E2 sandwich method rabbit monoclonal antibody mAb28 or antigen-binding fragment thereof, and preparation method and application thereof

Abstract

This invention belongs to the field of immunoassay technology, specifically relating to an E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment, its preparation method, and its applications. The E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment includes a light chain variable region (VL) and a heavy chain variable region (VH). The light chain variable region (VL) includes complementarity-determining regions (LCDR1, LCDR2, and LCDR3), and the heavy chain variable region (VH) includes complementarity-determining regions (HCDR1, HCDR2, and HCDR3). The monoclonal antibody involved in this invention can achieve highly specific binding to the E2 complex, making it suitable for immunoassay in a double-antibody sandwich configuration. When applied to a double-antibody sandwich ELISA or chemiluminescent immunoassay kit, the detection sensitivity can be as low as below 8 pg / mL when detecting the E2 standard antigen using a chemiluminescence platform. When using magnetochemiluminescence to detect clinical samples, this kit exhibits good correlation with clinical comparative data over a wide detection range of 0-15000 pmol / L, providing effective detection support for clinical health management in both women and men.

Description

Technical Field

[0001] This invention relates to the field of immunoassay technology, specifically to an E2 sandwich method rabbit monoclonal antibody mAb28 or its antigen-binding fragment, its preparation method, and its application. Background Technology

[0002] Estradiol (E2), the most physiologically active member of the estrogen family, is derived from cholesterol and is primarily produced by the female ovaries, with some secretion also occurring in the male testes and adrenal glands. Its physiological functions encompass multiple systems, including the reproductive, skeletal, cardiovascular, and neurocognitive systems: At the reproductive axis level, E2 drives endometrial proliferation, follicle maturation and ovulation, regulates the menstrual cycle, and promotes breast development and the formation of female secondary sexual characteristics; at the bone metabolism level, E2 maintains bone density by promoting calcium deposition and inhibiting bone resorption, and its levels decline sharply after menopause, a major cause of osteoporosis; at the cardiovascular level, E2 improves vascular endothelial function, dilates blood vessels, and possesses antioxidant and anti-inflammatory effects; furthermore, E2 also influences skin texture, mood, and cognitive function.

[0003] At the clinical level, E2 level testing is used not only to assess women's ovarian function, menstrual cycle, menopausal status, and assisted reproductive treatment, but also to assess men's health status and play an important role in the auxiliary diagnosis of estrogen-secreting tumors.

[0004] Although mass spectrometry is considered the gold standard for E2 detection, its cumbersome pretreatment, long detection cycle, and poor automation adaptability limit its large-scale application in routine clinical testing. Immunoassay, with its advantages of speed and convenience, has become an alternative route. However, E2, as a small molecule hormone, lacks sufficient antigenic epitopes, causing current mainstream immunoassays to still rely on competitive methods. This method has inherent drawbacks such as insufficient sensitivity, lack of specificity, high risk of false positives, and high process complexity.

[0005] To overcome this technical bottleneck, developing highly specific and sensitive monoclonal antibodies that support the sandwich method for E2 detection is not only the core path to breaking through the limitations of competitive methods, but also an urgent need to improve the accuracy and accessibility of E2 clinical testing. Summary of the Invention

[0006] To overcome the above-mentioned technical problems, the present invention provides E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment, which has good specificity and high affinity, and its application in immunoassay kits prepared by double antibody sandwich ELISA or chemiluminescence method.

[0007] In a first aspect, the present invention provides an E2 sandwich-type rabbit monoclonal antibody mAb28 or its antigen-binding fragment, comprising a light chain variable region VL. The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.1. The amino acid sequence of LCDR2 is AAS (SEQ ID NO.2) or a variant having one amino acid substitution, deletion, or addition compared to AAS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.3.

[0008] Furthermore, the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment further includes a heavy chain variable region VH. The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.6.

[0009] Furthermore, the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment can specifically bind to E2 hormone.

[0010] Further, the light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.8, wherein any one or more amino acids have been substituted, deleted, and / or added, and / or terminally modified.

[0011] Furthermore, amino acid sequences with 95%, 96%, 97%, 98%, or 99% identity.

[0012] Furthermore, the light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.8.

[0013] Furthermore, the antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, dsFv, and bispecific antibodies.

[0014] Secondly, the present invention also provides a biomaterial selected from polynucleotides, carriers, or cells. The polynucleotide encodes the E2 sandwich antibody mAb28 or its antigen-binding fragment as described in the first aspect; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment as described in the first aspect.

[0015] Furthermore, the cells are eukaryotic cells, preferably mammalian cells, more preferably 293 cells or CHO cells.

[0016] Thirdly, the present invention also provides a method for preparing the E2 sandwich method rabbit monoclonal antibody mAb28 or its antigen-binding fragment as described in the first aspect, comprising culturing cells as described in the second aspect, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a polynucleotide comprising the E2 sandwich method rabbit monoclonal antibody mAb28 or its antigen-binding fragment, the polynucleotide comprising a heavy chain expression plasmid and a light chain expression plasmid, and the transformation comprising co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

[0017] Fourthly, the present invention also provides the application of the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment described in the first aspect, or the biological material described in the second aspect, wherein the application is selected from one or more of the following: 1) Detection of E2 hormone for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting E2 hormone; 3) Used for purifying E2 hormone; 4) Prepare products for purifying E2 hormone.

[0018] In this invention, the applications are not for diagnostic or therapeutic purposes. Specifically, they can be used to explore the regulatory mechanisms of E2 on reproductive function, bone density, cardiovascular health, mood, and cognitive function, such as studying the effects of E2 on neuroprotection and neurotrophic function in animal models; athletes or fitness enthusiasts can use non-clinical E2 testing to understand the impact of exercise on hormone levels and assist in adjusting training plans; and in animal husbandry, E2 testing can be applied to monitor the estrous cycle of female animals and optimize reproductive efficiency, which falls under animal production management rather than human disease diagnosis and treatment.

[0019] Fifthly, the present invention also provides an E2 hormone immunoassay product, wherein the immunoassay product comprises the E2 sandwich antibody mAb28 or its antigen-binding fragment as described in the first aspect or the biological material as described in the second aspect.

[0020] Furthermore, the E2 hormone immunoassay product is a reagent or kit for detection using enzyme-linked immunosorbent assay (ELISA), Western blot, immunohistochemical staining (IHC), flow cytometry (FCM), immunoprecipitation, and magnetic microparticle chemiluminescence immunoassay.

[0021] Compared to existing technologies, the E2 sandwich method rabbit monoclonal antibody mAb28 of the present invention can bind to E2 with high specificity and has high affinity, with an affinity constant Ka reaching 6 × 10⁻⁶. 9 L / mol. The E2 sandwich rabbit monoclonal antibody of the present invention can also be used to prepare various immunoassay kits for detecting E2, especially for use in immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods. The double-antibody sandwich chemiluminescence platform for detecting the E2 standard antigen has a detection sensitivity of less than 8 pg / mL. Magnetochemiluminescence detection of clinical samples shows good correlation with clinical R within the sample range of 0-15000 pmol / L. Attached Figure Description

[0022] Figure 1 This is an electrophoresis diagram of the full-length amplification products of the mAb28 heavy and light chains, where M is the DNA molecular weight marker.

[0023] Figure 2 The mAb28 sandwich ELISA method was used to detect cross proteins and target antigen samples. The vertical axis represents the detected OD value.

[0024] Figure 3 The standard curve was used to detect E2 standard antigen using mAb28 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the concentration of E2 standard antigen (pg / mL), and the y-axis represents the detected luminescence value. The R-squared value of the standard curve is shown. 2 =0.9903, linear detection range 0-5 ng / mL, derive the sample concentration calculation formula: y = -0.02x2 +164.03x+12770.

[0025] Figure 4 The standard curve was used to detect clinical samples using mAb28 magnetic microparticle chemiluminescence immunoassay. The x-axis represents the free E2 concentration (pmol / L) of the clinical samples, and the y-axis represents the detected luminescence value. The R-value of the standard curve is... 2 =0.9707, linear detection range 0-15000 pmol / L, derived sample concentration calculation formula: y=34.071x+5965.9. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, conditions described in a laboratory manual, or conditions recommended by the manufacturer.

[0027] Example 1: Preparation of rabbit monoclonal antibody using the E2 sandwich method 1) Preparation of immunogens The E2 complex was prepared with an immunogen purity of over 90%, meeting the purity requirements for preparing monoclonal antibodies.

[0028] 2) Animal immunization The E2 complex prepared above was emulsified with complete Freund's adjuvant at a 1:1 volume ratio and administered subcutaneously to approximately 2 kg New Zealand white rabbits at a dose of 800 μg / rabbit. A second immunization was performed two weeks later, emulsified with incomplete Freund's adjuvant at a 1:1 volume ratio, at a dose of 400 μg / rabbit. Tail blood was collected after both immunizations and serum titers were determined using a serially diluted ELISA method. The OD450 at an ELISA titer of 128,000 was considered to be greater than 1.0. Based on the results, it was determined whether to collect PBMCs or continue immunization. Rabbits with the highest antibody titers were selected for PBMC collection.

[0029] 3) PBMC isolation, specific B cell sorting, and clonal recombination The rabbit was placed supine on the operating table. The fur around the heart was trimmed, and the skin was disinfected with alcohol. The area with the most prominent heartbeat was selected and punctured with a 50mL syringe. Blood flowed into the syringe immediately after the needle entered the heart. The needle was quickly withdrawn after obtaining the required amount of blood. The whole blood in the syringe was transferred into a sterile 50mL tube and mixed with an equal amount of PBS. The mixture was then slowly added dropwise to the lymphocyte separation medium. The mixture was centrifuged at 400×g for 30 minutes at room temperature. After centrifugation, the liquid surface separated into four layers from top to bottom: a yellow plasma layer, a white thin film layer (i.e., a mononuclear cell layer), a separation medium layer, and a red blood cell layer. The mononuclear cell layer was carefully aspirated and washed with PBS to remove platelets and lymphocyte separation medium, thus obtaining rabbit PBMCs.

[0030] Antigen-specific B cells were further sorted from rabbit PBMCs and cultured. Positive clones were selected from the B cell supernatant using antigen-coated ELISA plates. Cells from positive clones were collected, lysed, and RNA was extracted and reverse transcribed into cDNA. The full-length light and heavy chain sequences of naturally paired rabbit monoclonal antibodies were amplified from the cDNA of the corresponding positive clones. Rabbit monoclonal antibody expression vectors were constructed using clonal recombination methods, and the sequences were confirmed by sequencing. The results of the amplified full-length PCR products are shown below. Figure 1 .

[0031] 4) Preparation and purification of monoclonal antibodies To obtain multiple rabbit monoclonal antibodies recognizing the E2 small molecule, the heavy and light chain genes of rabbit monoclonal antibodies were loaded into an expression vector. The plasmid was transfected into KEK293 cells, and after 120-144 hours, the culture supernatant contained recombinant rabbit monoclonal antibodies recognizing the E2 small molecule. The cell suspension was collected, the supernatant was obtained by centrifugation, and the antibody was purified by affinity chromatography. The concentration of the purified monoclonal antibody was determined by the BCA method, and then aliquoted, lyophilized, and named rabbit monoclonal antibody mAb28.

[0032] Example 2: Identification of rabbit monoclonal antibodies using the E2 sandwich method 1) Specificity identification of rabbit monoclonal antibodies Indirect ELSA was used for detection. The ELISA plate was coated with cross-linked protein and E2 complex antigen at a concentration of 1 μg / mL and incubated overnight at 4°C. The plate was then blocked with PBST containing 1% BSA. 10 μg / mL of PBST was added. 4 The purified rabbit monoclonal antibody was diluted 10 times and reacted at 37°C for 50 min. The plate was washed 3 times with PBST, HRP-goat anti-rabbit IgG secondary antibody was added, and the plate was reacted at 37°C for 50 min. The plate was washed 5 times with PBST, TMB was added for color development for 10 min, stop solution was added, and the A450 was measured by microplate reader.

[0033] Figure 2 The results showed that the cross-linking protein reacted negatively with rabbit monoclonal antibody mAb28, and OD... 450 All values ​​were less than 0.1; the E2 complex reacted positively with mAb28 antibody, and the OD value was much higher than that of the cross protein, indicating that the E2 sandwich method rabbit monoclonal antibody of the present invention specifically recognizes the E2 complex.

[0034] 2) Determination of affinity constant of rabbit monoclonal antibody Affinity constant (Ka) was determined using a non-competitive ELISA method.

[0035] Coating: Dilute the antigen with carbonate buffer to concentrations of 1, 0.5, 0.1, and 0.05 μg / mL, add 100 μL / well to a 96-well microplate for coating, and incubate at 4°C for 24 h.

[0036] Blocking: Wash the plate 4 times with PBST, add BSA solution at 200 μL / well, and incubate at 37°C for 2 h.

[0037] Add monoclonal antibody: Wash the plate 4 times with PBST, serially dilute the rabbit monoclonal antibody with carbonate buffer starting at 100 μg / mL, add 100 μL to each well, and incubate at 37°C for 2 h.

[0038] Add enzyme-labeled secondary antibody: Wash the plate 4 times with PBST, add 100 μL of HRP enzyme-labeled goat anti-rabbit Ig secondary antibody diluted 1:10000 to each well, and incubate at 37℃ for 30 min.

[0039] Color development and termination: Wash the plate 4 times with PBST, add 100 μL of substrate color development solution to each well, and react at 37℃ in the dark for 15 min; add 50 μL of 1.0 mol / L H2SO4 stop solution to each well to terminate the reaction.

[0040] Detection: The absorbance value at a wavelength of 450 nm (A450nm) was measured.

[0041] Plotting the logarithm of antibody concentration on the x-axis and OD value on the y-axis, an S-shaped curve was constructed. The calculated affinity constant Ka for the E2 sandwich monoclonal antibody mAb28 was 6 × 10⁻⁶. 9 L / mol.

[0042] 3) Sandwich antibody pairing To select the optimal combination of coating and detection antibodies, E2-binding protein was coated onto an ELISA plate and incubated overnight at 4°C. The next day, the plate was removed, washed once with PBST, blocked with 1% BSA solution at 37°C for 2 hours, and washed three times with PBST. 100 μL of the E2 complex (20 ng / mL) was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was removed, washed three times with PBST, and HRP-labeled rabbit monoclonal antibody mAb28 was added as the detection antibody, and the plate was incubated at 37°C for 1 hour. The plate was washed five times with PBST, TMB substrate was added, and the plate was incubated at 37°C for 10 minutes. Finally, stop solution was added, and the OD was measured using an ELISA reader. 450 Readings. Based on the OD value of the sample and the background value of the negative control, the most ideal antibody pair was selected. The pairing screening results are shown in Table 1.

[0043] Table 1. Results of antibody pairing assay

[0044] As shown in Table 1, rabbit monoclonal antibody mAb28 was used as the detection antibody in the determination of OD on the ELISA reader. 450A reading greater than 3 indicates that the antibody pairing is feasible and that the antibody possesses good overall performance. The antibody mAb28 involved in this invention is optimally suited for sandwich assays.

[0045] Example 3: Analysis of the gene and amino acid sequence of the variable region of a monoclonal antibody Using the recombinant plasmid of the antibody as a DNA template, sequencing primers for the light chain variable region and heavy chain variable region were designed based on the vector sequences at the 5' ends of the light and heavy chains on the template. Sequencing was performed using an ABI 3730 sequencer. The nucleotide sequences of the light and heavy chain variable regions of the rabbit monoclonal antibody were obtained by sequencing.

[0046] Using the internet and the IMGT / V-QUEST analysis software at http: / / www.imgt.org, the nucleotide sequences of the light chain variable region and the heavy chain variable region were sequenced and analyzed. The amino acid sequence of the light chain variable region of rabbit monoclonal antibody mAb28 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0047] The VL is 113 amino acids long. The number of amino acids in the four domains of its FR are 26, 17, 36 and 11, respectively. The number of amino acids in the three domains of LCDR are 6, 3 and 14, respectively. The regions of LCDR1, LCDR2 and LCDR3 are 27aa-32aa, 50aa-52aa and 89aa-102aa, respectively. Their amino acid sequences are QTINSW (SEQ ID NO.1), AAS (SEQ ID NO.2) and QSYSGGILVSYSWA (SEQ ID NO.3), respectively.

[0048] The VH is 120 amino acids in length. The number of amino acids in the four domains of its FR are 25, 17, 38 and 11, respectively. The number of amino acids in the three domains of HCDR are 8, 7 and 14, respectively. HCDR1, HCDR2 and HCDR3 are 26aa-33aa, 51aa-57aa and 96aa-109aa, respectively. Their amino acid sequences are GFSLSNYA (SEQ ID NO.4), IGVGVSA (SEQ ID NO.5) and ARVGSNVDYNPLDI (SEQ ID NO.6), respectively.

[0049] Example 4: E2 sandwich method rabbit monoclonal antibody used for magnetic microparticle chemiluminescent immunoassay.

[0050] 1. Detection Principle and Method A magnetic microparticle chemiluminescence immunoassay technique based on the double-antibody sandwich method was employed. Biotin-labeled E2 antibody was used to immobilize the biotinylated binding protein with SA magnetic beads. ALP was conjugated to the E2 complex using a sandwich method with rabbit monoclonal antibody. Simultaneously, E2, ALP substrate, and corresponding buffer components were placed on a Cosmetic fully automated magnetic microparticle chemiluminescence analyzer, and the instrument program was set for detection. A positive result was interpreted as a signal-to-noise ratio (SNR) greater than 2.0. The magnitude of the luminescence value reflects the amount of bound enzyme-labeled antibody and is directly proportional to the concentration of E2 in the sample. A standard curve was plotted based on the measured luminescence values ​​of the standards, as shown below. Figure 3 The E2 concentration value in the sample to be tested can be obtained from the standard curve.

[0051] 2. Composition of the magnetic particle chemiluminescence detection kit for E2 detection

[0052] 1) SA magnetic beads bound to biotin-E2 binding protein: Take 50 μL of magnetic beads into a 0.5 mL centrifuge tube, place it on a magnetic rack, and remove the supernatant after 1 min; wash the magnetic beads 3 times with 0.5 mL of antibody dilution buffer; add a certain amount of biotin-labeled E2 binding protein and mix at room temperature for 60 min; after magnetic separation, resuspend in magnetic storage buffer at a working concentration of 0.5 mg / mL.

[0053] 2) mAb28 conjugation to ALP: First, the 2-IT antibody mAb28 is reduced; then, the ALP-SMCC intermediate is formed; finally, ALP-SMCC is conjugated to the reducing antibody. After conjugation, the ALP is diluted to the working concentration using ALP preservation buffer.

[0054] 3) Washing buffer: Standard pH 7.4 PBST containing 0.05% Proclin 300, prepared as a 20-fold concentrate.

[0055] 4) Chemiluminescent colorimetric solution: purchased from Aivid Biotechnology.

[0056] 5) Sample diluent: PBST containing 1% BSA and 0.05% Proclin 300, filtered for sterilization.

[0057] 6) Standard: E2 small molecule, diluted to 5 μg / mL with PBS containing 1% BSA, 5% sucrose, 10% glycerol and 0.05% Proclin 300 as diluent, filtered for sterilization and aseptically dispensed.

[0058] 3. Testing of E2 clinical samples Clinical samples with different E2 concentrations were processed. Using E2 antibody as the coating antibody and mAb28 antibody as the detection antibody, the above detection method was used to detect clinical samples of different concentrations. The detection results are shown below. Figure 4 .

[0059] Based on the results, the rabbit monoclonal antibody described in this invention, when used in a magnetic microparticle chemiluminescent immunoassay reagent, showed good correlation with clinical results within a sample range of 0-15000 pmol / L.

[0060] In summary, when the E2 sandwich rabbit monoclonal antibody mAb28 of this invention is applied to immunoassay kits prepared by double-antibody sandwich ELISA or chemiluminescence methods, the detection sensitivity of the E2 standard antigen on the double-antibody sandwich chemiluminescence platform is less than 8 pg / mL. When the magnetochemiluminescence method is used to detect clinical samples, the clinical composite rate is >0.97 in the sample range of 0-15000 pmol / L, which is significantly higher than that of traditional competitive detection methods. Moreover, the process is simple and breaks through the limitations of traditional competitive methods.

[0061] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An E2 sandwich-type rabbit monoclonal antibody mAb28 or its antigen-binding fragment, characterized in that, Including the light chain variable region VL, The light chain variable region VL includes LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 is the sequence shown in SEQ ID NO.1 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

1. The amino acid sequence of LCDR2 is AAS or a variant with one amino acid substitution, deletion, or addition compared to AAS. The amino acid sequence of LCDR3 is the sequence shown in SEQ ID NO.3 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

3.

2. The E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment according to claim 1, characterized in that, It also includes the heavy chain variable region VH, The heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is the sequence shown in SEQ ID NO.4 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

4. The amino acid sequence of HCDR2 is the sequence shown in SEQ ID NO.5 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

5. The amino acid sequence of HCDR3 is the sequence shown in SEQ ID NO.6 or a variant with one or two amino acid substitutions, deletions, or additions compared to the sequence shown in SEQ ID NO.

6.

3. The E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment according to claim 2, characterized in that, The light chain variable region VL comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.7, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids; the heavy chain variable region VH comprises an amino acid sequence having more than 95% homology to the amino acid sequence shown in SEQ ID NO.8, obtained by substitution, deletion, and / or addition of one or more amino acids and / or terminal modification of any one or more amino acids.

4. The E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment according to claim 3, characterized in that, The light chain variable region VL includes the amino acid sequence shown in SEQ ID NO.7, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO.

8.

5. The E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment according to claim 1, characterized in that, The antigen-binding fragment is one of F(ab')2, Fab', Fab, Fv, scFv, and dsFv.

6. A biomaterial, characterized in that, The biomaterial is a polynucleotide, a carrier, or a cell. The polynucleotide encodes the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment as described in any one of claims 1-5; The vector carries the polynucleotide; The cell carries the polynucleotide, or contains the vector, or is capable of expressing the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment as described in any one of claims 1-5.

7. The method for preparing the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment according to any one of claims 1-5, characterized in that, The method includes culturing the cells as described in claim 6, wherein the cells are prepared by transforming the cells with a polynucleotide encoding a rabbit monoclonal antibody mAb28 or an antigen-binding fragment thereof using the E2 sandwich method, wherein the polynucleotide includes a heavy chain expression plasmid and a light chain expression plasmid, and the transformation includes co-transforming the heavy chain expression plasmid and the light chain expression plasmid into the cells.

8. The application of the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment according to any one of claims 1-5, or the biological material according to claim 6, characterized in that, The application is selected from one or more of the following: 1) Detection of E2 hormone for purposes other than disease diagnosis and treatment; 2) Prepare immunoassay products for detecting E2 hormone; 3) Used for purifying E2 hormone; 4) Prepare products for purifying E2 hormone.

9. An E2 hormone immunoassay product, characterized in that, The immunoassay product comprises the E2 sandwich rabbit monoclonal antibody mAb28 or its antigen-binding fragment as described in any one of claims 1-5, or the biological material as described in claim 6.

10. The E2 hormone immunoassay product according to claim 9, characterized in that, The E2 hormone immunoassay products are reagents or kits for detection using enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunohistochemical staining, flow cytometry, immunoprecipitation, or magnetic microparticle chemiluminescence immunoassay.

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