CN112175925A - PIVKA-II epitope peptide, anti-PIVKA-II monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a PIVKA-II epitope peptide, an anti-PIVKA-II monoclonal antibody and application thereof. Based on the epitope peptide, the monoclonal antibody can accurately identify and combine PIVKA-II without combining with prothrombin, has good stability, and can be used as a key raw material of a diagnostic reagent; the invention also provides a PIVKA-II chemiluminescence immunoassay detection kit using the monoclonal antibody and a preparation method thereof.

Description

PIVKA-II epitope peptide, anti-PIVKA-II monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of immunology and biomedical engineering, in particular to PIVKA-II epitope peptide construction, anti-human PIVKA-II monoclonal antibody screening and expanded production, and application of the antibody in an in-vitro diagnosis kit.

Background

The qualitative diagnosis of liver cancer mainly refers to the level of tumor marker AFP in serum; however, studies have shown that AFP is less specific and sensitive, and not suggested as a sole diagnostic indicator, and need to be used in combination with other diagnostic methods to improve the reliability of the diagnosis.

The new liver cancer marker PIVKA-II has higher diagnosis specificity and is more and more concerned. PIVKA-II is an English abbreviation for vitamin K deficiency induced Protein-II (Protein induced by vitamin Kabsense-II), also known as abnormal prothrombin, and is a glycoprotein that coagulates blood and has a structure similar to prothrombin. Prothrombin is a protein consisting of 622 amino acid residues, 10 glutamic acid residues located near the amino terminus are gamma-carboxylated to form gamma-carboxyglutamic acid (Gla) residues, but when the organism is exposed to hepatocyte injury, liver failure, vitamin K deficiency, vitamin K antagonism or hepatoma, the corresponding carboxylation is disturbed resulting in partial or total lack of carboxylation of 10 glutamic acid residues, and this modified abnormal protein is called abnormal prothrombin DCP or VKPIA-II.

PIVKA-II was first studied as a tumor marker in 1984 by Liebeman. PIVKA-II has attracted attention in the field of liver cancer diagnosis and detection in recent years, and is recommended to be used for screening high-risk people, assisting liver cancer diagnosis, monitoring treatment effect and predicting prognosis and relapse in guidelines of Asia-Tai liver disease society and Japanese liver disease society. It has been found that the N-terminal carboxylation number of PIVKA-II is often more than 5 in patients with benign liver diseases such as chronic hepatitis, alcoholic liver disease or liver cirrhosis, and the corresponding carboxylation number is less than 4 in patients with hepatocellular carcinoma.

Due to the high similarity of PIVKA-II and normal thrombin and the variability of the carboxylation sites, the current monoclonal antibodies capable of specifically recognizing and binding PIVKA-II with high affinity are very rare. At present, only a few companies at home and abroad can provide the PIVKA-II monoclonal antibody, such as MU-3 used by Fujiebio and 3C10 used by Abbott ARCHITECT. Therefore, the development of the PIVKA-II monoclonal antibody with excellent performance and independent property rights has important significance for improving the performance of the PIVKA-II diagnostic reagent, improving the detection reliability and early discovering and treating liver cancer.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the problem that the existing monoclonal antibody capable of specifically recognizing and combining PIVKA-II with high affinity is rare due to the high similarity of PIVKA-II and normal thrombin and the variability of carboxylation sites in the prior art, and provide a monoclonal antibody of anti-human LP-PLA2 protein with high specificity and strong affinity and application thereof.

In order to solve the technical problems, the invention provides the following technical scheme:

a PIVKA-II epitope peptide has an amino acid sequence shown in SEQ ID NO. 1. The polypeptide sequence is based on PIVKA-II amino acid sequence 11-26, the glutamic acid is not gamma-carboxylated, and a unique 8 amino acid sequence is added at the tail end to increase the immunity. By utilizing the immune polypeptide, the invention provides a monoclonal antibody with high affinity, strong specificity and good stability, and establishes a PIVKA-II detection kit based on a chemiluminescence method so as to realize accurate detection of the concentration of the monoclonal antibody.

The high specificity and strong affinity antihuman PIVKA-II monoclonal antibody PV-Ab1, the monoclonal antibody PV-Ab1 heavy chain variable region amino acid sequence is shown as SEQ ID NO.2, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 3. Monoclonal antibody PV-Ab1 is a monoclonal antibody that specifically recognizes an amino-terminal lacking carboxylated modified epitope that binds PIVKA-II.

3. A kit comprising monoclonal antibody PV-Ab1 of claim 2.

4. The kit of claim 3, wherein the monoclonal antibody PV-Ab1 is used as a coating antibody or a labeled antibody.

5. The LP-PLA2 protein monoclonal antibody of claim 1 or the kit of claim 2, for use in an auxiliary diagnostic or detection reagent for liver cancer.

The PIVKA-II epitope peptide provided by the invention can better expose a PIVKA-II specific sequence and has stronger immunity; the monoclonal antibody for resisting PIVKA-II protein provided by the invention has excellent affinity, high specificity and good stability, can be used as a key material for PIVKA-II detection, and can be used for quickly developing a diagnostic reagent or a detection instrument based on antigen-antibody recognition and combination. The PIVKA-II kit has the advantages of good repeatability, wide linear range, high sensitivity, strong anti-interference capability and good reagent stability, and can well meet the requirements of clinical examination.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the correlation between the detection kit and Yapei control reagent in 116 clinical specimens.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 PIVKA-II epitope peptide construction and coupling with Carrier proteins

The epitope peptide used in the invention can be prepared by various ways, such as eukaryotic/prokaryotic expression and chemical synthesis, the polypeptide in the embodiment is synthesized by the national institute of bioscience and technology, Inc., and the purity is more than 98% by HPLC identification.

Coupling carrier protein:

1. 2mg of KLH keyhole limpet hemocyanin was dissolved in 200ul of 0.1M MES buffer (pH 5);

2. 2mg of PIVKA-II polypeptide was dissolved in 500ul of 0.1M MES buffer (pH 5); and adding it to the KLH protein solution;

3. weighing 10mg of EDC, dissolving in 1ml of ultrapure water, immediately mixing uniformly, adding 100ul of EDC into the polypeptide-KLH solution in the previous step, and shaking and mixing;

4. standing and reacting for 2 hours at room temperature;

5. and (3) removing the polypeptide which is not connected to the KLH by using ultrafiltration membrane centrifugal ultrafiltration, and collecting the coupling product as a subsequent animal immunogen.

Example 2 animal immunization and potency assay

1. Antigen immunization

The PIVKA-II immunogen protein with the concentration of 1.5mg/ml and the same volume of Sigma-Aldrich Freund's complete adjuvant are fully mixed and emulsified by an emulsifier to obtain oily emulsified antigen. The emulsified antigen was injected into ALB/c mice, each mouse was injected with a 0.2ml dose in the lower part of the body. 7 days after the first immunization, 1.5mg/ml of PIVKA-II immunogen protein and the same volume of Sigma-Aldrich Freund incomplete adjuvant are emulsified by an emulsifier, 0.2ml of dose immunity enhancing injection is given to each mouse from a tarsal joint after emulsification, tail vein blood sampling is carried out when the immunity is enhanced to three needles, the serum titer is detected by an indirect ELISA method, and the titer reaches the fusion requirement.

2. Cell fusion, screening and subcloning of positive hybridoma

Satisfactory mice were sacrificed to take lymphocytes. Using 50% PEG 4000 as a fusogenic agent, sp2/0 myeloma cells and lymphocytes were fused using conventional methods, at a ratio of 1:3 to 1: 5. The fused cells were selectively cultured in HAT conditioned medium. Cell placement into CO by fusion Chamber₂Culturing at 37 deg.C for 7-9 days in incubator, and observing fusion state. Screening was performed using indirect ELISA plates starting at 10 days. And selecting cell strains with strong positive and large thin numbers from the preliminarily screened positive clones, and carrying out subcloning for 4 times by using a limiting dilution method. Through the screening process, a cell strain capable of stably secreting PIVKA-II antibody is obtained.

3. Preparation of monoclonal antibodies

Selecting BALB/c mice growing and developing normally in 6-8 weeks, and firstly injecting 0.5ml Freund incomplete adjuvant into the abdominal cavity of each mouse; the injection is administered by 2x10 intraperitoneally after 9 days⁶And (3) hybridoma cells. Ascites can be generated 7-9 days after the mice are inoculated with the hybridoma cells, and the health condition and the ascites production quantity of the mice are observed; mice were sacrificed when ascites production was high and mice were less healthy, and the corresponding ascites were collected.

The collected mouse ascites fluid was diluted with 3 volumes of 0.05M pH 4.0 acetate buffer. Adding caprylic acid with the final concentration of 3% into the mixed solution to precipitate ascites impurities. Centrifuging at 14000rpm at 4 deg.C for 30min, and collecting supernatant. The pH was adjusted to 7.4 with 1M sodium hydroxide solution. The solution was passed through a equilibrated protein A/G column, washed with PBS solution and eluted with 0.1M Gly-HCl (pH 2.8) solution, and the eluted product was adjusted to pH 7.5 with 1M tris-HCl (pH 9.0).

Preparation of PIVIKA-II chemiluminescence detection kit

The kit is prepared based on the principles of a double-antibody sandwich method and an acridinium ester luminescence method, and the other labeled antibody in the application is a prothrombin antibody produced by the company with the product number AB 0009-4.

1. Preparation of streptavidin magnetic microparticles

Commercially available streptavidin magnetic particles from sequoise were diluted to 0.02% solids using magnetic particle buffer (50mM PB, 150mM Nacl, 3% trehalo, 0.1% Proclin300) as R1 reagent.

2. Acridinium ester labeled antibody

Performing reaction on the acridinium ester and the prothrombin antibody according to a molecular molar ratio of 3: 1 in PBS for 2 h; after the reaction is finished, carrying out ultrafiltration centrifugation by using a 30kDa ultrafiltration centrifugal tube to remove free acridinium ester; after centrifugation is finished, collecting the marker in the ultrafiltration tube and detecting the concentration of the acridinium ester-antibody by using a BCA method; diluting the acridinium ester-antibody to 1.5ug/ml by using an R2 preparation solution, wherein the diluted solution is an R2 reagent, and the formula of the R2 preparation solution is 20mM Tris, 200mM KCl, 2% BSA, 0.1% Tween20 and 0.1% proclin 300.

3. Biotin-labeled antibody

NHS esterified organisms (EZ-link NHS-PEG4-biotin) were mixed with the anti-PIVKA-II antibodies of the invention at a molar ratio of 5: 1 ratio in PBS for labeling for 1 h; after the reaction is finished, performing ultrafiltration centrifugation by using a 30kDa ultrafiltration centrifugal tube, adding PBS (phosphate buffer solution) into the upper part of the ultrafiltration tube after the centrifugation is finished, performing centrifugation again, and repeating twice; collecting biotin-antibody labeled products in the ultrafiltration tube, and detecting the antibody concentration by using a BCA method; the product was diluted to an antibody concentration of 2ug/ml using R3 formulation diluent as R3 reagent, and R3 formulation diluent was 20mM Tris, 50mM KCl, 2% Casein, 0.1% Tween20, 0.1% proclin 300.

4. Preparing calibrator and quality control product

The PIVKA-II antigen is respectively diluted to 0, 40, 100, 1000, 5000, 10000, 20000 and 30000mAU/ml by using antigen diluent, and is subpackaged into a calibrator tube.

And (3) respectively releasing the PIVKA-II antigen diluted solution to 40mAU/ml and 2000mAU/ml by using the antigen diluted solution, and subpackaging into quality control tubes.

The above antigen diluent formulation was 100mM PB, 150mM Nacl, 5% BSA, 10% Glycerin, 0.5% procin300, pH 8.0.

Comparison of the detection kit with similar products

At present, only a few companies at home and abroad have registered certificates of PIVKA-II detection kits for in vitro diagnosis, such as Yapeh and Fuji Riboehu, and the PIVKA-II chemiluminescence detection kit in the embodiment is subjected to correlation comparison on 116 clinical specimens of Yapeh company, and the data is shown in figure 1. The correlation coefficient was 0.9933. The kit can accurately detect the PIVKA-II content, provides basis for liver cancer diagnosis, and can fully meet the clinical in-vitro diagnosis and detection requirements.

Examination of sensitivity and precision of detection kit

The blank and detection limits of the kits of the invention were studied according to the clinical and laboratory standards institute sensitivity validation analysis guidelines document CLSI EP17-A2 and showed blank and detection limits of 0.53mAU/mL and 1.38mAU/mL, respectively.

According to the precision verification analysis guideline file CLSI EP5-A3 of the clinical and laboratory standards institute, the precision research is carried out on the kit, 3 batches of reagents and calibrators are used, 2 levels of quality control products are detected, the quality control products are detected twice each day in the morning and afternoon and are continuously examined for 5 days, and the result shows that the total precision in each batch is less than 5%.

Analysis of anti-interference ability of detection kit

Different concentrations of interferents were added to two serum samples with a concentration level of about 40mAU/mL and 500mAU/mL of PIVKA-II, respectively, and the maximum concentration levels of the different interferents (not exceeding the highest concentration seen in the clinic) were investigated, using the non-added group as a control and the relative deviation of both the added and control groups of less than 5% as an interference acceptance criterion, and the results showed that the concentrations in the following table did not significantly interfere with the test results.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

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<120> PIVKA-II epitope peptide, anti-PIVKA-II monoclonal antibody and application thereof

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Claims (5)

1. A PIVKA-II epitope peptide is characterized in that the amino acid sequence of the peptide is shown as SEQ ID NO. 1.

2. The monoclonal antibody PV-Ab1 of the anti-human PIVKA-II protein with high specificity and strong affinity is characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody PV-Ab1 is shown as SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 3.

3. A kit comprising monoclonal antibody PV-Ab1 of claim 2.

4. The kit of claim 3, wherein the monoclonal antibody PV-Ab1 is used as a coating antibody or a labeled antibody.

5. The monoclonal antibody against the LP-PLA2 protein of claim 1 or the kit of claim 2, for use in an auxiliary diagnostic or detection reagent for liver cancer.

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