Chemiluminescence detection reagent for detecting HCV antibody and use method thereof

Abstract

The invention discloses a chemiluminiscence detection reagent for detecting an HCV antibody and a use method of the chemiluminiscence detection reagent, and relates to the technical field of immunodetection. The detection reagent is combined with a sandwich method and an indirect method for detection. When in use, a sample is incubated with the first reagent and the sample treating fluid, so that the HCV antibody to be detected in the sample reacts with the HCV coating antigen in the first reagent to form an antigen-antibody compound; adding a second reagent, and reacting the formed antigen-antibody compound with the HCV labeled antigen labeled by acridinium ester and the anti-IgG antibody labeled by acridinium ester in the second reagent to form an antigen-antibody-antigen compound and an antigen-antibody-secondary antibody compound; and finally, adding excitation liquid to enable acridinium ester to emit light, and detecting the HCV antibody in the sample by detecting a light-emitting signal value. The technical scheme of the invention greatly improves the sensitivity and specificity of HCV antibody detection, and can be used for solving the problem of easy missing detection in HCV antibody detection.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, and in particular to a chemiluminescent detection reagent for detecting HCV antibodies and its method of use. Background Technology

[0002] Hepatitis C virus (HCV) is a small-enveloped, single-stranded, positive-sense RNA virus primarily transmitted through blood. It belongs to the Flaviviridae family and the Hepatitis Virus genus. A small percentage (15-50%) of people infected with HCV will clear the virus, while the majority (50-85%) will develop chronic infection. Chronic HCV infection can lead to chronic inflammation, necrosis, and fibrosis of the liver; some patients may eventually develop cirrhosis or even hepatocellular carcinoma (HCC). Currently, there is no effective vaccine for hepatitis C.

[0003] HCV can be transmitted through blood, sexual contact, mother-to-child transmission, and skin lesions. After infection, HCV RNA can generally be detected in peripheral blood within 1-3 weeks, with the core antigen usually appearing almost simultaneously with the RNA. HCV antibodies typically appear 50-70 days after infection, although in some patients, they may not be detectable until 6-9 months later. Furthermore, because some individuals automatically clear HCV after infection, some patients may test positive for HCV antibodies but negative for nucleic acid testing. HCV genotypes 1b and 2a are relatively common in my country, with genotype 1b being the predominant type. Currently, HCV antigen testing primarily focuses on detecting the core antigen. While this method can shorten the window period, the core antigen is present in very low concentrations in serum and has a short duration. Moreover, HCV Ag testing may have reduced sensitivity due to mutations in the HCV core region; therefore, HCV core antigen testing must be combined with HCV antibody testing results for a more accurate diagnosis of HCV.

[0004] Currently, HCV antibody testing mainly uses anti-core antigen antibodies, anti-NS3 antibodies, and anti-NS4 antibodies as the main detection markers. However, HCV antibody testing has always had the problem of false negatives, making it difficult to meet the requirements of clinical testing. Summary of the Invention

[0005] The main objective of this invention is to propose a chemiluminescent detection reagent for detecting HCV antibodies and its method of use, aiming to solve the problem of missed detection in existing HCV antibody detection methods.

[0006] To achieve the above objectives, this invention proposes a chemiluminescent detection reagent for detecting HCV antibodies, comprising: The first reagent includes HCV-coated antigen and first diluent; The second reagent includes acridinium ester-labeled HCV-labeled antigen, acridinium ester-labeled anti-IgG antibody, and a second dilution solution; and, Sample processing solution.

[0007] In one embodiment, the first reagent contains: The HCV-coated antigen contains HCV core, NS3, and NS4 fragments; and / or, The HCV-coated antigen comprises a solid-phase carrier and antigen coated on the solid-phase carrier, wherein the solid-phase carrier is a carboxyl magnetic bead with a particle size of 3-5 μm; and / or, The first diluent comprises a mixture of 50 mM Tris-HCl buffer, 9 g / L NaCl, 0.10% (w / v) Proclin 300, 0.50% (w / v) sodium casein and 4.0% (w / v) glycine.

[0008] In one embodiment, the solid support is a carboxyl magnetic bead, and the first reagent is prepared according to the following steps: A1. Provide carboxyl magnetic beads and HCV antigen for coating. Wash and activate the carboxyl magnetic beads sequentially to obtain activated magnetic beads. Mix the activated magnetic beads and HCV antigen for coating, and perform cross-linking reaction and blocking treatment sequentially to obtain HCV coated antigen. A2. Dilute the HCV-coated antigen with the first diluent to obtain the first reagent.

[0009] In one embodiment, in step A1, the mass ratio of the activated magnetic beads to the HCV antigen coating is 1000:(10~30); and / or, In step A1, the crosslinking reaction is carried out at a temperature of 37°C for 1.5 to 2.5 hours; and / or, In step A2, the volume ratio of the first diluent to the HCV-coated antigen is (40~60):1.

[0010] In one embodiment, the second reagent contains: The acridinium ester-labeled HCV-labeled antigen contains HCV core, NS3, and NS4 fragments; and / or, The second diluent comprises a mixture of 55 mM MES buffer, 9 g / L NaCl, 20 g / L bovine serum albumin, 10 g / L sodium casein, 10 g / L Tween 20, 1 g / L Proclin 300, and 1 mL / L BND.

[0011] In one embodiment, the second reagent is prepared according to the following steps: B1. Provide HCV antigen for labeling and anti-IgG antibody. Desalt and purify the HCV antigen for labeling and anti-IgG antibody separately to obtain desalted HCV antigen for labeling and desalted anti-IgG antibody. B2. The desalted HCV antigen and the desalted antiIgG antibody were mixed with acridinium ester solution, and then labeled, blocked and purified in sequence to obtain acridinium ester labeled HCV antigen and acridinium ester labeled antiIgG antibody. B3. The acridine ester-labeled HCV-labeled antigen, the acridine ester-labeled anti-IgG antibody, and the second dilution solution are mixed to obtain the second reagent.

[0012] In one embodiment, in step B2, the molar ratio of the desalted HCV antigen for labeling to the acridinium ester solution is (0.5~2):10; and / or, In step B2, the molar ratio of the desalted anti-IgG antibody to the acridinium ester solution is (0.5~2):10; and / or, In step B2, the labeling reaction is carried out at room temperature for a duration of 45 to 90 minutes.

[0013] In one embodiment, in step B3, the volume ratio of the acridine ester-labeled HCV-labeled antigen to the second dilution is 1:10000, and the volume ratio of the acridine ester-labeled anti-IgG antibody to the second dilution is 1:40000.

[0014] In one embodiment, the sample processing solution comprises a mixture of 55 mM MES buffer, 9 g / L NaCl, 10 g / L bovine serum albumin, 20 g / L sodium caseinate, 10 g / L Tween 20, 1 g / L Proclin 300, and 1 mL / L BND.

[0015] This invention proposes a method for using the detection reagent as described in the foregoing technical solution, comprising the following steps: S10. Obtain the sample to be tested; S20. Mix the sample to be tested, the first reagent and the sample processing solution, incubate at room temperature for 10 min, and then wash to obtain the first reactant. S30. Add the second reagent to the first reactant, continue incubation at room temperature for 10 min, and then perform a washing process to obtain the second reactant; S40. Add chemiluminescent excitation solution to the second reactant and perform detection; The volume ratio of the sample to be tested, the first reagent, the second reagent, and the sample processing solution is 25:50:100:50.

[0016] The present invention provides a chemiluminescent detection reagent for detecting HCV antibodies, comprising a first reagent, a second reagent, and a sample processing solution, wherein the first reagent comprises HCV-coated antigen and a first diluent, and the second reagent comprises acridinium ester-labeled HCV-labeled antigen, acridinium ester-labeled anti-IgG antibody, and a second diluent. During detection, the sample to be tested is first mixed with the first reagent and sample processing solution and incubated. This allows the HCV antibody in the sample to react with the HCV-coated antigen in the first reagent, forming an antigen-antibody complex. Then, the second reagent is added and incubated again, allowing the formed antigen-antibody complex to react with the acridil ester-labeled HCV-labeled antigen and the acridil ester-labeled anti-IgG antibody secondary antibody in the second reagent, forming antigen-antibody-antigen complexes and antigen-antibody-secondary antibody complexes. Next, a chemiluminescent excitation solution is added. This excitation solution decomposes and emits light under alkaline conditions. The number of photons produced is proportional to the concentration of Anti-HCV in the sample. By detecting the luminescence signal value of the sample, the HCV antibody in the sample can be detected. This invention combines the sandwich method with an indirect method, improving the sensitivity and specificity of HCV antibody detection compared to conventional sandwich methods, and can address the problem of missed detections in HCV antibody testing. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0019] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0020] HCV antigen testing primarily detects the core antigen. While this method can shorten the window period, the core antigen is present in very low concentrations in serum and has a short duration. Furthermore, HCV Ag testing may have reduced sensitivity due to mutations in the HCV core region. Therefore, HCV core antigen testing must be combined with HCV antibody testing results for a more accurate diagnosis of HCV.

[0021] Studies have shown that NS3 has strong antigenicity and immunogenicity, making it one of the main antigens for detecting HCV infection. In addition, non-structural proteins NS4 and NS5 also have strong immunogenicity. To enhance antibody detection sensitivity, anti-NS4 and / or NS5 antibodies are often used as targets for HCV antibody detection. However, with prolonged chronic HCV infection (follow-up monitoring for 10 years), antibodies against non-structural proteins such as NS4 and NS5 disappear in about half of the patients, but antibodies against the core proteins remain positive in all patients, although their titers decrease year by year.

[0022] Currently, HCV antibody testing primarily uses anti-core antigen antibodies, anti-NS3 antibodies, and anti-NS4 antibodies as the main detection markers. Some manufacturers also include anti-NS5 antibodies as one of the HCV antibody detection markers to improve detection sensitivity. However, studies have shown that adding NS5 antigen peptides does not improve the detection sensitivity of the reagents and may even cause false positives. Therefore, HCV antibody testing still faces the problem of false negatives and fails to meet the requirements of clinical testing.

[0023] Based on the above background, the present invention proposes a chemiluminescent detection reagent for detecting HCV antibodies, comprising: The first reagent includes HCV-coated antigen and first diluent; The second reagent includes acridinium ester-labeled HCV-labeled antigen, acridinium ester-labeled anti-IgG antibody, and a second dilution solution; and, Sample processing solution.

[0024] The present invention provides a chemiluminescent detection reagent for detecting HCV antibodies. The chemiluminescent detection reagent for detecting HCV antibodies includes a first reagent, a second reagent, and a sample processing solution. The first reagent includes HCV-coated antigen and a first diluent. The second reagent includes acridine ester-labeled HCV-labeled antigen, acridine ester-labeled anti-IgG antibody, and a second diluent. During testing, the sample to be tested is mixed with the first reagent and sample processing solution and incubated. This allows the HCV antibody in the sample to react with the HCV-coated antigen in the first reagent, forming an antigen-antibody complex. Then, the second reagent is added and incubated again, allowing the formed antigen-antibody complex to react with the acridil ester-labeled HCV-labeled antigen and the acridil ester-labeled anti-IgG antibody secondary antibody in the second reagent, forming antigen-antibody-antigen complexes and antigen-antibody-secondary antibody complexes. Next, a chemiluminescent excitation solution is added. This excitation solution decomposes and emits light under alkaline conditions. The number of photons produced is proportional to the concentration of Anti-HCV in the sample. By detecting the luminescence signal value of the sample, the HCV antibody in the sample can be detected. This invention combines the sandwich method with an indirect method, improving the sensitivity and specificity of HCV antibody detection compared to conventional sandwich methods, and can address the problem of missed detections in HCV antibody testing.

[0025] In an embodiment of the present invention, the HCV coating antigen in the first reagent contains HCV core, NS3, and NS4 fragments. Using an antigen containing HCV core, NS3, and NS4 fragments simultaneously as the coating antigen is beneficial for improving the sensitivity and specificity of HCV antibody detection, thereby reducing the probability of false negatives.

[0026] In an embodiment of the present invention, the first reagent comprises an HCV-coated antigen including a solid-phase support and antigen coated on the solid-phase support, wherein the solid-phase support is a carboxyl magnetic bead with a particle size of 3-5 μm. Magnetic beads typically have a large specific surface area, providing more binding sites, thereby improving the binding efficiency of antigen or antibody. Furthermore, magnetic beads can be rapidly separated from the reaction medium by an external magnetic field, facilitating subsequent washing steps, reducing non-specific binding, and improving the specificity and sensitivity of the detection. The technical solution of the present invention uses carboxyl magnetic beads with a particle size of 3-5 μm as the solid-phase support, ensuring sufficient specific surface area while avoiding the operational difficulties that may arise from excessively small particle sizes.

[0027] In embodiments of the present invention, the first diluent comprises a mixture of 50 mM Tris-HCl buffer, 9 g / L NaCl, 0.10% (w / v) Proclin 300, 0.50% sodium casein (w / v), and 4.0% glycine (w / v). The Tris-HCl buffer is used to maintain a stable pH of 7.4-8.0, ensuring the HCV antigen remains stable and preventing pH fluctuations from causing conformational changes or magnetic bead aggregation. NaCl provides ionic strength. Proclin 300, as a broad-spectrum preservative, prolongs the reagent's stability. Sodium casein blocks the remaining binding sites of the magnetic beads, reducing the false positive rate. Glycine, as a competitive protectant, competitively binds to the hydrophobic regions of the antigen using its amino / carboxyl groups, preventing the antigen from adsorbing onto the magnetic bead surface due to hydrophobic interactions and thus masking the active sites. The simultaneous use of casein and glycine forms a "double-blocking" system, further improving the stability of the magnetic beads in the Tris-HCl buffer. The technical solution of this invention uses the above-mentioned components to form the first diluent, which is beneficial to improving the coating efficiency and stability of HCV antigen, thereby improving the signal-to-noise ratio of HCV antibody detection.

[0028] In an embodiment of the present invention, the solid support is a carboxyl magnetic bead, and the first reagent is prepared according to the following steps: A1. Provide carboxyl magnetic beads and HCV antigen for coating. Wash and activate the carboxyl magnetic beads sequentially to obtain activated magnetic beads. Mix the activated magnetic beads and HCV antigen for coating, and perform cross-linking reaction and blocking treatment sequentially to obtain HCV coated antigen. A2. Dilute the HCV-coated antigen with the first diluent to obtain the first reagent.

[0029] The technical solution of this invention uses a conventional preparation process to prepare the first reagent. First, the magnetic beads are pretreated by cleaning and activation. Then, the pretreated magnetic beads and the HCV antigen to be coated are mixed and subjected to a cross-linking reaction to form a linker, thus obtaining the HCV-coated antigen. Subsequently, the HCV-coated antigen is diluted with a first diluent to obtain the first reagent.

[0030] In an embodiment of the present invention, in step A1, the mass ratio of the activated magnetic beads to the HCV antigen used for coating is 1000:(10~30). Exemplarily, the mass ratio of the activated magnetic beads to the HCV antigen used for coating can be 1000:10, 1000:20, or 1000:30. Too little antigen will result in insufficient utilization of the magnetic bead surface, leaving numerous unbound sites, leading to increased subsequent non-specific adsorption (such as binding to contaminating proteins in the sample), increased background, and decreased ability to capture the target antibody, thus reducing sensitivity. Too much antigen increases reagent costs without providing performance gains. The technical solution of the present invention, by controlling the amount of activated magnetic beads and HCV antigen used for coating within the above range, helps ensure sufficient antigen coating, improves detection sensitivity, specificity, and stability, while controlling costs.

[0031] In an embodiment of the present invention, in step A1, the temperature of the crosslinking reaction is 37°C, and the time of the crosslinking reaction is 1.5~2.5h. Exemplarily, the time of the crosslinking reaction can be 1.5h, 2h, or 2.5h.

[0032] In an embodiment of the present invention, in step A2, the volume ratio of the first diluent to the HCV-coated antigen is (40~60):1. Exemplarily, the volume ratio of the first diluent to the HCV-coated antigen can be 40:1, 50:1, or 60:1. The amount of the first diluent affects the final concentration (i.e., working concentration) of the HCV-coated antigen, directly impacting the capture efficiency and signal strength of the detection system. If the antigen concentration is too low, or if the amount of antigen coated on the magnetic beads is insufficient, it will be difficult to effectively capture the HCV antibody in the sample, resulting in a weak signal and decreased sensitivity. If the antigen concentration is too high, it may cause steric hindrance, hindering antibody binding, and may also lead to increased non-specific adsorption, causing a "hook effect." The technical solution of the present invention sets the amounts of the first diluent and the HCV-coated antigen within the above range, which helps to reduce the hook effect and optimize sensitivity and signal-to-noise ratio.

[0033] In an embodiment of the present invention, the acridil ester-labeled HCV-labeled antigen in the second reagent contains HCV core, NS3, and NS4 fragments. Using an antigen containing HCV core, NS3, and NS4 fragments simultaneously as the labeling antigen is beneficial for improving the sensitivity and specificity of HCV antibody detection, thereby reducing the probability of false negatives.

[0034] In an embodiment of the present invention, the second diluent comprises a mixture of 55 mM MES buffer, 9 g / L NaCl, 20 g / L bovine serum albumin (BSA), 10 g / L sodium casein, 10 g / L Tween 20, 1 g / L Proclin 300, and 1 mL / L BND. The MES buffer provides a suitable pH environment to reduce the hydrolysis of acridine esters and also provides low ionic strength. NaCl is used to maintain physiological ionic strength. BSA is used to reduce interference from heterophilic antibodies (HAMA, RF) and nonspecific IgG, and also serves as a protein reservoir to dilute locally high concentrations of the marker generated instantaneously, preventing signal degradation due to self-quenching of acridine esters. Casein carries a negative charge under slightly acidic conditions, which can increase colloidal stability and inhibit marker aggregation. Simultaneously, casein and BSA form a "double-blocking" effect, which helps reduce nonspecific adsorption and improves the sensitivity and accuracy of detection. Tween 20 is a nonionic surfactant that dissolves chylomicrons in lipemia samples, eliminating scattering interference caused by lipid turbidity; it also improves the luminescence efficiency of free acridine esters by blocking hydrophobic-hydrophobic interactions. Proclin 300 and BND are used as antibacterial agents to improve reagent stability.

[0035] In an embodiment of the present invention, the second reagent is prepared according to the following steps: B1. Provide HCV antigen for labeling and anti-IgG antibody. Desalt and purify the HCV antigen for labeling and anti-IgG antibody separately to obtain desalted HCV antigen for labeling and desalted anti-IgG antibody. B2. The desalted HCV antigen and the desalted antiIgG antibody were mixed with acridinium ester solution, and then labeled, blocked and purified in sequence to obtain acridinium ester labeled HCV antigen and acridinium ester labeled antiIgG antibody. B3. The acridine ester-labeled HCV-labeled antigen, the acridine ester-labeled anti-IgG antibody, and the second dilution solution are mixed to obtain the second reagent.

[0036] The technical solution of this invention employs a conventional preparation process to prepare the second reagent. First, the HCV antigen and anti-IgG antibody are desalted. Then, the desalted HCV antigen and anti-IgG antibody are reacted with an acridil ester solution to form conjugates: acridil ester-labeled HCV antigen and acridil ester-labeled anti-IgG antibody. Finally, the acridil ester-labeled HCV antigen and acridil ester-labeled anti-IgG antibody are mixed with a second diluent to obtain the second reagent.

[0037] It should be noted that, since free amines compete with acridine esters for the reaction, consuming labeling reagents and reducing labeling efficiency, the HCV antigen and anti-IgG antibody used for labeling are desalted to remove small molecule amines and ensure that acridine esters react specifically with the antibody / antigen.

[0038] In an embodiment of the present invention, in step B2, the molar ratio of the desalted HCV antigen for labeling to the acridine ester solution is (0.5~2):10. In the technical solution of the present invention, the acridine ester solution used is a solution formed by mixing acridine ester and N,N-dimethylformamide (DMF). Setting the amounts of the desalted HCV antigen for labeling and the acridine ester solution within the above range allows each HCV antigen molecule to be linked with an appropriate amount of acridine ester molecules, while the key antigenic epitopes of the antigen are not destroyed, allowing for effective binding of HCV antibodies. This results in a labeled product with high luminescence efficiency and stability, obtaining a sufficient luminescent signal, and retaining antigenic immunogenicity.

[0039] In an embodiment of the present invention, in step B2, the molar ratio of the desalted anti-IgG antibody to the acridine ester solution is (0.5~2):10. In the technical solution of the present invention, the acridine ester solution used is a solution formed by mixing acridine ester and N,N-dimethylformamide (DMF). If too much acridine ester solution is used, over-labeling may occur, potentially leading to steric hindrance or conformational changes in the anti-IgG antibody, reducing its affinity for the antigen; moreover, excessive acridine ester is prone to non-specific aggregation under hydrophobic interaction, affecting the uniformity and repeatability of the reagent. If too little acridine ester solution is used, weak luminescent signal and insufficient sensitivity will occur, resulting in low reagent utilization and poor cost-effectiveness. The technical solution of this invention sets the amounts of the desalted anti-IgG antibody and acridine ester solution within the above-mentioned range, which allows each anti-IgG antibody molecule to be linked with an appropriate amount of acridine ester molecule, while ensuring that the labeled antibody can still effectively recognize and bind to the target IgG, thus ensuring the sensitivity, specificity and stability of the final detection system, while controlling costs.

[0040] In an embodiment of the present invention, in step B2, the labeling reaction is carried out at room temperature, and the labeling reaction time is 45-90 minutes. Exemplarily, the labeling reaction time can be 45 minutes, 60 minutes, or 90 minutes.

[0041] In an embodiment of the present invention, in step B3, the volume ratio of the acridine ester-labeled HCV-labeled antigen to the second diluent is 1:10000, and the volume ratio of the acridine ester-labeled anti-IgG antibody to the second diluent is 1:40000. The present invention sets the amounts of the second diluent, the acridine ester-labeled HCV-labeled antigen, and the acridine ester-labeled anti-IgG antibody within the above-mentioned ranges, which helps to reduce the hook effect and optimize sensitivity and signal-to-noise ratio.

[0042] In an embodiment of the present invention, the sample processing solution comprises a mixture of 55 mM MES buffer, 9 g / L NaCl, 10 g / L bovine serum albumin, 20 g / L sodium caseinate, 10 g / L Tween 20, 1 g / L Proclin 300, and 1 mL / L BND.

[0043] This invention proposes a method for using a chemiluminescent detection reagent for detecting HCV antibodies, comprising the following steps: S10. Obtain the sample to be tested; S20. Mix the sample to be tested, the first reagent and the sample processing solution, incubate at room temperature for 10 min, and then wash to obtain the first reactant. S30. Add the second reagent to the first reactant, continue incubation at room temperature for 10 min, and then perform a washing process to obtain the second reactant; S40. Add chemiluminescent excitation solution to the second reactant and perform detection; The volume ratio of the sample to be tested, the first reagent, the second reagent, and the sample processing solution is 25:50:100:50.

[0044] During testing, the sample to be tested is mixed with the first reagent and sample processing solution and incubated first, allowing the HCV antibody in the sample to react with the HCV-coated antigen in the first reagent, forming an antigen-antibody complex. Then, the second reagent is added and incubated again, allowing the formed antigen-antibody complex to react with the acridil ester-labeled HCV-labeled antigen and the acridil ester-labeled anti-IgG antibody secondary antibody in the second reagent, forming antigen-antibody-antigen complexes and antigen-antibody-secondary antibody complexes. Finally, a chemiluminescent excitation solution is added to the above reaction system. The excitation solution allows the acridil ester to decompose and emit light in an alkaline environment. Since the number of photons generated is proportional to the concentration of Anti-HCV in the sample, the HCV antibody in the sample can be detected by detecting the luminescence signal value of the sample.

[0045] In an embodiment of the present invention, the chemiluminescent excitation solution is a mixture of sodium hydroxide and H2O2.

[0046] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0047] Experimental materials Carboxylated magnetic beads: particle size 3μm, solid content 10mg / mL, purchased from Merck; HCV antigen used for coating: purchased from Phytobio Biotechnology Co., Ltd., containing recombinant HCV core antigen and NS3 and NS4 antigens; HCV antigen for labeling: purchased from Phytobio Biotechnology Co., Ltd., containing recombinant HCV core, NS3, and NS4 antigens; Anti-human IgG antibody: purchased from Phytobio Biotechnology Co., Ltd., goat-derived, IgG purity ≥98%; Acridine ester: purchased from Mektar Corporation; when using, prepare a 10 mM acridine ester-DMF solution with anhydrous DMF before use.

[0048] Example 1 This embodiment provides a method for preparing a first reagent, specifically including the following steps: 1) Preparation of HCV coated antigen (magnetic bead coating stock solution): ① Magnetic bead washing: Take 1 mL of carboxylated magnetic bead suspension (containing 10 mg of magnetic beads), place it on a magnetic rack to separate the magnetic beads, and discard the supernatant; add 1 mL of PBST (PBS containing 0.05% Tween-20) to resuspend, mix well, and then perform magnetic separation again. Repeat the washing process 3 times.

[0049] ② Activation of magnetic beads: Resuspend the cleaned magnetic beads in 0.5 mL of MES buffer (0.1 M, pH 5.0), add 25 μL of EDC solution (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, final concentration 2.5 mg / mL), and shake for 15 min at room temperature.

[0050] ③ Magnetic bead crosslinking: Add 200 μL of HCV antigen solution for coating (containing 200 μg of antigen, i.e., magnetic beads: antigen = 1 mg: 20 μg) to the activated magnetic bead suspension, and make up to 1 mL with MES buffer. Incubate at room temperature in the dark with shaking for 2 h.

[0051] ④ Magnetic bead blocking: After the reaction, the supernatant was discarded by magnetic separation, and 1 mL of 1 mol / L ethanolamine solution was added. The mixture was then shaken at room temperature for 30 min to block the bead coating. After blocking, the magnetic beads were washed three times with PBST and finally resuspended in 1 mL of storage buffer (PBS containing 1% BSA and 0.1% Proclin 300, pH 7.4) to obtain the original magnetic bead coating solution. This solution was stored at 2-8℃ in the dark for later use.

[0052] 2) Preparation of the first reagent (magnetic bead working solution): ① Preparation of the first diluent: 1.15g Tris, 6.37g Tris HCl, 9g NaCl, 1g Proclin 300, 5g sodium caseinate and 40g glycine, add water to make up to 1L.

[0053] ② Take the original solution of magnetic bead coating prepared in step 1) and dilute it with the first diluent at a volume ratio of 1:50 to obtain the first reagent.

[0054] Example 2 This embodiment provides a method for preparing a second reagent, specifically including the following steps: 1) Preparation of acridine ester-labeled HCV-labeled antigen stock solution: ① Antigen desalting: The HCV antigen-labeled solution was loaded onto a pre-equilibrated PD-10 desalting column, eluted with PBS, and the elution peak containing the antigen was collected. The protein concentration was then measured and adjusted to 1.0 mg / mL.

[0055] ② Labeling reaction: Mix the desalted antigen with 5 μL of 10 mM acridinium ester-DMF solution (final molar ratio, acridinium ester stock solution: antigen = 10:1) and react at room temperature in the dark for 60 min.

[0056] ③ Blocking unreacted groups: Add 50 μL of 1M glycine solution and continue the reaction for 15 min to block the residual active ester.

[0057] ④ Label desalting and purification: Load the blocked reaction solution onto another PD-10 desalting column, elute with PBS, collect the elution peak of the labeled antigen, and obtain the acridinium ester-labeled HCV antigen stock solution, which is stored at 2~8℃.

[0058] 2) Preparation of acridinium ester-labeled anti-human IgG antibody stock solution: ① Antibody desalting: Load anti-human IgG antibody onto a pre-equilibrated PD-10 desalting column, elute with PBS, and collect the elution peak containing antibody.

[0059] ② Labeling reaction: Mix the desalted anti-human IgG antibody with 4 μL of 10 mM acridinium ester-DMF solution (final molar ratio, acridinium ester stock solution: antibody = 10:1) and react at room temperature in the dark for 60 min.

[0060] ③ Blocking unreacted groups: Add 40 μL of 1M glycine solution and continue the reaction for 15 min to block the residual active ester.

[0061] ④ Label desalting and purification: Load the blocked reaction solution onto another PD-10 desalting column, elute with PBS, collect the elution peak of the labeled antibody, and obtain the stock solution of acridine ester-labeled anti-human IgG antibody, which is stored at 2~8℃.

[0062] 3) Preparation of the second reagent: ① Second dilution preparation (1L): 10.66g MES H2O, 9g NaCl, 20g bovine serum albumin, 10g sodium caseinate, 10g Tween20, 1g Proclin300 and 1mL BND, add water to make up to 1L.

[0063] ② Take the acridine ester-labeled HCV-labeled antigen stock solution prepared in step 1), the acridine ester-labeled anti-human IgG antibody stock solution prepared in step 2), and the second dilution solution, and mix them to obtain the second reagent; The composition is as follows: the volume ratio of the acridinium ester-labeled HCV antigen stock solution to the second dilution is 1:10000, and the volume ratio of the acridinium ester-labeled anti-IgG antibody stock solution to the second dilution is 1:40000.

[0064] Example 3 This embodiment provides a chemiluminescent detection reagent for detecting HCV antibodies. The chemiluminescent detection reagent for detecting HCV antibodies includes a first reagent from Example 1, a second reagent from Example 2, and a sample processing solution. The first reagent includes HCV-coated antigen and a first diluent. The second reagent consists of acridinium-labeled HCV-labeled antigen, acridinium-labeled anti-IgG antibody, and a second diluent. The sample processing solution is prepared according to the following steps: 10.66g MESH2O, 9g NaCl, 10g bovine serum albumin, 20g sodium caseinate, 10g Tween20, 1g Proclin300, and 1mL BND, diluted to 1L with water.

[0065] Example 4 A chemiluminescent detection method for HCV antibodies, using the chemiluminescent detection reagent for detecting HCV antibodies described in Example 3, includes the following steps: 1) Add 25 μL of the sample to be tested to the reaction tube, add 50 μL of the first reagent (magnetic bead working solution) and 50 μL of the sample processing solution to the sample to be tested, and then incubate at 37°C for 10 min. After the reaction is completed, the magnetic field of the instrument's magnetic separation system attracts the magnetic beads, washes away the unbound substances, and obtains the first reactant. 2) Add 100 μL of the first reagent (acrididine ester working solution) to the first reactant obtained in step 1), and continue to incubate at 37°C for 10 min. After the reaction is completed, the magnetic field of the instrument's magnetic separation system attracts the magnetic beads, washes away the unbound substances, and obtains the second reactant. 3) Add chemiluminescent excitation solution (a mixture of NaOH and H2O2) to the second reactant and detect the luminescence signal value.

[0066] Comparative Example 1 This comparative example provides a method for detecting HCV antibodies using a sandwich method, specifically including the following steps: 1) Add 25 μL of the sample to be tested to the reaction tube, add 50 μL of the first reagent (magnetic bead working solution) and 50 μL of the third reagent (sample processing solution) to the sample to be tested, and then incubate at 37°C for 10 min. After the reaction is completed, the magnetic field of the instrument's magnetic separation system attracts the magnetic beads and washes away the unbound substances. 2) Add the chemiluminescent substrate to the reaction tube and detect the luminescence signal value.

[0067] Comparative Example 2 This comparative example provides a method for detecting HCV antibodies using an indirect method, specifically including the following steps: 1) Add 25 μL of the sample to be tested to the reaction tube, add 100 μL of the second reagent (acrididine ester working solution) and 50 μL of the third reagent (sample processing solution) to the sample to be tested, and then incubate at 37 °C for 10 min. After the reaction is completed, the magnetic field of the instrument's magnetic separation system attracts the magnetic beads and washes away the unbound substances. 2) Add the chemiluminescent substrate to the reaction tube and detect the luminescence signal value.

[0068] Performance testing SeraCare TM The hepatitis C serological conversion panel (catalog number: 22-554-537, batch number: 0610022) was used to detect HCV antibodies. The results are shown in Table 1.

[0069] Table 1. Detection results of sandwich method and sandwich method combined with indirect method.

[0070] The test results in Table 1 show that the combined sandwich method and indirect method (sandwich method + indirect method) can effectively detect the gray area of ​​the conversion disc and positive samples, while neither the sandwich method nor the indirect method alone can detect them. These results indicate that combining the sandwich method and indirect method can effectively solve the problem of missed detection of HCV antibodies, thereby improving the sensitivity and specificity of HCV antibody detection.

[0071] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A chemiluminescent detection reagent for detecting HCV antibodies, characterized in that, include: The first reagent includes HCV-coated antigen and first diluent; The second reagent includes acridinium ester-labeled HCV-labeled antigen, acridinium ester-labeled anti-IgG antibody, and a second dilution solution; and, Sample processing solution.

2. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 1, characterized in that, In the first reagent: The HCV-coated antigen contains HCV core, NS3, and NS4 fragments; and / or, The HCV-coated antigen comprises a solid-phase carrier and antigen coated on the solid-phase carrier, wherein the solid-phase carrier is a carboxyl magnetic bead with a particle size of 3-5 μm; and / or, The first diluent comprises a mixture of 50 mM Tris-HCl buffer, 9 g / L NaCl, 0.10% (w / v) Proclin 300, 0.50% (w / v) sodium casein and 4.0% (w / v) glycine.

3. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 2, characterized in that, The solid support is a carboxyl magnetic bead, and the first reagent is prepared according to the following steps: A1. Provide carboxyl magnetic beads and HCV antigen for coating. Wash and activate the carboxyl magnetic beads sequentially to obtain activated magnetic beads. Mix the activated magnetic beads and HCV antigen for coating, and perform cross-linking reaction and blocking treatment sequentially to obtain HCV coated antigen. A2. Dilute the HCV-coated antigen with the first diluent to obtain the first reagent.

4. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 3, characterized in that, In step A1, the mass ratio of the activated magnetic beads to the HCV antigen coating is 1000:(10~30); and / or, In step A1, the crosslinking reaction is carried out at a temperature of 37°C for 1.5 to 2.5 hours; and / or, In step A2, the volume ratio of the first diluent to the HCV-coated antigen is (40~60):

1.

5. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 1, characterized in that, In the second reagent: The acridinium ester-labeled HCV-labeled antigen contains HCV core, NS3, and NS4 fragments; and / or, The second diluent comprises a mixture of 55 mM MES buffer, 9 g / L NaCl, 20 g / L bovine serum albumin, 10 g / L sodium casein, 10 g / L Tween 20, 1 g / L Proclin 300, and 1 mL / L BND.

6. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 1, characterized in that, The second reagent is prepared according to the following steps: B1. Provide HCV antigen for labeling and anti-IgG antibody. Desalt and purify the HCV antigen for labeling and anti-IgG antibody separately to obtain desalted HCV antigen for labeling and desalted anti-IgG antibody. B2. The desalted HCV antigen and the desalted antiIgG antibody were mixed with acridinium ester solution, and then labeled, blocked and purified in sequence to obtain acridinium ester labeled HCV antigen and acridinium ester labeled antiIgG antibody. B3. The acridine ester-labeled HCV-labeled antigen, the acridine ester-labeled anti-IgG antibody, and the second dilution solution are mixed to obtain the second reagent.

7. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 6, characterized in that, In step B2, the molar ratio of the desalted HCV antigen and acrid ester solution for labeling is (0.5~2):10; and / or, In step B2, the molar ratio of the desalted anti-IgG antibody to the acridinium ester solution is (0.5~2):10; and / or, In step B2, the labeling reaction is carried out at room temperature for a duration of 45 to 90 minutes.

8. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 6, characterized in that, In step B3, the volume ratio of the acridine ester-labeled HCV-labeled antigen to the second dilution is 1:10000, and the volume ratio of the acridine ester-labeled anti-IgG antibody to the second dilution is 1:40000.

9. The chemiluminescent detection reagent for detecting HCV antibodies as described in claim 1, characterized in that, The sample processing solution comprises a mixture of 55 mM MES buffer, 9 g / L NaCl, 10 g / L bovine serum albumin, 20 g / L sodium casein, 10 g / L Tween 20, 1 g / L Proclin 300, and 1 mL / L BND.

10. A method of using a chemiluminescent detection reagent for detecting HCV antibodies as described in any one of claims 1 to 9, characterized in that, Includes the following steps: S10. Obtain the sample to be tested; S20. Mix the sample to be tested, the first reagent and the sample processing solution, incubate at room temperature for 10 min, and then wash to obtain the first reactant. S30. Add the second reagent to the first reactant, continue incubation at room temperature for 10 min, and then perform a washing process to obtain the second reactant; S40. Add chemiluminescent excitation solution to the second reactant and perform detection; The volume ratio of the sample to be tested, the first reagent, the second reagent, and the sample processing solution is 25:50:100:50.

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