Preparation and application of he4 monoclonal antibody

Abstract

The present application relates to the field of biological detection, in particular to preparation and application of HE4 monoclonal antibody. The present application provides two mouse monoclonal antibodies with high specificity and sensitivity, which can recognize and bind to different antigen epitopes of HE4 protein respectively. A preparation method of anti-HE4 antibody is also provided, in which HE4 recombinant antigen is used as immunogen to immunize mice, and then cell fusion, screening and cloning are carried out to obtain mouse hybridoma cell strain which can highly secrete anti-HE4 protein monoclonal antibody. The specific antibody prepared by the present application is derived from the same monoclonal cell, and has good batch repeatability. In addition, strict cloning screening is carried out to ensure high specificity and high affinity.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and in particular to an anti-HE4 antibody and its cell line, as well as a preparation method, which is suitable for the diagnosis of early-stage and recurrent ovarian cancer. Background Technology

[0002] Ovarian cancer is the deadliest type of female reproductive system tumor, mainly because its early symptoms are often subtle and difficult to detect, and early diagnosis is challenging due to a lack of effective screening methods. As the duration of cancer development increases, the 5-year survival rate drops from 70-90% to 20%. Therefore, early diagnosis is crucial for improving the cure rate of ovarian cancer.

[0003] Existing early diagnostic tools, such as imaging examinations and tumor marker tests, suffer from insufficient sensitivity and specificity, failing to meet the needs of early detection. CA125, a traditional biomarker widely used clinically, has limited effectiveness in the early detection of ovarian cancer. Firstly, it has a high false-positive rate, as positive expression can also occur in other cancers, benign gynecological diseases such as endometriosis and pelvic inflammatory disease, and under physiological conditions (such as menstruation and pregnancy). Secondly, only 50%–60% of patients with stage I ovarian cancer have elevated CA125 levels, resulting in a positive diagnostic rate of less than 10% using CA125 alone. Even when combined with other detection methods, such as ultrasound, the positive diagnostic rate can only be increased to 20%. Clinically, a more reliable diagnostic method is urgently needed to improve the accuracy of early ovarian cancer detection, reduce patient mortality, and improve the overall prognosis of the disease.

[0004] Human epididymal protein 4 (HE4) is a novel tumor marker and a secreted protein. Its gene was first extracted from the distal epididymal epithelium by Kirchhoff in 1991, hence the name HE4. The protein consists of two core structures: a native N-terminal glycosylated protein of approximately 25 kDa and two whey acidic protein core regions (WAP, composed of a core region of 4 disulfide bonds and 8 cysteine ​​residues).

[0005] HE4 is expressed in the normal female reproductive tract, breast, respiratory tract, distal convoluted tubules of the kidney, colon, and salivary glands. However, it is expressed at low levels or not at all in normal ovarian tissue. The positive expression rate in ovarian tissue is correlated with the histological type of ovarian cancer, with the highest expression in serous ovarian cancer. HE4 levels rise earlier than CA125 levels, making it more sensitive in identifying early-stage ovarian cancer. Therefore, it has a certain advantage in differentiating ovarian cancer from benign ovarian tumors. Summary of the Invention

[0006] In view of this, the first objective of the present invention is to provide an anti-HE4 monoclonal antibody, comprising a first antibody and a second antibody, for measuring the expression of HE4 protein in serum, for the early diagnosis of ovarian cancer, and for detecting the recurrence of ovarian cancer.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides an antibody, wherein:

[0009] Light chain CDR1 has:

[0010] (1) The amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 7; or

[0011] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0012] (3) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2).

[0013] and / or

[0014] Light chain CDR2 has:

[0015] (4) An amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 8; or

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

[0017] (6) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5).

[0018] and / or

[0019] Light chain CDR3 has:

[0020] (7) An amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 9; or

[0021] (8) An amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (7), and whose function is the same as or similar to that of (7); or

[0022] (9) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (7) or (8).

[0023] and / or

[0024] Heavy chain CDR1 has:

[0025] (10) An amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 10; or

[0026] (11) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (10), and whose function is the same as or similar to that of (10); or

[0027] (12) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (10) or (11).

[0028] and / or

[0029] Heavy chain CDR2 has:

[0030] (13) An amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 11; or

[0031] (14) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (13), and whose function is the same as or similar to that of (13); or

[0032] (15) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (13) or (14).

[0033] and / or

[0034] Heavy chain CDR3 has:

[0035] (16) An amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 12; or

[0036] (17) An amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (16), and whose function is the same as or similar to that of (16); or

[0037] (18) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (16) or (17).

[0038] The number of "several" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0039] In some specific embodiments of the present invention, the above-mentioned antibody:

[0040] The amino acid sequence of its light chain CDR1 is shown in SEQ ID NO: 1 or SEQ ID NO: 7; and / or

[0041] The amino acid sequence of its light chain CDR2 is shown in SEQ ID NO: 2 or SEQ ID NO: 8; and / or

[0042] The amino acid sequence of its light chain CDR3 is shown in SEQ ID NO: 3 or SEQ ID NO: 9; and / or

[0043] The amino acid sequence of its heavy chain CDR1 is shown in SEQ ID NO: 4 or SEQ ID NO: 10; and / or

[0044] The amino acid sequence of its heavy chain CDR2 is shown in SEQ ID NO: 5 or SEQ ID NO: 11; and / or

[0045] The amino acid sequence of its heavy chain CDR3 is shown in SEQ ID NO: 6 or SEQ ID NO: 12.

[0046] In some specific embodiments of the present invention, the antibody described above is a monoclonal antibody.

[0047] In some specific embodiments of the present invention, the antibody is a mouse IgG1κ subtype monoclonal antibody.

[0048] In some specific embodiments of the present invention, the above-mentioned antibody includes a first antibody and / or a second antibody;

[0049] The amino acid sequences of the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the first antibody are shown in SEQ ID NO: 1~6, respectively.

[0050] The amino acid sequences of the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the second antibody are shown in SEQ ID NO: 7~12.

[0051] In some specific embodiments of the present invention, the light chain variable region of the first antibody of the above-described antibody has:

[0052] (1) The amino acid sequence as shown in SEQ ID NO: 13; or

[0053] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0054] (3) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2).

[0055] In some specific embodiments of the present invention, the heavy chain variable region of the first antibody of the above-described antibody has:

[0056] (4) The amino acid sequence as shown in SEQ ID NO: 14; or

[0057] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0058] (6) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5).

[0059] In some specific embodiments of the present invention, the light chain variable region of the second antibody of the above-mentioned antibody has:

[0060] (7) The amino acid sequence as shown in SEQ ID NO: 15; or

[0061] (8) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (7), and whose function is the same as or similar to that of (7); or

[0062] (9) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (7) or (8).

[0063] In some specific embodiments of the present invention, the heavy chain variable region of the second antibody of the above-described antibody has:

[0064] (10) The amino acid sequence as shown in SEQ ID NO: 16; or

[0065] (11) An amino acid sequence obtained by substitution, deletion or addition of one or more residues as shown in (10), and whose function is the same as or similar to that of (10); or

[0066] (12) An amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (10) or (11).

[0067] In some specific embodiments of the present invention, the plurality of antibodies is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0068] In some specific embodiments of the present invention, the first antibody in the above-mentioned antibody is HE4-11#, the amino acid sequence of the light chain variable region is SEQ ID NO: 13, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 14.

[0069] In some specific embodiments of the present invention, the second antibody in the above-mentioned antibody is HE4-155#, the amino acid sequence of the light chain variable region is SEQ ID NO: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 16.

[0070] The present invention also provides an antibody combination comprising:

[0071] (i) the first antibody among the antibodies mentioned above; and / or

[0072] (ii) The second antibody among the antibodies mentioned above.

[0073] This invention also provides antibody derivatives, including modified ones:

[0074] (I) The above-mentioned antibodies; or

[0075] (II) The above antibody combination.

[0076] In some specific embodiments of the present invention, the modifications of the above-mentioned antibody derivatives include, but are not limited to, at least one of enzyme labeling, fluorescent labeling, biotin labeling, radioisotope labeling, PEGylation modification, glycosylation modification, antibody-drug conjugate modification, and protein tag modification.

[0077] The present invention also provides a nucleic acid molecule comprising:

[0078] (i) encoding the antibodies described above; and / or

[0079] (ii) Encoding the first antibody in the above antibody combination; and / or

[0080] (iii) The second antibody in the above antibody combination.

[0081] In some specific embodiments of the present invention, the above-mentioned nucleic acid molecule includes a first nucleic acid molecule or a second nucleic acid molecule;

[0082] The first nucleic acid molecule encodes the first antibody in the above antibody combination;

[0083] The second nucleic acid molecule encodes the second antibody in the above antibody combination.

[0084] The present invention also provides an expression vector comprising the above-described nucleic acid molecules.

[0085] In some specific embodiments of the present invention, the above expression vector expresses the above antibody.

[0086] In some specific embodiments of the present invention, the above expression vector expresses the above antibody combination.

[0087] In some specific embodiments of the present invention, the expression vector expresses the first antibody in the antibody combination.

[0088] In some specific embodiments of the present invention, the expression vector expresses the second antibody in the antibody combination.

[0089] The present invention also provides a host cell comprising:

[0090] (i) the above-mentioned nucleic acid molecules; or

[0091] (ii) The above-mentioned expression carriers.

[0092] In some specific embodiments of the present invention, the host cells described above do not possess totipotency.

[0093] In some specific embodiments of the present invention, the host cell is a hybridoma cell.

[0094] In some specific embodiments of the present invention, the hybridoma cells of the host cells described above are derived from mice.

[0095] The present invention also provides biological materials, including at least one of the above-mentioned nucleic acid molecules, the above-mentioned expression vectors, and the above-mentioned host cells.

[0096] In some specific embodiments of the present invention, the above-mentioned biomaterials are used in a broad sense, and specifically may refer to genetically engineered materials.

[0097] This invention also provides the use of any of the following in the preparation of products that capture human epididymal protein 4 or in the preparation of human epididymal protein 4 detection products:

[0098] (i) the antibodies mentioned above;

[0099] (ii) The above antibody combination;

[0100] (iii) The above-mentioned nucleic acid molecules;

[0101] (iv) The aforementioned expression carriers;

[0102] (v) The aforementioned host cells;

[0103] (vi) The above-mentioned biological materials.

[0104] The present invention also provides a product for capturing human epididymal protein 4, comprising any of the following:

[0105] (i) the antibodies mentioned above;

[0106] (ii) The above antibody combination.

[0107] In some specific embodiments of the present invention, the product described above that captures human epididymal protein 4 can be a product used to isolate and purify human epididymal protein 4.

[0108] This invention also provides a product for detecting human epididymal protein 4, characterized in that it includes any of the following:

[0109] (i) the antibodies mentioned above;

[0110] (ii) The above antibody combination.

[0111] In some specific embodiments of the present invention, the above-mentioned product for detecting human epididymal protein 4 adopts a double antibody sandwich method, wherein the first antibody of the product is the first antibody in the above-mentioned antibody combination, and the second antibody of the product is the second antibody in the above-mentioned antibody combination.

[0112] In some specific embodiments of the present invention, the above-mentioned detection of human epididymal protein 4 refers to a detection product targeting human epididymal protein 4.

[0113] In some specific embodiments of the present invention, the above-mentioned product for detecting human epididymal protein 4 can also be an ovarian cancer diagnostic kit.

[0114] The present invention also provides the application of the above-mentioned antibodies, combinations of the above-mentioned antibodies, or the above-mentioned products in the diagnosis of ovarian cancer.

[0115] The present invention also provides a method for detecting human epididymal protein 4, which is based on the above-mentioned antibody, the above-mentioned antibody combination or the above-mentioned product.

[0116] The present invention also provides a method for diagnosing ovarian cancer, which is based on the above-mentioned antibodies, combinations of the above-mentioned antibodies, or the above-mentioned products.

[0117] Compared with the prior art, the present invention has the following beneficial effects:

[0118] This invention provides two highly specific and sensitive mouse monoclonal antibodies, which can respectively recognize and bind to different antigenic epitopes of the HE4 protein. It also provides a method for preparing anti-HE4 antibodies, using recombinant HE4 antigen as an immunogen to immunize mice, followed by cell fusion, screening, and cloning to obtain a mouse hybridoma cell line that efficiently secretes anti-HE4 protein monoclonal antibodies. The specific antibodies prepared by this invention originate from the same monoclonal cell line, exhibiting good batch-to-batch reproducibility. Furthermore, rigorous clonal screening ensures high specificity and high affinity. Attached Figure Description

[0119] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0120] Figure 1 Results of a clinical comparison between the HE4 symmetric graph and Roche. Detailed Implementation

[0121] This invention discloses an anti-HE4 antibody and its cell line, as well as a preparation method, suitable for the diagnosis of early-stage and recurrent ovarian cancer. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0122] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0123] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0124] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0125] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0126] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0127] In practice, the anti-HE4 monoclonal antibody includes a first antibody HE4-11# and a second antibody HE4-155#, which measures the expression of HE4 protein in serum for early diagnosis of ovarian cancer and detection of ovarian cancer recurrence.

[0128] The first antibody HE4-11# includes light chain complementarity-determining regions CDR1-VL, CDR2-VL, and CDR3-VL, as shown in SEQ ID NO: 1-3, and heavy chain complementarity-determining regions CDR1-VH, CDR2-VH, and CDR3-VH, as shown in SEQ ID NO: 4-6.

[0129] The second antibody HE4-155# includes light chain complementarity-determining regions CDR1-VL, CDR2-VL, and CDR3-VL, as shown in SEQ ID NO: 7-9, and heavy chain complementarity-determining regions CDR1-VH, CDR2-VH, and CDR3-VH, as shown in SEQ ID NO: 10-12.

[0130] The above sequences are shown in Table 1.

[0131] Table 1: Amino acid sequences of each complementarity-determining region

[0132]

[0133] Furthermore, the first antibody HE4-11# includes a light chain variable region and a heavy chain variable region with amino acid sequences as shown in SEQ ID NO: 13~14;

[0134] The second antibody HE4-155# includes a light chain variable region and a heavy chain variable region, with amino acid sequences as shown in SEQ ID NO: 15~16.

[0135] The SEQ ID NO: 13 is:

[0136] DIQMTQSPASLSASVGETVTITCRPSENIYSYLAWYQQKPGKSPQLLVYNTKTLAEGVPSRFSGSGSGTQFFLKINNLQPEDFGDYYCQHHYAPPYTFGGGTKLEIK;

[0137] The SEQ ID NO: 14 is:

[0138] EVRLVESGGDLVKPGGSLKLSCAVSAFFTFSTYGMSWIRQTPDKRLEWVATISNGGNYTYYPDSLKGRFTIARDNVKNTLYLQMSSLKSEDTAMYYCASSYDYAWFVYWGQGTLVTVSA;

[0139] The SEQ ID NO: 15 is:

[0140] QIVLSQSPAILSASPGEKVTMTCRANSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSFTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK;

[0141] The SEQ ID NO: 16 is:

[0142] EVMLVESGGGLVKPGGSLKLSCVASGFTFSSYAMSWFRQTPEKRLEWVATISNGGTYTYSPDSVKGRFTISRDNAKNTLFLQMSSLRSEDTAMYYCARQYANYFDYWGQGTTLTVSS.

[0143] Furthermore, the monoclonal antibody is a mouse IgG1κ subtype monoclonal antibody.

[0144] Furthermore, the first antibody HE4-11# and the second antibody HE4-155# can specifically recognize the HE4 protein.

[0145] In another implementation, a mouse hybridoma cell line capable of secreting and expressing anti-HE4 antibodies is used.

[0146] Another implementation method uses an HE4 protein immunoassay kit.

[0147] Furthermore, the kit includes the HE4 monoclonal antibody and acceptable excipients.

[0148] Furthermore, the immunoassay kit employs a double-antibody sandwich method, wherein the first antibody HE4-11# serves as the coating antibody, and the second antibody HE4-155# serves as the labeling antibody.

[0149] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0150] The present invention will be further illustrated below with reference to the embodiments.

[0151] Example 1: Preparation of anti-HE4 monoclonal antibody

[0152] 1. Animal immunization

[0153] Using our self-developed, bioactive recombinant human HE4 protein as an immunogen, Bal / c mice were immunized via intraperitoneal injection. The initial immunization dose was 100 μg per mouse, fully emulsified with Freund's complete adjuvant. Two booster immunizations were administered at 2-3 weeks intervals, with a booster dose of 50 μg per mouse. After the three immunizations, serum titers were measured, and mice with higher titers were used for fusion cloning.

[0154] 2. Fusion Cloning

[0155] High-titer mouse spleens were mixed with NS-1 myeloma cells at a 5:1 ratio and fused using PEG1500. Cells that achieved complete fusion were then screened in HAT medium. Approximately 6-7 days post-fusion, the content of specific antibodies in the cell supernatant was detected using an indirect ELISA method. Highly reactive positive wells were selected and cloned using a limiting dilution method until the positive well rate for monoclonal cells reached 100%. Monoclonal cells were then picked for expansion culture and stored in liquid nitrogen. Two hybridoma cell lines stably secreting anti-human HE4 antibodies were obtained in this cloning operation and named HE4-11# and HE4-155#.

[0156] 3. Monoclonal antibody purification

[0157] The mouse hybridoma cells obtained in the previous step (5×10) 5 1 mL was injected into the peritoneal cavity of mice. Ascites fluid was collected 7 days later, purified by SPA purification method, and the purity and concentration of the antibody were identified. The antibody was then stored at -20℃.

[0158] Example 2: Subtype Identification

[0159] The recombinant HE4 antigen was coated onto the ELISA plate at a concentration of 2 μg / mL. The diluted antibody was added to the plate and reacted for 60 min. Then, the subtype identification reagent was added. The subtype identification reagent was selected from Beijing Yiqiao Biotechnology Co., Ltd. The subtype identification reagent was diluted to 1 / 200 with PBST enzyme dilution and reacted at 37℃ for 30 min. Then, HRP-goat anti-mouse (1 / 8000) was added and reacted at 37℃ for 30 min. The OD value was read using an ELISA reader.

[0160] Table 2: Subtype Identification Results (Beijing Yiqiao Subtype Reagent)

[0161]

[0162] As shown in Table 2, the antibody subtype is IgG1 type murine monoclonal antibody.

[0163] Example 3: Clinical relevance of monoclonal antibodies and Roche antibodies

[0164] Using a magnetic microparticle chemiluminescence platform, HE4-11# antibody was coated onto magnetic beads using a two-step carboxyl group method to prepare a magnetic bead suspension as the coating and capture antibody. HE4-155# was labeled with horseradish peroxidase using a sodium periodate oxidation method to serve as the detection antibody, thus establishing a double-antibody sandwich ELISA method for detecting HE4 protein. This method was tested alongside a benchmark product on 141 clinical samples. The clinical relevance results between the detection method established in this invention and the benchmark product are as follows: Figure 1 As shown.

[0165] The results showed that the detection method established in this invention had a high clinical relevance to the benchmark product, R 2 =0.9827.

[0166] Example 4: Stability of monoclonal antibodies

[0167] The HE4-11# magnetic bead suspension prepared in Example 3 and the horseradish peroxidase-labeled HE4-155# antibody were placed at 4℃ and 37℃ for 14 days, respectively, and the thermal stability of calibrators C1 and C2 and clinical samples P1~P9 was tested.

[0168] Table 3: Results of thermostability of monoclonal antibodies

[0169]

[0170] As shown in Table 3, the changes in the standard and clinical samples were consistent, and the changes were all within 10%, indicating that the stability met the requirements for production and use.

[0171] Example 5: Antibody Sequencing

[0172] Freshly cultured hybridoma cells were collected by centrifugation, and total RNA was extracted from the hybridoma cells using the Trizol method and reverse transcribed into cDNA. Using the cDNA as a template, the light chain variable region (VL) and heavy chain variable region (VH) genes of mouse monoclonal antibodies were amplified by PCR, constructed into expression vectors, and sequenced.

[0173] The measured sequence is shown above.

[0174] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody, characterized in that, include: Light chain CDR1 has: (1) The amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 7; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or (3) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (1) or (2); and / or Light chain CDR2 has: (4) An amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 8; or (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or (6) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (4) or (5); and / or Light chain CDR3 has: (7) An amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 9; or (8) An amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (7), and whose function is the same as or similar to that of (7); or (9) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (7) or (8); and / or Heavy chain CDR1 has: (10) An amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 10; or (11) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (10), and whose function is the same as or similar to that of (10); or (12) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (10) or (11); and / or Heavy chain CDR2 has: (13) An amino acid sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 11; or (14) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (13), and whose function is the same as or similar to that of (13); or (15) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (13) or (14); and / or Heavy chain CDR3 has: (16) An amino acid sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 12; or (17) An amino acid sequence obtained by substituting, deleting, or adding one or more residues to the amino acid sequence shown in (16), and whose function is the same as or similar to that of (16); or (18) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (16) or (17); The number of items is 2 to 50.

2. The antibody as described in claim 1, characterized in that, Including primary and / or secondary antibodies; The amino acid sequences of the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the first antibody are shown in SEQ ID NO: 1~6, respectively. The amino acid sequences of the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 of the second antibody are shown in SEQ ID NO: 7~12.

3. The antibody as described in claim 2, characterized in that, include: The light chain variable region of the first antibody has: (19) The amino acid sequence as shown in SEQ ID NO: 13; or (20) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (19), and whose function is the same as or similar to that of (19); or (21) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (19) or (20); The heavy chain variable region of the first antibody has: (22) The amino acid sequence as shown in SEQ ID NO: 14; or (23) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (22), and whose function is the same as or similar to that of (22); or (24) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (22) or (23); The light chain variable region of the second antibody has: (25) The amino acid sequence as shown in SEQ ID NO: 15; or (26) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (25), and whose function is the same as or similar to that of (25); or (27) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (25) or (26); The heavy chain variable region of the second antibody has: (28) The amino acid sequence as shown in SEQ ID NO: 16; or (29) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (28), and whose function is the same as or similar to that of (28); or (30) An amino acid sequence that is at least 90% homologous to the amino acid sequence shown in (28) or (29); The number of items is 2 to 50.

4. An antibody combination, characterized in that, include: (i) the first antibody as described in claim 2 or 3; and / or (ii) The second antibody in the antibody of claim 2 or 3.

5. An antibody derivative, characterized in that, Including the modified version: (I) The antibody according to any one of claims 1 to 3; or (II) The antibody combination according to claim 4.

6. The antibody derivative as described in claim 5, characterized in that, The modifications include, but are not limited to, at least one of the following: enzyme labeling, fluorescent labeling, biotin labeling, radioisotope labeling, PEGylation modification, glycosylation modification, antibody-drug conjugate modification, and protein tag modification.

7. A biomaterial, characterized in that, Including at least one of nucleic acid molecules, expression vectors, and host cells; The nucleic acid molecule: (i) Encoding the antibody according to any one of claims 1 to 3; and / or (ii) Encoding the first antibody in the antibody combination of claim 4; and / or (iii) Encoding the second antibody in the antibody combination of claim 4; The expression vector includes the nucleic acid molecule; The host cell includes the nucleic acid molecule or the expression vector.

8. The following applications in the preparation of products that capture human epididymal protein 4 or products that detect human epididymal protein 4: (i) the antibody according to any one of claims 1 to 3; (ii) The antibody combination as described in claim 4; (iii) The antibody derivative according to claim 5 or 6; (iv) The biomaterial as described in claim 7.

9. A product that captures human epididymal protein 4, characterized in that, Includes any of the following: (i) the antibody according to any one of claims 1 to 3; (ii) The antibody combination as described in claim 4; (iii) The antibody derivative according to claim 5 or 6.

10. A product for detecting human epididymal protein 4, characterized in that, Includes any of the following: (i) the antibody according to any one of claims 1 to 3; (ii) The antibody combination as described in claim 4; (iii) The antibody derivative according to claim 5 or 6.

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