阅读说明：本技术 杂交瘤细胞lcz8a3及其分泌的单克隆抗体和应用 (Hybridoma cell LCZ8A3, monoclonal antibody secreted by hybridoma cell LCZ8A3 and application of monoclonal antibody ) 是由 钱泽 孟洁 于 2019-09-29 设计创作，主要内容包括：本发明公开了一种杂交瘤细胞LCZ8A3及其分泌的单克隆抗体和应用,所述杂交瘤细胞LCZ8A3保藏于中国典型培养物保藏中心,保藏编号为CCTCC No：C2019144。本发明杂交瘤细胞能够稳定产生抗人前列腺小体外泄蛋白单克隆抗体,且细胞染色体稳定、抗体效价高,完全能够应用于人前列腺小体外泄蛋白的应用的研究中。(The invention discloses a hybridoma cell LCZ8A3, a monoclonal antibody secreted by the hybridoma cell LCZ8A3 and application of the monoclonal antibody, wherein the hybridoma cell LCZ8A3 is preserved in China center for type culture Collection with the preservation number of CCTCC No: C2019144. the hybridoma cell can stably produce the anti-human prostasomal exoprotein monoclonal antibody, has stable cell chromosome and high antibody titer, and can be completely applied to the research of the application of the human prostasomal exoprotein.)

1. A hybridoma cell LCZ8a3, wherein: the hybridoma LCZ8A3 is preserved in China center for type culture Collection with the preservation number of CCTCC No: C2019144.

2. a monoclonal antibody characterized by: it is secreted by the hybridoma cell LCZ8a3 of claim 1.

3. A kit, characterized in that: the kit comprises the hybridoma cell of claim 1 or the monoclonal antibody of claim 2.

4. The kit of claim 3, wherein: the kit is a chemiluminescence kit.

5. Use of the hybridoma cell of claim 1 or the monoclonal antibody of claim 2 in the preparation of a kit.

6. Use according to claim 5, characterized in that: the kit is based on immunoassay.

7. Use according to claim 6, characterized in that: the kit is a chemiluminescence kit.

8. Use according to claim 5, characterized in that: the kit is used for detecting the human prostatitis.

9. Use of the hybridoma cell of claim 1 or the monoclonal antibody of claim 2 for the preparation of a kit for the diagnosis of human prostatitis.

Technical Field

The invention belongs to the technical field of clinical diagnosis, and particularly relates to a hybridoma cell LCZ8A3 and a preparation method thereof, and application of a monoclonal antibody secreted by the hybridoma cell LCZ8A3 and a monoclonal antibody in a kit for detecting human prostatitis.

Background

Chronic Prostatitis (CP) is a common disease in adult men, and it is classified into four types: type I: acute bacterial prostatitis; type II: chronic bacterial prostatitis; type III: chronic prostatitis/chronic pelvic pain syndrome, which is divided into two subtypes, namely inflammatory (IIIA) and non-inflammatory (IIIB) according to the white blood cell count in the prostate massage liquid and semen; and IV, type: asymptomatic inflammatory prostatitis. The common clinical types of prostatitis are mainly type II, type IIIA and type IIIB. Although CP is one of the most frequent diseases in urology surgery, there is a strong clinical need for simple, convenient, non-invasive CP detection methods to aid and improve CP detection rate due to the diversity of patients' symptoms and complex etiology.

The prostasomes are subcellular structures secreted by human prostate epithelial cells, have an average diameter of 150nm (50-500nm), are secreted or leaked into the lumen by the prostate epithelial cells through exocytosis and exocytosis, can be located in the cysts, the acinar ducts, or the prostatic fluid and seminal fluid, and can also be found in the culture fluid of prostate cell lines. The major components of the prostasomes include sphingomyelin, phosphorylcholine, and ceramides. The ratio of cholesterol to phospholipids is 2:1, whereas this ratio is 1:1 on a typical mammalian cell membrane. Compared to other exosomes, prostasomes are rich in cholesterol, sphingomyelin, calcium, guanosine diphosphate, and a number of transmembrane proteins (CD13, CD46, CD55, CD59), CD59 being a membrane inhibitor of reactive cell lysis. Utlegg et al identified 139 proteins in prostasomes by high performance liquid chromatography electrospray ion mass spectrometry, which are classified into six major classes: enzyme (35%), transport structure protein (19%), guanosine triphosphate GTP-binding protein (14%), chaperone protein (6%), signal transduction protein (17%), derivative protein (9%). Poliakov et al identified 440 human prostasomal proteins by trypsin digestion and liquid chromatography/mass spectrometry as having antibacterial, antioxidant and multiple immunomodulating effects.

The antibacterial properties of the prostasomes may be due to the presence of antimicrobial protein, human cationic microbial protein 18(hcap-18), belonging to the family of antimicrobial peptides, on the membrane, which releases antimicrobial peptide LL 37. Reactive Oxygen Species (ROS) are the main cause of male idiopathic infertility, and increase of ROS is found in 40% of semen of sterile patients, while leukocyte infiltration, especially polymorphonuclear leukocytes, in the semen are the main cause of ROS generation. Unlike other antioxidants, prostasomes directly scavenge ROS, but transfer cholesterol, sphingomyelin, from the prostasomes to the cell membrane, inhibiting the NADPH oxidase activity of polymorphonuclear leukocytes, thereby reducing ROS responses. Because the prostasomes can be isolated from blood and urine, they can be used as a good prostate disease marker.

Disclosure of Invention

The invention mainly solves the technical problem of providing a hybridoma cell LCZ8A3 and a preparation method thereof, a monoclonal antibody secreted by the hybridoma cell and a kit for detecting human prostatitis.

In order to solve the technical problems, the invention adopts the following technical scheme:

a hybridoma cell LCZ8a3, wherein: the hybridoma LCZ8A3 is preserved in China center for type culture Collection with the preservation number of CCTCC No: C2019144.

the invention also provides a monoclonal antibody, wherein the monoclonal antibody is secreted and produced by the hybridoma cell LCZ8A 3.

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 the prostatitis.

The invention also provides the application of the hybridoma cell or the monoclonal antibody in preparing a kit for diagnosing the human prostatitis.

The invention has the following beneficial effects:

1. the hybridoma cell can stably produce the anti-human prostasomal exoprotein monoclonal antibody, has stable cell chromosome and high antibody titer, and can be completely applied to the research of the application of the human prostasomal exoprotein;

2. the monoclonal antibody of the anti-human prostasomal exocrine protein can be applied to the preparation of a human prostatitis diagnostic kit, and detection samples comprise human body samples such as human urine, blood, semen, prostate massage liquid and the like, so that the monoclonal antibody is used for diagnosing the human prostatitis, and greatly makes 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 LCZ8a3, comprising the steps of:

1. the establishment of the mouse anti-human prostasomal leakage protein hybridoma cell comprises the following steps:

a. selecting 9 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 72ug/ml prostasome exoprotein and Freund's complete adjuvant with equal volume stirring, and is injected intramuscularly. After two weeks, using 36ug/ml prostate corpuscle exoproteins and Freund's incomplete adjuvant to strengthen immunization for 2 times, and every 2 weeks, ELISA detecting serum antibody titer, wherein the titer of the immunized mice obviously reaches more than 1:150000, and preparing for strengthening immunization;

wherein, the ELISA detection method specifically comprises the following steps: adding the prostatic body exoprotein antigen diluted to 4ug/ml by using a coating buffer into a 96-well plate, adding 50u1 into each reaction well for 1 hour, washing by using a washing solution for 3 times, adding 100u 1/well blocking solution, blocking for 2 hours at 37 ℃, washing for 3 times, and patting to dry. Adding the serum to be detected, diluting the serum in the first hole at a ratio of 1:800, diluting the diluted serum in a gradient multiple ratio of 1:2, incubating the diluted serum for 30min at 37 ℃, washing the plate for 4 times, and patting the plate dry. Diluting goat anti-mouse IgG labeled with horseradish enzyme at a ratio of 1:2500, adding into reaction wells at a ratio of 100u 1/well, incubating at 37 ℃ for 30min, washing for 4 times, and patting dry. Finally adding TMB 100u 1/hole, and developing for 15-30 min. 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 was positive mice with a serum titer of 1:150000, which were used for cell fusion.

c. The prostate corpuscle exoproteins were injected intramuscularly at 44ug/ml to boost the immunity (without adjuvant), and after 3 days, blood was collected from the tail and the spleen was removed.

d. Spleen cells and myeloma cells were mixed at a ratio of about 10:1, and fused under the action of polyethylene glycol (PEG, molecular weight 1500) to select medium (50ml) of HAT-containing RPMI1640 medium. After 11 days, screening out positive hybridoma cells capable of reacting with the prostate corpuscle leakage protein by an indirect ELISA method, carrying out expanded culture on the preliminarily screened positive hybridoma cells, removing the labeled protein hybridoma cells, and re-screening out hybridoma cells which are not labeled and are directed to the prostate corpuscle leakage protein;

e. cloning the obtained positive hybridoma cells by using a limiting dilution method, and finally determining 8 hybridoma cells A-1, A-2, A-3, A-4, A-5, A-6, A-7 and A-8 which can stably secrete monoclonal antibodies through 7 times of screening.

The indirect ELISA method is used for detecting whether all positive 8 strains of cells can specifically recognize the prostasome exoprotein: a positive cell named A can be specifically combined with the prostate corpuscle excretion protein, which shows that A can specifically identify the prostate corpuscle excretion protein. The hybridoma cell A capable of stably secreting the monoclonal antibody of the anti-human prostasomal excretion protein is obtained by screening, is classified and named as hybridoma cell LCZ8A3, is preserved in China center for type culture Collection (CCTCC N0: C2019144) in 8-month and 28-month in 2019, and is addressed to Wuhan university in China.

2. Preparation of mouse anti-human prostasomal leakage protein monoclonal antibody and identification of monoclonal antibody subtype

The subtype of the monoclonal antibody is identified by a sigma typing kit by taking the culture supernatant of 1000u1 hybridoma LCZ8A3(8 hybridoma LCZ8A3-1, LCZ8A3-2, LCZ8A3-3, LCZ8A3-4, LCZ8A3-5, LCZ8A3-6, LCZ8A3-7 and LCZ8A3-8), and the result shows that: the heavy chain of the monoclonal antibody is of the IgGl type, and the light chain is of the Kappa type.

Example 2: the preparation and purification of the ascites monoclonal antibody of the mouse anti-human prostasome exoprotein comprises 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 10⁶8 monoclonal hybridoma cells of example 1, LCZ8A3-1, LCZ8A3-2, LCZ8A3-3, LCZ8A3-4, LCZ8A3-5, LCZ8A3-6, LCZ8A3-7 and LCZ8A3-8, two mice per monoclonal hybridoma cell;

b.7 days later, ascites can be extracted if the abdomen is obviously enlarged. 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 using sulfuric acid according to a precipitation and dialysis method.

Carrying out ELISA titer determination, molecular mass identification and concentration determination on the ascites monoclonal antibody:

1) measurement of ELISA Titers: the ascites monoclonal antibody purified by indirect ELISA was assayed, and 1ug/ml of antigen 100u 1/well was coated on a 96-well plate for ELISA, and the results showed a titer after purification of l: 260000.

2) The molecular mass of the antibody is identified by determining the monoclonal antibody by SDS-PAGE, which shows that the heavy chain of the monoclonal antibody is about 46KD and the light chain is about 25 KD.

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. As a result, the protein content was 18 mg/ml.

Example 3: clinical diagnosis of pathogens

1. Preparation of the kit

The kit adopts a chemiluminescence immunoassay method.

1) Coating a rabbit-derived anti-human prostasomal exocrine protein detection antibody (EliOnco USA) on an activated magnetic bead, taking 100ul of the activated Mag COOH magnetic bead, adding 100ug of the rabbit-derived anti-human prostasomal exocrine protein detection antibody, vertically mixing for 120 minutes at 25 ℃, carrying out magnetic separation and washing for 3 times, removing free antibodies, adding 300ul of 1% BSA (bovine serum albumin) sealing solution, vertically mixing for 60 minutes at 25 ℃, carrying out magnetic separation and washing for 3 times, and resuspending in 1ml of PBST solution;

2) acridinium esters were labeled on the anti-human prostasomal leakage protein monoclonal antibody LCZ8a 3. The labeling buffer 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: 1). 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 acridinium ester labeled anti-human prostasomal exocrine protein monoclonal antibody LCZ8A3, 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: the chemiluminescence substrate solution A and the chemiluminescence substrate solution B are respectively added with 50ul of solution, and the integration time is 3S. And drawing a standard curve according to the chemiluminescence values of the standard substances with different concentrations, and comparing the numerical value of the patient with the numerical value of the positive standard hole, thereby obtaining the actual concentration of the prostasomal exocrine protein 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 prostasomal Expressor protein content (ng/ml) in patients and in the Normal population

Study object grouping	Number of cases	Mean value of	Standard deviation of	Median number
					Patient group	200	4.63	1.251	2.41
Normal control group	200	0.48	0.215	0.38

*Note：Z＝P＜0.05

As can be seen from Table 1, the content of prostasome exoproteins in urine of prostatitis patients is obviously increased compared with that of normal people, and the statistics shows significant difference.

The above 200 samples were subjected to two tests of the clinical gold standard and the kit. And (3) calculating the statistical results of the sensitivity, specificity, total coincidence rate, positive expected value, negative expected value and the like of the clinical detection of the prostatic body exocrine protein detection kit, wherein the results are shown in a table 2.

TABLE 2 summary of clinical gold standards and prostasomal protein assay clinical data of this kit

Sensitivity a/(a + c) × 100% > -187/200 × 100% > -93.5%;

specificity d/(b + d) × 100% > -189/200 × 100% > -94.5%;

the total coincidence rate is (a + d)/(a + b + c + d) — 187+189)/400 × 100% — 94.0%.

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 93%.

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.