Preparation of golgi protein 73 scfv antibody and application thereof

Abstract

The present application relates to the field of antibody preparation and in vitro diagnostic reagent, and particularly relates to a GP73 protein scFV antibody prepared based on a phage display technology and an application of a magnetic microparticle chemiluminescence kit for detecting a GP73 content in human serum based on the antibody. Compared with the same type of product, the scFv antibody and the detection product developed based on the scFv antibody have a wider linear range and a better detection sensitivity, are simple to operate, have a detection time of no more than 30 minutes, and have a more stable and reliable detection result.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody preparation and in vitro diagnostic reagent, in particular to the preparation of GP73 protein scFV antibody based on phage display technology and the application of the magnetic microparticle chemiluminescence kit for detecting the content of GP73 in human serum based on the antibody. BACKGROUND

[0002] Golgi protein 73 (GP73) is a transmembrane protein discovered on the Golgi apparatus in recent years. Its relative molecular weight is 73 KD in protein electrophoresis, so it is named GP73. GP73 is mainly expressed in bile duct epithelial cells and is not expressed or expressed in very low amount in normal hepatocytes. In recent years, research data shows that the expression of GP73 is significantly up-regulated in the development of liver diseases such as cirrhosis. Specifically, the expression of GP73 gradually increases from acute and chronic hepatitis to liver cirrhosis, and the expression is the highest in liver cirrhosis tissue. Comprehensive research data shows that GP73 has the potential to become a new serum marker for liver cirrhosis.

[0003] Liver cirrhosis is an important cause of morbidity and mortality in patients with chronic liver disease. Liver cirrhosis can lead to hepatocellular carcinoma and liver decompensation, including ascites, hepatic encephalopathy and variceal rupture, cardiac dysfunction, etc., which is associated with 2.4% of global mortality per year. The main causes of liver cirrhosis include hepatitis B and C virus infection, alcohol-related liver disease and fatty liver disease. According to the analysis data of liver cirrhosis and chronic liver disease provided by the Global Burden of Disease (GBD) database, the estimated number of patients with compensated cirrhosis worldwide is 112 million. The disease burden of liver cirrhosis is still very large worldwide, and it is expected that the number of liver cirrhosis-related deaths and decompensated cirrhosis cases will continue to rise in the next ten years.

[0004] The accuracy of in vitro detection of Golgi protein 73 content is highly dependent on the quality of the GP73 antibody used. Almost all the GP73 antibodies mentioned in the existing patents are obtained by hybridoma technology. Although this technology is widely used, it still has some technical shortcomings, such as long preparation time, poor repeatability, low fusion efficiency of spleen cells and myeloma cells, unstable production of hybridoma cells, large batch-to-batch differences, etc. In addition, hybridoma technology cannot prepare antibodies for non-immune or highly toxic antigens, and the antibodies produced often need to be humanized, and the antibody preparation process is extremely tedious.

[0005] CN101735319B discloses a monoclonal antibody against GP73 protein and a preparation method thereof. The GP73 antibody is prepared by using common hybridoma technology, which has a huge time consumption and a complicated screening process. The cell fusion and hybridoma separation efficiency is low. The hybridoma cell strain is unstable in genetic culture. The prepared murine antibody is easily recognized by the human immune system, causing HAMA reaction. There are individual differences in immunizing animals with hybridoma cells, and the antibody difference between different batches is large. The detection time of the standard enzyme-linked immunoassay and chemiluminescence immunoassay mentioned in the patent is about 70 minutes. The overall operation process is complex, and it is a pure manual operation, which has a large human interference factor. In addition, the preparation of the GP73 monoclonal antibody in the patent, especially the covered epitope of the obtained GP73 monoclonal antibody lacks necessary description, but the coverage of the epitope has a very important role in the sensitivity and specificity of GP73 detection.

[0006] CN111378627B discloses a Golgi protein 73 monoclonal antibody, kit and application. The GP73 antibody mentioned in the patent is also prepared by using hybridoma cell method, and a polypeptide is used as an immunogen for animal immunization, which causes the prepared antibody to be unable to recognize all epitopes of the GP73 protein, affecting the types and specificity of the produced antibody. The detection method of the kit mentioned in the patent is enzyme-linked immunoassay, and the detection time is about 90 minutes. The overall operation is a manual operation, which is prone to human error. The linear range of the ELISA kit developed based on the antibody is 1 ng / ml-475 ng / ml, and the linear range is small. In addition, the patent also lacks necessary description of the covered epitope of the obtained GP73 monoclonal antibody.

[0007] Single-chain antibody (scFv) is a recombinant antibody formed by splicing the variable region genes of antibody heavy and light chains through a connecting peptide. The scFv has the advantages of small molecular weight, strong tissue penetration, short in vivo circulation half-life, low immunogenicity, easy genetic engineering improvement and the like, and plays an important role in clinical diagnosis, treatment and prevention of diseases. SUMMARY

[0008] The present application prepares two strains of GP73 protein scFv antibodies by phage display technology, which not only saves a lot of preparation time, but also has a wider linear range and better detection sensitivity compared with the same type of product based on the scFv antibody developed for the detection product. The operation is simple, the detection time is not more than 30 minutes, and the detection result is more stable and reliable. According to the experimental data, the GP73 protein scFv antibody prepared by phage display in the present application and the kit derived therefrom have more excellent performance.

[0009] The present application aims to provide two strains of GP73 protein scFV antibodies (SGA-1 and SGA-2) with high affinity prepared by phage display technology.

[0010] The present application provides a scFv antibody, characterized in that,

[0011] The sequences of CDR1 to CDR3 of the heavy chain variable region are TYAMS, VISSGSSYTYYPDSVKG, EGELHYYAMD respectively;

[0012] The sequences of CDR1 to CDR3 of the light chain variable region are VASSSVSSYLH, TTSSLASG, HQYHRSPYT respectively;

[0013] The sequences of CDR1 to CDR3 of the light chain variable region are VASSSVSSYLH, TTSSLASG, HQYHRSPYT respectively;

[0014] The sequences of CDR1 to CDR3 of the light chain variable region are KSSQSLLYSDGKTYLIWLI, LVSKLDSGVP, YQGTHFPYT respectively.

[0015] Preferably, the heavy chain variable region thereof is:

[0016] ESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRLTPEKRLEWVAVISSGSSYTYYPDSVKGRFTISRDNVKSTLYLQMSS LRSEDTAMYYCARNWEGELHYYAMDYWGQGTS;

[0017] The light chain variable region thereof is:

[0018] QSPAIMSASLGERVTMTCVASSSVSSYLHWYQQKPGSSPKLWVYTTSSLASGVPARFSGSGSGTSYSLTINNMEAEDAAT YFCHQYHRSPYTFGGGTK;

[0019] The heavy chain variable region thereof is:

[0020] QSGPALVKPGASVKMSCKASGYTFTSYVIHWVKQKPGQGLERIGWIWPYNDGTKFNEKFKGKATLTSDKSSSTAYMELSS LTSEDSAVYYCSSQQLAYWGQGTT;

[0021] The light chain variable region thereof is:

[0022] QTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLIWLIQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLRISRVEA EDLGVYYCYQGTHFPYTFGGGTK.

[0023] In particular, the Linker between the heavy chain and the light chain is GGSSRSS.

[0024] The present application also provides a kit containing the scFv antibody.

[0025] In particular, it is a chemiluminescence kit. Preferably, it is a magnetic microparticle chemiluminescence kit.

[0026] More particularly, it comprises immunomagnetic beads RM; coating antibody; detection antibody; calibrator 1; calibrator 2; quality control 1; quality control 2;

[0027] The coating antibody is a biotin-labeled mouse anti-GP73 protein scFv antibody, and the detection antibody is an acridinium ester-labeled mouse anti-GP73 protein scFv antibody.

[0028] The calibrator is a GP73 protein antigen liquid for calibrating the standard curve on the machine, and the quality control is a GP73 protein antigen liquid for quality control of the sample.

[0029] In particular, the concentration of the calibrator is 300 and 100 mg / ml, and the concentration of the quality control is 150 and 50 mg / ml.

[0030] In a specific embodiment, the immunomagnetic beads RM are superparamagnetic microparticles covalently combined with high-purity streptavidin and suspended in PBS buffer to prepare a magnetic microparticle solution, preferably with a concentration of 1 mg / mL of the magnetic microparticle solution.

[0031] More particularly, it comprises:

[0032] One bottle of immunomagnetic beads RM, 4.2 ml / bottle;

[0033] One bottle of coating antibody, 11.2 ml / bottle;

[0034] One bottle of detection antibody, 11.2 ml / bottle;

[0035] One bottle of calibrator 1, 1 ml / bottle;

[0036] One bottle of calibrator 2, 1 ml / bottle;

[0037] One bottle of quality control 1, 1 ml / bottle;

[0038] One bottle of quality control 2, 1 ml / bottle.

[0039] One of the keys to the present invention is the use of phage display methods to prepare GP73 protein scFV antibodies. Unlike the common hybridoma cell-based antibody production method on the market, this method not only saves a lot of time, but also the prepared antibodies have excellent antigen-binding properties as tested by us.

[0040] The present invention selects the complete human GP73 protein sequence as an immunogen to immunize mice by analyzing the full-length sequence of human Golgi protein 73 (GP73). After the immunization cycle, the B cells of the mice are collected, the total RNA is extracted and reverse transcribed to generate cDNA. The VH and VL genes are amplified using human-specific antibody primers, and the scFv gene fragments are prepared by overlapping PCR. After the scFv gene fragment is fused with the pCANTAB-5E phagemid, it is electrotransduced into TG1 Escherichia coli, and the helper phage VCSM13 is added for overnight culture to construct a GP73 protein scFv antibody phage display library. After multiple rounds of screening, phages that can recognize the GP73 protein antigen are retained, and the scFv antibodies expressed by these phages are identified by mass spectrometry. Combined with sequence analysis, the GP73 protein scFv antibodies that meet the requirements are expressed and purified for preliminary identification, and used to prepare a detection kit to detect GP73 in human serum.

[0041] The beneficial effects of the present invention are as follows: ① In the prior art, some immunogens are fragments / polypeptides, which will lead to limited types and specificity of antibodies produced. We use full-length fragments for immunization, which produces more types of antibodies and recognizes richer epitopes of GP73; ② The present invention adopts a phage display method to prepare antibodies. Compared with the hybridoma method on the market, it not only saves a lot of time, but also saves a lot of manpower and material resources. The antibodies produced by this method can avoid the HAMA effect. Simply put, it is more effective in recognizing human proteins; ③ The GP73 protein scFv antibody obtained in the present invention not only retains the antigen binding site of the original monoclonal antibody, has high affinity and specificity, but also has better tissue permeability and faster blood clearance due to its small molecular weight (usually 25-30kDa), and is suitable for the treatment and diagnosis of tumors or deep tissue targets. ④ The GP73 antibody obtained by phage display and its derived magnetic microparticle chemiluminescence kit has a wider linear range of 0.1-1000ng / ml (the linear range in CN111378627B patent is 1ng / ml-475ng / ml), and the operation time is shorter, 30 minutes (CN101735319B is 70 minutes, CN111378627B is 90 minutes) BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Linear range of the kit.

[0043] Figure 2 .ROC curve of the magnetic microparticle chemiluminescence kit for GP73scFv antibody. DETAILED DESCRIPTION

[0044] Example 1 Two GP73 protein scFv antibodies were prepared using phage display technology.

[0045] 1) Construction of GP73 protein immunogen.

[0046] Primers were designed based on the full-length human GP73 gene sequence, and the full-length GP73 sequence was amplified by PCR using the cDNA as a template. EcoR I and Xba I sites were introduced at both ends of the sequence, and the recombinant plasmid was recombined with the pcDNA3.1-His plasmid. The resulting recombinant plasmid was transformed into Escherichia coli, cultured, and harvested for induced expression. Purified soluble GP73 protein was obtained after nickel affinity chromatography for subsequent animal immunization.

[0047] 2) Construction of GP73 protein scFv phage antibody library.

[0048] Five BALB / c mice aged 6-8 weeks and weighing approximately 20g were immunized. The antigen content of the first immunization was controlled at 0.1mg / mouse. The next immunization was performed one week later, and this process was repeated for four rounds. The purpose was to stimulate the immune system to produce specific antibodies against the antigen. After the last immunization, the mouse serum was collected to test the antibody titer, which was 6×10 5 After immunization, 30 mg of mouse spleen tissue was collected, and RNA was extracted and reverse transcribed to obtain immune information containing all GP73 antigens in lymphocytes. VH and VL genes were amplified using specific primers for human antibodies, and the VH and VL fragments were purified and recovered. Then, overlap PCR was performed using linker primers to splice the recovered full set of VH and VL genes into scFv genes. The scFv genes were then connected to the pCANTAB-5E phagemid vector and introduced into Escherichia coli TG1 by electroporation, and finally a library with a capacity of 6.0×10 7 Antibody library of fully human GP73 protein.

[0049] The E. coli containing the GP73 antibody signal was mixed with phage at a specific concentration. For this step, 50 μl of VSCM13 helper phage was used, and the phage concentration should be 50 times the E. coli concentration. After infection, the cells were cultured overnight, and the supernatant was precipitated with PEG8000 / NaCl and collected for subsequent screening and identification.

[0050] 3) Screening and identification of anti-GP73 protein scFv antibodies.

[0051] ELISA plates were coated with previously purified GP73 protein, and the constructed antibody library was subjected to 3-5 cycles of adsorption-elution-enrichment. Single clones were randomly selected from the screened bacterial colonies and identified by phage-ELISA. Phage that recognized the GP73 protein antigen were retained, and after multiple rounds of panning, two scFv antibodies with different nucleic acid sequences were ultimately obtained, designated SGA-1 and SGA-2. (The antibody gene sequences are shown in Table 1.) Combined with sequence analysis, the GP73 protein scFv antibodies that met the requirements were expressed, purified, and initially identified. They were then used to prepare a detection kit for detecting GP73 expression in human serum.

[0052] Table 1. Amino acid sequences of two scFv antibodies

[0053]

[0054]

[0055] Example 2: A magnetic microparticle chemiluminescence kit comprising the above-mentioned GP73 protein scFv antibody.

[0056] In this example, a Golgi protein 73 (GP73) magnetic microparticle chemiluminescence immunoassay kit (50 servings) was prepared, comprising:

[0057] One bottle of immunomagnetic beads (RM), 4.2 ml / bottle;

[0058] One bottle of coating antibody (R1), 11.2 ml / bottle;

[0059] One bottle of detection antibody (R2), 11.2 ml / bottle;

[0060] Calibrator 1 (CAL1), 1 ml / bottle;

[0061] One bottle of calibrator 2 (CAL2), 1 ml / bottle;

[0062] Quality control product 1 (QC1) one bottle, 1ml / bottle;

[0063] Quality control product 2 (QC2) one bottle, 1ml / bottle.

[0064] (1) Immunomagnetic beads (RM):

[0065] The immunomagnetic beads (RM) are superparamagnetic particles covalently bound to high-purity streptavidin and suspended in PBS buffer to prepare a magnetic particle solution with a concentration of about 1 mg / mL.

[0066] (2) Biotin-labeled mouse anti-GP73 protein scFv antibody (R1):

[0067] The biotin-labeled mouse anti-GP73 protein scFv antibody (R1) is conjugated with the scFv antibody SGA-1 using biotin, and the two are added at a molecular ratio of 20:1. After incubation at room temperature for 1 hour, desalting and purification are performed to remove free biotin, and the R1 component is diluted to a concentration of 0.5-1 ug / mL and stored in the dark at 2-8°C.

[0068] (3) Acridinium ester labeled mouse anti-GP73 protein scFv antibody (R2):

[0069] The acridinium ester-labeled mouse anti-GP73 protein scFv antibody (R2) uses acridinium ester to bind to the amino group of the scFv antibody SGA-2 to generate an antibody with a fluorescent signal. The two are added at a molecular ratio of 5-10:1, incubated at room temperature for 1 hour, desalted and ultrafiltered to remove free acridinium ester, and diluted to a concentration of 0.5-1 ug / mL to prepare the R2 component, which is stored at 2-8°C in the dark.

[0070] (4) Protein calibrators (CAL1 and CAL2)

[0071] The protein calibrators are prepared by dissolving GP73 antigen at different concentrations in diluent, at 100 ng / ml and 300 ng / ml (referred to as CAL1 and CAL2, respectively). They are primarily used for calibration / correction of the master curve. The liquid protein standard contains a lyophilized product containing a protein stabilizer and preservative, with a pH of 7.2 ± 0.2.

[0072] (5) Protein controls (QC1 and QC2)

[0073] The protein control samples are prepared by dissolving GP73 antigen in diluent at different concentrations, 50 ng / ml and 150 ng / ml (referred to as QC1 and QC2, respectively). Their purpose is primarily for quality control, and the measured concentration of the quality control samples should be within the target range. The liquid protein standard contains a lyophilized product containing protein stabilizers and preservatives, with a pH of 7.2 ± 0.2.

[0074] Example 3 Quality detection of the magnetic microparticle chemiluminescence kit containing the above-mentioned GP73 protein scFv antibody.

[0075] (1) Evaluation of linearity: The high-value sample close to the upper limit of the linear interval was diluted to at least 5 concentrations according to a certain ratio, among which the low-value concentration sample must be close to the lower limit of the linear interval. Each concentration of the sample was tested 3 times, and the average value was calculated. The average value and the dilution ratio were fitted with a linear least square method, and the linear correlation coefficient r was calculated. The linear correlation coefficient r = 0.9998 was obtained, and the linear range was 0.1ng / ml to 1000ng / ml. The results are shown in Table 1 and Figure 1 .

[0076] Evaluation results of linearity

[0077]

[0078] (2) Evaluation of accuracy: A known high concentration of GP73 sample (A) was added to a low concentration of serum B, with a volume ratio of 1:9 between the added sample A and sample B. The above mixture and serum B were each repeatedly tested 3 times, and the average value was calculated to obtain a recovery rate of 99.81%. The results are shown in Table 2.

[0079] Evaluation of accuracy

[0080]

[0081]

[0082] (3) Precision evaluation of the kit: Three batches of reagents were used for precision experiments, and high / low value standards were used for evaluation, each measured 20 times to calculate the coefficient of variation of the measured concentration. The results are shown in Table 3. The results show that the coefficient of variation is less than 5%.

[0083] Evaluation results of precision

[0084]

[0085] (4) Evaluation of blank limit (LOB): The zero value calibrator was measured 20 times for relative light units (RLU), and the RLU values of the 20 measurements were obtained. The average value (M) and standard deviation (SD) were calculated, and the RLU value corresponding to M+2SD was obtained. The RLU value corresponding to M+2SD was substituted into the calibration curve equation of the calibrator used in the kit to obtain the corresponding concentration value as the blank limit. The blank limit is <0.04 ng / mL. The results are shown in Table 4.

[0086] Evaluation results of blank limit

[0087]

[0088] (5) Evaluation of detection limit (LOD): The low value serum sample was diluted to a theoretical concentration of 0.05, 0.1, 0.15, 0.2, and 0.25 ng / mL, which is 1-5 times the blank limit concentration, and was numbered as (B1, B2, B3, B4, B5). The GP73 chemiluminescence assay kit was used to test the 5 low concentration samples for 3 consecutive days, and each sample was tested 4 times. The test results were calculated to obtain the LoD value. The detection limit is <0.05 ng / mL. The results are shown in Table 5.

[0089] Evaluation results of detection limit

[0090]

[0091]

[0092] (6) Limit of Quantitation (LOQ) Evaluation: Low-value serum samples were diluted to theoretical concentrations of 0.05, 0.1, 0.2, 0.3, and 0.4 ng / mL, respectively, and numbered (C1, C2, C3, C4, and C5). Five low-concentration samples were tested using the GP73 chemiluminescence assay kit on three consecutive days, with each sample tested four times. The LoQ values ​​were calculated from the test results. The TE at 0.1 ng / mL met the accuracy target of ≤25%. The results are detailed in Table 6.

[0093] Table 6 Evaluation results of quantitation limit:

[0094]

[0095] (7) Specificity evaluation: The kit was used to detect human serum albumin at a concentration of 1 mg / ml, alpha-fetoprotein (AFP) at a concentration of 2700 ng / ml, and glypican 3 (GPc3) at a concentration of 500 ng / ml. The test was repeated three times for each concentration. The detection results were ≤1.0 ng / ml, with no significant interference. The results are shown in Table 7.

[0096] Table 7 Specificity evaluation results:

[0097]

[0098]

[0099] (8) Stability: The reagent was placed in an oven at 2-8°C and 37°C for 7 days and 14 days, and the relative luminescence value (RLU) decay rate of the test calibrators and clinical samples was <5%. The results are detailed in Table 8.

[0100] Table 8 Stability evaluation results:

[0101]

[0102] Example 4 Clinical sample validation of the magnetic microparticle chemiluminescence kit containing the above-mentioned GP73 protein scFv antibody.

[0103] A total of 1,852 clinical samples were collected in this study. A receiver operating characteristic (ROC) curve was used to confirm the cutoff value for the magnetic microparticle chemiluminescence kit for GP73 protein scFv antibody. The agreement between the optimal threshold for positive and negative samples determined by the gold standard and the gold standard was evaluated.

[0104] The detailed selection information is shown in Table 9.

[0105] Table 9 Description of research samples

[0106] Hospital name Normal sample number Cirrhosis sample number Total Hunan Provincial People's Hospital 544(53.1％) 480(46.9％) 1024 Hunan Provincial Tumor Hospital 464(55.9％) 364(44.0％) 828 Total 1008(54.4％) 844(45.6％) 1852

[0107] Table 10 Description of baseline demographic data

[0108]

[0109]

[0110] Note: Q1: 25th percentile; Q3: 75th percentile

[0111] Table 11 Descriptive analysis of GP73 measurement results:

[0112]

[0113] Note: Q1: 25th percentile; Q3: 75th percentile

[0114] SPSS Statistics 26.0 statistical software was used to calculate the ROC curve. Figure 2 , AUC area, optimal cutoff, 95% CI and other values ​​are shown in Table 12.

[0115] Table 12 Calculation results of ROC curve of magnetic microparticle chemiluminescence kit for GP73 monoclonal antibody

[0116] Number Statistical value Sample size Positive samples = 844, negative samples = 1008 Best threshold (Cutoff) 3.49 (sensitivity = 94.79%; specificity = 97.32%) Youden index J 0.923 AUC area 0.986 Standard error Se 0.0132 95% CI 0.982-0.990

[0117] *AUC: Area under the ROC curve

[0118] As shown in Table 12, the area under the ROC curve of the test reagent is 0.986, which has good diagnostic value. The highest Youden index J value is 0.923, and the corresponding concentration value is 3.49 ng / mL. The sensitivity at the optimal threshold of 3.49 ng / ml is 94.79%; the specificity is 97.32%.

[0119] Gold standard comparison, compliance rate, sensitivity and specificity analysis

[0120] Of the 844 liver cirrhosis samples tested in this experiment, 800 tested positive and 44 tested negative, resulting in a sensitivity of 800 / 844 = 94.79%. Of the 1008 healthy samples tested, 981 tested negative and 27 tested positive, resulting in a specificity of 981 / 1008 = 97.32%, and an overall coincidence rate of (981 + 800) / 1852 = 96.17%. The results are shown in Table 13.

[0121] Table 13 Results of paired test of gold standard and assessment reagents

[0122]

[0123] Table 14. Kappa consistency test statistical results

[0124] Number Statistical value PA, Pe, Kappa value 0.9276, 0.5048, 0.9226, Kappa value ≥ 0.75, good consistency Standard error 0.0202 95% CI upper limit 0.9622 95% CI lower limit 0.8830 Test value Z Z = 12.5027, probability value P < 0.0001 Test result P < 0.05, reject H0, Kappa value from the total population of non-0 .

Claims

1. A scFv antibody, characterized in that The sequences of CDR1 to CDR3 of the heavy chain variable region are TYAMS, VISSGSSYTYYPDSVKG, and EGELHYYAMD, respectively; The sequences of CDR1 to CDR3 of the light chain variable region are VASSSVSSYLH, TTSSLASG, and HQYHRSPYT, respectively; or the sequences of CDR1 to CDR3 of the heavy chain variable region are SYVIH, WIWPYNDGTKFNEKFKG, and QQLAY, respectively; The sequences of CDR1 to CDR3 of the light chain variable region are KSSQSLLYSDGKTYLIWLI, LVSKLDSGVP, and YQGTHFPYT, respectively.

2. The scFv antibody according to claim 1, wherein The heavy chain variable region is: ESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRLTPEKRLEWVAVISSGSSYTYYPDSVKGRFTISRDNVKSTLYLQMSSLRSEDTAMYYCARNWEGELHYYAMDYWGQGTS; Its light chain variable region is: QSPAIMSASLGERVTMTCVASSSVSSYLHWYQQKPGSSPKLWVYTTSSLASGVPARFSGSGSGTSYSLTINNMEAEDAATYFCHQYHRSPYTFGGGTK; Or its heavy chain variable region is: QSGPALVKPGASVKMSCKASGYTFTSYVIHWVKQKPGQGLERIGWIWPYNDGTKFNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCSSQQLAYWGQGTT; Its light chain variable region is: QTPLTLSVTIGQPASISCKSSQSLLYSDGKTYLIWLIQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLRISRVEAEDLGVYYCYQGTHFPYTFGGGTK.

3. The scFv antibody according to claim 1, wherein The linker between the heavy chain and the light chain is GGSSRSS.

4. A kit comprising the scFv antibody according to any one of claims 1 to 3.

5. The kit according to claim 4, wherein It is a chemiluminescent kit.

6. The kit according to claim 5, wherein It is a magnetic microparticle chemiluminescence kit.

7. The kit according to claim 6, wherein include Immunomagnetic beads RM; coating antibody; detection antibody; calibrator 1; calibrator 2; quality control product 1; quality control product 2; The coating antibody is a biotin-labeled mouse anti-GP73 protein scFv antibody, and the detection antibody is an acridinium ester-labeled mouse anti-GP73 protein scFv antibody; The calibrator is GP73 protein antigen liquid used to calibrate the standard curve on the machine, and the quality control is GP73 protein antigen liquid used for sample quality control.

8. The kit according to claim 7, wherein The concentrations of calibrators were 300 and 100 mg / ml, and the concentrations of controls were 150 and 50 mg / ml.

9. The kit according to claim 7 or 8, wherein The immunomagnetic beads RM are superparamagnetic particles covalently bound to high-purity streptavidin and suspended in PBS buffer to prepare a magnetic particle solution.

10. The kit according to claim 9, wherein The concentration of the magnetic particle solution is 1 mg / mL.

11. The kit according to claim 10, wherein include One bottle of immunomagnetic beads RM, 4.2 ml / bottle; One bottle of coating antibody, 11.2 ml / bottle; One bottle of detection antibody, 11.2ml / bottle; Calibrator 1, one bottle, 1 ml / bottle; One bottle of calibrator 2, 1 ml / bottle; Quality control product 1, one bottle, 1ml / bottle; Quality control product 2, one bottle, 1ml / bottle.

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