CN117903305A

CN117903305A - Monoclonal antibody against estradiol, detection reagent based on monoclonal antibody and application of monoclonal antibody

Info

Publication number: CN117903305A
Application number: CN202311665026.3A
Authority: CN
Inventors: 戴瞻; 董万春; 陶言梦; 高歌; 胡斌; 宋丹
Original assignee: Nanjing Oukai Biotechnology Co ltd
Current assignee: Nanjing Oukai Biotechnology Co ltd
Priority date: 2023-12-05
Filing date: 2023-12-05
Publication date: 2024-04-19

Abstract

The present invention relates to monoclonal antibodies or antigen binding fragments thereof directed against estradiol (E2), related nucleic acid products, cell products, detection reagents or kits, and methods of making and using the same. The monoclonal antibody or antigen binding fragment thereof can bind E2 with high specificity and high affinity, thereby improving the detection accuracy and sensitivity of E2; in addition, the invention also develops a high-performance paired antibody suitable for double-antibody sandwich method immunodetection, thereby breaking through the limitations of low sensitivity and poor clinical relevance of the traditional competitive method detection reagent, realizing the E2 double-antibody sandwich method immunodetection and having important significance for early diagnosis and treatment of ovarian diseases and domestic substitution of core antibody raw materials.

Description

Monoclonal antibody against estradiol, detection reagent based on monoclonal antibody and application of monoclonal antibody

Technical Field

The invention relates to the technical field of monoclonal antibody preparation and immunological detection, in particular to a monoclonal antibody aiming at estradiol (E2), a detection reagent based on the monoclonal antibody and application thereof.

Background

Estradiol (E2 for short in english) is a steroid hormone mainly secreted by the ovarian follicles, the corpus luteum and the placenta during pregnancy, and has a molecular weight of about 272Da. Estrone (E1 for short), estradiol (E2 for short) and estriol (E3 for short) are main forms of in-vivo estrogens, and E2 is the hormone with the highest content and the strongest biological activity in the estrogens, and has the main functions of promoting the transformation of endometrium into proliferation period and promoting the development of female secondary sex characteristics, and has important clinical significance in the aspect of evaluating ovarian function. In addition, E2 has obvious influence on endocrine system, cardiovascular system, metabolism of human body, bone growth and other aspects.

E1 and E2 can be mutually converted, E3 is a metabolite of E2, and the three are very similar in structure, so that the requirement on the specificity of the antibody is high. The E2 antibodies on the market at present have the problem of cross reaction, and particularly have stronger interference on E1. The existing E2 immunoassay method mainly comprises a chemiluminescence method and an immunochromatography method, the immunoassay methods mainly adopt the principle of a competition method, reagents are deficient in specificity and sensitivity, and the linear range is narrow, so that the graduation of a low-value end region and a high-value end region of a clinical sample is small, and the detection result is misjudged.

In view of the above problems in the prior art, development of a monoclonal antibody with high specificity and high affinity for E2 is currently urgently needed to meet the requirements of immunological detection, and in particular, development of a high-performance pairing antibody is particularly important to break through the limitations of the traditional competition method and realize detection of E2 by a double-antibody sandwich method.

Disclosure of Invention

Object of the Invention

In view of the problems or needs in the prior art, the present invention aims to provide a monoclonal antibody or an antigen binding fragment thereof with high specificity and high affinity for estradiol (E2 for short), and develop a high-performance pairing antibody suitable for the immunological detection by a double antibody sandwich method.

Solution scheme

In order to achieve the above purpose, the invention obtains monoclonal antibodies or antigen binding fragments thereof with high specificity and high affinity for estradiol (E2 for short), related nucleic acids and cell products thereof, a preparation method and application thereof, and an immunodetection reagent based on the same through a large number of screening, thereby providing a basis for the high-specificity and high-sensitivity immunodetection of E2.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides a monoclonal antibody or antigen-binding fragment thereof directed against estradiol, said monoclonal antibody or antigen-binding fragment thereof comprising:

(1) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 6, AAS and SEQ ID NO. 7, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(2) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 15, GAT and SEQ ID NO. 16, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(3) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 19, SEQ ID NO. 20 and SEQ ID NO. 21, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 24, LAS and SEQ ID NO. 25, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(4) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 33, SAS and 34, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(5) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 42, SAS, and SEQ ID NO. 43, LCDR1, LCDR2, and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(6) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 46, SEQ ID NO. 47 and SEQ ID NO. 48, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 51, ATS and SEQ ID NO. 52 for LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(7) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 60, NAK and SEQ ID NO. 61, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(8) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 64, SEQ ID NO. 65 and SEQ ID NO. 66, HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 69, AAS and SEQ ID NO. 70, LCDR1, LCDR2 and LCDR3, respectively; or alternatively, the first and second heat exchangers may be,

(9) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 73, SEQ ID NO. 74 and SEQ ID NO. 75 for HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO:78, QMS and SEQ ID NO:79 for LCDR1, LCDR2 and LCDR3, respectively;

(10) A heavy chain variable region having amino acid sequences shown as SEQ ID NO. 82, SEQ ID NO. 83 and SEQ ID NO. 84 as HCDR1, HCDR2 and HCDR3, respectively; and a light chain variable region having amino acid sequences as shown in SEQ ID NO. 87, AAS and SEQ ID NO. 88 for LCDR1, LCDR2 and LCDR3, respectively.

Preferably, the monoclonal antibody or antigen binding fragment thereof comprises:

(1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 4; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 8; or alternatively, the first and second heat exchangers may be,

(2) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 13; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 17; or alternatively, the first and second heat exchangers may be,

(3) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 22; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 26; or alternatively, the first and second heat exchangers may be,

(4) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 31; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 35; or alternatively, the first and second heat exchangers may be,

(5) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 40; and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO. 44; or alternatively, the first and second heat exchangers may be,

(6) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 49; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 53; or alternatively, the first and second heat exchangers may be,

(7) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 58; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 62; or alternatively, the first and second heat exchangers may be,

(8) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 67; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 71; or alternatively, the first and second heat exchangers may be,

(9) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 76; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 80; or alternatively, the first and second heat exchangers may be,

(10) A heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO. 85; and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 89.

In addition, the monoclonal antibody or antigen binding fragment thereof further comprises a constant region; preferably, the constant region is any one selected from the group consisting of: constant regions of IgG, igA, or IgM antibodies.

Further preferred, the monoclonal antibody or antigen binding fragment thereof comprises:

(1) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 5; and a light chain having an amino acid sequence as shown in SEQ ID NO. 9; or alternatively, the first and second heat exchangers may be,

(2) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 14; and a light chain having an amino acid sequence as shown in SEQ ID NO. 18; or alternatively, the first and second heat exchangers may be,

(3) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 23; and a light chain having an amino acid sequence as shown in SEQ ID NO. 27; or alternatively, the first and second heat exchangers may be,

(4) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 32; and a light chain having an amino acid sequence as shown in SEQ ID NO. 36; or alternatively, the first and second heat exchangers may be,

(5) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 41; and a light chain having an amino acid sequence as shown in SEQ ID NO. 45; or alternatively, the first and second heat exchangers may be,

(6) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 50; and a light chain having an amino acid sequence as shown in SEQ ID NO. 54; or alternatively, the first and second heat exchangers may be,

(7) A heavy chain having an amino acid sequence as shown in SEQ ID NO. 59; and a light chain having an amino acid sequence as shown in SEQ ID NO. 63; or alternatively, the first and second heat exchangers may be,

(8) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 68; and a light chain having an amino acid sequence as shown in SEQ ID NO. 72; or alternatively, the first and second heat exchangers may be,

(9) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 77; and a light chain having an amino acid sequence as shown in SEQ ID NO. 81; or alternatively, the first and second heat exchangers may be,

(10) A heavy chain having an amino acid sequence as set forth in SEQ ID NO. 86; and a light chain having an amino acid sequence as shown in SEQ ID NO. 90.

In the first aspect, the monoclonal antibodies defined in items (1) to (10) are hereinafter referred to as antibodies 1 to 10, respectively.

In some possible embodiments, the antigen binding fragment of the monoclonal antibody is selected from the group consisting of fab, fab ', F (ab') ₂, fd, fv, dAb, complementarity determining region fragments, single chain antibodies, human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.

In a second aspect, the present invention provides a polynucleotide encoding a monoclonal antibody or antigen binding fragment thereof as described in the first aspect above. The polynucleotide is not limited to the method of its production and may be obtained using genetic engineering recombinant techniques or chemical synthesis methods.

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

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

The vector of the present invention may be a cloning vector or an expression vector, and may be, for example, a plasmid, a cosmid, a phage, or the like.

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

In a fifth aspect, the present invention provides a transformed host cell into which a polynucleotide as described in the second aspect, a nucleic acid construct as described in the third aspect or a recombinant vector as described in the fourth aspect has been transformed;

Such host cells include, but are not limited to: prokaryotic cells, such as E.coli cells; eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, e.g., mouse cells, human cells, etc.). The host cell may also be a cell line, such as a 293T cell line.

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

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

In a possible embodiment, the sample is a biological sample of the subject, preferably a serum or plasma sample of the subject.

In a seventh aspect, the present invention provides the use of any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof as described in the first aspect above in the preparation of a detection reagent or kit for detecting estradiol.

Preferably, the two monoclonal antibodies or antigen binding fragments thereof are selected from the group consisting of:

(I) Antibody 1 or antigen-binding fragment thereof, and antibody 2 or antigen-binding fragment thereof;

(II) antibody 3 or antigen-binding fragment thereof, and antibody 4 or antigen-binding fragment thereof;

Wherein antibody 1 or an antigen-binding fragment thereof is defined in any of item (1) of the first aspect, antibody 2 or an antigen-binding fragment thereof is defined in any of item (2) of the first aspect, antibody 3 or an antigen-binding fragment thereof is defined in any of item (3) of the first aspect, and antibody 4 or an antigen-binding fragment thereof is defined in any of item (4) of the first aspect.

In an eighth aspect, the present invention provides a detection reagent for detecting estradiol comprising a monoclonal antibody or antigen binding fragment thereof as defined in the first aspect above, a polynucleotide as defined in the second aspect above, a nucleic acid construct as defined in the third aspect above, a recombinant vector as defined in the fourth aspect above and/or a transformed host cell as defined in the fifth aspect above.

Preferably, the detection reagent is a double antibody sandwich immunoassay kit comprising any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect above, preferably comprising two monoclonal antibodies or antigen binding fragments thereof selected from the group consisting of:

In a possible embodiment, the double-antibody sandwich immunoassay kit comprises an immunochromatographic test strip, which is based on the principle of double-antibody sandwich method, and takes any two monoclonal antibodies or antigen binding fragments thereof selected from the monoclonal antibodies or antigen binding fragments thereof described in the first aspect as a capture antibody and a detection antibody respectively;

In some preferred embodiments, the immunochromatographic test strip has (1) any one of antibody 1 or an antigen-binding fragment thereof and (2) antibody 2 or an antigen-binding fragment thereof as a capture antibody, and the other one as a detection antibody; further preferably, the immunochromatographic test strip uses an antibody 1 or an antigen-binding fragment thereof as a detection antibody and an antibody 2 or an antigen-binding fragment thereof as a capture antibody;

In other preferred embodiments, the immunochromatographic test strip has any one of (1) antibody 3 or an antigen-binding fragment thereof and (2) antibody 4 or an antigen-binding fragment thereof as a capture antibody, and the other one as a detection antibody; further preferably, the immunochromatographic test strip uses an antibody 3 or an antigen-binding fragment thereof as a detection antibody and an antibody 4 or an antigen-binding fragment thereof as a capture antibody;

preferably, the monoclonal antibody or antigen-binding fragment thereof as a detection antibody is labeled with a detectable label; such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials, enzymes (e.g., horseradish peroxidase), and the like.

In a preferred embodiment, the detectable label is a fluorescent microsphere, a latex microsphere or a colloidal gold particle, preferably a colloidal gold particle.

In a ninth aspect, the present invention provides a method of preparing a monoclonal antibody or antigen-binding fragment thereof as described in the first aspect above, the method comprising: allowing the transformed host cell of the fifth aspect described above to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.

In a tenth aspect, the present invention provides a method of detecting the presence or level of estradiol in a sample, said method comprising using a monoclonal antibody or antigen binding fragment thereof as defined in the first aspect, a polynucleotide as defined in the second aspect, a nucleic acid construct as defined in the third aspect, an expression vector as defined in the fourth aspect, a transformed host cell as defined in the fifth aspect and/or a detection agent as defined in the eighth aspect.

In a possible embodiment, the sample includes, but is not limited to, serum, plasma, and the like from a subject.

The method may be used for diagnostic purposes (e.g., the sample is a sample from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not a sample from a patient).

General methods for detecting the presence or level of an antigen of interest in a sample using monoclonal antibodies or antigen binding fragments thereof are well known to those skilled in the art. In certain preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method, and the like.

Advantageous effects

The monoclonal antibody aiming at the estradiol can be combined with the estradiol with high affinity and high specificity (the combination activity EC50 of the monoclonal antibody reaches ng/ml level, and KD is 10 ^-10M～10^-11 M), so that the high-accuracy and high-sensitivity detection of the estradiol is realized; in addition, the invention also develops a high-performance paired antibody suitable for the immunological detection of the double-antibody sandwich method, thereby breaking through the limitations of low sensitivity and poor clinical relevance of the traditional competitive detection reagent, realizing the immunological detection of the double-antibody sandwich method of the estradiol, and the detection method has higher specificity and sensitivity, and the clinical coincidence rate is more than 0.95.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1 is a gel diagram of SDS-PAGE detection of 10 anti-E2 monoclonal antibodies expressed and purified in example 3 of the present invention; wherein M represents the electrophoresis band of molecular weight Marker, lane 1 shows the electrophoresis band of each monoclonal antibody under reducing conditions, and lane 2 shows the electrophoresis band of each monoclonal antibody under non-reducing conditions.

FIG. 2 is a graph showing the results of detection of the binding activity of 10 anti-E2 monoclonal antibodies expressed and purified in example 3 and E2-coupled protein according to the present invention, as detected in example 4.

FIG. 3 is a schematic diagram of a colloidal gold immunochromatographic test strip prepared in example 5 of the present invention; wherein 1 is a PVC bottom plate; 2 is a sample pad; 3 is a nitrocellulose membrane; 4 is gold thread; 5 is a detection line; 6 is a quality control line; and 7 is a water absorption pad.

FIG. 4 is a graph showing the correlation between two colloidal gold immunochromatographic test strips and a Roche test reagent based on two paired antibodies of the present invention, as tested in example 6; fig. 4A is a graph showing the result of the colloidal gold immunochromatographic strip 1 based on the E2 mAb 1+e2mab 2 paired antibody, and fig. 4B is a graph showing the result of the colloidal gold immunochromatographic strip 2 based on the E2 mAb 3+e2mab 4 paired antibody.

Detailed Description

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.

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

In order that the invention may be more readily understood, certain technical and scientific terms are defined as follows. Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "about" when used in conjunction with a numerical value is intended to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ± 5%, ±2%, ±1% and ± 0.1%, as these variations are suitable for carrying out the disclosed methods.

The term "and/or" is understood to mean any one of the selectable items or a combination of any two or more of the selectable items.

As used herein, the term "or" should be understood to have the same meaning as "and/or" as defined above. For example, when items in a list are separated, "or" and/or "should be construed as inclusive, i.e., including at least one of the list of elements or amounts, but also including more than one, and optionally, additional unlisted items. To the extent that only one term is explicitly recited, such as "only one" or "exactly one" or "consisting of" is used in the claims, it will refer to only one number listed or an element of a list.

The term "percent (%) amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment may be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to obtain the maximum alignment for the full length of sequences compared.

The term "antibody" refers to any form of antibody that has the desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

"Antigen binding fragment" refers to antigen binding fragments of antibodies and antibody analogs, which generally comprise at least a portion of the antigen binding or variable regions of the parent antibody, e.g., one or more CDRs. Fragments of the antibodies retain at least some of the binding specificity of the parent antibody. Antigen binding fragments include peptides selected from the group consisting of fab, fab '-SH, fv, scFv, F (ab') 2, diabodies, cdrs comprising peptides, and the like.

"Fab fragment" consists of a light chain and a heavy chain CH1 and variable domains.

The "Fc" region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain.

"Fab ' fragments" contain a portion of one light chain and one heavy chain comprising a portion of the VH domain, CH1 domain and constant region between the CH1 and CH2 domains, with an inter-chain disulfide bond formed between the two heavy chains of the two Fab ' fragments to form a F (ab ') 2 molecule.

"F (ab') 2 fragments" contain two light chains and portions of two heavy chains comprising portions of the VH domain, CH1 domain, and constant region between CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks constant regions.

"Single chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the VH and VL domains of an antibody, which domains are contained in a single polypeptide chain. In general, scFv polypeptides comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

A "diabody" is a small antigen-binding fragment having two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using a linker that is so short that it is not possible to pair between two domains of the same strand, the domains pair with complementary domains of the other strand and form two antigen binding sites.

"Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art.

The term "high affinity" for IgG antibodies refers to KD of 1.0 x 10 ^-9 M or less for antigen. For other antibody subtypes, "high affinity" binding may vary.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

Example 1: preparation of monoclonal antibodies against E2

(1) Animal immunization:

the preparation method comprises the steps of taking coupled protein E2-BSA (purchased from Nanjing Ming Biotechnology Co., ltd., product No. MY-E2-001) as an antigen, fully emulsifying the antigen with an equivalent Freund's complete adjuvant, performing intraperitoneal injection to immunize mice (variety: balb/c, female, 6-8 weeks old) for boosting, performing total immunization for 3 times at a two-week time interval, taking trace tail blood 5-8 days after the last boosting, detecting serum titer by an ELISA method, and preparing spleen single cell suspension when the serum titer reaches a standard of more than 128K.

(2) Spleen single cell suspension preparation:

The mice were sacrificed by cervical dislocation, the small opening was cut in the middle of the left abdomen side of the mice, the skin was torn open, the abdominal wall was exposed, the peritoneum was lifted up on the underside of the spleen, the spleen was exposed after the cutting open, the spleen was lifted with forceps, and connective tissue under the spleen was separated by an ophthalmic scissors, and the spleen was taken out. The spleen was placed on a cell filter screen, and the spleen was gently ground with a syringe needle to obtain a cell suspension. Taking a 15mL sterile test tube, firstly adding a cell separating liquid Ficoll, sucking the freshly prepared cell suspension, slowly adding the cell suspension into the test tube, and the volume ratio of the separating liquid to the cell suspension is 1:2. the cells were resuspended after centrifugation at room temperature for 10 minutes at 1500 rpm in a centrifuge, after centrifugation for 20 minutes at room temperature, after which the test tubes were removed and the different layers were visible, the upper non-cellular layer was aspirated off, the mononuclear layer was aspirated in an additional 15mL sterile test tube.

(3) Obtaining single B cells:

and (3) using the prepared spleen single cell suspension to carry out cell screening by using a single cell chip screening platform through antigen marking so as to obtain the B cells with antigen specificity.

Through the above steps, 10 monoclonal antibodies against E2 were obtained in total, and identified as IgG antibodies, respectively designated as E2 mAb 1-E2 mAb10. In the following examples, the light and heavy chain gene sequences of 10 monoclonal antibodies were detected, and expression, purification and binding activity detection were performed.

Example 2: acquisition of the light and heavy chain Gene sequences of monoclonal antibodies against E2

Preparation of cDNA:

Respectively extracting RNA from 10B cells obtained in the embodiment 1, carrying out reverse transcription by taking the RNA as a template to obtain cDNA, and amplifying genes of light chains and heavy chains of the antibody by using a PCR (polymerase chain reaction) mode;

Acquisition of light and heavy chain genes:

Amplifying by using a murine specific primer, cloning the amplified DNA sequences into vectors, then carrying out conversion coating, picking cloning and extracting plasmids for sequencing to obtain light chain gene sequences and heavy chain gene sequences of 10 antibodies; deducing and obtaining the amino acid sequences of the light chain and the heavy chain of each antibody according to the detected light chain gene sequences and the heavy chain gene sequences of each antibody; specifically, the heavy chain amino acid sequences of the antibodies E2 mAb 1-E2 mAb10 are shown as SEQ ID NO. 5, 14, 23, 32, 41, 50, 59, 68, 77 and 86, and the light chain amino acid sequences are shown as SEQ ID NO. 9, 18, 27, 36, 45, 54, 63, 72, 81 and 90;

analysis of light and heavy chain variable region gene sequences:

and analyzing the VH and VL according to the sequencing result to finally obtain the amino acid sequence of the light chain variable region and the amino acid sequence of the heavy chain variable region.

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

E2 mAb1：

VH(SEQ ID NO:4)

QIQLIQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWINT ETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARGLGSYYFDYWGQ GSILTVSS;

VL(SEQ ID NO:8)

DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYAASALDS GVPKRFSGSGSGSDYSLTISSLESEDFADYYCLQYASYPLTFGAGTKLELK;

E2 mAb2：

VH(SEQ ID NO:13)

EVLLQQSGPELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPN NDVTIYNQKFKGKATLTVDMSSSTSYMELRSLTSEDTAVYYCARRHYYGLYYFDYW GQGTTLTVSS;

VL(SEQ ID NO:17)

DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLE IGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPWTFGGGTKLEIK;

E2 mAb3：

VH(SEQ ID NO:22)

QIQLVQSGPELKKPGETVKISCKTSGYTFTDYSMHWVKQAPGKGLKWMGWINT ETGEPTYEDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSRGFLLPYYFDYW GQGTTLTVSS;

VL(SEQ ID NO:26)

DIVLTQSPASLDVSLGQRVTISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLA SNLESGVPVRFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRGFPLTFGAGTALEVK;

E2 mAb4：

VH(SEQ ID NO:31)

QVQLQQSGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCAKETAYGSQSPWYFDVWGAGTTVTVSS;

VL(SEQ ID NO:35)

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRCSGVPDRFTGSGSGTDFTLTVTNVQSEDLAEYFCQQYNSYPFTFGSGTKLEIK;

E2 mAb5：

VH(SEQ ID NO:40)

QVQLQQSGAELVKPGASVKLSCKTSGYTFTSYWIQWVKQRPGQGLGWIGEIFPGTGTTYYNEKFKGKATLTIDTSSSTAYMHLSSLTSEDSAVYFCARGDYGSRSNYYAMDYWGQGTSVTVSS;

VL(SEQ ID NO:44)

DIVMTQSQKFVSTSVGDRVSVTCKASQNVGTTVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLTEYFCQQYSGYPYTFGGGTKLEIK;

E2 mAb6：

VH(SEQ ID NO:49)

QVQLQQSGADLVKPGASVKLSCGFTFSTYAIEWLKQRPGHGLEWIGQISGGASYTNYNAKFKDKATFTADTSSNTAYMQLLSLTSEDSGVYYCARHDYYGSNYDSFDYWGQGSTLTVSS;

VL(SEQ ID NO:53)

DIQMTQSPASLSASVGETVTITCSSLSYLAWYQQKQGKSPQLLVYATSTLADGVSSRFSGSGSGTQFSLKINSLQPEDFGSYYCQQWSSIPPTFGGGTKLEIK;

E2 mAb7：

VH(SEQ ID NO:58)

EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGVTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARRGYDGYYYFDYWGQGTTLTVSS;

VL(SEQ ID NO:62)

DIQMTQSPASLSASVGETVTITCRASGNIQNYLAWYQQKQGKSPQLLLYNAKALTDGVPSRFSGSGSGTQYYLKINSLQPEDFGTYYCQHFWSTPWTFGGGTKVEIK;

E2 mAb8：

VH(SEQ ID NO:67)

QAQLQQPGSVLVRPGASVKLSCKASGYTFTSSWMEWAKQRPGQGLEWIGEIHP NSGNTNYNEKFKGKATLTVDTSSSTAYVDLSSLTSEDSAVYYCARSGYDGSWFAYW GQGTLVTVSA;

VL(SEQ ID NO:71)

DIVLTQSPASLAVSLGQRATISCRASESVEYYGTSLMQWYHQKPGQPPKLLIYAA SNVESGVPARFSGSGSGTDFSLNIHPVEGDDIAMYFCQQSRLVPWTFGGGTKLEIK;

E2 mAb9：

VH(SEQ ID NO:76)

EVQLVESGGGLVKPGGSLKLSCAASGFIFSDYYMYWIRQTPEKRLEWVATISDG GSYTYYPDSVKGRFTISRDNAKNNLYLQMSSLKSEDTAIYYCARDRYNYDEGAFDY WGQGTTLTVSS;

VL(SEQ ID NO:80)

DIVMTQAAFSNPVTLGTSASISCRSSKNLLHSNGITYLYWYLQKPGQSPQLLIYQ MSNLASGVPDRFSNSGSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIK;E2 mAb10：

VH(SEQ ID NO:85)

EVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQTHGKSLEWIGDINPN SGITIYNQKFKGKATLTVDKSSGTAYMELRSLTSDDTAVYYCARGTTGTLYFDYWG QGTTLTVSS;

VL(SEQ ID NO:89)

DIQMIQSPSSLSASLGERVSLTCRTSQEISGFLSWLQQKPDGTIKRLIYAASALDSG VPKRFSGSGSGSDYSLTISSLESEDFADYYCLQYASYPPTFGGGTKLEIK.

Example 3: expression and purification of monoclonal antibodies against E2

(1) Culturing N293 suspension cells, and performing cell transfection when the density reaches (1-3) multiplied by 10 ⁶ cells/ml and the survival rate is more than 80-90%;

(2) Light and heavy chain genes of 10 monoclonal antibodies E2 mAb 1-E2 mAb10 are synthesized according to the light and heavy chain gene sequences of the 10 monoclonal antibodies E2 mAb 1-E2 mAb10, and the synthesized light and heavy chain genes are respectively constructed into pcDNA3.4 plasmids; then, the recombinant plasmid containing the antibody light and heavy chain genes is transfected into N293 cells under the following conditions: the mass ratio PEI/plasmid was 3:1, transfected at a ratio of 2. Mu.g plasmid/1 ml N293 cells;

(3) Collecting cell supernatant 5-7 days after transfection, performing affinity purification by using Protein A resin to obtain high-purity monoclonal antibody, replacing elution buffer solution with PBS by dialysis, and measuring antibody concentration by using Nanodrop;

next, the purity of the antibodies was checked by SDS-PAGE as follows:

SDS-PAGE detection:

Taking 2 mug of antibody to be detected, adding a proper amount of SDS-PAGE protein loading buffer to make the total volume be 20 mug; simultaneously preparing a reducing SDS-PAGE sample and a non-reducing SDS-PAGE sample, and then loading the samples; firstly, electrophoresis is carried out for 30min at 80V, and then electrophoresis is carried out at 120V until the separation of the strips is clear; and taking down the electrophoresis gel, performing coomassie brilliant blue dyeing, removing the dyeing liquid after 15min, washing with clear water, and then adding a decolorizing liquid for decolorizing until clear bands are seen.

The results are shown in FIG. 1, FIG. 1 showing: under reducing conditions, the 10 monoclonal antibodies all have two electrophoresis bands, and the corresponding molecular weights of the two electrophoresis bands are consistent with the sizes of the respective light chains and heavy chains; under non-reducing conditions, 10 monoclonal antibodies all have a single band, and the corresponding molecular weight of the electrophoretic band is consistent with the size of the respective intact antibody; these results indicate that the 10 monoclonal antibodies are expressed accurately.

In addition, all the electrophoresis bands were clear in edge and free of bands, indicating that the purity of the monoclonal antibodies obtained by the above-described expression and purification steps was high.

Example 4: antigen binding Activity and affinity detection of monoclonal antibodies against E2

In this example, antigen binding activity and affinity assays were performed on 10 monoclonal antibodies E2mAb 1-E2 mAb10 expressed and purified in example 3, respectively.

Antigen binding activity and affinity detection procedure:

① Antigen coating: the antigen (i.e., the aforementioned conjugated protein E2-BSA) was diluted to an appropriate concentration and then added to an empty ELISA plate in a volume of 100. Mu.l/well; placing the ELISA plate in a refrigerator at 4 ℃ and incubating overnight;

② Closing: washing the plate for 5 times by using a plate washing machine, buckling, adding sealing liquid into the ELISA plate, placing 150 μl of sealing liquid into a 37 ℃ incubator, and sealing for 1h; washing the board for 5 times by a board washing machine, and buckling;

③ Incubating primary antibodies: diluting the antibody to be tested to a proper concentration gradient by using 1 XPBS; adding diluted antibody to be detected into an ELISA plate, wherein each hole is 100 mu l, and the reaction time is as follows: 37 ℃/1h; washing the board for 5 times by a board washing machine, and buckling;

④ Incubating a secondary antibody: the second enzyme-labeled antibody is diluted according to a proper proportion, then added into an enzyme-labeled plate, and the reaction time is 100 mu l per hole: 37 ℃/30min; washing the board for 5 times by a board washing machine, and buckling;

⑤ Color development: adding the color developing solution into the ELISA plate, wherein each hole is 100 mu l, and the reaction time is as follows: 37 degrees/15 min;

⑥ Termination and reading: adding a stop solution, wherein the volume is 100 mu l/hole; detection wavelength setting: the detection wavelength is 450nm, the reference wavelength is 620nm, and the detection result is derived into an Excel table.

⑦ Data analysis: data analysis and mapping were performed using software, EC50 was fitted, and KD was deduced.

The binding activity and affinity of these 10 monoclonal antibodies to the antigen are shown in FIG. 2 and Table 1 below.

TABLE 1

Antibody numbering	EC50(ng/ml)	KD
			E2 mAb1	34.69	2.31*10^-10M
E2 mAb2	14.63	9.75*10^-11M
			E2 mAb3	19.47	1.30*10^-10M
E2 mAb4	25.83	1.72*10^-10M
			E2 mAb5	27.88	1.86*10^-10M
E2 mAb6	23.05	1.54*10^-10M
			E2 mAb7	14.23	9.49*10^-11M
E2 mAb8	26.9	1.79*10^-10M
			E2 mAb9	28.72	1.91*10^-10M
E2 mAb10	15.79	1.05*10^-10M

Fig. 2 and table 1 show: the EC50 of the 10 monoclonal antibodies combined with antigen reaches ng/ml grade, and KD values are 10 ^-10M～10^-11 M grade.

As mentioned above, for IgG antibodies, it is generally considered in the art that the antibody has high affinity when the KD value of the antibody is 10 ^-9 M, and the 10 monoclonal antibodies of the invention have the KD value of 10 ^-10M-10^-11 M, which is 10-100 times the above-defined value, so that it can be obtained: the monoclonal antibody of the invention has extremely strong antigen binding activity and affinity.

Example 5: preparation of colloidal gold immunochromatography test strip based on anti-E2 monoclonal antibody by double-antibody sandwich method

In this example, the two colloidal gold immunochromatography test strips for detecting E2 by using the two double-antibody sandwich method principle are prepared by using the E2 mAb1 and the E2 mAb2, the E2 mAb3 and the T4 mAb4 expressed and purified in example 3 as paired antibodies respectively, and the schematic construction of the test strips is shown in FIG. 3; the preparation method of the test strip comprises the following steps:

(1) Preparation of Au-anti-E2 monoclonal antibody Complex

① Taking 3ml of colloidal gold particles (colloid Jin Lijing: 20nm, maximum absorption peak wavelength is 529nm, absorbance is 2.0A), adding 12 μl of 0.1M K ₂CO₃, mixing well, adding 8 μg of anti-E2 antibody (i.e. detection antibody), and reacting at normal temperature for 1h;

② Adding 600 μl of 5% BSA blocking solution into the ① system, and blocking at room temperature for 30min;

③ After completion of the blocking, the mixture was centrifuged at 11000rpm at 10℃for 15min, the supernatant was discarded, and the precipitate was redissolved (i.e., concentrated 60-fold, 60X) with 50. Mu.l of a gold-labeled diluent to obtain an Au-anti-E2 monoclonal antibody complex; the gold standard diluent comprises 100mM Tris buffer solution with pH of 7.5-8.5, 5% sucrose, 1% trehalose and 5% BSA;

(2) Gold drawing

① Closing line is marked: taking 5% BSA solution, setting the water yield of the instrument to be 3 mu L/cm, and placing the scratched bottom plate in a 37 ℃ oven for drying for 2 hours;

② Diluting the final concentration of the Au-anti-E2 monoclonal antibody complex to 40X, setting the water yield to 2 mu L/cm, scribing at the position of a closed line, then placing a bottom plate in a 37 ℃ oven for drying for 2 hours, and then placing a marking pad in an aluminum foil bag with a desiccant added in advance at the humidity of not more than 36%, sealing and preserving for later use;

(3) Coating quality control line C and detection line T

① Preparing a coating liquid of a quality control line C and a detection line T: the goat anti-mouse antibody is diluted into 1.0mg/mL antibody solution by 10mM PBS buffer solution with pH of 7.4; another anti-E2 monoclonal antibody (i.e., capture antibody) was taken and diluted to 0.8mg/mL in 10mM PBS buffer, ph 7.4;

② Coating: coating the coating liquid of the quality control line C and the detection line T to the positions of the quality control line and the detection line of the nitrocellulose membrane respectively, wherein the intervals between C, T lines are 8mm, and the coating amount is 1 mu L/cm; placing the coated PVC bottom plate in a baking oven at 37 ℃ for 16 hours, then placing the PVC bottom plate in an aluminum foil bag with a desiccant added in advance under the humidity of not more than 36%, and sealing and preserving for later use;

(4) Pretreatment of sample pad

Cleaning a drying net, soaking and wetting a sample pad by using sample pad pretreatment liquid, draining by rotation until no water drops drip, then drying for 4 hours in a 37 ℃ oven, and then placing the sample pad into an aluminum foil bag which is pre-added with a drying agent at the humidity of not more than 36%, sealing and preserving for later use;

the sample pad pretreatment solution contains 10mM Tris buffer with pH of 8.5, 0.1% PVP 10, 0.1% PEG20000, 0.1% Tween20 and 0.5% sodium caseinate;

(5) Assembling and cutting film materials

Taking out the water absorption pad and the treated sample pad under the humidity of not higher than 36%, cutting according to the size of 10mm multiplied by 300mm, taking out the coated PVC base plate, and assembling according to the following procedures: firstly, attaching a sample pad on a PVC bottom plate, enabling a sample pad part to press a nitrocellulose membrane for 2mm, and finally attaching a water absorption pad, wherein the water absorption pad presses the nitrocellulose membrane for 2mm; cutting the assembled PVC bottom plate into 4mm to obtain the colloidal gold immunochromatographic test strip for detecting E2.

The antibody pairing mode of the test strip 1 is as follows: e2mAb 1 (for preparing Au-antibody complex) +e2mab 2 (for T-line coating); the antibody pairing mode of the test strip 2 is as follows: e2mAb 3 (used to make Au-antibody complex) +e2mab4 (used for T-wire coating).

Example 6: detection performance of colloidal gold immunochromatography test strip based on anti-E2 monoclonal antibody by double-antibody sandwich method

In this example, the two colloidal gold immunochromatographic test strips prepared in example 5 were tested for their detection performance for E2 in clinical serum samples, and compared with the detection performance of conventional Roche reagents.

Specifically, for the two colloidal gold immunochromatographic test strips prepared in example 5, a clinical serum sample is diluted by a sample diluent (the composition of which is 10mM Tris+1%BSA+0.2%Tween-20+0.1% procin 300, pH 7.0), reacted at room temperature for 10min, then 75 μl is added, and after 15min, the detection is performed by a colloidal gold immunoassay analyzer, the detection value is read, and the data is processed to obtain E2 concentration in the sample of each dilution; for the Roche test kit, the serum sample is diluted and then tested according to the specification flow, so as to obtain E2 concentration data. Then, the E2 concentration data detected by the two test strips are compared with the E2 concentration data (as a standard) detected by the Rogowski reagent, and a clinical correlation (i.e., a coincidence rate with the Rogowski detection result) curve is made.

The results are shown in FIG. 4; wherein, fig. 4A and 4B are graphs of the results of clinical relevance (i.e., coincidence rate with the roche test result) of the colloidal gold immunochromatographic test strip 1 and the test strip 2, respectively, which show that: the clinical relevance (R ² value) of both test strips was > 0.97.

The results show that the E2 colloidal gold immunochromatographic test strip prepared based on the paired antibody provided by the invention has very high coincidence rate (R ² is more than 0.97) with the Rogowski detection result, so that the paired antibody has very good clinical relevance.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof directed against estradiol, characterized in that said monoclonal antibody or antigen-binding fragment thereof comprises:

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 2, wherein the monoclonal antibody or antigen-binding fragment thereof comprises:

4. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the antigen-binding fragment is selected from the group consisting of fab, fab ', F (ab') ₂, fd, fv, dAb, a complementarity determining region fragment, a single chain antibody, a human antibody, a chimeric antibody or a bispecific or multispecific antibody.

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

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

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

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

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

10. Use of any two monoclonal antibodies or antigen binding fragments thereof selected from the group consisting of the monoclonal antibodies or antigen binding fragments thereof according to any one of claims 1-4 in the preparation of a detection reagent or kit for detecting estradiol.

11. The use according to claim 10, wherein the two monoclonal antibodies or antigen binding fragments thereof are selected from the group consisting of:

Wherein antibody 1 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (1), antibody 2 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (2), antibody 3 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (3), and antibody 4 or an antigen binding fragment thereof is defined in claim 1 to claim 4 as item (4).

12. A test agent for detecting estradiol comprising the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, the polynucleotide of claim 5, the nucleic acid construct of claim 6, the recombinant vector of claim 7 and/or the transformed host cell of claim 8.

13. The detection reagent according to claim 12, wherein the detection reagent is a double antibody sandwich immunoassay kit comprising any two monoclonal antibodies or antigen-binding fragments thereof selected from the monoclonal antibodies or antigen-binding fragments thereof according to any one of claims 1 to 4.

14. The detection reagent of claim 13, wherein the two monoclonal antibodies or antigen-binding fragments thereof are selected from the group consisting of:

15. The detection reagent according to claim 14, wherein the double-antibody sandwich immunoassay kit comprises a double-antibody sandwich immunochromatographic test strip in which:

Using the antibody 1 or the antigen binding fragment thereof as a detection antibody, and using the antibody 2 or the antigen binding fragment thereof as a capture antibody; or alternatively

Antibody 3 or an antigen-binding fragment thereof is used as a detection antibody, and antibody 4 or an antigen-binding fragment thereof is used as a capture antibody.

16. A method of making the monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, the method comprising: allowing the transformed host cell of claim 8 to express the monoclonal antibody or antigen-binding fragment thereof under conditions suitable for expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the expressed monoclonal antibody or antigen-binding fragment thereof from a culture of the host cell.