CN-122017239-A - Method for detecting abnormal prothrombin PIVKA-II

Abstract

The invention discloses a method for detecting abnormal prothrombin PIVKA-II, which belongs to the field of medicines and biotechnology and comprises the following steps of S1, preparing a luminous particle coated PIVKA-II antibody, S2, preparing a biotin-labeled PIVKA-II antibody, S3, preparing a PIVKA-II calibrator and establishing a standard curve, and S4, detecting. The detection method has the characteristics of high sensitivity, good specificity, strong stability and simple operation.

Inventors

• Alexei Kostyukov
• SHANG YIWEN
• YUAN ZHIGANG

Assignees

• 浙江夸克生物科技有限公司

Dates

Publication Date

20260512

Application Date

20260415

Claims (8)

1. 1. The method for detecting abnormal prothrombin PIVKA-II is characterized by comprising the following steps of: S1, preparing a luminous particle coated PIVKA-II antibody: Dialyzing PIVKA-II antibody to be coated to obtain treated protein to be coated, centrifuging aldehyde group luminous particles, discarding supernatant, adding reagent buffer solution, performing ultrasonic dispersion and centrifugation to obtain treated luminous particles, mixing the treated protein to be coated and the luminous particles according to a proportion, supplementing reaction buffer solution until the luminous particles reach reaction concentration, performing rotary reaction after uniformly mixing, adding prepared NaBH 4 solution after the reaction, uniformly mixing, performing rotary reaction after uniformly mixing, adding Gly solution, performing rotary reaction after uniformly mixing, centrifuging the reacted luminous particles, discarding supernatant, washing and centrifuging to obtain a mixed product, and diluting the mixed product to obtain PIVKA-II antibody coated with the luminous particles; S2, preparing a biotin-labeled PIVKA-II antibody: dissolving biotin with DMSO to obtain biotin solution, adding biotin solution into PIVKA-II antibody, mixing to obtain reaction product, dialyzing the reaction product, and diluting the dialyzate with biotin reagent buffer solution to obtain biotin-labeled PIVKA-II antibody; S3, preparing a PIVKA-II calibrator and establishing a standard curve: Adding bovine serum albumin and a preservative into Tris buffer solution to prepare calibrator buffer solution, carrying out gradient dilution on PIVKA-II antigen by using the calibrator buffer solution, adding PIVKA-II antibody coated by luminescent particles and biotin-labeled PIVKA-II antibody prepared in the step S1 and the step S2 into each part of PIVKA-II antigen gradient dilution pure product, and measuring to obtain PIVKA-II standard curve; S4, detecting: And mixing and incubating the serum sample to be detected, the PIVKA-II antibody coated by the luminous particles and the PIVKA-II antibody marked by biotin, adding a universal liquid after incubation, incubating again, measuring a luminous signal value, and calculating the concentration of the serum sample to be detected.
2. 2. The method according to claim 1, wherein in the step S1, the dialysis bag has a molecular weight cut-off of 14000D, a dialysis temperature of 2-8 ℃, the dialysis buffer is a PBS solution, the concentration of the dialysis buffer is 0.2mol/L, the Gly solution is 75mg/mL, and the concentration of the Gly solution is added in a proportion of 0.16mL of Gly solution per 10mg of microparticles.
3. 3. The method according to claim 1, wherein in the step S1, the reagent buffer is a CB buffer containing Tween-20 at a mass concentration of 0.1%, the concentration of the CB buffer is 0.05mol/L, and the concentration of the NaBH 4 solution is 8mg/mL.
4. 4. The method according to claim 1, wherein in the step S1, the mass ratio of the luminescent particles to the protein to be coated is 10:1.
5. 5. The method according to claim 1, wherein in the step S1, the mixed product is diluted to 10 μg/mL with 0.05mol/L HEPES buffer.
6. 6. The method according to claim 1, wherein in the step S2, 1mg of PIVKA-II antibody is measured and the molar ratio of PIVKA-II antibody to biotin is 1:20.
7. 7. The method according to claim 1, wherein in the step S2, the concentration of biotin in the biotin solution is 5mg/mL, the concentration of sodium bicarbonate buffer in the dialysate is 0.1mol/L, the concentration of biotin reagent buffer in the Tris buffer is 0.1mol/L, and the concentration of dialysis product dilution is 0.2 μg/mL.
8. 8. The method for detecting abnormal prothrombin PIVKA-II according to claim 1, wherein in the step S4, the volume ratio of the serum sample to be detected, the luminous particle coated PIVKA-II antibody to the biotin-labeled PIVKA-II antibody is 1:1:1, the luminous particle coated PIVKA-II antibody concentration is 10 μg/mL, the biotin-labeled PIVKA-II antibody concentration is 0.2 μg/mL, and a universal liquid containing 50 μg/mL streptavidin photosensitive particles is added after incubation.

Description

Method for detecting abnormal prothrombin PIVKA-II Technical Field The invention relates to the field of medicine and biotechnology, in particular to a method for detecting abnormal prothrombin PIVKA-II. Background Abnormal prothrombin PIVKA-II showed a specific elevation in liver cancer cells. The threshold value of PIVKA-II >40mAU/mL is adopted to diagnose the hepatocellular carcinoma (HCC), the sensitivity and the specificity are respectively 48.16% and 95.93%, and the comprehensive accuracy is as high as 71.6%. The positive rate of PIVKA-II in serum of HCC patient is 55.0%, which is higher than 45.0% positive rate of Alpha Fetoprotein (AFP), PIVKA-II has no correlation with AFP, and both have complementarity to diagnosis of HCC, and the sensitivity can be improved to 71% when combined with both for auxiliary diagnosis of HCC. PIVKA-II is used as a marker for detection in diagnosis of liver cell liver cancer and treatment effect judgment. Can be used for early diagnosis of liver cancer and dynamic monitoring and treatment effect evaluation of the diagnosed liver cancer patients. The concentration is not directly related to the tumor size, growth, malignancy and classification/stage. In addition, there are reports on the usefulness of the auxiliary diagnosis of recurrence. However, in some cases, such as administration of vitamin K antagonists (warfarin) or the use of antibiotics, the index may be increased, while administration of vitamin K may be decreased. Other liver diseases rarely cause PIVKA-II to rise, the serum half-life (40-72 hours) of PIVKA-II is shorter than the half-life (5-7 days) of AFP, and PIVKA-II can reflect the curative effect of HCC in time. Patients positive for PIVKA-II have a high incidence of intrahepatic metastasis, portal vein invasion and hepatic vein thrombosis, and capsule infiltration. Current methods for detecting PIVKA-II are Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and chemiluminescent immunoassay (CLEIA). The PIVKA-II kit generally comprises a magnetic particle PIVKA-II calibrator, a magnetic particle suspension coated with PIVKA-II antibody and a magnetic particle PIVKA-II enzyme conjugate to form an antibody-antigen-antibody-enzyme complex, and has high detection sensitivity, but high detection cost, long reaction time, no benefit for large-scale popularization and application and low sensitivity, or part of the PIVKA-II detection kit also comprises a detection paper card, wherein the paper card comprises a nitrocellulose membrane, a sample pad and a binding pad, and the binding pad is marked with an anti-PIVKA-II antibody, but the detection sensitivity of the kit is low, and the condition that the antibody is combined with an antigen nonspecific site easily occurs due to only one adsorption, so that false positive is generated. Therefore, the invention provides the method for rapidly detecting PIVKA-II, which has the advantages of high sensitivity, good specificity, simple and convenient operation and stable detection. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a method for detecting abnormal prothrombin PIVKA-II. In order to achieve the above purpose, the present invention adopts the following technical scheme: A method for detecting abnormal prothrombin PIVKA-II, comprising the steps of: S1, preparing a luminous particle coated PIVKA-II antibody: Dialyzing PIVKA-II antibody to be coated to obtain treated protein to be coated, centrifuging aldehyde group luminous particles, discarding supernatant, adding reagent buffer solution, performing ultrasonic dispersion and centrifugation to obtain treated luminous particles, mixing the treated protein to be coated and the luminous particles according to a proportion, supplementing reaction buffer solution until the luminous particles reach reaction concentration, performing rotary reaction after uniformly mixing, adding prepared NaBH 4 solution after the reaction, uniformly mixing, performing rotary reaction after uniformly mixing, adding Gly solution, performing rotary reaction after uniformly mixing, centrifuging the reacted luminous particles, discarding supernatant, washing and centrifuging to obtain a mixed product, and diluting the mixed product to obtain PIVKA-II antibody coated with the luminous particles; S2, preparing a biotin-labeled PIVKA-II antibody: dissolving biotin with DMSO to obtain biotin solution, adding biotin solution into PIVKA-II antibody, mixing to obtain reaction product, dialyzing the reaction product, and diluting the dialyzate with biotin reagent buffer solution to obtain biotin-labeled PIVKA-II antibody; S3, preparing a PIVKA-II calibrator and establishing a standard curve: Adding bovine serum albumin and a preservative into Tris buffer solution to prepare a calibrator buffer solution, carrying out gradient dilution on PIVKA-II antigen gradient diluent by using the calibrator buffer solution, adding the PIVKA-II antib