Method for preparing anti-oncofetal monoclonal antibody and application thereof

Abstract

The present application relates to the technical field of biology, and particularly relates to a preparation method and application of an anti-oncofetal antigen monoclonal antibody. The present application discloses a monoclonal antibody 12# with a heavy chain variable region shown in SEQ ID NO:1 and a light chain variable region shown in SEQ ID NO:2. The present application further discloses a preparation method and application of the antibody. The present application uses CEA extracted from liver cancer tissue after liver metastasis of intestinal cancer as an immunogen to immunize Balb / c mice, and uses hybridoma technology to screen and purify 12 strains of anti-CEA monoclonal antibodies. The obtained antibodies are labeled with horseradish peroxidase, and through cross pairing, one strain of antibody that can be used for labeling is selected and applied to detect CEA in human serum. The double antibody sandwich detection method established by using the labeled antibody has the advantages of high sensitivity, simple operation, short detection time and the like, and can provide key materials for subsequent development of commercial kits for CEA detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, 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 antigenic properties. It is present 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. CEA is a polysaccharide-protein complex. The CEA molecule is composed of 641 amino acids with a molecular weight of 180-200 kDa, 60% of which is sugar chains. This heterogeneity is due to the different sugar chains.

[0003] CEA is primarily found in rectal and colon cancer tissue and in the fetal intestinal mucosa. Tumors that secrete CEA are mostly located in hollow organs derived from the endoderm, such as the digestive tract and respiratory tract. It is a non-organ-specific tumor-associated antigen. Under normal circumstances, CEA is metabolized in the gastrointestinal tract and does not enter the circulatory system. However, CEA secreted by tumor cells can enter local body fluids, blood, or lymphatic circulation. Therefore, abnormally elevated CEA levels can be seen in the serum and body fluids of patients with these cancers. Studies have shown that serum CEA levels are often elevated in patients with certain benign diseases, particularly liver disease, but generally below 20 μg / L.

[0004] CEA is a broad-spectrum tumor marker often tested in combination with other tumor markers. Clinically, when CEA levels exceed 60 μg / L, colon, rectal, gastric, and lung cancers are considered. In early-stage, pre-metastatic primary colorectal cancer, the CEA positivity rate is approximately 50% to 75%. In patients with established metastases, the serum CEA positivity rate and concentration are significantly elevated. CEA concentrations generally correlate with disease progression. Increased concentrations indicate increased tumor infiltration, with approximately 70% of cases being metastatic. Decreased concentrations indicate suppressed growth of infiltrated cancer tissue. Therefore, CEA measurement can be used as a basis for evaluating therapeutic efficacy.

[0005] Serum CEA levels are often elevated in patients with primary liver cancer. When various primary cancers metastasize to the liver, CEA levels are higher than in those without liver metastasis. Studies have found that liver metastatic cancer tissues from various sources contain high levels of CEA. Therefore, CEA can also be used as a diagnostic indicator for liver cancer.

[0006] CEA levels increase during the first six months of pregnancy and then gradually decrease. After birth, the fetus's serum CEA level is already very low. Studies have found that CEA levels are somewhat elevated in smokers. Over 96% of healthy, non-smoking adults have serum CEA concentrations below 2.5 μg / L, while 20% to 40% of heavy smokers have serum CEA concentrations above 2.5 μg / L, and a few have concentrations as high as 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 chemiluminescence immunoassay technology. The detection sensitivity, specificity and environmental friendliness have been greatly improved. However, due to the limitations of the antibodies used in the detection, 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 antibodies against carcinoembryonic antigen and its application to solve the problems existing in the above-mentioned prior art. The antibodies prepared by this method can be used to detect carcinoembryonic antigen in human serum, providing key materials for the subsequent development of commercial kits 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 that is at least 90% homologous 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 used to label the antibody is horseradish peroxidase.

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

[0024] (i) the above-mentioned 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, kit or device for detecting carcinoembryonic antigen:

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

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

[0029] (iii) the above antibody combination.

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

[0031] (i) the above-mentioned 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 reagents, kits or devices.

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

[0039] This study used CEA as an immunogen to immunize Balb / c mice. Using hybridoma technology, 12 monoclonal antibodies against carcinoembryonic antigen were screened and purified. These antibodies were labeled with horseradish peroxidase (HRP), and a pair of capture and detection antibodies was selected through cross-pairing. These antibodies were then used to detect carcinoembryonic antigen in human serum. The detection method developed using these paired antibodies offers the advantages of simple operation, low-value correlation, and provides key materials for the subsequent development of commercial kits for carcinoembryonic antigen detection. DETAILED DESCRIPTION

[0040] The present invention discloses methods for preparing and applying antibodies against carcinoembryonic antigens. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify, alter, and combine the methods and applications described herein to implement and apply the technology of the present invention without departing from the content, spirit, and scope 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 the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region with the 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 colorectal cancer liver metastasis was emulsified with an equal volume of adjuvant and used to immunize mice three times. The spleens were harvested and spleen cells were hybridized and fused with myeloma cells.

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

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

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

[0047] The present invention also provides a use of the anti-carcinoembryonic antigen antibody in detecting human serum carcinoembryonic antigen.

[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 individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0053] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or 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 remains operable. Additionally, two or more steps or actions may 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 does not limit the scope of the present application. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present application.

[0056] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular 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 with reference to 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 third immunization, and boosted 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 using the following screening method:

[0064] The indirect ELISA method was used to determine the optimal antigen coating concentration and the optimal serum dilution using the matrix method to establish an ELISA screening method for monoclonal antibodies. The specific procedures are as follows:

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

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

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

[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.

[0069] 5) Add TMB substrate colorimetric solution at 100 μl / well, let the color 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 value using a microplate reader. 450nm .

[0070] 6) ELISA result judgment was performed, and the antigen coating concentration and serum dilution degree with the OD value of positive well closest to 1 and the OD value of negative well less than 0.1 and the P / N value greater than or equal to 2.1 were taken as the optimal working concentration.

[0071] The cells in the positive culture wells were clonally purified by limited dilution until all the clonal cells were 100% positive.

[0072] 1.3 Production and purification of monoclonal antibodies

[0073] Balb / c mice were injected intraperitoneally with 0.5 ml of sterilized paraffin oil per mouse, and 10 6 days later, 10 hybridoma cell strains of anti-carcinoembryonic antigen monoclonal antibodies were injected intraperitoneally per mouse, and 7 days later, the ascites were collected. The ascites were purified by the caprylic acid-ammonium sulfate method to obtain the monoclonal antibodies, which were stored at -20°C after concentration determination.

[0074] 1.4 Identification of monoclonal antibody subtypes

[0075] The Beijing Yiqiao antibody subtype kit was used to determine that the antibodies of the 12 monoclonal antibodies were IgG subtypes, and the light chains were κ chains.

[0076] 1.5 Titer detection of monoclonal antibodies

[0077] CEA was diluted to 1 μg / ml with 0.05 mmol / L CB buffer at pH=9.6, and 50 μl was added to each well of a 96-well enzyme immunoassay plate (Corning), which was coated overnight at 4°C. The next day, the plate was washed 3 times with PBST, and then blocked with 1% Casein at a concentration of 100 μl / well for 2 hours at 37°C. The purified 12 monoclonal antibodies (all at a concentration of 5 mg / ml) were diluted by a factor of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and so on, with 0.05 mmol / L CB buffer at pH=9.6. The diluted antibodies were added to the enzyme immunoassay plate coated with CEA, and 50 μl of 0.05 mmol / L CB buffer at pH=9.6 was added to the negative control wells. After 30 minutes of reaction at 37°C, the plate was washed 5 times with PBST, dried, and then 1:4000 diluted HRP-goat anti-mouse IgG (SIGMA) was added at 100 μl / well for 30 minutes of reaction at 37°C. The plate was washed 5 times with PBST, dried, and then the ordinary enzyme immunoassay substrate was added at 100 μl / well, and the reaction was carried out at room temperature for 10 minutes in the dark. 50 μl of 0.1 mol / L sulfuric acid was added to terminate the reaction, and the absorbance at 450 nm was measured. The detection results are shown in Table 1.

[0078] Table 1: Absorbance at different dilution factors

[0079]

[0080] As shown in 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] To identify antibody sites, a series of peptides were synthesized based on epitope predictions from various software. Peptides 1 and 2 corresponded to dominant epitopes, while peptides 3 and 4 were nonspecific epitopes. After conjugation, these peptides were coated on enzyme immunoassay plates for reactivity verification. The results showed that all 12 antibodies reacted with peptides conjugated to different epitopes of the F protein. Among them, 4# and 12# only recognized the peptide 1 epitope, while 6#, 7#, and 9# only recognized the peptide 2 epitope. The remaining antibodies all reacted with either peptide 3 or peptide 4, demonstrating 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 assay for carcinoembryonic antigen detection

[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 to 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 hour, wash 5 times with PBST, add luminescent substrate, and read the luminescence value on a luminometer.

[0090] Based on the paired test results, the low-value sample signal values ​​were higher when paired with enzyme-labeled antibody 12# and coated antibodies 6#, 7#, and 9#. Considering the good overall correlation, the 6# coating and 12# labeling pairing is preferred. Some paired test data are shown in Table 3.

[0091] Table 3

[0092]

[0093] 2.2 Magnetic particle coating

[0094] 30 μl of the mixed magnetic microparticle stock solution was washed five times with 300 μl of PBS buffer. The magnetic microparticles were then activated with 10% glutaraldehyde for one hour and then washed twice with PBS buffer (pH 7.2). Mouse monoclonal antibodies 6#, 7#, and 9# against carcinoembryonic antigen were added to the magnetic beads at a concentration of 0.3 μg / dose and coated at 4°C for two hours. Finally, the beads were blocked with a blocking buffer containing BSA for two hours.

[0095] 2.3 Clinical sample testing

[0096] Eighteen 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 detection signal values ​​of the paired detections using 6#, 7#, and 9# coatings and 12# labeling and the detection values ​​of the 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 detect the hospital clinical samples on a magnetic particle platform. The correlation between the paired detection signal values ​​of the 6#, 7#, and 9# coatings and the 12# labeling and the detection values ​​of the Roche CEA detection kit in this value range (3.7-72.27) reached 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 from hybridoma cells 6# and 12# was reverse transcribed into cDNA. PCR was performed using primers HF and HR for amplifying the heavy chain variable regions of monoclonal antibodies 6# and 12#, while primers LF and LR were used for amplifying the light chain variable regions of monoclonal antibodies 6# and 12#. Both PCR reactions used a hot start setting: 95°C for 5 minutes, followed by 30 cycles of 95°C for 15 seconds, 55°C for 45 seconds, and 72°C for 30 seconds, and finally 72°C for 7 minutes. PCR products were separated by 1% agarose gel electrophoresis and purified. The fragments were cloned into the pM18-T vector, transformed into Escherichia coli DH5α cells, and screened on LB solid plates. White plaques were then inoculated into LB liquid medium containing ampicillin for amplification. Positive clones were screened, plasmids were extracted using a QIAGEN plasmid extraction kit, and sequencing was performed to determine the heavy and light chain variable region sequences of monoclonal antibody 12#.

[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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An antibody, characterized in that Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, and its light chain variable region has the amino acid sequence shown in SEQ ID NO:

2.

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, wherein The enzyme is horseradish peroxidase.

6. An antibody combination, characterized in that These include antibodies against carcinoembryonic antigen, as well as: (i) The antibody of 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 according to claim 8.

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

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