Oestrone monoclonal antibody and application thereof

Abstract

The invention provides an oestrone monoclonal antibody and application thereof, sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the oestrone monoclonal antibody are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, sequences of CDR1, CDR2 and CDR3 of a light chain variable region of the oestrone monoclonal antibody are respectively shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, and the oestrone monoclonal antibody can be specifically combined with oestrone.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological immunology, and particularly relates to an estrone monoclonal antibody and its application. Background Art

[0002] Estradiol (E2) is a steroid estrogen, with α and β types, and the α type has strong physiological effects. The molecular formula of E2 is C 18 H 24 O2, with a molecular weight of 272.38. It is mainly secreted by ovarian follicles, corpus luteum, and placenta during pregnancy. It is the estrogen with the highest content and the strongest biological activity. Its main function is to promote the transformation of the endometrium into the proliferative phase and the development of female secondary sexual characteristics, and it has important clinical significance in evaluating ovarian function. In addition, E2 also has obvious effects on the endocrine system, cardiovascular system, human metabolism, bone growth, etc. Estrone (E1), also known as estrogen ketone and estrophenone, is also a steroid hormone compound and one of the natural estrogens in the human body and animals. The molecular formula of estrone is C 18 H 22 O2, with a molecular weight of 270.37. E1 has a wide range of applications in medicine and drugs, and patients may have an abnormal increase in the concentration of E1 in the body due to taking drugs.

[0003] E1 and E2 are two different estrogens. In terms of chemical structure, as Figure 1 shown, E2 has two hydroxyl (-OH) groups, while the molecular structure of E1 contains one hydroxyl group and one ketone group. In terms of activity difference, E2 is the most active estrogen, and its potency is 10 times that of E1. In addition to the differences, E1 and E2 also have a strong correlation. In the body, E2 is first secreted, then converted into E1 under the action of 17β-dehydrogenase, and then converted into estriol (E3) under the action of reductase. This indicates that E1 is an intermediate product in the metabolic process of E2.

[0004] Because their molecular structures are very similar and they are both small molecule compounds with only one antigen-binding site, most of the current E2 antibodies on the market will cross-react with E1, that is, the E2 immunoassay reagents have a large lack of specificity. If the sandwich method is used to detect E2, false positives may occur due to the cross-reaction of the first antibody with E1, resulting in misjudgment. The existing E2 immunoassay methods mainly adopt the principle of the competitive method. Summary of the Invention

[0005] The present invention provides an estrone monoclonal antibody and its application, which can effectively solve the above problems.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, the present invention provides an estrone monoclonal antibody, wherein the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown as SEQ.ID No.1, SEQ.ID No.2, and SEQ.ID No.3 respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown as SEQ.ID No.4, SEQ.ID No.5, and SEQ.ID No.6 respectively, and the estrone monoclonal antibody can specifically bind to estrone.

[0008] In a second aspect, the present invention provides the application of the above-mentioned estrone monoclonal antibody in an estrone immunoassay reagent.

[0009] In a third aspect, the present invention provides an estrone immunoassay reagent or kit, which includes the above-mentioned estrone monoclonal antibody.

[0010] In a fourth aspect, the present invention provides the application of the estrone monoclonal antibody in an estradiol immunoassay reagent, and the estrone monoclonal antibody blocks the cross-reaction between estrone and the detection antibody in the estradiol immunoassay reagent by specifically binding to estrone.

[0011] In a fifth aspect, the present invention provides an estradiol immunoassay reagent or kit, which includes the above-mentioned estrone monoclonal antibody, and the estrone monoclonal antibody is a blocking agent.

[0012] The beneficial effects of the present invention are as follows:

[0013] The present invention provides an estrone monoclonal antibody, which can specifically bind to estrone, has high affinity, and avoids cross-reaction and non-specific binding with estradiol.

[0014] The present invention provides an estradiol immunoassay kit, which can block the interference of small molecule compound E1 in the sample, improve the specificity of the kit, adding the estrone monoclonal antibody has no obvious effect on the detection value, and can improve the detection linearity compliance.

[0015] The estradiol immunoassay kit uses the sandwich method to detect E2. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 It is a molecular structural formula diagram of estrone and estradiol.

[0018] Figure 2 SDS-PAGE protein gel electrophoresis pattern of the purified E1 monoclonal antibody 8H2 of the embodiment of the present invention.

[0019] Figure 3 ELISA detection result chart of the activity of E1 antibody 8H2 of the embodiment of the present invention.

[0020] Figure 4 Linear graph of the detection of clinical samples of E2 immunofluorescence reagent without adding E1 antibody 8H2.

[0021] Figure 5 Linear graph of the detection of clinical samples of E2 immunofluorescence reagent with the addition of E1 antibody 8H2. Detailed implementation mode

[0022] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the present invention claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0023] Example 1

[0024] 1. Preparation of immunogen

[0025] Weigh 10 mg of estrone 3-O-aminosulfonate (CAS No.: 148672-09-7) and dissolve it in 2 mL of DMSO. Weigh 20 mg of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (ThermoFisher, product number: 22360) and dissolve it in 1 mL of DMSO. At room temperature, drop the above solution into 10 mg of maleimide-activated BlueCarrier protein (ThermoFisher, product number: 77664), and slowly stir and react for 2 - 4 h. After the reaction, dialyze and replace the product into PBS with 20 mM, pH 7.4, and 100 mM NaCl. The immunogen E1-KLH is obtained.

[0026] 2. Immunization of mice

[0027] Take the above immunogen E1-KLH, dilute it to 0.4 mg / mL with PBS, emulsify it evenly with an equal volume of Freund's Adjuvant, Complete (Thermo Fisher, catalog number: 77140). Take SPF-grade Balb / c mice aged 6 - 8 weeks (Fuzhou Wu's Animal Experiment Center), inject 200 μg per mouse subcutaneously at multiple points. After a 2-week interval, emulsify the antigen with an equal volume of Freund's Adjuvant, Incomplete (Thermo Fisher, catalog number: 77145), inject 100 μg per mouse subcutaneously at multiple points, boost the immunization 2 times, and inject 100 μg per mouse intraperitoneally 3 days before fusion for boosting.

[0028] 3. Preparation of feeder cells

[0029] Use Balb / c mouse peritoneal macrophages as feeder cells. One day before fusion, sacrifice the mouse by cervical dislocation, soak the whole body in 75% alcohol for disinfection for 5 min, perform aseptic operation in a laminar flow hood, cut open the abdominal skin with scissors to expose the peritoneum, inject 5 mL of RPMI1640 basal medium containing 1% Penicillin-Streptomycin into the peritoneal cavity with a syringe, rinse repeatedly, recover the rinsing solution, centrifuge at 1000 rpm for 5 minutes, keep the precipitate, resuspend it with RPMI1640 complete medium containing 1% HAT, adjust the cell concentration to 1×10 5 cells / mL, add it to 4 96-well cell culture plates, 150 μL per well, and culture overnight at 37°C and 5% CO2.

[0030] RPMI1640 basal medium containing 10% fetal bovine serum and 1% double antibiotics (fetal bovine serum, Penicillin-Streptomycin) is the complete medium.

[0031] Penicillin-Streptomycin: Gibco, catalog number: 15140122.

[0032] Fetal bovine serum: Shanghai Xiaopeng Biotechnology, catalog number: C04001-500.

[0033] RPMI1640 basal medium: Shanghai Yuanpei Biotechnology, catalog number: L210KJ.

[0034] HAT medium: HAT Media Supplement (50×), Thermo Fisher, catalog number: 21060017.

[0035] 4. Preparation of immune spleen cells

[0036] Three days after the last immunization of the above mice, the spleens were removed under sterile conditions, placed in a petri dish, rinsed once with RPMI1640 basal culture medium, ground and filtered through a nylon mesh (the nylon mesh was placed in a small beaker) to prepare a cell suspension. Centrifuge, discard the supernatant, resuspend with RPMI1640 basal culture medium, repeat this three times, and count.

[0037] 5. Cell fusion

[0038] (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium (Shanghai Yuanpei Biotechnology, product number: L310KJ), and 1 mL of 50% PEG (M12000, Sigma, product number: P7181), and pre-warm them in a 37°C water bath respectively;

[0039] (2) Take mouse myeloma cells Sp2 / 0 (2×10 7 cells) and the above immune spleen cells (1×10 8 cells) suspension, add them to a 50 mL centrifuge tube and mix well, and add DMEM serum-free culture medium to 40 mL. Centrifuge for 10 minutes, pour out the supernatant, and mix well;

[0040] (3) Place the centrifuge tube in the water pre-warmed at 37°C, take 0.7 mL of 50% PEG solution pre-warmed at 37°C, and let it stand for 90 seconds. Immediately add dropwise 15 mL of serum-free culture medium pre-warmed at 37°C;

[0041] (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, pour out the supernatant. Add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum FBS. Mix well with a pipette, and drop it into the small holes of the above 96-well cell culture plate containing feeder cells, 2 drops per well, and culture in an incubator at 37°C and 7% CO2.

[0042] 6. Selective culture of hybridoma cells

[0043] On the 1st, 3rd, 5th, and 7th days after cell fusion, change the culture medium with HAT culture medium containing 15%-20% fetal bovine serum. Among them, the surviving ones are hybridoma cells, and the non-hybridoma cells will die, and the true hybridoma cells are selected.

[0044] 7. Detection of specific antibodies and cloning of hybridoma cells

[0045] Aspirate the supernatant from each culture well, and use the indirect ELISA method to screen out the culture wells whose supernatant contains antibodies that specifically recognize E1-BSA but do not recognize E2-BSA. The culture wells with an OD490 value of the ELISA that recognizes E1-BSA greater than 2 and an OD490 value of the ELISA that recognizes E2-BSA less than 0.2 are positive hybridoma cell lines.

[0046] One day before cloning, feeder cells were prepared and plated according to Step 3; the positive hybridoma cells to be cloned were pipetted and mixed well, and diluted to 1 cell per well with HT medium (HT Media Supplement (50×), Thermo Fisher, catalog number: H0111067030), and cultured at 37 °C in a 5% CO₂ humidified environment for 7 - 10 days. Once clones visible to the naked eye appeared, the antibodies could be detected; observed under an inverted microscope, the wells with only single clones growing were marked, and finally, the monoclonal antibody hybridoma cell line 8H2 was preliminarily screened and obtained.

[0047] 8. Antibody Sequencing

[0048] The cloned antibody 8H2 cells were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The amino acid sequences of the monoclonal antibody 8H2 are as follows:

[0049] The heavy chain CDR1 sequence is: CTMGQA (SEQ.ID No.1)

[0050] The heavy chain CDR2 sequence is: NVYMDGRAQTLSSTQ (SEQ.ID No.2)

[0051] The heavy chain CDR3 sequence is: CGTVKPA (SEQ.ID No.3)

[0052] The light chain CDR1 sequence is: TPRQAKCB (SEQ.ID No.4)

[0053] The light chain CDR2 sequence is: MRQPF (SEQ.ID No.5)

[0054] The light chain CDR3 sequence is: YTSWVNK (SEQ.ID No.6)

[0055] The heavy chain variable region sequence is:

[0056] QVQLQQSAAELARPGASVKMSCKASGYTFCTMGQAWVKERPGQGLEWIGYINVYMDGRAQTLSSTQKTTLTADKSSSTTYMQLSSLTSEDSAVYYCVRCGTVKPAWGQGTTLTVSS (SEQ.ID No.7)

[0057] The light chain variable region sequence is:

[0058] DVLMTQTPLSLPVSLGDQASISCTPRQAKCBSNGNTYLEWYLQKPG QSPKLLIYMRQPFFSGVPDRFSGSESGTDFTLKISRVEAEDLGVYYCYTS WVNKYTFGGGTKLEIK(SEQ.ID No.8)

[0059] The heavy chain sequence is:

[0060] QVQLQQSAAELARPGASVKMSCKASGYTFCTMGQAWVKERPGQGLEWIGYINVYMDGRAQTLSSTQKTTLTADKSSSTTYMQLSSLTSEDSAVYYCVRCGTVKPAWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ.IDNo.9)

[0061] The light chain sequence is:

[0062] DVLMTQTPLSLPVSLGDQASISCTPRQAKCBSNGNTYLEWYLQKPGQSPKLLIYMRQPFFSGVPDRFSGSESGTDFTLKISRVEAEDLGVYYCYTSWVNKYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ.ID No.10)

[0063] 9. Antibody expression and purification

[0064] Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin. Ten days later, 1×10 hybridoma cell line 8H2 was inoculated with 6 After about 10 days, the abdomen of the mouse will begin to swell, and the mouse will be killed by pulling the neck, and the whole body will be soaked in 75% alcohol for 5 minutes for disinfection, and the ascites will be extracted once.

[0065] The ascites supernatant was separated and purified by Protein A affinity chromatography column (Protein A Resin, Changzhou Tiandi Renhe Biology, Catalog No.: SA023010) according to the Cytiva Company Handbook: Affinity Chromatography, Vol 1: Antibodies to purify and prepare E1 monoclonal antibody 8H2. The purification results are shown in Figure 2 As shown in the figure, it can be seen that the heavy and light chain bands of the purified antibody are clear and single, indicating that the purity is high.

[0066] Example 2

[0067] ELISA to detect the activity of E1 antibody 8H2

[0068] 1. Take an ELISA plate and add 100 μL of E1-BSA (Xi'an Qiyue Biological, Catalog No.: Q-0035933) diluted to 2 μg / mL with coating solution (10 mM phosphate buffer, pH 7.4) and 100 μL of E2-BSA (Medis, Catalog No. LA428) diluted to 2 μg / mL with coating solution to each well to coat the plate, cover the plate, and incubate overnight at 2-8°C.

[0069] 2. Aspirate the liquid in each well and add 200 μL of washing buffer (containing 0.05% Tween 20 mM phosphate buffered saline) to each well for washing once. After washing, invert the plate and tap gently on absorbent paper to remove residual liquid.

[0070] 3. Add 200 μL of blocking buffer (10% skim milk, prepared in PBST) to each well and block for 1 h at room temperature.

[0071] 4. Aspirate, invert the plate and tap gently on absorbent paper to remove any residual liquid.

[0072] 5. Add 100 μL of antibody 8H2 solution diluted to different concentrations to each well and place at room temperature for 1 hour. Use PBS for dilution.

[0073] 6. Aspirate the liquid in each well and add 200 μL of washing buffer to each well and wash 6 times.

[0074] 7. Add 100 μL of HRP-labeled goat anti-mouse antibody (Shanghai Sangon Biotech, catalog number: D110087) diluted 10,000-fold to each well and incubate at 37 °C for 30 min.

[0075] 8. Aspirate the liquid in each well and wash each well 6 times with 200 μL of washing buffer.

[0076] 9. Add 100 μL of freshly prepared substrate chromogenic solution (Sangon Biotech, catalog number: D110098) to each well and develop color at 37 °C for 15 min.

[0077] 10. Terminate the reaction with 2 mol / L H2SO4 and read the OD450 value on an enzyme-linked immunosorbent assay reader. The test results are shown in Table 1 and Figure 3 as follows.

[0078] Table 1 ELISA data of 8H2 with E1-BSA and E2-BSA

[0079]

[0080] As can be seen from Table 1 and Figure 3 the 8H2 antibody has basically no binding to E2-BSA. As the antibody concentration increases, OD450 remains basically unchanged, while the 8H2 antibody binds to E1-BSA with a good gradient, indicating that the 8H2 antibody is an E1-specific antibody and has no cross-reaction with E2.

[0081] The E1 monoclonal antibody 8H2 provided in the embodiment of the present invention is a highly specific and highly affinity monoclonal antibody against E1, which can meet the requirement of blocking the interference of E1 without affecting the detection of E2, and can be used for the development of E2 reagent blockers.

[0082] Example 3

[0083] 1. Blocking effect of 8H2 antibody on E2 immunofluorescence reagent

[0084] Take the small molecule compound E1 and dilute it to 10, 50, 100, 500, 1000 ng / mL respectively as samples.

[0085] Use the E2 immunofluorescence kit (Baotai Biotech, catalog number: 3.5.02.05.0001). Take the sample diluent and set three groups: add 1 μg / person 8H2 antibody, add 5 μg / person 8H2 antibody, and do not add 8H2 antibody, and test the above samples with different E1 concentrations.

[0086] The test results are shown in Table 2.

[0087] Table 2 Blocking effect of 8H2 antibody on E1 in E2 reagent

[0088]

[0089] As can be seen from Table 2, the 8H2 antibody at 1 μg / person dose can completely block the effect of 50 ng / mL of E1. When the dose of the 8H2 antibody is increased to 5 μg / person, it can basically block the effect of 500 ng / mL of E1, indicating that the 8H2 antibody has a good blocking effect and can be used to improve the specificity of the E2 detection kit, solving the problem of false positives caused by the influence of the small molecule compound E1 on the E2 detection.

[0090] 2. Influence of the 8H2 antibody on the E2 immunofluorescence reagent for testing clinical samples

[0091] Take the above-mentioned E2 immunofluorescence kit, and set the sample diluent into two groups: adding 5 μg / person of the 8H2 antibody and not adding the 8H2 antibody. Test 20 clinical samples in each group to investigate the influence of adding the 8H2 antibody on the linearity and fluorescence value of the clinical samples. The results are shown in Table 3 and Figure 4 、 Figure 5 as follows.

[0092] Table 3 Clinical detection results of the E2 immunofluorescence sandwich method

[0093]

[0094] As can be seen from Table 3 and Figure 4 、 Figure 5 as follows, adding the E1 antibody has no obvious influence on the overall fluorescence value of the reagent, and the linear compliance of the detection value with the addition of the E1 antibody is improved, and the R 2 value is better than that without addition.

[0095] In the embodiment of the present invention, the E1 monoclonal antibody 8H2 is used as a blocker and applied to the E2 sandwich immunofluorescence detection reagent. Compared with the reagent without adding the E1 antibody, it can improve the accuracy of the reagent for clinical sample detection, providing a new idea and method for the rapid and accurate detection of E2.

[0096] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An estrone monoclonal antibody, characterized in that: The sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown as SEQ.ID No.1, SEQ.ID No.2 and SEQ.ID No.3 respectively, and the sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as SEQ.ID No.4, SEQ.ID No.5 and SEQ.ID No.6 respectively. The estrone monoclonal antibody can specifically bind to estrone.

2. The estrone monoclonal antibody according to claim 1, characterized in that The sequence of the heavy chain variable region is shown in SEQ.ID No.7, and the sequence of the light chain variable region is shown in SEQ.ID No.

8.

3. The estrone monoclonal antibody according to claim 1, characterized in that The sequence of its heavy chain is shown in SEQ.ID No.9, and the sequence of its light chain is shown in SEQ.ID No.

10.

4. Use of the estrone monoclonal antibody according to any one of claims 1 to 3 in an estrone immunoassay reagent.

5. An estrone immunoassay reagent or kit, characterized in that: The invention comprises the estrone monoclonal antibody according to any one of claims 1 to 3.

6. Use of the estrone monoclonal antibody according to any one of claims 1 to 3 in an estradiol immunoassay reagent, characterized in that: The estrone monoclonal antibody specifically binds to estrone to block the cross reaction between estrone and the detection antibody in the estradiol immunoassay reagent.

7. An estradiol immunoassay reagent or kit, characterized in that: The invention comprises the estrone monoclonal antibody according to any one of claims 1 to 3, wherein the estrone monoclonal antibody is a blocking agent.

8. The estradiol immunoassay reagent or kit according to claim 7, characterized in that: The estrone monoclonal antibody is 4.8-5.2 μg / person.

9. The estradiol immunoassay reagent or kit according to claim 7, characterized in that: A sample diluent is included, wherein the sample diluent includes the estrone monoclonal antibody.

10. The estradiol immunoassay reagent or kit according to claim 7, characterized in that: The estradiol immunoassay reagent is a double antibody sandwich assay reagent, or the estradiol immunoassay kit is a double antibody sandwich assay kit.

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