CN114075281B - anti-Inhibin-alpha protein monoclonal antibody, cell line, preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of biological detection, and provides an anti-Inhibin-alpha protein monoclonal antibody, wherein the amino acid sequences of the heavy chain and light chain variable regions are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3. The inventor also provides a hybridoma cell line for secreting anti-Inhibin-alpha protein, wherein the cell line is a mouse hybridoma cell line 15E11D5E11 with the preservation number of: CGMCC NO.22320. The anti-Inhibin-alpha protein monoclonal antibody has high specificity and sensitivity, can specifically identify cells expressing Inhibin-alpha protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

anti-Inhibin-alpha protein monoclonal antibody, cell line, preparation method and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an Inhibin-alpha protein resisting monoclonal antibody, a cell line, a preparation method and application thereof.

Background

Inhibin is one of the important hormones regulated by the hypothalamic pituitary gonadal axis system. Inhibin belongs to a member of TGF-beta family, and can inhibit synthesis and secretion of adenohypophysis anterior lobe basic and FSH regulated by gonadotropin-releasing hormone by reducing transcription of follicle stimulating hormone gene and down-regulating gonadotropin-releasing hormone receptor in paracrine and autocrine manner. And has local regulating effect on gonadal steroid production. Inhibin consists essentially of inhibin A and inhibin B, both of which have alpha subunits, differing in the form of beta subunits. The Inhibin-alpha subunit has only one molecular structure, has a relative molecular mass of 18000, and is located on human chromosome 2q33.

Inhibin-alpha is produced by Sertoli cells and ovarian granulosa cells of the testis and can inhibit the production and secretion of pituitary gonadotropins. Inhibin-alpha is obviously increased in most epithelial ovarian cancers, is more obviously increased in granulocytic tumors and mucinous cystadenocarcinoma, has higher expression in the early stage of ovarian malignant tumors, and is commonly used for differential diagnosis of granulocytic ovarian tumors and cancers. In addition, inhibina is expressed in normal adrenal cortex, cortical nodular hyperplasia and cortical tumor, mainly expressed in the inner layer of the reticular zone and fasciculate zone of the cortex, and is mainly expressed in the cortical tumor (particularly cortical adenoma) in a diffuse way, thereby being helpful for judging normal or hyperplastic tissue or tumor tissue.

Disclosure of Invention

The inventor provides an anti-Inhibin-alpha protein monoclonal antibody, and the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.

Furthermore, the heavy chain and light chain variable region amino acid sequences of the monoclonal antibody are respectively encoded by the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.

Furthermore, in the preparation process of the monoclonal antibody, an antigen used by an immunized mouse is a recombinant protein which is coded by a nucleotide sequence shown in SEQ ID NO.1 and is expressed by escherichia coli recombination.

Further, the monoclonal antibody specifically recognizes the human Inhibin-alpha protein.

Further, the monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO.22320. The cell line is a mouse hybridoma cell line 15E11D5E11, and is classified and named as follows: a mouse hybridoma cell line which has been preserved in China general microbiological culture Collection center (CGMCC) at 29.04.2021, and is addressed to the institute of microbiology, national academy of sciences No.3, xilu No.1, beijing, chaoyang, north Chen.

Further, the anti-Inhibin-alpha protein is a mouse IgG1 subtype monoclonal antibody.

The inventor also provides a preparation method of the anti-Inhibin-alpha protein monoclonal antibody, and an antigen used by an immune mouse is a recombinant protein which is coded by a nucleotide sequence shown in SEQ ID NO.1 and is expressed by escherichia coli recombination.

The inventor also provides a hybridoma cell line for secreting anti-Inhibin-alpha protein, wherein the cell line is a mouse hybridoma cell line 15E11D5E11, the cell line is preserved in China general microbiological culture Collection center with the preservation number of: CGMCC NO.22320.

The inventor also provides the application of the anti-Inhibin-alpha protein monoclonal antibody in human Inhibin-alpha protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor further provides a human Inhibin-alpha protein immunoassay reagent, and the immunoassay reagent contains any one of the above Inhibin-alpha protein monoclonal antibodies as an effective component.

Different from the prior art, according to the technical scheme, based on the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and a secondary structure of Inhibin-alpha protein, the technical scheme selects amino acid fragments from 128 th position to 300 th position of the Inhibin-alpha protein and corresponding nucleotide fragments, carries out recombinant expression through escherichia coli, immunizes a mouse, and obtains a monoclonal cell line 15E11D5E11 efficiently secreting the monoclonal antibody against the Inhibin-alpha protein through cell fusion, screening and cloning, and carries out in vitro culture on the monoclonal cell line 15E11D5E11, and obtains the monoclonal antibody against the Inhibin-alpha protein secreted by the cell line. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing Inhibin-alpha protein, and is suitable for immunological detection, particularly immunohistochemical detection.

The above description of the present invention is only an overview of the technical solutions of the present application, and in order to make the technical solutions of the present application more clearly understood by those skilled in the art, the present invention may be further implemented according to the content described in the text and drawings of the present application, and in order to make the above objects, other objects, features, and advantages of the present application more easily understood, the following description is made in conjunction with the detailed description of the present application and the drawings.

Drawings

FIG. 1 is a graph showing the results of electrophoresis of the purified Inhibin-alpha monoclonal antibody in example 3; m: and (4) marking molecular weight.

FIG. 2 is an immunoblot of detection of human Inhibin-alpha protein by Inhibin-alpha monoclonal antibody.

FIG. 3 is a graph showing immunohistochemical staining results for ovarian solitary stromal tumors.

Detailed Description

To explain in detail the possible application scenarios, technical principles, and practical embodiments of the present application, and to achieve the objectives and effects thereof, the following detailed description is given with reference to the accompanying drawings. The embodiments described herein are merely for more clearly illustrating the technical solutions of the present application, and therefore, the embodiments are only used as examples, and the scope of the present application is not limited thereby.

Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the application. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or related to other embodiments specifically defined. In principle, in the present application, the technical features mentioned in the embodiments can be combined in any manner to form a corresponding implementable solution as long as there is no technical contradiction or conflict.

Unless otherwise defined, technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the use of relational terms herein is intended to describe specific embodiments only and is not intended to limit the present application.

In the description of the present application, the term "and/or" is a expression for describing a logical relationship between objects, meaning that three relationships may exist, for example a and/or B, meaning: there are three cases of A, B, and both A and B. In addition, the character "/" herein generally indicates that the former and latter associated objects are in a logical relationship of "or".

In this application, terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation without necessarily requiring or implying any actual such relationship or order between such entities or operations.

Without further limitation, in this application, the use of "including," "comprising," "having," or other similar expressions in phrases and expressions of "including," "comprising," or "having," is intended to cover a non-exclusive inclusion, and such expressions do not exclude the presence of additional elements in a process, method, or article that includes the recited elements, such that a process, method, or article that includes a list of elements may include not only those elements but also other elements not expressly listed or inherent to such process, method, or article.

As is understood in the examination of the guidelines, the terms "greater than", "less than", "more than" and the like in this application are to be understood as excluding the number; the expressions "above", "below", "within" and the like are understood to include the present numbers. In addition, in the description of the embodiments of the present application, "a plurality" means two or more (including two), and expressions related to "a plurality" similar thereto are also understood, for example, "a plurality of groups", "a plurality of times", and the like, unless specifically defined otherwise.

EXAMPLE 1 preparation of recombinant Inhibin-alpha protein fragments

1. Antigen fragment selection

The Inhibin-alpha protein sequence with the number P05111 was selected as the standard sequence from the Uniprot database (http:// www.uniprot.org). The invention selects an amino acid fragment from 128 th site to 300 th site of human Inhibin-alpha protein as an antigen, and designs a specific upstream Primer of the Inhibin-alpha protein of 5 'GGATCCTCCTGCCCAGCTGTGGTTTCATAC' (SEQ ID NO.6 restriction site, bamH I) and a downstream Primer by using software Primer 5.0 according to a corresponding base sequence (SEQ ID NO. 1): 5'TATCTCGAGCGGGATGTGCAGACCAC 3' (SEQ ID NO.7, restriction site, xho I), and reverse transcription amplifying gene fragment SEQ ID NO.1 coding for amino acids from position 128 to position 300.

And separating the PCR product by agarose gel electrophoresis, recovering, performing BamH I and Xho I double enzyme digestion on the recovered target gene and a plasmid vector pET28a for expression respectively, performing electrophoresis recovery again, and connecting by T4 DNA ligase. And transforming the connecting product into an escherichia coli competent cell Rosetta, selecting clone on a plate, inoculating, expanding and culturing, extracting plasmid DNA, and performing PCR identification. The clone with positive target gene shown by PCR is sequenced and analyzed, and the clone with completely correct sequence is used for the next step.

2. Expression and purification of recombinant Inhibin-alpha protein fragment

100ul of competent cells (Rosetta) stored at-80 ℃ were taken out, thawed, and 5ul of Inhibin- α plasmid DNA was added thereto, followed by standing on ice for 30min. After heat shock at 42 ℃ for 90 seconds and ice bath for 5min, 800. Mu.L of non-resistant LB medium was added. Resuscitating and culturing at 37 deg.C for 60min, plating, and culturing overnight. The transformed plate was picked up and monocloned into 4ml of LB liquid medium, cultured overnight at 37 ℃ and 200rpm, and then maintained.

4ul of the bacterial liquid is taken to be put into 4ml of LB liquid culture medium and cultured overnight for recovery. The cultured bacterial suspension was transferred to 200ml of LB liquid medium, mixed, cultured at 37 ℃ and 200rpm until OD =0.6-0.8, and then IPTG (0.5 mM) was added thereto to induce overnight at low temperature. The cells were collected by centrifugation in a 400ml large centrifuge bowl at 4000rpm for 10min and the supernatant was discarded. The precipitate was dispersed in 20-30ml of 10mM Tris-HCl (pH 7.0) and NaCl at a final concentration of 0.5M, and the cells were disrupted by sonication. After centrifugation at 12000rpm for 20 minutes, the centrifuged supernatant was applied to a Ni-NTA nickel column (QIAGEN) for purification, and then eluted with 10mM Tris-HCl (pH 7.0) containing 50mM imidazole, 250mM imidazole and 500mM imidazole, respectively, containing 0.5M sodium chloride, and the protein peaks were collected and detected by electrophoresis, respectively, for use.

EXAMPLE 2 establishment of the 15E11D5E11 hybridoma cell line

1. Immunization

The Inhibin-alpha protein fusion protein obtained in example 1 was mixed and emulsified with an equal volume of complete Freund's adjuvant (CFA, sigma) and 18-20g of Balb/c mice (purchased from Wu's laboratory animal, fuzhou) were immunized by abdominal injection at a dose of 50. Mu.g/mouse. Thereafter, every 14 days of booster immunization, the antigen was emulsified with Freund's incomplete adjuvant (IFA, sigma Co.) at a dose of 25. Mu.g/mouse. The multi-antibody titer of the anti-immunogen in the mouse serum is detected by indirect ELISA (wavelength of 450 nm) 14 days after the 2 nd boosting immunization, the mouse with the highest titer is subjected to impact immunization by tail vein injection, and the antigen is uniformly mixed by PBS solution, and the dosage is 50 mu g/mouse.

2. Cell fusion:

aseptically preparing mouse splenocyte suspension with standard immunity, mixing with mouse myeloma cell sp2/0 (ATCC) at a ratio of 5:1, centrifuging at 1000rpm for 10min, discarding supernatant, adding 1mL of PEG (Sigma) solution preheated to 37 ℃ from slow to fast within 1 min, and slightly rotating the centrifuge tube during the adding process to ensure that the cells are fully contacted with the PEG. Standing at room temperature for 90s, adding 4mL of serum-free DMEM (Hyclone company) culture medium preheated to 37 ℃ from slow to fast within 2min, then adding 10mL of preheated serum-free DMEM culture medium within 2min, finally adding the rest preheated serum-free DMEM culture medium within 2min, and fixing the volume to 50mL, wherein a centrifugal tube needs to be slowly shaken in the whole adding process to ensure uniform mixing, so that the damage to cells is reduced. Standing at room temperature for 10min, centrifuging (1000rpm, 5 min), discarding the supernatant, resuspending the cells in 10-20mLHAT (Sigma) medium, and diluting with HAT medium to a final concentration of 0.5X 10 ⁶ cells/mL, all solutions were transferred to 96-well plates at 200. Mu.L/well and labeled. Transferring the 96-well culture plate carefully to 37 deg.C, 5% ₂ Culturing in an incubator. The growth state and potential pollution of cells are regularly checked, and the incubator is opened and closed as little as possible to ensure the stability of the culture environment. On day 5 post-fusion, plates were supplemented with HAT medium at 50. Mu.L/well.

3. Cloning and ELISA screening positive hybridoma cells:

when the fused cell diameter is as large as about 1-2mm, 50-200. Mu.L of culture supernatant is aspirated for the first cell screening (ELISA, IHC-P, and other assays), and HAT medium is added to the culture well to 200. Mu.L. And (3) detecting the supernatant of the culture solution by ELISA, transferring all cell culture solutions in culture wells with positive results obtained by detection to a 24-well culture plate, supplementing HT medium (HT medium) for 2 mL/well, and culturing for 3 days.

And repeatedly screening each cell line in the 24-well plate, and removing culture well cells which are not positive results to obtain culture well cells with better positive results. Performing subclone screening on the positive well cells obtained from the 24-well culture plate by using a limiting dilution method, namely adding the cell sap obtained by the limiting dilution method into a 96-well culture plate, and transferring to CO ₂ Culturing in an incubator for 11 days, and repeating cell screening when the diameter of the cloned cell is 1-2 mm. According to the detection result, 4 well-grown monoclonal positive culture wells are selected from each subcloned cell line, and transferred to a 24-well plate for continuous culture. After a period of time, a positive clone cell line obtained by cloning in the 24-well plate is screened again, and the positive clone cell line is the hybridoma cell line 15E11D5E11 secreting the specific monoclonal antibody. The cell line is transferred into a T-75 culture flask to be amplified to a logarithmic growth phase for seed preservation or subsequent experiments are carried out.

EXAMPLE 3 preparation of monoclonal antibodies by in vitro culture

1. In vitro culture

After obtaining the stable hybridoma cell line, obtaining the monoclonal antibody by adopting an in vitro culture method.

The hybridoma cells were cultured in DMEM complete medium containing 10% fetal bovine serum, and then centrifuged at low speed to collect the culture supernatant, which was stored at 4 ℃ for further use.

2. Purification of monoclonal antibodies

Antibody purification using rProtein A sepharose Fast Flow (GE) affinity column: (1) loading the column, loading a proper amount of purchased ProteinA filler into a gravity chromatography column, and washing with an equilibrium buffer (1.5M Tris solution containing 1.5M NaCl, pH 7.0) until equilibrium is reached; (2) loading, namely adding cell supernatant filtered by a 0.22-micron filter membrane into a packed chromatographic column, and controlling the flow rate to be 1 drop/second; (3) balancing, and washing the sample solution to balance by using a balance buffer solution after the sample solution is loaded; (4) eluting, adding elution buffer (0.1M citric acid solution, pH4.5) to wash the column and collecting eluate; (5) regenerating, adding an equilibrium buffer solution to wash the column to balance after the elution is finished, washing the column with 2 times of column volume of 20% ethanol, and storing at 4 ℃. And finally, identifying the purity of the antibody by adopting an SDS-PAGE method. The result is shown in figure 1, the polyacrylamide gel electrophoresis picture of the purified Inhibin-alpha monoclonal antibody has the purity of more than 95 percent, and the concentration of the antibody is measured by an ultraviolet micro-spectrophotometer method and reaches more than 3.0 mg/mL.

EXAMPLE 4 characterization of monoclonal antibodies

1. Subtype identification

The antigen proteins were diluted to 1. Mu.g/mL coated ELISA plates, 100. Mu.L per well, coated overnight at 4 ℃, the liquid was decanted, the plates were washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. Mu.L of blocking solution (PBS-T solution containing 2% BSA) was added per well, and incubated at 37 ℃ for 1h. The liquid was decanted and washed 3 times with PBS-T. 0.1mL of culture supernatant of hybridoma cell line diluted 5-fold was added to each well and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Using a confining liquid 1: HRP-labeled goat anti-mouse (kappa,. Lamda., igM, igG1, igG2a, igG2b, igG) was diluted 400 ₃ IgA) antibody (Southern Biotech) was added in an amount of 0.1mL per well and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. 100 mu LTMB (Hiroshi, huzhou) substrate (A, B equal volume mixed solution) is added into each hole for color development, the reaction is carried out for 15min at room temperature, 50 mu L of 1N HCl solution is added into each hole to stop the color development reaction, and then an enzyme-labeling instrument is used for measuring the OD value at the wavelength of 450 nm. The results show that the monoclonal antibody of the invention is an IgG1 type murine monoclonal antibody.

2. Determination of affinity constant

Inhibin-alpha protein was coated at 100. Mu.g/ml, 100. Mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. Mu.l of blocking solution to each well and block at 37 ℃ for 1h, wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was diluted to the following concentrations (unit: ng/mL): 2000. 500, 125, 62.5, 31.25, 15.625, 3.125, 0.625, incubation at 37 ℃ for 1h, PBS-T wash 3 times. HRP-labeled goat anti-mouse secondary antibody 1:5000 dilution, 100. Mu.l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. 100. Mu.l of TMB (Biotechnology, inc., england, huzhou) color developing solution was added to each well, and the reaction was stopped by adding 100. Mu.l of 1.0N hydrochloric acid solution after color development for 13 min. The absorbance at a wavelength of 450nm was measured with a microplate reader. Drawing the curve of OD value corresponding to the antibody dilution factor, and finding out the antibody concentration corresponding to 1/2' platform OD valueA. The affinity constant was calculated to be 6.93X 10 using the following formula ⁹ 。

3. Reaction specificity and application effect of monoclonal antibody

Selecting immunogen solution, detecting the recognition specificity of the monoclonal antibody by an immunoblotting method, and carrying out 15% polyacrylamide gel electrophoresis on Inhibin-alpha protein. The gel proteins were transferred to PVDF membrane using conventional wet transfer methods. The membrane was placed in a 5% bsa-TBST solution (protein side down) and shaker blocked at 37 ℃ for 1h to eliminate non-specific background. After blocking was completed, 5% of BSA-TBST was washed away with TBST, and the monoclonal antibody against Inhibin- α prepared in the present invention was added thereto, followed by incubation for 1 hour in a decolorizing shaker. After washing the membrane with TBST, a secondary antibody was added, and the membrane was incubated for 1 hour in a decolorization shaker to allow the secondary antibody to bind fully to the primary antibody. The membrane was washed again with TBST and ECL color development kit was added. The experimental results are shown in the immunoblotting chart of detecting human Inhibin-alpha protein by Inhibin-alpha monoclonal antibody in FIG. 2: the single band and darker color indicate the distinct specific reaction between the antibody and the antigen.

Example 5 tissue chip staining and characterization

1. Tissue wax block preparation

HE section staining was performed on the sample tissue to determine the tumor lesion site. And (5) circling the lesion site and preparing to punch. When the receptor wax block is manufactured, a plastic frame is placed on a mold, melted paraffin (the melting point is 56-58 ℃) is poured into the mold, the tissue block is placed into wax liquid in the mold, then a proper amount of wax liquid is added to completely embed the tissue block in the wax liquid, the mold is placed into a refrigerator with the temperature of-20 ℃ for 6min after being cooled to room temperature, and the wax block is taken out of the mold and sliced or placed into a refrigerator with the temperature of 4 ℃ for storage for later use. Slicing continuously, slicing to 4 μm thickness, rinsing the slices in 40% ethanol, naturally spreading, transferring the slices into 45 deg.C warm water, spreading for 30 s, mounting the slices with 2% APES acetone solution treated glass slide, baking the obtained tissue chip in 60 deg.C oven for 2 hr, taking out, cooling at room temperature, and storing in-4 deg.C refrigerator.

2. IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each time with 100%, 95%, 85% gradient ethanol hydration for 3 minutes each time and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3min with PBS. Dropwise adding 3%H ₂ O ₂ Incubate for 10min and wash with PBS for 3 x 3 min. The PBS was spun off and the peroxidase blocker was added dropwise and incubated for 10 minutes at room temperature. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 20 seconds, PBS turns blue. Dehydration was carried out in sequence according to an alcohol gradient of 85% (3 min), 95% (3 min), 100% (3 min) and 100% (3 min), and finally two times xylene was cleared for 10min, followed by neutral gum blocking.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+++".

4. Samples were negative and marked "-".

3. And (3) sample detection results:

the results of the detection of the anti-Inhibin-alpha protein monoclonal antibody (15E 11D5E 11) prepared by the invention in 21 cases of mesothelioma, 25 cases of ovarian interstitial tumor and 1 case of adrenocortical adenoma are shown in the following table:

the result shows that the staining localization of the anti-Inhibin-alpha protein monoclonal antibody (15E 11D5E 11) is accurate, the staining is clear, the non-specific staining is avoided, the background is clean, and the specificity of the anti-Inhibin-alpha protein monoclonal antibody (15E 11D5E 11) is strong. FIG. 3 is a graph showing immunohistochemical staining results for ovarian solitary stromal tumors.

Finally, it should be noted that, although the above embodiments have been described in the text and drawings of the present application, the scope of the patent protection of the present application is not limited thereby. All technical solutions generated by replacing or modifying the equivalent structure or the equivalent flow described in the text and the drawings of the present application and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields and the like based on the substantial idea of the present application are included in the scope of the patent protection of the present application.

SEQUENCE LISTING

<110> Fuzhou mai New Biotechnology development Co., ltd

<120> anti-Inhibin-alpha protein monoclonal antibody, cell line, preparation method and application thereof

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Claims (6)

1. The monoclonal antibody for resisting Inhibin-alpha protein is characterized in that the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are encoded by the nucleotide sequences set forth in SEQ ID No.4 and SEQ ID No.5, respectively.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody is produced by a hybridoma cell line having a collection number of CGMCCNO.22320.

4. The monoclonal antibody of claim 1, wherein said anti-Inhibin- α protein monoclonal antibody is a mouse IgG1 subtype monoclonal antibody.

5. A hybridoma cell line for secreting an anti-Inhibin-alpha protein monoclonal antibody, wherein the cell line is a mouse hybridoma cell line 15E11D5E11, the cell line is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO.22320.

6. An immunoassay reagent for human Inhibin-alpha protein, characterized in that the immunoassay reagent contains the anti-Inhibin-alpha protein monoclonal antibody of any one of claims 1 to 4 as an active ingredient.

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