CN107746430B - Preparation and application of GP 73C-terminal antigen - Google Patents

Abstract

The invention provides GP 73C-terminal protein, so that a monoclonal antibody resisting GP 73C-terminal is prepared, and aims to overcome the defect that a GP73 quantitative detection kit is lack of as a means for early diagnosis and detection of liver cirrhosis clinically at present, the invention also aims to provide a detection kit which is simple, convenient to operate, accurate, sensitive, stable in quality and capable of being produced in a large scale and a determination method thereof, so that the GP73 expression content can be observed from the protein level, an effective comprehensive treatment scheme can be adopted as soon as possible, and further malignant change of liver cirrhosis can be effectively prevented.

Description

Preparation and application of GP 73C-terminal antigen

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to preparation and application of GP 73C-terminal antigen.

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 causes of liver cirrhosis are many, and can be viral liver cirrhosis, alcoholic liver cirrhosis, metabolic liver cirrhosis, cholestatic liver cirrhosis, hepatic vein reflux resistant liver cirrhosis, autoimmune liver cirrhosis, toxic and drug-induced liver cirrhosis, malnutritional liver cirrhosis, cryptogenic liver cirrhosis, and the like.

The Child-Pugh classification standard is a classification standard which is commonly used in clinic for quantitative evaluation of liver reserve function of cirrhosis patients, and is firstly proposed by Child in 1964, and Child divides different states of 5 indexes (including general conditions, ascites, serum bilirubin, serum albumin concentration and prothrombin time) of the patients into three levels, respectively recording the three levels by 1 point, 2 points and 3 points, and adds the scores of the 5 indexes, wherein the sum is divided into 5 points at the lowest and 15 points at the highest, so that the liver reserve function is divided into A, B, C levels according to the number of the sum, and liver damage with three different degrees of severity is predicted (the higher the score is, the worse the liver reserve function is). However, since the general condition of patients is often not easily scored, Pugh subsequently proposes to replace the general condition with the presence and extent of hepatic encephalopathy, a Child-Pugh modified classification method commonly used in clinics today.

Cirrhosis can be divided into compensatory and decompensated cirrhosis. Compensatory liver cirrhosis corresponds to grade A in the Child-Pugh standard, and decompensation corresponds to grades B and C.

Cirrhosis disease presents a chronic process. At any stage of Child-Pugh, or at compensatory stage and decompensated stage, the pathological histology of the liver has extensive hepatocyte necrosis, nodular regeneration of residual hepatocytes, connective tissue hyperplasia and fibroseptal formation, which results in the structural damage of hepatic lobules and the formation of pseudolobules, and the liver gradually deforms and hardens to develop into cirrhosis.

Hepatic fibrosis is a continuously progressive process in disease evolution, and is also an intermediate inevitable stage in the progression of chronic liver disease into cirrhosis.

Clinically, it is difficult to clearly distinguish liver fibrosis from liver cirrhosis, but the two are fundamentally different. Liver fibrosis is not an independent disease, but is a liver disorder accompanied by various chronic liver diseases caused by excessive deposition of diffuse extracellular matrix (particularly collagen) in the liver, and liver cirrhosis caused by excessive fibrosis to make the liver atrophy and harden.

Liver fibrosis is a pre-stage lesion of liver cirrhosis, the early stage of which is reversible, while liver cirrhosis is the result of further development of liver fibrosis, the terminal manifestation of various liver diseases, and is generally difficult to reverse.

The pathological features of hepatic fibrosis are that there is a lot of fibrous tissue hyperplasia and deposition in the region of the junction and the lobules, but the lobular space is not formed yet, the cirrhosis has the formation of false lobules, the central venous region and the junction region have the space, the normal structure of the liver is destroyed, and the further development of hepatic fibrosis is cirrhosis.

In summary, cirrhosis is a chronic progressive liver disease. Early diagnosis is helpful for effective treatment in hepatic fibrosis stage, so that liver diseases become reversible, and the prognosis of liver cirrhosis is poor if the liver diseases are not developed.

The current auxiliary detection method for diagnosing liver cirrhosis mainly comprises the following steps: imaging examinations, liver biopsies and laboratory serological examinations. The result of the imaging examination is reliable, but the result can be found only when the tissue structure of the liver is obviously changed, so the early diagnosis of the liver cirrhosis is not facilitated. Liver biopsy can establish a definitive diagnosis of cirrhosis, but is less acceptable to patients due to its invasiveness. At present, serological detection in a laboratory is hepatic fibrosis detection, and clear diagnosis of cirrhosis is difficult to provide. Taking four examinations (type iii procollagen, type iv collagen, laminin, and hyaluronidase) in the hepatic fibrosis stage as an example, the four examinations in hepatic fibrosis are not highly specific and sensitive because they are greatly affected by inflammation of the liver.

GP73, also known as Golm1 or Golph2, was the first type ii golgi transmembrane protein discovered by researchers in 2000. The molecular weight is about 45KD, and the sequence number is 51280 in GeneBank after glycosylation is about 73 KD. Because 7.3X 10 is displayed during electrophoresis⁴Thus designated GP 73. The gene for coding GP73 is located on chromosome 9, has total 3080 nucleotides, contains 1200bp open reading frame in the gene, codes 402 amino acids and has 5 glycosylation sites. GP73 is a transmembrane protein composed of three regions, intracellular, transmembrane and extracellular. Immunohistochemical studies confirmed that: many cells express GP73, while normal hepatocytes express little to no expression. GP73 is normally an integral membrane protein on the golgi membrane and is secreted intracellularly and on the cell surface in disease cases. The researchers confirmed that: GP73 is secreted extracellularly by the action of the cleavage enzyme into the peripheral blood.

Further, researchers find that: the level of GP73 in liver tissues of patients with chronic hepatitis B, chronic hepatitis C, alcoholic hepatitis and autoimmune hepatitis is 70 times higher than that of normal people. GP73 expression was elevated in various liver diseases suggesting that GP73 levels were not associated with viral infection. Further research shows that: all liver diseases induced elevation of GP73 in all cells, but were particularly prominent around connective tissue and at sites of cirrhosis nodules.

According to the data retrieval carried out by the inventor, no other reports about the double-monoclonal antibody sandwich ELISA method for detecting the GP73C end exist at home and abroad.

Disclosure of Invention

In order to investigate the sensitivity and specificity of the monoclonal antibody for detecting GP73 protein in the diagnosis of liver cirrhosis, the present invention has performed a large number of clinical trials. The result shows that the serum GP73 can be used as an auxiliary diagnosis index of the liver cirrhosis, and has more important clinical diagnosis value particularly in the process of converting the chronic hepatitis into the liver cirrhosis.

The invention aims to provide GP73C terminal protein, the amino acid sequence of which is SEQ ID NO. 1.

The invention also aims to provide a preparation method of GP 73C-terminal protein, which mainly comprises the following steps:

(1) and (3) PCR amplification: PCR amplifying GP 73C-terminal gene (1019-1336 bp) by using a primer containing BamHI and EcoRI enzyme cutting sites, connecting the PCR amplified gene with PGEX-4T-2 plasmid subjected to BamHI and EcoRI enzyme cutting after enzyme cutting purification, and converting the PCR amplified gene into escherichia coli BL 21;

(2) determining the expression form: randomly selecting positive clones, chemically inducing with 1% isopropyl-beta-D-thiogalactopyranoside (IPTG) at 25 ℃, cracking with 1% NP-40, repeatedly freezing and thawing at-80 ℃ for 3 times, centrifuging, taking supernatant and precipitate, respectively carrying out SDS-polyacrylamide gel electrophoresis (SDS-PAGE) identification, and determining an expression form;

(3) and (3) amplification culture and purification: selecting positive clone for amplification culture, cracking by 1% NP-40, repeatedly freezing and thawing at-80 ℃ for 3 times, and purifying GP73C terminal antigen by chromatography.

Wherein, the upstream primer in the step (1) is GC GCT GGA TCC CAG CTG GCC TCA (5 '-3', and the downstream primer is GC GAA TTC TGA TGTGAG ATGATT.

The invention also aims to provide a preparation method of the GP 73C-terminal protein monoclonal antibody, which mainly comprises the following steps:

(1) animal immunization

Mixing 300mg/L of recombinant GP73C terminal antigen protein with the same amount of complete Freund's adjuvant to prepare an emulsifier, and immunizing 5 Balb/c mice together; the 1 st immunization is carried out at a dose of 100 mug/mouse, subcutaneous multiple injection is carried out, the 2 nd immunization is carried out after 2 weeks by using incomplete Freund's adjuvant, and the dose and the route are the same as those of the 1 st immunization; after 2 weeks, tail vein blood is taken to determine the potency, and the potency reaches 1: 1000-1: 5000 hours for fusion; 3 days before fusion, the antigen is directly used for strengthening immunity of the abdominal cavity and the spleen region for 1 time;

(2) measurement of potency by Indirect ELISA

The concentration of purified recombinant GP73C end antigen of the coated polystyrene plate is 5mg/L, an enzyme-labeled antibody is goat anti-mouse IgG (a product of Sigma company, the working concentration is 1: 5000), and the OD value is measured by an enzyme-labeling instrument at the wavelength of 450 nm; the P/N value is more than or equal to 2.1 times positive and less than 2.1 times negative (the N value is less than 0.05, calculated according to 0.05);

(3) preparation of immune spleen cells

Taking an immunized Balb/c mouse, and discharging blood from an eyeball for detecting an antibody; taking out spleen under aseptic operation, gently cleaning and removing connective tissue, placing on copper net, squeezing, grinding, squeezing into solution through mesh, centrifuging for 5min at 1000r/min, discarding supernatant, suspending in solution to obtain spleen cell suspension, and adding trypan blue dye solution for cell counting;

(4) preparation of myeloma cell broth

Place myeloma cells at 37 ℃ 5% CO₂Carrying out amplification culture in an incubator; collecting SP2/O myeloma cells in logarithmic growth phase on the day of fusion, centrifuging for 5min at 1000r/min, discarding supernatant, and performing cell counting after resuspension in liquid;

(5) cell fusion

Spleen cell and myeloma cell suspensions were aspirated separately as 5: 1, mixing, placing the mixture in a centrifugal tube, fully and uniformly mixing, centrifuging, removing supernatant, and lightly flicking the bottom of the tube to ensure that precipitated cells are loosened and uniformly mixed into paste; adding 0.7ml of 50% polyethylene glycol (PEG) into the mixed cells to promote fusion, strictly controlling the action time within 2-3 min, and immediately adding incomplete culture solution to dilute so as to stop the action of the PEG; centrifuging at 800r/min for 7min, and discarding the supernatant; adding 10mL HAT culture solution to prepare cell suspension, adding 0.1mL of the cell suspension into a 96-well plate paved into a feeder cell layer, culturing in a 5% CO2 incubator at 37 ℃ for 4-5 d, changing the solution for 1 time, taking the supernatant when 8-10 d, measuring the titer by an ELISA method, performing primary screening, and performing subcloning on positive holes;

(6) cloning of hybridoma cells (limiting dilution method) and ascites preparation

Repeatedly and uniformly blowing hybridoma cells to be cloned in a 96-well plate by using a sample injector, and then taking a small amount of cell suspension to place in another sterile vial; serial dilution of this cell suspension was performed accurately until 10 cells per ml; the diluted cell suspension was inoculated into a 96-well plate containing 0.1mL of feeder cells per well, i.e., one cell per well, and placed at 37 ℃ in 5% CO₂Culturing in an incubator, and observing the cell clone growth condition under an inverted microscope for about 5 days; repeating cloning for 3 times to determine positive monoclonal cell strain, numbering the cloned cell strain, and storing in liquid nitrogen.

Another object of the present invention is to provide an agent for diagnosing liver diseases, which comprises a monoclonal antibody against the C-terminal protein GP 73.

Another object of the present invention is to provide a kit for diagnosing liver diseases, which comprises a monoclonal antibody against GP 73C-terminal protein.

It is another object of the present invention to provide the use of the above-mentioned reagent and/or kit for the preparation of a product for diagnosing liver diseases.

Specifically, 3 pairs of monoclonal antibodies with better pairing are screened by the invention aiming at different epitopes of the ectodomain of GP 73.

Moreover, the invention discloses the application of the monoclonal antibody i) in the aspect of a kit for detecting the GP73 content in serum by an ELISA method; ii) the application of the kit for detecting the GP73 content by a magnetic bead chemiluminescence method; iii) the application of the kit for detecting the content of fucosylated GP73 in serum by an ELISA method.

The quantitative detection method of GP 73C-terminal protein sandwich ELLSA is characterized in that two monoclonal antibodies aiming at different epitopes of GP 73C-terminal protein are respectively used as a coating antibody and a detection antibody, GP73 protein is detected through substrate color development, and the content of GP73 protein is determined through a standard curve prepared by a calibrator.

Wherein, the coated antibody coats the ELISA plate, and the detection antibody marks horseradish peroxidase.

It should be noted that: the C-terminal peptide of GP73 has good hydrophilicity and accessibility, encodes amino acids located in the extracellular domain, contains no other transmembrane domains, and is unique among the human protein repertoire; meanwhile, serum GP73 was released extracellularly by furin cleavage at amino acid 55, with an intact C-terminus. Therefore, a sequence at the C terminal of GP73 is used as an epitope, a GP73C terminal protein is prokaryotic expressed, and the monoclonal antibody prepared by the method is directed at the GP73C terminal and used for producing a kit, so that the detection rate of GP73 in a sample can be improved.

The software analysis determines that the amino acids are 302-401 amino acids, and the result shows that the antigen binding site of the fragment is more exposed, so that the fragment has stronger antigenicity and is hydrophilic and soluble, and the fragment contains 100 amino acids, has larger molecular weight and is easy to store stably for a long time. The GP 73C-terminal protein is prepared by a genetic engineering method, which comprises the following steps: amplifying a DNA sequence for coding and expressing GP73C terminal by adopting a PCR method, then inserting the sequence into a multiple cloning site of an expression vector, constructing a recombinant expression plasmid, then transforming to an expression host bacterium, constructing an engineering strain, and obtaining GP73C terminal recombinant protein by the induced expression, separation and purification of the engineering strain. And (3) immunizing Balb/C mice with the expression and synthesis of GP 73C-end recombinant protein to prepare and screen hybridoma cell strains, prepare ascites and purify the monoclonal antibody.

A determination kit aiming at GP73 antigen is prepared by a double antibody sandwich method, and GP73 in human serum is quantitatively detected. The monoclonal antibody at the GP73C end is used for coating an enzyme label plate, serum to be detected is added, another enzyme conjugate of the monoclonal antibody at the GP73C end is added after reaction, the enzyme conjugate reacts with a TMB substrate, and the color development intensity is related to the concentration of GP73 in a sample. HRP-mAb2 is used to substitute for the binding reaction of HRP-rabbit polyclonal antibody, or rabbit polyclonal antibody and HRP-goat anti-rabbit antibody.

The invention has the beneficial effects that the GP 73C-terminal protein is provided, so that the monoclonal antibody resisting GP73C terminal is prepared, in order to solve the defect that the GP73 quantitative detection kit is lack of as the early diagnosis and detection means of liver cirrhosis clinically at present, the invention also aims to provide the detection kit which is simple and convenient to operate, accurate and sensitive, has stable quality and can be produced in a large scale and the determination method thereof, so that the GP73 expression content can be observed from the protein level, an effective comprehensive treatment scheme can be taken as early as possible, and the further malignant change of liver cirrhosis can be effectively prevented;

in addition, the detection limit of the GP73C end kit provided by the invention is less than or equal to 1.00 ng/ml; linear range: 1.00-450.00 ng/ml, and the correlation coefficient is more than or equal to 0.9900; the variation coefficient CV between batches is less than or equal to 15 percent; the recovery rate is 85-115%; cross-reaction value with AFP (alpha fetoprotein), GPC3 (phosphatidylinositol proteoglycan 3) and human serum albumin is less than or equal to 1.00 ng/ml; can be stably stored for 9 months at the temperature of 2-8 ℃.

Drawings

FIG. 1 is a standard curve plotting the log concentration and the log OD value of GP73 calibrator in the examples.

FIG. 2 is ROC curve analysis of liver cirrhosis specimen.

Detailed Description

The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not indicated in the examples below, are generally carried out under conventional conditions, or conditions which can be inferred within the knowledge of those skilled in the art, or according to conditions recommended by the manufacturers. The reagents and instrumentation referred to in the examples below are generally commercially available products or products obtainable by other published routes.

Example 1 preparation of GP 73C-terminal antigen

In order to prepare a monoclonal antibody with good specificity and high affinity for further application in a clinical GP73 detection kit, a GP73C terminal antigen is expressed first. The GP73 gene is located on chromosome 9 with a total length of 3042bp, contains a unique open reading frame (1200bp) and has a code of 402 amino acids. GP73 is structurally different from serum GP73(sGP 73). GP73 consists of an N-terminal myristoylated region in the cytoplasm, a single transmembrane region, and a long C-terminal extracellular domain. Its N-terminus is hydrophobic, glycosylated, contains a single transmembrane region and a signal peptidase cleavage site (aa28-aa29), and comprises two helix-helix domains. The C-terminal of the polypeptide is rich in acidic amino acids, contains tetradecanoylation (GLGNGRRS) continuous sequences and has 5 glycosylation sites. Next to the transmembrane region there are several coiled coils which are involved in the interaction between proteins. These properties suggest that the protein interacts with other proteins through the extracellular domain. The C-terminal peptide of GP73 has good hydrophilicity and accessibility, encodes amino acids located in the extracellular domain, contains no other transmembrane domains, and is unique among the human protein repertoire; meanwhile, serum GP73 was released extracellularly by furin cleavage at amino acid 55, with an intact C-terminus. Therefore, a sequence at the end of GP73C is used as an epitope, a GP73C terminal protein is expressed in a prokaryotic mode, and the monoclonal antibody prepared by the method is directed at the GP73C terminal and used for producing a kit, so that the detection rate of GP73 in a sample can be improved. The software analysis determines that the amino acids are 302-401 amino acids, and the result shows that the antigen binding site of the fragment is more exposed, so that the fragment has stronger antigenicity and is hydrophilic and soluble, and the fragment contains 100 amino acids, has larger molecular weight and is easy to store stably for a long time.

Among a plurality of protein expression systems, the prokaryotic expression system has the advantages of easy operation, rapid growth, high yield, low cost and the like, so the experiment adopts the prokaryotic expression system. The selection of proper carrier and thallus is very important for expressing exogenous gene, and the thallus can regulate the expression of its gene according to the change of external environment factors to make its growth and reproduction in optimum state. In this example, PGEX-4T-2 was used as an expression vector carrying a GST. Tag, which was used for purification of the expressed protein.

The preparation method comprises the following steps:

1. analysis of antigenic epitopes:

the nucleotide and amino acid full sequences of GP73 were downloaded from the NCBI website, the sequences were aligned using Biosun biology software, epitopes were analyzed and representative epitope segments were screened. Determining 302-401 amino acids, finding out the nucleotide sequence corresponding to the segment, and carrying out the next synthesis. The result of software analysis shows that the fragment has more exposed antigen binding sites, thus having stronger antigenicity and being hydrophilic and soluble, and the fragment contains 100 amino acids, has larger molecular weight and is easy to store stably for a long time.

2. Obtaining GP73C end antigen gene:

according to the GP73 gene sequence, a primer aiming at the GP 731019-1336 bp sequence is designed. An upstream primer: GC GCT GGA TCC CAG CTG GCC TCA (5 '-3' downstream primer: GC GAA TTC TGA TGTGAG ATGATT (5 '-3' uses cDNA plasmid as template, synthesizes gene fragment by PCR, recovers and purifies PCR product gel, transforms and connects into PMD19T (sequencing vector) by conventional method, carries out sequencing verification, after PCR amplification, 1.5% agarose electrophoresis identification, obtains 300bp amplified fragment, is consistent with expected result, obtains a plurality of positive clones after connecting and transforming with T vector, and analyzes sequencing result (Gen-Bank accession number AP002069 is completely matched), gene sequence is correct.

3. Cloning, expressing and purifying GP73C terminal antigen

(1) Connection transformation: adopting a primer containing BamHI and EcoRI enzyme cutting sites to amplify GP 73C-terminal genes (1019-1336 bp), carrying out enzyme cutting purification, connecting the purified gene with PGEX-4T-2 plasmid subjected to enzyme cutting by BamHI and EcoRI by a conventional method, and transforming the plasmid into BL 21.

(2) Determining the expression form: randomly selecting positive clones, chemically inducing with 1% isopropyl-beta-D-thiogalactopyranoside (IPTG) at 25 ℃, cracking with 1% NP-40, repeatedly freezing and thawing at-80 ℃ for 3 times, centrifuging, taking supernatant and precipitate, respectively carrying out SDS-polyacrylamide gel electrophoresis (SDS-PAGE) identification, and determining the expression form. The results show that purified GP 73C-terminal antigen is expressed in large amounts after induction, mainly in the form of soluble protein.

(3) And (3) amplification culture and purification: selecting positive clone for amplification culture, cracking by 1% NP-40, repeatedly freezing and thawing at-80 ℃ for 3 times, and purifying GP73C terminal antigen by chromatography.

4. And (3) detecting a purified product:

the GP73 antigen from Abnova was used as a control, and subjected to conventional electrophoresis with a gel concentration of 12% together with the GP73C terminal antigen purified in this example, followed by membrane transfer. 5% skimmed milk powder is sealed at room temperature for 1h, and diluted with Golgi protein polyclonal antibody A01, monoclonal antibodies M06 and M04 as primary antibodies, and incubated at 4 deg.C overnight. Membranes were washed 3 times at room temperature with wash (Tris salt buffer, TBST, 0.05% Tween 20). And (3) taking horseradish peroxidase-labeled goat anti-mouse IgG as a secondary antibody, incubating at room temperature for 1h, washing the membrane at room temperature for 3 times by TBST, presenting the result by electrochemical hypersensitivity luminescence (ECL), and detecting by a Western blot analysis technology (Western blot). The results show that the GP73C terminal antigen has better reactivity to 3 antibodies, and the reactivity of the two antigens has no obvious difference.

5. And (3) detecting the immune activity of the antigen:

detection was performed by enzyme-linked immunosorbent assay (ELISA). And detecting the immunocompetence of the purified antigen by using the Golgi protein polyclonal antibody A01, the monoclonal antibodies M06 and M04 as primary antibodies and using horseradish peroxidase-labeled goat anti-mouse IgG as a secondary antibody. Diluting the purified GP73C terminal antigen to 2 mu g/ml by using carbonate buffer solution to coat an ELISA microplate, discarding the solution after each well is 100 mu l and overnight at 4 ℃, washing the plate for 3 times by using washing solution (phosphate buffer solution containing 0.05 percent Tween-20, PBS), discarding the solution, and patting dry; adding blocking solution (100 μ l of phosphate buffer solution containing bovine serum albumin, BSA-PBS, standing overnight at 4 ℃, discarding solution, adding 50 μ l of primary antibody with the concentration of 100ng/ml into each well, uniformly mixing, carrying out warm bath at 37 ℃ for 1h, washing the plate for 5 times by using washing solution, patting dry, adding 50 μ l of 1 ten thousand times and 2 ten thousand times diluted horse radish peroxidase labeled goat anti-mouse IgG into each well, carrying out warm bath at 37 ℃ for 30min, washing the plate for 5 times by using the washing solution, patting dry, adding 100 μ l of single-component color developing agent, carrying out warm bath at 37 ℃ for 10min in a dark place, adding 50 μ l of stop solution into each well, reading the light absorption value of 450nm by using a microplate reader, taking normal mouse serum as negative control for the primary antibody, taking the light absorption value of negative control with the cut-off value of 2.1 times, and judging that the detection result is greater than the cut-off value to be.

When the horseradish peroxidase-labeled monoclonal antibody mouse IgG is diluted by 2 ten thousand times, the reactivity of the purified GP73C terminal antigen and the purchased antigen to A01 is weak, and the reactivity to other antibodies is good. When the monoclonal antibody mouse IgG marked by horseradish peroxidase is diluted by 1 ten thousand times, the purified GP73C terminal antigen and the purchased antigen have good reactivity to 3 antibodies, which indicates that the expressed GP73C terminal protein has stronger immunocompetence.

EXAMPLE 2 preparation of monoclonal antibody against GP73C terminus

1. Animal immunization

300mg/L of recombinant GP73C terminal antigen protein is mixed with complete Freund's adjuvant in equal amount to prepare an emulsifier, and 5 Balb/c mice are immunized together. 100 mug/mouse of the 1 st immunization, subcutaneous multi-point injection, and the 2 nd immunization with incomplete Freund's adjuvant after 2 weeks, wherein the dosage and the route are the same as the 1 st immunization. After 2 weeks, tail vein blood is taken to determine the potency, and the potency reaches 1: 1000-1: it is used for fusion at 5000 deg.C. 3 days before fusion, the splenic region of the abdominal cavity was boosted 1 time with antigen directly.

2. The titer is determined by adopting a conventional indirect ELISA method

The concentration of purified recombinant GP73C end antigen of the coated polystyrene plate is 5mg/L, the enzyme-labeled antibody is goat anti-mouse IgG (product of Sigma, working concentration is 1: 5000), and the OD value is measured by a 450nm wavelength of an enzyme-labeling instrument. The P/N value is more than or equal to 2.1 times positive and less than 2.1 times negative (the N value is less than 0.05, calculated according to 0.05).

3. Preparation of immune spleen cells

Immunized Balb/c mice are taken, and eyeball blood is discharged for detecting antibodies. Taking out spleen under aseptic operation, gently cleaning and removing connective tissue, placing on copper net, squeezing, grinding, squeezing into solution through mesh, centrifuging for 5min at 1000r/min, discarding supernatant, suspending in solution to obtain spleen cell suspension, and adding trypan blue dye solution for cell counting.

4. Preparation of myeloma cell broth

Place myeloma cells at 37 ℃ 5% CO₂And (5) performing amplification culture in an incubator. SP2/O myeloma cells were collected at the logarithmic growth phase on the day of fusion, centrifuged at 1000r/min for 5min, the supernatant was discarded, and the cells were counted after being resuspended in liquid.

5. Cell fusion

Spleen cell and myeloma cell suspensions were aspirated separately as 5: 1, mixing, placing in a centrifugal tube, fully and uniformly mixing, centrifuging, removing supernatant, and lightly flicking the bottom of the tube to ensure that precipitated cells are loosened and uniformly formed into paste. Adding 0.7ml of 50% polyethylene glycol (PEG) into the mixed cells to promote fusion, strictly controlling the action time within 2-3 min, and immediately adding incomplete culture solution to dilute so as to stop the action of the PEG. Centrifuging at 800r/min for 7min, and discarding the supernatant. Adding HAT culture medium 10mL to obtain cell suspension, adding into 96-well plate with feeder cell layer, placing 0.1mL per well, and placing at 37 deg.C with 5% CO₂Culturing in an incubator, changing the culture solution for 1 time after 4-5 days, taking the supernatant when 8-10 days later, measuring the titer by an ELISA method, performing primary screening, and performing subcloning on positive holes.

6. Cloning of hybridoma cells (limiting dilution method) and ascites preparation

And repeatedly and uniformly blowing the hybridoma cells to be cloned in the 96-well plate by using a sample injector, and then taking a small amount of cell suspension to be placed in another sterile vial. This cell suspension was accurately serially diluted until it contained 10 cells per ml. The diluted cell suspension was inoculated into a 96-well plate containing 0.1mL of feeder cells per well, i.e., one cell per well, and placed at 37 ℃ in 5% CO₂Culturing in an incubator, and observing the growth condition of the cell clone under an inverted microscope for about 5 days. Repeating cloning for 3 times to determine positive monoclonal cell strain, numbering the cloned strain, and storing in liquid nitrogen. The titer results of the monoclonal antibody cell lines are shown in Table 1.

TABLE 16 measurement results of the potency of GP73McAb

Monoclonal antibody numbering	Ascites antibody titer
		GP73C end-1	1:120000
GP73C terminal-2	1:3200000
		GP 7C terminal 3-3	1:1280000
GP73C terminal-4	1:640000
		GP73C terminal-5	1:1600000
GP73C terminal-6	1:800000

ELISA results show that the monoclonal antibody of GP73C terminal has high sensitivity to antigen recognition, and the highest titer reaches 1: 3200000. The obtained 6 strains GP73McAb only react with GP73 antigen protein, have no cross reaction with other unrelated proteins, and can well recognize natural GP73 protein in immunofluorescence experiments of HepG2 cells, thereby prompting that the prepared GP73McAb has good specificity and immunocompetence.

7. Subclass identification

96 well plates were coated with GP 73C-terminal antigen (2.5mg/L) overnight at 4 ℃. After being sealed by 2% BSA-PBS-Tween20, different cell culture supernatants (0.1 ml/hole) are added, the room temperature is 2h, rabbit immune serum resisting mouse Ig and subclasses thereof is added after washing, the rabbit immune serum is incubated for 1h at 37 ℃, enzyme-labeled goat anti-rabbit Ig antibody is added after washing, the temperature is 37 ℃ for 1h, and the results are washed, developed and judged.

Subclass identification results:

the 6 anti-GP 73McAb supernatants are respectively subjected to IgG subclass identification, the 6 monoclonal antibodies all belong to IgG1 and K chain (Kapa chain), and can still stably secrete antibodies after being stored in liquid nitrogen for multiple resuscitation passages, and the titer is not reduced by ELISA determination.

8, monoclonal antibody epitope analysis of GP73C end

And (3) coating an enzyme label plate (2mg/L) with a monoclonal antibody at the GP73C end, adding GP73C end expression antigen, and detecting GP73 by a double antibody sandwich method. Screening 3 pairs of monoclonal antibodies with better matching, aiming at different epitopes of the GP73 extracellular domain, respectively: GP73C terminal-1 to GP73C terminal-2, GP73C terminal-1 to GP73C terminal-5, GP73C terminal-3 to GP73C terminal-6. The GP73C end-1-GP 73C end-2 is the best pair, the 2 monoclonal antibody pairs are also used in the subsequent kit production, GP73C end-1 is used as a coating antibody to coat an enzyme label plate, and GP73C end-2 is used as a detection antibody to label horseradish peroxidase.

Example 3 preparation of HRP-labeled monoclonal antibody (enzyme-labeled antibody) against GP73C

The HRP-labeled monoclonal antibody is prepared by adopting an improved sodium periodate method, and the method comprises the following specific steps:

1. dissolving 5mg HRP in 0.5ml of double distilled water, adding newly prepared 0.06Mol/L NaIO₄Aqueous solution (10ml double distilled water +128mg NaIO)₄)0.5ml, mixing evenly, and placing for 30min at 4 ℃;

2. taking out, adding 0.16Mol/L ethylene glycol aqueous solution (10 mlH)₂O +0.09ml ethylene glycol) 0.5ml, standing at room temperature for 30 min;

3. adding 1ml of water solution containing 5mg of monoclonal antibody of purified GP73C end, mixing, filling into a dialysis bag, and dialyzing for 6h (or overnight) with 0.05Mol/L of pH 9.5 carbonate buffer solution under slow stirring for binding;

4. adding intoNaBH₄0.2ml of solution (5mg/ml) is mixed evenly and placed at 4 ℃ for 2 h;

5. slowly adding equal volume of saturated ammonium sulfate solution into the above solution, mixing, at 4 deg.C for 30min, centrifuging, removing supernatant, dissolving the precipitate with 0.02Mol/L PBS (pH7.4PBS), placing into dialysis bag, dialyzing with the same solution at 4 deg.C, and desalting overnight;

6. centrifuging the next day to remove insoluble substances to obtain enzyme-antibody (HRP-IgG) conjugate, and adding 0.02Mol/L PBS (pH 7.4) to 5 ml;

7. after the titer is determined to be qualified, adding equivalent high-quality glycerol, subpackaging the obtained product into small bottles, and storing the obtained product at low temperature.

Example 4 Main production materials and production technology of kit

Production process of GP73C end antibody coated plate

1.1 picking up

And (4) obtaining corresponding amounts of coating solution, antibody stock solution, an ELISA plate and a sealing plate film according to the production instruction.

1.2 coating

Coating the purified GP73C end-1 on an enzyme label plate according to the concentration of 2 mu g/mL, and incubating for not less than 16 hours at the temperature of 2-8 ℃.

1.3 sealing

And (3) throwing away the coating solution in the ELISA plate, washing for 1 time by using a washing solution (phosphate buffer solution, PH 7.2), throwing away the washing solution, adding a confining solution, and incubating for not less than 16 hours at 2-8 ℃.

1.4 drying

And (4) throwing off the confining liquid in the ELISA plate, drying, then carrying out vacuum sealing, and storing in an intermediate product temporary storage chamber at 2-8 ℃.

2. Production process of kit

2.1 picking up

The required reagents and consumables are obtained according to the production instructions and the reagent formula, which are shown in the table 2.

TABLE 2 summary of materials for the production Process of the kit

In the embodiment, single-component TMB color developing solution (2 substrate solutions in a domestic commercial kit are separately prepared by AB solution, and the mixing method before use is complex in operation and large in batch difference) is adopted, so that the operation steps are reduced.

In the embodiment, horseradish peroxidase (HRP) and labels (enzyme-labeled antibody, antigen, polypeptide and the like) thereof are unstable and are easy to inactivate at low concentration. Therefore, dilution of HRP and its marker and preparation of stable working solution have made higher demands on researchers.

The horseradish enzyme marker diluent prepared in the embodiment contains a plurality of HRP stabilizers, has strong protective effect on HRP, the marker and the antibody activity, and ensures that diluted reagents such as the HRP, the enzyme marker and the antibody are still stable when used at room temperature under extremely low concentration (ng level), thereby meeting the requirement of scientific researchers engaged in experiments such as ELISA for preparing the enzyme and the marker stable working solution.

In the embodiment, although the ELISA detection method has simple operation steps, the influence factors are more, and the blocking is one of the most important influence factors. The sensitivity and specificity of an ELISA detection method can be directly influenced by improper blocking, and a false positive result is generated; the good sealing liquid can also play the roles of a stabilizer and a protective agent, so that the storage life of the ELISA plate is improved; poor confining liquids can also interfere with the reaction of the various reagents.

The blocking solution prepared in this example was recoated with a high concentration of irrelevant protein solution to allow the irrelevant proteins to fill the uncoated gaps, thereby reducing the re-adsorption of interfering substances in the subsequent step of ELISA.

2.2 weigh

And calculating the dosage of the batch of products according to the production instruction, and accurately weighing/absorbing the required reagent.

Note that: the concentrated enzyme conjugate belongs to a pollution-prone reagent and is stored in a dark place.

The sulfuric acid solution is corrosive and should avoid splashing of the liquid onto the skin and clothing.

2.3 preparation of

The reagents are dissolved in sequence, the volume is determined by deionized water, and the pH value is measured. The blocking solution, enzyme stock solution, concentrated enzyme conjugate dilution and calibrator dilution were filtered through 0.22 μm filters. And (3) labeling the container with a label, noting name, batch number, capacity, expiration date and the like, and storing the container in an intermediate product temporary storage room at the temperature of 2-8 ℃.

2.4 subpackaging the semi-finished products

2.4.1 Tie Material

And (3) taking corresponding amounts of semi-finished products and raw materials according to production instructions, standing the semi-finished products at room temperature (18-28 ℃) for balancing for 1 hour, and subpackaging according to the component table of the specification.

Note: all blanks have passed the blank inspection.

3. Requirements of the main raw materials

3.1 antigens, antibodies

3.1.1 sources

The recombinant GP73C terminal protein (GP 73C terminal antigen) and the GP73C terminal monoclonal antibody are Tianjin Jinhong biotech development Co., Ltd.

TABLE 3 use and sources of the main raw materials

Name (R)	Use of	Source
			GP73C terminal antigen	Formulating a calibrator	Prokaryotic expression of Escherichia coli
GP73C end-1	Coated enzyme label plate	Balb/c mice
			GP73C end-2	Labeling of Horseradish peroxidase	Balb/c mice

3.1.2 appearance

The raw materials should be clear and transparent without sediment when observed by naked eyes.

3.1.3 purity and molecular weight

The purity was determined by SDS-PAGE and the loading was 10. mu.g. The purity of GP73C end antigen is more than 90%, and the molecular weight is about 12 KD; the antibody purity is more than 90%, and a color band is respectively shown at about 25KD and 55 KD.

3.1.4 protein concentration

The protein concentration in the GP73C end antigen was determined by BCA method. The OD of the purified GP73C end-1 and the GP73C end-2 marked by Horse Radish Peroxidase (HRP) are measured by an ultraviolet spectrophotometer₂₈₀Push-push (OD)₂₈₀X 0.625) ═ mg/mL protein concentration was calculated.

The protein concentration of the GP73C terminal antigen and the purified GP73C terminal monoclonal antibody is more than 0.5mg/mL, and the molar ratio of the monoclonal antibody at the GP73C terminal marked by the HRP is between 1 and 2.

3.1.5 Linear

3.1.5.1 GP73C terminal antigen linear assay

The enzyme label plate is coated by purified GP73C end-1 according to 2ug/mL, and a calibrator is prepared by expressing purified GP73C end antigen to prepare a kit for detection.

The linear range is 1.00-450.00 ng/mL, and the correlation coefficient is not lower than 0.9900.

3.1.6 ascites titer of monoclonal antibody at GP73C end

Injecting the mouse abdominal cavity with monoclonal cell strain No. 1 and No. 2 of GP73C end, diluting the obtained ascites, coating an ELISA plate with the antigen expressing purified GP73C end, and detecting by ELISA indirect method. 2.1 times of the negative control OD value is a threshold value for judging positive, and the maximum dilution of the positive reaction is the ascites titer of the monoclonal antibody.

The ascites titer of the monoclonal antibody of GP73C end is not less than 1:10⁵。

EXAMPLE 5 Performance indices and testing protocols for kits-related finished and semi-finished products

First, performance index and inspection rule of finished product

1. Performance index

1.1 appearance

The appearance of the kit is complete and is not damaged; the enzyme-labeled lath is smooth, has no flash, good surface finish, and no ripple or scratch at the bottom; the liquid component should be clear and transparent, free of floc, and free of leakage; the label is clear and easy to identify.

1.2 detection Limit

The detection limit is less than or equal to 1.00 ng/mL.

1.3 Linear

Within the range of 1.00 ng/mL-450.00 ng/mL, the correlation coefficient is more than or equal to 0.9900.

1.4 in-batch repeatability

Coefficient of variation CV is less than or equal to 15 percent.

1.5 run-to-run repeatability

Coefficient of variation CV is less than or equal to 15 percent.

1.6 accuracy

The recovery rate is in the range of 85-115%.

1.7 stability

And (3) taking the expiration date kit, detecting appearance, detection limit, linearity, repeatability and accuracy, wherein the result meets the requirements of 1.1-1.4 and 1.6.

1.8 traceability

The product calibrator can be traced to recombinant GP73C terminal protein (hereinafter referred to as GP73C terminal antigen).

2. Inspection method

2.1 appearance

The test method comprises the following steps: the result of visual inspection should meet the requirement of 1.1.

The test results are shown in Table 4.

TABLE 4

2.2 detection Limit

The test method comprises the following steps: detecting with zero concentration calibrator as sample, repeating measurement for 20 times to obtain 20 times of measurement results (X value), calculating average value (M) and Standard Deviation (SD) to obtain X value corresponding to M +2SD, substituting X value into standard curve equation, and calculating corresponding concentration value as detection limit, wherein the result is in accordance with 1.2.

The test results are shown in Table 5.

TABLE 5

2.3 Linear

The test method comprises the following steps: diluting at least 5 dilution concentration (xi) points in a proportion with a sample that is at or near the upper concentration (activity) of the linear range, wherein the lower concentration should be near the lower limit of the linear range. The test was carried out with the test kit, 3 times for each dilution concentration, and the mean value (yi) of the measurement results was determined. The linear regression equation was calculated using the dilution concentration (xi) as an independent variable and the measurement result mean (yi) as a dependent variable. The correlation coefficient (r) of the linear regression is calculated according to equation (1).

The result should meet the requirement of 2.3.

The test results are shown in Table 6.

TABLE 6

Batch number	First batch	Second batch	Third batch
				Linearity	0.9942	0.9919	0.9948

2.4 in-batch repeatability

The test method comprises the following steps: the kit is used for testing three samples with high (100.00 ng/mL-300.00 ng/mL), medium (30.00 ng/mL-100.00 ng/mL) and low (1.00 ng/mL-30.00 ng/mL) horizontal concentrations, the test is repeated for 20 times (n is 20), 20 measurement results (X values) are obtained, the average value (M) and the Standard Deviation (SD) of 20 measurement values of each concentration sample are respectively calculated, the Coefficient of Variation (CV) is calculated, and the result meets the requirement of 2.4.

The test results are shown in Table 7.

TABLE 7

Batch number	Low concentration CV (%)	Middle concentration CV (%)	High concentration CV (%)
				First batch	5.33	3.53	4.55
Second batch	5.94	8.53	5.02
				Third batch	2.56	3.34	1.69

2.5 run-to-run repeatability

The test method comprises the following steps: taking three kits of different batches, respectively testing the same sample (the concentration is 30.00 ng/mL-100.00 ng/mL), repeatedly testing each batch for 10 times to obtain 30 measurement results (X values), calculating the average value (M) and the Standard Deviation (SD) of 30 measurement values, and calculating the Coefficient of Variation (CV), wherein the result meets the requirement of 2.5.

The test results are shown in Table 8.

TABLE 8

Batch number	First, second and third batches
		Middle concentration CV (%)	8.33

2.6 accuracy

The test method comprises the following steps: adding a certain low-concentration serum sample into a high-concentration standard solution or a pure product (the volume ratio of the standard solution to the sample is not more than 1:9 or the volume ratio of the standard solution to the sample does not change, the total concentration of the sample must be in a linear range of the kit measurement after the standard solution is added), repeating the measurement for 3 times for each concentration, calculating the recovery rate according to a formula (4), and ensuring that the result meets the requirement of 2.6.

In the formula: r-recovery rate;

v-adding volume of standard solution;

v0 — volume of low concentration sample;

c, measuring the concentration of the low-concentration sample after the low-concentration sample is added into the standard solution;

c0-measured concentration of low concentration sample;

cs — concentration of standard solution.

The test results are shown in Table 9.

TABLE 9

Batch number	First batch	Second batch	Third batch
				Recovery (%)	109.04	105.71	108.46

2.7 stability

And (3) detecting 2.1-2.4 and 2.6 items by using the expiration date kit, wherein the result meets the requirement of 1.7.

Second, the performance index and inspection rule of semi-finished product

3.1 GP73C end antibody coated plate

And (3) appearance inspection: the GP73C end antibody coating batten is flat, has no flash, has good surface finish, and has no ripple and scratch at the bottom.

And (3) taking the tested kit and the GP73C end antibody coating plate of the semi-finished product to be tested to jointly test 6 bottles of calibrator in the kit, respectively repeating the test for 3 times on each bottle of the two GP73C end antibody coating plates, and taking the mean value of OD values of each point, wherein the relative deviation of the OD values of each concentration point of the calibrator is within the range of +/-15%. Vacuum packaging and preserving at 2-8 ℃.

3.2 appearance of the reagent

And (3) appearance inspection: the liquid component should be clear and transparent, free of flocs, and free of leakage.

3.3 reagent testing, dispensing and preservation

3.3.1 calibrators

And (3) taking the tested kit and the calibrator to be tested, respectively testing two calibrators, 6 bottles each, repeating the test for 3 times in each bottle, and taking the average value of the OD values of all points, wherein the relative deviation of the OD values of all concentration points of the calibrator is within the range of +/-15%.

The packaging amount of the calibrator of each concentration of each kit is 0.5mL, and the calibrator is stored at 2-8 ℃.

3.3.3 enzyme conjugates

And (3) taking the tested kit and the reagent to be tested to test 6 bottles of calibrators in the kit together, wherein the two reagents are tested repeatedly for 3 times in each bottle respectively, the average value of the OD values of all points is taken, and the relative deviation of the OD values of all concentration points of the calibrators is within the range of +/-15%.

The packaging amount of each kit is 12mL, and the kit is stored at 2-8 ℃.

3.3.4 concentrated Wash (20X)

The washing solution in each kit is 20 times of concentrated phosphate buffer solution, the packaging amount is 50mL, and the kit is stored at 2-8 ℃.

3.3.5 Single-component TMB color developing solution

And (3) taking the tested kit and the reagent to be tested to test 6 bottles of calibrators in the kit together, wherein the two reagents are tested repeatedly for 3 times in each bottle respectively, the average value of the OD values of all points is taken, and the relative deviation of the OD values of all concentration points of the calibrators is within the range of +/-15%.

The packaging amount of each kit is 12mL, and the kit is stored at 2-8 ℃ in a dark place.

3.3.6 stop solutions

The packaging amount of each kit is 7mL, and the kit is stored at room temperature (18-28 ℃).

Example 6 results of clinical experiments

A double-antibody sandwich method for measuring the GP73 level in serum is established by Tianjin Jinhong biotechnology development limited company through a large number of experiments. The method adopts the monoclonal antibody as a raw material, optimizes the traditional process, and has higher sensitivity (1.00ng/ml) and specificity. The detection method is simple, convenient and quick, and the time is less than 1.5 hours. The linear relation of the standard curve is good, and the correlation coefficient r is more than or equal to 0.9900, as shown in figure 1.

ROC curves were plotted as GP73 concentrations for 858 cirrhosis serum samples and 1111 serum samples from health examination populations. The area under the ROC curve is 0.959, and the sensitivity and specificity are optimal when 8.52ng/ml is cut-off for GP73 diagnosis of cirrhosis, 81.4% and 95.0%, respectively, as shown in FIG. 2. The concentration of GP73 is more than 4.53ng/ml, and moderate liver injury indicates that liver diseases with more than obvious liver fibrosis exist; GP73 concentrations >8.52ng/ml indicate the potential for cirrhosis. At the same time, the following findings are provided: the serum GP73 is slightly increased during chronic persistent hepatitis, is obviously increased during liver cirrhosis, and the level of the serum GP73 is positively correlated with the degree of liver lesion. Therefore, the kit can assist in prompting the early hepatic fibrosis of the liver cirrhosis on the basis of clinical auxiliary diagnosis of the liver cirrhosis.

This study showed significant statistical differences in serum GP73 concentrations between different Child-Pugh fractions in cirrhosis patients. With a decline in liver function, serum GP73 levels are also correspondingly elevated. Patients with Child-Pugh grade C (32.58ng/mL + -16.52 ng/mL) were significantly higher than those with grade B (19.37ng/mL + -10.23 ng/mL) and grade A (12.28ng/mL + -6.09 ng/mL).

The positive rate and concentration of GP73 in the hepatitis C disease are both obviously higher than those of hepatitis B related liver disease (P <0.001), which shows that GP73 expression is up-regulated in HCV infected cells, GP73 is a key substance in the HCV secretion process, and the method is consistent with the literature report.

The concentration of GP73 in 6 non-liver malignant tumor specimens is 1.0ng/ml which is obviously lower than that of a liver cirrhosis specimen (P <0.05), and the GP73 is suggested to have good specificity and can be used as a serum marker only aiming at liver cirrhosis.

158 cases of liver cirrhosis and 160 cases of plasma samples of healthy physical examination people were examined. The result shows that the GP73 content of the plasma sample of the cirrhosis people is (9.10ng/mL +/-8.71 ng/mL), and the GP73 content of the plasma sample of the health examination people is (1.16ng/mL +/-0.25 ng/mL), which indicates that the kit of the embodiment can be simultaneously used for detecting the plasma sample.

In a word, the quantitative detection reagent for GP73 in serum of the embodiment has the advantages of simple and rapid method and low cost, and clinical application results show that GP73 can be used as a marker for assisting clinical diagnosis of liver cirrhosis, has a monitoring effect on hepatitis, hepatic fibrosis and the progress of liver cirrhosis, and has good clinical application value.

Example 7 preparation and use of kit for detecting GP73 by magnetic bead chemiluminescence method

The magnetic immunoassay technology established by taking the magnetic nano-microspheres as the solid phase carrier is an important immunoassay technology at home and abroad in recent years. Coupling the magnetic microspheres with chemically modified surfaces to antibody molecules through covalent bonds; the huge specific surface area can be coupled with more antibodies, and the antigen binding sites of the antibodies are fully exposed, so that the steric effect is reduced; in addition, the nano magnetic microspheres have superparamagnetism, and are uniformly dispersed in a solution without an external magnetic field, so that the antigen-antibody reaction is quicker and more efficient.

In the embodiment, a biotin-labeled GP73C end monoclonal antibody is coupled with streptavidin modified magnetic beads, serum to be detected is added to react with an enzyme conjugate of another GP73C end antibody, and then the reaction product reacts with a chemiluminescent substrate luminol to judge the result according to the luminescent value.

The specific operation is as follows:

1. sample application

Add 50. mu.l of magnetic beads, 20. mu.l of calibrator or serum sample and 50. mu.l of enzyme conjugate to the respective wells, and mix well by shaking for 30 seconds.

2. Incubation of

The plates were sealed with a sealing plate and incubated in a 37 ℃ constant temperature water bath for 15 minutes.

3. Washing machine

Carefully remove the coversheet and wash it 6 times with a plate washer, adding at least 300. mu.l of wash solution per well.

4. Adding a substrate

Adding 50 mul of chemiluminescence substrate A liquid into each hole, adding 50 mul of chemiluminescence substrate B liquid into each hole, avoiding generating bubbles when adding the substrates, shaking and mixing uniformly for 30 seconds, and placing a sealing plate and a membrane sealing plate in a back position for 5 minutes at 37 ℃ in a dark place.

5. Measurement of

Starting the chemiluminescence detector and the main control computer, and setting the measurement mode and parameters. The luminescence value RLU of each well was measured at the 5 th minute after the addition of the substrate, and the measurement time per well was 1 second.

6. Calibration curve fitting and sample concentration calculation

A regression equation of GP73 concentration versus RLU was constructed from the RLU measurements for each calibrator using a log-log fit equation plotted on the ordinate (Y-axis) against the log of the concentration of each calibrator and on the abscissa (X-axis) against the log of the chemiluminescence value of each calibrator (minus the chemiluminescence value of S0). And (4) substituting each sample RLU value into the calibration curve, and calculating to obtain a sample GP73 concentration value.

Example 8 Experimental results of fucosylated GP73 in diagnosis of liver cirrhosis

Glycosylation (glycosylation) is one of the most common modes of post-translational modification of proteins, with approximately 50% of proteins having glycosylation modifications. Studies have shown that the degree of glycosylation of glycoproteins changes during the development and progression of many diseases. With the development of liver lesions, GP73 develops core fucosylation to varying degrees. According to the characteristic that the lentil lectin can be specifically bound with the core fucosylated glycoprotein, Fuc-GP73 (fucosylated GP73) is obtained by eluting through an agarose gel affinity adsorption centrifuge tube coupled with the lentil lectin, and the content of Fuc-GP73 in the serum of a patient is quantitatively detected by combining an ELISA method.

The specific method comprises the following steps:

1. preparation of LCA-coupled agarose gel

(1) 1.7g Sepharose CL 6B was soaked in 1mmol/L HCl to swell, transferred to a sand core funnel and washed with 350mL 1mmol/L HCl for 25 minutes.

(2) 5mg of LCA was weighed, dissolved in 17mL of coupling buffer, combined with washed Sepharose CL 6B, mixed by inverting in a stoppered 20mL tube and allowed to stand at room temperature for 2.5 hours. The column was loaded and unconjugated LCA was washed off with 15mL of coupling buffer.

(3) The coupled LCA was blocked with 0.2mol/L glycine.

(4) Washing with 15mL of 0.1mol/L acetate buffer solution and 0.1mol/L Tris buffer solution for 3 times, washing with PBS-BSA for 1 time, and storing at 4 deg.C.

Fuc-GP73 purification step

(1) Completely centrifuging the serum to be detected; taking out the pre-packed centrifugal column stored at 4 ℃, and discarding the liquid in the lower collecting tube.

(2) Diluting the blood sample, namely sucking 200 mu L of serum into a test tube, adding 300 mu L of cleaning solution for dilution, and gently shaking and mixing the solution uniformly.

(3) And (3) sample adding, namely sucking 400 mu L of diluted sample, adding the sample into an upper centrifuge tube, and placing the upper centrifuge tube into a 37 ℃ thermostat, wherein in the step, the cover of the centrifuge tube is not required to be covered, and the diluent is waited to completely flow into a lower collecting tube for about 25 minutes.

(4) The liquid in the lower collection tube was discarded.

(5) Add 500. mu.L of wash solution to the upper centrifuge tube, wait for the wash solution to flow completely into the lower collection tube (about 5 minutes), cover the centrifuge tube lid, and centrifuge for 1 minute at 3000 rpm at room temperature.

(6) The liquid in the lower collection tube was discarded.

(7) Repeating the steps (5) and (6) once.

(8) 400 μ L of the solution was added to the upper centrifuge tube, and after the eluent was dropped into the lower collection tube, the centrifuge tube lid was closed and incubated in a 37 ℃ incubator for 30 minutes.

(9) The tube was removed and centrifuged at 3000 rpm for 1 min at room temperature.

(10) The liquid (containing GP73 heteroplasmon) flowing into the lower collecting pipe is collected for standby.

(11) And (3) detecting the content of GP73 heteroplasmon in the collection liquid, and detecting the content of GP73 in the serum by the ELISA method.

156 patients with liver cirrhosis, 160 patients with chronic hepatitis and 205 patients with physical examination of serum Fuc-GP73 were tested. The results show that: Fuc-GP73 level in the cirrhosis group was significantly higher than that in the chronic hepatitis (P <0.01) and the healthy control group. By drawing a ROC curve, the area under the Fuc-GP73 detection liver cirrhosis curve is 0.908, the sensitivity is 76.9%, the specificity is 93.1%, and the positive judgment value is 2.55 ng/ml.

The present invention is described in detail with reference to the embodiments, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the invention shall fall within the scope of the patent coverage of the invention.

Sequence listing

<110> Tianjin Jinhong Biotechnology development Co., Ltd

<120> preparation and application of GP 73C-terminal antigen

<130> 2017

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 100

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Gln Ala Ala Leu Ser Val Ser Gln Glu Asn Pro Glu Met Glu Gly

1 5 10 15

Pro Glu Arg Asp Gln Leu Val Ile Pro Asp Gly Gln Glu Glu Glu Gln

20 25 30

Glu Ala Ala Gly Glu Gly Arg Asn Gln Gln Lys Leu Arg Gly Glu Asp

35 40 45

Asp Tyr Asn Met Asp Glu Asn Glu Ala Glu Ser Glu Thr Asp Lys Gln

50 55 60

Ala Ala Leu Ala Gly Asn Asp Arg Asn Ile Asp Val Phe Asn Val Glu

65 70 75 80

Asp Gln Lys Arg Asp Thr Ile Asn Leu Leu Asp Gln Arg Glu Lys Arg

85 90 95

Asn His Thr Leu

100

Claims (10)

1. The amino acid sequence of GP73C terminal protein is SEQ ID NO. 1.

2. A method for preparing the GP 73C-terminal protein according to claim 1, wherein, firstly, a DNA sequence coding for expressing GP 73C-terminal is amplified by a PCR method; then, inserting the sequence into the multiple cloning site of an expression vector to construct a recombinant expression plasmid; then, transforming the strain into an expression host bacterium to construct an engineering strain, and performing induced expression on the engineering strain; finally, the GP73C terminal recombinant protein is obtained by separation and purification.

3. The method for preparing GP 73C-terminal protein according to claim 2, which mainly comprises the following steps:

(1) and (3) PCR amplification: PCR amplifying GP73C terminal gene by using a primer containing BamHI and EcoRI enzyme cutting sites, wherein the GP73C terminal group is connected with PGEX-4T-2 plasmid after enzyme cutting by BamHI and EcoRI after enzyme cutting purification because of the gene of 1019-1336 bp, and is transformed into escherichia coli BL 21;

(2) determining the expression form: randomly selecting positive clones, chemically inducing with 1% isopropyl-beta-D-thiogalactopyranoside (IPTG) at 25 ℃, cracking with 1% NP-40, repeatedly freezing and thawing at-80 ℃ for 3 times, centrifuging, taking supernatant and precipitate, respectively carrying out SDS-polyacrylamide gel electrophoresis (SDS-PAGE) identification, and determining an expression form;

(3) and (3) amplification culture and purification: selecting positive clone for amplification culture, cracking by 1% NP-40, repeatedly freezing and thawing at-80 ℃ for 3 times, and purifying GP73C terminal antigen by chromatography.

4. The method for preparing GP 73C-terminal protein according to claim 3, wherein the upstream primer in the step (1) is 5 '-3': GC GCT GGA TCC CAG CTG GCC TCA, the downstream primer is 5 '-3': GC GAA TTC TGA TGTGAG ATGATT are provided.

5. The method for preparing the monoclonal antibody by using the GP 73C-terminal protein according to claim 1, wherein hybridoma cell lines are prepared and screened by immunizing Balb/C mice with the expression and synthesis GP 73C-terminal recombinant protein, ascites is prepared, and the monoclonal antibody is purified.

6. Use of the monoclonal antibody produced according to claim 5 for the production of a reagent for diagnosing liver diseases.

7. Use of the monoclonal antibody produced according to claim 5 for the production of a kit for diagnosing liver diseases.

8. The kit as claimed in claim 7, wherein the detection process of the kit comprises a process of detecting the GP73 content/fucosylated GP73 content in serum by ELISA with the monoclonal antibody.

9. The kit of claim 7, wherein the detection process comprises a process of detecting the GP73 content of the monoclonal antibody by using a magnetic bead chemiluminescence method.

10. The kit as claimed in claim 8, wherein the ELISA method for detecting GP73 content in serum comprises using two monoclonal antibodies against different epitopes of GP 73C-terminal protein as a coating antibody and a detection antibody, respectively, detecting GP73 protein by substrate color development, and determining GP73 protein content by a standard curve prepared from a calibrator.

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