CN113087802B

CN113087802B - Kit for detecting cancer by combining nucleic acid detection with antibody

Info

Publication number: CN113087802B
Application number: CN202110373230.2A
Authority: CN
Inventors: 张海涛; 王振
Original assignee: Zhejiang Gress Sonwin Technology Co ltd
Current assignee: Zhejiang Gress Sonwin Technology Co ltd
Priority date: 2021-04-07
Filing date: 2021-04-07
Publication date: 2022-10-11
Anticipated expiration: 2041-04-07
Also published as: CN113087802A

Abstract

The invention relates to a kit for detecting cancer by combining nucleic acid detection and antibody detection. The kit contains the monoclonal antibody for resisting the non-small cell lung cancer, has better affinity property and specificity, and identifies the specific amino acid sequences of the light chain variable region and the heavy chain variable region of the antibody. The designed PCR detection is matched with the PCR detection, so that the accuracy and the precision of sample detection can be effectively ensured, the method can be used for diagnosing the non-small cell lung cancer, and has better clinical application prospect.

Description

Kit for detecting cancer by combining nucleic acid detection with antibody

Technical Field

The application relates to the field of detection, in particular to a kit for detecting cancer by combining nucleic acid detection and antibody.

Disclosure of Invention

Lung cancer is one of the most common malignant tumors worldwide, seriously threatens human health, and the morbidity and mortality of the lung cancer are ranked first. Non-small cell lung cancer (NSCLC) accounts for about 80% of lung cancer patients, the 5-year survival rate of NSCLC is less than 20%, and the 5-year survival rate of timely discovered and treated 1A-stage NSCLC patients can reach about 80%. Therefore, the survival rate of NSCLC patients can be greatly improved by timely discovering and treating early lung cancer.

Currently, the most common diagnostic methods for lung cancer mainly include sputum cast-off cell examination, bronchoscopy, chest X-ray and other imaging examinations. However, the bronchoscope examination method is invasive, highly traumatic to the patient, and cancer cells are not necessarily detected by brushing every time. Sputum cast cells and chest X-ray examination are less sensitive to the auxiliary diagnosis of lung cancer, and are not ideal diagnosis methods. Although the researchers at present suggest that the conventional screening of lung cancer can be performed by using the method of low-dose helical CT, the low-dose helical CT has no standardized scheme for screening lung cancer, and the clinical application value of the low-dose helical CT is still in the discussion stage. The blood tumor marker has important clinical value in tumor screening, and has the advantages of convenient material acquisition, small wound, relatively low price, easy reinspection and the like. However, the sensitivity of the currently clinically used tumor markers (CEA, NSE, cyfra21-1, etc.) is low, especially the sensitivity in detecting early lung cancer (stage I + stage II) is only about 15%, and the tumor markers have great limitation in the auxiliary diagnosis of NSCLC. Therefore, there is a great clinical need for lung cancer tumor markers with good specificity and sensitivity, especially for detecting early stage lung cancer.

The research finds that the expression of PGP in NSCLC is closely related to the neuroendocrine differentiation. The research shows that the expression rate of the transcription factor SOX2 in the lung squamous carcinoma tissues detected by an immunohistochemical method can reach 69%, the expression rate in the adenocarcinoma tissues can reach 4.5%, and the expression rate in the SCLC can reach 70%. GBU4-5 is taken as one of the markers for early screening of the lung cancer, and the combination of the GBU4-5 and CAGE with other different lung cancer standards can obviously improve the diagnosis efficiency of the lung cancer. Tumor-testis antigen (NY-ESO-1), tumor protein P53 (P53), cyclin-dependent protein kinase 2 (CDK 2), annexin A2 (annexin 2), insulin-like growth factor mRNA binding protein 1 (IMPl), ubiquitin (ubiquilin), and phosphoglycerate mutase (PGAMl) a total of 7 autoantibodies were tested in NSCLC with a sensitivity of 68.7% and a specificity of 91.1%.

RT-PCR technology is the most commonly used technology for diagnosing NSCLC micrometastasis in recent years, and the sensitivity of micrometastasis detection is 10-100 times higher than that of the traditional immunocytochemistry method, and can be 10 times or less ⁶ —10 ⁷ 1-10 tumor cells were detected from normal cells. At present, due to the lack of specific tumor markers for detection and the occurrence of false positive in tissue specific markers, the research results are inconsistent. The key to improving the specificity and reliability of the micrometastasis detection is to screen better tumor tissue specific markers.

The monoclonal antibody with specific reactivity to the lung cancer can be used for diagnosing and researching tumors and also becomes the focus of pharmaceutical research in the aspect of tumor biotherapy at present. Although there are many studies on monoclonal antibodies against lung cancer, few studies have been conducted to screen monoclonal antibodies against human non-small cell lung cancer. .

Furthermore, antibody detection and viral nucleic acid detection are the most important diagnostic criteria in terms of etiological diagnosis. At present, for the diagnosis of lung cancer, few inventions are available for combined detection of nucleic acid and antibody, and research is insufficient.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a kit for effectively detecting lung cancer. The kit contains a nucleic acid detection reagent and an antibody detection reagent.

On one hand, the invention provides a specific detection primer pair, and the sequences of the primer pair are CYP2A 6F 1 tcatgaagatcagtgagcgc and CYP2A 6R 1 ggtggcgatggagaagcgcc.

The invention also provides a detection kit, which contains the primer pair and other detection reagents.

The invention provides a monoclonal antibody for non-small cell lung cancer, wherein the heavy chain variable region sequence and the light chain variable region sequence of the monoclonal antibody are respectively shown as SEQ ID NO:1 or 2.

In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells, or by ascites in mice. The hybridoma method comprises collecting supernatant of hybridoma cell culture, extracting IgG by saturated ammonium sulfate precipitation method, and purifying the antibody by affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells were inoculated into the abdominal cavity of the sensitized mice, and significant abdominal distension was observed within 2-4 weeks. Ascites is extracted, and after the crude extraction by saturated ammonium sulfate precipitation, the antibody of the crude extraction is purified by an affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is provided with a detectable label. More preferably, the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.

The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody is fluorescently labeled, and a fluorescently labeled monoclonal antibody is obtained.

The last purpose of the invention is to provide an enzyme linked immunosorbent assay kit for detecting or assisting the non-small cell lung cancer cells in a sample to be detected.

The enzyme linked immunosorbent assay kit for detecting or assisting the non-small cell lung cancer cells in a sample to be detected comprises the monoclonal antibody and an enzyme-labeled secondary antibody which are independently packaged.

In the kit, the enzyme is any enzyme suitable for labeling enzyme-linked immunosorbent assay, such as horseradish peroxidase, alkaline phosphatase or glucose oxidase; the enzyme is specifically horseradish peroxidase.

The kit also comprises a substrate, wherein the substrate is o-phenylenediamine, dianisidine, 5-aminosalicylic acid, o-tolidine or 3,3',5,5' -methylbenzidine; the substrate is specifically 3,3',5,5' -methyl benzidine.

The application of the kit in at least one of the following 1) to 5) also belongs to the protection scope of the invention:

1) Detecting or assisting in detecting the non-small cell lung cancer in a sample to be detected;

2) Screening for early stage lung cancer solid tumors and/or lung cancer;

3) Preparing a product for screening early-stage lung cancer solid tumors and/or lung cancer;

in the above application or the above kit, the sample to be tested is urine and/or serum and/or plasma and/or cancer tissue.

The antibody of the present invention can be used alone or in combination with a PCR detection reagent.

The antibody may contain a label. Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Advantageous effects

The monoclonal antibody for resisting the non-small cell lung cancer has better affinity property and specificity, and specific amino acid sequences of a light chain variable region and a heavy chain variable region of the antibody are identified. The designed PCR detection is matched with the PCR detection, so that the accuracy and the precision of sample detection can be effectively ensured, the method can be used for diagnosing the non-small cell lung cancer, and has better clinical application prospect.

Drawings

FIG. 1 is a graph showing the results of measurement of subtype of monoclonal antibody

FIG. 2 is a graph showing the results of protein recognition by antibody specificity (A is capable of binding specifically, B is not capable of binding specifically)

FIG. 3 is a graph showing the results of the cell separation ratio

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the invention thereto. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

EXAMPLE 1 preparation of nucleic acid detecting reagent

Extracting total RNA of HOP-92 human small cell lung cancer cells according to Trizol Reagent instructions, and storing at-70 ℃ for later use.

RNA-cDNA specific sequence reverse transcription, experimental conditions: 10min at 30 ℃, 35min at 42 ℃, 5min at 99 ℃ and 5min at 4 ℃; centrifuge instantaneously for l0 seconds. Carrying out gradient dilution on the cDNA to test the PCR detection precision;

the specific primer sequence is obtained by the applicant through specific screening and optimization, and the specific sequence is shown in table 1 and synthesized by Shanghai biological engineering Co. PCR conditions were 95 ℃ for 1min; (95 ℃ 1min,56 ℃ 55s,72 ℃ 1 min) for 40 cycles; cooling at 72 deg.C for 10min, and storing at 4 deg.C.

TABLE 1 primer List

Name of primer	Primer sequences
		CYP2A6 F1	tcatgaagatcagtgagcgc
CYP2A6 R1	ggtggcgatggagaagcgcc
		CYP2A 6F 2 control	acaccaagtttcgggatttc
CYP2A 6R 2 control	aacagtaccgctttccgatg
		β-actin F	ctcattgtcttctacgggctgttag
β-actin R	ctttatgccgagagggatggt

Electrophoresis is carried out on 2% agarose gel, electrophoresis is carried out for 30min under the voltage of 100V, ethidium bromide is used for staining for 30min, and photographing and recording are carried out under an ultraviolet analyzer. The results are shown in Table 2.

TABLE 2 result of PCR detection accuracy (+ indicates that there is a band in the PCR result, and-indicates that there is no band)

As can be seen from the results in Table 2, the redesigned CYP2A6 primer has better detection precision, improves the detection advantage on the order of magnitude level compared with the primer pair in the prior art, and has better effect.

The CYP2A 6F 1 and CYP2A 6R 1 primers are prepared into a kit for later use according to a conventional method in the field.

Example 2 screening and preparation of monoclonal antibodies specific for Lung cancer

After lung cancer cell HOP-92 as immunogen is mixed with adjuvant (Freund's complete/incomplete) in equal volume, the mixture is put into a three-way valve glass emulsifier for emulsification until one drop of the lung cancer cell HOP-92 is agglomerated in water without diffusion, thus completing emulsification.

The emulsified immunogen was used to immunize 3 BALB/c mice, which were about 6 weeks old. The first immunization antigen amount is 50ug, and subcutaneous injection is adopted for immunization. After 14d, carrying out secondary immunization, wherein the antigen amount is the same as that of the first immunization, but the adjuvant is Freund incomplete adjuvant; the third immunization was performed at 28d, and the immunization method and the amount of antigen were the same as the second immunization. Tail blood is collected about seven days after the three-immunization, the serum antibody titer is detected by using an indirect ELISA method, and BALB/c mice with high titer are selected for boosting. The amount of antigen for boosting immunity is 100ug, and no adjuvant is added for immunization.

Taking immune No. 2 mouse with high antibody titer, taking eyeball blood, removing neck, killing, soaking in 75% alcohol for 10min, and sterilizing. Mice were fixed on a dissecting plate and the skin and peritoneum were cut open with sterile scissors, leaving the spleen exposed. The tissue around the spleen was detached, the removed spleen was washed with serum-free 1640, and then placed in a 200 mesh sterilized copper mesh placed in a dish, the spleen was washed with serum-free 1640 while being crushed, and the collected spleen cell suspension was added to a centrifuge tube and centrifuged at 1000rpm for 10min. Resuspend pellet with serum-free 1640 and count cells.

The spleen cells and SP2/0 cells 10 were placed in a 50mL centrifuge tube and centrifuged at 1000rpm for 10min. Discarding the supernatant, gently bouncing the cell sediment at the bottom of the centrifuge tube, placing the centrifuge tube in a 37 ℃ water bath, preheating the cell fusion agent, sucking 1mL of the cell fusion agent, slowly adding the cell fusion agent at the speed of 1mL/min, gently shaking the centrifuge tube while adding the cell fusion agent so as to fully contact and uniformly mix the cells and the fusion agent, and placing the centrifuge tube in the 37 ℃ water bath for standing for 90s. 1mL of serum-free 1640 was added and the tube was gently shaken while dropping at a rate of 1mL/min and the process was repeated once more. Then, 1mL of serum-free 1640 was added and added dropwise at a rate of 1mL/30s, and finally 7mL of serum-free 1640 was added within 2min, and the tube was gently shaken while adding dropwise. Centrifuge at 1000rpm for 10min and discard the supernatant. Add preheated HAT medium for cell resuspension, add 100 μ L/well to 96-well cell plates plated with feeder cells, and culture in 5% co2 cell incubator at 37 ℃. On day 7 post-fusion, half-changes were made with pre-warmed HT medium. Observing the cell state every day, detecting the cell supernatant by using an indirect-ELISA method when the hybridoma cells grow to 1/2 of the bottom of a 96-well cell plate, setting a negative control hole and a positive control hole, carrying out three times of detection before subcloning, and carrying out subcloning on the hybridoma cell holes of which all the three times of detection are positive. Four subclones were made to achieve 100% positivity. Two hybridoma cell strains 4F5 and 5D2 with the strongest ELISA positive reaction are selected for preparing ascites.

The abdominal cavity of 8-week-old BALB/c mice was injected with 0.5 mL/mouse with sterilized liquid paraffin. After 10 days, the hybridoma cells were resuspended in serum-free 1640 and counted at 1X10 ⁶ Individual hybridoma cells/mL, 0.5 mL/mouse were injected intraperitoneally. After inoculation, the state of the mice is observed, the abdomen of the mice begins to bulge after about one week, and ascites begins to be collected when the abdominal circumference of the mice swells and the movement of the mice is slow. The liquid in the abdominal cavity of the mouse is extracted 2-3 times by a syringe. And (3) centrifuging the ascites at 2000rpm for 10min, sucking the supernatant, removing grease and cells, purifying by using a purification column to obtain 4F5 and 5D2 monoclonal antibodies respectively, and adjusting the concentration to be 1mg/mL for later use.

EXAMPLE 3 4F5 monoclonal antibody titre assay

The antibody titer of the 4F5 ascites is detected by an indirect ELISA method, negative and positive controls are set, the ascites is subjected to multiple dilution detection, and the mouse ascites titer of the 4F5 monoclonal antibody is identified to be 1.

Example 4 subtype determination of the F5 monoclonal antibody, dissociation constant identification and variable region sequence identification

And (3) identifying by using a monoclonal Ig class/subclass identification kit, and carrying out specific experimental operation according to the instruction. The results are shown in FIG. 1. The antibodies of the invention are of the IgG1 subtype.

Sequencing of the antibody was performed by Nanjing Kingsrei Biotech, inc., which includes the steps of: culturing hybridoma cells with RPMI1640 and 20% serum for 24 hr, centrifuging, discarding supernatant, resuspending cells with TriZol, and culturing with 10% serum ⁶ Individual cells/mL. After DNA extraction, the heavy chain and light chain genes are amplified by RT-PCR, cloned to a T vector and then subjected to DNA sequencing. Obtaining the light chain and the heavy chain of the antibodyThe variable regions are shown below.

SEQ ID NO:1: heavy chain variable region sequences

EVKLVESGGGLVKPGGSLKLYCAASGFTFSGYSMSWVRQIPEKRLEWVAEISPFQSLYFSSRVTTRFTISRDNARNICYLQMNSLRSDATAMYYCARAVQVDSQYASRWGQGTTLTVSS

The amino acid sequence of SEQ ID NO:2: light chain variable region sequence

DIVLTQSPASLAVQLGQRATISCAAQQSVENDGTSLDRWYQQKPGQPPKLLIYARSNVCRGVPARFSGSGSGTDFSRNIHPVEEDDIAMYFCSKSRKGPKLFGAGTKLELK

Indicating that the plasma resonance analysis is carried out by using Biacore 8K, and the specific steps are as follows: a proteinA chip was selected, and the aforementioned antibody was immobilized on the chip by affinity between proteinA and antibody Fc, and the immunogen cells were diluted by a 10mM HEPES solution at a pH of 150mM NaCl 7.4 in a double ratio and loaded one by one from low to high concentrations. The corresponding binding utilization constants were calculated using the corresponding software, and the results are shown in table 3.

TABLE 3 kinetic constants of the antibodies

Name of antibody	KD(M)
		4F5	4.52E-10

Example 5 antibody detection and nucleic acid detection experiments in hepatoma cells

Take 1x10 ⁶ 0.5ml of a cell lysate containing 1% of Triton X-100,1mM EDTA,50mM NaCl,0.1 (m/v) SDS,1% sodium deoxyholate, 1mM PMSF, 2. Mu.g/ml Aprotinin, 1. Mu.g/ml Leutepptin and 1. Mu.g/ml pepstatin was added to each of HOP-92 human small cell lung cancer cells and normal human lung cells, and the cells were lysed on ice for 30minThen, the mixture was centrifuged at 10000rpm at 4 ℃ for 15min, and the supernatant was collected to obtain the target protein extract.

2. Mu.g of monoclonal antibody 4F5,4 ℃ was added to the cell lysis supernatant and incubated for two hours, and 15. Mu.l of protein A/G beads were added thereto and incubated overnight at 4 ℃. On the next day, after washing the beads twice with the cell lysate containing no protease inhibitor, 1xSDS loading buffer was added for immunoblotting detection, and the membrane was transferred for 35min with NC membrane at 100V. After blocking with 5% skim milk for 2h, antibody diluted with 5% skim milk was added at a concentration of 2 μ g/ml, incubated overnight at 4 ℃, washed 4 times with 0.1% tween in TBS, anti-Mouse IgG (Fc specific) -peroxidase antibody (1.

As can be seen from FIG. 2, the 4F5 antibody specifically recognized lung cancer cell surface protein (A), but did not bind lung cell protein (B).

Example 6 tumor cell detection assay with fluorescently labeled antibody in a simulated blood environment

The control antibody was a non-small cell lung carcinoma monoclonal antibody, clone No. TFS-4, MAB4337, eimeria Abnova.

Labeling of fluorescent antibody: labeling with FITC antibody labeling kit, which comprises adding 20 μ l monoclonal antibody (5 μ g/μ l) into reaction column, centrifuging at 14000rpm/min and 4 deg.C for 3min, and discarding the separated liquid; mixing 90. Mu.l antibody and 10. Mu.l 10 × reaction solution; transferring all the liquid into a fluorescent dye tube, uniformly mixing, and reacting for 30min at room temperature in a dark place; transferring all the reacted liquid to a storage liquid pipe, mixing uniformly, subpackaging and storing at-20 ℃.

Respectively taking HOP-92 human small cell lung cancer cell, pancreatic cancer cell PANC-1, BEAS-2B human normal lung epithelial cell (1 × 10) ⁴ Ml), and the blood volume of healthy human peripheral blood (WBC: 6X 10 ⁹ /mL) are mixed according to the proportion of 1; adding 2ml PBS,1500rpm/min, centrifuging for 5min, and repeating for 2 times; adding 1ml PBS resuspended mixed peripheral blood cells, washing by hemolysis, and sorting with flow cytometerThe results are shown in FIG. 3 by taking the ratio of the number of cells in the mixture as the identification.

As can be seen from FIG. 3, the antibody of the present invention was able to isolate non-small cell lung cancer cell HOP-92 with a separation efficiency of (98.45. + -. 1.1)% which is higher than that of the control antibody (89.66. + -. 2.0)%. And neither antibody can be effectively used for separating PANC-1 cells and BEAS-2B cells, and has better specificity.

In addition, the results of PCR amplification using the PCR method of example 1 for each of the three cells are shown in Table 4.

TABLE 4 expression of CYP2A6mRNA in non-small cell lung carcinoma and other cells

Group of	CYP2A6 mRNA(△△CT)
		HOP-92 human small cell lung carcinoma cell	18.46±0.64
Pancreatic cancer cell PANC-1	16.16±0.47
		BEAS-2B cells	15.86±0.52

The results in table 4 show that the expression level for non-small cell lung cancer is significantly higher than that of non-small cell lung cancer cells, and has very significant difference, and can be used for effectively and synergistically distinguishing non-small cell lung cancer, and in the detection, the delta CT is more than 17, and can be effectively used for distinguishing human small cell lung cancer from other cells.

While the invention has been described and illustrated in detail as being sufficient to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications thereof and other uses will occur to those skilled in the art. Such modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

Sequence listing

<110> Beijing Song Biotechnology Ltd

<120> nucleic acid detection and antibody combined cancer detection kit

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ggtggcgatg gagaagcgcc 20

Claims

1. A 4F5 monoclonal antibody characterized by: the heavy chain variable region sequence is shown as SEQ ID NO:1, the light chain variable region sequence is shown as SEQ ID NO:2, respectively.

2. Use of the antibody of claim 1 for the preparation of a kit for detecting non-small cell lung cancer cells.

3. The use according to claim 2, wherein said non-small cell lung cancer cell is a HOP-92 cell.

4. Use of the antibody of claim 1 in the preparation of a kit for detecting non-small cell lung cancer cells together with a PCR detection reagent, wherein the PCR detection reagent comprises SEQ ID NO:3 and SEQ ID NO: 4.

5. Use according to claim 2 or 4, characterized in that the antibody contains a detectable label.

6. Use according to claim 5, characterized in that the detectable label is a luminescent label, a radioactive label, an MRI or CT contrast agent.

7. Use according to claim 5, characterized in that the detectable label is an enzyme which is a detectable product.

8. Use according to claim 4, characterized in that it contains the corresponding PCR reagents for the PCR reaction.