CN115806620B

CN115806620B - Monoclonal antibody against HE4 protein, cell strain, preparation method and application thereof

Info

Publication number: CN115806620B
Application number: CN202211554200.2A
Authority: CN
Inventors: 陈泳; 杨清海; 陈惠玲; 王小亚
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2022-12-06
Filing date: 2022-12-06
Publication date: 2023-12-05
Anticipated expiration: 2042-12-06
Also published as: CN115806620A

Abstract

The application relates to a monoclonal antibody capable of recognizing human HE4 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunodetection. According to the technical scheme, 31-124 amino acids of HE4 protein are selected as antigen peptides, codon optimization is carried out, the gene fragment suitable for expression in escherichia coli BL21 is formed, and finally the obtained recombinant protein comprises a TRX protein tag, an HE4 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and cell fusion, screening and subcloning are carried out to obtain a mouse hybridoma cell strain 3G10 which can efficiently secrete the HE4 protein monoclonal antibody and the HE4 protein monoclonal antibody secreted by the cell strain. The antibody obtained by the scheme has high specificity and sensitivity, can specifically identify cells expressing HE4 protein, and is suitable for immunological detection, in particular immunohistochemical detection.

Description

Monoclonal antibody against HE4 protein, cell strain, preparation method and application thereof

Technical Field

The application relates to the field of biomedical engineering, in particular to a monoclonal antibody for resisting HE4 protein, a cell strain, a preparation method and application thereof.

Background

Human epididymal protein (HE 4) is a secreted protein, and its gene cDNA was first extracted from the distal end of epididymal epithelium by Kirchhoff in 1991, and was solidly named human epididymal protein. Because of the highly conserved characteristic WAP domain in the molecular structure, the HE4 gene is also known as WAP-4-disulfide core domain 2 (WFDC 2), also known as WFDC2.WAP domain is a conserved sequence of approximately 50 amino acids containing 8 cysteines with 4 disulfide bonds (4-DSC) as core region, and the encoded protein has high homology with extracellular protease inhibitor, but HE4 has not been found to have the effect of inhibiting protease. The HE4 gene is located on human chromosome 20q12-13.1, has a total length of about 12kb and consists of 5 exons and 4 introns, and has various shearing forms and encodes small molecule secretion proteins. HE4 is expressed in the genital tract, breast, respiratory tract, kidney distal tubule, colon, salivary glands of human normal females, but is expressed low or not in normal ovarian tissue, whereas the positive expression rate in ovarian tissue is correlated with the tissue type of ovarian cancer, with serous ovarian cancer being the highest. Studies have shown that HE4 may inhibit cell proliferation in vitro through PI3K/AKT signaling pathways, and from the premise that HE4 may play a protective role by inhibiting cancer cell proliferation.

The method for detecting the expression condition of HE4 in different ovarian tissues by using an immunohistochemical method shows that HE4 is highly expressed in high-grade serous ovarian cancer and has statistical significance with the expression difference between normal ovarian tissues and benign ovarian tumor tissues. The positive expression rate of HE4 increases with the progress of clinical stage, and the difference between the III-IV stage and the I-II stage has statistical significance.

Disclosure of Invention

The inventor provides a monoclonal antibody of anti-HE 4 protein, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Further, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown as SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown as SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes HE4 protein.

Further, the monoclonal antibody is a mouse IgG1 kappa subtype monoclonal antibody.

Further, the monoclonal antibody is produced by a hybridoma cell strain with a preservation number of CGMCCNO 21972.

The inventor also provides a preparation method of the anti-HE 4 protein monoclonal antibody, wherein an antigen used for immunizing mice is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombination way and comprises a TRX protein tag, an HE4 protein fragment and a histidine protein tag.

Further, the HE4 protein fragment is an amino acid fragment from 31 st to 124 th positions of HE4 protein, and the amino acid sequence of the HE4 protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting HE4 protein resistant molecules, wherein the cell strain is a mouse hybridoma cell strain 3G10, and the cell strain is preserved in China general microbiological culture collection center (CGMCCNO) 21972 at 2021, 3 months and 24 days, and is provided at the national institute of microbiological culture, national institute of sciences, 1, 3, beijing, chaoyang, north.

The inventor also provides an HE4 protein immunodetection reagent, which contains an amino acid sequence of a heavy chain variable region, wherein the amino acid sequence is shown as SEQ ID NO. 4; the monoclonal antibody of the HE4 protein with the amino acid sequence shown in SEQ ID NO.5 as the light chain variable region is taken as an effective component.

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

Compared with the prior art, the application has the following beneficial technical effects: according to the technical scheme, 31-124 amino acids of HE4 protein are selected as antigen peptides, codon optimization is carried out, the gene fragment suitable for expression in escherichia coli BL21 is formed, and finally the obtained recombinant protein comprises a TRX protein tag, an HE4 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and cell fusion, screening and subcloning are carried out to obtain a mouse hybridoma cell strain 3G10 which is used for efficiently secreting the HE4 protein monoclonal antibody and the HE4 protein monoclonal antibody secreted by the cell strain. The antibody obtained by the scheme has high specificity and sensitivity, can specifically identify cells expressing HE4 protein, and is suitable for immunological detection, in particular immunohistochemical detection.

Drawings

FIG. 1 is a diagram of purified pectin from recombinant HE4 proteins fused with histidine tag of example 1, wherein M represents Marker;1 is a supernatant sample; 2 is the post-ultrasound pellet sample.

FIG. 2 is a graph comparing results of immunohistochemical staining of ovarian serous tumors; wherein the left part is HE4 secreted by 3G10, and the right part is commercial HE4.

Detailed Description

In order to describe the possible application scenarios, technical principles, practical embodiments, and the like of the present application in detail, the following description is made with reference to the specific embodiments and the accompanying drawings. The embodiments described herein are only for more clearly illustrating the technical aspects of the present application, and thus are only exemplary and not intended to limit the scope of the present application.

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

Unless defined otherwise, technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains; the use of related terms herein is for the purpose of describing particular embodiments only and is not intended to limit the application.

In the description of the present application, the term "and/or" is a representation for describing a logical relationship between objects, which means that three relationships may exist, for example a and/or B, representing: there are three cases, a, B, and both a and B. In addition, the character "/" herein generally indicates that the front-to-back associated object is an "or" logical relationship.

In the present application, terms such as "first" and "second" are used merely to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual number, order, or sequence of such entities or operations.

Without further limitation, the use of the terms "comprising," "including," "having," or other like terms in this specification is intended to cover a non-exclusive inclusion, such that a process, method, or article of manufacture that comprises a list of elements does not include additional elements but may include other elements not expressly listed or inherent to such process, method, or article of manufacture.

As in the understanding of "review guidelines," the expressions "greater than", "less than", "exceeding" and the like are understood to exclude this number in the present application; the expressions "above", "below", "within" and the like are understood to include this number. Furthermore, in the description of embodiments of the present application, the meaning of "a plurality of" is two or more (including two), and similarly, the expression "a plurality of" is also to be understood as such, for example, "a plurality of" and the like, unless specifically defined otherwise.

EXAMPLE 1 preparation of recombinant HE4 protein fragments

1. Gene optimization and synthesis

HE4 selects the 31-124 amino acid protein fragment according to the protein sequence with accession number Q14508 in Uniprot database, and directly optimizes the gene fragment suitable for expression in escherichia coli BL 21. Respectively adding at the 5 'and 3' ends of the gene during PCR _BamHI And an XhoI cleavage site.

The PCR product was recovered after agarose gel electrophoresis separation, bamHI and XhoI digestion were performed on the recovered fusion protein gene and plasmid vector pET30a for expression, respectively, and the recovered product was again electrophoretically recovered and ligated with T4DNA ligase. And (4) transforming the connection product into escherichia coli competent cells BL21, picking up clones on a flat plate, and carrying out bacterial liquid PCR identification. Clones positive to the PCR result were selected for sequencing analysis and clones with the correct sequence were used.

The selection of different antigens for immunization may produce antibodies with different binding characteristics, which molecule may have multiple variants due to variable cleavage at the same time, ultimately resulting in different antibodies with different recognition capacities and patterns for antigen expressing cells. HE4 molecules are analyzed according to published sequences, according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids on cell membranes and secondary structure, a region suitable for soluble expression and having good immunogenicity is selected for recombinant expression, 31-124 amino acid residues of HE4 are selected for codon optimization, and the molecular weight is about 42kDa. And obtaining the HE4 protein by utilizing a prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of HE4 protein fragment with antigenicity and protein tag for recombinant protein purification, wherein the protein tag is TRX and HIS.

2. Protein expression and purification

The overnight bacteria cultured by single colony are transferred to 100mLLB culture medium according to the proportion of 1:100, kanamycin with the final concentration of 10 mug/mL is added, the culture medium is subjected to shaking culture at 37 ℃ until the OD600 is 0.6-0.8, IPTG with the concentration of 0.5mmol/L is added, the culture medium is subjected to shaking culture at 37 ℃ for 2 hours, and the bacteria are harvested by ultrasonic disruption. The recombinant protein has a histidine tag, and affinity purification of the protein is performed using a nickel column. Elution was performed with 300mmol/L imidazole and detection was performed by SDSPAGE separation.

FIG. 1 is a diagram of purified pectin from recombinant HE4 proteins fused to histidine tags. The protein concentration is 2.3mg/mL, and can be used for animal immunization and the requirements of antibody screening and identification.

Example 2 establishment of hybridoma cell lines

1. Immunization

The recombinant protein of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881) and immunized with 4-6 week old female ICR mice (purchased from Peking Vitre Liwa laboratory animal technologies Co., ltd.) at a dose of 20 μg/mouse by abdominal subcutaneous injection at 6 points per mouse. Once every 14 days, the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg/dose. The polyclonal antibody titer of the anti-immunogen in the serum of the mice was detected by indirect ELISA (wavelength 450 nm) 7 days after the 3 rd booster immunization, the mice with the highest titer were immunized by tail vein injection, and the antigens were uniformly mixed with physiological saline at a dose of 20 mug/mouse.

2. Cell fusion

Sterile preparation of immune-up to-standard mouse spleen cell suspension, mixing with mouse myeloma cell sp2/0 (ATCC NumberCRL-8287) at a ratio of 5:1, centrifuging at 1500rpm for 5min. The supernatant was discarded, the tube was placed in a 37℃water bath, 1ml of PEG1500 (Roche Co.) was slowly added over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, centrifuged at 1000rpm for 5min. After the supernatant was discarded, 10ml of serum (PAA company) was added and the cells were carefully blown up, and 5ml of thymocytes mixed with 10xHAT (Sigma company) were added and mixed. 25ml of semisolid medium containing 2.1% nitrocellulose (Sigma Co.) was added and mixed well, and then poured into 20 cell culture dishes uniformly. Placing the cell culture dish into a wet box, placing 5% CO at 37deg.C ₂ Culturing in an incubator.

3. Cloning and ELISA screening positive hybridoma cells

The size and density of the cloned cell mass are moderate 7 days after fusion, round, solid and large clone mass is sucked into a 96-hole culture plate which is prepared with culture medium in advance under a dissecting lens, and is put into 5% CO at 37 DEG C ₂ Culturing in an incubator. After 3 days, the cell mass was approximately 2/3 of the floor space, and 100. Mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely changed and 200. Mu.L of complete medium containing feeder cells and 1% HT (Sigma Co.) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates in which medium (containing feeder cells and HT) was prepared in advance. Five days later, 100 μl of the supernatant was subjected to a third ELISA screening, and positive clones were successively transferred into 6-well plates and cell culture flasks for expansion culture and frozen storage. EXAMPLE 3 preparation of monoclonal antibodies by ascites Induction

1. Ascites preparation

Cells in logarithmic growth phase were washed and suspended in serum-free medium and counted approximately 5X 10 ⁵ 1ml. Suspended cells were intraperitoneally injected into mice previously sensitized with paraffin oil. Ascites collection was started after 7 days. The ascites removed was centrifuged at 4000rpm at 4℃for 10min. Carefully aspirate the middleThe ascites is collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

2. Purification of monoclonal antibodies

Antibodies were purified from ascites fluid using HiTraprProteinAFF (GE company) affinity chromatography as described. SDS-PAGE gel was used to identify purity and concentration was determined by the Bradford method. Purified antibodies were stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

1. Subclass identification

The coated sheep anti-mouse IgG (Abies sinensis Biotechnology Co., ltd., beijing) was diluted to 0.5. Mu.g/ml with 100mM PBS (pH 7.4), 100. Mu.l/well, 4℃overnight. The liquid was emptied and washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. Mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well and incubated for 1h at 37 ℃. The liquid was emptied and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37℃for 1h. The liquid was emptied and washed 3 times with PBS-T. With blocking liquid 1: HRP-labeled goat anti-mouse (κ, λ) antibody or 1:2000 dilution of HRP-labeled goat anti-mouse (IgM, igG1, igG2a, igG2b, igG3, igA) antibodies (southern Biotech) 0.1ml per well were added to the appropriate wells and incubated for 1h at 37 ℃. The liquid was emptied and washed 3 times with PBS-T. Mu.l of a mixture containing 0.15% ABTS (southern Biotech) and 0.03% H was added to each well ₂ O ₂ The reaction was developed with a citric acid buffer (pH 4.0) and the OD at 405nm was measured in 10-20 min.

The results showed that the monoclonal antibody of the present application was an IgG1 kappa-type murine monoclonal antibody.

2. Affinity constant determination

HE4 recombinant protein prepared in example 1 was coated at a concentration of 2. Mu.g/ml, 100. Mu.l/well, coated overnight at 4℃and washed 3 times with PBS-T. Mu.l of blocking solution was added to each well and blocked at 37℃for 2h, and PBS-T was washed 3 times. The monoclonal antibodies purified in example 3, from 1:200 beginning 2-fold gradient dilution, leaving 1 well blank for control, incubating for 1h at 37℃and washing 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: diluted 20000, incubated at 37℃for 1h in 100. Mu.l/well and washed 3 times with PBS-T. Mu.l of 0.1% TMB (Sigma Co.) and 0.03% H were added to each well ₂ O ₂ Is developed for 10min in citric acid-phosphoric acid buffer solution, and 50 mu.l of 0.5M sulfur is addedThe acid solution terminated the reaction. The absorbance at a wavelength of 450nm was measured with a microplate reader. Drawing a curve of OD value corresponding to dilution factor of antibody, finding out dilution factor A corresponding to half of maximum binding OD value, and calculating affinity constant of the antibody to 1.92×10 by using the following formula ⁹ 。

Monoclonal antibody reaction specificity and application effect

The HE4 recombinant protein prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the application by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. Gel protein bands were transferred onto PVDF membranes (Millipore Corp.) in a Bio-Rad electrotransfer system according to conventional methods. The membranes were placed in TBS-T blocking solution containing 5% nonfat milk powder overnight at 4 ℃. Monoclonal antibodies (1:1000 dilution) to the antibodies secreted by the 41B1 hybridoma were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, 1: sheep anti-mouse secondary antibody (fir gold bridge biotechnology limited in beijing) diluted at 5000 was incubated for 1 hour at room temperature. The membrane was again washed with TBST, ECL hypersensitivity developing solution (Beijing pride Gene technologies Co., ltd.) was added, and chemiluminescent image data was collected using a ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequence of antibodies

Taking fresh hybridoma cells, taking supernatant, performing antigen binding characteristic verification, confirming that cell strain used for cloning can indeed secrete required antibody, and centrifuging and collecting 10 after confirming the result ⁶ The above hybridoma cells. Trizol method is used for extracting total RNA of hybridoma cells, 9 mu L of total RNA is taken, 2.5 mu oligo (dT) 12-18primer (10 mM) and 5 mu L dNTPs are added, and the mixture is uniformly mixed, and the mixture is kept at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or the denaturation operation is carried out according to the reverse transcriptase used. Subsequently, 5. Mu.LRTbuffer (5X), 2.5. Mu.LDTT (0.1M) and 1. Mu.L reverse transcriptase were added and reacted at 42℃for 1 hour. The reaction was terminated by incubating at 70℃for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA obtained was amplified by PCR, and each primer was added to a 50. Mu.L reaction system25pmol of primers for amplifying the heavy chain variable region and the light chain variable region were designed and synthesized according to the sequence of the murine monoclonal antibody primer in the book of recombinant antibody (scientific Press, 2005) mainly edited by Shen Beifen.

The rest dNTPs and buffer solution are added according to the conventional method, and finally 1 mu L of cDNA template and 1U of hot start Taq DNA polymerase are added. The PCR amplification procedure was set to 94℃for 40 seconds, 52℃for 40 seconds, 72℃for 40 seconds, 20 to 25 cycles were performed, and finally 72℃was extended for 3 minutes, and the product was allowed to stand by at 4℃or directly electrophoresed. The 20 mu LPCR product is taken for electrophoresis analysis, separated on 1.5% agarose gel, the length of the light chain (kappa light chain) is 320-340bp, the length of the heavy chain is 340-370bp, the specific product in the region is recovered by cutting gel, and cloned to a T vector or an expression vector for sequencing.

EXAMPLE 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE section staining was performed on each sample to determine tumor sites. The tissue chip was fabricated using a full-automatic tissue chip instrument from 3 DHISTECH. Putting the prepared tissue chip wax block into a wax block manufacturing mould, putting the mould into a 68 ℃ oven for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking out the mould from the oven, cooling the paraffin in a semi-fused state for about 30 minutes at room temperature, putting the mould into a-20 ℃ refrigerator for 6 minutes, taking out the tissue chip wax block from the mould, and slicing or putting the mould into a 4 ℃ refrigerator for preservation for later use. After trimming, continuous slicing is carried out, the thickness is set to be 3 mu m, the continuous slicing is floated in 40% alcohol, the continuous slicing is naturally unfolded, then the separated slices are transferred into warm water with the temperature of 50 ℃ for 30 seconds, the slices are pasted on a glass slide which is treated by polylysine, the prepared tissue chips are placed into an oven with the temperature of 68 ℃ for baking the slices for 2 hours, and the tissue chips are taken out, cooled at room temperature and placed into a refrigerator with the temperature of minus 4 ℃ for preservation.

IHC staining and analysis

Conventional xylenes were dewaxed 3 times for 6 minutes each, hydrated in 100%, 95%, 85% gradient ethanol for 3 minutes each, and finally rinsed with tap water. Antigen retrieval was performed and the sections were then placed in a wet box and rinsed 3 x 3 min in PBS. Dropwise adding 3%H ₂ O ₂ Incubate for 10min, rinse with PBS for 3 x 3 min. The sections were spun dry, incubated for 1 hour at room temperature (25 ℃) with primary antibody diluted in the appropriate ratio (the dilution ratio of the antibody was designed according to the concentration of the antibody for the first time), washed 3X 3 minutes with PBS, incubated for 15-30 minutes with secondary antibody at room temperature, washed 3X 3 minutes with PBS, spun off the PBS, and developed for 3-10 minutes with freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds and PBS returns to blue for 30 seconds. Sequentially dehydrating according to an alcohol gradient of 85% (3 minutes) to 95% (3 minutes) to 100% (3 minutes), and finally making xylene transparent for 3 minutes, and sealing the gel.

Immunohistochemical staining results were divided into: positive and negative. Positive expression must be positive at the cell and tissue specific antigenic sites. Under the conditions of clear tissue staining distribution and accurate cell positioning, the staining results are further divided according to the difference of staining intensity, and the specific steps are as follows:

1. the sample is weak positive; marked as "+";

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

3. the sample was highly positive; marked as' ++ ".

4. The sample was negative, labeled "-".

Third, data statistics

1. Tumor tissue chip detection results:

the present antibody HE4 (3G 10) and the commercial antibody HE4 (OTI 1B 9) were synchronously detected in 32 cases of ovarian mucinous adenocarcinoma and 36 cases of ovarian serous tumors, and the detection results were compared.

The immunohistochemical results for HE4 were counted. The whole test process adopts double-blind design, and the statistical result is as follows:

the result shows that the anti-HE 4 protein monoclonal antibody secreted by the 3G10 cell strain has accurate staining and positioning, clear staining, no nonspecific staining and clean background. In immunohistochemical detection, the positive rate was comparable to that of commercially available antibodies, but the positive intensity was higher than that of commercially available antibodies. The HE4 secreted by the 3G10 cell strain is higher in sensitivity, and false negative results are effectively avoided.

FIG. 2 is a comparison of results of immunohistochemical staining of ovarian serous tumors (left HE4 secreted by 3G10, right commercially available HE 4).

2. Normal tissue chip detection results:

the normal tissue chip comprises 30 normal tissue samples, wherein the normal tissue samples are mainly selected from fresh and timely fixed surgical specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsils, skeletal muscle, thymus (young children), skin, bone marrow, peripheral nerves, lung, mesothelial cells.

The antibody (3G 10) and the commercial antibody are synchronously detected on a normal tissue chip, and the detection results of yin and yang are consistent, which shows that the specificity of the antibody in normal tissues is equivalent to that of the commercial antibody.

Finally, it should be noted that, although the embodiments have been described in the text and the drawings, the scope of the application is not limited thereby. The technical scheme generated by replacing or modifying the equivalent structure or equivalent flow by utilizing the content recorded in the text and the drawings of the specification based on the essential idea of the application, and the technical scheme of the embodiment directly or indirectly implemented in other related technical fields are included in the patent protection scope of the application.

Claims

1. A monoclonal antibody against HE4 protein, characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID No. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2 and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes HE4 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG1 kappa subtype monoclonal antibody.

5. A monoclonal antibody against HE4 protein is produced by hybridoma cell strain with collection number of CGMCC NO 21972.

6. A hybridoma cell strain secreting HE4 protein resisting molecules, wherein the cell strain is a mouse hybridoma cell strain 3G10, and the cell strain is preserved in China general microbiological culture Collection center with the preservation number of: CGMCC NO 21972.

7. An HE4 protein immunodetection reagent, characterized in that it contains the monoclonal antibody against HE4 protein of claim 1 as an active ingredient.

8. The immunoassay reagent of claim 7, wherein said immunoassay comprises immunohistochemistry, immunoblotting and enzyme-linked immunoassay.