CN110031629B - ELISA kit for detecting human serum DLK1 protein and its use - Google Patents

Abstract

The invention discloses an ELISA kit for detecting human serum DLK1protein and application thereof. The antigen of the ELISA kit adopts recombinant human DLK1 antigen. The detection method has high sensitivity and specificity, can quickly detect the expression of DLK1 and liver cancer and hepatoblastoma, and has wide application prospect in prevention, treatment and diagnosis of liver cancer and hepatoblastoma.

Description

ELISA kit for detecting human serum DLK1 protein and its use

technical field

The invention relates to the technical field of biological detection, in particular to an ELISA kit for detecting human serum DLK1 protein and use thereof.

Background technique

Hepatocellular carcinoma (HCC) has a high incidence in my country and is the fifth most common malignant tumor in the world. It lacks early preventive and treatment measures, has a high degree of malignancy, progresses rapidly, and has a poor prognosis. Treatment is difficult to cure. In my country, the incidence and mortality of liver cancer ranks second among cancers. The occurrence of liver cancer is the result of a multi-stage, multi-factor accumulation, as well as the involvement and mutation of multiple genes, and its specific etiology and pathogenesis are not yet clear. Various known possible external factors involved in primary liver cancer include viral hepatitis, liver cirrhosis, aflatoxin, drinking water pollution and some chemical carcinogens such as nitrosamines. In addition, gene mutations, chromosomal rearrangements, and epigenetic modifications within the body are all involved in the occurrence and development of liver cancer. Therefore, it has become the common goal of medical workers in various countries to search for possible genes related to the occurrence and development of liver cancer and to explore the corresponding possible diagnosis and treatment methods. AFP is the only serological marker for the diagnosis of liver cancer at present, with a detection rate of only 60-70%, and there are still a considerable number of people who cannot rely on serological detection for diagnosis.

Hepatoblastoma (HB) is the most common primary liver malignancy in children, accounting for 90% of pediatric liver malignancies. Metastases are lung, abdominal cavity, lymph nodes and brain. 90% of hepatoblastomas occur in children under 3 years of age, and 60% in children under 1 year of age. Because the pathogenesis of HB is still unclear and the treatment is difficult, the existing treatment options include surgery, intervention, chemotherapy, liver transplantation and other multidisciplinary collaborative treatments. Surgical resection is currently the most important treatment for hepatoblastoma, and chemotherapy helps convert inoperable tumors into surgically resectable tumors. But clinically, more than half of the patients have lost the opportunity for surgical treatment when they go to the doctor. The comprehensive treatment of patients who can complete surgical resection and postoperative chemotherapy can significantly improve the survival rate and quality of life of children.

Therefore, the focus of HB treatment should be to improve the complete tumor resection rate, standardize postoperative chemotherapy, and improve the tumor cure rate. In this process, it is particularly important to closely track the indicators that detect tumors.

AFP is a serum protein that is expressed at a high level in normal newborns, and the level of AFP gradually decreases after birth, and the level of AFP does not drop to the normal level until the age of 1. AFP can be significantly increased in most HB patients, and it is found that AFP is positive in 80% to 90% of HB children by clinical testing, so it is now widely adopted as a clinical detection index. Following complete resection of the HB tumor, AFP levels should theoretically drop significantly, approaching normal values within a few weeks. Failure to reach normal values may indicate residual tumor. In children who cannot be resectable and received chemotherapy, the rate of decline in AFP levels after chemotherapy is related to prognosis. The American COG reported that after the start of 4 courses of chemotherapy, 75% of patients with AFP levels decreased by 2 logarithms survived; If the AFP level decreased by less than 2 log before surgery, the survival rate was extremely low. However, in clinical practice, there are some children with remission of clinical symptoms after surgery and chemotherapy and no residual lesions on imaging examinations, and their AFP levels remain high. For these children and HB infants younger than 1 year old, the AFP test results after surgery and chemotherapy often cause great distress to clinicians and their parents, and it is impossible to accurately assess the effect of tumor on treatment and monitor whether the disease recurs.

Therefore, AFP, as the only serum monitoring index for liver cancer and hepatoblastoma, has its clinical limitations. Therefore, finding a specific and reliable clinical detection index has become an urgent clinical problem to be solved.

Early diagnosis of liver cancer and hepatoblastoma is still an important means to prevent and treat them.

Serological indicators of liver cancer: Alpha-fetoprotein heterogenous body (FucAFP): At present, the lentil agglutinin (LCA) affinity cross-autoimmunity method is used to determine the diagnostic value of AFP heterogenous body. There are two isoforms, LCA non-binding (AFP-N-L) and bound (AFP-R-L). Liver cancer contained AFP-N-L in an average of 49.13±27.20% (0-100%), <75% was the diagnostic standard for liver cancer, and the positive rate was 86.0%, which decreased with the deterioration of the disease.

The non-cancer liver disease AFP-N-L was 93.30±7.66% with a false positive rate of 1.6%.

Serological index of liver cancer aldolase isoenzyme A (ALD-A): when ALD-A appears and increases >800ng/ml in liver cancer, it is helpful for diagnosis, and the positive rate of AFP-negative liver cancer is 73.6%.

Serum fucosidase (AFu): Afu is a lysosomal acid hydrolase, and its main physiological function is to participate in the catabolism of bioactive macromolecules such as fucosylated glycoproteins and glycolipids. Primary liver cancer should be considered when Afu exceeds 110Kat/L. Domestic reports indicate that the positive rate of Afu in diagnosing primary liver cancer is 81.2%, and the positive rates for AFP-negative liver cancer and small liver cancer are 76.1% and 70.8%, respectively. Liver mass lesions were all negative, but the false positives of liver cirrhosis and chronic hepatitis were higher.

To sum up, liver cancer markers have certain auxiliary significance for the diagnosis of primary liver cancer, especially AFP-negative cases, but they still cannot replace the status of AFP in the diagnosis of liver cancer. In the past, it was always believed that AFP was a specific tumor marker for the diagnosis of primary liver cancer, and it had the functions of establishing diagnosis, early diagnosis and differential diagnosis. However, clinical studies in recent years have found that some patients with liver cirrhosis will have AFP values reaching thousands for a long time. But there are no signs of liver cancer for many years, and about 20% of patients with advanced liver cancer have been found to have an AFP of less than 10 until death. At present, chemiluminescence, enzyme labeling, enzyme labeling electrophoresis, and radioimmunoassay are commonly used for detection. According to practical experience, serum AFP detection combined with 1 or 2 liver cancer markers can significantly improve the positive detection rate of primary liver cancer. In clinical analysis, comprehensive judgment should be combined with medical history, diagnostic imaging or histological data to draw accurate conclusions.

DLK1 is a liver cancer stem cell marker gene that we discovered earlier, and it is expressed on the surface of stem cell membranes. Human DLK1 protein is a transmembrane protein composed of 383 amino acids (385 in mouse and 383 in rat), and its structure and amino acid sequence are similar to those of Delta, Notch, Serrate of invertebrate Drosophila, Lin-12 and glpl of C. elegans. , and the corresponding structures of mammalian Notch proteins have a high degree of homology. DLK1 is characterized by 6 strings of epidermal growth factor (EGF)-like repeats and a signal sequence in the extracellular region. The repeat segment is a domain composed of 35-40 amino acids, with a conservative interval of 6 cysteine. The size of the sugar chain attached to the N-terminus of DLK1 varies so that its molecular weight is distributed between 45 and 60 kDa. FA1 (fetal antigen 1) was first isolated from human amniotic fluid. It is a single-chain glycoprotein containing 225-262 amino acids. It is considered to be the proteolytic hydrolysis product of a large fragment of the extracellular part of the DLK1 protein, that is, its dissolved form.

The secreted fragment of DLK1 is secreted into human serum and may be detected in the serum of patients with liver cancer or hepatoblastoma.

At present, the main immunodiagnostic detection methods for DLK1 are: immunoenzyme-linked adsorption assay (ELISA).

The enzyme-linked immunosorbent assay (ELISA) is a detection method using enzyme-labeled antibodies or antigens as the main reagents. It belongs to a labeled immune technology. Among various diagnostic methods, ELISA is the most detailed a kind of. Currently available DLK1 detection kits on the market are Human Pref-1/DLK-1/FA1Quantikine ELISA Kit (Cat. No.: DPRF10, discontinued) from R&D Company and DLK1, SOLUBLE(human) ELISA KIT, (Cat. No.AG-45A-0032EK-KI01).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an ELISA kit for detecting human serum DLK1 protein and its application. The specific technical problems to be solved are as follows:

One of the technical problems to be solved by the present invention is to provide a monoclonal antibody that can specifically bind to native DLK1 or denatured DLK1.

The second technical problem to be solved by the present invention is to provide a hybridoma cell line that secretes and produces the above-mentioned monoclonal antibody.

The third technical problem to be solved by the present invention is to provide a method for preparing the monoclonal antibody of the present invention.

The fourth technical problem to be solved by the present invention is to provide an ELISA kit for rapid detection of human serum DLK1 protein with high sensitivity and specificity comprising the monoclonal antibody of the present invention.

The fifth technical problem to be solved by the present invention is to provide the coating liquid of the above-mentioned ELISA kit using 1*PBS.

The sixth technical problem to be solved by the present invention is to provide the coating liquid of the above-mentioned ELISA kit through 8.18g sodium chloride, 0.2g potassium chloride, 3.58g 12 water disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, using hydrogen It is prepared by adjusting the pH of sodium oxide to 7.3 and then diluting to 1000ml.

The seventh technical problem to be solved by the present invention is to provide 1.0N hydrochloric acid for the enzyme reaction termination solution described in the above-mentioned ELISA kit.

The eighth technical problem to be solved by the present invention is to provide the detection method of the above-mentioned ELISA kit.

The ninth technical problem to be solved by the present invention is to provide the application of the above-mentioned ELISA kit.

In order to solve the above-mentioned technical problems, the present invention is realized through the following technical solutions:

In the present invention, the extracellular end (24aa-303aa) of the recombinant human DLK1 protein is selected as the immunogen, and mice that secrete high-titer anti-DLK1 monoclonal antibody are obtained by repeatedly subcutaneously immunizing mice with small doses; The spleen cells were taken from mice, and multi-strains of hybridoma monoclonal cells that stably secreted anti-human DLK1 antibodies were established by in vitro fusion with mouse myeloma cells, drug screening and subcloning. Two of the hybridoma cell lines, codenamed 29A7D11C8B7-019 and 27B1C2E3G8-020, were identified by ELISA, western blotting, flow cytometry, immunohistochemistry and other methods, and it was confirmed that the monoclonal antibodies secreted by them can specifically recognize and binds to human DLK1.

The first aspect of the present invention provides two hybridoma cell lines that can secrete DLK1 monoclonal antibody, the hybridoma cell lines are SP2/0 and Balb/c mouse splenocyte monoclonal antibody hybridoma cell lines; deposit number CGMCC No. 11596 or CGMCC No. 11595, respectively.

In the second aspect of the present invention, two DLK1 monoclonal antibodies that can specifically bind to DLK1 protein are provided, which are secreted and produced by the hybridoma cell lines with the above-mentioned deposit numbers of CGMCC No. 11596 or CGMCC No. 11595.

In a third aspect of the present invention, a method for preparing the above-mentioned monoclonal antibody is provided, comprising the steps of:

Step 1. Prepare recombinant human DLK1 protein as an immunogen;

Step 2. Animal immunization: mice that secrete high-titer anti-human DLK1 monoclonal antibody were obtained by repeatedly subcutaneously immunizing mice with small doses;

Step 3. Pick mice from them, take their spleen cells, and fuse them with mouse myeloma cells in vitro;

Step 4. Screening hybridoma cells with positive antibody secretion by enzyme-linked immunosorbent assay;

Step 5. The positive hybridoma cells are identified by subcloning to obtain multiple hybridoma monoclonal cells that stably secrete anti-human DLK1 monoclonal antibody;

Step 6. The hybridoma cells are expanded and cultured, the culture medium is collected, and the anti-human DLK1 monoclonal antibody is separated and purified by affinity chromatography.

Wherein, step 1 is specifically as follows: inserting the fragment kappa sp-DLK1soluble reg-His6tag, cloning it into the mammalian expression vector pCPC vector, obtaining the expression plasmid pCPC-DLK1-His, transfecting 293-E/CHOE cells, and screening out high-efficiency The engineered cell line is expressed, and after the cell line is cultured and expanded, and the supernatant is collected for separation and purification, the DLK1 protein with a purity of more than 95% is obtained.

Wherein, the method for subcutaneous immunization of mice described in step 2 is as follows: mixing the purified recombinant human DLK1 protein with Freund's complete adjuvant, and subcutaneously injecting mice at multiple points.

Wherein, the affinity chromatography method described in step 6 is specifically: loading the supernatant of the hybridoma cell filtrate containing the monoclonal antibody on the Ni-EF affinity chromatography pre-filled with the covalently cross-linked recombinant human DLK1 protein After loading the sample, the affinity chromatography column was eluted with 0.1M Tris-HCL Ph8.0 to remove impurity proteins, and then the antibody protein adsorbed by the Ni-EF column was eluted with citric acid, and then dialyzed; the sample after dialysis The purified anti-DLK1 monoclonal antibody was obtained after filtration.

In a fourth aspect of the present invention, an ELISA kit for detecting human serum DLK1 protein is provided, comprising: a solid-phase carrier coated with a DLK1 monoclonal antibody, an enzyme-labeled DLK1 monoclonal antibody, an enzyme substrate, and a negative control The DLK1 antibody coated on the solid phase carrier is derived from recombinant antibody protein 020.

The coating solution used 1*PBS.

Described coating liquid is prepared by 8.18g sodium chloride, 0.2g potassium chloride, 3.58g 12 water disodium hydrogen phosphate, 0.24g potassium dihydrogen phosphate, and adjusting pH to 7.3 with sodium hydroxide and then settling to 1000ml. have to.

The enzyme-labeled antigens are divided into two types, including:

No. 1 enzyme conjugate, using biotin-labeled mouse anti-human DLK1 monoclonal antibody secreted by the hybridoma cell line with deposit number CGMCC No. 11596; working solution configuration of No. 1 enzyme conjugate: sample diluent Dilute it at a ratio of 1:10000 and mix it well, that is, it is configured as a working solution of No. 1 enzyme conjugate, which needs to be prepared immediately for use here;

No. 2 enzyme conjugate, using horseradish peroxidase streptavidin (Horseradish Peroxidase Streptavidin) labeled mouse anti-human DLK1 monoclonal antibody secreted by the hybridoma cell line with deposit number CGMCC No. 11595 ; Working solution configuration of No. 2 enzyme conjugate: Dilute with sample diluent at a ratio of 1:5000 and mix well, that is, configure No. 2 enzyme conjugate working solution, which needs to be prepared immediately.

The enzyme reaction termination solution adopts 1.0N hydrochloric acid solution.

In a fifth aspect of the present invention, a detection method for detecting human serum DLK1 is provided, comprising the steps of:

Step 1. Sample dilution: Dilute the serum to be tested by 1:20 with the sample diluent (if the content is particularly high, the dilution ratio should be increased);

Step 2. Sample addition reaction:

(1) Add 50ul of diluted sample serum to each well of the sample testing well, and make two or more duplicate wells for each sample in parallel. At the same time, set 2 wells for negative, positive and blank controls, and take negative and positive controls. 100ul of each was added to the reaction wells, and only 100ul of sample diluent was added to the blank control wells;

(2) After 60 minutes of reaction in the dark at 37°C, shake off the liquid in the wells, fill each well with sample diluent or washing solution, shake off immediately, then fill with sample diluent or washing solution, let stand for 2 minutes, and shake off Liquid, repeat washing 3 times, each time need to stand for 2 minutes, shake off and pat dry for the last time;

Step 3. Enzyme reaction:

(1) Add 50ul of the working solution of No. 1 enzyme conjugate to each well, react in the dark at 37°C for 60 minutes, shake off the liquid in the well, wash as above, and pat dry;

(2) Add 50 ul of the working solution of No. 2 enzyme conjugate to each well, shake off the liquid in the well after reacting in the dark at 37°C for 30 minutes, repeat washing as above for 5 times, and pat dry;

Step 4. Color reaction: add 100ul of the prepared substrate coloring solution TMB to each well, react at room temperature for 10-15 minutes, add 50ul/well of enzyme reaction stop solution, mix well, terminate the reaction, and immediately read with a microplate reader;

Step 5. Measure the reading of the microplate reader: immediately measure and read the microplate plate that has terminated the reaction, and use the blank control to zero to read the OD value at 450 nm.

In a sixth aspect of the present invention, there is provided the application of the above ELISA kit in preparing a product for detecting liver cancer and hepatoblastoma. The applications do not include applications in disease diagnosis and treatment.

A hybridoma cell line of the present invention that can secrete DLK1 monoclonal antibody is code-named 29A7D11C8B7-019, and is classified as SP2/0 and Balb/c mouse splenocyte monoclonal antibody hybridoma cell lines. On the 24th, it was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (referred to as CGMCC), the deposit number is CGMCC No. 11596, and the deposit address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

Another hybridoma cell line of the present invention that can secrete DLK1 monoclonal antibody is codenamed 27B1C2E3G8-020, and is classified as SP2/0 and Balb/c mouse splenocyte monoclonal antibody hybridoma cell line. It was deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee (referred to as CGMCC) on March 24, and the deposit number is CGMCC No. 11595. The deposit address is: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

Compared with the prior art, the present invention has the following beneficial effects:

1. The ELISA kit and detection method for detecting human serum DLK1 protein of the present invention have high sensitivity and specificity, and can quickly detect the expression of DLK1 and liver cancer and hepatoblastoma; It plays an important role in the serum of patients;

2. The technical advantage of the present invention is that compared with the existing DLK1 detection kits on the market, the detection sensitivity of DLK1 protein is improved, and lower concentrations of DLK1 protein in serum can be detected, providing a more reliable reference for clinical diagnosis. significance.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

Fig. 1 is the standard curve of the identification and detection of 019/02 hybridoma cell supernatant binding to recombinant DLK1 protein by ELISA;

2 is a schematic diagram showing the comparison of the standard curve of the ELISA detection kit and the existing commercial kit in Example 5 of the present invention; wherein, mAb020 (0.25ug/ml) refers to the standard curve of the kit of the present invention, and Kit refers to the standard curve of the kit of the present invention. is the standard curve of the control commercial kit (Human Pref-1/DLK-1/FA1Quantikine ELISA Kit);

Figure 3 is a graph showing the results of detecting liver cancer samples using a commercial control kit;

4 is a graph of the detection results of DLK1 in the serum of 40 normal children using the kit of the present invention;

FIG. 5 is a graph showing the results of detecting liver cancer serum samples using the kit of the present invention.

Detailed ways

The present invention will be described in detail below with reference to the embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several adjustments and improvements can be made without departing from the inventive concept. These all belong to the protection scope of the present invention. The experimental methods for which specific conditions are not indicated in the examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

Example 1. Establishment, screening and identification of hybridoma cell lines that stably secrete anti-DLK1 monoclonal antibodies

Step 1. Preparation of recombinant human DLK1 protein (immune antigen)

Using PCR technology, the BGH sequence was deleted from the 5' end of the self-built plasmid vector pcDNA3.1B-DLK1 (pcDNA3.1B sequence was deleted, T7 promoter, XhoI, NotI, EcoRV and EcoRI restriction sites were added, and three flags were introduced. tag, the modified vector is more convenient for gene expression) to amplify the gene fragment encoding the human DLK1 secretory region sequence (DLK1 soluble region, 24-303aa), the kappa signal peptide sequence is added in the front, and the His 6tag tag sequence is added in the back. The insert fragment kappa sp-DLK1soluble reg-His 6tag was identified correctly by sequence determination, and after treatment with restriction endonuclease, it was cloned into the expression vector pCPC(KpnI/NotI) (purchased). The expression plasmid pCPC-DLK1-His was obtained and transfected into 293-E/CHOE cell line, and the high-expression cell line was screened. The high-efficiency expression cell line is amplified and cultured, and after expression, separation and purification, the DLK1-His protein with a purity of more than 95% is obtained.

Step 2. Animal immunization:

The recombinant human DLK1 protein purified above was mixed with Freund's complete adjuvant, and 5 Balb/c and 5 SJL mice were subcutaneously injected at multiple points (100ul/mice, a total of 100ug DLK1 protein). 2-3 weeks after the first immunization, the mice were further boosted by subcutaneous injection of DLK1 protein and incomplete adjuvant mixture. After boosting immunization for 2-3 times, take a small amount of mouse serum, use 96-well microtiter plate coated with DLK1 protein to detect the titer of anti-DLK1 protein antibody in mouse serum by ELISA, and the spleen cells of mice with higher titer for the next step of cell fusion.

Step 3. Cell fusion:

Three days after the last immunization with DLK1 protein, mouse spleen cell suspension was aseptically prepared, and S/P 20 mouse myeloma cells (purchased from the Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) were in a ratio of 10:1 at 50% Fusion under the action of PEG-1500 (product of Sigma Company, USA). The fusion was performed according to the conventional method (Kohler G. and Milstein C: Nature 1975; 256: 495-497), and the amount of PEG was 1 ml, which was slowly added within 1 minute. After 90 seconds of reaction, the reaction was terminated with serum-free RPMI-1640 medium, centrifuged at 1000 rpm for 10 min, and the supernatant was removed. T thymidine, a product of Sigma company), the cell concentration was adjusted to 1×10 ⁶ cells/ml in RPMI 1640-10% FCS medium, added to a 96-well flat-bottomed cell culture plate (200ul per well), at 37°C, Culture in a 5% CO ₂ incubator for 2-3 weeks.

Step 4. Enzyme-linked immunosorbent assay (ELISA) to screen hybridoma cells with positive antibody secretion:

The ELISA plate was coated with recombinant human DLK1 protein (1 μg/ml, pH 7.4, PBS solution), and coated at 37°C for 2 hours or at 4°C overnight; blocked with 2% bovine serum albumin (BSA) and kept at 4°C overnight. After washing with PBS-0.1% Tween20 solution, add the culture supernatant of the hybridoma cells to be tested (using unfused S/P 20 myeloma cell culture supernatant as negative control) and incubate at 37°C for 2 hours; After washing, horseradish peroxidase (HRP)-labeled goat anti-mouse Ig (product of Sigma) was added, and incubated at 37°C for 1 hour; after thorough washing with PBS-0.1% Tween20 solution, TMB substrate solution was added to show The reaction was terminated with 0.1M HCl for 10-15min. Read the OD value at 450 nm in a plus384 microplate reader (product of M.D Company). Hybridoma cells with a measured OD 450 value 5-10 times higher than that of the negative control were re-cloned and expanded and cryopreserved.

Step 5. Subcloning of positive hybridoma cells-limiting dilution method

The positive cells obtained from the primary screening above were diluted with RPMI-1640-10% FCS medium to 1-10 cells per well, plated on a 96-well cell culture plate, and cultured in a 37°C, 5% CO ₂ incubator for 2 -3 weeks. After the clones were grown, the supernatant was taken to detect the secretion of anti-DLK1 antibody again by ELISA. After detection and identification, a number of antibody secretion-positive cell lines were obtained. Among them, after subcloning and identification again, two hybridoma cell lines with the codes 29A7D11C8B7-019 and 27B1C2E3G8-020 (referred to as 019 and 020 for short) were obtained that stably secreted anti-DLK1 monoclonal antibodies, respectively corresponding to the deposit number of CGMCCNo.11596 , CGMCC No.11595 hybridoma cell line. Figure 1 shows the identification and detection of the binding of the 019/020 hybridoma supernatant to the recombinant DLK1 protein by ELISA. The results show that the hybridoma supernatant contains a high titer of monoclonal antibody against DLK1 protein. The monoclonal antibodies were identified as IgG2a and IgG1 classes, respectively. The hybridoma cell line was further amplified in large quantities, subcultured for a long time and cryopreserved. Table 1 below is the data corresponding to the curve in FIG. 1 .

Table 1

bovine serum group BSA group r2 0.9855 0.9943 Scope of application 0.02-5ng/ml 0.02-5ng/ml

Example 2. In vitro preparation and purification of anti-DLK1 monoclonal antibody

The established 019/020 hybridoma cells were expanded and cultured in serum-free medium, and the addition of liquid was stopped when the cell concentration reached 10 ⁵ cells/ml or more, and the culture was continued until the cell culture medium turned yellow. The culture medium was collected, centrifuged at 1500 rpm for 10 minutes, and the supernatant was filtered through a 0.45 μm filter membrane and stored at 4°C or -20°C for later use or directly used for the next step of separation and purification of monoclonal antibodies.

Step 6. The hybridoma cells are expanded and cultured, the culture medium is collected, and the anti-human DLK1 monoclonal antibody is separated and purified by affinity chromatography.

In this example, the anti-DLK1 monoclonal antibody (019/020) was isolated and purified by affinity chromatography. The purification steps are: loading the supernatant of the hybridoma cell filtrate containing the 019/020 monoclonal antibody on an affinity chromatography column (200ml/5ml chromatography column) pre-filled with DLK1ProteinA (GE product); The affinity chromatography column was eluted with 0.1M Tris-HCL Ph8.0 to remove impurity proteins, and then the adsorbed antibody proteins were eluted with low pH (2.7) citric acid (0.1M) solution. The eluent was adjusted to pH 7.0 with 1mol/LTris (pH 9.0), and then dialyzed against 10 times the volume of 1x PBS for 12-16 hours (the medium was changed 2-3 times during the period), and the dialyzed samples were filtered through 0.22 μm. The purified anti-human DLK1 monoclonal antibody was obtained after membrane filtration.

Example 3. Preparation of human serum DLK1 protease-linked detection kit

1. Antigen-coated solid support

The solid phase carrier can be selected from polystyrene or polyvinyl chloride in the form of a 96-well plate or a microtiter plate.

The anti-human DLK1 monoclonal antibody (019/020) 100ul/well (0.25ug/ml) prepared in Example 2 was coated in polystyrene or polyvinyl chloride reaction wells.

2. Solution preparation

2.1 Preparation of coating buffer

The coating solution used 1*PBS. The coating solution was prepared by adjusting the pH to 7.3 with sodium hydroxide and then adjusting the volume to 1000ml by 8.18g sodium chloride, 0.2g potassium chloride, 3.58g 12 water disodium hydrogen phosphate and 0.24g potassium dihydrogen phosphate. .

2.2 Preparation of enzyme conjugates (enzyme-labeled antigens)

There are two types of enzyme-labeled antigens, including:

No. 1 enzyme conjugate, using biotin-labeled DLK1 antibody (code 019).

Working solution configuration of No. 1 enzyme conjugate: Dilute with sample diluent at a ratio of 1:10000, mix well, and configure No. 1 enzyme conjugate working solution, which needs to be prepared immediately.

No. 2 enzyme conjugate, using horseradish peroxidase streptavidin Horseradish Peroxidase Streptavidin labeled DLK1 antibody (code 020). (SIGMA, S2438-250UG)

Working solution configuration of No. 2 enzyme conjugate: Dilute with sample diluent at a ratio of 1:5000, mix well, and configure No. 2 enzyme conjugate working solution, which also needs to be prepared immediately.

2.3 Enzyme substrate solution (chromogenic solution)

Two-component TMB (Huzhou Yingchuang Biotechnology, TMB-S-003), ready-to-use and ready-to-use, when using, mix solution A and solution B at a ratio of 1:1.

2.4 Sample Diluent or Wash Solution Configuration

Select the following samples and weights: 1% BSA

Configured as a sample diluent.

2.5 Configuration of stop solution

Add 10ml of HCl (36-38%, 7647-01-0, 12M) to 110ml of distilled water, the mixing process needs to be carried out slowly, and it is configured into 1N HCl as a stop solution.

Embodiment 4, detection method and operation procedure

1. Sample Dilution

Dilute the serum to be tested by 1:20 with sample diluent, for example, add 100ul of diluent to 5ul of serum, and mix thoroughly. The yin and yang reference substances do not need to be diluted.

2. Sample addition reaction

2.1 Add 50ul of diluted sample serum to each well of the sample testing well. It is recommended to test 2 wells of each sample in parallel. At the same time, 2 wells of negative, positive and blank controls were set up, and 50ul of negative and positive controls were added to the reaction wells respectively, and only 50ul of sample diluent was added to the blank control wells.

2.2 After 60 minutes of reaction in the dark at 37°C, shake off the liquid in the well, fill each well with sample diluent or washing solution, shake off immediately, then fill with sample diluent or washing solution, let stand for 2 minutes, shake off the liquid, Repeat the washing 3 times, each time need to stand for 2 minutes, the last time to shake off and pat dry.

3. Enzyme reaction

3.1 Add 50ul of the working solution of No. 1 enzyme conjugate (ready to use) to each well, react in the dark at 37°C for 60 minutes, shake off the liquid in the well, wash as above, and pat dry.

3.2 Add 50ul of No. 2 enzyme conjugate working solution (ready-to-use) to each well, react at 37°C in the dark for 30 minutes, shake off the liquid in the well, repeat the above washing three times, and pat dry.

4. Color development reaction: add the prepared substrate color development solution (currently prepared) TMB to each well, 100ul/well, and react at room temperature for 15 minutes. Add stop solution 1.0N HCL 50ul/well, mix well, and stop the reaction. Read immediately with a microplate reader.

5. Measure the reading of the microplate reader: immediately measure and read the microplate plate that has terminated the reaction, and use the blank control to zero to read the OD value at 450nm.

It should be noted here that the color reaction of this system is fast. When developing, terminating and measuring the reading, each block must be carried out one by one, otherwise errors are easily caused.

6. Result judgment

Instrument judgment: the OD value of the well to be tested is greater than or equal to 2.0 times of the negative control as positive. When the OD value of the negative control is lower than 0.05, it is calculated as 0.05.

7. Precautions

(1) When using the row gun to add samples, it is necessary to avoid the pipette tip touching the inner wall of the ELISA plate hole;

(2) The kit should be stored at 2-8°C, in which No. 1 enzyme conjugate (No. 1 solution), substrate (No. 3 solution), negative control substance, and positive control substance should be stored at -20°C, and each time they are taken out Equilibrate to room temperature before use;

(3) When washing, fill the hole with diluent or washing liquid, and shake off the liquid in the hole after parking for 2 minutes each time. Disable other water sources such as tap water.

Example 5. The ratio of the human serum DLK1 protease-linked detection kit of the present invention to the existing commercial kit Compare.

The standard curve was compared between the human serum DLK1 protease-linked detection kit of the present invention and the existing commercial kit (Item No.: DPRF10, Human Pref-1/DLK-1/FA1Quantikine ELISA Kit).

Figure 2 is a schematic diagram comparing the standard curve of the ELISA detection kit in this embodiment and the existing commercial kit; as shown in Figure 2, the results show that the sensitivity of the kit of the present invention is higher than that of the commercial kit.

The r2 and detection range of the kit of the present invention are shown in Table 2 below:

Table 2

in BSA r2 0.9943 Scope of application 0.02-5ng/ml

The r2 and application range of commercial kits are shown in Table 3 below:

table 3

r2 0.9935 Scope of application 0.156-10ng/ml

Embodiment 6, further test the kit of the present invention with human serum to obtain the test of the kit effect and clinical application prospect.

1. To evaluate the sensitivity of recombinant antibodies #019 and #020 for tumor (liver cancer, hepatoblastoma) ELISA diagnosis

Blood collection method: Patients with hepatoblastoma, children with reproductive system tumors (testicular cancer, yolk sac tumor, etc.), infant hepatitis, and normal children are the blood collection objects

(1) Select a certain number of patients with hepatoblastoma, children with reproductive system tumors (testicular cancer, yolk sac tumor, etc.), infant hepatitis, normal children, and collect blood samples before and after surgery or during outpatient physical examination or chemotherapy. Blood samples must have AFP clinical test values

All blood samples were renumbered to meet the requirements of single-blind and double-blind methods.

2. ELISA detection

2.1 Human serum DLK1 detection kit: Human Pref/DLK1-1/FA1Quantikine ELISA kit produced by R&D systems was used as a control.

A commercial control kit was used to detect liver cancer samples, and some serum samples could be detected to be positive for DLK1. As shown in Figure 3, 6 of the 40 liver cancer samples could be detected to be positive for DLK1 expression.

2.2 Laboratory testing:

Diagnostic antibodies: #019, #020 2 antibodies are used in combination

ELISA: Biotin/Streptavidin

Negative standard: take the serum of normal children as the reference.

Positive control: DLK1 is an embryonic expression gene, similar to AFP, and newborn babies still have a high level of DLK1 expression, which is a physiologically high expression. Therefore, infant serum less than 1 month old can also be used as a positive control.

Detection of DLK1 in serum of 40 normal children using this patented kit (10 infants less than 1 month old, 10 infants from 1 month to 1 year old, 10 infants from 1 year old to 3 years old, 10 e.g. children older than three years). As shown in Figure 4, the results show that this kit can distinguish the expression of DLK1 at different ages. The serum DLK1 protein expression of infants less than 1 month old was the highest, and 10 samples were positive; only 4 cases of DLK1 protein expression were positive in the serum samples of infants from 1 month to 1 year old; DLK1 protein expression was barely detectable in serum samples from three-year-old children.

Serum samples of liver cancer were detected using the patented kit; as shown in Figure 5, serum samples with elevated DLK1 expression could be detected. The serum samples of 20 cases of liver cancer were diluted 1:4 and 1:10 respectively. The results showed that compared with the 1:4 serum dilution concentration, the expression of DLK1 protein could still be detected in the serum samples after 1:10 dilution.

3. Statistical results

The blood samples collected from hepatoblastoma and other patients were detected by ELISA using single-blind method and double-blind method respectively. The total number of samples detected was 79 cases (Table 4).

The results show that DLK1 and AFP have consistency and complementarity in the detection of patient serum, and are expected to be used as supplementary diagnostic indicators for some patients with negative clinical AFP.

Table 4 Blind evaluation results of 019 and 020 antibody ELISA in serum of patients with hepatoblastoma and other patients

diagnostic antigen Number of cases Proportion (%) Elevated AFP 37 46.83 Elevated DLK1 28 35.44 Elevated AFP, normal DLK1 19 24.05 AFP is normal, DLK1 is elevated 10 12.66 AFP is elevated, DLK1 is elevated 18 22.78

4. Result analysis

At present, the detection of childhood hepatoblastoma, gonadal tumor, and infant hepatitis mainly relies on the serological examination of AFP, but the detection of AFP also has a certain missed detection rate. Due to the sensitivity of the method or the influence of other interference factors, the detection of this method The results cannot faithfully reflect the actual number of patients. Therefore, the development of additional diagnostic test kits with higher sensitivity and specificity is particularly important for the diagnosis of hepatoblastoma, gonadal tumors, and infantile hepatitis. The present invention obtains a target gene DLK1 with diagnostic value through the research on liver cancer and hepatoblastoma related genes, and obtains a target gene DLK1 with diagnostic value by using molecular biology laboratory technology. Serum detection of tumors, infant hepatitis and normal children, the results show that the recombinant antibody of DLK1 can be used as a diagnostic kit for serum detection of patients, its sensitivity is slightly higher than that of mature kits, and its specificity is close to mature kits. Therefore, these two recombinant antibodies have the prospect of being further developed into diagnostic kits.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention.

Claims (5)

1. a hybridoma cell line that secretes DLK1 monoclonal antibody, is characterized in that, described hybridoma cell line is SP2/0 and Balb/c mouse splenocyte monoclonal antibody hybridoma cell line; Deposit number is respectively CGMCC No. 11596 or CGMCC No. 11595. 2 . A DLK1 monoclonal antibody, characterized in that, the DLK1 monoclonal antibody is secreted and produced by the hybridoma cell line according to claim 1 . 3. an ELISA kit for detecting human serum DLK1 protein, characterized in that the kit comprises: a solid phase coated with any one of the DLK1 monoclonal antibodies described in claim 2 or the combination of both Carrier, enzyme-labeled DLK1 monoclonal antibody according to claim 2 any one or the combination of both, enzyme substrate, negative control substance and positive control substance, coating solution, washing solution and enzyme reaction termination solution . 4. the ELISA kit of detecting human serum DLK1 protein according to claim 3, is characterized in that, the DLK1 monoclonal antibody of described enzyme label is the combination of two, comprising: Using biotin-labeled mouse anti-human DLK1 monoclonal antibody secreted by the hybridoma cell line with deposit number CGMCC No. 11596; A mouse anti-human DLK1 monoclonal antibody secreted from a hybridoma cell line with deposit number CGMCC No. 11595, labeled with horseradish peroxidase streptavidin, was used. 5. A use of the ELISA kit as claimed in claim 3 or 4 in the preparation of a product for detecting liver cancer and hepatoblastoma.

Images