Anti-estradiol monoclonal antibodies or their antigen-binding fragments and their applications

Abstract

This invention relates to monoclonal antibodies against estradiol or their antigen-binding fragments and their applications. The invention provides a monoclonal antibody against estradiol or its antigen-binding fragment that binds to estradiol (E2) with high specificity and high affinity. The invention also develops high-performance paired antibodies suitable for double-antibody sandwich immunoassay, thus overcoming the limitations of traditional competitive assay reagents in terms of low sensitivity and poor clinical relevance. This enables double-antibody sandwich immunoassay for E2, and significantly improves detection accuracy and low-end sensitivity compared to CN 117903305.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody preparation and immunological detection technology, specifically to anti-estradiol monoclonal antibodies or their antigen-binding fragments and their applications. Background Technology

[0002] Estradiol (E2) is a steroid hormone primarily secreted by ovarian follicles, the corpus luteum, and the placenta during pregnancy, with a molecular weight of approximately 272 Da. E2 is the most abundant and biologically active estrogen, playing a crucial role in the development of the female reproductive system and the emergence of secondary sexual characteristics during reproductive years. It also influences various aspects of life, including bone health, cardiovascular health, brain function, and metabolism. Estrone (E1), estradiol (E2), and estriol (E3) are the main forms of estrogen in the body. E1 and E2 can interconvert, and E3 is a metabolite of E2. These three hormones are structurally very similar, thus requiring high antibody specificity. Common methods for detecting E2 include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), colloidal gold assay, chemiluminescence immunoassay, and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Radioimmunoassay has been gradually phased out due to the risk of radioactive contamination. Enzyme-linked immunosorbent assay (ELISA) and colloidal gold assay have limited sensitivity and specificity. Currently, the mainstream clinical method for detecting E2 is chemiluminescent immunoassay, which has high sensitivity and is less expensive than mass spectrometry. The first-generation E2 sandwich antibody disclosed in prior art 1 (CN 117903305) has significantly improved sensitivity and specificity compared to competing methods, but there is still room for optimization in terms of low-end sensitivity. Summary of the Invention

[0003] Purpose of the invention

[0004] In view of the problems or needs existing in the prior art, the purpose of this invention is to provide a monoclonal antibody or antigen-binding fragment of estradiol (E2) with high specificity and high affinity, and to develop a high-performance paired antibody suitable for double antibody sandwich immunological detection.

[0005] Technical solution

[0006] To achieve the above objectives, this invention has obtained highly specific and high-affinity monoclonal antibodies or antigen-binding fragments against estradiol (E2), their related nucleic acids and cell products, their preparation methods and applications, and immunoassay reagents based thereon through extensive screening, thereby providing a foundation for highly specific and sensitive immunoassay of E2.

[0007] Specifically, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides an anti-estradiol monoclonal antibody or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment thereof comprising:

[0009] (1) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 6, YVS and SEQ ID NO: 7, respectively, LCDR1, LCDR2 and LCDR3; or,

[0010] (2) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 15, GAT and SEQ ID NO: 16, respectively, LCDR1, LCDR2 and LCDR3; or,

[0011] (3) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 24, STS and SEQ ID NO: 25, respectively, LCDR1, LCDR2 and LCDR3; or,

[0012] (4) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 33, NAK and SEQ ID NO: 34, respectively, LCDR1, LCDR2 and LCDR3; or,

[0013] (5) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 42, NAK and SEQ ID NO: 43, respectively, LCDR1, LCDR2 and LCDR3; or,

[0014] (6) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 51, NAK and SEQ ID NO: 52, respectively, LCDR1, LCDR2 and LCDR3; or,

[0015] (7) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 60, RAN and SEQ ID NO: 61, respectively, LCDR1, LCDR2 and LCDR3; or,

[0016] (8) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 69, YAS and SEQ ID NO: 70, respectively, LCDR1, LCDR2 and LCDR3; or,

[0017] (9) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 78, RAN and SEQ ID NO: 79, respectively, LCDR1, LCDR2 and LCDR3; or,

[0018] (10) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO: 87, HGT and SEQ ID NO: 88, respectively, LCDR1, LCDR2 and LCDR3.

[0019] Preferably, the monoclonal antibody or its antigen-binding fragment comprises:

[0020] (1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:4; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:8; or,

[0021] (2) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:13; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:17; or,

[0022] (3) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:22; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:26; or,

[0023] (4) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:31; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:35; or,

[0024] (5) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:40; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:44; or,

[0025] (6) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:49; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:53; or,

[0026] (7) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:58; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:62; or,

[0027] (8) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:67; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:71; or,

[0028] (9) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:76; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO:80; or,

[0029] (10) Heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:85; and light chain variable region having an amino acid sequence as shown in SEQ ID NO:89.

[0030] In addition, the monoclonal antibody or its antigen-binding fragment further includes a constant region; preferably, the constant region is selected from any one of the following: the constant region of IgG, IgA or IgM antibody.

[0031] More preferably, the monoclonal antibody or its antigen-binding fragment comprises:

[0032] (1) A heavy chain having the amino acid sequence shown in SEQ ID NO:5; and a light chain having the amino acid sequence shown in SEQ ID NO:9; or,

[0033] (2) A heavy chain having the amino acid sequence shown in SEQ ID NO:14; and a light chain having the amino acid sequence shown in SEQ ID NO:18; or,

[0034] (3) a heavy chain having the amino acid sequence shown in SEQ ID NO:23; and a light chain having the amino acid sequence shown in SEQ ID NO:27; or,

[0035] (4) a heavy chain having the amino acid sequence shown in SEQ ID NO:32; and a light chain having the amino acid sequence shown in SEQ ID NO:36; or,

[0036] (5) a heavy chain having the amino acid sequence shown in SEQ ID NO:41; and a light chain having the amino acid sequence shown in SEQ ID NO:45; or,

[0037] (6) a heavy chain having the amino acid sequence shown in SEQ ID NO:50; and a light chain having the amino acid sequence shown in SEQ ID NO:54; or,

[0038] (7) A heavy chain having the amino acid sequence shown in SEQ ID NO:59; and a light chain having the amino acid sequence shown in SEQ ID NO:63; or,

[0039] (8) A heavy chain having the amino acid sequence shown in SEQ ID NO:68; and a light chain having the amino acid sequence shown in SEQ ID NO:72; or,

[0040] (9) A heavy chain having the amino acid sequence shown in SEQ ID NO:77; and a light chain having the amino acid sequence shown in SEQ ID NO:81; or,

[0041] (10) a heavy chain having an amino acid sequence as shown in SEQ ID NO:86; and a light chain having an amino acid sequence as shown in SEQ ID NO:90.

[0042] In the first aspect above, the monoclonal antibodies defined by items (1) to (10) are named antibody 1 to 10 respectively below.

[0043] In some feasible implementations, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies, human antibodies, chimeric antibodies, bispecific or multispecific antibodies.

[0044] Secondly, the present invention provides a polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect above. This polynucleotide is not limited to any particular method of its production and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.

[0045] Thirdly, the present invention provides a nucleic acid construct comprising a polynucleotide as described in the second aspect above, and at least one expression regulatory element operatively linked to the polynucleotide.

[0046] Fourthly, the present invention provides a recombinant vector comprising a polynucleotide as described in the second aspect above, or a nucleic acid construct as described in the third aspect above.

[0047] The vector of the present invention can be a cloning vector or an expression vector, for example, a plasmid, a granule, a bacteriophage, etc.

[0048] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0049] Fifthly, the present invention provides a transformed host cell, wherein the transformation comprises a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, or a recombinant vector as described in the fourth aspect above;

[0050] The host cells include, but are not limited to: prokaryotic cells, such as Escherichia coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells can also be cell lines, such as the 293T cell line.

[0051] Preferably, the host cell is a eukaryotic cell, and more preferably a mammalian cell.

[0052] In a sixth aspect, the present invention provides the use of monoclonal antibodies or antigen-binding fragments thereof as described in the first aspect above, polynucleotides as described in the second aspect above, nucleic acid constructs as described in the third aspect above, recombinant vectors as described in the fourth aspect above, and / or transformed host cells as described in the fifth aspect above in the preparation of detection reagents or kits for detecting estradiol.

[0053] In a feasible implementation, the sample is a biological sample of the subject, preferably a serum or plasma sample of the subject.

[0054] In a seventh aspect, the present invention provides a method for detecting the presence or level of estradiol in a sample, the method comprising using a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, a polynucleotide as described in the second aspect above, a nucleic acid construct as described in the third aspect above, an expression vector as described in the fourth aspect above, a transformed host cell as described in the fifth aspect above, and / or a detection reagent as described in the eighth aspect above.

[0055] In feasible implementations, the sample includes, but is not limited to, serum, plasma, etc., from the subject.

[0056] This method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).

[0057] The general methods for detecting the presence or level of a target antigen in a sample using monoclonal antibodies or their antigen-binding fragments are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.

[0058] Beneficial effects

[0059] The anti-estradiol monoclonal antibody of the present invention can bind to estradiol with high affinity and high specificity (its binding activity EC50 reaches the ng / ml level, and its KD is above 10). -10 M~10 -11 This invention achieves high accuracy and sensitivity in the detection of estradiol (M). Furthermore, it develops a high-performance paired antibody suitable for double-antibody sandwich immunoassay, overcoming the limitations of low sensitivity and poor clinical relevance of traditional competitive assay reagents, thus realizing the double-antibody sandwich immunoassay for estradiol. Compared to the first-generation sandwich antibody of prior art 1, the antibody of this invention has a greater advantage in low-end sensitivity, resulting in more accurate results when detecting samples. Attached Figure Description

[0060] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0061] Figure 1 This is a gel image obtained by SDS-PAGE analysis of 10 anti-E2 monoclonal antibodies expressed and purified in this invention. In the image, M represents the electrophoretic band of the molecular weight marker; lane 1 shows the electrophoretic bands of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoretic bands of each monoclonal antibody under non-reducing conditions. Specifically, a is the band corresponding to E2 mAb1, b is the band corresponding to E2 mAb2, c is the band corresponding to E2 mAb3, d is the band corresponding to E2 mAb4, e is the band corresponding to E2 mAb5, f is the band corresponding to E2 mAb6, g is the band corresponding to E2 mAb7, h is the band corresponding to E2 mAb8, i is the band corresponding to E2 mAb9, and j is the band corresponding to E2 mAb10.

[0062] Figure 2 The graph shows the results of the binding activity assay of 10 anti-E2 monoclonal antibodies expressed and purified in this invention with E2 conjugate protein.

[0063] Figure 3 The standard curves for the chemiluminescent reagents prepared in this invention are shown, with the horizontal axis representing the standard dilution concentration and the vertical axis representing the signal values ​​read by the chemiluminescence instrument for different concentrations of the standard. In this curve, A represents paired antibodies based on E2 mAb1 + E2 mAb2, and B represents paired antibodies based on E2 mAb3 + E2 mAb4.

[0064] Figure 4 The results are shown in the diagrams below, which illustrate the correlation between two chemiluminescent reagents based on the two paired antibodies of this invention and Roche's detection reagents in clinical samples. Specifically, A represents the results of chemiluminescent reagent 1 based on the paired antibodies E2 mAb1+E2 mAb2, B represents the results of chemiluminescent reagent 2 based on the paired antibodies E2 mAb3+E2 mAb4, and C represents the results of chemiluminescent reagents prepared from monoclonal antibodies in the prior art 1. Detailed Implementation

[0065] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising" shall be understood to include the stated elements or components without excluding other elements or other components.

[0066] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0067] To facilitate a better understanding of this invention, certain technical terms are specifically defined as follows. Unless otherwise expressly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0069] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0070] Example 1: Preparation of anti-E2 monoclonal antibody

[0071] (1) Animal immunization:

[0072] Using conjugated protein E2-BSA (purchased from Nanjing Mingyan Biotechnology Co., Ltd., catalog number MY-E2-001) as the antigen, mice (Bullet: Balb / c, female, 6-8 weeks old) were immunized by intraperitoneal injection after thorough emulsification with an equal volume of Freund's complete adjuvant. Booster immunizations were administered at two-week intervals, for a total of three immunizations. Five to eight days after the last booster immunization, a small amount of tail blood was collected, and serum titer was detected by ELISA. When the serum titer reached >128K, spleen single-cell suspensions were prepared. Specific detection information is shown in Tables 1 and 2.

[0073] Table 1. Experimental Information

[0074]

[0075] Table 2. Serum titer data detected by ELIAS method

[0076]

[0077] Mice with a serum titer of 512K were selected to prepare spleen single-cell suspensions.

[0078] (2) Preparation of spleen single-cell suspension:

[0079] Mice were euthanized by cervical dislocation. A small incision was made in the middle of the left ventral side of the mouse, the skin was torn open to expose the abdominal wall, the peritoneum was lifted below the spleen, cut open and turned upwards to expose the spleen, the spleen was lifted with forceps, and the connective tissue beneath the spleen was separated with ophthalmic scissors to remove the spleen. The spleen was placed on a cell filter and gently crushed with a syringe needle core to obtain a cell suspension. In a separate 15 mL sterile test tube, Ficoll cell separation medium was added first, and the freshly prepared cell suspension was slowly added to the test tube at a volume ratio of 1:2. The test tube was centrifuged at 2000 rpm for 20 minutes at room temperature. After centrifugation, the test tube was removed, and different layers were observed. The upper non-cellular layer was aspirated and discarded, and the single nuclear layer was aspirated into another 15 mL sterile test tube and centrifuged at 1500 rpm for 10 minutes at room temperature. The supernatant was carefully removed and the cells were resuspended.

[0080] (3) Obtaining a single B cell:

[0081] Using a prepared spleen single-cell suspension, cells were screened using a single-cell screening platform after antigen labeling to obtain antigen-specific B cells.

[0082] Through the above steps, a total of 123 monoclonal antibodies against folic acid and its binding protein complex were obtained. These were identified as IgG antibodies, and their names are shown in Tables 3A and 3B. In the following examples, the light and heavy chain gene sequences of the monoclonal antibodies were detected and expressed. The transfected cell supernatants were placed in 96-well plates according to the layout in Tables 3A and 3B for binding activity assay.

[0083] Table 3A. Clone numbers of 88 antibody supernatants

[0084]

[0085] Table 3B. Clone numbers of 35 antibody supernatants

[0086]

[0087] The negative control is blank cell supernatant, i.e., the supernatant of directly cultured cells that have not been transfected with plasmids.

[0088] The positive control was the immune serum of mice.

[0089] Example 2: Specific detection of monoclonal antibodies against E2

[0090] The supernatant of the cell clones in Example 1 was subjected to antigen-specific detection using ELISA.

[0091] ① Antigen coating: Dilute E2-BSA to 2µg / ml, then add 100μl / well to an empty microplate, place the microplate in a 4°C refrigerator and incubate overnight;

[0092] ② Blocking: Wash the plate 5 times with a plate washer, dry it, add 150 μl of blocking solution to each well of the microplate, place it in a 37℃ incubator and block for 1 hour; wash the plate 5 times with a plate washer and dry it.

[0093] ③ Incubation with primary antibody: Add 100 μl of culture supernatant from the 96-well plate to each well of the microplate and incubate at 37°C for 1 hour; wash the plate 5 times with a plate washer and blot dry.

[0094] ④ Incubation with secondary antibody: Dilute HRP-labeled goat anti-mouse secondary antibody at a ratio of 1:10000, then add 100 μl to each well of the microplate and incubate at 37°C for 30 min; wash the plate 5 times with a plate washer and blot dry.

[0095] ⑤ Color development: Add 100 μl of color development solution to each well of the microplate, and react for 15 min at 37°C.

[0096] ⑥ Termination and reading: Add stop solution, volume 100 μl / well; Detection wavelength setting: detection wavelength 450 nm, reference wavelength 620 nm, the detection results are shown in Table 4.

[0097] Table 4. Specific detection results

[0098]

[0099] ⑦ Data Analysis: By comparing the signal strength, 10 clones with stronger response signals were selected (i.e., E2 mAb1, E2mAb2, E2 mAb3, E2 mAb4, E2 mAb5, E2 mAb6, E2 mAb7, E2 mAb8, E2 mAb9 and E2 mAb10, clone numbers 4M1B4, 3M6B7, 3M1B2, 3M1E3, 3M6B12, 3M1E8, 3M6B11, 3M5F10, 3M6D11 and 4M1G7, respectively) for subsequent amplification, expression and purification.

[0100] Example 3: Obtaining the light and heavy chain gene sequences of a monoclonal antibody targeting E2

[0101] cDNA preparation:

[0102] RNA was extracted from the 10 B cell lines obtained in Example 1, and cDNA was obtained by reverse transcription using RNA as a template. The genes of the light and heavy chains of the antibody were then amplified by PCR.

[0103] Obtaining light and heavy chain genes:

[0104] Amplification was performed using mouse-specific primers. The amplified DNA sequences were cloned into vectors, transformed, plated, and clones were picked, plasmids were extracted, and sequenced to obtain the light and heavy chain gene sequences of 10 antibodies. Based on the obtained light and heavy chain gene sequences of each antibody, the amino acid sequences of the light and heavy chains of each antibody were deduced. Specifically, the heavy chain amino acid sequences of antibodies E2mAb1 to E2 mAb10 are shown in SEQ ID NO:5, 14, 23, 32, 41, 50, 59, 68, 77, and 86, respectively, and their light chain amino acid sequences are shown in SEQ ID NO:9, 18, 27, 36, 45, 54, 63, 72, 81, and 90, respectively.

[0105] Analysis of light and heavy chain variable region gene sequences:

[0106] Based on the sequencing results, VH and VL were analyzed, and the amino acid sequences of the light chain variable region and the heavy chain variable region were finally obtained.

[0107] The amino acid sequences of the heavy chain variable region and light chain variable region of 10 monoclonal antibodies are shown below, where the underlined regions are their CDR regions.

[0108] E2 mAb1:

[0109] VH (SEQ ID NO:4)

[0110] EVQLQHSGTVLARPGASVKMSCKAS GFTFSNYG MHWVKQRPGQGLEWVGV ISGADSFT SYNQKFKGKTKLTAVTSASTASMELSSLTNEDSAVYYC ARQGYDWAFAY WGQGTSVTVSS;

[0111] VL (SEQ ID NO:8)

[0112] DIVLTQSPASLAVSLGQRATLSCKAS QNISNN MNWYQQKPGQPPKLLIY YVS NLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQSHSWPHYT FGGGTELEIN;

[0113] E2 mAb2:

[0114] VH (SEQ ID NO:13)

[0115] EVQLVESGGDLVKPGGSLKLSCAAS GYSFTSYWMSWIRQTPDKRLEWVAT VYPGNSDT YCPDSVKGRFTISRDIAKNTLYLQMSSLKSEDTAMYYC SKGQLGPTIHYSAMDY WGQGTLVTVSA:

[0116] VL (SEQ ID NO:17)

[0117] DIVLAQSPATLSVTPTNSVSLSCRAS QSVDYDGDSY LHWYQQKSHESPRLLIK AAS QSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFC QQSNEDPPT FGGGTKLEIK:

[0118] E2 mAb3:

[0119] VH (SEQ ID NO:22)

[0120] QVQLQQSGAELMCPGASVCISCEAT GYIFTDYE IEWVNQRPGHGFEWIGE IDPETGGT NYNDKFKGKATFTADTSNSNTAMQLSSLTSEDSAVYYC TRWVYLYSMDY WGQGTLVTVSA:

[0121] VL (SEQ ID NO:26)

[0122] DIQMTQSPASLSASSVGETVTIRCRAS SSVSSIY LVWYQQKQGKSPQLLVY STS NLASGVPARFSGSGSGTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QQYSGYPLT FGSGTCLEARS;

[0123] E2 mAb4:

[0124] VH (SEQ ID NO:31)

[0125] QGQLQQSGAELVRPGASVTLSCKAS GYTFSTYW MHWVKQTPVHGLEWIGA ILPGSGRT AYNQKFKGKATLTADKSSSTAYMEFRLTYEDSAVYC ARGLYRFEFAY WGQGTSVTVSS:

[0126] VL (SEQ ID NO:35)

[0127] ENVLTQSPAIMSASPGEKVTMTCRVS ENINSY LHWYQQKSGASPKLWIY NAK NLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYC QHHYGTPFT FGAGTKLELK;

[0128] E2 mAb5:

[0129] VH (SEQ ID NO:40)

[0130] EVMLVESGGGLVKPGGSLKLSCGAS GYTFSTYW MSWVRQTPEKRLEWVAT ILPGNGRT YYRDSVQGRFTVSRDNAKNTLYLQKSSLRSGDTAIYYC ARGLYRFEFAF WGQGTSVTVSS;

[0131] VL (SEQ ID NO:44)

[0132] DIVLTQSPATLSVTPGDSVSLSCRAS ENIHNY LHWFQQKSHESPRLLIK NAK QSISGIPSRISGSGSGTDFTLSINSVETEDFGMYFC QHHYGTPFT FGTGTKLELK;

[0133] E2 mAb6:

[0134] VH (SEQ ID NO:49)

[0135] DVPLVESGGGLVQPGGSRKLSCAAS GYTFSTYW MHWVRQAPEKGLEWVAY ILPGSDST YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYC ARGLYRFEFAY WGQGTTLTVSS;

[0136] VL (SEQ ID NO:53)

[0137] DIKMTQSPSSMFASLGERVAITCRAS ENINSY LSWFQQKPGKSPKTLIY NAKRLVDGVPSRFSGSGSGQDYSLTISSLEEDGIYYC QHHYGTPFT FGGGTKLEIK:

[0138] E2 mAb7:

[0139] VH (SEQ ID NO:58)

[0140] QVQLQQSGAELMCPGASVCISCEAT GFTFSSFG IEWVNQRPGHGLEWIGE ISSGSSSI NYNDKFKGQATFTADTSNSNTAMQLSSLTSEDSAVYYC ARDRYDEGDFDY WGQGTLVTVSA:

[0141] VL(SEQ ID NO:62)

[0142] DIQMTQSPASLSASSVGETVTIRCRAS QDINTY LVWYQQKQGKSPQLLVY RAN TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC LQYDEFPWT FGSGTKLEIK;

[0143] E2 mAb8:

[0144] VH (SEQ ID NO:67)

[0145] QVQLQQSGAELMCPGASVCISCEAT GLTFNNYA IEWVNQRPGHGLEWIGE ISSGGTYT NYNEKFKGKATFTADTSNSNTAMQLSSLTSEDSAVYC ARHHYDFAMDS WGQGTLVTVSA:

[0146] VL(SEQ ID NO:71)

[0147] DIQMTQSPASLSASSVGETVTIRCRAS QNISNN LVWYQQKQGKSPQLLVY YAS TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QQSNSWPQLT FGSGTKLEIK:

[0148] E2 mAb9:

[0149] VH (SEQ ID NO:76)

[0150] QVQLQQSGADLLKPGASVKISCKAT GFTFSSFG IEWVKQRPGHGLEWIGE INSGSSTI KYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYC ARDRYDEGDFDY WGAGTTVTVSS;

[0151] VL (SEQ ID NO:80)

[0152] DILMTQSPSSMSVSLGDTVNITCHAS QDINSY IGWLQQKPGKSFKGLIY RAN NLEDGVPSRFSGSGSAADYSLTISSLESEDFADYYC LQYDEFPWT FGGGTKLEIK;

[0153] E2 mAb10:

[0154] VH (SEQ ID NO:85)

[0155] DVQLVESGGGLVQPGGSRKLSCAAS GYTFSSYW MHWVRQAPEKGVEWVAY ILPGSGST YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYC ARFPLYYGSSHWYFDV WGQGTTLTVSS;

[0156] VL (SEQ ID NO:89)

[0157] DIKMTQSPSSMFAFLGESVSITCKAS QGISSN LSWFQQKPGKSPKTLIY HGT RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC VQYAQFPYT FGGGTKLEIK.

[0158] Example 4: Expression and purification of monoclonal antibodies against E2

[0159] (1) Culture N293 suspension cells until the density reaches (1-3) × 10⁻⁶. 6 Cell transfection is performed when the cell count is 100 cells / ml and the viability is >80-90%.

[0160] (2) The light and heavy chain genes of the 10 monoclonal antibodies E2 mAb1 to E2 mAb10 obtained in Example 2 were synthesized, and the synthesized light and heavy chain genes were constructed into pcDNA3.4 plasmids respectively. Then, the recombinant plasmids containing the antibody light and heavy chain genes were transfected into N293 cells. The transfection conditions were: the mass ratio of PEI / plasmid was 3:1, and the transfection ratio was 2 μg plasmid / 1 ml N293 cells.

[0161] (3) 5-7 days after transfection, cell supernatant was collected and affinity purified with Protein A resin to obtain high-purity monoclonal antibody. The elution buffer was replaced with PBS by dialysis and the antibody concentration was determined using Nanodrop.

[0162] Next, the purity of the antibody was detected by SDS-PAGE, and the specific method is as follows:

[0163] SDS-PAGE testing:

[0164] Take 2 μg of the antibody to be tested and add an appropriate amount of SDS-PAGE protein loading buffer to make a total volume of 20 μl. At the same time, prepare reducing SDS-PAGE samples and non-reducing SDS-PAGE samples, and then load them. First, electrophoresis at 80V for 30 min, and then electrophoresis at 120V until the bands are clearly separated. Remove the PAGE gel after electrophoresis, stain it with Coomassie brilliant blue, remove the staining solution after 15 min, rinse it with water, and then add destaining solution to destain until clear bands are seen.

[0165] The results are as follows Figure 1 As shown, Figure 1 The results show that under reducing conditions, all 10 monoclonal antibodies exhibited two electrophoretic bands, and the molecular weights of these two bands corresponded to the sizes of their respective light and heavy chains. Under non-reducing conditions, all 10 monoclonal antibodies exhibited a single band, and the molecular weight of this band corresponded to the size of their respective intact antibodies. These results indicate that the expression of these 10 monoclonal antibodies was accurate.

[0166] In addition, all electrophoretic bands had clear edges and no extraneous bands, indicating that the monoclonal antibody obtained by the above expression and purification steps had high purity.

[0167] Example 5: Detection of antigen-binding activity and affinity of monoclonal antibodies against E2

[0168] In this embodiment, the antigen-binding activity and affinity of the 10 monoclonal antibodies E2 mAb1 to E2 mAb10 expressed and purified in Example 3 were detected.

[0169] Antigen binding activity and affinity assay procedure:

[0170] ① Antigen coating: Dilute the antigen (i.e., the aforementioned coupled protein E2-BSA) to an appropriate concentration, and then add it to an empty ELISA plate, 100 μl / well; place the ELISA plate in a 4°C refrigerator and incubate overnight;

[0171] ② Blocking: Wash the plate 5 times with a plate washer, dry it, add 150 μl of blocking solution to each well of the ELISA plate, and place it in a 37℃ incubator for 1 hour; wash the plate 5 times with a plate washer and dry it.

[0172] ③ Incubation with primary antibody: Dilute the antibody to be tested to a suitable concentration gradient with 1×PBS; add 100 μl of the diluted antibody to each well of the ELISA plate; reaction time: 37℃ / 1h; wash the plate 5 times with a plate washer and dry.

[0173] ④ Incubate the secondary antibody: Dilute the enzyme-labeled secondary antibody at an appropriate ratio, then add 100 μl to each well of the microplate. Reaction time: 37℃ / 30 min; wash the plate 5 times with a plate washer and blot dry.

[0174] ⑤ Color development: Add 100 μl of color development solution to each well of the microplate, and react for 15 min at 37°C.

[0175] ⑥ Termination and reading: Add stop solution, 100 μl / well; set detection wavelength: detection wavelength 450 nm, reference wavelength 620 nm, and export the detection results to an Excel spreadsheet.

[0176] ⑦ Data Analysis: Use software to perform data analysis and graphing, fit EC50, and derive its KD.

[0177] The results of the binding activity and affinity tests of these 10 monoclonal antibodies to the antigen are as follows: Figure 2 And as shown in Table 5 below.

[0178] Table 5

[0179]

[0180] Figure 2 As shown in Table 5, the EC50 of these 10 monoclonal antibodies bound to the antigen all reached the ng / ml level, and their KD values ​​were all above 10. -10 M~10 -11 M level.

[0181] As mentioned earlier, for IgG antibodies, it is generally accepted in the art that when the KD value for binding to the antigen reaches 10... -9 At M, it is considered to have high affinity, and the KD values ​​of the 10 monoclonal antibodies of this invention all reached 10. -10 M-10-11 The number of M is 10-100 times that defined above, which leads to the conclusion that the monoclonal antibody of the present invention has extremely strong antigen-binding activity and affinity.

[0182] Example 6: Preparation of a double-antibody sandwich chemiluminescent reagent based on anti-E2 monoclonal antibody

[0183] In this embodiment, E2 mAb1 and E2 mAb2, and E2 mAb3 and E2mAb4, expressed and purified in Example 4, were used as paired antibodies to prepare two chemiluminescent reagents for detecting E2 based on the double-antibody sandwich method. The acridil ester chemiluminescent system was used in this experiment. The preparation method of the detection reagents is as follows:

[0184] (1) Preparation of magnetic bead coating of anti-E2 monoclonal antibody

[0185] ① Place the magnetic bead concentrate solution on a roller mixer and mix at room temperature. Then, pipette 1 ml of the magnetic bead concentrate solution into a centrifuge tube (10 mg / ml).

[0186] ② Place the centrifuge tube against the magnet surface and magnetically attract it for 5 minutes to remove the internal solution. Add 1 ml of 0.1 M MES buffer (pH 5.0) and mix with a vortex mixer. Continue mixing and reacting at room temperature for 30 minutes.

[0187] ③ Add 0.2 ml of antibody (E2 mAb1, E2 mAb3) (concentration 1 mg / ml) and mix at room temperature for 3 h. Place the centrifuge tube against the magnet for 5 min to remove the inner solution, add 1 ml of 0.1 M MES buffer (pH 5.0) and mix with a vortex mixer, then add 1 ml of 1% BSA aqueous solution and mix at room temperature for 6 h.

[0188] ④ Repeat step 2 twice to obtain magnetic beads coated with anti-E2 monoclonal antibody at a concentration of 10 mg / ml.

[0189] (2) Preparation of acridinium ester-labeled anti-E2 monoclonal antibody

[0190] ① Weigh an appropriate amount of acridine ester and dissolve it in anhydrous DMSO to a concentration of 2 mg / ml. Dilute the anti-E2 monoclonal antibodies (E2 mAb2, E2 mAb4) to 2 mg / ml using 35 mM sodium bicarbonate and 15 mM anhydrous sodium carbonate (pH 9.0). Add 2 mg / ml acridine ester to the 2 mg / ml antibody solution (molar ratio of acridine ester to antibody is 15:1), mix for 30 seconds, and allow to react at room temperature in the dark for 1 hour.

[0191] ② Add an equal volume of 1M / L glycine buffer and mix for 30 seconds, then mix and react at room temperature in the dark for 0.5 hours.

[0192] ③ Add 200 μl of 35 mM sodium bicarbonate and 15 mM anhydrous sodium carbonate (pH 9.0), and purify it using an ultrafiltration tube (12000 rpm, room temperature, 20 min). Repeat three times.

[0193] ④ Collect and measure the final product, add an equal volume of glycerol, mix thoroughly, and store at -20℃;

[0194] (3) Preparation of reagents

[0195] ① Preparation of R1: Dilute the acridinium ester-labeled E2 mAb2 to 0.5 ug / ml with 10 mM PBS, and denote it as reagent component R1.

[0196] ② Preparation of R2: Dilute the acridinium ester-labeled E2 mAb4 to 0.5 ug / ml with 10 mM PBS, and denote it as reagent component R2.

[0197] (4) Preparation of calibrators

[0198] The pure E2 solution was diluted to five different concentrations using 10 mM PBS, and these concentrations, along with the 0 concentration dilution, were designated S1-S6. The signal values ​​were measured using prepared reagents R1 and R2. The concentrations and signal values ​​are shown in Tables 6-1 and 6-2, and the fitted curves are shown below. Figure 3 A in Figure 3 As shown in B in the diagram.

[0199] Table 6-1

[0200]

[0201] Table 6-2

[0202]

[0203] Example 7: Detection performance of acridinium ester chemiluminescent reagent based on anti-E2 monoclonal antibody sandwich method

[0204] In this embodiment, the detection performance of the double-antibody sandwich acridine ester chemiluminescent reagent for anti-E2 monoclonal antibody prepared in Example 4 was tested and compared with the detection performance of the traditional Roche detection reagent.

[0205] Specifically, 43 serum samples were tested using the two chemiluminescent reagents prepared in Example 6 and the chemiluminescent reagent prepared from the monoclonal antibody in prior art 1, and the detection values ​​were read and substituted into... Figure 3In the calibration curve, the E2 concentration in samples of each dilution was obtained. For the Roche test kit, the operation and testing were performed according to its instructions to obtain E2 concentration data, as shown in Tables 7-1, 7-2, and 7-3. Then, the E2 concentration data detected by the three reagents were compared with the E2 concentration data detected by the Roche reagent (as a standard) to create a clinical relevance (i.e., the concordance rate with Roche test results) curve.

[0206] The results are as follows Figure 4 As shown; where, Figure 4 A in Figure 4 B and Figure 4 The graph shows the clinical relevance (i.e., the concordance rate with Roche detection results) of the chemiluminescent reagents prepared from monoclonal antibodies in chemiluminescent reagent 1, reagent 2, and prior art 1, respectively. It illustrates the clinical relevance (RC) of the two reagents of this invention. 2 The clinical relevance (R value) of the chemiluminescent reagent prepared from monoclonal antibodies in prior art 1 is all >0.99. 2 (Value) < 0.99.

[0207] The above results show that, compared with the first-generation E2 antibody in prior art 1, the E2 chemiluminescent detection reagent prepared by the paired antibody of the present invention has better low-end sensitivity, that is, the results are more accurate when detecting low values, and therefore the concordance rate with Roche detection results is very high (R 2 (Above 0.99) indicates good clinical relevance.

[0208] Table 7-1

[0209]

[0210] Table 7-2

[0211]

[0212] Table 7-3

[0213]

[0214] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-estradiol monoclonal antibody or its antigen-binding fragment, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (4) Heavy chain variable region having amino acid sequences as shown in SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively, HCDR1, HCDR2 and HCDR3; and light chain variable region having amino acid sequences as shown in SEQ ID NO:33, NAK and SEQ ID NO:34, respectively, LCDR1, LCDR2 and LCDR3.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (4) Heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO:31; and light chain variable region, the amino acid sequence of which is shown in SEQ ID NO:

35.

3. The monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises: (4) Heavy chain, the amino acid sequence of which is shown in SEQ ID NO:32; and light chain, the amino acid sequence of which is shown in SEQ ID NO:

36.

4. The monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3, characterized in that, The antigen-binding fragments are selected from Fab, Fab', F(ab')2, Fd, and Fv.

5. A polynucleotide encoding a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-4.

6. A nucleic acid construct comprising the polynucleotide as described in claim 5, and at least one expression regulatory element operatively linked to said polynucleotide.

7. A recombinant vector comprising the polynucleotide of claim 5 or the nucleic acid construct of claim 6.

8. A transformed host cell comprising the polynucleotide of claim 5, the nucleic acid construct of claim 6, or the recombinant vector of claim 7.

9. The use of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the polynucleotide as described in claim 5, the nucleic acid construct as described in claim 6, the recombinant vector as described in claim 7, and / or the transformed host cell as described in claim 8 in the preparation of a detection reagent or kit for detecting estradiol.

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