-labeled AFP capture antibody at a concentration of 0.5 to 5 μg / mL; the signal-labeled AFP detection antibody reagent contains a signal-labeled AFP detection antibody at a concentration of 0.01 μg / mL to 1.0 μg / mL.

[0021] In some embodiments of the present invention, the first reagent group includes a lectin magnetic bead reagent and a streptavidin magnetic bead reagent; the lectin magnetic bead reagent contains a volume fraction of 5% to 50% of lectin magnetic beads; the streptavidin magnetic bead reagent contains a concentration of 0.1 to 5 mg / mL of streptavidin magnetic beads.

[0022] In some embodiments of the present invention, the second reagent group includes a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent and an eluent; the biotin-labeled AFP capture antibody reagent contains a biotin-labeled AFP capture antibody with a concentration of 0.5 to 5 μg / mL; the signal-labeled AFP detection antibody reagent contains a signal-labeled AFP detection antibody with a concentration of 0.01 μg / mL to 1.0 μg / mL.

[0023] Preferably, in any of the compositions, the eluent comprises methyl glucoside, manganese chloride, magnesium chloride and calcium chloride.

[0024] The present invention also provides a kit for measuring alpha-fetoprotein isoforms, which comprises any of the aforementioned compositions for measuring alpha-fetoprotein isoforms.

[0025] Preferably, the kit further comprises a washing solution for washing the magnetic beads and an excitation solution for exciting the signal substance to generate a signal.

[0026] The composition for detecting alpha-fetoprotein isoforms provided by the present invention can complete the determination of AFP-L3 concentration in a sample in a two-step incubation reaction. The first step involves capturing AFP-L3 in the sample using a first reagent set. The second step involves simultaneously eluting AFP-L3 (separating AFP-L3 from lectin) and determining AFP-L3 concentration in the same reaction system using a second reagent set.

[0027] In some embodiments of the present invention, the first reagent group consists of a lectin magnetic bead reagent and a sample diluent; the second reagent group consists of an AFP capture magnetic bead reagent, a signal-labeled AFP detection antibody reagent, and an eluent; the AFP capture magnetic bead reagent contains or does not contain an eluent, the signal-labeled AFP detection antibody reagent contains or does not contain an eluent, and the eluent contains an eluent.

[0028] In some embodiments of the present invention, the first reagent group consists of a lectin magnetic bead reagent and a sample diluent; the second reagent group consists of an AFP capture magnetic bead reagent and a signal-labeled AFP detection antibody reagent; at least one of the AFP capture magnetic bead reagent and the signal-labeled AFP detection antibody reagent contains an eluent.

[0029] In some embodiments of the present invention, the first reagent group consists of a lectin magnetic bead reagent and a sample diluent; the second reagent group consists of a streptavidin magnetic bead reagent, a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent and an eluent; the streptavidin magnetic bead reagent contains or does not contain an eluent, the biotin-labeled AFP capture antibody reagent contains or does not contain an eluent, the signal-labeled AFP detection antibody reagent contains or does not contain an eluent, and the eluent contains an eluent.

[0030] In some embodiments of the present invention, the first reagent group consists of a lectin magnetic bead reagent and a sample diluent; the second reagent group consists of a streptavidin magnetic bead reagent, a biotin-labeled AFP capture antibody reagent and a signal-labeled AFP detection antibody reagent; at least one of the streptavidin magnetic bead reagent, the biotin-labeled AFP capture antibody reagent and the signal-labeled AFP detection antibody reagent contains an eluent.

[0031] In some embodiments of the present invention, the first reagent group consists of a lectin magnetic bead / streptavidin magnetic bead reagent and a sample diluent; the second reagent group consists of a biotin-labeled AFP capture antibody reagent, a signal-labeled AFP detection antibody reagent and an eluent; the biotin-labeled AFP capture antibody reagent contains or does not contain an eluent, the signal-labeled AFP detection antibody reagent contains or does not contain an eluent, and the eluent contains an eluent.

[0032] In some embodiments of the present invention, the first reagent group consists of a lectin magnetic bead / streptavidin magnetic bead reagent and a sample diluent; the second reagent group consists of a biotin-labeled AFP capture antibody reagent and a signal-labeled AFP detection antibody reagent; at least one of the biotin-labeled AFP capture antibody reagent and the signal-labeled AFP detection antibody reagent contains an eluent.

[0033] In some embodiments of the present invention, in the first reagent set, the lectin magnetic bead reagent contains 20% lectin magnetic beads by volume; the sample diluent is a 10mM tris solution; and the volume ratio of the lectin magnetic bead reagent to the sample diluent is 1:10. In the second reagent set, the AFP capture magnetic bead reagent contains 0.5mg / mL AFP capture magnetic beads; the signal-labeled AFP detection antibody reagent contains 0.1μg / mL acridinium ester-labeled AFP detection antibody; the eluent contains 500mM methyl glucoside, 1.0mM manganese chloride, 1.0mM magnesium chloride, and 1.0mM calcium chloride; and the volume ratio of the AFP capture magnetic bead reagent, the signal-labeled AFP detection antibody reagent, and the eluent is 1:5:5.

[0034] In some embodiments of the present invention, in the first reagent set, the lectin magnetic bead reagent contains 20% lectin magnetic beads by volume; the sample diluent is a 10mM tris solution; and the volume ratio of the lectin magnetic bead reagent to the sample diluent is 1:10. In the second reagent set, the streptavidin magnetic bead reagent contains 0.5mg / mL streptavidin magnetic beads; the biotin-labeled AFP capture antibody reagent contains 1.0μg / mL biotin-labeled AFP capture antibody; the signal-labeled AFP detection antibody reagent contains 0.1μg / mL acridinium ester-labeled AFP detection antibody; the eluent contains 500mM methyl glucoside, 1.0mM manganese chloride, 1.0mM magnesium chloride, and 1.0mM calcium chloride; and the volume ratio of the streptavidin magnetic bead reagent, the biotin-labeled AFP capture antibody reagent, the signal-labeled AFP detection antibody reagent, and the eluent is 1:5:5:5.

[0035] In some embodiments of the present invention, in the first reagent set, the lectin magnetic beads / streptavidin magnetic beads reagent contains 50% lectin magnetic beads and 0.5 mg / mL streptavidin magnetic beads by volume; the sample diluent is a 10 mM tris solution; and the volume ratio of the lectin magnetic beads / streptavidin magnetic beads reagent to the sample diluent is 1:5. In the second reagent set, the biotin-labeled AFP capture antibody reagent contains 1.0 μg / mL biotin-labeled AFP capture antibody; the signal-labeled AFP detection antibody reagent contains 0.1 μg / mL acridinium ester-labeled AFP detection antibody; the eluent contains 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride, and 1.0 mM calcium chloride; and the volume ratio of the biotin-labeled AFP capture antibody reagent, the signal-labeled AFP detection antibody reagent, and the eluent is 1:1:1.

[0036] To further improve the performance of the composition, we optimized the type of groups attached to the magnetic beads, the concentration and size of the lectin beads, and the concentration of the eluent in the reaction system. Results showed that 10-30 μm magnetic beads with NHS groups performed better, with an optimal working concentration of 10% (mass fraction). Eluent (methyl glucoside) concentrations between 0.5 and 5 M performed best, but for cost considerations, the optimal concentration was 0.5 M.

[0037] The composition provided herein is suitable for use with various fully automated chemiluminescence immunoassay analyzers that support two-step reactions, such as the ACL2800, Smart6500, Shine2910, and Shine1910. If the instrument only supports one magnetic bead component, the first reagent set can utilize lectin magnetic beads / streptavidin magnetic beads, leaving the second reagent set free of magnetic beads, further improving the compatibility of the composition with the instrument.

[0038] In some embodiments of the present invention, the steps for determining AFP-L3 in a sample using the composition provided by the present invention are as follows: (1) taking 50 μL of the sample, adding each reagent of the first reagent group, incubating at 37° C. for 10 min; washing three times with a cleaning solution; (2) adding each reagent of the second reagent group, incubating at 37° C. for 10 min; washing three times with a cleaning solution; adding a pre-excitation solution and an excitation solution, and measuring the luminescence intensity.

[0039] The present invention also provides a method for determining alpha-fetoprotein isoforms. The method uses the composition provided by the present invention to determine the concentration of AFP-L3 in a sample, comprising the following steps:

[0040] Step 1 (Incubation 1): Take 50 μL of sample, add each reagent of the first reagent set, incubate at 37°C for 10 min; wash three times with cleaning solution;

[0041] Step 2 (incubation 2): add each reagent of the second reagent group and incubate at 37°C for 10 min; wash three times with cleaning solution; add pre-excitation solution and excitation solution, and measure the luminescence intensity.

[0042] The method provided by the present invention uses lectin magnetic beads in a first reagent set to capture AFP-L3 in a sample. A second reagent set is then used to separate AFP-L3 from the lectin and simultaneously measure the AFP-L3 concentration. This method requires only two steps to complete the determination of AFP-L3 concentration in a sample, boasting high instrument versatility, short detection time, and high detection efficiency. To verify the feasibility of the method, we compared the linearity, blank limit, and correlation of the calibrator with different composition patterns (Table 1). The results demonstrated high consistency in the test results (Table 2).

[0043] In summary, the present invention provides a composition and method for detecting alpha-fetoprotein isomers with high specificity and sensitivity (the positive detection rate for primary liver cancer can reach 91.7%, and the specificity for healthy people reaches 100%). This can greatly improve the detection efficiency of AFP-L3. The instrument has strong versatility and has important application value for the early diagnosis and treatment of liver cancer. DETAILED DESCRIPTION

[0044] The present invention is further described below with reference to the following examples. It should be understood that the following examples are only intended to explain and illustrate the present invention and are not intended to limit the scope of the present invention in any form.

[0045] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be obtained commercially or prepared according to conventional methods in the art, and the specifications are laboratory pure grade. Unless otherwise specified, the methods used in the following examples are all conventional methods in the art, and reference can be made to the relevant experimental manuals or manufacturer's instructions.

[0046] Example 1. Preparation of a composition for two-step determination of AFP-L3

[0047] 1. Reagent Preparation

[0048] Tris-HCl buffer (10 mM, pH 7.4): Weigh 1.2 g of tris (hydroxymethylaminomethane) (Tris) (Sigma, V900483), add 600 mL of ultrapure water, and after complete dissolution, adjust the pH to 7.4 and make up to 1000 mL.

[0049] Cleaning solution: Weigh 1.2 g of tris (hydroxymethylaminomethane) (Tris) (Sigma, V900483), 8 g of sodium chloride (NaCl) (Sinopharm, 10019308), and 500 μL of Tween-20 (Sigma, 44112). Add 600 mL of ultrapure water, dissolve completely, adjust the pH to 7.4, and dilute to 1000 mL.

[0050] 1% BSA solution: Weigh 1.2 g of Tris (Sigma, V900483), 10 g of bovine serum albumin (Amresco, S12003C01), 1.0 mM manganese chloride (Sinopharm, XW77730154), 1.0 mM magnesium chloride (Sinopharm, 10012818), and 1.0 mM calcium chloride (Sinopharm, 10005817). Add 600 mL of ultrapure water, dissolve completely, adjust the pH to 8.0, and dilute to 1000 mL.

[0051] Phosphate buffer (20 mM PB, pH 7.4): Weigh 0.6 g of sodium dihydrogen phosphate (NaH2PO4) (Sinopharm, 20040799) and 5.8 g of disodium hydrogen phosphate (Na2HPO4) (Sinopharm, 100203008). Add 600 ml of ultrapure water, dissolve completely, adjust the pH to 7.4, and bring the volume to 1000 ml.

[0052] Pre-excitation solution: concentrated hydrochloric acid (Sinopharm, 10011018) 8 mL, hydrogen peroxide (Sinopharm, 10011208) 5 mL, dilute to 1000 mL with pure water.

[0053] Excitation solution: NaOH 8 g (Sinopharm, 10019762), Tween-20 (Sigma, 44112) 10 mL, dilute to 1000 mL with pure water.

[0054] 50 mM MES solution (pH 5.5): Weigh 10.7 g of MES (Aladdin reagent, M105074), dissolve it in 900 mL of pure water, adjust the pH to 5.5, and dilute to 1000 mL with pure water.

[0055] 3M ethanolamine solution: Weigh 183 g of ethanolamine (Aladdin reagent, E808764), add 900 mL of pure water to dissolve, adjust the pH to 9.0, and dilute to 1000 mL with pure water.

[0056] R1 (lectin magnetic beads reagent): contains 20% (v / v) lectin magnetic beads, diluted with 1% BSA solution. The preparation method refers to BeaverBeads TM The instructions for Magrose NHS magnetic beads (70705) are as follows: After mixing the magnetic beads, take 100 μL of magnetic beads (BeaverBeads TM Magrose NHS magnetic beads, product number 70705) were placed in a 1 mL centrifuge tube, and the supernatant was removed by magnetic separation. 200 μL of 1 mM hydrochloric acid at 2-8°C was added to the centrifuge tube and vortexed for 15 seconds to mix the magnetic beads evenly. After magnetic separation, the supernatant was removed; 200 μg of lectin (Vector, product number L-1040) was added and gently mixed; the beads were coupled at 25°C for 2 hours, and the magnetic beads were kept suspended during the coupling period. The centrifuge tube was placed on a magnetic separator and the supernatant was removed after separation. 200 μL of ethanolamine solution (3 M, pH 9.0) was added to resuspend the magnetic beads, and the supernatant was removed by magnetic separation. After repeating twice, 200 μL of ethanolamine solution was added and reacted at 25°C for 2 hours, during which the magnetic beads were kept suspended. The beads were washed once with 200 μL of 1% BSA solution, the supernatant was removed, and 100 μL of 1% BSA solution was added to resuspend the beads and stored at 2-8°C.

[0057] R2 (streptavidin magnetic beads reagent): contains 0.5 mg / mL streptavidin magnetic beads (GE, 30-1029-61), prepared by diluting with 1% BSA solution.

[0058] R3 (AFP capture magnetic bead reagent): Contains 0.5 mg / mL AFP capture magnetic beads, prepared by diluting with 1% BSA solution. The AFP capture magnetic beads are conjugated to magnetic beads using an AFP capture antibody purchased from Feipeng Biotechnology, catalog number 2AFP-27. The magnetic beads are Magnosphere MS300 / Carboxyl (3 μm), developed by JSR Life Sciences Co., Ltd. The AFP capture magnetic beads were prepared according to the Magnosphere MS300 / Carboxyl instructions as follows: Adjust the antibody concentration to 0.5 mg / mL with 20 mM PB (pH 7.4) to prepare an antibody solution. Weigh the carboxyl magnetic beads and dissolve them in 50 mM MES (pH 5.5) to a final concentration of 10 mg / mL, mixing thoroughly. Add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) to the magnetic bead solution to a final concentration of 0.5 mg / mL. Mix thoroughly and incubate at room temperature for 30 minutes to activate the beads. Add the above antibody solution to the activated magnetic bead solution at a ratio of 10 μg antibody to 1 mg magnetic beads. Mix thoroughly and allow to react at room temperature for 120 minutes. Place the magnetic bead solution on a magnetic rack, replace the solution with 1% BSA solution, and store at 2-8°C.

[0059] R4 (biotin-labeled AFP capture antibody reagent): contains 1.0 μg / mL biotin-labeled AFP capture antibody, prepared by diluting with 1% BSA solution. The AFP capture antibody is from Feipeng Bio, item number 2AFP-27; biotin is purchased from ThermoFisher, item number 21335. The labeling method of the AFP capture antibody refers to the instruction manual of biotin, as follows: adjust the antibody concentration with 20 mM PB (pH 7.4). Weigh biotin, dissolve it in purified water, mix thoroughly and set aside. Add biotin to the antibody solution, mix well, and react at room temperature. Remove excess biotin by dialyzing with 20 mM PB (pH 7.4). Remove the labeled material, add an equal volume of glycerol and freeze at -20°C.

[0060] R5 (Signal-labeled AFP detection antibody reagent): Contains 0.1 μg / mL acridinium ester-labeled AFP detection antibody, prepared by diluting with 1% BSA solution. The AFP detection antibody is from Feipeng Bio, item number 2AFP-28. Acridinium ester was purchased from Helison, item number HS-11015005. The labeling method of the AFP detection antibody refers to the instruction manual of acridinium ester, which is as follows: Add acridinium ester to the solution of the substance to be labeled, mix well, and react at room temperature. Remove excess acridinium ester by dialyzing with 10 mM Tris buffer (containing 0.05% Tween-20, pH 7.4). Remove the labeled material, add an equal volume of glycerol, and freeze at -20°C.

[0061] R6 (eluent): Weigh 1.2 g of tris (hydroxymethylaminomethane) (Tris) (Sigma, V900483), 500 mM methyl glucoside (Sigma, M9376), 1.0 mM manganese chloride (Sinopharm, XW77730154), 1.0 mM magnesium chloride (Sinopharm, 10012818), and 1.0 mM calcium chloride (Sinopharm, 10005817). Add 800 mL of ultrapure water, dissolve completely, adjust the pH to 7.4, and bring the volume to 1000 mL.

[0062] R7 (sample diluent): 10 mM Tris (hydroxymethylaminomethane) solution. Prepare by weighing 1.21 g of Tris (hydroxymethylaminomethane) (Sigma, V900483), adding 600 mL of ultrapure water, and dissolving completely. Adjust the pH to 7.4 and dilute to 1000 mL.

[0063] R12 (Lectin Magnetic Beads / Streptavidin Magnetic Beads Reagent): Prepared with a 1% BSA solution, containing 50% (v / v) lectin magnetic beads and 0.5 mg / mL streptavidin magnetic beads. The lectin magnetic beads were prepared using the same method as R1.

[0064] R26: Prepared with 1% BSA solution, containing 0.5 mg / mL streptavidin magnetic beads (GE, 30-1029-61), 500 mM methyl glucoside (Sigma, M9376), 1.0 mM manganese chloride (Sinopharm, XW77730154), 1.0 mM magnesium chloride (Sinopharm, 10012818) and 1.0 mM calcium chloride (Sinopharm, 10005817).

[0065] R36: Prepared with 1% BSA solution, containing 0.5 mg / mL AFP capture magnetic beads, 500 mM methyl glucoside,

[0066] 1.0 mM manganese chloride (Sinopharm, XW77730154), 1.0 mM magnesium chloride (Sinopharm, 10012818) and 1.0 mM calcium chloride (Sinopharm, 10005817). The preparation method of AFP capture magnetic beads is the same as R3.

[0067] R46: Prepared with a 1% BSA solution containing 1.0 μg / mL biotinylated AFP capture antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride (Sinopharm, XW77730154), 1.0 mM magnesium chloride (Sinopharm, 10012818), and 1.0 mM calcium chloride (Sinopharm, 10005817). The biotinylated AFP capture antibody was prepared using the same method as R4.

[0068] R56: Prepared with a 1% BSA solution containing 0.1 μg / mL acridinium ester-labeled AFP detection antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride (Sinopharm, XW77730154), 1.0 mM magnesium chloride (Sinopharm, 10012818), and 1.0 mM calcium chloride (Sinopharm, 10005817). The preparation method for the acridinium ester-labeled AFP detection antibody is the same as for R5.

[0069] The above R1, R2, R3, R4, R5, R6, R7, R12, R26, R36, R46, and R56 are reagent numbers.

[0070] 2. Configuration of the composition

[0071] As shown in Table 1, the reagent composition for the two-step AFP-L3 assay provided by the present invention includes a first reagent set and a second reagent set. The first reagent set is used to capture AFP-L3 from a sample, while the second reagent set is used to simultaneously separate AFP-L3 from a lectin and determine the AFP-L3 concentration in the same reaction system. The various reagents comprising the first reagent set can be added to the reaction system individually or mixed before addition. The various reagents comprising the second reagent set can be added to the reaction system individually or mixed before addition.

[0072] Table 1. Compositions used for the two-step AFP-L3 assay

[0073]

[0074] Example 2. Establishment of a two-step method for determining AFP-L3

[0075] 1. Two-step determination steps

[0076] Step 1 (capture AFP-L3): Take 50 μL of sample, add the first reagent group, incubate at 37°C for 10 minutes, and wash three times with the cleaning solution.

[0077] Step 2 (separation of AFP-L3 from lectin and determination of AFP-L3 concentration): Add the second reagent set and incubate at 37°C for 10 min; wash three times with cleaning solution; add 100 μL each of pre-excitation solution and excitation solution, and measure the luminescence intensity.

[0078] 2. Two-step method validation

[0079] Preparation of an AFP-L3 gradient sample: A high-AFP sample was diluted with 1% BSA solution to obtain an AFP-L3 gradient sample with concentrations of 0 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, 100 ng / mL, 300 ng / mL, and 1000 ng / mL. High-AFP samples were obtained from Qingdao Bodhi Huisheng Medical Testing Co., Ltd. AFP-L3 concentrations in the samples were measured using the μTASWako AFP-L3 Immunofluorescence Assay Kit.

[0080] Using each composition mode in Table 1, the prepared AFP-L3 gradient samples were measured separately according to the above two-step measurement steps using a smart6500 instrument (Chongqing Cosmay). The 0 ng / mL sample was also repeatedly measured 20 times. The linear correlation coefficient r and blank limit for different composition modes were calculated (the RLU average value of the 0 ng / mL sample plus 2 times the standard deviation was substituted into the calibration curve for calculation). Calibration curve drawing: The calibration curve for each composition mode was drawn with the concentration of the AFP-L3 gradient sample as the horizontal axis and the RLU (relative light unit) value of the AFP-L3 gradient sample as the vertical axis, where the RLU value of each sample was the average of three repeated experiments.

[0081] At the same time, set the following control mode (mode 15):

[0082] Step 1 (capture of AFP-L3): Take 50 μL of sample, add the first reagent set [R1 (10 μL), R7 (100 μL)], incubate at 37°C for 10 min; wash three times with cleaning solution;

[0083] Step 2 (separation of AFP-L3 and lectin): add eluent (R6, 50 μL) and incubate at 37°C for 10 min;

[0084] Step 3 (AFP-L3 concentration determination): Add AFP determination reagent [R3 (10 μL), R5 (50 μL)] and incubate at 37°C for 10 min; wash three times with cleaning solution; add 100 μL each of pre-excitation solution and excitation solution, and measure the luminescence intensity.

[0085] The results are shown in Table 2. The linear correlation coefficients and blank limits for the different reaction modes were consistent with those for the control mode. Modes 11, 12, 13, and 14 premixed two magnetic beads, eliminating one magnetic bead reagent compared to other modes and improving compatibility between the reagent and the instrument (some instruments only support one magnetic bead component).

[0086] Table 2. Linear correlation coefficients and blank limits for AFP-L3 determination using different compositions

[0087]

[0088]

[0089] 3. Magnetic bead group selection

[0090] Magnetic beads containing different groups were coupled with lectin (Vector, Cat. No. L-1040) to obtain lectin magnetic beads. Magnetic beads with different groups include NHS magnetic beads (Beaver Biotech, product number 70702), amino magnetic beads (Beaver Biotech, product number 70203-50; Beaver Biotech, product number BK2021062301; Primemag, product number PMAG018; Qiyue Biotech, product number Q-0021571), hydroxyl magnetic beads (Beaver Biotech, product number 70802-10; Beaver Biotech, product number BK2021062109; Qiyue Biotech, product number Q-QJ21553), carboxyl magnetic beads (Beaver Biotech, product number 70103-50), epoxy magnetic beads (Xiamen Primemag, product number PMAG014; Qiyue Biotech, product number Q-0021582), and aldehyde magnetic beads (Xiamen Primemag, product number PMAG012). Composition model 11 was used to measure an AFP-L3 gradient sample (0 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, 100 ng / mL, 300 ng / mL, and 1000 ng / mL). The AFP-L3 gradient sample preparation method was the same as above. R12 (lectin magnetic beads / streptavidin magnetic beads reagent) in composition model 11 was prepared using lectin magnetic beads with different groups, so that the mass fraction of lectin magnetic beads in the first step reaction system was 10%. The remaining reagents were the same as those in composition model 11 in Table 1. The 0 ng / mL sample was measured 20 times, and the linear correlation coefficient r was calculated. The results are shown in Table 3, with NHS magnetic beads being the optimal.

[0091] Table 3. Linear correlation coefficients of AFP-L3 determination using magnetic beads with different groups

[0092]

[0093] 4. Optimal concentration and particle size of lectin magnetic beads

[0094] Magnetic beads with a particle size of 10-30 μm (Beaver Biotechnology, Cat. No. 70113-50) were coupled with lectin (Vector, Cat. No. L-1040) to produce 10-30 μm lectin magnetic beads. Magnetic beads with a particle size of 30-150 μm (Beaver Biotechnology, Cat. No. 70103-50) were coupled with lectin (Vector, Cat. No. L-1040) to produce 30-150 μm lectin magnetic beads.

[0095] AFP-L3 gradient samples (0 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, 100 ng / mL, 300 ng / mL, and 1000 ng / mL) were measured using composition model 11. The AFP-L3 gradient sample preparation method was the same as above. R12 (lectin magnetic beads / streptavidin magnetic beads reagent) in composition model 11 was prepared using lectin magnetic beads with particle sizes of 10-30 μm and 30-150 μm, respectively, with the mass fractions of lectin magnetic beads in the first step being 3% / 10% / 30%, respectively. The remaining reagents were the same as those in composition model 11 in Table 1. The 0 ng / mL sample was measured 20 times, and the 10 ng / mL sample was measured 10 times. The linear correlation coefficient r and repeatability were calculated. A linear fit was performed using the RLU values ​​corresponding to two points in the AFP-L3 gradient sample (0 ng / mL and 3 ng / mL). The RLU obtained by adding the mean RLU value of the 0 ng / mL sample plus two times the standard deviation was then substituted into the calibration curve to calculate the blank limit. The results are shown in Tables 4 and 5. 10-30 μm magnetic beads performed better, with an optimal working concentration of 10% (w / w).

[0096] Table 4. Linear correlation coefficients and blank limits for the determination of AFP-L3 using lectin magnetic beads of different particle sizes and concentrations

[0097]

[0098] Table 5. Repeatability of AFP-L3 assay using lectin magnetic beads of different particle sizes and concentrations

[0099]

[0100] 5. Optimization of eluent concentration

[0101] R46 and R56 in composition pattern 11 were prepared with 1% BSA solution so that the reaction system in the second step contained different concentrations (0.05M, 0.15M, 0.5M, 1.5M, 5M) of the eluent (methyl glucoside).

[0102] Using composition model 11, an AFP-L3 gradient sample (0 ng / mL, 3 ng / mL, 10 ng / mL, 30 ng / mL, 100 ng / mL, 300 ng / mL, and 1000 ng / mL) was measured at different eluent concentrations (reaction system concentration). The AFP-L3 gradient sample preparation method was the same as above. The 0 ng / mL sample was measured 20 times, and the 10 ng / mL sample was measured 10 times. The linear correlation coefficient r, repeatability, and blank limit were calculated (the RLU mean of the 0 ng / mL sample plus two standard deviations of the RLU were substituted into the calibration curve). The results are shown in Tables 6 and 7. The measurement effect was best when the eluent concentration ranged from 0.5 to 5 M. Considering cost factors, the optimal condition was 0.5 M.

[0103] Table 6. Linear correlation coefficients and blank limits for the determination of AFP-L3 at different methyl glucoside concentrations

[0104]

[0105] Table 7. AFP-L3 concentrations measured at different methyl glucoside concentrations

[0106]

[0107] 6. Clinical performance

[0108] Clinical samples included 121 serum samples from patients with primary liver cancer, 353 serum samples from healthy individuals, 85 serum samples from patients with cirrhosis, 81 serum samples from patients with hepatitis, and 35 serum samples from patients with other cancers (lung cancer, breast cancer, and prostate cancer). All clinical samples were provided by Qingdao Bodhi Huisheng Medical Testing Co., Ltd.

[0109] The two-step method of the present invention was used to measure AFP-L3 in the above clinical samples. The details are as follows:

[0110] Step 1: Take 50 μL of sample, add the first reagent group, incubate at 37°C for 10 minutes, and wash three times with cleaning solution;

[0111] Step 2: Add the second reagent group and incubate at 37°C for 10 minutes; wash three times with cleaning solution; add 100 μL each of pre-excitation solution and excitation solution, and measure the luminescence intensity.

[0112] Mode 11 was used for the determination. The first reagent group consisted of R12 (20 μL) and R7 (100 μL), wherein the lectin magnetic beads in R12 were prepared using NHS magnetic beads (Beaver Bio, product number 70702), and the mass fraction of the lectin magnetic beads in R12 was 10%; the second reagent group consisted of R46 (75 μL) and R56 (75 μL), wherein the concentration of methyl glucoside was 0.5 M.

[0113] At the same time, an AFP kit (produced by Tongxin Biotechnology) was used to detect AFP in the above clinical samples.

[0114] The test results are shown in Table 8. The method of the present invention has a positive detection rate of 91.7% for primary liver cancer, a specificity of 100% for healthy people, a specificity of 94.1% and 97.5% for cirrhosis and hepatitis in high-risk groups for liver cancer, respectively, and a specificity of 100% for other cancers.

[0115] Table 8

[0116]

Claims

1. A composition for measuring alpha-fetoprotein heterogeneity, characterized in that: The method comprises a first reagent group for capturing alpha-fetoprotein isoforms and a second reagent group for separating and determining alpha-fetoprotein isoforms in the same reaction system; The first reagent set consists of R12 lectin magnetic beads / streptavidin magnetic beads reagent and R7 sample diluent, wherein the volume ratio of R12 to R7 is 1:5; the second reagent set consists of R46 biotin-labeled AFP capture antibody reagent and R56 signal substance-labeled AFP detection antibody reagent, wherein the volume ratio of R46 to R56 is 1:1; The R12 is prepared with a 1% BSA solution, containing 50% (v / v) lectin magnetic beads and 0.5 mg / mL streptavidin magnetic beads; the R7 is a 10 mM tris solution; the R46 is prepared with a 1% BSA solution, containing 1.0 μg / mL biotin-labeled AFP capture antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride and 1.0 mM calcium chloride; the R56 is prepared with a 1% BSA solution, containing 0.1 μg / mL acridinium ester-labeled AFP detection antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride and 1.0 mM calcium chloride.

2. A composition for measuring alpha-fetoprotein heterogeneity, characterized in that: The method comprises a first reagent group for capturing alpha-fetoprotein isoforms and a second reagent group for separating and determining alpha-fetoprotein isoforms in the same reaction system; The first reagent set consists of R12 lectin magnetic beads / streptavidin magnetic beads reagent and R7 sample diluent, wherein the volume ratio of R12 to R7 is 1:5; the second reagent set consists of R46 biotin-labeled AFP capture antibody reagent and R5 signal substance-labeled AFP detection antibody reagent, wherein the volume ratio of R46 to R5 is 2:1; The R12 is prepared with a 1% BSA solution, containing 50% (v / v) lectin magnetic beads and 0.5 mg / mL streptavidin magnetic beads; the R7 is a 10 mM tris (hydroxymethyl)aminomethane solution; the R46 is prepared with a 1% BSA solution, containing 1.0 μg / mL biotin-labeled AFP capture antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride and 1.0 mM calcium chloride; the R5 is prepared with a 1% BSA solution, containing 0.1 μg / mL acridinium ester-labeled AFP detection antibody.

3. A composition for measuring alpha-fetoprotein heterogeneity, characterized in that: The method comprises a first reagent group for capturing alpha-fetoprotein isoforms and a second reagent group for separating and determining alpha-fetoprotein isoforms in the same reaction system; The first reagent set consists of R12 lectin magnetic beads / streptavidin magnetic beads reagent and R7 sample diluent, wherein the volume ratio of R12 to R7 is 1:5; the second reagent set consists of R4 biotin-labeled AFP capture antibody reagent and R56 signal substance-labeled AFP detection antibody reagent, wherein the volume ratio of R4 to R56 is 1:2; The R12 is prepared with a 1% BSA solution, containing 50% (v / v) lectin magnetic beads and 0.5 mg / mL streptavidin magnetic beads; the R7 is a 10 mM tris solution; the R4 is prepared with a 1% BSA solution, containing 1.0 μg / mL biotin-labeled AFP capture antibody; the R56 is prepared with a 1% BSA solution, containing 0.1 μg / mL acridinium ester-labeled AFP detection antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride and 1.0 mM calcium chloride.

4. A composition for measuring alpha-fetoprotein heterogeneity, characterized in that: The method comprises a first reagent group for capturing alpha-fetoprotein isoforms and a second reagent group for separating and determining alpha-fetoprotein isoforms in the same reaction system; The first reagent group is composed of R12 lectin magnetic beads / streptavidin magnetic beads reagent; the second reagent group is composed of R46 biotin-labeled AFP capture antibody reagent and R56 signal substance-labeled AFP detection antibody reagent, wherein the volume ratio of R46 to R56 is 1:1; The R12 is prepared with a 1% BSA solution, containing 50% (v / v) lectin magnetic beads and 0.5 mg / mL streptavidin magnetic beads; the R46 is prepared with a 1% BSA solution, containing 1.0 μg / mL biotin-labeled AFP capture antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride and 1.0 mM calcium chloride; the R56 is prepared with a 1% BSA solution, containing 0.1 μg / mL acridinium ester-labeled AFP detection antibody, 500 mM methyl glucoside, 1.0 mM manganese chloride, 1.0 mM magnesium chloride and 1.0 mM calcium chloride.

5. The composition according to any one of claims 1 to 4, characterized in that The magnetic beads are NHS magnetic beads.

6. The composition according to any one of claims 1 to 4, characterized in that The particle size of the magnetic beads is 10-30 μm.

7. A kit for determining alpha-fetoprotein heterogeneity, characterized in that: The invention comprises the composition for determining alpha-fetoprotein isoforms according to any one of claims 1 to 6.

8. The kit according to claim 7, characterized in that It also includes a washing solution for washing the magnetic beads and an excitation solution for exciting the signal substance to generate a signal.

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