Single-chain antibody and compound antibody for sandwich detection of estradiol and application of single-chain antibody and compound antibody

Abstract

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

Description

Technical Field

[0001] The present invention relates to a single-chain antibody and a complex antibody for sandwich detection of estradiol and their applications, belonging to the technical field of estradiol detection. Background Art

[0002] Estradiol (E2) is a steroid estrogen, and it is the most abundant and most active one among estrogens. Estradiol is mainly secreted by ovarian follicular granulosa cells, corpus luteum and placenta during pregnancy. In adult women, estradiol shows cyclic changes with the menstrual cycle; while in men, estradiol is mainly synthesized and secreted by testicular interstitial cells.

[0003] The main physiological functions of estradiol include promoting the development of female reproductive organs and secondary sexual characteristics, regulating the functions 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 also used to diagnose and differentiate whether a woman ovulates. In normal women, estrogen shows cyclic changes, while there is no cyclic change in hormones without ovulation.

[0004] Clinically, the detection of estradiol has certain value for diagnosing endocrine and gynecological diseases such as precocious puberty and dysplasia. It can be used for the diagnosis of early pregnancy, ectopic pregnancy, threatened abortion, the diagnosis and monitoring of female endocrine, breast and gynecological diseases, and the auxiliary diagnosis of some diseases.

[0005] Currently, the mainstream methodology for detecting E2 clinically is immunoassay. Since E2 belongs to a small molecule compound with a single antigenic determinant and cannot be detected by sandwich assay with two different antibodies, competitive assay is generally used in clinical immunoassay. However, the competitive assay itself has methodological defects such as insufficient precision, insufficient accuracy, susceptibility to interference, and narrow linear range.

[0006] Currently, the more feasible method is the complex antibody sandwich assay. The specific detection principle is as follows: an anti-E2 antibody (the first antibody) forms an immune complex with the small molecule to be detected, and another anti-complex antibody (the second antibody) binds to the aforementioned immune complex to form an immune sandwich complex. The recognition site is the new epitope formed after the first antibody binds to the small molecule, and it is required that the complex antibody does not recognize the free first antibody and the small molecule. By using the complex antibody sandwich assay, the sensitivity and accuracy of small molecule detection can be greatly improved. However, due to the difficulty in screening complex antibodies, there is still a lack of highly active E2 complex antibodies in the prior art. Summary of the Invention

[0007] The present invention provides a single-chain antibody, a complex antibody for sandwich detection of estradiol and their applications, which can effectively solve the above problems.

[0008] The present invention is implemented as follows:

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

[0010] A complex antibody for sandwich detection of estradiol, the sequences of CDR1, CDR2, and CDR3 in its 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 its 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 complex antibody for sandwich detection of 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 sequence of the heavy chain of the complex antibody for sandwich detection of estradiol is shown in SEQ.IDNo.10, and the sequence of the light chain is shown in SEQ.ID No.11.

[0013] A reagent for detecting estradiol by complex antibody sandwich method, comprising the single-chain antibody and the complex antibody.

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

[0015] The beneficial effects of the present invention are:

[0016] The single-chain antibody of the present invention has unique specific binding ability, can accurately bind to the small molecule estradiol, and thus form a stable immune complex. The complex antibody of the present invention can not only specifically recognize and tightly bind to this immune complex, but also does not bind to the single-chain antibody and small molecule estradiol in the free state. Based on this property, the complex antibody of the present invention can be widely used in the complex antibody sandwich detection technology for the accurate detection of estradiol. Through this technical means, the sensitivity and accuracy of the detection of small molecule estradiol can be greatly improved, providing a more reliable and efficient detection tool for scientific research and clinical applications in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 thus 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.

[0018] Figure 1 Result diagram of expressing and purifying 24H6-scFv antibody in 293F cells for 24 hours.

[0019] Figure 2 Result diagram of expressing and purifying 55C7 antibody in 293F cells. Detailed implementation mode

[0020] 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 in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to 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 claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0021] An embodiment of the present invention provides a single-chain antibody for sandwich detection of estradiol, and its specific amino acid sequence is detailedly listed in SEQ.ID No.1. This single-chain antibody has high specificity and can precisely bind to the small molecule estradiol, thereby forming a stable immune complex, providing a solid foundation for subsequent detection work.

[0022] The embodiment of the present invention further provides a complex antibody for sandwich detection of estradiol. The heavy chain variable region of this complex antibody contains three complementary determining regions (CDRs), and the amino acid sequences of its CDR1, CDR2, and CDR3 are detailedly listed in SEQ.ID No.2, SEQ.ID No.3, and SEQ.ID No.4 respectively; the light chain variable region also contains three complementary determining regions (CDRs), and the amino acid sequences of its CDR1, CDR2, and CDR3 are detailedly listed in SEQ.ID No.5, SEQ.ID No.6, and SEQ.ID No.7 respectively. This complex antibody has high specificity and can precisely bind to the aforementioned immune complex without cross-recognizing free single-chain antibody or small molecule estradiol, thus ensuring the accuracy and reliability of the detection.

[0023] In the screening process of complex antibodies, the E2 antibody 24H6 was first modified to construct the 24H6-scFv-avi-His format. The purpose of this modification is to reduce the interference of the first antibody during the complex immunization process and ensure the smooth progress of the screening process. At the same time, an avi tag was added to the C-terminus of the antibody. This design facilitates the subsequent site-specific labeling of biotin at the C-terminus, making the binding site of the immune complex easier to expose, avoiding being covered by other substances during the detection process, thereby improving the sensitivity and accuracy of the detection. In addition, through the application of the two-step detection method, the presence or absence of anti-complex antibodies can be further confirmed, which greatly improves the efficiency and accuracy of the screening process and ensures that the final complex antibody has the best detection performance.

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

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

[0026] The embodiment of the present invention aims to provide an efficient reagent for detecting estradiol based on the complex antibody sandwich method, the core components of which include a specially prepared single-chain antibody and a complex antibody matched therewith. Specifically, the single-chain antibody of the present invention has a high degree of specificity, and can accurately identify and tightly bind to small molecule estradiol, thereby forming a stable immune complex. At the same time, the complex antibody of the present invention also has excellent specificity, and it can specifically identify and tightly bind to the immune complex formed above, while it does not cross-react at all with the single-chain antibody and small molecule estradiol in the free state. This unique design enables the reagent to perform well in the application of the complex antibody sandwich method for detecting estradiol, and can significantly improve the sensitivity and accuracy of the detection of small molecule estradiol, thereby providing more reliable technical support for scientific research and clinical diagnosis in related fields.

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

[0028] The extraction kit for the target plasmid is a high-purity plasmid miniprep midiprep kit (DP107), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the 293F cells are from Xiamen University; the amplification primers are synthesized by Guangzhou Tsingke Biotechnology Co., Ltd.; the Escherichia coli DH5a competent strain is purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the LB liquid medium reagent is purchased from sigma company; the molecular amplification reagent and cloning ligation kit are purchased from Takara; the 96-well plate for PCR is purchased from Axygen; the fresh medium is OPM293 CD05 medium, purchased from Shanghai OPMI Biotechnology Co., Ltd.; the feeding medium is OPM293ProFeed medium, purchased from Shanghai OPMI Biotechnology Co., Ltd.; the optiMEM medium is purchased from Thermo Fisher Scientific (China) Co., Ltd.; the nickel column and protein G column are purchased from Huiyan Biotechnology Co., Ltd.; the lymphocyte separation solution is purchased from Dayou; the BirA biotin ligase is purchased from Yijin Biology; the SA is purchased from Tiandi Renhe Biotechnology Co., Ltd.; the goat anti-mouse IgG-HRP secondary antibody is purchased from sigma; the Elisa plate and cell plate are purchased from Guangzhou Jet Bio-Filtration Co., Ltd.; the eluent and preservation solution reagents are purchased from Sinopharm Group; the analysis methods are SDS-PAGE protein gel electrophoresis, Elisa detection and agarose gel electrophoresis methods.

[0029] Example 1

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

[0031] S1.1 Immunize mice

[0032] Purchase E2-KLH (Creative Diagnostics) as the immunogen. After dissolution, 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 points. After 2 weeks, emulsify the antigen with Freund's incomplete adjuvant and inject 100 μg / mouse subcutaneously at multiple points. Boost the immunization 2 times, and perform a boost injection intraperitoneally 3 days before fusion.

[0033] S1.2 Cell fusion and subclonal screening

[0034] The spleen of immunized mice was taken, ground and separated to obtain dispersed single spleen cells. The spleen cells and myeloma cells were fused using an electrofusion apparatus. After standing, the cells were taken and placed into a culture medium and plated in a 96-well plate. The medium was changed after one week, and the supernatant was taken for ELISA detection. E2-BSA (Creative Diagnostics) was coated, and goat anti-mouse IgG-HRP (sigma) was used as the enzyme-labeled secondary antibody to evaluate the cell supernatant. Positive wells were selected and further subcloned by the limited dilution method. After culturing for one week, ELISA detection was continued and repeated 3 - 4 times until all the detection wells were positive and the cells in the wells were single colonies. The specific hybridoma cell line 24H6 was obtained by expansion culture. The detection results are shown in Table 1.

[0035] Table 1

[0036]

[0037] Retrieval of S1.3 gene

[0038] The 24H6 hybridoma cell line was expanded, mRNA was extracted, and cDNA products were obtained by reverse transcription. After the addition of A reaction to the products using Taq DNA polymerase, they were inserted into the pMD-19T vector and transformed into DH5α competent cells. Ten plaques each of the heavy chain and light chain gene clones were sent to a gene sequencing company for sequencing.

[0039] Sequence analysis of S1.4 antibody gene

[0040] The gene sequences obtained from the above sequencing were analyzed in the IMGT antibody database, and the snapgene software was used for analysis to determine the correct heavy chain and light chain variable region genes.

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

[0042] Using pcDNA3.1 as the vector, an scFv recombinant antibody expression vector was constructed through 3 GGGGS flexible linkers. According to the antibody variable region gene sequencing results in the above pMD-19T, specific primer pairs for heavy and light chains for homologous recombination were designed, and the light chain and heavy chain gene fragments were obtained by overlapping PCR amplification. Homologous primers were designed for the pcDNA3.1 vector with existing signal peptide, avi, and His tags. After PCR amplification, the vector fragment was recovered by electrophoresis. The gene fragment and the vector fragment were ligated homologously, transformed into DH5α competent cells, and positive colonies were verified by colony PCR and then sent for sequencing. Normal colonies were picked for expanded culture, and the vector plasmid ligated with the heavy chain and light chain variable regions was extracted, simply referred to as pcDNA3.1-24H6-scFv-avi-His.

[0043] Expression of S1.6 recombinant antibody

[0044] The target plasmid obtained in the above steps was transfected into 293F cells by the PEI transfection method. After successful transfection, the cells were cultured in an incubator for 24 h, and then fresh OPM-293CD05 medium with the same volume as the culture medium was added. When the cell density reached 4×10 6 cells / mL, 1% (v / v) OPM-293ProFeed feeding medium was added every day, and the cells were returned to an incubator at 37 °C, 8% CO 2 , and cultured at 120 rpm for another 96 h to obtain the 293F cell culture solution expressing the 24H6-scFv recombinant antibody.

[0045] Purification of the S1.7 recombinant antibody

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

[0047] Sequencing showed that the sequence information of the anti-small molecule antibody 24H6-scFv was as follows:

[0048] 24H6-scFv chain

[0049] QVQLQQSAAEVARPGASVKMSCKASGYNFWVKQRPGQGLEWIGYINPSSGHTDYNQKFKDKTTLTADKFSNTAYMQLTSLTSEDSAVYYCASPYYRYSAWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKVLIYRASNLESGIPARFSGSGSRTDFTLTIKPVEADDVASYYCQQTNEDPWTFGGGTKLEIKRA(SEQ.ID No.1)

[0050] Example 2

[0051] Preparation process of the anti-complex antibody (Ab2)

[0052] S2.1 Immunization of mice

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

[0054] The immunogen was emulsified evenly with an equal volume of Freund's complete adjuvant (Sigma). SPF-grade Balb / c mice at 6 - 8 weeks of age were subcutaneously injected with 200 μg / mouse at multiple points. Two weeks later, the antigen was emulsified with Freund's incomplete adjuvant and subcutaneously injected with 100 μg / mouse at multiple points for two booster immunizations. Three days before fusion, an intraperitoneal injection was given for boosting.

[0055] S2.2 Cell Fusion and Subcloning Screening

[0056] The spleen of the immunized mice was taken, ground and separated to obtain dispersed single spleen cells. The spleen cells and myeloma cells were fused using an electrofusion instrument. After standing, the cells were taken and put into a culture medium and plated in a 96-well plate. The medium was changed after one week, and the supernatant was taken for Elisa detection.

[0057] The detection method was divided into two steps. First step: SA (Tian Di Ren He, 1 μg / ml) was used as the coating antigen. After blocking, 24H6-scFv-bio (indicating that 24H6 was labeled with biotin) or the complex of 24H6-scFv-bio + E2 was added respectively, and then the cell supernatant was added. The chromogenic agent was the secondary antibody goat anti-mouse IgG-HRP. Finally, the positive wells with low reactivity to 24H6-scFv-bio and strong reactivity to 24H6-scFv-bio + E2 were selected, and the presence or absence of anti-complex antibodies was further confirmed by the competitive method.

[0058] Second step: SA (Tian Di Ren He, 1 μg / ml) was used as the coating antigen. After blocking, 24H6-scFv-bio or the complex of 24H6-scFv-bio + E2 was added respectively. The cell supernatant was first reacted with 24H6-scFv at 37°C for 1 h, and then the reaction solution was taken and added to the above plate. The chromogenic agent was the secondary antibody goat anti-mouse IgG-HRP. Finally, the positive wells with no reactivity to 24H6-scFv-bio but strong reactivity to 24H6-scFv-bio + E2 were selected.

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

[0060] Table 2

[0061]

[0062] Further subcloning was carried out by the limiting dilution method. After culturing for one week, ELISA detection was continued. Positive wells that showed no reaction with Bio-Fab but strong reaction with Bio-Fab+E2 were selected for limiting dilution and repeated 3-4 times until all the detection wells were positive and the cells in the wells were single colonies. Then, the cell wells that showed no reaction with Bio-Fab and the strongest reaction with Bio-Fab+E2 were selected for expansion culture to obtain the specific hybridoma cell line 55C7.

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

[0064] Table 3

[0065]

[0066] Ascites preparation of S2.3 positive cell line

[0067] The single colony cells were expanded in culture and injected into mice pre-inoculated with IFA to prepare ascites. The ascites were collected to obtain the complex antibody, which was affinity purified through a protein G column.

[0068] Retrieval of S2.4 gene

[0069] The 55C7 hybridoma cell line was expanded in culture, mRNA was extracted, and cDNA products were obtained through reverse transcription. After the addition of A reaction to the products with Taq DNA polymerase, they were inserted into the pMD-19T vector and transformed into DH5α competent cells. Ten plaques were taken from each of the heavy chain and light chain gene clones and sent to a gene sequencing company for sequencing.

[0070] Sequence analysis of S2.5 antibody gene

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

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

[0073] Using pTT5 with an existing signal peptide and constant region as a vector, a recombinant antibody expression vector was constructed. According to the antibody variable region gene sequencing results in the above pMD-19T, heavy and light chain specific primer pairs for homologous recombination were designed (the primers are shown in Table 4). The light and heavy chain gene fragments were obtained by PCR amplification. Homologous primers were designed for the pTT5 vector with the existing signal peptide and constant region, and the vector fragment was recovered by electrophoresis after PCR amplification. The gene fragment and the vector fragment were ligated homologously, transformed into DH5α competent cells, and after verifying positive by colony PCR, it was sent for sequencing. Normal colonies were picked for enlarged culture, and the vector plasmids ligated with the heavy and light chain variable regions were extracted, simply named pTT5-55C7 H chain and pTT5-55C7 L chain.

[0074] Table 4 Primer Sequence Table

[0075]

[0076] Note: The lowercase letter sequences are the homologous ligation parts, H is the heavy chain, and L is the light chain

[0077] S2.7 Recombinant Antibody Expression

[0078] The target plasmid obtained in the above step was transfected into 293F cells by the PEI transfection method. After successful transfection, it was placed in an incubator for 24 h, and then fresh OPM-293CD05 medium with the same volume as the culture medium was added. When the cell density reached 4×10 6 cells / mL, 1% (v / v) of OPM-293ProFeed feeding medium was added every day, and it was returned to the incubator at 37 °C, 8% CO2, and 120 rpm for continued culture for 96 h to obtain the 293F cell culture solution expressing the recombinant anti-complex antibody 55C7.

[0079] S2.8 Recombinant Antibody Purification

[0080] The culture medium of the 293F cells expressing the product obtained in the above steps was centrifuged at 9000 rpm for 15 min, and the supernatant was collected, filtered through a 0.22-μm filter membrane, and Protein G was selected for protein purification; before use, the Protein G column was equilibrated with 5 column volumes of an equilibration buffer containing 0.02 M PB and 0.15 M NaCl, with a pH of 7.4. Then, the supernatant obtained by centrifugation was passed through the column, and the column was rinsed with 5 column volumes of an equilibration buffer containing 0.02 M PB and 0.15 M NaCl, with a pH of 7.4. Subsequently, it was eluted with 5 column volumes of an elution buffer containing 0.1 M Glycine-HCl, with a pH of 2.7, and the eluate was immediately neutralized with a neutralization solution of 1.0 M Tris-HCl, with a pH of 9.0. Finally, the purified 55C7 antibody was dialyzed into PBS with a pH of 8.0 to obtain the recombinant anti-complex antibody 55C7. The purification results are as Figure 2 shown.

[0081] Sequencing showed that the sequence information of the anti-complex antibody 55C7 was 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] QVQLQQSGAELVRPGASVKMSCKAAGFTFTNYWIAWVRQRPGHGLEWIGDIYPGGIYTNYNEKFKGQATLTADTSSSTAYMQLSSLTSEDSAIYYCARDDEFAYWGQGTLVTVSA(SEQ.ID No.9)

[0092] Light chain:

[0093] DIVMTQSHKFMSTSVGDRVSITCKASQDVGTSVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSNYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ.IDNo.10)

[0094] Heavy chain:

[0095] QVQLQQSGAELVRPGASVKMSCKAAGFTFTNYWIAWVRQRPGHGLEWIGDIYPGGIYTNYNEKFKGQATLTADTSSSTAYMQLSSLTSEDSAIYYCARDDEFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ.IDNo.11)

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

Claims

1. A single-chain antibody for sandwich detection of estradiol, characterized in that: Its sequence is shown as SEQ.ID No.

1.

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

9.

4. The sandwich complex 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 by a complex antibody sandwich method, characterized in that: It comprises the single-chain antibody according to claim 1 and the complex antibody according to any one of claims 2 to 4.

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

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