Monoclonal antibody against HLA-G2 and HLA-G6 molecules and its use

Abstract

The present invention discloses a monoclonal antibody against HLA-G2 and HLA-G6 molecules and its uses. The antibody (YWHG-26) is produced by a hybridoma with a deposit number of CCTCC NO: C202240. It uses the consensus sequence of HLA-G2 and HLA-G6 isoforms, and the antigenic peptide (RGYYNQSEAKPPKTHVTHHPV) located in the α1 and α3 connecting regions as an immunogen. The present invention provides nucleotide sequences encoding the YWHG-26 antibody and the encoded amino acid sequences thereof. The present invention also provides the use of the YWHG-26 antibody for the detection of HLA-G2 and HLA-G6 isoforms by immunoblotting and immunohistochemistry.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the following aspects of an antibody (YWHG-26) against HLA-G2 and HLA-G6 isoforms: using the consensus sequence of HLA-G2 and HLA-G6 isoforms and an antigenic peptide (RGYYNQSEAKPPKTHVTHHPV) located in the α1 and α3 connecting regions as an immunogen to prepare a monoclonal antibody (YWHG-26) against HLA-G molecules; nucleotides encoding the YWHG-26 antibody of the present invention and the amino acid sequence encoded therein; and use of the antibody (YWHG-26) for the detection of HLA-G2 and HLA-G6 isoforms by immunoblotting and immunohistochemistry. Background Art

[0002] The human leukocyte antigen-G (HLA-G) gene, 6.0 kb in length, is located on the distal short arm of human chromosome 6, at position 6p21.3. During protein translation, exon 1 of the HLA-G mRNA encodes the signal peptide, while exons 2, 3, and 4 encode the extracellular α1, α2, and α3 domains, respectively. Exon 5 encodes the transmembrane region. Exon 6 encodes the intracellular portion of the HLA-G molecule, consisting of only six amino acid residues. Due to the presence of a stop codon within exon 6, exon 7 is not transcribed. Exon 8 corresponds to the 3′ UTR of the HLA-G gene. The primary HLA-G transcript undergoes alternative splicing, resulting in seven mature mRNAs encoding seven molecular isoforms of varying molecular weight (HLA-G1, -G2, -G3, -G4, -G5, -G6, and HLA-G7). HLA-G1, HLA-G2, HLA-G3, and HLA-G4 contain transmembrane domains and are membrane-bound isoforms, while HLA-G5, HLA-G6, and HLA-G7 lack transmembrane domains and are soluble isoforms. The molecular weights of HLA-G1 through -G7 isoforms are 39kD, 31kD, 22kD, 30kD, 34kD, 23kD, and 16kD, respectively.

[0003] HLA-G1 is encoded by the full-length HLA-G mRNA and consists of the extracellular α1, α2, and α3 domains, a transmembrane region, and an intracellular domain. HLA-G2 lacks the α2 domain and consists of the extracellular α1 and α3 domains, a transmembrane region, and an intracellular domain. HLA-G3 lacks the α2 and α3 domains and consists of the extracellular α1 domain, a transmembrane region, and an intracellular domain. HLA-G4 lacks the α3 domain and consists of the extracellular α1 and α2 domains, a transmembrane region, and an intracellular domain. The extracellular domains are identical to those of HLA-G1 and HLA-G2, respectively, but they are encoded by HLA-G mRNA containing intron 4. Because intron 4 contains a stop codon, the encoded protein lacks the transmembrane region encoded by exon 5, resulting in a soluble HLA-G molecule. Since the mRNA encoding HLA-G7 contains a stop codon in intron 2, the extracellular region only contains the α1 domain and the two amino acid residues encoded by intron 2 ( Figure 1 ).

[0004] Under normal physiological conditions, HLA-G molecules are exclusively expressed in the extravillous trophoblasts at the maternal-fetal interface, maintaining maternal-fetal immune tolerance during pregnancy. Under pathological conditions, HLA-G expression can be induced in tumor cells and virally infected tissues, closely implicated in the development and progression of disease. HLA-G is a key immune tolerance-inducing molecule and a crucial immune checkpoint molecule. Its immunosuppressive function primarily occurs by binding to the immunosuppressive receptors immunoglobulin-like transcript-2 (ILT2 / LILRB1 / CD85j) and immunoglobulin-like transcript-4 (ILT4 / LILRB2 / CD85d), transmitting inhibitory signals and inducing immune tolerance. Its specific mechanisms of action include: ① Binding to ILT-2 expressed on T cells, NK cells, and B cells, inhibiting the cytotoxic activity of T and NK cells, as well as B cell proliferation and antibody secretion. ② Binding to ILT-2 and ILT-4 expressed on dendritic cells (DCs), inhibiting DC maturation and differentiation, thereby inducing the generation of tolerant DCs. ③ It binds to ILT-2 and ILT-4 expressed on myeloid-derived suppressor cells (MDSCs) and macrophages (Mф), inducing the differentiation of pro-inflammatory, anti-tumor M1 macrophages into immunotolerant M2 macrophages. Therefore, HLA-G plays a crucial role in the development and progression of cancer and other diseases. Several tumor immunotherapy treatments targeting HLA-G have entered Phase I clinical trials in the United States and other countries.

[0005] HLA-G binds to the receptors ILT-2 and ILT-4 with molecular structural specificity. The binding site for ILT-2 and ILT-4 receptors is the α3 domain of the HLA-G extracellular region. ILT-2 binds exclusively to the HLA-G / β2m complex, while ILT-4 can bind not only to the HLA-G / β2m complex but also to free HLA-G molecules without β2m. Due to differences in expression mechanisms and molecular structures, HLA-G1, -G2, -G3, -G4, -G5, -G6, and HLA-G7 isoforms can bind to HLA-G1 and HLA-G, while ILT-4 can bind to HLA-G1, HLA-G2, HLA-G5, and HLA-G6 isoforms. For example, HLA-G3, -G4, and HLA-G7 isoforms lack the α3 domain in their extracellular regions and are therefore unable to bind to ILT-2 and ILT-4. Different HLA-G isoforms can exert specific immunological effects in pathophysiological processes. Currently, targeted therapies based on HLA-G and ILT-related tumors have been gradually implemented. HLA-G isoform expression exhibits extensive heterogeneity, and the expression of different HLA-G isoforms has specific clinical significance. Therefore, analyzing the expression of specific HLA-G isoforms and the expression profiles of different HLA-G isoforms is of great significance for elucidating the biological functions and clinical significance of specific HLA-G isoforms.

[0006] Currently, the antibodies used for HLA-G immunohistochemistry and immunoblotting in China and abroad include 4H84, MEM-G1, and MEM-G2. Antibody 4H84, whose recognition site is located in the α1 domain of the extracellular region common to all HLA-G molecules, can detect the seven known HLA-G isoforms containing the α1 domain (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7), but cannot distinguish the expression of specific HLA-G isoforms in immunohistochemistry. Antibodies MEM-G1 and MEM-G2 are obtained by immunizing mice with full-length HLA-G heavy chains, and their specific recognition sites are unpredictable. Theoretically, antibodies MEM-G1 and MEM-G2 are similar to antibody 4H84 and can recognize the aforementioned HLA-G isoforms, but similarly cannot distinguish the expression of specific HLA-G isoforms.

[0007] Currently, there are no specific monoclonal antibodies against HLA-G2 and HLA-G6 isoforms. Therefore, in the context of targeted precision medicine, there is an urgent need to develop more monoclonal antibodies against HLA-G molecules with better specificity and affinity. Summary of the Invention

[0008] Given that there are currently no specific monoclonal antibodies against HLA-G2 and HLA-G6 isoforms, the present invention aims to provide a specific monoclonal antibody (YWHG-26) against HLA-G2 and HLA-G6 isoforms, a nucleotide sequence encoding the antibody (YWHG-26) of the present invention and the amino acid sequence encoded therein; and the use of the YWHG-26 antibody for detection such as immunoblotting and immunohistochemistry.

[0009] The present invention uses the consensus sequence of HLA-G2 and HLA-G6 isoforms, and the antigen peptide (RGYYNQSEAKPPKTHVTHHPV) located in the α1 and α3 connecting regions as the immunogen, and its amino acid sequence is the amino acid sequence shown in SEQ ID No. 19 (RGYYNQSEAKPPKTHVTHHPV) ( Figure 1 Based on this, a specific monoclonal antibody (YWHG-26) against HLA-G2 and HLA-G6 isoforms was prepared.

[0010] Based on the inventors' research results, the present invention provides a monoclonal antibody (YWHG-26) against HLA-G2 and HLA-G6 isoforms, comprising at least one or more of the light chain hypervariable regions CDR1, CDR2, and CDR3, and / or one or more of the heavy chain hypervariable regions CDR1, CDR2, and CDR3.

[0011] The amino acid sequence of the light chain of the monoclonal antibody (YWHG-26) is as shown in SEQ ID No. 1, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 1 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the antibody light chain hypervariable region CDR1 is the sequence QSIVHSNGNTY shown in SEQ ID No. 2, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 2 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the light chain hypervariable region CDR2 is the sequence KVS shown in SEQ ID No. 3, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 3 formed by replacing, deleting or adding one or more amino acids, and the amino acid sequence of the light chain hypervariable region CDR3 is the sequence FQGSHVPLT shown in SEQ ID No. 4, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 4 formed by replacing, deleting or adding one or more amino acids.

[0012] The amino acid sequence of the heavy chain of the monoclonal antibody (YWHG-26) is as shown in SEQ ID No. 5, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 5 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the heavy chain hypervariable region CDR1 of the antibody is the sequence GYAFSTYW shown in SEQ ID No. 6, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 6 formed by replacing, deleting or adding one or more amino acids, the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence IYPGDGDT shown in SEQ ID No. 7, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 7 formed by replacing, deleting or adding one or more amino acids, and the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence ARVYYGNGFAY shown in SEQ ID No. 8, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No. 8 formed by replacing, deleting or adding one or more amino acids.

[0013] Furthermore, the monoclonal antibody (YWHG-26) further includes a light chain framework region (Framework region, FR) and a heavy chain framework region; wherein the light chain framework region includes one or more of light chain FR1, FR2, FR3 and FR4, and the amino acid sequence of the light chain FR1 is the sequence DIMLTQTPLSLPVSLGDQASISCRSS shown in SEQ ID No.9, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No.9 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the light chain FR2 is the sequence LEWYLQKPGQSPKLLIY shown in SEQ ID No.10, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No.10 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the light chain FR3 is the sequence NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC shown in SEQ ID No.11, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No.12 formed by replacing, deleting or adding one or more amino acids. No. 11; the amino acid sequence of the light chain FR4 is the sequence FGAGTKLELK shown in SEQ ID No. 12, or an amino acid sequence having equivalent functions to the sequence shown in SEQ ID No. 12 formed by replacing, deleting or adding one or more amino acids; the heavy chain framework region includes one or more of heavy chain FR1, FR2, FR3 and FR4, wherein: the amino acid sequence of the heavy chain FR1 is the sequence LGQLQESGAELVRPGSSVKISCKAS shown in SEQ ID No. 13, or an amino acid sequence having equivalent functions to the sequence shown in SEQ ID No. 13 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the heavy chain FR2 is the sequence MNWVKQRPGQGLEWIGQ shown in SEQ ID No. 14, or an amino acid sequence having equivalent functions to the sequence shown in SEQ ID No. 14 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the heavy chain FR3 is the sequence SEQ ID The sequence RYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFC shown in No.15, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No.15 formed by replacing, deleting or adding one or more amino acids; the amino acid sequence of the heavy chain FR4 is the sequence WGQGTLLTVSA of SEQ ID No.16, or an amino acid sequence having equivalent function to the sequence shown in SEQ ID No.16 formed by replacing, deleting or adding one or more amino acids.

[0014] Furthermore, the nucleotide sequence encoding the light chain in the monoclonal antibody (YWHG-26) is the sequence shown in SEQ ID No. 17, or a nucleotide sequence having equivalent function to the sequence shown in SEQ ID No. 17 formed by replacing, deleting or adding one or more nucleotides in the sequence, and the nucleotide sequence encoding the heavy chain in the monoclonal antibody (YWHG-26) is the sequence shown in SEQ ID No. 18, or a nucleotide sequence having equivalent function to the sequence shown in SEQ ID No. 18 formed by replacing, deleting or adding one or more nucleotides in the sequence.

[0015] According to one aspect of the present invention, the present invention provides a preferred monoclonal antibody (YWHG-26) against HLA-G2 and HLA-G6 isoforms. The monoclonal antibody (YWHG-26) is produced by a hybridoma with a deposit number of CCTCC NO: C202240. The hybridoma cell line is classified and named YWHG-26. The depository institution is: China Center for Type Culture Collection, and the deposit date is March 8, 2022. The address of China Center for Type Culture Collection is Wuhan University, Wuhan, China, Postal Code 430072.

[0016] The present invention also provides the use of the anti-HLA-G molecule antibody (YWHG-26) for HLA-G molecule immunoblotting and immunohistochemistry detection, which has the characteristics of high specificity and strong affinity.

[0017] To provide a clearer understanding of the inventive concept and technical solutions of this application, the present application will be further explained below through specific examples and accompanying drawings. The technical solutions described in the embodiments are merely preferred embodiments and should not be construed as limiting the scope of this application. Those skilled in the art may make several improvements and adjustments without departing from the technical principles of this application, and such improvements and adjustments should also be considered to fall within the scope of protection of this application.

[0018] Unless otherwise defined, all technical and scientific terms used herein shall be deemed to have the same meaning as understood by one of ordinary skill in the art. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to the 20 common amino acids.

[0019] The light chain hypervariable region or heavy chain hypervariable region mentioned in the present invention, wherein the "hypervariable region" is also called the complementarity determining region (CDR).

[0020] The "sequences" mentioned in the present invention may refer to amino acid sequences or "conservative substitutions" that contain certain biologically equivalent functions, and "other sequences" may contain functionally non-equivalent amino acids or "non-conservative substitutions" that are genetically engineered to improve the properties of CDRs or CDR-containing antibodies. Without substantially affecting the activity of the antibody, those skilled in the art can manipulate the sequences in the present application, i.e., replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to obtain variants of the antibody or its functional fragment sequence. They should be considered to be included within the scope of protection of this application. For example, amino acids with similar properties are substituted in the variable region. The sequences of the variants mentioned in the present invention may have at least 95%, 96%, 97%, 98% or 99% identity with their source sequences. Sequence identity can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, is used with default parameters. The various amino acid sequences described herein are detailed in the sequence listing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a diagram of the molecular structure of 7 different HLA-G isoforms and the location of immune antigen peptide segments.

[0022] Figure 2 The heavy and light chain subclass identifications of the antibody (YWHG-26) are shown.

[0023] Figure 3 The antibody (YWHG-26) was tested by SDS-PAGE for purity.

[0024] Figure 4 The antibody affinity constant of the antibody (YWHG-26) was determined by ELISA.

[0025] Figure 5 The antibody (YWHG-26) is used for immunoblotting detection of HLA-G2 and HLA-G6 molecules.

[0026] Figure 6 The antibody (YWHG-26) is used for immunohistochemical detection of HLA-G2 and HLA-G6 molecule expression in colorectal cancer tissues. DETAILED DESCRIPTION

[0027] 1. Preparation of anti-HLA-G monoclonal antibody (YWHG-26)

[0028] ① Antigen peptide synthesis

[0029] An antigenic peptide (SEQ No. 19 RGYYNQSEAKPPKTHVTHHPV) located in the consensus sequence of HLA-G2 and HLA-G6 isoforms and located in the α1 and α3 connecting regions was synthesized as an immunogen.

[0030] ② Mouse immunization

[0031] Four SPF-grade BALB / c female mice were immunized initially with 60ug / mouse. The first booster immunization was performed with 30ug / mouse. The second booster immunization was performed with 30ug / mouse. The third booster immunization was performed with 30ug / mouse. Blood was collected from the eye sockets and the serum titer was measured. The plates were coated with "SEQ No.19RGYYNQSEAKPPKTHVTHHPV" and the titer of the immunized mice was determined by ELISA. The plates were coated with "SEQ No.19RGYYNQSEAKPPKTHVTHHPV" at 2ug / ml overnight at 4°C; then blocked with 2% milk at 37°C for 2h; the serum was serially diluted 2-fold starting from 200-fold. The blank control was PBS, and the negative control was a 200-fold dilution of negative serum. Before fusion, the mice were shock-immunized with 50ug of the "SEQ No.19RGYYNQSEAKPPKTHVTHHPV" immunogen. In the fusion experiment, mouse spleen cells and SP2 / 0 cells were fused using the PEG method. After the fusion, the cells were screened and cultured using a semi-solid culture medium (containing HAT).

[0032] ③Monoclonal cell screening

[0033] 93 single clones from 10 plates were selected and cultured in 96-well cell culture plates (previously seeded with thymocytes, 100 μl / well). Plates were coated with "SEQ No. 19RGYYNQSEAKPPKTHVTHHPV" and diluted with coating buffer to a final concentration of 2 μg / ml. 100 μl / well was added and incubated at 4°C overnight. The plates were then washed three times with wash buffer. The plates were blocked with 2% milk blocking buffer (200 μl / well) and incubated at 37°C for 2 hours. The plates were then washed three times with wash buffer. Primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000-fold in PBS) were added at 100 μl / well. The plates were incubated at 37°C for 1 hour. The plates were then washed three times with wash buffer. Add secondary antibody diluted 20,000-fold with PBS, 100 μl / well, incubate at 37°C for 1 hour; remove and wash three times with washing solution. Develop color with 100 μl / well of color development solution for approximately 5 minutes. Add 50 μl of stop solution to each well to terminate the reaction. Measure absorbance at dual wavelengths (450, 630). Perform the first screening of the selected clones using ELISA to obtain positive hybridoma cell lines. Re-coat the positive cell lines with "SEQ No. 19RGYYNQSEAKPPKTHVTHHPV" and perform the second screening using ELISA to obtain positive hybridoma cell lines. After multiple screenings, a monoclonal antibody against the SEQ No. 19RGYYNQSEAKPPKTHVTHHPV fragment was obtained and named YWHG-26 (deposit number: CCTCCNO: C202240).

[0034] ④ Identification of subclasses of anti-HLA-G monoclonal antibody (YWHG-26)

[0035] Subtype identification was performed on the 10 positive cell lines screened. The coated antibody was diluted to 0.5 μg / ml in 100 mM PBS (pH 7.4), and 0.1 ml was added to each well incubated at 4°C overnight. The cells were washed twice with PBS-T, and 200 μl of blocking solution was added to each well, incubated at 37°C for 2 hours. The cells were washed three times with PBS-T, and 100 μl of hybridoma supernatant was added to each well, incubated at 37°C for 1 hour. The cells were washed three times with PBS-T, and 0.1 ml of HRP-conjugated antibody diluted 1:10,000 (κ, λ) or 1:20,000 (other) in blocking solution was added to the appropriate wells. The cells were incubated at 37°C for 1 hour. The cells were washed three times with PBS-T, and 50 μl of substrate solution was added to each well. The absorbance was measured at dual wavelengths (450 and 630 nm) within 10-20 minutes. The antibody subtype was again determined using Thermo Fisher Scientific's rapid mouse antibody subtype identification method (Pierce Rapid Isotyping Kits-Mouse, Catalog" #26178) to confirm that the monoclonal antibody subtype produced by the cell line was IgG2a heavy chain and kappa (κ) light chain ( Figure 2 ).

[0036] ⑤Antibody purification

[0037] Sample Pretreatment: Dilute the sample 1:3 with the corresponding coupling buffer, centrifuge at 12,000 rpm at 4°C for 10 minutes, and filter through a 0.22 μm filter to remove fat, cellular debris, and small particulate matter. Equilibration: Equilibrate the column with 10 column volumes of the corresponding coupling buffer, maintaining a flow rate of 1 ml / min. Sample Loading: Inject the sample into the upper port of the column and collect the flow-through, maintaining a flow rate of 1 ml / min. Wash: Pass the column with 5 column volumes of coupling buffer, maintaining a flow rate of 1 ml / min. Elution: Elute the antibody with 5 column volumes of elution buffer, collect the solution in the aforementioned EP tube, maintaining a flow rate of 1 ml / min. Immediately adjust the pH to 7.0 with 1 M Tris-HCl buffer, pH 9.0. Equilibrate: Equilibrate the column back to pH 7.0 with 10 column volumes of coupling buffer, maintaining a flow rate of 1 ml / min. Dialysis: Dialyze the antibody overnight against 0.01 M PBS, changing the buffer three times.

[0038] ⑥SDS-PAGE to detect antibody purity

[0039] Prepare SDS-PAGE gel, the concentration of separation gel is 12%. Sample preparation: add sample buffer to the sample and boil it in boiling water for 10 minutes. Sample loading: 10ul per well. Gel running: 80V for 30 minutes for concentrated gel and 120V for 60 minutes for separation gel. Stop electrophoresis when the front of bromophenol blue reaches the bottom of the glass plate and take out the gel. Staining and destaining: immerse the gel in Coomassie brilliant blue staining solution and shake it slowly on a shaker for more than 30 minutes (the staining time needs to be adjusted appropriately according to the thickness of the gel). Take out the gel and rinse it in water several times, then add Coomassie brilliant blue destaining solution and shake it. Destain the gel until the bands are roughly visible for 1 hour. For complete destaining, the destaining solution needs to be replaced 2 to 3 times and shaken for more than 24 hours. After destaining, the gel can be scanned and recorded by the ECL gel imaging system. The purity of the purified antibody YWHG-26 is >90% ( Figure 3 ).

[0040] ⑦ Determination of affinity constant of antibody by ELISA

[0041] Antigen (SEQ No. 19RGYYNQSEAKPPKTHVTHHPV) was diluted with coating buffer to a final concentration of 2 μg / ml, at 100 μl / well, at 4°C overnight. The cells were then washed twice with wash buffer. Blocking buffer was applied, at 200 μl / well, at 37°C for 2 hours, followed by a single wash with wash buffer. Purified antibody was serially diluted two-fold starting at 200-fold in PBS, with a blank control containing PBS at 100 μl / well. The cells were incubated at 37°C for 1 hour, followed by three washes with wash buffer. Secondary antibody diluted 20,000-fold in PBS was added, at 100 μl / well, at 37°C for 1 hour, followed by three washes with wash buffer. Color development was performed with 100 μl / well of colorimetric solution for 5-15 minutes. Stopping was performed by adding 50 μl of stop buffer to each well. Absorbance was measured at dual wavelengths (450 and 630 nm) and analyzed graphically. According to the logistic fitting equation, the affinity constant is ≈150,000×A / antibody concentration (A is the antibody dilution factor corresponding to the 1 / 2 OD value; dilution factor = 25,600; YWHG-26 antibody concentration = 2.7 mg / mL). The affinity constant of antibody YWHG-26 is 1.42×10 9 L / mol( Figure 4 ).

[0042] 2. Application Examples

[0043] Example 2.1. Immunoblotting to detect HLA-G isoform recognition specificity of antibodies.

[0044] After electroporation of HLA-G isoform standard proteins HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7, the membrane was blocked with 5% skim milk powder at room temperature for 4 hours and washed with 0.2% TBS (Teween-20 PBS). The antibody (YWHG-26, 1.0 μg / ml) was added to detect its recognition specificity, incubated at 4°C overnight, and washed. HRP-labeled rabbit anti-mouse IgG antibody was added and incubated at room temperature for 30 minutes. After washing, the membrane was incubated with Dako REAL TM EnVision TM The results showed that the antibody (YWHG-26) can specifically recognize HLA-G2 and HLA-G6 standard proteins, and has no cross-reaction with other HLA-G isoform molecules, and has good specificity for distinguishing HLA-G2 and HLA-G6 isoform molecules ( Figure 5 ).

[0045] Example 2.2. The antibody (YWHG-26) is used for immunohistochemical detection of HLA-G2 and HLA-G6 molecule expression in colorectal cancer tissues.

[0046] The colorectal cancer tissues were fixed in 10%-12% neutral formalin and embedded in paraffin. The tissue sections were subjected to conventional preparation procedures such as baking, dewaxing, hydration and antigen retrieval. An appropriate amount of 1% BSA was added to the tissue, covering the tissue and 2mm of the tissue edge, and incubated at room temperature for 10 minutes for blocking. The antibody (YWHG-26) (1mg / mL, 1:500 dilution) was added and kept in a 4°C refrigerator humid chamber overnight (16-20h). Rinse with TBS buffer, add secondary antibody (TBS diluted antibody goat anti-mouse ratio 1:300), and incubate in a 37°C constant temperature box for 30 minutes. Rinse with TBS buffer, add color developer DAB working solution, and after the tissue is completely colored, place the slide in running water for 5 minutes and soak in distilled water for 5 minutes. After HE counterstaining, dehydration, transparency, and sealing, observe the situation of each field of view of the tissue section under an optical microscope. The brown-colored cells are HLA-G positive cells in gastric cancer tissues. The intensity of HLA-G expression can be determined based on the depth of brown-colored cells. Figure 6 (A) and (B) are HLA-G2 / 6 negative samples of colorectal cancer, (C) and (D) are HLA-G2 / 6 positive samples of colorectal cancer. Figure 6 ).

Claims

1. A monoclonal antibody against HLA-G2 and HLA-G6 molecules, wherein the monoclonal antibody YWHG-26 is produced by a hybridoma with a deposit number of CCTCC NO: C2022240.

2. A monoclonal antibody against HLA-G2 and HLA-G6 molecules, characterized in that: The monoclonal antibody YWHG-26 comprises at least light chain hypervariable regions CDR1, CDR2 and CDR3, and heavy chain hypervariable regions CDR1, CDR2 and CDR3; The amino acid sequence of the light chain variable region of the monoclonal antibody YWHG-26 is shown in SEQ ID No. 1; the amino acid sequence of the light chain hypervariable region CDR1 of the antibody is shown in SEQ ID No. 2; the amino acid sequence of the light chain hypervariable region CDR2 is shown in SEQ ID No. 3, and the amino acid sequence of the light chain hypervariable region CDR3 is shown in SEQ ID No. 4; The amino acid sequence of the heavy chain variable region of the monoclonal antibody YWHG-26 is shown in SEQ ID No. 5; the amino acid sequence of the heavy chain hypervariable region CDR1 of the antibody is the sequence shown in SEQ ID No. 6, the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence shown in SEQ ID No. 8.

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