Preparation method and application of anticancer embryonic antigen monoclonal antibody

Abstract

The invention relates to the technical field of biology, in particular to a preparation method and application of an anticancer embryonic antigen monoclonal antibody. The invention discloses a monoclonal antibody 12 #, which has a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 2. The invention also discloses a preparation method and application of the antibody. CEA extracted from liver cancer tissue after intestinal cancer liver metastasis is used as an immunogen to immunize Balb / c mice, 12 anti-CEA monoclonal antibodies are obtained through screening and purification by using a hybridoma technology, horse radish peroxidase is labeled with the obtained antibodies, and an antibody for labeling is selected through cross pairing and is applied to detection of CEA in human serum. A double-antibody sandwich detection method established by using the labeled antibody has the advantages of high sensitivity, simple operation and short detection time, and can provide a key material for subsequent development of commercial kits applied to CEA detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a carcinoembryonic antigen antibody and a preparation method thereof, as well as application of the antibody in a carcinoembryonic antigen detection kit. Background Art

[0002] Carcinoembryonic antigen (CEA) is an acidic glycoprotein with human embryonic antigen characteristics. It exists on the surface of cancer cells differentiated from endoderm cells and is a structural protein of the cell membrane. Its encoding gene is located on chromosome 19. It is a polysaccharide-protein complex. The CEA molecule is composed of 641 amino acids with a molecular weight of 180~200kDa, of which 60% are sugar chains. Due to the different sugar chains, CEA has heterogeneity.

[0003] CEA is mainly found in rectal and colon cancer tissues and fetal intestinal mucosa. Tumors that secrete CEA are mostly located in hollow organs developed from the endoderm, such as the digestive tract and respiratory tract, and are non-organ-specific tumor-associated antigens. Under normal circumstances, CEA is metabolized by the gastrointestinal tract and does not enter the circulatory system, while CEA secreted by tumor cells can enter local body fluids, blood or lymphatic circulation. Therefore, abnormal increases in CEA may occur in the serum and body fluids of patients with the above-mentioned cancers. Studies have shown that serum CEA is often elevated in patients with certain benign diseases, especially those with liver disease, but is generally less than 20μg / L.

[0004] CEA is a broad-spectrum tumor marker and is often detected in combination with other tumor markers. Clinically, when CEA is greater than 60 μg / L, colon cancer, rectal cancer, gastric cancer, and lung cancer are considered. In the early stages of primary colorectal cancer without metastasis, the CEA positivity rate is about 50% to 75%. The serum CEA positivity rate and concentration of patients with metastasis are significantly increased. The concentration of CEA is usually related to the course of the disease. When the concentration increases, it indicates that the degree of tumor infiltration increases, of which about 70% are metastatic cancers; when the concentration decreases, it indicates that the growth of infiltrated cancer tissue is inhibited. Therefore, the determination of CEA can be used as one of the bases for observing the efficacy of the treatment.

[0005] The serum CEA level of patients with primary liver cancer is often elevated. When various primary cancers metastasize to the liver, the CEA level is higher than that of patients without liver metastasis. Studies have found that liver metastatic cancer tissues from various sources contain a large amount of CEA. Therefore, CEA can also be used as an indicator for the diagnosis of liver cancer.

[0006] The CEA level increases in the first 6 months of pregnancy, and then gradually decreases. After the fetus is born, its serum CEA level is already very low. Studies have found that CEA levels in smokers are partially elevated, with more than 96% of healthy adults with no smoking history having serum CEA levels less than 2.5 μg / L, and 20% to 40% of heavy smokers having serum CEA levels greater than 2.5 μg / L, and a few even reaching 5 μg / L.

[0007] With the development of detection technology, the detection method of CEA has been developed from enzyme-linked immunosorbent assay technology, radioimmunoassay technology to chemiluminescent immunoassay technology. The detection sensitivity, specificity and environmental friendliness have been greatly improved. However, it is limited by the antibodies used in the detection, and the correlation of the low value range of the detection still has room for improvement. Summary of the invention

[0008] In view of this, the present invention provides a method for preparing an antibody against carcinoembryonic antigen and its application to solve the problems existing in the above-mentioned prior art. The antibody prepared by this method can be used to detect carcinoembryonic antigen in human serum, providing key materials for the subsequent development of a commercial kit for carcinoembryonic antigen detection.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides an antibody, the heavy chain variable region of which has:

[0011] (1) the amino acid sequence shown in SEQ ID NO: 1; or

[0012] (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0013] (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2);

[0014] Its light chain variable region has:

[0015] (4) the amino acid sequence shown in SEQ ID NO: 2; or

[0016] (5) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or

[0017] (6) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (4) or (5);

[0018] The plurality is 2 to 5.

[0019] The present invention also provides a nucleic acid molecule encoding the above antibody.

[0020] The present invention also provides an expression vector, comprising the above nucleic acid molecule.

[0021] The present invention also provides an enzyme-labeled antibody, which is the above-mentioned antibody.

[0022] In some specific embodiments of the present invention, the enzyme of the enzyme-labeled antibody is horseradish peroxidase.

[0023] The present invention also provides an antibody combination, comprising an anti-carcinoembryonic antigen antibody, and:

[0024] (i) the above antibodies; or

[0025] (ii) the above enzyme-labeled antibody.

[0026] The present invention also provides the use of any of the following in the preparation of a reagent, a kit or a device for detecting carcinoembryonic antigen:

[0027] (i) the above-mentioned antibodies;

[0028] (ii) the above enzyme-labeled antibody;

[0029] (iii) The above antibody combination.

[0030] The present invention also provides a reagent, comprising:

[0031] (i) the above antibodies; or

[0032] (ii) the above enzyme-labeled antibody; or

[0033] (iii) The above antibody combination.

[0034] The present invention also provides a kit, comprising the above reagents.

[0035] The present invention also provides a device carrying the above reagent.

[0036] The present invention also provides a diagnostic method, comprising capturing the antigen with the above-mentioned antibody, the above-mentioned enzyme-labeled antibody or a combination of antibodies, obtaining a detection signal, and obtaining a diagnostic result.

[0037] The present invention also provides a diagnostic method, including diagnosis based on the above reagent, kit or device.

[0038] The method for preparing the anti-carcinoembryonic antigen antibody and the application of the present invention have the following effects:

[0039] The present invention uses CEA as an immunogen to immunize Balb / c mice, uses hybridoma technology to screen and purify 12 monoclonal antibodies against carcinoembryonic antigen, labels the obtained antibodies with horseradish peroxidase (HRP), selects a pair of capture antibodies and detection antibodies through cross-pairing, and applies them to the detection of carcinoembryonic antigen in human serum. The detection method established using the paired antibodies has the advantages of simple operation and high low-value correlation, and can provide key materials for the subsequent development of commercial kits for carcinoembryonic antigen detection. DETAILED DESCRIPTION

[0040] The present invention discloses a method for preparing an antibody against carcinoembryonic antigen and its application. A person skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the above. It should be particularly noted that all similar substitutions and modifications are obvious to a person skilled in the art and are considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0041] The present invention discloses a mouse monoclonal antibody against carcinoembryonic antigen (CEA), wherein the mouse monoclonal antibody is 12#, and the antibody has a heavy chain variable region with an amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region with an amino acid sequence shown in SEQ ID NO: 2.

[0042] The present invention provides a method for preparing the anti-carcinoembryonic antigen monoclonal antibody, comprising the following steps:

[0043] Step 1: CEA extracted from liver cancer tissue after liver metastasis of colorectal cancer was emulsified with an equal volume of adjuvant, and mice were immunized three times. The spleens were collected and spleen cells were hybridized and fused with myeloma cells.

[0044] Step 2: Using traditional hybridoma technology, after three rounds of screening, monoclonal antibody hybridoma cells were obtained;

[0045] Step 3: Inject the monoclonal antibody hybridoma cell line into the peritoneal cavity of Balb / c mice, and collect the ascites 7 days later;

[0046] Step 4: Purify the ascites using the caprylic acid-ammonium sulfate method to obtain the monoclonal antibody.

[0047] The present invention also provides an application of the anti-CEA antibody in detecting CEA in human serum.

[0048] The heavy chain variable region sequence of antibody 12# involved in the present invention is:

[0049] GFSLTRYGVH WVRQSPGKGL EWLGVIWAGG STNYNSGLMS RLSISKDNSK SQVFLKMNSLQTDDTAMYYC ASYYWQRDGM DC (SEQ ID NO: 1).

[0050] The light chain variable region sequence of antibody 12# involved in the present invention is:

[0051] QGIRGNLDWY QQKPGGTIKL LIYSTSNLNS GVPSRFSGSG SGSDYSLTIS SLESEDFADYYCLQRNAYPY T (SEQ ID NO: 2).

[0052] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0053] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0054] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0055] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0056] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0057] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0058] The present invention will be further described below in conjunction with the embodiments.

[0059] Example 1: Screening and preparation of anti-carcinoembryonic antibodies

[0060] 1.1 Animal immunization

[0061] CEA extracted from liver cancer tissue after liver metastasis of colorectal cancer was subcutaneously injected into 6-8 week old Balb / c mice at a dose of 100 μg / mouse for a total of 3 immunizations, with an interval of 2 weeks between each immunization. Cell fusion was performed 5 days after the three immunizations, and booster immunization was performed again 3 days before cell fusion at a dose of 100 μg / mouse.

[0062] 1.2 Screening of positive hybridoma cells

[0063] Cell fusion was performed according to conventional methods. Hybridoma cells were screened 7 days after fusion. The screening method was as follows:

[0064] According to the indirect ELISA method, the optimal antigen coating concentration and the optimal serum dilution were determined by the matrix method to establish the ELISA screening method for monoclonal antibodies. The specific operation is as follows:

[0065] 1) Use CEA as the coating antigen, dilute it to 16μg / ml, 8μg / ml, 4μg / ml, 2μg / ml, 1μg / ml, 0.5μg / ml with coating solution, and coat the ELISA plate at a volume of 50μl / well. After coating at 4℃ for 12-16h, discard the coating solution and wash the plate twice with PBST solution.

[0066] 2) Block the ELISA plate with blocking solution containing 1% Casein, 150 μl / well, at 37°C for 2 h, shake off the blocking solution, and dry at 37°C.

[0067] 3) Dilute the negative and positive sera by 1:100, 1:200, 1:400, 1:800, and 1:1600, respectively, 100 μl / well, react at 37°C for 1 h, and wash the plate 5 times with PBST solution.

[0068] 4) Add 1:4000 diluted HRP-goat anti-mouse IgG+IgM, 100 μl / well, react at 37°C for 1 h, and wash the plate 5 times with PBST solution.

[0069] 5) Add TMB substrate colorimetric solution at 100 μl / well, let it develop at room temperature for 10 min, add 50 μl / well stop solution (2 mol / L H2SO4) to terminate the reaction, and read the OD with a microplate reader. 450nm .

[0070] 6) Determine the ELISA results and take the antigen coating concentration and serum dilution with the positive well OD value closest to 1, the negative well OD value less than 0.1, and the P / N value ≥ 2.1 as the optimal working concentration.

[0071] The cells in the positive culture wells were cloned and purified by limiting dilution until all cloned cells were 100% positive.

[0072] 1.3 Production and purification of monoclonal antibodies

[0073] Balb / c mice were intraperitoneally injected with 0.5 ml of sterile paraffin oil. Seven days later, 10 hybridoma cells containing monoclonal antibodies against carcinoembryonic antigen were intraperitoneally injected. 6 The ascites was collected after 7 days. The ascites was purified by the caprylic acid-ammonium sulfate method to obtain monoclonal antibodies, which were stored at -20°C after the concentration was determined.

[0074] 1.4 Identification of monoclonal antibody subtypes

[0075] The antibodies of the 12 monoclonal antibodies measured by Beijing Sino-Qiao Antibody Subtyping Kit were all IgG subtypes, and the light chains were κ chains.

[0076] 1.5 Monoclonal Antibody Titer Detection

[0077] CEA was diluted to 1 μg / ml with 0.05 mmol / L, pH=9.6 CB buffer, and 50 μl was added to each well of a 96-well ELISA plate (Corning), and coated overnight at 4°C. The next day, the plate was washed three times with PBST, and then blocked with 1% Casein, 100 μl / well, at 37°C for 2 hours. The purified 12 monoclonal antibodies (all at a concentration of 5 mg / ml) were diluted in 0.05 mmol / L, pH=9.6 CB buffer at a ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, etc. The diluted antibodies were added to the ELISA plate coated with CEA, and 0.05 mmol / L, pH=9.6 CB buffer was added to the negative control wells, 50 μl / well. After reacting at 37°C for 30 minutes, wash the plate 5 times with PBST, pat dry, add 1:4000 diluted HRP-goat anti-mouse IgG (SIGMA), 100μl / well, and react at 37°C for 30 minutes. Wash the plate 5 times with PBST, pat dry, add common enzyme-immune substrate, 100μl / well, react at room temperature in the dark for 10 minutes, add 50μl 0.1mol / L sulfuric acid to terminate the reaction, and measure the absorbance at 450nm. The test results are shown in Table 1.

[0078] Table 1: Absorbance at different dilution factors

[0079]

[0080] As can be seen from Table 1, the average OD value of the negative control was 0.05, and the titers of the 12 monoclonal antibodies all showed high titers.

[0081] 1.6 Site identification of monoclonal antibodies

[0082] In order to identify the antibody site, a series of peptides were synthesized according to the antigen epitopes predicted by different software. The epitopes corresponding to peptide 1 and peptide 2 were dominant epitopes; peptide 3 and peptide 4 were non-specific epitopes. After coupling, these peptides were coated with enzyme immunoassay plates for reactivity verification. The results showed that all 12 antibodies could react with peptides coupled with different epitopes of F protein, among which 4# and 12# only recognized the epitope of peptide 1, 6#, 7#, and 9# only recognized the epitope of peptide 2, and the remaining antibodies reacted with peptide 3 or peptide 4, which proved that 4# and 12# could be paired with 6#, 7#, and 9#, respectively. The test results are shown in Table 2.

[0083] Table 2

[0084]

[0085] Example 2: Application of carcinoembryonic antigen antibody in detecting human carcinoembryonic antigen

[0086] 2.1 Double antibody sandwich method for detecting carcinoembryonic antigen

[0087] (1) ELISA plates were coated with 12 carcinoembryonic antigen antibodies at a concentration of 5 μg / ml at 4°C overnight. The plates were washed twice with PBST the next day, patted dry with absorbent paper, and blocked with 1% Casein blocking solution for 2 h.

[0088] (2) CEA was diluted to 1000 ng / ml, and then serially diluted into 8 concentrations. 100 μl of each concentration of antigen was added to the antibody-coated plate per well, incubated at 37°C for 1 h, washed 5 times with PBST solution, and patted dry with absorbent paper.

[0089] (3) According to the site classification, add HRP-labeled antibodies (1:1000 dilution) with different sites from the coated antibody, incubate at 37°C for 1 h, wash 5 times with PBST, add luminescent substrate, and read the luminescence value on a luminometer.

[0090] According to the paired test results, the low-value sample signal value of 12# as the enzyme-labeled antibody paired with the coated antibodies 6#, 7#, and 9# is higher. Considering the overall good correlation, the 6# coated and 12# labeled pairing is preferred. Some paired test data are shown in Table 3.

[0091] Table 3

[0092]

[0093] 2.2 Magnetic particle coating

[0094] Take 30 μl of the mixed magnetic microparticle stock solution and wash it 5 times with 300 μl of PBS buffer, then activate the magnetic microparticles with 10% glutaraldehyde for 1 hour, and then wash the activated magnetic microparticles twice with PBS buffer at pH 7.2. Mouse monoclonal antibodies 6#, 7#, and 9# against carcinoembryonic antigen are added to the magnetic beads at a rate of 0.3 μg / person, and coated at 4°C for 2 hours. Finally, block with a blocking solution containing BSA for 2 hours.

[0095] 2.3 Clinical sample testing

[0096] 18 samples of carcinoembryonic antigen-positive samples from a hospital were collected and tested using the double antibody sandwich method of the present invention. Two paired antibodies were used to detect hospital clinical samples on a magnetic particle platform. The overall correlation between the paired detection signal values ​​of 6#, 7#, 9# coating and 12# labeling and the detection value of Roche CEA detection kit reached above 0.95, as shown in Table 4.

[0097] Table 4

[0098]

[0099] 2.4 Low-value clinical sample testing

[0100] 40 samples with low carcinoembryonic antigen values ​​in the range of 3.7-72.27 were collected from a hospital and tested using the double antibody sandwich method of the present invention. Two paired antibodies were used to test hospital clinical samples on a magnetic particle platform. The paired detection signal values ​​of 6#, 7#, 9# coating and 12# labeling were correlated with the detection values ​​of the Roche CEA detection kit in this value range (3.7-72.27) to be above 0.95, as shown in Table 5.

[0101] Table 5

[0102]

[0103] Example 3: Monoclonal Antibody Sequencing

[0104] The following primers were synthesized based on the constant region sequence of the antibody gene:

[0105] LF: 5′-GACATTGTGATGACCCAGTCTCCT-3′ (SEQ ID NO: 3);

[0106] LR: 5′-TGGACACTGTTGGGGCCGCATCGGCCCT-3′ (SEQ ID NO: 4);

[0107] HF: 5′-CAGGTGCAGCTGCAGGAGTCAGGA-3′ (SEQ ID NO: 5);

[0108] HR: 5′-GATAGACAGATGGGGGTGTCGTTTTGGC-3′ (SEQ ID NO: 6).

[0109] 3 × 10 6 Total RNA of hybridoma cell 6# and hybridoma cell 12# was reverse transcribed into cDNA. HF and HR were used as primers for PCR amplification of the heavy chain variable region of monoclonal antibody 6# and 12#, and LF and LR were used as primers for PCR amplification of the light chain variable region of monoclonal antibody anti-6# and 12#. The PCR reaction was hot-started, and the reaction conditions were: 95℃ 5 minutes; 95℃ 15 seconds, 55℃ 45 seconds, 72℃ 30 seconds, 30 cycles; 72℃ 7 minutes. The PCR product was separated by 1% agarose gel electrophoresis and the target fragment was recovered and purified. It was cloned into pM18-T vector, transformed into Escherichia coli DH5α cells, and screened on LB solid plates. White plaques were inoculated in LB liquid medium containing ampicillin for amplification. Positive clones were screened, plasmids were extracted with QIAGEN's plasmid extraction kit and sequenced, and the heavy chain and light chain variable region sequences of monoclonal antibody 12# were determined.

[0110] The heavy chain variable region of monoclonal antibody 12# has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antibody, characterized in that Its heavy chain variable region has: (1) the amino acid sequence shown in SEQ ID NO: 1; or (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2); Its light chain variable region has: (4) the amino acid sequence shown in SEQ ID NO: 2; or (5) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or (6) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (4) or (5); The plurality is 2 to 5.

2. A nucleic acid molecule encoding the antibody of claim 1.

3. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 2.

4. An enzyme-labeled antibody, characterized in that The antibody is the antibody according to claim 1.

5. The enzyme-labeled antibody according to claim 4, characterized in that The enzyme is horseradish peroxidase.

6. An antibody combination, characterized in that These include antibodies against carcinoembryonic antigen, as well as: (i) the antibody according to claim 1; or (ii) The enzyme-labeled antibody according to claim 4 or 5.

7. Use of any of the following in the preparation of a reagent, kit or device for detecting carcinoembryonic antigen: (i) The antibody according to claim 1; (ii) the enzyme-labeled antibody according to claim 4 or 5; (iii) The antibody combination according to claim 6.

8. A reagent, characterized in that include: (i) The antibody according to claim 1; or (ii) the enzyme-labeled antibody according to claim 4 or 5; or (iii) The antibody combination according to claim 6.

9. A kit, characterized in that Comprising the reagent described in claim 8.

10. The device, characterized in that Contains the reagent according to claim 8.

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