WO2023109061A1 - Monoclonal antibody for resisting hla-g isomer molecules hla-g5 and hla-g6, and use thereof - Google Patents

Abstract

Disclosed in the present invention are a monoclonal antibody for resisting HLA-G isomer molecules HLA-G5 and HLA-G6, and a use thereof. The monoclonal antibody (YWHG-5) is generated by hybridoma with an accession number of CCTCC NO: 202122, and an antigen peptide (EGLPEPLMLRWSKEGDGGIMS) having an HLA-G molecular heavy chain located in an α3 structural domain and an HLA-G5/6 molecule-specific transmembrane region amino acid sequence is used as an immunogen. The present invention provides a nucleotide for encoding a YWHG-5 antibody of the present invention, and an amino acid sequence encoding the YWHG-5 antibody. The present invention further provides a use of a YWHG-5 antibody for detection of immunohistochemistry, immunoblotting, flow cytometry and the like of HLA-G isomer molecules HLA-G5 and HLA-G6.

Description

Monoclonal antibody against HLA-G isoform molecules HLA-G5 and HLA-G6 and application thereof technical field

The invention belongs to the field of biomedicine, and relates to the following aspects of an anti-HLA-G isoform antibody (YWHG-5): the amino acid sequence of the heavy chain α3 domain of the HLA-G molecule and the unique transmembrane region of the HLA-G5/6 molecule The antigenic peptide (EGLPEPLMLRWSKEGDGGIMS) is an immunogen to prepare antibodies (YWHG-5) against HLA-G isomers HLA-G5 and HLA-G6; the nucleic acid molecule and amino acid sequence encoding the YWHG-5 antibody of the present invention, and the The above antibody (YWHG-5) is used for HLA-G5 and HLA-G6 immunohistochemistry, western blot and flow cytometry detection and other purposes.

Background technique

Human leukocyte antigen-G (human leukocyte antigen-G, HLA-G) gene, with a total length of 6.0kb, is located at 6p21.3 on the distal short arm of human chromosome 6. During protein translation, the first exon of HLA-G mRNA encodes the signal peptide, the second, third, and fourth exons encode the α1, α2, and α3 domains of the extracellular region, respectively, and the fifth exon encodes Transmembrane region; exon 6 encodes the intracellular segment of HLA-G molecule containing only 6 amino acid residues; exon 7 is not transcribed because exon 6 contains a stop codon; exon 8 corresponds to the 3'UTR of the HLA-G gene. Alternative splicing of HLA-G initial transcription products can produce 7 mature mRNAs, which encode 7 isoform molecules with different molecular weights (HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA -G7). Among them, HLA-G1, HLA-G2, HLA-G3 and HLA-G4 contain transmembrane regions and are membrane-bound isomers; HLA-G5, HLA-G6 and HLA-G7 lack transmembrane structures and are soluble isomers . The molecular weights of HLA-G1～-G7 isomers are 39kD, 31kD, 22kD, 30kD, 34kD, 23kD and 16kD, respectively.

HLA-G1 is encoded by full-length HLA-G mRNA and consists of the extracellular domain α1, α2 and α3 domains, the transmembrane domain and the intracellular domain. HLA-G2 lacks the α2 domain, which consists of the extracellular region α1 and α3 domains, the transmembrane region and the intracellular domain; HLA-G3 lacks the α2 and α3 domains, which consists of the extracellular region α1 domain, the transmembrane region and the intracellular domain Intracellular domain composition; HLA-G4 lacks the α3 domain, which is composed of the extracellular region α1 and α2 domains, the transmembrane region and the intracellular domain; G1 and HLA-G2 are the same, but they are encoded by HLA-G mRNA containing intron 4. Because intron 4 contains a stop codon, the encoded protein molecule lacks the transmembrane region encoded by exon 5 , forming soluble HLA-G molecules. 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 are connected (Figure 1).

Under normal physiological conditions, HLA-G molecules are only expressed in the extravillous trophoblast cells at the maternal-fetal interface, maintaining maternal-fetal immune tolerance during pregnancy. Under pathological conditions, HLA-G molecules can be induced to express in pathological tissue cells such as tumor cells and virus infection, and are closely related to the occurrence and progression of diseases. HLA-G molecule is an important immune tolerance molecule in the body, and it is also an important immune checkpoint molecule. Its immunosuppressive function is mainly through binding to the immunosuppressive receptor immunoglobulin-like transcript-2 2, ILT2/LILRB1/CD85j), immunoglobulin-like transcript-4 (ILT4/LILRB2/CD85d), transmit inhibitory signals and induce immune tolerance. The specific mechanism of action is as follows: ① It binds to ILT2 expressed on T cells, NK cells and B cells, inhibits the immune killing activity of T cells and NK cells, and B cell proliferation and antibody secretion. ② Combine with ILT2 and ILT4 on dendritic cells (DC), inhibit the maturation and differentiation of DC cells, and induce the generation of tolerant DC cells. ③Combine with ILT2 and ILT4 on myeloid-derived suppressor cells (MDSC) and macrophages (Mф), and induce the differentiation of pro-inflammatory and anti-tumor M1 cells into immune-tolerant M2 cells, etc. Therefore, HLA-G plays an important role in the occurrence and development of diseases such as tumors. Tumor immunotherapy based on HLA-G targeting has entered phase I clinical trials in the United States.

HLA-G binds to receptors ILT2 and ILT4, and has molecular structure specificity. The receptor ILT2, ILT4 binds to the HLA-G α3 domain of the extracellular region of HLA-G. ILT2 only binds to the HLA-G/β2m complex, while ILT4 not only binds to HLA-G/ _β2m , but also binds to free HLA-G molecules without _β2m . Due to the differences in expression mechanism and molecular structure of HLA-G1, -G2, -G3, -G4, -G5, -G6 and HLA-G7, such as HLA-G3, -G4, and HLA-G7 isoforms extracellular The region does not contain the α3 domain and cannot bind to ILT2 and ILT4. Different HLA-G isoform molecules can exert specific immunological effects in pathophysiological processes. In different pathological conditions, especially in tumor tissue cells, there is extensive heterogeneity in the expression of HLA-G isoforms, and the expression of different HLA-G molecular isoforms has specific clinical significance. Therefore, analyzing the expression of specific HLA-G isoform molecules and the expression profiles of different HLA-G isoform molecules is of great significance for elucidating the biological functions and clinical significance of specific HLA-G isoform molecules.

The antibodies currently used for HLA-G immunohistochemistry and western blotting mainly include 4H84, MEM-G1, MEM-G2, 2A12 and 5A6G7. Among them, the antibody 4H84, whose recognition site is located in the α1 domain of the extracellular region that all 7 kinds of HLA-G isoform molecules have, can specifically detect the 7 kinds of HLA-G isoforms that contain the α1 structure domain. Molecules (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7), but the expression of specific HLA-G isoform molecules cannot be distinguished in immunohistochemistry . Antibodies MEM-G1 and MEM-G2 are obtained from mice immunized with full-length HLA-G heavy chains, and the specific recognition sites cannot be predicted. These two antibodies are theoretically similar to antibody 4H84 and can recognize 7 HLA-G isoforms Molecules (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7), but also cannot distinguish the expression of specific HLA-G isoform molecules. Antibodies 2A12 and 5A6G7 are obtained by immunizing mice with 22 amino acid residues located at the carboxy-terminal of HLA-G5 and HLA-G6 molecules, and can recognize HLA-G5 and HLA-G6 molecules. However, antibodies 2A12 and 5A6G7 can also recognize other unknown non-HLA-G5 and HLA-G6 molecules with a molecular weight in the range of 36-175kDa. There may be cross-reactions in the application of immunohistochemistry, false positives, and incorrect HLA - G5 and HLA-G6 molecule expression. The monoclonal antibodies used to detect HLA-G molecules by flow cytometry include 87G and MEM-G/9, but only HLA-G1 and HLA-G5 molecules can be detected.

Therefore, in the context of targeted precision medicine, the existing antibodies for detecting HLA-G molecules are not all satisfactory, and there is an urgent need to develop more monoclonal antibodies targeting different HLA-G isoform molecules.

Contents of the invention

In view of the above-mentioned deficiencies in the existing HLA-G antibodies, the purpose of the present invention is to provide a specific monoclonal antibody (YWHG-5) against HLA-G5 and HLA-G6 isoform molecules, encoding the antibody of the present invention ( The nucleic acid molecule and amino acid sequence of YWHG-5); and the YWHG-5 antibody is used for HLA-G5 and HLA-G6 immunohistochemistry, immunoblotting and flow detection.

The present invention adopts the antigenic peptide of the heavy chain α3 domain of HLA-G molecules and the amino acid sequence of the transmembrane region unique to HLA-G5/6 molecules as an immunogen, and its amino acid sequence is the amino acid sequence shown in SEQ ID No.17 (EGLPEPLMLRWSKEGDGGIMS) . The EGLPEPMLLRWSKEGDGGIMS peptide is located in the heavy chain α3 domain of HLA-G molecule and the unique transmembrane region of HLA-G5/6 molecule (the region shown in the virtual box in Figure 1), and based on this, the anti-HLA-G5 and HLA-G6 isomerization Monoclonal antibody specific to the body molecule (YWHG-5).

According to the research results of the inventors, the present invention provides a monoclonal antibody (YWHG-5) against HLA-G isoform molecules HLA-G5 and HLA-G6, which at least includes heavy chain hypervariable regions CDR1, CDR2 and CDR3 One or more of, or/and one or more of light chain hypervariable region CDR1, CDR2 and CDR3;

The amino acid sequence of the antibody light chain of the monoclonal antibody (YWHG-5) is as shown in SEQ ID No.1 or has equivalent functions to the sequence shown in SEQ ID No.1 formed by replacing, deleting or adding one or more amino acids Amino acid sequence; the amino acid sequence of the antibody light chain hypervariable region CDR1 is the sequence QSLLHSNGDTY shown in SEQ ID No.2 or the sequence shown in SEQ ID No.2 formed by replacing, deleting or adding one or more amino acids Amino acid sequence with equivalent functions; the amino acid sequence of the light chain hypervariable region CDR2 is the sequence NIS shown in SEQ ID No.3 or the sequence NIS shown in SEQ ID No.3 by replacing, deleting or adding one or more amino acids The sequence shown has the amino acid sequence of the same function, and the amino acid sequence of the hypervariable region CDR3 of the light chain is the sequence FQGSYVPYT shown in SEQ ID No.4 or the sequence FQGSYVPYT formed by replacing, deleting or adding one or more amino acids and SEQ ID The sequence shown in No.4 has an amino acid sequence with equivalent functions;

The nucleotide sequence of the antibody heavy chain of the monoclonal antibody (YWHG-5) is as shown in SEQ ID No.5 or is formed by replacing, deleting or adding one or more amino acids with the sequence shown in SEQ ID No.5 The amino acid sequence of the same function; the amino acid sequence of the antibody heavy chain hypervariable region CDR1 is the sequence GFSLTSYG shown in SEQ ID No.6 or the sequence GFSLTSYG shown in SEQ ID No.6 by replacing, deleting or adding one or more amino acids The sequence shown has an amino acid sequence with equivalent functions, and the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence IWAGGNT shown in SEQ ID No. 7 or the sequence IWAGGNT shown in SEQ ID No. 7 or replaced, deleted or added with one or more amino acids. .7 The sequence shown in 7 has an amino acid sequence with the same function, and the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence ARDRAGTARFYYYALDN shown in SEQ ID No.8 or the combination formed by replacing, deleting or adding one or more amino acids The sequence shown in SEQ ID No.8 has an amino acid sequence with equivalent functions.

Further, the monoclonal antibody (YWHG-5) also includes a light chain framework region (Framework region, FR) and a heavy chain framework region; wherein, the light chain framework region includes one of light chain FR1, FR2 and FR3 one or more, the amino acid sequence of the light chain FR1 is the sequence DIVMTQDELSLPVSLGDQASIPCGSS shown in SEQ ID No.9 or the sequence is replaced, deleted or added with one or more amino acids and has the same sequence as the sequence shown in SEQ ID No.9 Amino acid sequence with equivalent function; the amino acid sequence of the light chain FR2 is the sequence LHWFLQTPGQSPKLLRY shown in SEQ ID No.10 or the sequence shown in SEQ ID No.10 is equivalent to that formed by substitution, deletion or addition of one or more amino acids Functional amino acid sequence; the amino acid sequence of the light chain FR3 is the sequence NRFSGVPDRFSGSGSGSGTDFTLKISRVEGEDLGVYYC shown in SEQ ID No.11 or the sequence shown in SEQ ID No.11 has the same function as that formed by replacing, deleting or adding one or more amino acids The amino acid sequence of the heavy chain framework region includes one or more of heavy chain FR1, FR2 and FR3, wherein: the amino acid sequence of the heavy chain FR1 is the sequence EVKLQESGPSLVAPSQSLSIACTVS shown in SEQ ID No.12 or replaced An amino acid sequence having equivalent functions to the sequence shown in SEQ ID No.12 formed by deletion or addition of one or more amino acids; the amino acid sequence of the heavy chain FR2 is the sequence VHRIRQPPGKGLEWLGI shown in SEQ ID No.13 or replaced, An amino acid sequence having the same function as the sequence shown in SEQ ID No.13 formed by deletion or addition of one or more amino acids; the amino acid sequence of the heavy chain FR3 is the sequence KYNSALMSRLCISKDNSKSQAFLKMNSLQTDDTAMYYC shown in SEQ ID No.14 or replaced or deleted Or add one or more amino acids to form an amino acid sequence with the same function as the sequence shown in SEQ ID No.14.

Further, the nucleotide sequence encoding the light chain variable region in the monoclonal antibody (YWHG-5) is the sequence shown in SEQ ID No.15 or the sequence is replaced, deleted or added with one or more nucleosides The nucleotide sequence having the same function as the sequence shown in SEQ ID No.15 formed by acid, the nucleotide sequence encoding the heavy chain variable region in the monoclonal antibody (YWHG-5) is shown in SEQ ID No.16 The sequence shown or the sequence is substituted, deleted or added one or more nucleotides to form a nucleotide sequence with the same function as the sequence shown in SEQ ID No.16.

According to one aspect of the present invention, the present invention provides a preferred monoclonal antibody (YWHG-5) against HLA-G isomer HLA-G5 and HLA-G6 molecules, said monoclonal antibody (YWHG-5) Produced by the hybridoma with the deposit number CCTCC NO:202122, the depository institution is: China Center for Type Culture Collection, and the deposit time is August 11, 2021; the address of China Center for Type Culture Collection is Wuhan University, Wuhan City, Hubei Province, China , zip code 430072.

The present invention also provides the anti-HLA-G5 and HLA-G6 isoform molecule antibody (YWHG-5) used for HLA-G isoform HLA-G5 and HLA-G6 molecule immunohistochemistry, immunoblotting and flow cytometry The use of cytometry and other detections has the characteristics of high specificity and strong affinity.

For a clearer understanding of the inventive concepts and technical solutions of the application, the application will be further explained below through specific examples and accompanying drawings, wherein the technical solutions in the implementation modes are only preferred implementation modes and should not be construed as limiting the application range. Those skilled in the art can make some improvements and adjustments without departing from the technical principles of the present application, and these improvements and adjustments should also be deemed to fall within the protection scope of the present application.

Unless otherwise defined, all technical and scientific terms used herein should be deemed to have the same meaning as understood by one of ordinary skill in the art. Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to the 20 commonly used amino acids.

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

The "sequence" mentioned in the present invention may refer to amino acid sequences or "conservative substitutions" that contain certain biological functions equivalent, and "other sequences" may contain functionally non-equivalent amino acids or "non-conservative substitutions", which are genetically Engineering to improve the properties of CDRs or CDR-containing antibodies. Under the premise of not substantially affecting the activity of the antibody, those skilled in the art can operate on the sequences in the application, that is, replace, add and/or delete one or more (such as 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequence thereof. They should be considered included in the protection scope of this application. For example, amino acids with similar properties are substituted in variable regions. The sequence of the variant mentioned in the present invention may have at least 95%, 96%, 97%, 98% or 99% identity with its source sequence. Sequence identity can be measured using sequence analysis software. For example the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. The various amino acid sequences described herein are detailed in the Sequence Listing.

Description of drawings

Figure 1 is a schematic diagram of the molecular structure of seven different HLA-G isoforms and the position of the immune peptide.

Figure 2 is the identification of heavy chain and light chain subclasses of the antibody (YWHG-5).

Fig. 3 is the detection of antibody purity by SDS-PAGE of the antibody (YWHG-5).

Fig. 4 is the determination of the antibody affinity constant of the antibody (YWHG-5) by ELISA method.

Fig. 5 is the immunoblotting detection of HLA-G5 and HLA-G6 molecules by the antibody (YWHG-5).

Fig. 6 is the detection of HLA-G5 molecular concentration by the ELISA method of the antibody (YWHG-5).

Fig. 7 is the flow cytometry HLA-G5 and HLA-G6 molecular detection of the antibody (YWHG-5).

Fig. 8 shows the application of the antibody (YWHG-5) in immunohistochemical detection of HLA-G5 and HLA-G6 molecular expression in tissues.

Detailed ways

1. Preparation of anti-HLA-G monoclonal antibody (YWHG-5)

① Antigenic peptide synthesis

Synthesize the antigenic peptide of the heavy chain α3 domain of HLA-G molecule and the amino acid sequence of the transmembrane region unique to HLA-G5/6 molecule, SEQ No.17 EGLPEPLMLRWSKEGDGGIMS.

② Mouse immunization

Initially immunize 4 SPF grade BALB/c female mice, 60ug/mouse. For the first booster immunization mice, 30ug/only. Mice were boosted for the second time, 30ug/only. The third booster immunization mice, 30ug/only. Orbital blood was taken to measure the serum titer. Use "SEQ No.17 EGLPEPLMLRWSKEGDGGIMS" to coat the plate, and ELISA to measure the titer of immunized mice. Coat with "SEQ No.17 EGLPEPLMLRWSKEGDGGIMS", 2ug/ml, overnight at 4°C; 2% milk, block at 37°C for 2h; serum was serially diluted 2 times from 200 times, blank control (blank) was PBS, negative control (negative control) ) is a 200-fold dilution of negative serum. Before fusion, the mice were immunized with 50ug of "SEQ No.17 EGLPEPLMLRWSKEGDGGIMS" immunogen. For the fusion experiment, mouse splenocytes and SP2/0 cells were taken and fused by the PEG method, and the fused cells were screened and cultured in a semi-solid medium (containing HAT).

③ Monoclonal cell screening

Pick 10 plates × 93 cell monoclonals and culture them in 96-well cell culture plates (thymocytes have been plated in advance, 100ul/well). Cover the plate with "SEQ No.17 EGLPEPLMLRWSKEGDGGIMS", dilute "SEQ No.17 EGLPEPLMLRWSKEGDGGIMS" with the coating solution, the final concentration is 2ug/ml, 100ul/well, 4°C, overnight; then wash 3 times with washing solution. Block with 2% milk blocking solution, 200ul/well, incubator at 37°C for 2 hours; then wash with washing solution for 3 times. Add primary antibody (cell culture supernatant), negative control (SP2/0 culture supernatant), blank control (PBS), positive control (1000-fold dilution of positive serum in PBS), all 100ul/well, incubator at 37°C for 1h ;Finally wash 3 times with lotion. Add secondary antibody diluted 20,000 times in PBS, 100ul/well, incubator at 37°C for 1 hour; take it out and wash it 3 times with washing solution. Color development, color development solution 100ul/well, color development time is about 5min. Add 50ul of stop solution to each well to stop. Dual wavelength (450, 630) to measure the absorbance value. The selected clones were screened for the first time by ELISA method to obtain positive hybridoma cell lines. The positive cell lines were re-coated with "SEQ No. 17 EGLPEPLMLRWSKEGDGGIMS", and the ELISA method was used for the second screening to obtain positive hybridoma cell lines. After multiple screenings, a monoclonal antibody against the fragment of SEQ No.17 EGLPEPLMLRWSKEGDGGIMS was obtained, named YWHG-5 (preservation number CCTCC NO: 202122).

④ Anti-HLA-G monoclonal antibody (YWHG-5) subclass identification

Subtype identification was carried out on the 10 screened positive cell lines, and the coating antibody was diluted to 0.5ug/ml with 100mM PBS (pH7.4), and 0.1ml was added to each well, and left overnight at 4°C. Wash twice with PBS-T, add 200ul blocking solution to each well, and incubate at 37°C for 2h. Wash 3 times with PBS-T; add 100ul hybridoma supernatant to each well, and incubate at 37°C for 1h. Wash 3 times with PBS-T; add 0.1ml of HRP-labeled antibody diluted with blocking solution 1:10000 (κ, λ) or 1:20000 (others) to each well, add to appropriate wells, and incubate at 37°C for 1h. Wash 3 times with PBS-T; add 50ul of substrate solution to each well, and measure absorbance at dual wavelengths (450, 630) within 10-20min. The antibody subtype was determined again by using the mouse antibody subtype rapid identification method (Pierce Rapid Isotyping Kits–Mouse, Catalog”#26178) of Thermo Company to determine that the subtype of the monoclonal antibody produced by the cell line was IgG1 for the heavy chain and kappa for the light chain (κ) type (Figure 2).

⑤ Antibody purification

Sample pretreatment: dilute 1:3 with the corresponding coupling buffer, centrifuge at 12000rpm at 4°C for 10min, and filter with a 0.22μm filter membrane to remove fat, cell residues and small particles. Equilibration: equilibrate the column with 10 times the column volume of the corresponding coupling buffer, and keep the flow rate at 1ml/min. Sample loading: inject the sample into the upper port of the column, collect the effluent, and keep the flow rate at 1ml/min. Washing: Use 5 times the column volume of coupling buffer to pass through the column, and keep the flow rate at 1ml/min. Elution: Elute the antibody with 5 times the column volume of elution buffer, collect it in the above EP tube, and keep the flow rate at 1ml/min. Immediately adjust the pH to 7.0 with 1M Tris-HCl buffer, pH 9.0. Equilibration: equilibrate the column back to pH 7.0 with 10 column volumes of coupling buffer, and keep the flow rate at 1ml/min. Dialysis: Dialyze the antibody against 0.01M PBS buffer overnight and change the medium 3 times.

⑥ SDS-PAGE detection of antibody purity

SDS-PAGE glue was configured, and the concentration of the separating gel was 12%. Sample preparation: After adding the sample buffer to the sample, boil it in boiling water for 10 minutes. Sample loading: 10ul per well. Gel running: stacking gel 80V, 30min; separating gel 120V, 60min. Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and remove the gel. Staining and decolorization: Immerse the gel in Coomassie Brilliant Blue staining solution and shake slowly on a shaker for more than 30 minutes (staining time should be adjusted according to the thickness of the gel). Take out the gel and rinse it in water for several times, then add Coomassie Brilliant Blue destaining solution and shake. Decolorize the gel until the bands can be seen clearly for 1 hour. For complete decolorization, the decolorization solution needs to be replaced 2 to 3 times, and the shaking lasts for more than 24 hours. After gel decolorization, it can be scanned and recorded by ECL gel imaging system. The purified antibody YWHG-5 had a purity >90% ( FIG. 3 ).

⑦ Determination of the affinity constant of the ELISA detection antibody

Dilute the antigen with coating solution, SEQ No.17 EGLPEPLMLRWSKEGDGGIMS, the final concentration is 2ug/ml, 100ul/well, 4°C, overnight; then wash twice with washing solution. Block with blocking solution, 200ul/well, incubator at 37°C, 2h; then wash once with washing solution. The purified antibody was serially diluted 2 times (PBS) starting from 200 times, and the blank control (blank) was PBS, both at 100ul/well, in a 37°C incubator for 1 hour; and then washed 3 times with washing solution. Add secondary antibody diluted 20,000 times in PBS, 100ul/well, incubator at 37°C for 1 hour; take it out and wash it 3 times with washing solution. For color development, the color development solution is 100ul/well, and the color development time is 5-15min. Add 50ul of stop solution to each well to stop. Double wavelength (450, 630) to measure the absorbance value, graph analysis. According to the Logistic fitting equation, the affinity constant ≈150000×A/antibody concentration (A is the antibody dilution factor corresponding to the 1/2OD value; dilution factor=12800; YWHG-5 antibody concentration=1.9mg/mL), antibody YWHG- The affinity constant for 5 is 1.01×10 ⁹ L/mol ( FIG. 4 ).

⑧The HLA-G isoform recognition specificity of the antibody was detected by immunoblotting.

HLA-G isoform standard proteins HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, HLA-G7 and HLA-G molecular standard proteins lacking in the α1 domain were electrotransferred to the membrane After that, it was blocked with 5% non-fat milk powder at room temperature for 4 hours, and washed with 0.2% TBS (Teween-20PBS). Add the antibody (YWHG-5, 1.0ug/ml) to detect its recognition specificity, incubate overnight at 4°C, and wash; add HRP-labeled rabbit anti-mouse IgG antibody, incubate at room temperature for 30min, after washing, use Dako REAL ^TM EnVision ^TM The detection system (DAKO) was incubated for 1-3min. The results show that: the results show that the antibody (YWHG-5) can specifically recognize HLA-G5 and HLA-G6 standard proteins, and there is no cross-reaction with other HLA-G isoform molecules ( FIG. 5 ).

2. Application examples

Example 2.1. The antibody (YWHG-5) immunoblotting detection of HLA-G5 and HLA-G6 molecular detection

HLA-G isoform standard protein HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6 and HLA-G7 molecular standard protein, denatured PAGE electrophoresis. After semi-dry electrotransfer, the membrane was blocked with 5% non-fat milk powder at room temperature for 4 hours, and washed with 0.2% TBS (Teween-20PBS). Add the antibody (YWHG-5) to detect its recognition specificity, incubate overnight at 4°C, wash; add HRP-labeled rabbit anti-mouse IgG antibody, incubate at room temperature for 30min, after washing, use Dako REAL ^TM EnVision ^TM detection system (DAKO) Incubate for 1-3min. The results show that: the results show that the antibody (YWHG-5) can specifically recognize HLA-G5 and HLA-G6 standard proteins, and there is no cross-reaction with other HLA-G isoform molecules ( FIG. 5 ).

Example 2.2. The antibody (YWHG-5) ELISA method for HLA-G5 molecular concentration detection

Dilute the HLA-G5 protein in a 2-fold gradient with the coating solution (0.1M, pH=9.6NaHCO3), and prepare the final concentrations as 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml and 0.0625ug/ml, with the coating solution as the blank control. 100ul/well packaged plate, 4°C, overnight; then washed twice with washing solution. 200ul/well 1% BSA blocked, 37 ℃ incubator, 2h; then washed 3 times with washing solution.

After washing, add biotin-labeled antibody (YWHG-5) at a final concentration of 1.0ug/ml, 100ul/well, in a 37°C incubator, for 1 hour; then wash 3 times with washing solution. Add PBS to dilute 1.0ug/ml peroxidase-conjugated streptavidin (Peroxidase-conjugated Streptavidin), 100ul/well, incubator at 37°C for 1h; remove and wash 3 times with washing solution. Color development, TMB color development solution 100ul/well, color development time is 5-15min. Add 50ul of stop solution to each well to stop. The wavelength (450) was used to measure the absorbance value, and graphed and analyzed. The OD ₄₅₀ of 4ug/ml, 2ug/ml, 1ug/ml, 0.5ug/ml, 0.25ug/ml, 0.125ug/ml, 0.0625ug/ml and the coating solution blank control were 0.449, 0.396, 0.247, 0.165, 0.11, 0.076, 0.069 and 0.055. The results showed that the antibody (YWHG-5) specifically recognized HLA-G5 and had a significant correlation with the concentration of HLA-G5 [Y (concentration) = 0.034 x (OD) ² + 0.1747x (OD); R ² = 0.9988] (Figure 6).

Example 2.3. The antibody (YWHG-5) flow cytometry HLA-G5 and HLA-G6 molecular detection

Cell cultures expressing HLA-G5 and HLA-G6 molecular standard protein K562-HLA-G5 and K562-HLA-G6 in the logarithmic growth phase were taken respectively. HLA-G5/6 is a soluble HLA-G molecule, detected by intracellular flow cytometry.

Flow cytometric detection of intracellular HLA-G5 and HLA-G6 molecular expression: collect K562-HLA-G5 and K562-HLA-G6 cell cultures by flow cytometry tube, centrifuge and wash twice with 2% BSA/PBS (250g), add 250ul cells Membrane breaking agent (BD Cytofix/Cytoperm ^TM ), mixed thoroughly, and placed in a refrigerator at 4°C for 20 minutes. Add 1ml BD Perm/Wash ^TM and centrifuge twice (250g). After washing, resuspend K562-HLA-G5 and K562-HLA-G6 cells in 100ul BD Perm/Wash ^TM , add FITC-labeled purified antibody (YWHG-5) containing 1ul (1.0mg/ml), and incubate at 4°C for 30 Minutes, centrifuge and wash 3 times with 1ml 1×BD Perm/Wash ^TM (250g); resuspend the cells into 300ul cell suspension with 1×BD Perm/Wash ^TM , and perform flow cytometry detection (Figure 7).

Example 2.4. The antibody (YWHG-5) was applied to immunohistochemical detection of HLA-G5/6 molecule expression in gastric cancer tissue.

The gastric cancer tissues were fixed in 10%-12% neutral formalin and embedded in paraffin. Tissue sections go through routine production processes such as baking, dewaxing, hydration, and antigen retrieval. An appropriate amount of 1% BSA was added dropwise on the tissue, covering the tissue and the edge of the tissue by 2 mm, and incubated at room temperature for 10 min to seal. Add anti-HLA-G5/6 isoform molecule antibody (YWHG-5) (1 mg/mL, 1:500 dilution) dropwise, and overnight (16-20h) in a humid box at 4°C. Rinse with TBS buffer, drop secondary antibody (diluted antibody goat-to-mouse ratio 1:300 in TBS), and incubate at 37°C for 30min. Rinse with TBS buffer solution, add the chromogenic agent DAB working solution dropwise, and after the tissue color development is complete, rinse the slide in running water for 5 minutes, and soak in distilled water for 5 minutes. After HE counterstaining, dehydration, transparency, and mounting, the optical microscope was used to observe the conditions of each field of view of the tissue section, and count the total number of cells and the number of brown stained cells in each field of view. The brown stained cells are positive expression of HLA-G5/6 molecules in gastric cancer tissue cells, and the positive intensity of HLA-G5/6 molecules can be judged according to the depth of brown staining of cells. Figure 8 (A), (B) is positive for HLA-G5/6 molecule in gastric cancer tissue; Figure 8 (C), (D) is negative for HLA-G5/6 molecule in gastric cancer tissue (Figure 8).

Claims (5)

A monoclonal antibody (YWHG-5) against HLA-G isomer molecules HLA-G5 and HLA-G6, said monoclonal antibody (YWHG-5) is produced by a hybridoma with a deposit number of CCTCC NO: 202122; The preservation institution is: China Center for Type Culture Collection, and the preservation time is August 11, 2021. An anti-HLA-G isoform molecule HLA-G5 and HLA-G6 monoclonal antibody (YWHG-5), characterized in that the monoclonal antibody (YWHG-5) at least includes light chain hypervariable regions CDR1, CDR2 and one or more of CDR3, or/and one or more of heavy chain hypervariable region CDR1, CDR2 and CDR3; The amino acid sequence of the antibody light chain of the monoclonal antibody (YWHG-5) is as shown in SEQ ID No.1 or has the same function as the sequence shown in SEQ ID No.1 through replacement, deletion or addition of one or more amino acids The amino acid sequence of the antibody light chain hypervariable region CDR1 is the sequence QSLLHSNGDTY shown in SEQ ID No.2 or the sequence QSLLHSNGDTY shown in SEQ ID No.2 through substitution, deletion or addition of one or more amino acids The sequence has an amino acid sequence with equivalent functions; the amino acid sequence of the light chain hypervariable region CDR2 is the sequence NIS shown in SEQ ID No.3 or the sequence NIS formed by replacing, deleting or adding one or more amino acids and SEQ ID No.3 The sequence shown has an amino acid sequence with the same function, and the amino acid sequence of the hypervariable region CDR3 of the light chain is the sequence FQGSYVPYT shown in SEQ ID No.4 or the sequence formed by substitution, deletion or addition of one or more amino acids and SEQ ID NO. The sequence shown in ID No.4 has an amino acid sequence with equivalent functions; The nucleotide sequence of the antibody heavy chain of the monoclonal antibody (YWHG-5) is as shown in SEQ ID No.5 or is formed by replacing, deleting or adding one or more amino acids with the sequence shown in SEQ ID No.5 The amino acid sequence of the same function; the amino acid sequence of the antibody heavy chain hypervariable region CDR1 is the sequence GFSLTSYG shown in SEQ ID No.6 or the sequence GFSLTSYG shown in SEQ ID No.6 by replacing, deleting or adding one or more amino acids The sequence shown has an amino acid sequence with equivalent functions, and the amino acid sequence of the heavy chain hypervariable region CDR2 is the sequence IWAGGNT shown in SEQ ID No. 7 or the sequence IWAGGNT shown in SEQ ID No. 7 or replaced, deleted or added with one or more amino acids. .7 The sequence shown in 7 has an amino acid sequence with the same function, and the amino acid sequence of the heavy chain hypervariable region CDR3 is the sequence ARDRAGTARFYYYALDN shown in SEQ ID No.8 or the combination formed by replacing, deleting or adding one or more amino acids The sequence shown in SEQ ID No.8 has an amino acid sequence with equivalent functions. The monoclonal antibody (YWHG-5) of a kind of anti-HLA-G isomer molecule HLA-G5 and HLA-G6 according to claim 2, is characterized in that, described monoclonal antibody (YWHG-5) also comprises Light chain framework region (Framework region, FR) and heavy chain framework region; Wherein, described light chain framework region comprises one or more in light chain FR1, FR2 and FR3, and the aminoacid sequence of described light chain FR1 is SEQ The sequence DIVMTQDELSLPVSLGDQASIPCGSS shown in ID No.9 or the amino acid sequence with the same function as the sequence shown in SEQ ID No.9 formed by replacing, deleting or adding one or more amino acids to the sequence; the amino acid sequence of the light chain FR2 is: The sequence LHWFLQTPGQSPKLLRY shown in SEQ ID No.10 or the amino acid sequence having the same function as 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 SEQ ID No.10 The sequence NRFSGVPDRFSGSGSGTDFTLKISRVEGEDLGVYYC shown in ID No.11 or the amino acid sequence having the same function as the sequence shown in SEQ ID No.11 formed by substitution, deletion or addition of one or more amino acids; the heavy chain framework region includes heavy chain FR1, One or more of FR2 and FR3, wherein: the amino acid sequence of the heavy chain FR1 is the sequence EVKLQESGPSLVAPSQSLSIACTVS shown in SEQ ID No. 12 or the sequence of SEQ ID No. The sequence shown in 12 has an amino acid sequence with equivalent functions; the amino acid sequence of the heavy chain FR2 is the sequence VHRIRQPPGKGLEWLGI shown in SEQ ID No.13 or the sequence VHRIRQPPGKGLEWLGI which is formed by replacing, deleting or adding one or more amino acids with the same sequence as SEQ ID No .13 amino acid sequence with the same function as shown in the sequence; the amino acid sequence of the heavy chain FR3 is the sequence KYNSALMSRLCISKDNSKSQAFLKMNSLQTDDTAMYYC of SEQ ID No.14 or the sequence shown in SEQ ID No.14 formed by substitution, deletion or addition of one or more amino acids The sequence has the same function as the amino acid sequence. The monoclonal antibody (YWHG-5) of a kind of anti-HLA-G isomer molecule HLA-G5 and HLA-G6 according to claim 2, is characterized in that, the coding in described monoclonal antibody (YWHG-5) The nucleotide sequence of the light chain variable region is the sequence shown in SEQ ID No.15 or the sequence formed by replacing, deleting or adding one or more nucleotides has the same function as the sequence shown in SEQ ID No.15 The nucleotide sequence of the heavy chain variable region in the monoclonal antibody (YWHG-5) is the sequence shown in SEQ ID No.16 or the sequence is replaced, deleted or added one or more A nucleotide sequence having the same function as the sequence shown in SEQ ID No.16 formed by nucleotides. The monoclonal antibody (YWHG-5) of the anti-HLA-G isomer molecule HLA-G5 and HLA-G6 described in any one of claim 1-5 is used for HLA-G5 and HLA-G6 immunohistochemistry, immune Uses in blotting and flow cytometry.

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