CN111378627A

CN111378627A - Monoclonal antibody of Golgi protein 73, kit and application

Info

Publication number: CN111378627A
Application number: CN202010164717.5A
Authority: CN
Inventors: 王卫东
Original assignee: Tianjin Jinhong Biotech Co ltd; Hainan Zhongsen Biotechnology Co ltd
Current assignee: Tianjin Jinhong Biotech Co ltd; Hainan Zhongsen Biotechnology Co ltd
Priority date: 2020-03-11
Filing date: 2020-03-11
Publication date: 2020-07-07
Anticipated expiration: 2040-03-11
Also published as: CN111378627B

Abstract

The invention relates to a Golgi protein 73 monoclonal antibody, a kit containing the antibody and application of the monoclonal antibody in detection of Golgi protein 73. The invention also relates to a method for detecting the level of the golgi protein 73 in the serum. The detection kit containing the GP73 monoclonal antibody is used for detecting GP73 in human serum, and has high sensitivity and strong specificity. In addition, the kit and the method provided by the invention can be clinically used for auxiliary diagnosis of liver cirrhosis.

Description

Monoclonal antibody of Golgi protein 73, kit and application

Technical Field

The invention relates to the field of biological detection, in particular to a monoclonal antibody of Golgi protein 73, a kit containing the monoclonal antibody and application of the monoclonal antibody in detection of the Golgi protein 73.

Background

Cirrhosis is a diffuse hepatic lesion formed by long-term or repeated action of one or more etiologies in the late stage of the development of various chronic liver diseases. In China, the majority of the patients are posthepatitic cirrhosis, and the minority of the patients are alcoholic cirrhosis and schistosomiasis cirrhosis. Histopathology includes extensive hepatocyte necrosis, nodular regeneration of residual hepatocytes, connective tissue hyperplasia and fibrosepta formation, which results in structural destruction of hepatic lobules and formation of pseudolobules, and the liver gradually deforms and hardens to develop cirrhosis. The liver compensation function is strong in the early stage, no obvious symptom exists, liver function damage and portal hypertension are mainly shown in the later stage, multiple systems are involved, and complications such as upper gastrointestinal hemorrhage, hepatic encephalopathy, secondary infection, splenic hyperfunction, ascites, canceration and the like often appear in the later stage.

The current methods for diagnosing cirrhosis mainly include: imaging examination, liver biopsy, and laboratory serological examination. The result of the imaging examination is reliable, but the result can be found only when the liver tissue structure is obviously changed, so the early diagnosis is not facilitated. Liver biopsy can establish a definitive diagnosis, but is poorly accepted by patients due to its invasiveness. However, the current laboratory serological detection method is difficult to provide clear diagnosis of liver cirrhosis, and takes four examinations (type III procollagen, type IV collagen, laminin and hyaluronic acid) in the stage of hepatic fibrosis as an example, because the four examinations are greatly influenced by liver inflammation, the specificity and the sensitivity of the four examinations are not high, and the requirements of clinical diagnosis of liver cirrhosis cannot be met.

Golgi protein 73(Golgi protein 73, GP73) was first discovered by Kladney et al in the year 2000 when studying the etiology of adult cytomegalohepatitis. GP73 is a Golgi transmembrane glycoprotein and has a relative molecular weight of 73kD when electrophoretically shown, and is therefore designated GP 73. The gene for coding GP73 protein is located on chromosome 9, the total length is 3080 nucleotides, the coding region is located on 199-1404 nt genes, and a unique open reading frame (1200-1430 bp) is contained in the coding region. GP73 is located anterogradely in the golgi, is glycosylated in vivo and is rich in acidic amino acids, and is mainly divided into 5 parts: amino acids 1 to 12 of the N-terminus are cytoplasmic domains, located in the cytoplasm; amino acids 12 to 35 are transmembrane domains; amino acids 36 to 205 are coiled-coil structures responsible for interactions with other proteins; amino acids 206 to 348 are amorphous domains; the amino acids 349 to 402 are acidic fragment regions and have hydrophilicity. The N end is a hydrophobic end and is mainly positioned in a Golgi body to form a transmembrane region and a signal peptide cutting site. The C end is positioned outside the Golgi body and comprises a tetradecanoylation continuous sequence, five glycosylation sites, an acid structural domain and a coiled coil structure.

It was found that GP73 is expressed predominantly in biliary epithelial cells and not or very little in hepatocytes. The expression level tends to increase gradually in the process of benign liver diseases, and the expression level of GP73 is increased gradually from acute and chronic hepatitis to liver cirrhosis, and the expression level is highest in liver cirrhosis tissues. The GP73 expression pathway may have different mechanisms: one is triggered by acute hepatocyte injury, while the other is present during the progression of chronic liver disease. The GP73 protein is thought to act on other proteins through its extracellular coiled coil region, is an important substance for maintaining the growth and development of liver, kidney and other internal organs, and plays an important role in the process of prenatal maturation of mammalian and amphibian embryos.

Disclosure of Invention

The invention selects GP73aa180-aa280 to express through analyzing the full-length sequence of the Golgi protein 73 (which can be abbreviated as GP 73). Preparing hybridoma cells by using the expressed GP73aa180-aa280 antigen, and further obtaining the monoclonal antibody aiming at the GP73aa180-aa280 fragment epitope. The GP73 monoclonal antibody is used for preparing a detection kit and detecting GP73 in human serum, and has high sensitivity and strong specificity.

Specifically, the method comprises the following steps:

the first purpose of the invention is to provide a Golgi protein 73 monoclonal antibody hybridoma cell strain.

The first Golgi protein 73 monoclonal antibody hybridoma cell strain provided by the invention, namely GP73 monoclonal antibody cell strain-1, is deposited in China general microbiological Culture Collection Center (CGMCC), No. 3 of Beijing university sunny West Lu No.1 of the south-facing-the-sun district, institute of microbiology, China academy of sciences, zip code 100101, with the preservation number of CGMCC No.19282, with the preservation date of 2020, 1 month and 2 days, and is named by classification: hybridoma cell lines.

The second strain of the hybridoma cell strain of the monoclonal antibody of the golgi protein 73, which is GP73 monoclonal antibody cell strain-2, is deposited in the common microorganism Center of the China committee for Culture Collection of microorganisms (China general microbiological Culture Collection Center, CGMCC), north cheng west road No.1 hospital No. 3, the institute for microbiology of China academy of sciences, zip code 100101, the Collection number is CGMCC No.19283, the Collection date is 2020, 1 month and 2 days, and the classification is named: hybridoma cell lines.

It is a second object of the present invention to provide a monoclonal antibody to Golgi protein 73. The antibody is secreted by the hybridoma cell strain.

It is a third object of the present invention to provide a kit comprising a monoclonal antibody to the Golgi protein 73.

The kit can be a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme linked immunosorbent assay kit or a fluorescence immunoassay kit.

As a preferred scheme of the invention, the kit is an enzyme linked immunosorbent assay kit.

As a preferred scheme of the invention, the enzyme linked immunosorbent kit comprises: an ELISA plate coated by the Golgi protein 73 monoclonal antibody, and a conjugate of the Golgi protein 73 monoclonal antibody and horseradish peroxidase. Compared with a polyclonal antibody, the monoclonal antibody has the characteristics of high specificity, high uniformity, high repeatability and the like, and the prepared kit has high stability.

As a preferable scheme of the invention, the calibrator contained in the enzyme linked immunosorbent assay kit is a liquid protein standard prepared by dissolving GP73 protein in a diluent. The liquid protein standard is preferably added with 0.03-0.07% of thimerosal and 0.03-0.07% of Proclin300 preservative, so that GP73 protein can be stably stored in liquid at the temperature of 2-8 ℃ for a long time, and the quality guarantee period can reach 9 months. Most of the calibrators in other similar kits are freeze-dried products or need to be stored at-20 ℃, and need to be diluted before use, so that the operation is complex and errors are easy to generate.

As a preferable scheme of the invention, the color developing agent contained in the enzyme linked immunosorbent assay kit is a single-component TMB color developing solution. The TMB, namely 3,3',5,5' -tetramethyl benzidine, is white crystal powder, is odorless and tasteless, is insoluble in water, is soluble in organic solvents such as acetone, diethyl ether, dimethyl sulfoxide and dimethylformamide, and is a novel safe chromogen reagent. The invention adopts the single-component developing liquid, can avoid the defects of complex operation, large batch difference and the like caused by separately preparing the two-component developing liquid and mixing the two-component developing liquid before use, reduces the operation steps and improves the accuracy.

The kit can further comprise washing solution, stop solution, a sealing plate membrane and other components.

The fourth purpose of the invention is to provide the application of the hybridoma cell strain, the monoclonal antibody or the kit in detecting the serum albumin 73.

The fifth purpose of the invention is to provide a method for detecting the serum middle-JIA 73 by adopting the hybridoma cell strain, the monoclonal antibody or the kit.

In order to improve the detection accuracy, the invention preferably adopts a method comprising the following steps of: collecting venous blood, standing for more than half an hour for full coagulation, horizontally centrifuging at 1300-1500 rpm for 5-10 min, and taking upper serum.

In a preferred embodiment of the present invention, the method employs a double antibody sandwich method to quantitatively detect a target substance (i.e., golgi protein 73). In actual operation, a sample to be detected is added into an enzyme label plate coated by a monoclonal antibody of the Golgi protein 73 for reaction, a conjugate of the monoclonal antibody of the Golgi protein 73 and horseradish peroxidase is added, and then the reaction is performed with a color developing agent, wherein the color developing intensity is related to the concentration of a target substance in the sample to be detected.

The method provided by the invention is used for measuring the GP73 content in human serum, the reaction time is short, and compared with the similar ELISA kit, the operation time is reduced by 50 minutes. The GP73 in the serum is the only serological marker for liver cirrhosis at home and abroad at present, so the kit and the method provided by the invention can be clinically used for the auxiliary diagnosis of liver cirrhosis.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

This example provides a stock solution of monoclonal antibody to Golgi protein 73.

The antibody stock solution is obtained by injecting hybridoma cells preserved by the invention into Balb/c mice to prepare ascites and then purifying Protein G.

Specifically, the hybridoma with the preservation number of CGMCC No.19282 is injected into a Balb/c mouse to prepare ascites, and then Protein G is purified to obtain GP73 monoclonal antibody stock solution-1. Injecting hybridoma with the preservation number of CGMCC No.19283 into Balb/c mouse to prepare ascites, and purifying Protein G to obtain GP73 monoclonal antibody stock solution-2.

Example 2

This example provides kits for detecting human serum GP73 levels. The specification of the kit is 96 parts per box, and the kit comprises the following components:

(1) GP73 antibody coated plate (96 well plate × 1 in each kit)

The coated plate is prepared by the following method: the GP73 monoclonal antibody stock solution-1 provided by the embodiment 1 is coated on a 96-hole enzyme label plate according to the concentration of 2 mug/ml, and is incubated for not less than 16 hours at the temperature of 2-8 ℃; removing the coating solution in the ELISA plate, washing with phosphate buffer solution for 1 time, adding confining liquid, and incubating at 2-8 deg.C for no less than 16 hr; and (4) throwing off the confining liquid in the ELISA plate, drying and storing at 2-8 ℃.

(2) GP73 calibrator (0.5 mL × 8 bottles per kit)

The GP73 calibrator is prepared by expressing recombinant GP73 protein (GP 73 antigen for short) in escherichia coli by a genetic engineering method, and then adding 0.02M phosphate buffer solution to prepare a working calibrator solution with specific concentration.

(3) GP73 enzyme conjugate (12 mL × 1 bottles per kit)

The GP73 enzyme conjugate, namely the GP73 monoclonal antibody-horseradish peroxidase conjugate, is prepared by combining the monoclonal antibody stock solution-2 obtained in the embodiment 1 with horseradish peroxidase by a periodate oxidation method to obtain an enzyme-labeled antibody, configuring the enzyme-labeled antibody into a specific concentration by using an enzyme conjugate diluent, and storing the enzyme-labeled antibody at the temperature of 2-8 ℃ in a dark place.

(4) The washing solution was concentrated (20 ×) (50 mL × 1 bottles in each kit), which contained 0.4M phosphate buffer and Tween-20.

(5) Single-component TMB color developing solution (12 mL × 1 bottles in each kit).

(6) Stop solution (7 mL × 1 bottles in each kit) was composed of 2M sulfuric acid.

(7) Sealing plate film (3 pieces in each kit)

The kit is stored in a finished product warehouse at the temperature of 2-8 ℃, and the validity period is 12 months.

Example 3

This example provides a method for detecting human serum GP73 levels using the kit provided in example 2.

1. Collecting samples: collecting venous blood, placing for more than half an hour, fully coagulating, horizontally centrifuging at 1500rpm for 5-10 min, and taking upper serum.

In order to ensure the accuracy of the detection result, the invention requires that fresh samples are used as much as possible, and repeated freeze thawing is avoided; the fresh sample can be stored for 7 days at the temperature of 2-8 ℃, the storage life at the temperature of-20 ℃ is not more than 3 months, and if the fresh sample is stored for a long time, the fresh sample is placed at the temperature of-80 ℃ and is not more than 2 years; the sample is transported under the condition of freezing and is in accordance with the related biological safety regulation; samples with severe hemolysis, hyperlipidemia or severe contamination cannot be used; the samples could not use sodium azide as a preservative. If the sample is frozen, the sample is equilibrated at room temperature (18-28 ℃) for at least 30min before detection.

2. Sample adding: add 50. mu.l of calibrator (S0-S7) and serum sample (blank well not) to the respective wells, mix well by shaking for 30S.

The method provided by the invention can complete the determination without adding a sample diluent, and has strong reaction specificity; in comparison, the similar kits require the addition of a sample diluent to reduce non-specific reactions, and are complex to operate.

3. And (3) incubation: sealing the plate with sealing plate film, and incubating in 37 deg.C constant temperature water bath tank for 30 min.

4. Washing: carefully remove the sealing film, wash with a plate washer 6 times, add at least 300 μ l of washing solution per well each time, and dry on clean absorbent paper after the last wash.

5. Adding an enzyme conjugate: add 100. mu.l of enzyme conjugate per well (blank wells not) and mix well with shaking for 30 s.

6. And (3) incubation: the same as step 3.

7. Washing: the same as step 4.

8. Color development: adding 100 μ l of single-component TMB color developing solution into each well, oscillating for 30s, sealing with a sealing plate membrane, and incubating at 37 deg.C in dark for 10 min.

9. And (3) determination: add 50. mu.l of stop solution to each well, mix well by shaking for 30s, and measure the absorbance (OD value) of each well at a wavelength of 450nm using a microplate reader. The microplate reader does not need blank holes for zero adjustment. The measurement should be carried out within 10min after termination of the reaction.

10. Data processing: data were processed using a log-log linear fitting method. The logarithm of the concentration of each calibrator was plotted on the ordinate (Y-axis) and the logarithm of the OD value of each calibrator (minus the OD value of S0) on the abscissa (X-axis) to create a standard curve. And (3) measuring the OD value of the sample by a single-wavelength (450nm) microplate reader, subtracting the OD value of the blank hole, taking a logarithm, and calculating the GP73 concentration of the sample from the standard curve. The OD value of the sample measured by a dual-wavelength (450nm and 630nm) microplate reader is directly logarithmized, and the GP73 concentration of the sample is calculated from the standard curve.

The detection limit of the method provided by the invention is less than or equal to 1.00 ng/mL; the linearity is good, the correlation coefficient is more than or equal to 0.9900 within the range of 1.00 ng/mL-475.00 ng/mL; the repeatability in batches is good, and the coefficient of variation CV is less than or equal to 15 percent; the repeatability among batches is good, and the coefficient of variation CV is less than or equal to 15 percent; high accuracy and high recovery rate (85-115%).

Example 4

This example was subjected to a clinical trial using the kit provided in example 2.

The test collects 1300 samples in total, wherein the effective sample 1299 meeting the sample selection condition accounts for 99.9 percent of the total samples, and the detailed selection is shown in table 1.

TABLE 1 study sample Condition description

The specific reasons for the 1 invalid sample are detailed in table 2.

TABLE 2 knockout case description

Center of a ship	Serial number	Sex	Age (year of old)	Reason for rejection
					2	629	Woman	57	Undetected results

TABLE 3 Baseline demographic data description (FAS)

1Q1, 25 decitex; 2Q3 75 percentile

TABLE 4 descriptive analysis (FAS) of the GP73 measurements

1Q1, 25 decitex; 2Q3 75 percentile

TABLE 5 Baseline demographic data description (PPS)

1Q1, 25 decitex; 2Q3 75 percentile

TABLE 6 descriptive analysis of the GP73 measurements (PPS)

1Q1, 25 decitex; 2Q3 75 percentile

Evaluation of diagnostic accuracy

And comparing with a gold standard, describing the sensitivity, specificity, coincidence rate and the like of the assessment reagent, giving a 95% credible interval of the corresponding sensitivity and specificity, and comparing the sensitivity and specificity with a target value by using Z test. And analyzing the diagnosis threshold value and the diagnosis accuracy of the assessment reagent by adopting an ROC curve. The analysis was performed on both FAS and PPS datasets.

As can be seen from the following table, the area under the ROC curve of the assessment reagent is 0.926, and the assessment reagent has better diagnostic value. The sensitivity at the optimal threshold and the 3.65ng/ml threshold was 89.50%, significantly higher than the target value of 0.75; the specificity was 95.57%, significantly higher than the target value of 0.70.

TABLE 7 evaluation reagent area under ROC curve (PPS)

AUC	Standard error of	Lower 95% CI of AUC	95% CI Upper Limit of AUC	Optimal threshold (mm)
					0.926	0.009	0.909	0.944	3.54

AUC: area under ROC curve

TABLE 8 diagnostic accuracy description (case distribution) of the assessment reagents at optimal thresholds (PPS)

TABLE 9 diagnostic accuracy index (PPS) of the assessment reagent at optimal threshold

Index (I)	Sensitivity of the probe	Degree of specificity	Rate of agreement	Kappa coefficient
					Estimated value	0.8950	0.9557	0.9276	0.8539
Standard error of	0.0125	0.0078	0.0072	0.0145
					Lower limit of 95% CI	0.8705	0.9404	0.9135	0.8255
Upper limit of 95% CI	0.9195	0.9709	0.9417	0.8823
					Comparison with the target value-Z	11.6000	32.7821	-	-
Comparison with the target value-P	<0.0001	<0.0001	-	-

TABLE 10 diagnostic accuracy description (case distribution) of the assessment reagent at a threshold of 3.65ng/ml (PPS)

TABLE 11 diagnostic accuracy index (PPS) for assessment reagent at 3.65ng/ml threshold

Conclusion

The analysis shows that the Golgi protein 73 kit (enzyme linked immunosorbent assay) provided by the invention is effective in the determination result of GP73 in human serum. The kit meets the requirements of clinical determination and can be widely used for clinical detection.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. The Golgi protein 73 monoclonal antibody hybridoma cell strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.19282 or CGMCC No. 19283.

2. A monoclonal antibody against Golgi protein 73, which is secreted by the hybridoma cell line of claim 1.

3. A kit comprising a monoclonal antibody to golgi protein 73 according to claim 2.

4. The kit according to claim 3, wherein the kit is a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme linked immunosorbent assay kit or a fluorescent immunoassay kit.

5. The kit of claim 4, wherein the kit is an enzyme linked immunosorbent kit comprising: an ELISA plate coated by the Golgi protein 73 monoclonal antibody, and a conjugate of the Golgi protein 73 monoclonal antibody and horseradish peroxidase.

6. The kit of claim 5, wherein the ELISA kit further comprises a liquid protein calibrator prepared by dissolving Golgi protein 73 in a diluent;

and/or the enzyme linked immunosorbent assay kit also comprises a color developing agent which is a single-component TMB color developing solution.

7. The hybridoma cell strain of claim 1, the monoclonal antibody of claim 2 or the kit of any one of claims 3 to 6, for use in detecting serum albumin 73.

8. A method for detecting the serum middle-JI protein 73 by using the hybridoma cell strain of claim 1, the monoclonal antibody of claim 2 or the kit of any one of claims 3 to 6.

9. The method of claim 8, wherein the sample to be tested is obtained by a method comprising the steps of: collecting venous blood, standing for more than half an hour for full coagulation, horizontally centrifuging at 1300-1500 rpm for 5-10 min, and taking upper serum.

10. Method according to claim 8 or 9, characterized in that it comprises the following steps: adding a sample to be detected into an ELISA plate coated by the monoclonal antibody of the Golgi protein 73 for reaction, adding a conjugate of the monoclonal antibody of the Golgi protein 73 and horseradish peroxidase, and reacting with a color developing agent, wherein the color developing intensity is related to the concentration of a target substance in the sample to be detected.