Kit for detecting AFP-L3 levels in alpha-fetoprotein isoforms

Abstract

This invention relates to the field of in vitro diagnostics, specifically to a kit for detecting the content of alpha-fetoprotein isoform AFP-L3. This invention provides a kit for detecting the content of alpha-fetoprotein isoform AFP-L3. After the kit sample is treated with SDS as a denaturing agent, the secondary structure of the AFP antigen is opened, and fucosylated AFP (AFP-L3) is fully exposed, making it easier for AFP to recognize and bind to LCA in the liquid phase, forming an AFP-L3-LCA complex. Simultaneously, we are introducing Ca2+ into the buffer system for the first time. 2+ and Mn 2+ Sodium dextran sulfate, along with two metal ions, plays an important role in maintaining LCA activity.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic technology, specifically to a kit for detecting the content of alpha-fetoprotein isoform AFP-L3. Background Technology

[0002] Hepatocellular carcinoma (HCC) is insidious in its onset, with over 80% of HCC patients diagnosed at an advanced stage. Therefore, early screening and monitoring of high-risk groups are crucial. Currently, the most commonly used clinical method for early HCC screening is the detection of the serum biomarker alpha-fetoprotein (AFP). A serum AFP level >400 ng / mL, after excluding pregnancy, chronic or active liver disease, germ cell tumors of the gonads, and gastrointestinal tumors, is highly suggestive of HCC. However, AFP testing for HCC has insufficient sensitivity. Furthermore, some benign liver diseases (including chronic hepatitis, fatty liver, and cirrhosis), benign liver tumors, reproductive system tumors, or pregnancy can all cause elevated AFP levels, resulting in insufficient specificity for AFP testing in HCC.

[0003] AFP is a glycoprotein that can be classified into three isoforms based on its different glycosylation forms. These are further classified as AFP-L1, AFP-L2, and AFP-L3 according to their ability to bind lentil lectin (LCA). AFP-L1 mainly originates from benign liver disease cells and exhibits LCA non-binding; AFP-L2 mainly originates from yolk sac tumors and pregnant women, exhibiting weak LCA binding; AFP-L3 exhibits strong LCA binding because it has a fucosylation site specifically recognized by LCA at the N-acetylglucosamine end of the biantennary-shaped glycan chain. The concentrations of total AFP and its isoform AFP-L3 in the samples were measured, and the ratio of AFP-L3 to total AFP (AFP-L3 / AFP) was calculated. A 10% cut-off was used to confirm the result; AFP-L3% ≥ 10% was considered positive, and AFP-L3% < 10% was considered negative. Since characteristic core fucosylation leads to changes in protein function, and AFP-L3 is usually derived from liver cancer cells, AFP-L3 can serve as an effective tumor marker for the early detection of liver cancer.

[0004] The "Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022 Edition)" states that serum AFP is a commonly used and important indicator for diagnosing liver cancer and monitoring treatment efficacy. For AFP-negative individuals, early diagnosis using abnormal prothrombin (PIVKA II), cell-free plasma microRNA, and AFP-L3 can effectively improve the sensitivity and specificity of liver cancer diagnosis. The U.S. Food and Drug Administration (FDA) approved AFP-L3 detection reagents and methods for clinical liver cancer early warning in 2005. Therefore, establishing a rapid, highly sensitive, and highly specific AFP-L3 detection method is essential.

[0005] Currently, all clinical methods for detecting AFP-L3 in China are based on the affinity adsorption of LCA (Liquid Crystal Acid). AFP-L3 affinity adsorption centrifuge tubes require manual single-sample handling. The sample is diluted, passed through a column for adsorption, and then washed to elute AFP-L3 before being quantified using an AFP detection kit. While AFP-L3 detection kits can automate the process, both affinity adsorption centrifuge tubes and kits are indirect methods for AFP-L3 detection, requiring extraction before quantification. AFP-L3 affinity adsorption centrifuge tubes are cumbersome, and human error is difficult to avoid, resulting in low detection efficiency. AFP-L3 detection kits also involve AFP-L3 pretreatment. The original sample is eluted by AFP-L3 separation beads to obtain the AFP-L3 sample. Both the original sample and the AFP-L3 sample require quantification using AFP detection beads to calculate the AFP-L3 percentage. The AFP-L3 separation magnetic beads in the AFP-L3 detection kit differ from conventional bio-based magnetic beads with a diameter of approximately 1–3 μm. To achieve maximum loading, agarose magnetic beads with a diameter of around 100 μm are required, leading to increased costs. Furthermore, the coupling efficiency of LCA is difficult to guarantee, requiring special preparation, which further increases detection costs. Existing methods for AFP-L3 detection do not involve sample denaturation. Without denaturation treatment with sodium dodecyl sulfate (SDS), the fucosylated AFP sites are small and easily folded or overlapped by the secondary structure of the AFP protein, affecting LCA recognition and causing incomplete affinity adsorption. Repeated extraction of AFP-L3 extract does not increase the proportion of AFP-L3, which is the fundamental reason for the poor reproducibility of affinity adsorption centrifuge tubes and AFP-L3 detection kits.

[0006] Wako's liquid-phase immunoaffinity electrophoresis (LC-AAE) method uses a direct approach. After capturing total AFP in a double-antibody sandwich in the liquid phase, the specific adsorption capacity of LCA for AFP-L3 is used to identify and separate the AFP-L3 component in the total AFP. This method can achieve automated direct quantification of AFP-L3. However, the affinity electrophoresis platform is not suitable for the widespread application of the detection technology in this project. Summary of the Invention

[0007] In view of this, the present invention provides a kit for detecting the content of alpha-fetoprotein isoform AFP-L3.

[0008] This invention provides a kit for detecting the content of alpha-fetoprotein isoform AFP-L3. After the kit samples are treated with SDS as a denaturing agent, the secondary structure of the AFP antigen is opened, and the fucosylated AFP (AFP-L3) is fully exposed, making it easier for AFP to recognize and bind to LCA in the liquid phase, forming an AFP-L3-LCA complex. Simultaneously, we are introducing Ca2+ into the buffer system for the first time. 2+ and Mn 2+ Sodium dextran sulfate, along with two metal ions, plays an important role in maintaining LCA activity.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a reagent combination, including a sample preparation agent and a sample diluent:

[0011] The sample processing agent comprises: 0.06–1% (w / v) SDS and buffer B;

[0012] The sample diluent comprises: 1.25–40 μg / mL LCA, 0.6–10% (w / v) sodium dextran sulfate and buffer B;

[0013] The buffer solution B comprises: 0.15 mol / L Tris-NaCl, 1‰ (w / v) Proclin 300, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100.

[0014] In some specific embodiments of the present invention, the sample processing agent comprises: 0.125–0.5% (w / v) SDS and the buffer solution B;

[0015] The sample diluent comprises: 5–20 μg / mL LCA, 1–5% (w / v) sodium dextran sulfate, and buffer B.

[0016] In some specific embodiments of the present invention, the sample processing agent comprises: 0.25% (w / v) SDS and the buffer B;

[0017] The sample diluent comprises: 10 μg / mL LCA, 2.5% (w / v) sodium dextran sulfate, and buffer B.

[0018] In some specific embodiments of the present invention, the preparation method of the sample diluent includes: preparing buffer B: 0.15 mol / L Tris-NaCl, pH 8.0 buffer solution, adding 1‰ (w / v) Proclin 300, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100 in sequence, mixing well and storing at 2-8℃ for later use.

[0019] Weigh 5 mg of LCA and dissolve it in 1 mL of 0.15 mol / L Tris-NaCl at pH 8.0 to prepare a 5 mg / mL LCA stock solution. Dilute the LCA stock solution to 5–20 μg / mL with buffer B and add 1.25–5% (w / v) of sodium dextran sulfate to obtain the sample dilution solution.

[0020] In some specific embodiments of the present invention, the preparation method of the sample processing agent includes: weighing 0.1g SDS and dissolving it in 1mL of pure water to prepare a 10% (w / v) SDS stock solution, and diluting the SDS stock solution with buffer B to 0.125% to 0.5% (w / v) to obtain the sample processing agent.

[0021] In some specific embodiments of the present invention, the reagent combination further includes a magnetic microparticle suspension;

[0022] The magnetic microparticle suspension comprises activated JSR carboxyl magnetic beads, LCA monoclonal antibody, and sealing solution;

[0023] The sealing solution comprises: 0.05 mol / L Tris-NaCl buffer, 1‰ (w / v) Proclin 300, 1% (w / v) bovine serum albumin, 5% (w / v) sucrose and 0.5‰ (w / v) Tritonx-100.

[0024] In some specific embodiments of the present invention, the preparation method of activated JSR carboxyl magnetic beads in the magnetic microparticle suspension includes: washing JSR carboxyl magnetic beads with PBS buffer, activating the magnetic beads with 25 mg / mL LEDC and 25 mg / mL NHS for 1 to 1.5 h to obtain the activated JSR carboxyl magnetic beads.

[0025] In some specific embodiments of the present invention, the method for preparing the magnetic microparticle suspension includes: washing the activated JSR carboxyl magnetic beads with MES buffer, then mixing and shaking with 2.5 mg / mL LCA monoclonal antibody for 3 h, terminating the reaction with ethanolamine for 0.5-1 h, and then blocking with blocking solution three times to obtain the magnetic microparticle suspension.

[0026] In some specific embodiments of the present invention, the reagent combination further includes calibrators;

[0027] The calibrators include AFP-L3 antigen, 0.05 mol / L Tris-NaCl, 1‰ (w / v) Proclin 300 and 1% (w / v) bovine serum albumin;

[0028] The concentration of the AFP-L3 antigen is 0–1000 ng / mL.

[0029] In some specific embodiments of the present invention, the concentration of the AFP-L3 antigen is 0 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL and 1000 ng / mL.

[0030] In some specific embodiments of the present invention, the reagent combination further includes an enzyme conjugate reaction solution;

[0031] The enzyme conjugate reaction solution includes an HRP-labeled AFP-L3 monoclonal antibody Fab fragment and buffer A;

[0032] The buffer A comprises: 0.02 mol / L PBS, 1‰ (w / v) Proclin 300, 1‰ (w / v) casein, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100.

[0033] In some specific embodiments of the present invention, the preparation method of the AFP-L3 monoclonal antibody Fab fragment includes: dialyzing AFP-L3 monoclonal antibody at a concentration of 5 mg / mL in 0.2 mol / L sodium acetate buffer (pH 4.0) at 4°C for 16 h, then adding AFP-L3 monoclonal antibody: pepsin = 1:0.04 (w / w), incubating in a water bath at 37°C for 6 h, adjusting the pH to 7.4 with 3 mol / L Tris solution; dialyzing in PBS at 4°C for 16 h; after dialyzing, passing through a PBS-equilibrated Seghadex G-100 antibody purification column, collecting the first flow-through peak liquid; adding a final concentration of 10 mmol / L mercaptoethanol and passing through a PBS-equilibrated Seghadex G-25 column, collecting the protein peak liquid, and detecting the protein content at 2.7 mg / mL using a UV spectrophotometer to obtain the AFP-L3 monoclonal antibody Fab fragment.

[0034] In some specific embodiments of the present invention, the preparation method of the enzyme conjugate reaction solution includes: taking 5.4 mg HRP, preparing a concentration of 10 mg / mL with purified water, adding an equal volume of sodium periodate (12.8 mg / mL, dissolved in purified water, freshly prepared and used), and reacting at 4°C in the dark for 30 min; adding an equal volume of 1% ethylene glycol solution (freshly prepared and used) to the HRP solution, and reacting at 4°C in the dark for 45 min; adding 2.7 mg AFP-L3 monoclonal antibody and mixing, and desalting using a PD SpinTrap G-25 centrifugal desalting column. During the desalting process, the desalting column is first pretreated with PBS. The mixture in the centrifuge tube is collected, and sodium borohydride solution (5 mg / mL, dissolved in purified water, freshly prepared and used) is added at a rate of 20 μL per mg antibody. The mixture is reacted at 4°C in the dark for 2 h; slowly adding an equal volume of saturated ammonium sulfate solution, reacting at 4°C for 2 h, centrifuging (4°C, 8000 rpm, 0.5 h), and discarding the supernatant; dissolving the precipitate with an appropriate amount of PBS, and reacting with PD... The desalting process was performed using a SpinTrap G-25 centrifugal desalting column. The desalting column was first pretreated with PBS, and the liquid in the centrifuge tube was collected to obtain the HRP-labeled AFP monoclonal antibody Fab fragment.

[0035] The HRP-labeled AFP monoclonal antibody Fab fragment was added to buffer A at a volume ratio of 1:1000 to prepare the enzyme conjugation reaction solution; the concentration of the enzyme conjugation reaction solution was 1.9 μg / mL.

[0036] The present invention also provides the application of the reagent combination in the preparation of a kit for detecting liver cancer.

[0037] In some specific embodiments of the present invention, the detection of liver cancer includes the detection of AFP-L3.

[0038] Based on the above research, the present invention also provides a kit comprising the reagent combination and acceptable excipients or adjuvants.

[0039] In some specific embodiments of the present invention, the method for detecting AFP-L3 using the kit includes: taking a serum sample after centrifugation (10000 rpm, 5 min) to obtain the supernatant; mixing 25 μL of the supernatant with an equal volume of sample processing reagent and incubating at 37°C for 15 min; adding 25 μL of sample diluent containing LCA and 20 μL of magnetic microparticle suspension to a reaction vessel, incubating at 37°C for 15 min, and washing 5 times; adding 50 μL of enzyme conjugation reaction solution, incubating at 37°C for 15 min, and washing 5 times; adding the luminescent substrate, mixing well, and then detecting the signal value.

[0040] This invention provides a kit for detecting the content of alpha-fetoprotein isoform AFP-L3. The kit uses LCA monoclonal antibody magnetic beads and HRP-labeled AFP-L3 monoclonal antibody Fab fragments to sandwich the AFP-L3-LCA complex, allowing direct quantification of AFP-L3 concentration in the sample. Compared to methods that indirectly extract and then quantify AFP-L3, this avoids the manual process of AFP-L3 separation, extraction, and re-detection, enabling fully automated instrument operation with low error and high accuracy. The kit has no special requirements for instruments or magnetic beads, exhibits high sensitivity and specificity, is low-cost, and can be used for single-person or batch processing. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0042] Figure 1 The standard curve of the alpha-fetoprotein isoform AFP-L3 calibrator in Example 5 is shown. Detailed Implementation

[0043] This invention discloses a kit for detecting the content of alpha-fetoprotein isoform AFP-L3. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0044] The purpose of this invention is to provide a faster and more convenient kit for the direct detection of alpha-fetoprotein isoform AFP-L3 using a double-antibody sandwich method.

[0045] The kit of this invention conjugates LCA monoclonal antibodies to carboxyl magnetic beads via the carbodiimide method. After the sample is treated with SDS as a denaturant, the secondary structure of the AFP antigen is opened, and the fucosylated AFP (AFP-L3) is fully exposed, making it easier for it to recognize and bind to LCA in the liquid phase, forming the AFP-L3-LCA complex. Simultaneously, we are introducing Ca2+ into the buffer system for the first time. 2+ and Mn 2+Sodium dextran sulfate, along with two metal ions, plays a crucial role in maintaining LCA activity. A double-antibody sandwich technique was used to encapsulate the AFP-L3-LCA complex using LCA monoclonal antibody magnetic beads and HRP-labeled AFP-L3 monoclonal antibody Fab fragments. This avoided the influence of the Fc-terminal glycosylation site of the AFP-L3 monoclonal antibody on the LCA, and the AFP-L3 content was accurately determined using HRP-catalyzed luminescence.

[0046] The kit of this invention uses LCA monoclonal antibody magnetic beads and HRP-labeled AFP-L3 monoclonal antibody Fab fragment to sandwich the AFP-L3-LCA complex with a double antibody, allowing direct quantification of AFP-L3 concentration in the sample. The labeled antibody used in this invention is the AFP-L3 monoclonal antibody Fab fragment, which effectively avoids the non-specific binding effect of its Fc-terminal glycosylation site on LCA. Compared to methods that indirectly extract and then quantify AFP-L3, this method avoids the manual process of AFP-L3 separation, extraction, and re-detection, enabling fully automated instrument operation with low error and high accuracy.

[0047] The kit of this invention conjugates LCA monoclonal antibodies to carboxyl magnetic beads via the carbodiimide method and introduces an appropriate amount of LCA into the sample dilution solution, thereby achieving the effective formation of the AFP-L3-LCA complex. This invention does not have special requirements for magnetic beads and ensures that the amount of LCA used is minimized, thus reducing detection costs.

[0048] The reagent kit of this invention is applicable to all models of the Antu chemiluminescence platform, and is simple to operate and inexpensive.

[0049] After the kit samples of this invention are treated with SDS as a denaturant, the secondary structure of the AFP antigen is opened, and the fucosylated AFP (AFP-L3) is fully exposed, making it easier for AFP to recognize and bind to LCA in the liquid phase to form the AFP-L3-LCA complex. Simultaneously, we are introducing Ca2+ into the buffer system for the first time. 2+ and Mn 2+ In addition, dextran sulfate, along with two metal ions and sodium dextran sulfate, plays an important role in maintaining LCA activity.

[0050] The kit of this invention uses LCA monoclonal antibody magnetic beads and HRP-labeled AFP-L3 monoclonal antibody Fab fragments to sandwich the AFP-L3-LCA complex, allowing direct quantification of AFP-L3 concentration in samples. Compared to methods that indirectly extract and then quantify AFP-L3, this avoids the manual process of AFP-L3 separation, extraction, and re-detection, enabling fully automated instrument operation with minimal error and high accuracy. The kit has no special requirements for instruments or magnetic beads, exhibits high sensitivity and specificity, is low-cost, and can be used for single-person or batch processing.

[0051] A kit for detecting alpha-fetoprotein isoform AFP-L3 includes: sample processing agent, magnetic microparticle suspension, enzyme conjugate, sample diluent and AFP-L3 calibrator;

[0052] The sample processing agent contains 0.125% to 0.5% (w / v) of SDS buffer B;

[0053] The magnetic microparticle suspension comprises a magnetic microparticle suspension coated with LCA monoclonal antibody; the preparation method is as follows: after thoroughly mixing and washing the magnetic microparticle stock solution, EDC and NHS activator are added, mixed and shaken, washed, and then LCA monoclonal antibody (provided by Imeno) is added, mixed and shaken; the reaction is terminated with ethanolamine, and finally blocked with sealing solution and stored at 2-8℃ for later use;

[0054] The enzyme conjugation reaction solution contains a buffer solution containing an HRP-labeled AFP-L3 monoclonal antibody Fab fragment. The specific preparation method for the AFP-L3 monoclonal antibody Fab fragment is as follows: AFP monoclonal antibody is digested with pepsin under acidic conditions at a mass ratio of 1:0.04 (AFP-L3 monoclonal antibody to pepsin), digested at 37°C for 6–24 h, separated by column chromatography, and reduced with mercaptoethanol. The preparation method for the HRP-labeled AFP-L3 monoclonal antibody Fab fragment is as follows: The HRP-labeled AFP-L3 monoclonal antibody Fab fragment is placed in buffer A at a ratio of 1 / 1500 to 1 / 2000 and stored at 2–8°C until use.

[0055] The sample diluent contains 5–20 μg / mL LCA and 1–5% (w / v) sodium dextran sulfate buffer B;

[0056] The calibrators include alpha-fetoprotein isoform AFP-L3 antigen and a series of calibrators with AFP-L3 antigen concentrations ranging from low to high (0-1000 ng / mL) at multiple different concentration points, such as 0.05 mol / L Tris-NaCl, 1‰ (w / v) Proclin 300, and 1% (w / v) bovine serum albumin.

[0057] The buffer A comprises: 0.02 mol / L PBS, 1‰ (w / v) Proclin 300, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100;

[0058] Buffer B consists of: 0.15 mol / L Tris-NaCl, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 1‰ (w / v) Proclin 300, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100;

[0059] The specific sample addition steps are as follows: Before use, the serum sample is centrifuged (10000 rpm, 5 min) and the supernatant is collected; 25 μL of the processed sample is extracted by the fully automated chemiluminescence analyzer and an equal volume of sample processing reagent is added to the reaction vessel and incubated at 37°C for 15 min; 25 μL of sample diluent containing LCA and 20 μL of magnetic microparticle suspension are added to the reaction vessel and incubated at 37°C for 15 min, followed by 5 washes; 50 μL of enzyme conjugation reaction solution is added and incubated at 37°C for 15 min, followed by 5 washes; the luminescent substrate is added, mixed well, and the signal value is detected.

[0060] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The LCA monoclonal antibody and AFP-L3 monoclonal antibody were both from Imeno Biotech. This kit is prepared using a magnetic microparticle chemiluminescence platform and is paired with Antu Biotech's fully automated immunoassay analyzer, enabling rapid and automated detection of alpha-fetoprotein isoform AFP-L3. It is convenient and easy to use.

[0061] The raw materials and reagents used in the kit for detecting the content of alpha-fetoprotein isoform AFP-L3 provided by this invention are all commercially available.

[0062] The present invention will be further illustrated below with reference to the embodiments:

[0063] Example 1: Preparation of an AFP-L3 Detection Kit for Liver Cancer Diagnosis

[0064] 1. Preparation of magnetic microparticle suspension

[0065] Preparation of sealing solution: Add 1‰ (w / v) Proclin 300, 1% (w / v) bovine serum albumin, 5% (w / v) sucrose and 0.5‰ (w / v) Tritonx-100 to 0.05 mol / L Tris-NaCl buffer in sequence, mix well and store at 2-8℃ for later use.

[0066] Take 30 μL of commercially available JSR carboxyl magnetic beads, wash three times with PBS buffer (pH 7.2), add 30–60 μL of 25 mg / mL EDC and 30–60 μL of 25 mg / mL NHS, mix and vortex for 1–1.5 h; wash four times with 100–200 μL of MES buffer; add 20–30 μL of 2.5 mg / mL LCA monoclonal antibody, mix and vortex for 3 h; add 200–300 μL of ethanolamine to stop the reaction for 0.5–1 h; magnetically remove the supernatant, add 300–500 μL of blocking solution, wash three times; add 3 mL of blocking solution, store at 2–8 °C for later use.

[0067] 2. Preparation of AFP-L3 monoclonal antibody Fab fragment

[0068] Prepare buffer A: Add 1‰ (w / v) Proclin 300, 1‰ (w / v) casein, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100 to 0.02 mol / L PBS, pH 7.2, mix well and store at 2-8°C until use.

[0069] AFP-L3 monoclonal antibody at a concentration of 5 mg / mL was dialyzed in 0.2 mol / L sodium acetate buffer (pH 4.0) at 4°C for 16 h. Then, AFP-L3 monoclonal antibody was added at a ratio of AFP-L3 monoclonal antibody to pepsin of 1:0.04 (w / w), and the mixture was incubated at 37°C for 6 h. The pH was adjusted to 7.4 with 3 mol / L Tris solution. The mixture was then dialyzed in PBS at 4°C for 16 h. After dialyzing, the solution was passed through a PBS-equilibrated Seghadex G-100 antibody purification column, and the first flow-through peak was collected. After adding 10 mmol / L mercaptoethanol, the solution was passed through a PBS-equilibrated Seghadex G-25 column, and the protein peak was collected. The protein concentration was measured to be 2.7 mg / mL using a UV spectrophotometer, indicating that this was the AFP-L3 monoclonal antibody Fab fragment.

[0070] 3. Preparation of enzyme-conjugated reaction solution

[0071] Take 5.4 mg of horseradish peroxidase (HRP) and dissolve it in purified water to a concentration of 10 mg / mL. Add an equal volume of sodium periodate (12.8 mg / mL, dissolved in purified water, freshly prepared) to the HRP solution and react at 4°C in the dark for 30 min. Add an equal volume of 1% ethylene glycol solution (freshly prepared) to the HRP solution and react at 4°C in the dark for 45 min. Add 2.7 mg of AFP-L3 monoclonal antibody and mix. Desalt the mixture using a PD SpinTrap G-25 centrifugal desalting column. During desalting, pre-treat the desalting column with PBS. Collect the mixture in the centrifuge tube and add sodium borohydride solution (5 mg / mL, dissolved in purified water, freshly prepared) at a rate of 20 μL per mg of antibody. React at 4°C in the dark for 2 h. Slowly add an equal volume of saturated ammonium sulfate solution and react at 4°C for 2 h. Centrifuge (4°C, 8000 rpm, 0.5 h) and discard the supernatant. The precipitate was dissolved in an appropriate amount of PBS and desalted using a PD SpinTrap G-25 centrifugal desalting column. During the desalting process, the desalting column was first pretreated with PBS. The liquid in the centrifuge tube was collected, an equal volume of glycerol was added, and the mixture was mixed and stored at -20°C.

[0072] HRP-labeled AFP monoclonal antibody Fab fragment was added to buffer A at a volume ratio of 1:1000 to prepare an enzyme conjugation reaction solution with a concentration of 1.9 μg / mL.

[0073] 4. Preparation of sample diluent

[0074] Prepare buffer B: Add 1‰ (w / v) Proclin 300, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100 to a 0.15 mol / L Tris-NaCl, pH 8.0 buffer solution in sequence. Mix well and store at 2-8℃ until use.

[0075] Weigh 5 mg of LCA and dissolve it in 1 mL of 0.15 mol / L Tris-NaCl at pH 8.0 to prepare a 5 mg / mL LCA stock solution. Dilute the LCA stock solution with buffer B to 5–20 μg / mL and then add 1.25–5% (w / v) of sodium dextran sulfate.

[0076] 5. Preparation of sample processing reagents

[0077] Weigh 0.1 g of SDS and dissolve it in 1 mL of pure water to prepare a 10% (w / v) SDS stock solution. Dilute with buffer B to 0.125%–0.5% (w / v).

[0078] 6. Preparation of calibrators

[0079] High concentrations of alpha-fetoprotein isoform AFP-L3 antigen, 1‰ (w / v) Proclin 300, and 1% (w / v) bovine serum albumin were added to 0.05 mol / L Tris-NaCl. The concentrations of alpha-fetoprotein isoform AFP-L3 were set at 0 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0080] Example 2: Test method of the reagent kit of the present invention

[0081] 1. Using a fully automated chemiluminescence analyzer (AutoLumo A2000Plus), extract 25 μL of serum sample and 25 μL of sample processing reagent, add them to the reaction vessel, and incubate at 37℃ for 15 min.

[0082] 2. Add 25 μL of sample diluent containing LCA and sodium dextran sulfate and 20 μL of magnetic microparticle suspension to the reaction vessel, incubate at 37°C for 15 min, and wash 5 times.

[0083] 3. Add 50 μL of enzyme conjugation reaction solution, incubate at 37°C for 15 min, and wash 5 times;

[0084] Add the luminescent substrate (luminol + H2O2), mix well, and then detect the signal value.

[0085] Calculation: This kit recommends using a four-parameter fitting method, establishing a calibration curve with the calibrator concentration value on the x-axis and the logarithmic value of the calibrator on the y-axis. The corresponding concentration value is then calculated back from the luminescence value of the test sample. The instrument's automatic operating system can automatically calculate the sample test results using the stored calibration curve and the luminescence values ​​obtained from the sample tests.

[0086] Example 3: Effect of SDS concentration in sample treatment agent on reactivity

[0087] Buffer B was used to dilute 10% (w / v) SDS stock solution to 0.06%, 0.125%, 0.25%, 0.5%, and 1% (w / v) to prepare sample preparations with different SDS concentrations. Samples with AFP-L3 concentrations of 50, 100, and 200 ng / mL were selected. Following the steps in Example 2 (where the LCA concentration was 10 μg / mL and the sodium dextran sulfate concentration was 2.5%), 30 μL of the processed sample was extracted using a fully automated chemiluminescence analyzer. Equal volumes of the sample preparations with different SDS concentrations were added to the reaction vessel, and the mixture was incubated at 37°C for 15 min. The remaining steps were the same. The effect of different SDS concentrations in the sample preparations on reactivity was compared, and the results are shown in Table 1.

[0088] Table 1. Effect of SDS concentration on reactivity

[0089]

[0090] As shown in Table 1, the highest luminescence values ​​were obtained when the SDS concentration in the sample processing reagent was between 0.125% and 0.5% for different concentrations of AFP-L3 (50, 100, and 200 ng / mL). Both excessively high and low SDS concentrations will affect the luminescence values.

[0091] Example 4: Effect of LCA and sodium dextran sulfate concentrations in the sample dilution on reactivity

[0092] Buffer B was used to dilute 5 mg / mL LCA stock solution to concentrations of 40, 20, 10, 5, 2.5, and 1.25 μg / mL. 10%, 5%, 2.5%, 1.25%, and 0.6% (w / v) sodium dextran sulfate were added to each LCA concentration solution to prepare sample diluents with different LCA and sodium dextran sulfate concentrations. A sample with an AFP-L3 concentration of 100 ng / mL was selected. After the sample treatment agent (wherein the SDS concentration was 0.25% (w / v)) was applied, equal volumes of sample diluents with different LCA and sodium dextran sulfate concentrations and 20 μL of magnetic microparticle suspension were added to reaction vessels according to the steps in Example 2. The mixture was incubated at 37°C for 15 min; the remaining steps were the same. The effects of different LCA and sodium dextran sulfate concentrations in the sample diluents on reactivity were compared, and the results are shown in Table 2.

[0093] Table 2. Effects of different LCA and sodium dextran sulfate concentrations in sample dilutions on reactivity.

[0094]

[0095] As shown in Table 2, the luminescence value of the AFP-L3 100 ng / mL sample gradually increased with the increase of LCA concentration in the sample diluent, reaching a plateau in the range of 5–20 μg / mL. The luminescence value decreased when the concentration exceeded 20 μg / mL, indicating that excessively high LCA concentrations affected the binding with AFP-L3. Simultaneously, it was found that the luminescence value of the AFP-L3 100 ng / mL sample was higher when the sodium dextran sulfate concentration in the sample diluent was between 1.25% and 5% (w / v). Concentrations exceeding this range decreased the luminescence value. Therefore, the optimal concentrations of LCA and sodium dextran sulfate in the sample diluent were determined to be 5–20 μg / mL and 1.25%–5% (w / v).

[0096] Example 5: Performance Evaluation of the AFP-L3 Alpha-Fetoprotein Isoform Detection Kit

[0097] Linear range:

[0098] The method described in Example 2 (wherein the sample processing agent was 0.25% (w / v) SDS, and the sample diluent included 10 μg / mL LCA and 2.5% (w / v) sodium dextran sulfate) was used to detect the alpha-fetoprotein isoform AFP-L3 calibrator, and the standard curve was plotted as shown below. Figure 1 As shown. Linearity analysis was performed on calibrators with concentrations of 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL, and the linear correlation coefficient r was calculated. 2 =0.9989. The linear range of this kit for detecting alpha-fetoprotein isoform AFP-L3 samples is 0.5–1000 ng / mL. The lower limit of the linear range is the blank limit of 0.12 ng / mL, and the upper limit is the highest value of the standard of 1000 ng / mL. Specific data sources are available for both blank limit detection and linear correlation detection.

[0099] Blank limit detection:

[0100] The calibrator dilution solution was used as a sample for testing. The test was repeated 20 times to obtain the RLU value (relative luminescence value) of the 20 measurements. The mean (M) and standard deviation (SD) of the RLU value were calculated to obtain M+2SD. The RLU value of M+2SD was substituted into the calibration curve equation to obtain the corresponding concentration value, which is the blank limit.

[0101] Table 3. AFP-L3 Blank Limit Test Data

[0102] 1 4288 2 4210 3 4970 4 4851 5 5110 6 4247 7 4652 8 4489 9 5355 10 5212 11 4412 12 5041 13 4930 14 4365 15 5201 16 4791 17 4852 18 5049 19 5291 20 4141 Measurement of mean AV 4773 Standard deviation SD 393 AV+2SD 5560 Blank limit (ng / mL) 0.12

[0103] As shown in Table 3, the blank limit of the AFP-L3 alpha-fetoprotein isoform detection kit was calculated, and the blank limit was found to be 0.12 ng / mL.

[0104] Repeatability testing:

[0105] Ten tests were performed on each of the two alpha-fetoprotein isoforms (AFP-L3) at concentrations of 10 ng / mL and 100 ng / mL, and the repeatability of the kit was calculated.

[0106] Table 4 AFP-L3 Repeatability Test Data

[0107]

[0108]

[0109] As shown in Table 4, the repeatability of the assay for 10 ng / mL AFP-L3 samples was 2.09%, and the repeatability of the assay for 100 ng / mL AFP-L3 samples was 1.68%, indicating that the repeatability of the kit was less than 10%, which is good.

[0110] Example 6: Clinical Validation of the AFP-L3 Alpha-Fetoprotein Isoform Detection Kit

[0111] To determine the consistency between the detection of liver cancer using the kit of this invention and clinical detection in liver cancer patients, 168 clinical serum samples were collected, including 63 liver cancer samples, 54 normal human serum samples, 11 cirrhosis samples, and 40 hepatitis serum samples. The clinical diagnostic results were evaluated in parallel using the kit of this invention and the commercially available AFP-L3 detection reagent. The positive rates are shown in Table 5.

[0112] Table 5. Comparison of clinical diagnostic results between the reagent kit of this invention and the commercially available AFP-L3 reagent kit.

[0113]

[0114] The verification results are shown in Table 5. The positive rate of the kit of the present invention in detecting primary liver cancer was 74.6%, the positive rate of liver cirrhosis was 18.18%, and the positive rate of hepatitis was 25%. This shows that the kit of the present invention can effectively differentiate liver cancer and distinguish between benign and malignant liver diseases, and has good clinical applicability.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A reagent combination, characterized in that, Includes sample processing reagents and sample diluents: The sample processing agent comprises: 0.06~1% (w / v) SDS and buffer B; The sample diluent comprises: 1.25–40 μg / mL LCA, 0.6–10% (w / v) sodium dextran sulfate and buffer B; The buffer solution B comprises: 0.15 mol / L Tris-NaCl, 1‰ (w / v) Proclin 300, 1 mmol / L CaCl2, 1 mmol / L MnCl2, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100; The reagent combination also includes a magnetic microparticle suspension; The magnetic microparticle suspension comprises activated JSR carboxyl magnetic beads, LCA monoclonal antibody, and sealing solution; The sealing solution comprises: 0.05 mol / L Tris-NaCl buffer, 1‰ (w / v) Proclin 300, 1% (w / v) bovine serum albumin, 5% (w / v) sucrose and 0.5‰ (w / v) Tritonx-100; The preparation method of activated JSR carboxyl magnetic beads in the magnetic microparticle suspension includes: washing JSR carboxyl magnetic beads with PBS buffer, activating the magnetic beads with 25 mg / mL LEDC and 25 mg / mL NHS for 1~1.5 h to obtain the activated JSR carboxyl magnetic beads. The preparation method of activated JSR carboxyl magnetic beads in the magnetic microparticle suspension includes: washing JSR carboxyl magnetic beads with PBS buffer, activating the magnetic beads with 25 mg / mL LEDC and 25 mg / mL NHS for 1~1.5 h to obtain the activated JSR carboxyl magnetic beads. The preparation method of activated JSR carboxyl magnetic beads in the magnetic microparticle suspension includes: washing JSR carboxyl magnetic beads with PBS buffer, activating the magnetic beads with 25 mg / mL LEDC and 25 mg / mL NHS for 1~1.5 h to obtain the activated JSR carboxyl magnetic beads. The reagent combination also includes an enzyme conjugate reaction solution; The enzyme conjugate reaction solution includes an HRP-labeled AFP-L3 monoclonal antibody Fab fragment and buffer A; The buffer A comprises: 0.02 mol / L PBS, 1‰ (w / v) Proclin 300, 1‰ (w / v) casein, 1% (w / v) bovine serum albumin and 5‰ (w / v) Tritonx-100.

2. The reagent combination as described in claim 1, characterized in that, The sample processing agent comprises: 0.125~0.5% (w / v) SDS and the buffer solution B; The sample diluent comprises: 5-20 μg / mL LCA, 1-5% (w / v) sodium dextran sulfate and buffer B.

3. The reagent combination as described in claim 2, characterized in that, The sample processing agent comprises: 0.25% (w / v) SDS and the buffer B; The sample diluent comprises: 10 μg / mL LCA, 2.5% (w / v) sodium dextran sulfate, and buffer B.

4. The reagent combination according to any one of claims 1 to 3, characterized in that, It also includes calibrators; The calibrators include AFP-L3 antigen, 0.05 mol / L Tris-NaCl, 1‰ (w / v) Proclin 300 and 1% (w / v) bovine serum albumin; The concentration of the AFP-L3 antigen is 0~1000 ng / mL.

5. The use of the reagent combination as described in any one of claims 1 to 4 in the preparation of a kit for detecting liver cancer.

6. The application as described in claim 5, characterized in that, The detection of liver cancer includes the detection of AFP-L3.

7. A reagent kit, characterized in that, It includes the reagent combination as described in any one of claims 1 to 4, as well as acceptable excipients or adjuvants.

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