Anti-estradiol monoclonal antibody or antigen binding fragment and application thereof

Abstract

The invention relates to an estradiol-resistant monoclonal antibody or an antigen binding fragment thereof and application. The invention provides an estradiol-resistant monoclonal antibody or an antigen-binding fragment thereof. The monoclonal antibody or the antigen-binding fragment thereof can be bound with estradiol (E2) with high specificity and high affinity; the invention also develops a high-performance pairing antibody suitable for double-antibody sandwich method immunodetection, so that the limitation that a traditional competition method detection reagent is low in sensitivity and poor in clinical correlation is broken through, double-antibody sandwich method immunodetection of E2 is realized, and the detection accuracy and the low-end sensitivity are greatly improved compared with those of CN 117903305.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibody preparation and immunological detection, and in particular to anti-estradiol monoclonal antibody or antigen-binding fragment thereof and application thereof. Background Art

[0002] Estradiol (E2) is a steroid hormone that is mainly secreted by ovarian follicles, corpus luteum and placenta during pregnancy, with a molecular weight of about 272Da. E2 is the hormone with the highest content and strongest biological activity among estrogens. It is crucial to the development of the female reproductive system and the appearance of secondary sexual characteristics during the childbearing period. It also affects bones, cardiovascular and cerebrovascular, brain and metabolism. Estrone (E1), estradiol (E2) and estriol (E3) are the main forms of estrogen in the body. E1 and E2 can be converted into each other. E3 is a metabolite of E2. The three are extremely similar in structure, so the antibody specificity requirements are relatively high. Common methods for detecting E2 include radioimmunoassay, enzyme-linked immunosorbent assay, colloidal gold method, chemiluminescence immunoassay and liquid chromatography-tandem mass spectrometry. Radioimmunoassay has been gradually phased out due to the risk of radioactive contamination. The sensitivity and specificity of enzyme-linked immunosorbent assay and colloidal gold method are limited. The current mainstream clinical method for detecting E2 is chemiluminescence immunoassay, which has higher sensitivity and lower cost than mass spectrometry. The sensitivity and specificity of the first-generation E2 sandwich antibody (CN 117903305) disclosed in prior art 1 have been greatly improved compared to the competitive method, but there is still room for improvement in low-end sensitivity. Summary of the invention

[0003] Purpose of the Invention In view of the problems or needs existing in the prior art, the purpose of the present invention is to provide a highly specific and high-affinity monoclonal antibody or an antigen-binding fragment thereof for estradiol (E2 in English), and to develop a high-performance paired antibody suitable for double antibody sandwich immunological detection.

[0004] Technical Solution

[0005] In order to achieve the above objectives, the present invention has obtained, through a large number of screenings, highly specific and high affinity monoclonal antibodies or antigen-binding fragments thereof against estradiol (E2 for short), their related nucleic acids and cell products, their preparation methods and applications, and immunoassay reagents based thereon, thereby providing a basis for highly specific and highly sensitive immunoassays of E2.

[0006] Specifically, the present invention provides the following technical solutions: In a first aspect, the present invention provides an anti-estradiol monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises: (1) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 6, YVS and SEQ ID NO: 7, respectively; or, (2) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 15, GAT and SEQ ID NO: 16, respectively; or, (3) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 24, STS and SEQ ID NO: 25, respectively; or, (4) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 33, NAK and SEQ ID NO: 34, respectively; or, (5) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 42, NAK and SEQ ID NO: 43, respectively; or, (6) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 51, NAK and SEQ ID NO: 52, respectively; or, (7) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, respectively; or, (8) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 69, YAS and SEQ ID NO: 70, respectively; or, (9) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 79, respectively; or, (10) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 87, HGT and SEQ ID NO: 88, respectively.

[0007] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:8; or, (2) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 17; or, (3) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 22; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 26; or, (4) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 31; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 35; or, (5) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:40; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:44; or, (6) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:49; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:53; or, (7) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 62; or, (8) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 67; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 71; or, (9) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 76; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 80; or, (10) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 85; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 89.

[0008] In addition, the monoclonal antibody or antigen-binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the following: a constant region of an IgG, IgA or IgM antibody.

[0009] Further preferably, the monoclonal antibody or antigen-binding fragment thereof comprises: (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, (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, (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, (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, (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, (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, (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, (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, (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, (10) a heavy chain having the amino acid sequence shown in SEQ ID NO: 86; and a light chain having the amino acid sequence shown in SEQ ID NO: 90.

[0010] In the above first aspect, the monoclonal antibodies defined in items (1) to (10) are hereinafter respectively named as antibodies 1 to 10.

[0011] In some feasible embodiments, the antigen-binding fragment of the monoclonal antibody is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody, human antibody, chimeric antibody, bispecific or multispecific antibody.

[0012] In a second aspect, the present invention provides a polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect. The polynucleotide is not limited by the method of its production and can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0013] In a third aspect, the present invention provides a nucleic acid construct comprising the polynucleotide as described in the second aspect above, and at least one expression control element operably linked to the polynucleotide.

[0014] In a fourth aspect, the present invention provides a recombinant vector comprising the polynucleotide as described in the second aspect above, or the nucleic acid construct as described in the third aspect above.

[0015] The vector of the present invention may be a cloning vector or an expression vector, for example, it may be a plasmid, a cosmid, a phage, etc.

[0016] In some preferred embodiments, the recombinant vector is a recombinant expression vector, preferably a eukaryotic expression vector.

[0017] In a fifth aspect, the present invention provides a transformed host cell, wherein the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, or the recombinant vector as described in the fourth aspect is transformed; The host cell includes, but is 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 cell can also be a cell line, such as a 293T cell line.

[0018] Preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell.

[0019] In a sixth aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the nucleic acid construct as described in the third aspect, the recombinant vector as described in the fourth aspect and / or the transformed host cell as described in the fifth aspect in the preparation of a detection reagent or kit for detecting estradiol.

[0020] In a feasible embodiment, the sample is a biological sample of a subject, preferably a serum or plasma sample of a subject.

[0021] In the seventh aspect, the present invention provides a method for detecting the presence or level of estradiol in a sample, the method comprising using the monoclonal antibody or its antigen-binding fragment as described in the first aspect above, the polynucleotide as described in the second aspect above, the nucleic acid construct as described in the third aspect above, the expression vector as described in the fourth aspect above, the transformed host cell as described in the fifth aspect above and / or the detection reagent as described in the eighth aspect above.

[0022] In a feasible embodiment, the sample includes but is not limited to serum, plasma, etc. from a subject.

[0023] The method can be used for diagnostic purposes (eg, the sample is a sample from a patient), or for non-diagnostic purposes (eg, the sample is a cell sample rather than a sample from a patient).

[0024] The general method of using monoclonal antibodies or their antigen binding fragments to detect the presence of target antigens in samples or their levels is well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method and similar detection methods.

[0025] Beneficial Effects

[0026] The anti-estradiol monoclonal antibody of the present invention can bind to estradiol with high affinity and high specificity (the EC50 of its binding activity is at the ng / ml level, and the KD is at 10 -10 M~10 -11 M), achieving high accuracy and high sensitivity detection of estradiol; and the present invention also develops high-performance paired antibodies suitable for double antibody sandwich immunological detection, thereby breaking through the limitations of low sensitivity and poor clinical relevance of traditional competitive detection reagents, and realizing double antibody sandwich immunological detection of estradiol. Compared with the first-generation sandwich antibody of prior art 1, the antibody of the present invention has greater advantages in low-end sensitivity, and the results are more accurate when detecting samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] One or more embodiments are exemplarily described by the pictures in the accompanying drawings, and these exemplary descriptions do not constitute limitations on the embodiments. The special word "exemplary" here means "used as an example, embodiment or illustrative". Any embodiment described as "exemplary" here is not necessarily interpreted as being superior or better than other embodiments.

[0028] Figure 1 The figure is a gel diagram of SDS-PAGE detection of 10 anti-E2 monoclonal antibodies expressed and purified by the present invention; wherein, M represents the electrophoresis band of the molecular weight Marker, lane 1 shows the electrophoresis bands of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoresis bands of each monoclonal antibody under non-reducing conditions; wherein, 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 E2mAb6, 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.

[0029] Figure 2 The figure is a graph showing the binding activity test results of 10 anti-E2 monoclonal antibodies expressed and purified by the present invention and E2 coupling protein.

[0030] Figure 3 The standard curve measured by the chemiluminescent reagent prepared by the present invention, the horizontal axis is the dilution concentration of the standard product, and the vertical axis is the signal value read by the chemiluminescent instrument with different concentrations of the standard product. Among them, A is based on E2 mAb1 + E2 mAb2 as paired antibodies, and B is based on E2 mAb3 + E2 mAb4 as paired antibodies.

[0031] Figure 4 The figures are result graphs of the correlation between the two chemiluminescent reagents based on the two paired antibodies of the present invention and the Roche detection reagent for clinical samples; wherein A is the result graph of the chemiluminescent reagent 1 based on the paired antibodies of E2 mAb1+E2 mAb2, B is the result graph of the chemiluminescent reagent 2 based on the paired antibodies of E2 mAb3+E2 mAb4, and C is the result graph of the chemiluminescent reagent prepared from the monoclonal antibodies in the prior art 1. DETAILED DESCRIPTION

[0032] Unless explicitly stated otherwise, throughout the specification and claims, the term “comprise” or variations such as “include” or “comprising” will be understood to include the stated elements or components but not to exclude other elements or components.

[0033] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0034] In order to make the present invention more easily understood, some technical terms are specifically defined as follows. Unless otherwise clearly defined in other parts of this document, the technical terms used herein have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs.

[0035] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0036] The term "and / or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0037] Example 1: Preparation of anti-E2 monoclonal antibodies (1) Animal immunization: The coupled protein E2-BSA (purchased from Nanjing Mingyan Biotechnology Co., Ltd., catalog number MY-E2-001) was used as an antigen, and after being fully emulsified with an equal amount of Freund's complete adjuvant, mice (species: Balb / c, female, 6-8 weeks old) were immunized by intraperitoneal injection. The booster immunization was performed at intervals of two weeks for a total of 3 times. 5-8 days after the last booster immunization, a trace amount of tail blood was collected, and the serum titer was detected by ELISA. When the serum titer reached the standard of >128K, the spleen single cell suspension was prepared. The specific detection information is shown in Tables 1 and 2.

[0038] Table 1. Experimental information

[0039] Table 2. Serum titer data detected by ELIAS method

[0040] Mice with a serum titer of 512K were selected to prepare spleen single cell suspension.

[0041] (2) Preparation of spleen single cell suspension: The mice were killed by cervical dislocation. A small incision was made in the middle of the left ventral side of the mouse, the skin was torn open, the abdominal wall was exposed, the peritoneum was lifted up on the lower side of the spleen, and the spleen was exposed after being cut open. The spleen was lifted with forceps, and the connective tissue under the spleen was separated with ophthalmic scissors, and the spleen was removed. The spleen was placed on a cell filter, and the spleen was gently crushed with a syringe needle to obtain a cell suspension. Another 15mL sterile test tube was taken, and the cell separation solution Ficoll was first added. The cell suspension just prepared was slowly added to the test tube. The volume ratio of the separation solution to the cell suspension was 1:2. Place it in a centrifuge at 2000 rpm and centrifuge at room temperature for 20 minutes. After centrifugation, the test tube was taken out, and different layers could be seen. The upper non-cellular layer was aspirated and discarded, and the single nuclear layer was aspirated in another 15mL sterile test tube. Centrifuge at 1500 rpm at room temperature for 10 minutes, carefully remove the supernatant and resuspend the cells.

[0042] (3) Obtaining single B cells: The prepared spleen single cell suspension is used to label the antigen and perform cell screening using a single cell screening platform to obtain antigen-specific B cells.

[0043] Through the above steps, a total of 123 monoclonal antibodies against folic acid and its binding protein complex were obtained, and they were all IgG antibodies after identification, and their names are shown in Table 3A and Table 3B. In the following examples, the light and heavy chain gene sequences of the monoclonal antibodies were detected and expressed, and the transfected cell supernatant was placed in a 96-well plate according to the layout of Table 3A and Table 3B for binding activity detection.

[0044] Table 3A. 88 antibody supernatant clone numbers

[0045] Table 3B. 35 antibody supernatant clone numbers

[0046] The negative control is the blank cell supernatant, that is, the supernatant of cells directly cultured without transfection of plasmid. The positive control was mouse immune serum Example 2: Specific detection of monoclonal antibodies against E2 The supernatant of the cell clones in Example 1 was tested for antigen specificity using ELISA: ① Antigen coating: dilute E2-BSA to 2µg / ml, then add 100μl / well to an empty ELISA plate, place the ELISA plate in a 4℃ refrigerator, and incubate overnight; ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry; ③ Incubate with primary antibody: add the culture supernatant of 96-well plate to the wells of ELISA plate, 100 μl per well, incubate at 37°C for 1 hour; wash the plate 5 times with a plate washer and dry; ④ Incubate with secondary antibody: dilute HRP-labeled goat anti-mouse secondary antibody at 1:10000, then add to the ELISA plate, 100 μl per well, incubate at 37°C for 30 min; wash the plate 5 times with a plate washer and dry; ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37℃ / 15min; ⑥ Stop and read: add stop solution, volume 100 μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, the detection results are shown in Table 4.

[0047] Table 4. Specificity test results

[0048] ⑦Data analysis: By comparing the signal strengths, 10 clones with stronger reaction signals were finally 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 are 4M1B4, 3M6B7, 3M1B2, 3M1E3, 3M6B12, 3M1E8, 3M6B11, 3M5F10, 3M6D11 and 4M1G7, respectively) for subsequent amplification, expression and purification.

[0049] Example 3: Obtaining the light and heavy chain gene sequences of monoclonal antibodies against E2 cDNA preparation: RNA was extracted from the 10 B cells obtained by sorting in Example 1, and reverse transcription was performed using RNA as a template to obtain cDNA, and the genes of the light and heavy chains of the antibody were amplified by PCR.

[0050] Acquisition of light and heavy chain genes: Amplification was performed using mouse-specific primers, and the amplified DNA sequences were cloned into vectors, respectively, and then transformed and plated. Clones were picked, plasmids were extracted and sequenced to obtain the light and heavy chain gene sequences of 10 antibodies; based on the measured 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 NOs: 5, 14, 23, 32, 41, 50, 59, 68, 77, and 86, respectively, and the light chain amino acid sequences are shown in SEQ ID NOs: 9, 18, 27, 36, 45, 54, 63, 72, 81, and 90, respectively.

[0051] Analysis of light and heavy chain variable region gene sequences: VH and VL were analyzed according to the sequencing results, and finally the amino acid sequences of the light chain variable region and the heavy chain variable region were obtained.

[0052] The amino acid sequences of the heavy chain variable regions and light chain variable regions of the 10 monoclonal antibodies are shown below, wherein the CDR regions are underlined.

[0053] E2 mAb1: VH (SEQ ID NO:4) EVQLQHSGTVLARPGASVKMSCKAS GFTFSNYG MHWVKQRPGQGLEWVGV ISGADSFT SYNQKFKGKTKLTAVTSASTASMELSSLTNEDSAVYYC ARQGYDWAFAY WGQGTSVTVSS; VL (SEQ ID NO:8) DIVLTQSPASLAVSLGQRATLSCKAS QNISNN MNWYQQKPGQPPKLLIY YVS NLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQSHSWPHYT FGGGTELEIN; E2 mAb2: VH (SEQ ID NO:13) EVQLVESGGDLVKPGGSLKLSCAAS GYSFTSYW MSWIRQTPDKRLEWVAT VYPGNSDT YCPDSVKGRFTISRDIAKNTLYLQMSSLKSEDTAMYYC SKGQLGPTIHYSAMDY WGQGTLVTVSA; VL (SEQ ID NO:17) DIVLAQSPATLSVTPTNSVSLSCRAS QSVDYDGDSY LHWYQQKSHESPRLLIK AAS QSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFC QQSNEDPPT FGGGTKLEIK: E2 mAb3: VH (SEQ ID NO:22) QVQLQQSGAELMCPGASVCISCEAT GYIFTDYE IEWVNQRPGHGFEWIGE IDPETGGT NYNDKFKGKATFTADTSNSNTAMQLSSLTSEDSAVYYC TRWVYLYSMDY WGQGTLVTVSA: VL (SEQ ID NO:26) DIQMTQSPASLSASSVGETVTIRCRAS SSVSSIY LVWYQQKQGKSPQLLVY STS NLASGVPARFSGSGSGTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QQYSGYPLT FGSGTCLEARS; E2 mAb4: VH (SEQ ID NO:31) QGQLQQSGAELVRPGASVTLSCKAS GYTFSTYW MHWVKQTPVHGLEWIGA ILPGSGRT AYNQKFKGKATLTADKSSSTAYMEFRLTYEDSAVYC ARGLYRFEFAY WGQGTSVTVSS: VL(SEQ ID NO:35) ENVLTQSPAIMSASPGEKVTMTCRVS ENINSY LHWYQQKSGASPKLWIY NAK NLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYC QHHYGTPFT FGAGTKLELK; E2 mAb5: VH (SEQ ID NO:40) EVMLVESGGGLVKPGGSLKLSCGAS GYTFSTYW MSWVRQTPERLEWVAT ILPGNGRT YYRDSVQGRFTVSRDNAKNTLYLQKSSLRSGDTAIYYC ARGLYRFEFAF WGQGTSVTVSS: VL (SEQ ID NO:44) DIVLTQSPATLSVTPGDSVSLSCRAS ENIHNY LHWFQQKSHESPRLLIK NAK QSISGIPSRISGSGSGTDFTLSINSVETEDFGMYFC QHHYGTPFT FGTGTKLELK; E2 mAb6: VH (SEQ ID NO:49) DVPLVESGGGLVQPGGSRKLSCAAS GYTFSTYW MHWVRQAPEKGLEWWAY ILPGSDST YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYC ARGLYRFEFAY WGQGTTLTVSS: VL(SEQ ID NO:53) DIKMTQSPSSMFASLGERVAITCRAS ENINSY LSWFQQKPGKSPKTLIY NAK RLVDGVPSRFSGSGSGQDYSLTISSLEEDGIYYC QHHYGTPFT FGGGTKLEIK: E2 mAb7: VH (SEQ ID NO:58) QVQLQQSGAELMCPGASVCISCEAT GFTFSSFG IEWVNQRPGHGLEWIGE ISSGSSSI NYNDKFKGQATFTADTSNSNTAMQLSSLTSEDSAVYYC ARDRYDEGDFDY WGQGTLVTVSA: VL(SEQ ID NO:62) DIQMTQSPASLSASSVGETVTIRCRAS QDINTY LVWYQQKQGKSPQLLVY RAN TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC LQYDEFPWTFGSGTKLEIK; E2 mAb8: VH (SEQ ID NO:67) QVQLQQSGAELMCPGASVCISCEAT GLTFNNYA IEWVNQRPGHGLEWIGE ISSGGTYT NYNEKFKGKATFTADTSNSNTAMQLSSLTSEDSAVYC ARHHYDFAMDS WGQGTLVTVSA: VL(SEQ ID NO:71) DIQMTQSPASLSASSVGETVTIRCRAS QNISNN LVWYQQKQGKSPQLLVY YAS TLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QQSNSWPQLT FGSGTKLEIK; E2 mAb9: VH (SEQ ID NO:76) QVQLQQSGADLLKPGASVCKISCCAT GFTFSSFG IEWVKQRPGHGLEWIGE INSGSSTI KYNEKFKGKATFTADTSNSNTAMQLSSLTSEDSAVYC ARDRYDEGDFDY WGAGTTVTVSS: VL(SEQ ID NO:80) DILMTQSPSSMSVSLGDTVNITCHAS QDinsy IGWLQQKPGKSFKGLIY RAN NLEDGVPSRFSGSGSAADYSLTISSLESEDFADYYC LQYDEFPWT FGGGTKLEIK: E2 mAb10: VH (SEQ ID NO:85) DVQLVESGGGLVQPGGSRKLSCAAS GYTFSSYW MHWVRQAPEKGVEWWAY ILPGSGST YYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYC ARFPLYYGSSHWYFDV WGQGTTLTVSS: VL(SEQ ID NO:89) DIKMTQSPSSMFAFLGESVSITCKAS QGISN LSWFQQKPGKSPKTLIY HGT RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC VQYAQFPYT FGGGTKLEIK.

[0054] Example 4: Expression and purification of monoclonal antibodies against E2 (1) Cultivate N293 suspension cells until the density reaches (1-3)×10 6 When the cell count is 10 cells / ml and the survival rate is >80-90%, cell transfection is performed; (2) The light and heavy chain genes of the 10 monoclonal antibodies E2 mAb1 to E2 mAb10 measured in Example 2 were synthesized, and the synthesized light and heavy chain genes were respectively constructed into the pcDNA3.4 plasmid; then, the recombinant plasmid containing the antibody light and heavy chain genes was transfected into N293 cells, and 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; (3) 5-7 days after transfection, the cell supernatant was collected and affinity purified using Protein A resin to obtain high-purity monoclonal antibodies. The elution buffer was replaced with PBS by dialysis, and the antibody concentration was measured using Nanodrop; Next, the purity of the antibody was tested by SDS-PAGE as follows: SDS-PAGE detection: Take 2 μg of the antibody to be tested, add an appropriate amount of SDS-PAGE protein loading buffer to make the total volume 20 μl; prepare reducing SDS-PAGE samples and non-reducing SDS-PAGE samples at the same time, and then load them; first, electrophoresis at 80V for 30 minutes, and then electrophoresis at 120V until the bands are clearly separated; remove the PAGE gel after electrophoresis, stain with Coomassie Brilliant Blue, remove the staining solution after 15 minutes, rinse with clean water, and then add decolorizing solution for decolorization until clear bands are seen.

[0055] The results are as follows Figure 1 As shown, Figure 1 The results showed that under reducing conditions, all 10 monoclonal antibodies had two electrophoretic bands, and the corresponding molecular weights of the two electrophoretic bands were consistent with the sizes of their respective light and heavy chains; under non-reducing conditions, all 10 monoclonal antibodies had a single band, and the corresponding molecular weights of the electrophoretic bands were consistent with the sizes of their respective complete antibodies; these results indicated that the expression of these 10 monoclonal antibodies was accurate.

[0056] In addition, all electrophoresis bands had clear edges and no foreign bands, indicating that the monoclonal antibodies obtained by the above expression and purification steps had high purity.

[0057] Example 5: Antigen Binding Activity and Affinity Detection of Monoclonal Antibodies Against E2 In this example, the antigen binding activity and affinity of the 10 monoclonal antibodies E2 mAb1 to E2 mAb10 expressed and purified in Example 3 were tested respectively.

[0058] Antigen Binding Activity and Affinity Testing Procedure: ① Antigen coating: dilute the antigen (i.e., the aforementioned coupling protein E2-BSA) to an appropriate concentration, and then add it to an empty ELISA plate at a volume of 100 μl / well; place the ELISA plate in a 4°C refrigerator and incubate overnight; ② Blocking: Wash the plate 5 times with a plate washer, tap dry, add blocking solution to the ELISA plate, 150 μl per well, place in a 37°C incubator, block for 1 hour; wash the plate 5 times with a plate washer, tap dry; ③ Incubate the primary antibody: dilute the antibody to be tested to a suitable concentration gradient with 1×PBS; add the diluted antibody to be tested to the ELISA plate, 100 μl per well, reaction time: 37℃ / 1h; wash the plate 5 times with a plate washer and dry; ④ Incubate the secondary antibody: dilute the enzyme-labeled secondary antibody in an appropriate ratio, and then add it to the ELISA plate, 100 μl per well, reaction time: 37°C / 30 min; wash the plate 5 times with a plate washer and dry; ⑤ Color development: add the color development solution to the ELISA plate, 100 μl per well, reaction time: 37℃ / 15min; ⑥ Stop and read: add stop solution, volume 100μl / well; detection wavelength setting: detection wavelength 450nm, reference wavelength 620nm, export the detection results into Excel table.

[0059] ⑦Data analysis: Use software to analyze and plot data, fit EC50, and derive its KD.

[0060] The results of the binding activity and affinity test of these 10 monoclonal antibodies to antigens are as follows Figure 2 and as shown in Table 5 below.

[0061] Table 5

[0062] Figure 2 Table 5 shows that the EC50 of the 10 monoclonal antibodies binding to the antigen all reached the ng / ml level, and the KD values ​​were all above 10 -10 M~10 -11 M level.

[0063] As mentioned above, for IgG antibodies, it is generally believed in the art that when the KD value of binding to the antigen reaches 10 -9 M is considered to have high affinity, and the KD values ​​of the 10 monoclonal antibodies of the present invention for binding to the antigen all reached 10 -10 M-10 -11 M is 10-100 times the above-defined value, from which it can be concluded that the monoclonal antibody of the present invention has extremely strong antigen binding activity and affinity.

[0064] Example 6: Preparation of double antibody sandwich chemiluminescent reagent based on anti-E2 monoclonal antibody In this example, E2 mAb1 and E2 mAb2, E2 mAb3 and E2mAb4 expressed and purified in Example 4 were used as paired antibodies to prepare two chemiluminescent reagents for detecting E2 using the double antibody sandwich method. This experiment used an acridinium ester chemiluminescent system; the preparation method of the detection reagent is as follows: (1) Preparation of magnetic beads coated with anti-E2 monoclonal antibody ① Place the concentrated magnetic bead solution on a roller mixer and mix at room temperature. Pipette 1 ml of the concentrated magnetic bead solution into a centrifuge tube (10 mg / ml). ② Place the wall of the centrifuge tube close to the surface of the magnet for 5 minutes to remove the inner solution, add 1 ml of 0.1 M MES buffer (PH 5.0) and mix with a vortex mixer, and continue mixing at room temperature for 30 minutes; ③ Add 0.2 ml of antibody (E2 mAb1, E2 mAb3) (concentration of 1 mg / ml) and mix at room temperature for 3 hours. Place the centrifuge tube wall close to the surface of the magnet for 5 minutes to absorb the inner solution, add 1 ml of 0.1 M MES buffer (PH5.0) and mix with a vortex mixer, then add 1 ml of 1% BSA aqueous solution and mix at room temperature for 6 hours; ④ Repeat step 2 twice to obtain 10 mg / ml magnetic beads coated with anti-E2 monoclonal antibody.

[0065] (2) Preparation of acridinium ester-labeled anti-E2 monoclonal antibody ① Weigh an appropriate amount of acridinium ester and dissolve it in anhydrous DMSO to 2 mg / ml. Dilute anti-E2 monoclonal antibodies (E2 mAb2, E2 mAb4) to 2 mg / ml with 35 mM sodium bicarbonate and 15 mM anhydrous sodium carbonate (PH 9.0). Add 2 mg / ml acridinium ester to 2 mg / ml antibody solution (the molar ratio of acridinium ester to antibody is 15:1), mix for 30 seconds, and react at room temperature in the dark for 1 hour; ② Add an equal volume of 1M / L glycine buffer and mix for 30 seconds, then mix and react for 0.5 hours at room temperature in the dark; ③ Add 200 μl of 35 mM sodium bicarbonate and 15 mM anhydrous sodium carbonate (PH 9.0), purify it using an ultrafiltration tube (12000 rpm, room temperature, 20 min), and repeat three times; ④ Collect and measure the final product, add an equal volume of glycerol, mix thoroughly and store at -20℃; (3) Preparation of reagents ① Preparation of R1: Dilute the acridinium ester-labeled E2 mAb2 to 0.5 ug / ml with 10 mM PBS, which is recorded as reagent component R1.

[0066] ② Preparation of R2: Dilute the acridinium ester-labeled E2 mAb4 to 0.5 ug / ml with 10 mM PBS, which is recorded as reagent component R2.

[0067] (4) Preparation of calibrants The pure E2 was diluted to 5 different concentrations with 10mM PBS, and the 0 concentration dilutions were recorded as S1-S6. The signal values ​​were measured with the prepared R1 and R2 reagents. The concentrations and signal values ​​are shown in Table 6-1 and Table 6-2, and the fitting curve is shown in Figure 3 A in Figure 3 As shown in B.

[0068] Table 6-1 Table 6-2

[0069] Example 7: Detection performance of double antibody sandwich method acridinium ester chemiluminescent reagent based on anti-E2 monoclonal antibody In this example, the detection performance of the double antibody sandwich method acridinium ester chemiluminescent reagent of the anti-E2 monoclonal antibody prepared in Example 4 was tested and compared with the detection performance of the traditional Roche detection reagent.

[0070] Specifically, the two chemiluminescent reagents prepared in Example 6 and the chemiluminescent reagent prepared by the monoclonal antibody in the prior art 1 were tested on 43 serum samples, and the test values ​​were read and brought into Figure 3 In the calibration curve, the E2 concentration in the sample of each dilution was obtained; for the Roche test kit, the operation and detection were carried out according to the instructions to obtain the E2 concentration data, and the results are shown in Table 7-1, Table 7-2, and Table 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 produce a clinical correlation (i.e., the coincidence rate with the Roche test results) curve.

[0071] The results are as follows Figure 4 As shown; among them, Figure 4A in Figure 4 B and Figure 4 C in the figure are the clinical relevance (i.e., the coincidence rate with the Roche test result) result graphs of the chemiluminescent reagent 1, reagent 2 and the chemiluminescent reagent prepared by the monoclonal antibody in the prior art 1, which show: the clinical relevance (R 2 The clinical relevance of the chemiluminescent reagent prepared by the monoclonal antibody in the prior art 1 (R 2 value)<0.99.

[0072] The above results show that compared with the first generation E2 antibody in the prior art 1, the E2 chemiluminescent detection reagent prepared by the paired antibodies of the present invention has better low-end sensitivity, that is, the result is more accurate when detecting low values, and therefore has a high consistency rate with the Roche test results (R 2 above 0.99), which has good clinical relevance.

[0073] Table 7-1

[0075] Table 7-2

[0076] Table 7-3

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An anti-estradiol monoclonal antibody or an antigen-binding fragment thereof, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 6, YVS and SEQ ID NO: 7, respectively; or, (2) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 15, GAT and SEQ ID NO: 16, respectively; or, (3) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 24, STS and SEQ ID NO: 25, respectively; or, (4) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 33, NAK and SEQ ID NO: 34, respectively; or, (5) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 42, NAK and SEQ ID NO: 43, respectively; or, (6) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 51, NAK and SEQ ID NO: 52, respectively; or, (7) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62, respectively; or, (8) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 69, YAS and SEQ ID NO: 70, respectively; or, (9) a heavy chain variable region having HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 73, SEQ ID NO: 74 and SEQ ID NO: 75, respectively; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 79, respectively; or, (10) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 87, HGT and SEQ ID NO: 88, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:4; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:8; or, (2) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 17; or, (3) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 22; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 26; or, (4) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 31; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 35; or, (5) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:40; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:44; or, (6) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:49; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:53; or, (7) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 58; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 62; or, (8) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 67; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 71; or, (9) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 76; and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 80; or, (10) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 85; and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

89.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The monoclonal antibody or antigen-binding fragment thereof comprises: (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, (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, (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, (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, (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, (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, (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, (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, (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, (10) a heavy chain having the amino acid sequence shown in SEQ ID NO: 86; and a light chain having the amino acid sequence shown in SEQ ID NO:

90.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that: The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, human antibody, chimeric antibody, bispecific or multispecific antibody.

5. A polynucleotide encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

6. A nucleic acid construct comprising the polynucleotide according to claim 5, and at least one expression control element operably linked to the polynucleotide.

7. A recombinant vector comprising the polynucleotide according to claim 5, or the nucleic acid construct according to claim 6.

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

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

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