CN113234159B - anti-LAG 3 protein monoclonal antibody, cell strain thereof, preparation method and application - Google Patents

Abstract

The invention relates to a monoclonal antibody capable of recognizing human LAG3 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. According to the technical scheme, amino acids from 32 th site to 51 th site at the C terminal of LAG3 protein are selected as antigenic peptides, and the immunogen obtained after coupling KLH is used for immunizing a mouse, and a mouse hybridoma cell line 22G3 capable of efficiently secreting the anti-LAG 3 protein monoclonal antibody and the anti-LAG 3 protein monoclonal antibody secreted by the cell line are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing LAG3 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-LAG 3 protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-LAG 3 protein monoclonal antibody, a cell strain, a preparation method and an application thereof.

Background

Lymphocyte activation gene 3 (LAG 3) is an important class of immunosuppressive molecules, LAG3 is considered as the third immune checkpoint following PD-1, CTLA-4, which was first discovered by Triebel and colleagues in 1990. LAG3 is a 498 amino acid type I transmembrane protein on the surface of human activated NK and T cells, and LAG3 gene is located on human chromosome 12, adjacent to CD4 gene, both of which have identical exons and introns, thus LAG3 has high homology with CD4 molecule. MHC class II molecules are ligands of LAG3, and compared with CD4, LAG3 has higher affinity with MHC class II molecules, and inhibits the cytotoxic effect of T cells by combining with MHC class II molecules on the surface of antigen presenting cells to secrete negative cytokines such as IL-10 and TGF-beta, but the specific signal transduction mechanism in the cells is still unknown at present.

There have been numerous studies investigating the prognostic value of LAG3 in different tumors. A recent meta analysis of the prognostic value of LAG3 in tumors showed that high LAG3 expression was associated with longer overall survival, and sub-layer analysis showed no difference between different tumors. The expression and prognostic value research results of LAG3 in esophageal squamous cell carcinoma also indicate that the high expression of LAG3 is positively correlated with the overall survival and progression-free survival. A total of 30 patients were enrolled in the anti-PD-1 monoclonal antibody nivolumab study for the treatment of advanced gastric cancer, which showed that the LAG3+ CD4+/CD8+ T cell ratio increased and progression-free survival correlated significantly positively with the LAG3+ CD4+/CD8+ T cell ratio as the disease progressed. In the expression and prognosis value research of LAG3 in the treatment of triple negative breast cancer, the research result shows that the LAG3 level before neoadjuvant chemotherapy is obviously related to the pathological remission rate, and LAG3 is highly expressed in the tumor residual tissues, particularly the expression of PD-1 is related to poor prognosis. In the immunohistochemical analysis research on 4322 breast cancer primary excision specimens, multi-factor variable analysis finds that the breast cancer patients with LAG3+ ITILs have obviously improved specific survival rate of breast cancer, and the research shows that the LAG3+ ITILs are rich in estrogen receptor negative breast cancer and are independent favorable prognostic factors. LAG3 is also valuable for prognosis of NSCLC, and the research collects 553 primary tumor tissues and 143 corresponding metastatic lymph nodes of patients with NSCLC in stage I-IIIb, and the research shows that LAG3+ TILs are related to improving disease specific survival rate and are independent positive predictors for NSCLC patients in stage I-IIIb. A single-center retrospective analysis showed that LAG3 expression was an independent, adverse prognostic contributor to melanoma.

Disclosure of Invention

The inventor provides an anti-LAG 3 protein monoclonal antibody, wherein 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.

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

Further, the monoclonal antibody specifically recognizes LAG3 protein.

Further, the monoclonal antibody is a mouse IgG2a kappa subtype monoclonal antibody.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 20774.

The inventor also provides a preparation method of the anti-LAG 3 protein monoclonal antibody, which selects an amino acid sequence (32 th position to 51 th position) shown in SEQ ID NO.1 in LAG3 protein, adds a cysteine at the carboxyl terminal thereof, and then couples the amino acid sequence with a carrier protein KLH to be used as immunogen.

The inventor also provides a hybridoma cell strain secreting LAG-resistant 3 protein molecules, wherein the cell strain is mouse hybridoma cell strain 22G3 which is preserved in China general microbiological culture Collection center (CGMCC NO 20774) at 9 and 17 months of 2020, and the accession number is CGMCC NO 20774, and the cell strain is the institute of microbiology of China academy of sciences No.3, North Cheng West Lu No.1 institute of Tokyo Yang-ward, Beijing.

The inventor also provides the application of the monoclonal antibody in the immunoassay of the LAG3 protein.

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

The inventor finally provides an immunohistochemical detection reagent of the LAG3 protein, wherein the immunohistochemical detection reagent contains an amino acid sequence shown as SEQ ID NO.4 in the amino acid sequence of a heavy chain variable region; the monoclonal antibody of anti-LAG 3 protein with the amino acid sequence of the light chain variable region as shown in SEQ ID NO.5 is the effective component.

Different from the prior art, the invention has the beneficial technical effects that: according to the technical scheme, amino acids from 32 th site to 51 th site at the C terminal of LAG3 protein are selected as antigenic peptides, and the immunogen obtained after coupling KLH is used for immunizing a mouse, and a mouse hybridoma cell line 22G3 capable of efficiently secreting the anti-LAG 3 protein monoclonal antibody and the anti-LAG 3 protein monoclonal antibody secreted by the cell line are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing LAG3 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a comparison of immunohistochemical staining of tonsil granulocytes (LAG 3 secreted by 22G3 on the left and commercial LAG3 on the right).

FIG. 2 is a comparison of the results of immunohistochemical staining of part of appendix with granulocyte (left 22G 3-secreted LAG3 and right commercial LAG 3).

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

EXAMPLE 1 preparation of immunogen

First, immunogen preparation

By analyzing the sequence and the secondary structure of the protein sequence with the accession number P18627 in Uniprot, the molecular weight of the LAG3 protein with the total length of 260 amino acids is about 39 kDa. According to the parameters of secondary structure (secondary structure) and Surface Accessibility (Surface Accessibility) of the protein predicted by the online server http:// www.cbs.dtu.dk/services/NetSurfP/and by the analysis of its antigenicity index (Jameson BA, Wolf H. the antigenicity index: a novel homology for predicting antigenic derivatives. Compound application biosci.1988,4(1):181-6.), the amino acid sequence SEQ ID NO.1 at positions 32-51 is selected as the antigenic peptide for chemical synthesis, and for coupling, a cysteine is added at the carboxyl end of the polypeptide to provide thiol coupling.

Coupling and purification of polypeptide

The equine activated keyhole limpet hemocyanin kit from Thermo Scientific (cat # 77653) was selected and operated according to the protocol provided for the kit. For the polypeptide to be conjugated, the free thiol group in the polypeptide is first detected with Ellman's reagent (Thermo Scientific, cat # 22582): adding 100 mu L Ellman reagent stock solution into a 96-well plate, adding 10 mu L polypeptide solution, measuring the ultraviolet absorption value of the solution by a Nano Drop spectrophotometer at the lambda of 412nm, and performing the next step if the OD value is more than 0.15; the OD value is less than 0.15 and more than 0.05, and the polypeptide is added until the requirement is met; and the OD value is less than 0.05, and the polypeptide is returned to the polypeptide synthesis step for quality control. To begin conjugation, 200. mu.L of deionized water was added to each mcKLH package to make up 10mg/mL of KLH solution, 2mg of hapten was dissolved in 500. mu.L of Imject EDC conjugation buffer, 500. mu.L of polypeptide solution was added to 200. mu.L of carrier protein solution, 1mL of deionized water was added to one package of EDC (10mg) and shaken slowly to dissolve completely, 50. mu.L of polypeptide solution was added to mcKLH polypeptide solution and after 2 hours of reaction, unconjugated crosslinker and salts were removed by desalting column treatment to give LAG 3-KLH.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The crosslinked polypeptide of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and ICR mice (purchased from Beijing Wintotonghua laboratory animal technology Co., Ltd.) were immunized and injected ventrally at 6-point per mouse at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

Aseptically preparing mouse spleen with up-to-standard immunityThe cell suspension was mixed with mouse myeloma cells sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly 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, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 characterization of monoclonal antibodies

First, subclass identification

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, 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 at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 h. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted at 2000 in 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is a murine monoclonal antibody of the IgG2a kappa type.

Second, determination of affinity constant

The LAG3 polypeptide 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. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 3 was purified from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction mixture was developed in a citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Plotting the OD value against the dilution factor of the antibody, finding half the maximum binding OD valueThe dilution factor A, the affinity constant of the antibody was calculated to be 9.6X 10 using the following formula⁸。

Reaction specificity and application effect of monoclonal antibody

The LAG3 polypeptide prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and subjected to 12% polyacrylamide gel electrophoresis. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 22G3 hybridoma (1: 1000 dilution) were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

Example 5 determination of variable region sequences of antibodies

Collecting fresh hybridoma cells, collecting supernatant, performing antigen binding property verification to confirm that the cell strain for cloning can indeed secrete required antibody, and centrifuging to collect 10⁶The hybridoma cells described above. Total RNA of hybridoma cells was extracted by Trizol method, and 9. mu.L of total RNA was added with 2.5. mu.L of oligo (dT) 12-18 primer (10mM) and 5. mu.L of dNTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then, 5. mu.L of RT buffer (5X), 2.5. mu.L of DTT (0.1M) and 1. mu.L of reverse transcriptase were added and reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The first strand cDNA thus obtained was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.L reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were determined in accordance with the sequence of the murine monoclonal antibody described in "recombinant antibody" by Hippocampus (science publishers, 2005 publications)Primer sequence design and synthesis.

The rest dNTPs and buffer 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. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 20-25 cycles, final extension at 72 deg.c for 3 min, and setting the product at 4 deg.c for use or direct electrophoresis. 20 μ L of PCR product was analyzed by electrophoresis, separated on 1.5% agarose gel, the length of light chain (. kappa.light chain) was between 320-340bp and the length of heavy chain was between 340-370bp, and the region-specific product was recovered by gel cutting and cloned into T-vector or expression vector for sequencing.

Example 6 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ 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 the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, and adding diluted primary antibody (the first dilution of antibody is designed according to antibody concentration)Dilution ratio) was incubated at room temperature (25 ℃ C.) for 1 hour, washed with PBS for 3X 3 minutes, a secondary antibody was added dropwise thereto and incubated at room temperature for 15 to 30 minutes, washed with PBS for 3X 3 minutes, the PBS was thrown off, and developed with a freshly prepared DAB developing solution for 3 to 10 minutes. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

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. The sample was negative and marked "-".

Data statistics

1. Evaluating the detection result of the tissue chip:

the present antibody LAG3(22G3) and the commercial antibody LAG3(EPR20261) evaluation tissue chips (20 cases of tonsils and 30 cases of appendices) were subjected to simultaneous examination and the examination results were compared.

Immunohistochemical results for LAG3 were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-LAG 3 protein secreted by the 22G3 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In a positive test, the number of stained cells and staining intensity of the LAG3 antibody secreted by 22G3 were significantly higher than those of the commercial LAG 3; the fact that the LAG3 antibody secreted by 22G3 is more sensitive and effectively avoids false negative results.

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation 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, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (22G3) and the commercial antibody (E3) were detected on the normal tissue chip synchronously, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in the normal tissue was equivalent to that of the commercial antibody.

FIG. 1 is a comparison of immunohistochemical staining of tonsil granulocytes (LAG 3 secreted by 22G3 on the left and commercial LAG3 on the right).

FIG. 2 is a graph showing a comparison of the results of immunohistochemical staining of part of the appendix (LAG 3 secreted from 22G3 on the left and commercially available LAG3 on the right)

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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Claims (9)

1. The monoclonal antibody for resisting the LAG3 protein is characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence 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.

2. The monoclonal antibody according to claim 1, wherein the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2, and the coding DNA sequence of 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, which specifically recognizes LAG3 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG2a kappa subtype monoclonal antibody.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 20774.

6. A hybridoma cell strain secreting anti-LAG 3 protein molecules is a mouse hybridoma cell strain 22G3, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 20774.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of a LAG3 protein immunoassay reagent.

8. The use according to claim 7, wherein said immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

9. An immunohistochemical detection reagent for LAG3 protein, comprising the anti-LAG 3 monoclonal antibody of claim 1 as an active ingredient.

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