A single-chain antibody for detecting estradiol, a complex antibody and application thereof

Abstract

The application provides a single-chain antibody for sandwich detection of estradiol and a complex antibody thereof and application thereof, wherein the sequence of the single-chain antibody for sandwich detection of estradiol is shown in SEQ.ID No.1; the sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the complex antibody for sandwich detection of estradiol are shown in SEQ.ID No.2, SEQ.ID No.3 and SEQ.ID No.4 respectively; and the sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown in SEQ.ID No.5, SEQ.ID No.6 and SEQ.ID No.7 respectively. The single-chain antibody of the application can specifically combine with small molecule estradiol to form an immune complex, the complex antibody of the application can specifically combine with the immune complex, and the single-chain antibody and the small molecule estradiol are not recognized, and can be applied to complex antibody sandwich method for detection of estradiol, and the sensitivity and accuracy of detection of small molecule estradiol are greatly improved.

Description

Technical Field

[0001] This invention relates to a sandwich antibody for detecting estradiol, a complex antibody and their applications, and belongs to the field of estradiol detection technology. Background Technology

[0002] Estradiol (E2) is a steroid estrogen, and it is the most abundant and most active estrogen. Estradiol is primarily secreted by the granulosa cells of the ovarian follicles, the corpus luteum, and the placenta during pregnancy. In adult women, estradiol levels fluctuate cyclically with the menstrual cycle; while in men, it is mainly synthesized and secreted by the interstitial cells of the testes.

[0003] The main physiological functions of estradiol include promoting the development of female reproductive organs and secondary sexual characteristics, regulating the function of the hypothalamus and pituitary gland through positive and negative feedback, promoting bone growth, accelerating bone fusion, and affecting the body's lipoprotein and water and salt metabolism. In addition, estradiol is used to diagnose whether a woman is ovulating; in normal women, estrogen levels fluctuate cyclically, while in anovulation, hormone levels do not fluctuate cyclically.

[0004] In clinical practice, estradiol testing is valuable for diagnosing endocrine and gynecological diseases such as precocious puberty and developmental delays. It can be used to diagnose early pregnancy, ectopic pregnancy, and threatened miscarriage; to diagnose and monitor female endocrine, breast, and gynecological diseases; and as an adjunct diagnostic tool for some conditions.

[0005] Currently, the mainstream clinical method for detecting E2 is immunoassay. Because E2 is a small molecule compound with a single antigenic determinant, it cannot be detected by sandwich assays using two different antibodies. Therefore, clinical immunoassays generally employ competitive assays. However, competitive assays themselves have methodological limitations, such as insufficient precision, inadequate accuracy, susceptibility to interference, and narrow linear range.

[0006] Currently, the most feasible method is the complex antibody sandwich assay. The specific detection principle is as follows: one anti-E2 antibody (primary antibody) forms an immune complex with the target small molecule, and another anti-complex antibody (secondary antibody) binds to the aforementioned immune complex to form an immune sandwich complex. The recognition site is a new epitope formed after the primary antibody binds to the small molecule, and it is required that the complex antibody does not recognize either the free primary antibody or the small molecule. The use of the complex antibody sandwich assay can greatly improve the sensitivity and accuracy of small molecule detection. However, due to the difficulty in screening complex antibodies, current technology still lacks highly active E2 complex antibodies. Summary of the Invention

[0007] This invention provides a sandwich-type single-chain antibody, a complex antibody, and their applications for detecting estradiol, which can effectively solve the above-mentioned problems.

[0008] This invention is implemented as follows:

[0009] A sandwich single-chain antibody for detecting estradiol, the sequence of which is shown in SEQ.ID No.1.

[0010] A sandwich antibody for detecting estradiol, wherein the sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ.ID No.2, SEQ.ID No.3, and SEQ.ID No.4, respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ.ID No.5, SEQ.ID No.6, and SEQ.ID No.7, respectively.

[0011] In some embodiments, the sequence of the heavy chain variable region of the sandwich antibody for detecting estradiol is shown in SEQ.ID No. 8, and the sequence of the light chain variable region is shown in SEQ.ID No. 9.

[0012] In some embodiments, the heavy chain sequence of the sandwich antibody for detecting estradiol is shown in SEQ ID No. 10, and the light chain sequence is shown in SEQ ID No. 11.

[0013] A reagent for detecting estradiol using a complex antibody sandwich method comprises the single-chain antibody and the complex antibody.

[0014] A reagent for detecting estradiol, comprising the single-chain antibody described above.

[0015] The beneficial effects of this invention are:

[0016] The single-chain antibody of this invention possesses unique specific binding ability, enabling it to precisely bind to small molecule estradiol, thereby forming a stable immune complex. This complex antibody not only specifically recognizes and tightly binds to this immune complex, but also does not bind to free single-chain antibodies or small molecule estradiol. Based on this characteristic, the complex antibody of this invention can be widely used in complex antibody sandwich assays, specifically for the accurate detection of estradiol. This technique significantly improves the sensitivity and accuracy of small molecule estradiol detection, providing a more reliable and efficient detection tool for research and clinical applications in related fields. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 The image shows the results of purifying 24H6-scFv antibody via expression in 293F cells.

[0019] Figure 2 The image shows the results of 55C7 antibody expression and purification in 293F cells. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] This invention provides a single-chain antibody for sandwich detection of estradiol, the specific amino acid sequence of which is detailed in SEQ ID No. 1. This single-chain antibody exhibits high specificity, accurately binding to small molecule estradiol to form a stable immune complex, providing a solid foundation for subsequent detection.

[0022] This invention further provides a complex antibody for sandwich detection of estradiol. The heavy chain variable region of this complex antibody contains three complementarity-determining regions (CDRs), the amino acid sequences of which are detailed in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively. The light chain variable region also contains three complementarity-determining regions (CDRs), the amino acid sequences of which are detailed in SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7, respectively. This complex antibody exhibits high specificity, accurately binding to the aforementioned immune complex without cross-recognition with free single-chain antibodies or small-molecule estradiol, thereby ensuring the accuracy and reliability of the detection.

[0023] In the screening process for complex antibodies, the E2 antibody 24H6 was first modified to a 24H6-scFv-avi-His form. This modification aimed to reduce interference from the primary antibody during complex immunization, ensuring a smooth screening process. Simultaneously, an avi tag was added to the C-terminus of the antibody. This design facilitates subsequent site-specific labeling of biotin at the C-terminus, making the binding site of the immune complex more easily exposed and preventing it from being masked by other substances during detection, thereby improving the sensitivity and accuracy of the detection. Furthermore, the application of a two-step detection method further confirms the presence of the anti-complex antibody, significantly improving the efficiency and accuracy of the screening process and ensuring that the final complex antibody possesses optimal detection performance.

[0024] In some embodiments, the sequence of the heavy chain variable region of the sandwich antibody for detecting estradiol is shown in SEQ.ID No. 8, and the sequence of the light chain variable region is shown in SEQ.ID No. 9.

[0025] In some embodiments, the heavy chain sequence of the sandwich antibody for detecting estradiol is shown in SEQ ID No. 10, and the light chain sequence is shown in SEQ ID No. 11.

[0026] This invention aims to provide a highly efficient reagent for detecting estradiol based on a complex antibody sandwich method. The core components of this reagent include a specially formulated single-chain antibody and a corresponding complex antibody. Specifically, the single-chain antibody of this invention possesses high specificity, accurately recognizing and tightly binding to small molecule estradiol to form a stable immune complex. Simultaneously, the complex antibody of this invention also exhibits excellent specificity, specifically recognizing and tightly binding to the aforementioned immune complex, while showing no cross-reactivity with free single-chain antibody and small molecule estradiol. This unique design makes the reagent perform exceptionally well in the application of the complex antibody sandwich method for detecting estradiol, significantly improving the sensitivity and accuracy of small molecule estradiol detection, thereby providing more reliable technical support for research and clinical diagnosis in related fields.

[0027] The reagents used in the embodiments of the present invention are as follows:

[0028] The plasmid extraction kit was a high-purity plasmid mini-extraction kit (DP107), purchased from Tiangen Biotech (Beijing) Co., Ltd.; the 293F cells were from Xiamen University; the amplification primers were synthesized by Guangzhou Qingke Biotechnology Co., Ltd.; the Escherichia coli DH5α competent strain was purchased from Tiangen Biotech (Beijing) Co., Ltd.; the LB liquid culture medium was purchased from Sigma-Aldrich; the molecular amplification reagents and cloning ligation kit were purchased from Takara; the 96-well plates used for PCR were purchased from Axygen; and the fresh culture medium was OPM293. CD05 medium was purchased from Shanghai OPMI Biotechnology Co., Ltd.; the feed medium was OPM293 ProFeed medium, purchased from Shanghai OPMI Biotechnology Co., Ltd.; the OptiMEM medium was purchased from Thermo Fisher Scientific (China) Co., Ltd.; the nickel column and protein G column were purchased from Huiyan Biotechnology Co., Ltd.; the lymphocyte separation medium was purchased from Dayou; the BirA biotin ligase was purchased from Yijin Biotechnology; the SA was purchased from Tiandi Renhe Biotechnology Co., Ltd.; the goat anti-mouse IgG-HRP secondary antibody was purchased from Sigma; the ELISA plate and cell plate were purchased from Guangzhou Jetech Biofiltration Co., Ltd.; the elution buffer and preservation solution were purchased from Sinopharm Group; the analytical methods were SDS-PAGE protein gel electrophoresis, ELISA detection, and agarose gel electrophoresis.

[0029] Example 1

[0030] E2 single-chain antibody preparation process (Ab1)

[0031] S1.1 immunized mice

[0032] Purchase E2-KLH (Creative Diagnostics) as the immunogen, dissolve it, and emulsify it evenly with an equal volume of Freund's complete adjuvant (Sigma). Take 6-8 week old SPF-grade Balb / c mice and inject 200 μg / mouse subcutaneously at multiple sites. Two weeks later, emulsify the antigen with Freund's incomplete adjuvant and inject 100 μg / mouse subcutaneously at multiple sites. Administer two booster immunizations. Three days before fusion, administer a shock immunization via intraperitoneal injection.

[0033] S1.2 Cell Fusion and Subcloning Screening

[0034] Spleens were harvested from immunized mice, and dispersed single spleen cells were obtained by grinding and separation. Spleen cells and myeloma cells were fused using an electrofusion apparatus. After standing, the cells were transferred to culture medium and seeded into 96-well plates. The medium was changed after one week, and the supernatant was used for ELISA detection. The supernatant was coated with E2-BSA (Creative Diagnostics), and goat anti-mouse IgG-HRP (sigma) was used as the enzyme-labeled secondary antibody. The cell supernatant was evaluated, and positive wells were selected for further limiting dilution subcloning. ELISA detection was repeated after one week of culture, repeated 3-4 times until all wells were positive and the cells in the wells were single colonies. The specific hybridoma cell line 24H6 was obtained through expansion. The detection results are shown in Table 1.

[0035] Table 1

[0036]

[0037] S1.3 gene retrieval

[0038] The 24H6 hybridoma cell line was expanded, mRNA was extracted, and cDNA product was obtained by reverse transcription. The product was inserted into the pMD-19T vector after being subjected to an A-addition reaction with Taq DNA polymerase. The cells were then transformed into DH5α competent cells, and 10 plaques of heavy chain and light chain gene clones were sent to a gene sequencing company for sequencing.

[0039] Sequence analysis of the S1.4 antibody gene

[0040] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the correct heavy and light chain variable region genes were identified using snapgene software.

[0041] Construction of S1.5 scFv recombinant antibody expression plasmid

[0042] A recombinant antibody expression vector for scFv was constructed using pcDNA3.1 as the vector via three GGGGS flexible linkers. Based on the sequencing results of the variable region gene in pMD-19T, light and heavy chain specific primer pairs for homologous recombination were designed. Light and heavy chain gene fragments were obtained by overlapping PCR amplification. Homologous primers were designed for the existing pcDNA3.1 vector with signal peptide, avi, and His tags. Vector fragments were recovered by electrophoresis after PCR amplification. Homologous ligation of the gene and vector fragments was performed, and the cells were transformed into DH5α competent cells. After positive colony PCR verification, the cells were sequenced. Normal colonies were selected for expansion culture, and the vector plasmid containing the heavy and light chain variable regions was extracted and abbreviated as pcDNA3.1-24H6-scFv-avi-His.

[0043] S1.6 recombinant antibody expression

[0044] The target plasmid obtained in the above steps was transfected into 293F cells using the PEI transfection method. After successful transfection, the cells were incubated in an incubator for 24 hours, and then an equal volume of fresh OPM-293CD05 culture medium was added. The cells were allowed to grow to a size of 4 × 10⁶ cells / year. 6 At a density of cells / mL, 1% OPM-293ProFeed supplemental medium was added daily, and the cells were returned to an incubator at 37°C, 8% CO2, and 120 rpm for 96 hours to obtain 293F cell culture medium expressing the 24H6-scFv recombinant antibody.

[0045] S1.7 Recombinant Antibody Purification

[0046] The 293F cell culture medium expressing the 24H6-scFv recombinant antibody obtained in the above steps was centrifuged at 9000 rpm for 15 min. The supernatant was collected, filtered through a 0.22 μm filter membrane, and purified using a nickel column. Before use, the nickel column was equilibrated with 50 column volumes of equilibration buffer. The supernatant obtained by centrifugation was then passed through the column, and eluted sequentially with 20 column volumes of elution buffer containing 0 mM, 50 mM, 150 mM, and 300 mM imidazole. Elution buffers with different imidazole concentrations were collected, and the eluent containing the target protein was selected. This eluent was dialyzed into pH 6.0 PBS to obtain the 24H6-scFv antibody. The purification results are as follows. Figure 1 As shown.

[0047] The sequence information of the anti-small molecule antibody 24H6-scFv, obtained through sequencing, is as follows:

[0048] 24H6-scFv chain

[0049] QVQLQQSAAEVARPGASVKMSCKASGYNFWVKQRPGQGLEWIGYINPSSGHTDYNQKFKDKTTLTADKFSNTAYMQLTSLTSEDSAVYYCASPYYRYSAWFAYWGQGTLVTVSAGGGGSGGGGS GGGGSDIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKVLIYRASNLESGIPARFSGSGSRTDFTLTIKPVEADDVASYYCQQTNEDPWTFGGGTKLEIKRA(SEQ.ID No.1)

[0050] Example 2

[0051] Anti-complex antibody preparation process (Ab2)

[0052] S2.1 immunized mice

[0053] Both the E2 natural standard (Sigma) and the E2 antibody (24H6-scFv) were dialyzed into 0.01M PBS at pH 7.4 and incubated at a molar ratio of ≥20:1 at 37°C for 1 hour. The mixture was then filtered into 0.01M PBS to remove excess small molecules and used as the immunogen.

[0054] Immunogen was emulsified with an equal volume of Freund's complete adjuvant (Sigma) until homogeneous. 200 μg / mouse was injected subcutaneously at multiple sites in 6-8 week old SPF-grade Balb / c mice. Two weeks later, 100 μg / mouse was injected subcutaneously at multiple sites in the antigen emulsified with Freund's incomplete adjuvant. Two booster immunizations were given. Three days before fusion, a shock immunization was administered via intraperitoneal injection.

[0055] S2.2 Cell Fusion and Subcloning Screening

[0056] Spleens were taken from immunized mice, and dispersed individual spleen cells were obtained by grinding and separation. Spleen cells and myeloma cells were fused using an electrofusion instrument. After standing, the cells were transferred into culture medium and plated into 96-well plates. The medium was changed after one week, and the supernatant was collected for ELISA detection.

[0057] The detection method consists of two steps. In the first step, SA (Tiandi Renhe, 1 μg / ml) is used as the coating antigen. After blocking, 24H6-scFv-bio (referring to 24H6 labeled with biotin) or a complex of 24H6-scFv-bio+E2 is added separately. Then, cell supernatant is added, and the chromogenic agent is goat anti-mouse IgG-HRP secondary antibody. Finally, the positive wells that show low reactivity with 24H6-scFv-bio and strong reactivity with 24H6-scFv-bio+E2 are selected. The presence or absence of the anti-complex antibody is further confirmed by a competitive method.

[0058] Step 2: SA (Heaven, Earth, and Human, 1 μg / ml) was used as the coating antigen. After blocking, 24H6-scFv-bio or 24H6-scFv-bio+E2 complexes were added respectively. The cell supernatant was first reacted with 24H6-scFv at 37℃ for 1 hour. Then, the reaction solution was added to the plate. The chromogenic agent was goat anti-mouse IgG-HRP secondary antibody. Finally, the positive wells that did not react with 24H6-scFv-bio but reacted strongly with 24H6-scFv-bio+E2 were selected.

[0059] The test results are shown in Table 2.

[0060] Table 2

[0061]

[0062] Subcloning was further performed using the limiting dilution method. After one week of culture, ELISA was performed again. Positive wells that did not react with Bio-Fab but reacted strongly with Bio-Fab+E2 were selected for limiting dilution. This process was repeated 3-4 times until all wells were positive and the cells in the wells were monocolony. Then, cell wells that did not react with Bio-Fab but reacted strongly with Bio-Fab+E2 were selected for expansion culture to obtain the specific hybridoma cell line 55C7.

[0063] The test results are shown in Table 3.

[0064] Table 3

[0065]

[0066] Preparation of ascites fluid from S2.3 positive cell lines

[0067] Single-colony cells were expanded and cultured, then injected into mice pre-vaccinated with IFA to prepare ascites. The ascites was collected to obtain complex antibodies, which were then purified by affinity chromatography using a protein G column.

[0068] S2.4 gene retrieval

[0069] The 55C7 hybridoma cell line was expanded, mRNA was extracted, and cDNA product was obtained by reverse transcription. The product was inserted into the pMD-19T vector after being subjected to an A-addition reaction with Taq DNA polymerase. The cells were then transformed into DH5α competent cells, and 10 plaques of heavy chain and light chain gene clones were sent to a gene sequencing company for sequencing.

[0070] Sequence analysis of the S2.5 antibody gene

[0071] The gene sequences obtained from the sequencing were analyzed in the IMGT antibody database, and the correct heavy and light chain variable region genes were identified using snapgene software.

[0072] Construction of S2.6 recombinant antibody expression plasmid

[0073] A recombinant antibody expression vector was constructed using the existing signal peptide and constant region pTT5 as the vector. Based on the sequencing results of the variable region gene in pMD-19T antibody, light and heavy chain specific primer pairs for homologous recombination were designed (primers are shown in Table 4). Light and heavy chain gene fragments were obtained by PCR amplification. Homologous primers were designed for the existing signal peptide and constant region pTT5 vector. After PCR amplification, the vector fragments were recovered by electrophoresis. Homologous ligation of the gene fragments and vector fragments was performed, and the cells were transformed into DH5α competent cells. After positive colony PCR verification, the cells were sent for sequencing. Normal colonies were picked and expanded, and vector plasmids with the heavy and light chain variable regions were extracted and abbreviated as pTT5-55C7 H chain and pTT5-55C7 L chain.

[0074] Table 4 Primer sequence list

[0075]

[0076] Note: Lowercase letter sequences represent homologous links; H indicates a heavy chain, and L indicates a light chain.

[0077] S2.7 recombinant antibody expression

[0078] The target plasmid obtained in the above steps was transfected into 293F cells using the PEI transfection method. After successful transfection, the cells were incubated in an incubator for 24 hours, and then an equal volume of fresh OPM-293CD05 culture medium was added. The cells were allowed to grow to a size of 4 × 10⁶ cells / year. 6 At a density of cells / mL, 1% (v / v) of OPM-293ProFeed supplemental medium was added daily, and the cells were returned to an incubator at 37°C, 8% CO2, and 120 rpm for 96 hours to obtain 293F cell culture medium expressing recombinant anti-complex antibody 55C7.

[0079] S2.8 recombinant antibody purification

[0080] The 293F cell culture medium obtained in the above steps was centrifuged at 9000 rpm for 15 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. Protein G was used for protein purification. Before use, the Protein G column was equilibrated with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl at pH 7.4. The supernatant obtained by centrifugation was then passed through the column, and the column was washed with 5 column volumes of equilibration buffer containing 0.02 M PB and 0.15 M NaCl at pH 7.4. Elution was then performed with 5 column volumes of elution buffer containing 0.1 M Glycine-HCl at pH 2.7, and the elution buffer was immediately neutralized with 1.0 M Tris-HCl at pH 9.0. Finally, the purified 55C7 antibody was dialyzed into PBS at pH 8.0 to obtain the recombinant anti-complex antibody 55C7. The purification results are as follows. Figure 2 As shown.

[0081] The sequence information of the anti-complex antibody 55C7, obtained through sequencing, is as follows:

[0082] L-CDR1:KASQDVGTSVA(SEQ.ID No.2)

[0083] L-CDR2: WASTRHT (SEQ.ID No. 3)

[0084] L-CDR3:QQYSNYPYT(SEQ.ID No.4)

[0085] H-CDR1: NYWIA (SEQ.ID No. 5)

[0086] H-CDR2:DIYPGGIYTNYNEKFKG(SEQ.ID No.6)

[0087] H-CDR3: DDEFAY (SEQ.ID No.7)

[0088] Light chain variable region VL:

[0089] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTSVAWYQQKPGQSPKLLI YWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSNYPYTFGG GTKLEIKRA(SEQ.ID No.8)

[0090] Heavy chain variable region VH:

[0091] QVQLQQSGAELVRPGASVKMSCKAAGFTFTNYWIAWVRQRPGHGLE WIGDIYPGGIYTNYNEKFKGQATLTADTSSSTAYMQLSSLTSEDSAIYYCARD DEFAYWGQGTLVTVSA(SEQ.ID No.9)

[0092] Light chain:

[0093] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSNYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ.IDNo.10)

[0094] Heavy chain:

[0095] QVQLQQSGAELVRPGASVKMSCKAAGFTFTNYWIAWVRQRPGHGLEWIGDIYPGGIYTNYNEKFKGQATLTADTSSSTAYMQLSSLTSEDSAIYYCARDDEFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ.IDNo.11)

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A sandwich-type single-chain antibody for detecting estradiol, characterized in that, Its sequence is shown in SEQ.ID No.

1.

2. A sandwich antibody for detecting estradiol, characterized in that, The sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ.ID No.2, SEQ.ID No.3, and SEQ.ID No.4, respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ.ID No.5, SEQ.ID No.6, and SEQ.ID No.7, respectively.

3. The sandwich antibody for detecting estradiol according to claim 2, characterized in that, The sequence of its heavy chain variable region is shown in SEQ.ID No.8, and the sequence of its light chain variable region is shown in SEQ.ID No.

9.

4. The sandwich antibody for detecting estradiol according to claim 2, characterized in that, The sequence of its heavy chain is shown in SEQ.ID No.10, and the sequence of its light chain is shown in SEQ.ID No.

11.

5. A reagent for detecting estradiol using a complex antibody sandwich method, characterized in that, It includes the single-chain antibody of claim 1 and the complex antibody of any one of claims 2 to 4.

6. A reagent for detecting estradiol, comprising the single-chain antibody of claim 1.

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