CN112574955B - Hybridoma cell LCZ9H4, monoclonal antibody secreted by same and application of monoclonal antibody - Google Patents
Hybridoma cell LCZ9H4, monoclonal antibody secreted by same and application of monoclonal antibody Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention discloses a hybridoma cell LCZ9H4, a monoclonal antibody secreted by the hybridoma cell LCZ9H4 and application of the monoclonal antibody, wherein the hybridoma cell LCZ9H4 is preserved in China center for type culture Collection with the preservation number of CCTCC No: C2019145. the invention can stably generate the anti-delta-catenin monoclonal antibody, has stable cell chromosome and high antibody titer, and can be completely applied to the research of the application of delta-catenin.
Description
Technical Field
The invention belongs to the technical field of clinical diagnosis, and particularly relates to a hybridoma cell LCZ9H4 and a preparation method thereof, and an application of a monoclonal antibody secreted by the hybridoma cell LCZ9H4 and a monoclonal antibody in a kit for detecting human prostate cancer.
Background
Treatment and prognosis of prostate cancer is largely dependent on early diagnosis. Although testing of men generally at risk for prostate cancer is not mandated, the american cancer society recommends that men perform prostate-specific antigen (PSA) testing and Digital Rectal Examination (DRE) from the age of 50. Men at higher risk for prostate cancer should begin testing at age 45. Over the past decades, the 5-year survival rate of prostate cancer patients has increased dramatically since early screening for prostate cancer has allowed early detection of prostate cancer at both early and locally advanced stages, which is also well indicative of the importance of early detection and diagnosis of prostate cancer.
Prostate cancer is screened clinically primarily using Prostate Specific Antigen (PSA), fPSA/tPSA and Digital Rectal Examination (DRE), and patients with suspected prostate cancer are usually initially treated with digital rectal examination or serum PSA examination before taking a biopsy for prostate aspiration. Prostate cancer patients are diagnosed by histopathological diagnosis, primarily by systemic needle biopsy of the prostate gland.
However, a large body of data suggests that elevated serum PSA levels may be the result of factors other than prostate cancer, such as benign prostatic hyperplasia. Thus, false positives are a major drawback of PSA screening and may lead to unnecessary biopsies or other painful interventions for the patient. In addition, preoperative serum PSA levels are not always correlated with cancer volume or prostate cancer Gleason score. In addition, a considerable part of patients are in a diagnosis gray area (serum tPSA is between 4 and 10ng/ml, and domestic fPSA/tPSA >0.16 is recommended as a normal reference value), but even if fPSA/tPSA >0.25, the probability of prostate cancer is still about 8 percent, so that a clinically considerable part of patients cannot be diagnosed at an early stage. Many studies estimate that PSA examinations fail to detect more than 20% to 30% of prostate cancer patients. Recently, some investigations of clinical data in the united states have concluded that PSA screening does not detect prostate cancer well.
Therefore, it becomes important to develop more reliable biomarkers to complement PSA screening, which can improve the accuracy of risk assessment and prediction of the progression of prostate cancer, and avoid over-diagnosis and treatment of patients. The development of prostate cancer biomarkers independent of PSA detection not only can improve the accuracy of early diagnosis, but also can provide good guidance for treatment protocols.
Disclosure of Invention
The invention mainly solves the technical problem of providing a hybridoma cell LCZ9H4 and a preparation method thereof, a monoclonal antibody secreted by the hybridoma cell and a kit for detecting human prostate cancer.
In order to solve the technical problem, the invention adopts the following technical scheme:
a hybridoma cell LCZ9H4, characterized by: the hybridoma LCZ9H4 is preserved in China center for type culture Collection with the preservation number of CCTCC No: C2019145.
the invention also provides a monoclonal antibody, wherein the monoclonal antibody is secreted and produced by the hybridoma cell LCZ9H 4.
The invention also provides a kit, which comprises the hybridoma cell or the monoclonal antibody.
Further, the kit is a chemiluminescence kit.
The invention also provides the application of the hybridoma cell or the monoclonal antibody in preparing a kit.
Further, the kit is an immunoassay-based kit.
Further, the kit is a chemiluminescence kit.
Further, the kit is used for detecting human prostate cancer.
The invention also provides the application of the hybridoma cell or the monoclonal antibody in preparing a kit for diagnosing human prostate cancer.
The invention has at least the following advantages:
1. the hybridoma cell can stably generate an anti-delta-catenin monoclonal antibody, has stable cell chromosome and high antibody titer, and can be completely applied to research on application of delta-catenin;
2. the delta-catenin-resistant monoclonal antibody can be applied to the preparation of a human prostate cancer diagnostic kit, and detection samples comprise human urine, blood, semen, prostate massage liquid and other human samples, and is used for the diagnosis of human prostate cancer, thereby greatly making up the defects of the existing clinical diagnosis technology.
3. The detection method of the invention is not limited to the chemiluminescence immune technology, but also comprises various similar immune technologies such as a colloidal gold immune chromatography technology, a fluorescence immune chromatography technology and the like.
Detailed Description
The following detailed description of the preferred embodiments of the present invention is provided to enable those skilled in the art to more readily understand the advantages and features of the present invention, and to clearly and unequivocally define the scope of the present invention.
Example 1: preparation of hybridoma cell LCZ9H4 comprising the steps of:
1. the establishment of the mouse anti-delta-catenin hybridoma cell comprises the following steps:
a. selecting 8 healthy female BALB/C mice with age of 8-12 weeks and weight of about 20g, breeding for 1 week, and collecting negative blood as control;
b. the primary immunization is emulsified by 60ug/ml delta-catenin protein and Freund's complete adjuvant with equal volume stirring, and is injected intramuscularly. After two weeks, strengthening immunization for 2 times by using 30ug/ml delta-catenin protein and Freund incomplete adjuvant, and detecting the titer of serum antibodies by ELISA (enzyme-linked immuno sorbent assay) at intervals of 2 weeks, wherein the titer of an immunized mouse obviously reaches more than 1;
wherein, the ELISA detection method specifically comprises the following steps: the delta-catenin protein antigen was diluted to 3ug/ml with coating buffer added to a 96-well plate, 50u1,1 h was added to each reaction well, washed 3 times with wash solution, blocked with 100u 1/well blocking solution at 37 ℃ for 2h, washed 3 times, and patted dry. The test serum was added, diluted in the first well 1 at 800, diluted down to a gradient multiple of 1. And (3) diluting horseradish enzyme labeled goat anti-mouse IgG according to the proportion of 1 to 2500, adding the diluted goat anti-mouse IgG into a reaction hole at a rate of 100u 1/hole, incubating for 30min at 37 ℃, washing for 4 times, and patting dry. Finally adding TMB 100u 1/hole, and developing for 15-30min. Stop solution, 50u 1/well, was then added. The OD value of each well is measured at a single wavelength of 450nm, and a positive control well is used as a positive judgment. The result is a positive mouse with a serum titer of 1.
c. The delta-catenin protein of 40ug/ml is used for strengthening the immunity through intramuscular injection (without adjuvant), the tail part is sampled after 3 days, the serum antibody titer is detected by ELISA, and meanwhile, the spleen is taken.
d. Spleen cells and myeloma cells were mixed at about 10 cell numbers and fused under the action of polyethylene glycol (PEG, molecular weight 1500) to select a medium of HAT-containing RPMI1640 medium (50 ml). After 11 days, screening out positive hybridoma cells capable of reacting with delta-catenin protein by an indirect ELISA method, carrying out expanded culture on the preliminarily screened positive hybridoma cells, removing the hybridoma cells with the labeled protein, and re-screening out hybridoma cells which are not labeled and are directed against the delta-catenin protein;
e. cloning the obtained positive hybridoma cells by a limiting dilution method, and finally determining 5 hybridoma cells A-1, A-2, A-3, A-4 and A-5 which can stably secrete monoclonal antibodies through 6 times of screening.
Detecting whether all positive 5 strains of cells can specifically recognize delta-catenin by an indirect ELISA method: a positive cell named A can be specifically combined with delta-catenin, which shows that A can specifically recognize delta-catenin. The hybridoma cell A capable of stably secreting the anti-human delta-catenin monoclonal antibody is obtained by screening, is classified and named as hybridoma cell LCZ9H4, is preserved in China Center for Type Culture Collection (CCTCC) at the university of Wuhan, china at 8 months and 28 days in 2019, and has the preservation number of CCTCC N0: C2019145.
2. Preparation of mouse anti-human delta-catenin monoclonal antibody and identification of monoclonal antibody subtype
The culture supernatants of 1000u1 hybridoma cell LCZ9H4 (5 hybridoma cells LCZ9H4-1, LCZ9H4-2, LCZ9H4-3, LCZ9H4-4, and LCZ9H4-5) were taken, and the subtypes of the monoclonal antibody were identified by a sigma typing kit, and the results showed that: the heavy chain of the monoclonal antibody is of the IgGl type, and the light chain is of the Kappa type.
Example 2 preparation and purification of ascites monoclonal antibody against delta-catenin comprising the following steps:
a. selecting healthy female BALB/C mice with age of 8-12 weeks and weight of about 20g, and injecting sterilized liquid paraffin into the abdominal cavity, wherein each female BALB/C mouse is 0.5m 1; 7 days later, each mouse was injected intraperitoneally with 1X 10 6 5 monoclonal hybridoma cells of example 1, LCZ9H4-1, LCZ9H4-2, LCZ9H4-3, LCZ9H4-4, LCZ9H4-5, each monoclonal hybridoma cell injected into two mice;
b.7 days later, the ascites production condition of the mice is observed, and if the abdomen is obviously enlarged, the ascites can be extracted. And (3) after ascites collection, centrifuging at 13000rpm for l0min, removing grease and precipitates, and collecting supernatant, namely the ascites monoclonal antibody. The ascites monoclonal antibody is purified by sulfuric acid by precipitation and dialysis.
Carrying out ELISA titer determination, molecular mass identification and concentration determination on the ascites monoclonal antibody:
1) Measurement of ELISA Titers: an indirect ELISA is adopted to determine the purified ascites monoclonal antibody, 1ug/ml antigen 100u 1/well is coated on a 96-well plate to perform ELISA, and the result shows that the titer is l:210000 after purification.
2) Molecular mass characterization of the antibodies: the monoclonal antibody was assayed by SDS-PAGE and showed that the heavy chain was approximately 46kD and the light chain was approximately 25kD.
3) Measuring the concentration of the monoclonal antibody, namely measuring the absorbance values A280 and A260 of the monoclonal antibody at 280nm and 260nm by using an ultraviolet spectroscopy method. As a result, the protein content was 21mg/ml.
Example 3: clinical diagnosis of pathogens
1. Preparation of the kit
The kit adopts a chemiluminescence immunoassay method.
1) Rabbit anti-delta-catenin detection antibody (eliononco, usa) was coated on magnetic beads that had been activated. 100ul of activated Mag COOH magnetic beads are taken, 100ug of rabbit source anti-delta-catenin detection antibody is added, vertical mixing is carried out for 120 minutes at 25 ℃, magnetic separation and washing are carried out for 3 times, and free antibody is removed. Blocking with additional 300ul 1%;
2) Acridinium ester is labeled on the anti-delta-catenin monoclonal antibody LCZ9H 4. The labeling buffer solution is a CB system, the pH is 19.5, the feeding ratio (molar ratio) is 20:1 (acridine/antibody), and lysine was added to terminate the reaction (lysine/acridine ratio 140. Purifying the labeled antibody by sephadex G-50 sephadex column separation;
3) 50ul of the coupled magnetic beads are added into each reaction hole, and 50ul of the standard substance or urine to be detected is correspondingly added into each hole. Incubate in a 37 ℃ incubator for 30 minutes, and wash the plate 3 times with magnetic separation. Adding an acridinium ester marked anti-delta-catenin monoclonal antibody LCZ9H4, incubating for 30 minutes in a constant temperature box at 37 ℃, carrying out magnetic separation and plate washing for 3 times, and then measuring by using a chemiluminescence detector. The instrument parameters are as follows: 50ul of chemiluminescent substrate solution A and solution B are added respectively, and the integration time is 3S. Drawing a standard curve according to the chemiluminescence values of the standard substances with different concentrations, and comparing the value of the patient with the value of the positive standard hole, thereby obtaining the actual concentration of the tumor marker delta-catenin of the urine sample of the tested person.
2. Result detection
The kit is used for detecting prostate cancer and normal human urine samples. And (3) detecting true positive a, false positive b, false negative c and true negative d according to the clinical samples, and solving the detection sensitivity, specificity and total coincidence rate.
TABLE 1 comparison of tumor marker delta-catenin content (ng/ml) in patients and normal populations
| Study object grouping | Number of cases | Mean value of | Standard deviation of | Median number |
| Patient(s) is/are | 100 | 8.47 | 2.144 | 5.47 |
| Normal control | 100 | 0.79 | 0.287 | 0.58 |
*Note：Z＝P＜0.05
As can be seen from Table 1, the content of the tumor marker delta-catenin in urine of cancer patients is obviously increased compared with that of normal people, and the content is statistically significantly different.
The above 200 samples were tested for both clinical gold standard and the kit. And (3) calculating the statistical results of the clinical detection sensitivity, specificity, total coincidence rate, positive expected value, negative expected value and the like of the tumor marker delta-catenin detection kit, wherein the results are shown in a table 2.
TABLE 2 summary of clinical data for the delta-catenin assay for the clinical gold standards and kits
Sensitivity = a/(a + c) × 100% =89/100 × 100% =89.0%;
specificity = d/(b + d) × 100% =92/100 × 100% =92.0%;
total in-compliance rate = (a + d)/(a + b + c + d) = (89 + 92)/200 × 100% =90.5%.
As can be seen from Table 2, the sensitivity and specificity of the anti-human prostasomal excretion protein antibody secreted by the cell to the detection of prostasomal excretion protein can reach more than 89%.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structural changes made by using the contents of the present specification, or any other related technical fields directly or indirectly, are included in the scope of the present invention.
Claims (8)
1. A hybridoma cell LCZ9H4, wherein: the hybridoma LCZ9H4 is preserved in China center for type culture Collection with the preservation number of CCTCC No: C2019145.
2. a kit, characterized in that: the kit comprises the hybridoma cell of claim 1 or a monoclonal antibody secreted by the hybridoma cell.
3. The kit of claim 2, wherein: the kit is a chemiluminescence kit.
4. Use of the hybridoma cell of claim 1 or a monoclonal antibody secreted by said hybridoma cell for the preparation of a kit.
5. Use according to claim 4, characterized in that: the kit is based on immunoassay.
6. Use according to claim 5, characterized in that: the kit is a chemiluminescence kit.
7. Use according to claim 4, characterized in that: the kit is used for detecting human prostate cancer.
8. Use of the hybridoma cell of claim 1 or a monoclonal antibody secreted by said hybridoma cell in the preparation of a kit for the diagnosis of human prostate cancer.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910932913.XA CN112574955B (en) | 2019-09-29 | 2019-09-29 | Hybridoma cell LCZ9H4, monoclonal antibody secreted by same and application of monoclonal antibody |
| PCT/CN2019/129882 WO2021056907A1 (en) | 2019-09-29 | 2019-12-30 | Hybridoma cell lcz9h4, monoclonal antibody secreted therefrom, and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102998455A (en) * | 2012-02-14 | 2013-03-27 | 昂科生物医学技术(苏州)有限公司 | Kit for detecting or diagnosing prostatic cancer |
| CN104357404A (en) * | 2014-11-10 | 2015-02-18 | 昂科生物医学技术(苏州)有限公司 | Hybridoma cell and monoclonal antibody secreted by hybridoma cell, and applications |
| CN104897900A (en) * | 2014-03-03 | 2015-09-09 | 昂科生物医学技术(苏州)有限公司 | Prostateexosomal protein antigen, antibody and application thereof |
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| CA2678180A1 (en) * | 2007-02-13 | 2008-12-04 | Northeastern University | Methods and compositions for improving immune responses |
| CN104034902B (en) * | 2014-05-09 | 2015-11-25 | 赤峰学院 | Utilize the kit of 4 albumen associated prediction esophageal cancer patients prognosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102998455A (en) * | 2012-02-14 | 2013-03-27 | 昂科生物医学技术(苏州)有限公司 | Kit for detecting or diagnosing prostatic cancer |
| CN104897900A (en) * | 2014-03-03 | 2015-09-09 | 昂科生物医学技术(苏州)有限公司 | Prostateexosomal protein antigen, antibody and application thereof |
| CN104357404A (en) * | 2014-11-10 | 2015-02-18 | 昂科生物医学技术(苏州)有限公司 | Hybridoma cell and monoclonal antibody secreted by hybridoma cell, and applications |
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