CN112430583B - Monoclonal antibody against HPV E7, cell strain and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody of anti-HPV E7, a cell strain and application thereof, relating to the technical field of biological detection. The monoclonal antibody resisting HPV E7 is secreted by a hybridoma cell strain with the preservation number of CCTCC NO: C2020152 or CCTCC NO: C2020154, and the invention also provides application of the monoclonal antibody in preparation of an HPV E7 protein detection kit. The monoclonal antibody provided by the invention can be combined with E7 proteins of various HPV types, has strong specificity, can be used for detecting human HPV E7 protein, improves the specificity of cervical cancer screening, reduces missed diagnosis and avoids over diagnosis and treatment.

Description

Monoclonal antibody against HPV E7, cell strain and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a monoclonal antibody for resisting HPV E7, a cell strain and application thereof.

Background

Cervical cancer is the only cancer of definite etiology among all cancers at present, and its main cause is persistent infection with high-risk HPV. It is reported that the female suffers from cervical cancer, which has become an important disease threatening the health of the female, and the reduction of quality of life and death caused thereby have raised concerns of the world health organization. Therefore, early diagnosis and prevention of cervical cancer are becoming increasingly important. Early screening for cervical cancer has become widespread in some developed countries. The reason is attributed to the wide application of the pap smear, so that the development of cervical cancer screening projects is greatly promoted, however, the technology has limitations, and clinical diagnosis effects are influenced, such as unstable cell quantity obtained, unrepeatable diagnosis results due to the influence of material obtaining, and the like. The problem of insufficient cell amount is improved along with the clinical application of liquid-based cytology in recent years, however, the method depends on the subjective judgment of cytopathologists like pap smears, and the diagnosis level of the cytopathologists is inconsistent, so that the sensitivity of cytological diagnosis is reduced. In addition, the abnormal change of the cytological morphology is late, so that the pathological change condition cannot be predicted early, and the early diagnosis significance is limited. Based on the above factors, HPV detection becomes the entry point for early cervical cancer screening, which is also recognized by domestic and foreign peers.

With the popularization of the HPV HC2 method, the high detection sensitivity and the detection stability of the HPV HC2 method make the HPV HC2 the gold standard of the American FDA-approved detection, which plays an irreplaceable role in the cervical cancer screening. However, the prevalence of HPV and the lifetime cumulative risk of infection have reached 80%, making this method less specific for the diagnosis of cervical lesions. Many reports show that the detection specificity is low, false positives are easily caused, the result is often confused by further treatment of clinicians, and a considerable part of the clinicians can adopt excessive treatment modes, so that a lot of harm is brought to the body and the mind of patients. The root cause of low specificity is the infection universality and the characteristic of one-time elimination after infection, and the detection positive result can only prove the infection once, whether the virus is eliminated in the body at present and whether the infection is continued or not, which cannot be verified. Even if the virus is still present in the body, whether it is integrated into the host gene, which leads to intraepithelial lesions, and whether active replication and transcription are possible, cannot be determined from the experimental results, and thus the method has limitations. Under the premise, whether the detection can be a new biomarker or not is detected, whether the detection can reflect the activity condition of the virus in vivo or not is detected, and therefore the clinical significance is important to be discussed.

The circular HPV genome comprises seven early regions and two late regions, wherein oncogenic proteins encoded by E6 and E7 genes are important factors for cervical epithelial canceration. In the initial stage of HPV infection, HPV virus is eliminated with cell apoptosis, but if the HPV virus is infected continuously, the virus may be caused to integrate E6/E7 gene into host cells randomly, and thus the E6/E7 oncoprotein is over-expressed. The E7 oncoprotein can be combined with cancer suppressor Rb to replace the action mechanism of cyclin kinase CDK4/6, so that the DNA synthesis phase of cells is out of control, and the cells are cancerated. Therefore, the E7 oncoprotein is the most important biomarker of cervical epithelial canceration, wherein the E7 protein is closely related to early tumors.

The CFDA approved aptma HPV E7 mRNA detection in 2015 is an important milestone for the application of E7 as a specific biomarker for cervical cancer screening. However, mRNA is susceptible to degradation in vivo, and its detection requires expensive instruments, a rigorous laboratory environment, and cumbersome procedures, so tests based on the detection of E7 mRNA may have limited clinical application in routine gynecological practice. The reagent based on the detection of the E7 protein has advantages over the detection of HPV DNA or HPVE7 mRNA, but only E7 protein detection reagents aiming at HPV types such as HPV16 and 18 are available in the market at present, and the requirement of cervical cancer screening cannot be met.

Therefore, a new HPV E7 protein detection kit needs to be researched, so that the specificity of cervical cancer screening is improved, missed diagnosis is reduced, and excessive diagnosis and treatment are avoided while multiple HPV types are simultaneously detected.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the HPV E7 protein detection kit, which can be used for simultaneously detecting E7 proteins of various HPV types, improves the specificity of cervical cancer screening, reduces missed diagnosis and avoids over diagnosis and treatment.

In order to realize the aim, the invention provides a hybridoma cell strain capable of secreting the anti-HPV E7 monoclonal antibody, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2020152 or CCTCC NO: C2020154.

The invention also aims to provide the HPV E7 monoclonal antibody produced by the hybridoma cell strain capable of secreting the anti-HPV E7 monoclonal antibody.

The invention also aims to provide application of the HPV E7 monoclonal antibody in preparation of an HPV E7 protein detection kit.

The invention also aims to provide an HPV E7 protein detection kit, which comprises an HPV E7 monoclonal antibody, a quality control sheet, a sample diluent and a secondary antibody detection system; the monoclonal antibody is generated by hybridoma cell strains with the preservation number of CCTCC NO: C2020152 or the preservation number of CCTCC NO: C2020154.

According to the HPV E7 protein detection kit provided by the invention, the HPV E7 monoclonal antibody is generated by a hybridoma cell strain with the preservation number of CCTCC NO: C2020152.

According to the HPV E7 protein detection kit provided by the invention, the sample diluent comprises Tris (2-carboxyethyl) phosphine hydrochloride and Tris buffer.

According to the HPV E7 protein detection kit provided by the invention, the secondary antibody detection system is an SP method detection system, and biotin is adopted to mark goat anti-mouse IgG or rabbit anti-mouse IgG; preferably, the secondary antibody detection system employs a substance labeled goat anti-mouse IgG.

According to the HPV E7 protein detection kit provided by the invention, the quality control sheet comprises a positive quality control sheet and a negative quality control sheet; the positive control plate uses one of CaSki, SiHa, HeLa or MS751 as a cell, and the negative control plate is an HPV negative cervical cancer cell line C-33A; preferably, the cells used in the positive quality control wafer are CaSki.

The invention also provides application of the HPV E7 protein detection kit in detection of HPV type E7 protein.

Wherein the HPV types comprise high-risk types and low-risk types; the high-risk types comprise HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59 and HPV-68; the low-risk types include HPV-6, HPV-11, HPV-42, HPV-44, HPV-53, HPV-54, HPV-55 and HPV-56.

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

1. the kit provided by the invention has high sensitivity, strong specificity and good stability for detecting cervical intraepithelial neoplasia (CIN2 and above), has great advantages compared with the HPV E7 protein detection reagent adopting a single subtype in the existing market, and can greatly improve the sensitivity of the kit;

2. due to the particularity of the cervical exfoliated cell sample, most of the sample contains interfering substances such as tissue mucus and the like, and the kit is added with the sample treatment solution for the first time, so that the sample can be dispersed before detection, nonspecific staining is reduced, and subsequent sample result judgment is facilitated;

3. the substance-labeled goat anti-mouse IgG adopted by the kit secondary antibody system only identifies the Fc segment of mouse IgG, and has no cross reaction with human IgG, bovine IgG and equine IgG, thereby being more beneficial to reducing the nonspecific staining of samples;

4. the cytoplasm control tablet is added for the first time, so that the detection of each batch is controllable.

Drawings

FIG. 1 is a schematic view of the color development of the quality control chip detection kit provided by the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The detection principle of the invention is as follows: the specific binding principle of immunological antigen-antibody molecules due to the complementary structure and mutual affinity is utilized, and the enzyme color developing agent for marking the antibody is developed through oxidation-reduction chemical reaction to determine the tissue intracellular antigen, and the tissue intracellular antigen is positioned and characterized. Firstly, a mouse anti-human HPV E7 monoclonal antibody is connected with an HPV E7 antigen on a cell; second, the biotin-labeled goat anti-mouse IgG recognizes the HPV E7 antibody already attached; thirdly, adding peroxidase-labeled streptavidin to identify biotin; fourthly, adding a substrate, and catalyzing H in the DAB color developing solution by the horseradish peroxidase part on the polymer₂O₂Decomposing to oxidize benzidine into benzimide and enable the antigen sites in the cell sedimentation sheets to be yellow or brownish yellow colored; and finally, counterstaining and mounting the sample. And (4) observing the chromogenic condition through a microscope, and deducing the existence site and condition of the HPV E7 protein on the cell sediment sheet.

Based on the principle, a specific anti-human HPV E7 monoclonal antibody is firstly screened out, then a cervical exfoliated cell sample is detected by using an immunocytochemistry method, and the HPV infected cervical exfoliated cell sample related by the invention is a volunteer case.

The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof. The experimental procedures under specific conditions not specifically described in the following examples were carried out by a conventional experimental procedure.

EXAMPLE 1 screening of monoclonal antibodies against human HPV E7

After a BALB/C mouse is immunized by using HPV 16E 7 or HPV 18E 7 according to a normal immunization program (patent US8865162 of the company), the serum titer of the mouse is detected, when the serum titer reaches the optimal state, monoclonal hybridoma cells are produced and screened according to a standard hybridoma cell fusion program, cell supernatants are screened by using HPV 16E 7 and HPV 18E 7 of the screened monoclonal cell strains, double positive monoclonal cell strains are selected for expansion, 4 monoclonal cell strains with high antibody titer and good cell states (respectively named as Mab3, Mab4 and Mab8 according to the screening sequence) are selected to produce ascites according to the standard monoclonal cell strains, and the collected ascites is purified by using ProteinG according to a standard antibody purification program.

The three antibodies were diluted to 4. mu.g/ml using an antibody diluent (10mM PBS, 4% BSA, 0.05% Tween-20) to detect Caski, C-33A, and 10 samples, respectively. The results are shown in Table 1.

Table 1:

	mab3 (ratio%, color rendering)	Mab4 (ratio%, color rendering)	Mab8 (ratio%, color rendering)
				Caski	90％+++	40％+++	90％++
C-33A	N	N	N
				Sample 1	20％+	20% + (white blood cells 100% +)	20％+
Sample 2	N	N	N
				Sample 3	30％++	20％++	30％+
Sample 4	10％+	40％+	10％+
				Sample 5	50％+	50％+	50％+
Sample 6	N	100％+	N
				Sample 7	100+	100+	100+
Sample 8	N	N	N
				Sample 9	5％++	50% + (white blood cells 100% +)	5％+
Sample 10	N	N (white blood cell 100% +)	N

Based on the above results, the Mab4 antibody detected cells, which caused non-specific staining of leukocytes, as well as non-specific staining of some samples. Mab3 and Mab8 are basically consistent in color development, the requirements of the kit are met, and Mab8 is weaker in color development than Mab3, so that the Mab3 antibody is preferably used as a primary anti-reagent of the kit, and the Mab3 antibody is preferably used. China Wuhan university China type culture Collection (CCTCC) is delivered 24 days 08 and 24 days 2020, and Mab3 submits preservation information as follows, culture name (classification name): hybridoma cell line P1F5-3-B10, accession number: CCTCC NO: C2020152. Mab8 submitted the deposit information as, culture name (classification name): hybridoma cell strain P3C1-H5-1-C11, accession number: CCTCC NO: C2020154.

EXAMPLE 2 Positive control plate and negative control plate production

1) Cell recovery and passage: recovering CaSki, HeLa (SiHa and MS751 can also be selected to make positive quality control tablet) and C-33A, then passaging, after the cells reach the required cell amount, digesting with trypsin, and centrifuging;

2) cell fixation: adding the cell sediment into cell preservation solution (HEER, type I) and uniformly mixing, and fixing for more than 2 hours at the temperature of 2-8 ℃;

3) and (3) settling: centrifuging the fixed cells, adding a buffer solution (Tris, pH8.0) into the cells, mixing the mixture uniformly, and settling the mixture in a 10mm sleeve for 15 minutes;

4) cell fixation protection: fixing the settled slide in a cell protection solution (1% PEG, 50% ethanol) for 30 minutes;

5) and (3) drying: and (3) putting the fixed slide into a drying box for drying overnight, and then putting the slide into a slide box for vacuum packaging.

The quality control chip prepared according to the method is consistent with fresh cell color development according to the detection of a kit detection process, as shown in figure 1 (figure 1A is C-33A, HPV, 10 x; figure 1B is CaSki, HPV16 +, 500-600copies, 10 x; figure 1C is Hela, HPV18 +, 10-50copies, 10 x), the quality control chip can be stored for 6 months at 2-8 ℃, and the clinical quality control requirements can be completely met.

Example 3 preparation of Mab3 test kit

A kit for detecting HPV E7 protein, comprising an anti-HPV E7 monoclonal antibody as described in example 1 above, the positive and negative control discs of example 2, and a sample diluent and a secondary antibody detection system.

The sample diluent is 20mM Tris buffer solution and 3% TCEP.4HCl; the primary anti-reagent is Mab3 antibody diluted at a ratio of 1:200, the diluent is 4% bovine serum albumin, 10mM PBS buffer; the secondary antibody detection system comprises a confining liquid (1% goat serum, 5% bovine serum albumin, 10mM PBS), a biotin-labeled goat anti-mouse IgG reagent (1:1000 dilution, 1% goat serum, 5% bovine serum albumin, 10mM PBS), a peroxidase-labeled streptavidin reagent (1:500 dilution, the same diluting liquid as the confining liquid), a DAB color reagent A liquid (0.1% Tween 20, 0.05% carbamide peroxide, 20mM imidazole), and a DAB color reagent B liquid (1% DAB, 10mM PBS).

Example 4 preparation of Mab8 test kit

The difference from example 3 is that the primary anti-agent was Mab8 antibody.

Experimental example 1 detection of cervical exfoliated cells Using the kit of the present invention

The kit prepared in examples 3 and 4 was used for detection, specifically as follows:

1. detecting required instruments and equipment:

the device comprises a horizontal dye vat, a pipettor, an immunohistochemical oil pen, a timer, an incubation box, a staining rack, a cover glass, an optical microscope (10X-40X), a slide making plate, a wash bottle, a glass slide, a 10mm sleeve, a centrifugal machine, a constant temperature water bath incubator and a refrigerator.

2. Solution preparation:

see the specification of each product for PBS solution and PBST solution; preparing a DAB color developing solution: the DAB developing solution needs to be prepared within 30 minutes before use. The preparation method comprises the following steps: sequentially adding 1mL of DAB color developing agent A solution and 40 mu L of DAB color developing agent B solution into the prepared small test tube; the prepared DAB color developing solution is uniformly mixed and then stored in a dark place, and is effective within 30 minutes.

3. Laboratory temperature conditions: 22-28 ℃.

4. The test steps are as follows:

1) an appropriate amount of cells was centrifuged to remove the supernatant, and 500. mu.L of the supernatant was added to resuspend the mucus specimen (after the mucus specimen was treated with type III sample diluent, the mucus specimen was sedimented).

2) Settle for 15 minutes in a 10mm thimble.

3) After the sedimentation is finished, the mixture is washed twice by 50 percent ethanol, and the sleeve is taken down. (the experiment steps avoid drying the cells of the slide)

4) The settled cell slide was placed in a horizontal staining jar and washed 5 minutes × 2 times with a PBS solution.

5) The PBS solution was discarded and fixed in 95% ethanol for 10 minutes.

6) Blocking endogenous peroxidase

The PBS solution was discarded, and endogenous peroxidase blocking agent was added and blocked for 10 minutes at room temperature. The PBS solution was washed 5 min X3 times.

7) Adding a sealing liquid: the PBS solution was discarded, and 150. mu.l of blocking solution was added to the test cell area delineated by the oil pen, followed by incubation in an incubator at 37 ℃ for 1 hour.

8) Adding a primary anti-reagent: the blocking solution was discarded, 150. mu.L of primary antibody reagent was added thereto, and the mixture was left overnight (12 to 16 hours) in a refrigerator at 4 ℃.

9) Antibody liquid was discarded and PBST solution washed 5 min x 3 times.

10) Adding a biotin marker: the wash solution was discarded, 150. mu.L of biotin-labeled goat anti-mouse IgG was added, incubated at room temperature or 25 ℃ for 15 minutes in an incubator, and washed 5 minutes X3 times with PBST solution.

11) Adding enzyme-labeled streptavidin: the wash solution was discarded, 150. mu. LHRP enzyme-labeled streptavidin was added, incubated at room temperature or 25 ℃ for 15 minutes in an incubator, and washed 5 minutes × 3 times with PBST solution.

12) Color development: removing the cleaning solution, adding 150 mu L of freshly prepared DAB developing solution, incubating for 5-10 minutes, and observing the dyeing result by a light microscope for generally not more than 10 minutes. (Low temperature effects color development, Can be incubated wet box preheating in advance)

13) Counterdyeing: and (3) putting the cell sheet into a staining rack, washing the cell sheet for 4 minutes by using tap water, then immersing the cell sheet into hematoxylin staining solution for 3 minutes, washing the cell sheet for 3 minutes by using the tap water, then immersing the cell sheet into hydrochloric acid alcohol solution for 5-15 seconds, then taking the cell sheet out, immersing the cell sheet in secondary buffer solution for 1 minute, and washing the cell sheet for 3-4 minutes by using the tap water.

14) Dehydrating, transparent and sealing.

5. And (5) judging a result:

the staining solution precipitates in the region where the corresponding antigen is present, and a brownish yellow substance appears. The staining result is observed and judged by qualified pathologist under biological microscope.

Experimental example 2 determination of sensitivity and specificity of kit

Using the kits of examples 3 and 4, 300 samples of exfoliated cervical cells were tested according to the test method of Experimental example 1, of which 109 samples followed the biopsy result, and these samples were tested for HPV DNA using commercially available HPV DNA test reagents simultaneously, using the biopsy result as a gold standard, and the sensitivity and specificity of the kit of the present invention were calculated.

The results of the measurements are shown in tables 2 to 4 below.

Table 2: sensitivity and specificity of Mab3 kit

Table 3: sensitivity and specificity of Mab8 kit

Table 4: sensitivity and specificity of commercially available HPV DNA kit

From the data, the sensitivity and specificity of the detection kit in the embodiment 3 are 97% and 62% respectively (table 2), and compared with the existing cervical cancer screening reagent HPV DNA detection, the sensitivity is consistent, and the specificity is improved by more than one time. The sensitivity and specificity of the detection kit in the embodiment 4 of the invention are 91% and 68% respectively (Table 3), and compared with the HPV DNA detection (Table 4) of the existing cervical cancer screening reagent, the sensitivity is slightly lower, but the specificity is improved by more than one time. Therefore, the antibody and the kit provided by the invention can greatly reduce panic and over diagnosis of patients in clinical application.

The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.

Claims (10)

1. A hybridoma cell strain capable of secreting monoclonal antibody against HPV E7 is deposited in China center for type culture Collection with a collection number of CCTCC NO: C2020152 or CCTCC NO: C2020154.

2. An anti-HPV E7 monoclonal antibody produced by the hybridoma cell line of claim 1.

3. The use of the monoclonal antibody of claim 2 in the preparation of an HPV E7 protein detection kit.

4. An HPV E7 protein detection kit is characterized by comprising an anti-HPV E7 monoclonal antibody, a quality control sheet, a sample diluent and a secondary antibody detection system; the monoclonal antibody is generated by hybridoma cell strains with the preservation number of CCTCC NO: C2020152 or the preservation number of CCTCC NO: C2020154.

5. The HPV E7 protein detection kit according to claim 4, wherein the anti-HPV E7 monoclonal antibody is produced by a hybridoma cell line with a preservation number of CCTCC NO: C2020152.

6. The HPV E7 protein detection kit of claim 4, wherein the sample diluent comprises Tris (2-carboxyethyl) phosphine hydrochloride and Tris buffer.

7. The HPV E7 protein detection kit of claim 4, wherein the secondary antibody detection system is an SP method detection system and adopts biotin labeled goat anti-mouse IgG or rabbit anti-mouse IgG.

8. The HPV E7 protein detection kit according to claim 7, wherein the secondary antibody detection system employs biotin labeled goat anti-mouse IgG.

9. The HPV E7 protein detection kit of claim 4, wherein the quality control chip comprises a positive quality control chip and a negative quality control chip; the positive quality control chip uses one of CaSki, SiHa, HeLa or MS751 as a cell, and the negative quality control chip uses a HPV negative cervical cancer cell line C-33A as a cell.

10. The HPV E7 protein detection kit according to claim 9, wherein the cells used for the positive control panel are CaSki.

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