Method for detecting abnormal prothrombin PIVKA-II

Abstract

The invention discloses a method for detecting abnormal prothrombin PIVKA-II, and belongs to the technical field of medicine and biologica.The method comprises the following steps that S1, a PIVKA-II antibody coated with luminous particles is prepared; s2, a biotin labeled PIVKA-II antibody is prepared; s3, preparing a PIVKA-II calibrator and establishing a standard curve; and S4, detecting. The detection method disclosed by the invention has the characteristics of high sensitivity, good specificity, strong stability and simplicity in operation.

Description

Technical Field

[0001] This invention relates to the fields of medicine and biotechnology, and more specifically, to a method for detecting abnormal prothrombin PIVKA-II. Background Technology

[0002] Abnormal prothrombin II (PIVKA-II) shows a specific elevation in hepatocellular carcinoma (HCC) cells. Using a cutoff value of PIVKA-II > 40 mAU / mL for diagnosing HCC, the sensitivity and specificity were 48.16% and 95.93%, respectively, with an overall accuracy as high as 71.6%. The positive rate of PIVKA-II in the serum of HCC patients was 55.0%, higher than the 45.0% positive rate of alpha-fetoprotein (AFP). PIVKA-II and AFP are not correlated; therefore, they are complementary in diagnosing HCC. Combining both for auxiliary diagnosis of HCC can improve the sensitivity to 71%.

[0003] PIVKA-II is used as a biomarker in the diagnosis and treatment evaluation of hepatocellular carcinoma (HCC). It can be used for the early diagnosis of HCC, as well as for dynamic monitoring and treatment efficacy assessment of diagnosed HCC patients. Its concentration is not directly related to tumor size, growth, malignancy, or grade / stage. Furthermore, its usefulness in the auxiliary diagnosis of recurrence has been reported. However, in certain situations, such as when taking vitamin K antagonists (warfarin) or antibiotics, this indicator may be elevated; while when taking vitamin K, it may be decreased.

[0004] Other liver diseases rarely cause elevated PIVKA-II levels. The serum half-life of PIVKA-II (40-72 hours) is shorter than that of AFP (5-7 days), making PIVKA-II a more timely indicator of HCC treatment efficacy. Patients with positive PIVKA-II have a higher incidence of intrahepatic metastasis, portal vein invasion, hepatic vein tumor thrombosis, and capsular infiltration.

[0005] Currently, methods for detecting PIVKA-II include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay (CLEIA). PIVKA-II kits typically include magnetic microparticle PIVKA-II calibrators, magnetic microparticle suspensions coated with PIVKA-II antibodies, and magnetic microparticle PIVKA-II enzyme conjugates to form antibody-antigen-antibody-enzyme complexes. These kits offer high sensitivity but are costly, have long reaction times, and are not suitable for large-scale deployment and application due to their lower sensitivity. Alternatively, some PIVKA-II detection kits also include test strips containing a nitrocellulose membrane, a sample pad, and a conjugate pad labeled with anti-PIVKA-II antibodies. However, these kits have lower sensitivity because they only allow for a single adsorption, making them prone to antibody binding to non-specific antigen sites, leading to false positives.

[0006] Therefore, this invention provides a rapid detection method for PIVKA-II that is highly sensitive, specific, easy to operate, and stable. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting abnormal prothrombin PIVKA-II.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for detecting abnormal prothrombin PIVKA-II specifically includes the following steps:

[0010] S1. Preparation of PIVKA-II antibody coated with luminescent microparticles:

[0011] The PIVKA-II antibody to be coated was dialyzed to obtain the treated protein to be coated; the aldehyde-based luminescent microparticles were centrifuged, the supernatant was discarded, reagent buffer was added, and the mixture was sonicated and centrifuged to obtain the treated luminescent microparticles; the treated protein to be coated and the luminescent microparticles were mixed in a certain proportion, and reaction buffer was added until the luminescent microparticles reached the reaction concentration. After mixing, a rotational reaction was performed, and then a prepared NaBH4 solution was added, mixed, and another rotational reaction was performed. Then, Gly solution was added, mixed, and the rotational reaction was performed; the reacted luminescent microparticles were centrifuged, the supernatant was discarded, washed, and centrifuged to obtain the mixed product; the mixed product was diluted to obtain the luminescent microparticle-coated PIVKA-II antibody;

[0012] S2. Preparation of biotin-labeled PIVKA-II antibody:

[0013] Biotin was dissolved in DMSO to obtain a biotin solution; the biotin solution was added to the PIVKA-II antibody and mixed to obtain the reaction product; the reaction product was dialyzed and diluted with biotin reagent buffer to obtain biotin-labeled PIVKA-II antibody.

[0014] Preparation of S3 and PIVKA-II calibrators and establishment of standard curves:

[0015] Bovine serum albumin and preservatives were added to Tris buffer to prepare calibration buffer. The PIVKA-II antigen gradient dilution was serially diluted with calibration buffer. PIVKA-II antibody coated with luminescent microparticles and biotin-labeled PIVKA-II antibody prepared in steps S1 and S2 were added to each pure PIVKA-II antigen gradient dilution. The values ​​were measured to obtain the PIVKA-II standard curve.

[0016] S4. Detection:

[0017] The serum sample to be tested, the PIVKA-II antibody coated with luminescent microparticles and the biotin-labeled PIVKA-II antibody were mixed and incubated. After incubation, a universal solution was added and incubated again. The luminescence signal value was measured and the concentration of the serum sample to be tested was calculated.

[0018] Furthermore, in step S1, the molecular weight cutoff of the dialysis bag is 14000D, the dialysis temperature is 2-8℃, the dialysis buffer is PBS solution with a concentration of 0.2mol / L, and the Gly solution is 75mg / mL, added at a ratio of 0.16mL of Gly solution per 10mg of microparticles.

[0019] Further, in step S1, the reagent buffer is a CB buffer containing 0.1% Tween-20 by mass, the concentration of the CB buffer is 0.05 mol / L, and the concentration of the NaBH4 solution is 8 mg / mL.

[0020] Furthermore, in step S1, the mass ratio of the luminescent microparticles to the protein to be coated is 10:1.

[0021] Further, in step S1, the diluent for the mixed product is 0.05 mol / L HEPES buffer, and the mixed product is diluted to 10 μg / mL.

[0022] Further, in step S2, 1 mg of PIVKA-II antibody is measured, and the molar ratio of PIVKA-II antibody to biotin is 1:20.

[0023] Further, in step S2, the biotin concentration in the biotin solution is 5 mg / mL, the dialysate is 0.1 mol / L sodium bicarbonate buffer, the biotin reagent buffer is 0.1 mol / L Tris buffer, and the concentration of the diluted dialysate is 0.2 μg / mL.

[0024] Further, in step S4, the volume ratio of the serum sample to be tested, the PIVKA-II antibody coated with luminescent microparticles, and the biotin-labeled PIVKA-II antibody is 1:1:1, the concentration of the PIVKA-II antibody coated with luminescent microparticles is 10 μg / mL, the concentration of the biotin-labeled PIVKA-II antibody is 0.2 μg / mL, and after incubation, a universal solution containing 50 μg / mL streptavidin photosensitive microparticles is added.

[0025] In summary, the present invention has the following beneficial effects:

[0026] 1. High sensitivity: The participation of multi-stage energy transfer and high-energy intermediates (such as dioxane) leads to the appearance of luminescent signals, which increases the sensitivity of detection.

[0027] 2. High specificity: The reaction components ensure that the luminescence signal mainly comes from the target analyte, reducing background noise.

[0028] 3. Stability: Enzyme-free systems avoid instability issues associated with enzymatic reactions, such as decomposition over time or sensitivity to environmental conditions.

[0029] 4. Simple operation: The absence of enzymes simplifies the analysis procedure and the operation and maintenance of the system. Attached Figure Description

[0030] Figure 1 The standard curve of PIVKA-II obtained in Comparative Example 1;

[0031] Figure 2 The standard curve of PIVKA-II obtained in Example 1;

[0032] Figure 3 The correlation graph between Comparative Example 1 and the measured values ​​of Fuji Rebio reagent is shown.

[0033] Figure 4 The graph shows the correlation between the measured values ​​of Example 1 and the values ​​measured by Fuji Rebiol reagent. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0035] Example 1

[0036] A kit for detecting abnormal prothrombin (PIVKA-II) includes PIVKA-II antibody-coated luminescent microparticles, biotin-labeled PIVKA-II antibody, PIVKA-II calibrators and quality controls, supplemented with a universal solution (containing streptavidin-sensitive microparticles). Under homogeneous conditions, this kit employs a double-antibody sandwich immunophotocatalytic chemiluminescence assay for the quantitative detection of PIVKA-II in human serum. The quality controls are provided at two concentration levels: 50 mAU / mL and 10000 mAU / mL.

[0037] Both the calibrators and quality control samples include the PIVKA-II antigen to be tested; the PIVKA-II antibody coated with luminescent microparticles and the biotin-labeled PIVKA-II antibody are both monoclonal antibodies, and the molecular weight of the PIVKA-II antibody is 150,000D, while the molecular weight of the biotin is 244.31D.

[0038] Step 1: Preparation of PIVKA-II antibody coated with luminescent microparticles

[0039] 1. 1 mg of the PIVKA-II antibody to be coated (BXA027 from Beijing Kaijing Gene Technology Co., Ltd. was used in Example 1; A09H05-D40 from Baixinyi can also be used if needed) was placed in a dialysis bag with a molecular weight cutoff of 14000D and dialyzed at 2-8℃ in dialysis buffer (PBS buffer with pH=7.4 and 0.2mol / L). Dialysis was performed 3 times in total. The volume of the dialysis buffer was not less than 100 times the volume of the solution to be dialyzed (the PIVKA-II antibody to be coated) each time, and the dialysis time was not less than 4 hours each time. The treated protein to be coated was obtained. After dialysis, a sample was taken and the protein concentration of the PIVKA-II antibody was measured to be 1.27 mg / mL.

[0040] 2. Centrifuge 422 μL of aldehyde-based luminescent microparticle reagent at 4℃ and 16000 rpm for 30 min (in a 50 mL centrifuge tube), discard the supernatant, add CB buffer (4 mL of 0.05 mol / L CB buffer containing 0.1% Tween-20), and disperse under sonication at 500 W. Repeat the centrifugation and sonication process under the same conditions to obtain the treated luminescent microparticles. Samples were taken, and the particle size of the luminescent microparticles was measured to be 278.3 ± 44 nm.

[0041] Preparation method of aldehyde-based luminescent microparticle reagent:

[0042] Take the gel filtration chromatography packing material (purchased from Cytiva (GE Healthcare), product number: Sepharose CL-4B), wash it three times with deionized water in a sintered glass funnel, each time using 5 times the volume of water, and finally resuspend the gel filtration chromatography packing material in deionized water to prepare a 50% (v / v) gel filtration chromatography packing material suspension (i.e., equal volume of settled microspheres and water).

[0043] Preparation of NaIO4 solution (prepare fresh and protect from light): Weigh NaIO4 and dissolve it in 0.1 mol / L acetate buffer (pH=5.0) to prepare a 20 mmol / L periodic acid solution;

[0044] Mix the gel filtration chromatography packing material suspension with the periodic acid solution at a 1:1 volume ratio (total volume 20 mL), stir gently to ensure uniform distribution, and react at 4°C in the dark with shaking for 2 hours.

[0045] After the reaction is complete, add 2 mL of ethylene glycol solution (5% v / v) to the reaction system and continue to shake in the dark for 30 minutes to remove excess NaIO4 and prevent further oxidation.

[0046] Washing: The microspheres were washed sequentially with deionized water (3 times) and PBS solution (pH=7.4, 0.1mol / L) (2 times) by filtration to remove unreacted periodic acid solution and byproducts. Finally, the microspheres were resuspended in an equal volume of PBS (pH=7.4, 0.1mol / L) and stored at 4°C.

[0047] 3. Mix the luminescent microparticles with the treated protein to be coated at a mass ratio of 10:1, add CB buffer (CB buffer concentration is 0.05mol / L) until the luminescent microparticles reach a reaction concentration of 25μg / mL, mix quickly, and rotate at 37±2℃ and 35rpm for 16 hours.

[0048] 4. Reduction reaction: Prepare a NaBH4 solution with a concentration of 8 mg / mL using 0.05 mol / L CB buffer. Add the NaBH4 solution to the rotating reaction mixture at a ratio of 2 μL of NaBH4 solution to 1 mg of luminescent particles, and mix rapidly. Rotate the mixture at 37°C and 35 rpm for 2 hours.

[0049] 5. Blocking: Add Gly solution to the mixture at a ratio of 0.16 mL of 75 mg / mL Gly solution (glycine dissolved in 0.05 mol / L CB buffer) per 10 mg of luminescent microparticles, mix well, place in a chromatography cabinet at 4℃, and rotate vertically to mix at 40 rpm for 1 h.

[0050] 6. Washing: Centrifuge the reacted luminescent microparticles at 2-8℃ for 30 min at 16000 rpm. Discard the supernatant, add 1 mL of luminescent reagent washing solution (0.05 mol / L CB buffer), and sonicate (500 W) at 1 second / cycle for a total of 30 cycles. Repeat the centrifugation and sonication process as above, using 500 W at 1 second / cycle for a total of 90 cycles (add 200 mg / mL Tween-20 solution before the last sonication, at a ratio of 3.2 μL of Tween-20 solution per 10 mg of luminescent microparticles) to obtain the mixed product.

[0051] 7. The mixed product was diluted to 10 mg / mL with 0.05 mol / L HEPES (4-hydroxyethylpiperazine ethanesulfonic acid) buffer containing 1.6 wt% BSA by weighing. Then it was diluted to 10 μg / mL with 0.05 mol / L HEPES buffer containing 1.6 wt% BSA to obtain PIVKA-II antibody coated with luminescent microparticles.

[0052] Step 2: Preparation of biotin-labeled PIVKA-II antibody

[0053] 1) Measure 1 mg of PIVKA-II antibody (in this example 1, Kaijing BXA027 is used, but Baixinyi A09H05-D70 can also be used as needed).

[0054] 2) Weigh biotin (purchased from Merck Darmstadt, Germany, product number B4501) according to the molar ratio of PIVKA-II antibody to biotin of 1:20, and dissolve biotin in dimethyl sulfoxide (DMSO) to a final concentration of 5 mg / mL to obtain a biotin solution.

[0055] 3) Add biotin solution to PIVKA-II antibody, mix thoroughly, and react at room temperature for 2 hours to obtain the reaction product;

[0056] 4) Dialyze the reaction product using a dialysis bag for 16 hours. The volume of the dialysis solution should be 200 times that of the dialysis product. Dialyze with 1 mL of 0.1 mol / L sodium bicarbonate buffer. Adjust the volume of the dialyzed reaction product to 1 mg / mL with 0.1 mol / L Tris buffer (pH=7.4). Then dilute with 0.1 mol / L Tris buffer to a concentration of 0.2 μg / mL to obtain biotin-labeled PIVKA-II antibody.

[0057] Step 3: Preparation of PIVKA-II calibrators and establishment of standard curve

[0058] 1) Preparation of PIVKA-II calibrator buffer: Bovine serum albumin (purchased from Jiangsu Enmoasai Biotechnology Co., Ltd., catalog number CCS30014.01) and preservative (proclin-300, purchased from Merck Darmstadt, Germany, catalog number 48914-U) were added to 0.05 mol / L Tris buffer with pH=7.4 to prepare the calibrator buffer. The mass concentration of bovine serum albumin was 0.5% and the volume concentration of preservative was 0.2%.

[0059] 2) The purified PIVKA-II antigen (purchased from Nanjing Ainodi Biotechnology Co., Ltd., catalog number AMP0108) was serially diluted with calibration buffer to obtain abnormal prothrombin solutions with concentrations of 0 mAU / mL, 5 mAU / mL, 50 mAU / mL, 500 mAU / mL, 10000 mAU / mL, and 30000 mAU / mL. To each 25 μL aliquot of abnormal prothrombin solution at different concentrations, 25 μL of PIVKA-II antibody coated with luminescent microparticles prepared in steps 1 and 2 and 2 of biotin-labeled PIVKA-II antibody were added. After sufficient reaction, the luminescence signal value (RLU) in the mixed system was measured and recorded using a LOCI 1000 / 1600 fully automated homogeneous chemiluminescence immunoassay analyzer. A PIVKA-II standard curve was generated using cubic spline interpolation with PIVKA-II concentration as the x-axis and luminescence signal value (RLU) as the y-axis. (See figure). Figure 2 As shown.

[0060] Step 4: Testing

[0061] 1) Mix the serum sample to be tested (25 μL), the PIVKA-II antibody coated with luminescent microparticles (25 μL), and the biotin-labeled PIVKA-II antibody (25 μL), and incubate for 8 min to obtain a mixed system; collect human blood in standard test tubes, and obtain the serum sample to be tested after centrifugation (store at 2~8℃ after sampling, and complete the test within 24 h).

[0062] 2) After incubation, add 100 μL of universal solution (the concentration of streptavidin photosensitive particles in the universal solution is 50 μg / mL, and the solvent is morpholine ethanesulfonic acid reagent (MES) with a concentration of 50 mmol / L)) to the mixed system, incubate for 4 min, and obtain the substrate to be tested. Use a LOCI1000 / 1600 fully automated homogeneous chemiluminescence immunoassay analyzer to measure and record the luminescence signal value (RLU) of the sample, and calculate the concentration of the serum sample to be tested using the standard curve obtained in step 3.

[0063] Comparative Example 1

[0064] Preparation of PIVKA-II calibration buffer: Bovine serum albumin and preservative (proclin-300) were added to 0.05 mol / L Tris buffer with pH=7.4 to prepare the calibration buffer. The mass concentration of bovine serum albumin was 0.5% and the volume concentration of preservative was 0.2%.

[0065] The purified PIVKA-II antigen was serially diluted with calibration buffer to obtain abnormal prothrombin solutions with concentrations of 0 mAU / mL, 5 mAU / mL, 50 mAU / mL, 500 mAU / mL, 10000 mAU / mL, and 30000 mAU / mL. Biotin-labeled PIVKA-II antibody (30 μL, purchased from Feipeng Biotechnology Co., Ltd., catalog number PIVKA-II-REAB-C1-003) and alkaline phosphatase-labeled PIVKA-II antibody (30 μL, purchased from Feipeng Biotechnology Co., Ltd., catalog number PIVKA-II-REAB-C1-004) were added to each 30 μL of abnormal prothrombin solution at different concentrations. After sufficient reaction, the luminescence signal value (RLU) was measured using an instrument. A standard curve for PIVKA-II was fitted using cubic spline interpolation, with PIVKA-II concentration as the x-axis and luminescence signal value (RLU) as the y-axis. Figure 1 As shown.

[0066] The existing biotinylated abnormal prothrombin monoclonal antibody-streptavidin magnetic bead system was used for detection: Abnormal prothrombin sample (30 μL), biotinylated PIVKA-II antibody (30 μL), and alkaline phosphatase-labeled PIVKA-II antibody (30 μL) were mixed and incubated for 15 min to obtain a mixed system. Then, 30 μL of universal buffer (containing streptavidin magnetic microparticles, with a streptavidin magnetic microparticle concentration of 50 μg / mL and 50 mmol / L morpholine ethanesulfonic acid reagent (MES) as the solvent) was added to the mixed system. The mixture was incubated for 5 min, followed by magnetic separation for 2 min. The supernatant was discarded to remove impurities and substances formed by the binding of streptavidin magnetic microparticles and biotinylated PIVKA-II antibody, resulting in a precipitate. The precipitate was washed three times, each time with 300 μL of washing buffer (Tris-HCl). 30 mmol / L), to obtain the substrate to be tested. Each time, 200 μL of the substrate to be tested is aspirated and measured using a fully automated chemiluminescence immunoassay analyzer (Shine i1910, Shenzhen Yingkai Biotechnology Co., Ltd.).

[0067] Based on the hospital's clinical testing needs, 20 serum samples were collected (serum samples were stored at 2–8℃ and tested within 24 hours of sampling; for long-term storage, it is recommended to aliquot and store at -20℃ for 14 days, avoiding repeated freeze-thaw cycles). Each serum sample was divided into three aliquots, and the PIVKA-II test was performed using the methods described in Example 1, Comparative Example 1, and the Fuji Rebio reagent test. The results were compared with the given values ​​(Fuji Rebio reagent values), and the data are shown in Table 1. The Fuji Rebio reagent is the gold standard reagent for the PIVKA-II test. A result greater than 40 mAU / mL is considered a positive sample. The manufacturer of the Fuji Rebio reagent used is Rebio (Shanghai) Diagnostic Technology Co., Ltd., product number: 233352.

[0068] The Fuji Rebio reagent assay uses a double-antibody sandwich method, employing two specific monoclonal antibodies (MAb) to capture and detect the PIVKA-II antigen. The test should be performed according to the manufacturer's instructions. The test method is as follows:

[0069] 1. Solid-phase capture: The serum sample to be tested is mixed with magnetic microparticles coated with anti-PIVKA-II antibody. The antibody binds to PIVKA-II in the serum, forming a "magnetic bead-antibody-antigen" complex.

[0070] 2. First wash: Add washing buffer (0.15 mol / L NaCl-Tris buffer), mix for 30 seconds, magnetically separate for 1 minute, discard the supernatant, and remove unbound substances (such as serum proteins and normal prothrombin). Key point: This step determines specificity and can effectively distinguish between PIVKA-II and normal prothrombin.

[0071] 3. Enzyme labeling: Add alkaline phosphatase (ALP) labeled anti-PIVKA-II antibody (which recognizes different epitopes) to form a complete sandwich structure of "magnetic bead-antibody-antigen-enzyme-labeled antibody".

[0072] 4. Second wash: Add washing buffer, mix for 30 seconds, magnetically separate for 1 minute, discard the supernatant to remove free enzyme-labeled antibody. Ensure that the final luminescent signal comes only from the bound PIVKA-II.

[0073] 5. Luminescence detection: Add AMPPD substrate solution; ALP catalyzes substrate decomposition and luminescence. Take readings after 5 minutes. The photomultiplier tube instrument detects the relative luminescence units (RLU). RLU is positively correlated with PIVKA-II concentration; the instrument's built-in standard curve is automatically calculated.

[0074] like Figure 3 and Figure 4 The figures show the correlation between the measured values ​​of Comparative Example 1, Example 1, and the Fuji Rebio reagent. The results show that the correlation coefficients between the detection results of Example 1 and Comparative Example 1 and the given values ​​(Fuji Rebio) are all above 0.95, indicating good correlation. Furthermore, the correlation value of Example 1 is greater than that of Comparative Example 1, indicating that Example 1 has higher detection accuracy than Comparative Example 1. Simultaneously, Example 1 (12 min) is significantly better than Comparative Example 1 in terms of detection time.

[0075] Table 1. Comparison of measured values ​​from Comparative Example 1 and Example 1 with those from Fuji Rebio reagent.

[0076]

[0077] Additionally, perform abnormal prothrombin PIVKA-II testing according to the Abbott reagent instructions (Abbott Diagnostics Products (Shanghai) Co., Ltd., product number: 2P48-25). The test method is as follows:

[0078] 1. Step 1 (Incubation): The serum sample to be tested is mixed with magnetic microparticles (coated with anti-PIVKA-II antibody) to capture PIVKA-II onto the magnetic beads.

[0079] 2. Washing: Add 300μL of washing solution, mix for 20s, perform magnetic separation for 30s, discard the supernatant, and repeat 3-4 times. The magnetic beads will be adsorbed, and the unbound substances will be washed away.

[0080] 3. Second step (labeling): Add 50 μL of acridine ester-labeled anti-PIVKA-II antibody (to recognize different epitopes), incubate for 4 min at 37°C to form a "magnetic bead-antibody-PIVKA-II-acidine ester antibody" sandwich complex.

[0081] 4. Wash again: Add 300 μL of washing solution, mix for 20 s, perform magnetic separation for 90 s, discard the supernatant, and wash away the free acridine ester label.

[0082] 5. Luminescence Detection: Add 100 μL of pre-excitation solution (acidic H2O2 solution) and 100 μL of excitation solution (excitation solution is NaOH solution, concentration 0.35 mol / L). Acridinium ester is instantaneously (1 s) oxidized in alkaline peroxide, generating photons (RLU). The reading is taken immediately after excitation, and the RLU is detected by a photomultiplier tube (PMT). The RLU is positively correlated with the concentration of PIVKA-II, and the instrument automatically calculates the value using a built-in 6-point calibration curve.

[0083] Table 2 shows the performance comparison results of Example 1 and Abbott's reagent detection method.

[0084] Table 2 Performance comparison of Example 1 and Abbott reagent detection method

[0085]

[0086] Data analysis shows that the method in Example 1 is superior to the Abbott reagent detection method in both sensitivity and precision, and is simpler to operate with a shorter reaction time. In terms of specificity, its correlation with clinical samples is also above 0.95, significantly better than the Abbott reagent detection method. In summary, a method for detecting PIVKA-II with high specificity and high sensitivity, characterized by short detection time and simple operation, has been obtained.

[0087] The technical effects achieved by the present invention are as follows: both the luminescent microparticle coating and the biotin-labeled PIVKA-II antibody used are primary antibodies, resulting in higher specificity; the binding between streptavidin photosensitive microparticles and biotin has extremely high affinity, and the reaction is highly specific; since streptavidin does not contain glycosyl groups and has an isoelectric point close to neutral, streptavidin has a lower level of non-specific binding in detection applications. Therefore, while improving sensitivity, it does not increase non-specific interference, and the binding characteristics are not affected by the high dilution of the reaction reagents, thus minimizing the non-specific effects of the reaction reagents in practical applications.

[0088] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting abnormal prothrombin PIVKA-II, characterized in that, Specifically, the following steps are included: S1. Preparation of PIVKA-II antibody coated with luminescent microparticles: The PIVKA-II antibody to be coated was dialyzed to obtain the treated protein to be coated; the aldehyde-based luminescent microparticles were centrifuged, the supernatant was discarded, reagent buffer was added, and the mixture was sonicated and centrifuged to obtain the treated luminescent microparticles; the treated protein to be coated and the luminescent microparticles were mixed in a certain proportion, and reaction buffer was added until the luminescent microparticles reached the reaction concentration. After mixing, a rotational reaction was performed, and then a prepared NaBH4 solution was added, mixed, and another rotational reaction was performed. Then, Gly solution was added, mixed, and the rotational reaction was performed; the reacted luminescent microparticles were centrifuged, the supernatant was discarded, washed, and centrifuged to obtain the mixed product; the mixed product was diluted to obtain the luminescent microparticle-coated PIVKA-II antibody; S2. Preparation of biotin-labeled PIVKA-II antibody: Biotin was dissolved in DMSO to obtain a biotin solution; the biotin solution was added to the PIVKA-II antibody and mixed to obtain the reaction product; the reaction product was dialyzed and diluted with biotin reagent buffer to obtain biotin-labeled PIVKA-II antibody. Preparation of S3 and PIVKA-II calibrators and establishment of standard curves: Bovine serum albumin and preservatives were added to Tris buffer to prepare calibration buffer. PIVKA-II antigen was serially diluted with calibration buffer. PIVKA-II antibody coated with luminescent microparticles and biotin-labeled PIVKA-II antibody prepared in steps S1 and S2 were added to each pure PIVKA-II antigen serial dilution solution. The values ​​were measured to obtain the PIVKA-II standard curve. S4. Detection: The serum sample to be tested, the PIVKA-II antibody coated with luminescent microparticles and the biotin-labeled PIVKA-II antibody were mixed and incubated. After incubation, a universal solution was added and incubated again. The luminescence signal value was measured and the concentration of the serum sample to be tested was calculated.

2. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S1, the molecular weight cutoff of the dialysis bag is 14000D, the dialysis temperature is 2-8℃, the dialysis buffer is PBS solution with a concentration of 0.2mol / L, and the Gly solution is 75mg / mL, added at a ratio of 0.16mL of Gly solution per 10mg of microparticles.

3. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S1, the reagent buffer is a CB buffer containing 0.1% Tween-20 by mass, the concentration of the CB buffer is 0.05 mol / L, and the concentration of the NaBH4 solution is 8 mg / mL.

4. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S1, the mass ratio of luminescent microparticles to the protein to be coated is 10:

1.

5. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S1, the diluent for the mixed product is 0.05 mol / L HEPES buffer, and the mixed product is diluted to 10 μg / mL.

6. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S2, 1 mg of PIVKA-II antibody is measured, and the molar ratio of PIVKA-II antibody to biotin is 1:

20.

7. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S2, the biotin concentration in the biotin solution is 5 mg / mL, the dialysate is 0.1 mol / L sodium bicarbonate buffer, the biotin reagent buffer is 0.1 mol / L Tris buffer, and the concentration of the diluted dialysate is 0.2 μg / mL.

8. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, characterized in that, In step S4, the volume ratio of the serum sample to be tested, the PIVKA-II antibody coated with luminescent microparticles, and the biotin-labeled PIVKA-II antibody is 1:1:

1. The concentration of the PIVKA-II antibody coated with luminescent microparticles is 10 μg / mL, and the concentration of the biotin-labeled PIVKA-II antibody is 0.2 μg / mL. After incubation, a universal solution containing 50 μg / mL streptavidin photosensitive microparticles is added.

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