A monoclonal antibody against HLA-G1, HLA-G4 and HLA-G5 isoforms and its uses

Abstract

This invention discloses an antibody (YWHG-4) against HLA-G isoform molecules HLA-G1, HLA-G4, and HLA-G5, and its uses. The antibody (YWHG-4) is produced from a hybridoma with accession number CCTCC NO:2021204, using the antigenic peptide (RGYYNQSEASSHTLQWMIGC) of amino acid position 106-126 of the HLA-G molecule heavy chain as the immunogen. This invention provides the nucleotide encoding the YWHG-4 antibody and its encoding amino acid sequence. Furthermore, this invention provides the uses of the YWHG-4 antibody for the detection of HLA-G isoform molecules HLA-G1, HLA-G4, and HLA-G5 using Western blotting, immunohistochemistry, ELISA, and flow cytometry.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the following aspects of anti-HLA-G isoform antibody (YWHG-4): using the antigenic peptide (RGYYNQSEASSHTLQWMIGC) of the 106th to 126th amino acid sequence of the HLA-G molecular heavy chain as an immunogen to prepare antibodies (YWHG-4) against HLA-G isoforms HLA-G1, HLA-G4 and HLA-G5; the nucleic acid molecule and amino acid sequence encoding the YWHG-4 antibody of this invention; and the use of the antibody (YWHG-4) for the detection of HLA-G1, HLA-G4 and HLA-G5 by Western blotting, immunohistochemistry, ELISA and flow cytometry. Background Technology

[0002] The human leukocyte antigen-G (HLA-G) gene, 6.0 kb in length, is located on the distal side of the short arm of human chromosome 6, at 6p21.3. During protein translation, exon 1 of the HLA-G mRNA encodes a signal peptide; 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 segment of the HLA-G molecule, containing only 6 amino acid residues; exon 7 is not transcribed due to the stop codon in exon 6; and exon 8 corresponds to the 3′UTR of the HLA-G gene. The initial HLA-G transcript undergoes alternative splicing to produce seven mature mRNAs, each encoding a different molecular weight isoform (HLA-G1, -G2, -G3, -G4, -G5, -G6, and HLA-G7). 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 the HLA-G1 to -G7 isomers are 39 kD, 31 kD, 22 kD, 30 kD, 34 kD, 23 kD, and 16 kD, respectively.

[0003] HLA-G1 is encoded by full-length HLA-G mRNA and consists of extracellular α1, α2, and α3 domains, a transmembrane domain, and an intracellular domain. HLA-G2 lacks the α2 domain and consists of extracellular α1 and α3 domains, a transmembrane domain, and an intracellular domain. HLA-G3 lacks both α2 and α3 domains and consists of an extracellular α1 domain, a transmembrane domain, and an intracellular domain. HLA-G4 lacks the α3 domain and consists of extracellular α1 and α2 domains, a transmembrane domain, and an intracellular domain. The extracellular domains of HLA-G5 and HLA-G6 are the same as 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 molecules lack the transmembrane domain encoded by exon 5, forming soluble HLA-G molecules. The mRNA encoding HLA-G7 contains a stop codon in intron 2, and its extracellular region consists only of an α1 domain and two amino acid residues encoded by intron 2. Figure 1 ).

[0004] Under normal physiological conditions, HLA-G molecules are expressed only on 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 tissues such as tumor cells and viral infections, and are closely related to the occurrence and progression of diseases. HLA-G molecules are an important immune tolerance molecule in the body and also an important immune checkpoint molecule. Their immunosuppressive function is mainly achieved by binding to immunosuppressive receptors immunoglobulin-like transcript-2 (ILT2 / LILRB1 / CD85j) and immunoglobulin-like transcript-4 (ILT4 / LILRB2 / CD85d), transmitting inhibitory signals, and inducing immune tolerance. Specific mechanisms of action include: ① Binding to ILT2 expressed on T cells, NK cells, and B cells, inhibiting the immune killing activity of T cells and NK cells, as well as the proliferation and antibody secretion of B cells. ② Binding to ILT2 and ILT4 on dendritic cells (DCs), inhibiting the maturation and differentiation of DCs, and inducing the production of tolerant DCs. ③ It binds to ILT2 and ILT4 on bone marrow-derived suppressor cells (MDSCs) and macrophages (Mф), inducing the differentiation of pro-inflammatory and anti-tumor M1 cells into immune-resistant M2 cells. Therefore, HLA-G plays an important role in the development and progression of diseases such as tumors. HLA-G-targeted tumor immunotherapy has entered Phase I clinical trials in the United States.

[0005] HLA-G binds to receptors ILT2 and ILT4 with molecular structure specificity. The binding site for ILT2 and ILT4 is the α3 domain of the extracellular region of HLA-G. ILT2 binds only to the HLA-G / β2m complex, while ILT4 can bind not only to HLA-G / β2m but also to free HLA-G molecules without β2m. Due to differences in expression mechanisms and molecular structures among HLA-G1, -G2, -G3, -G4, -G5, -G6, and HLA-G7, isoforms such as HLA-G3, -G4, and HLA-G7, which lack the α3 domain in their extracellular regions, cannot bind to ILT2 and ILT4. Different HLA-G isoforms can exert specific immunological effects during pathophysiological processes. Under different pathological conditions, especially in tumor tissues and cells, HLA-G isoform expression exhibits wide heterogeneity, 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.

[0006] Currently, the main antibodies used for HLA-G immunohistochemistry and immunoblotting both domestically and internationally are 4H84, MEM-G1, MEM-G2, 2A12, and 5A6G7. Among them, antibody 4H84 recognizes the extracellular α1 domain, which is present in all seven HLA-G isoforms. Specifically, it 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). However, it cannot distinguish the expression of specific HLA-G isoforms in immunohistochemistry. Antibodies MEM-G1 and MEM-G2 were obtained by immunizing mice with the full-length HLA-G heavy chain. The specific recognition sites cannot be predicted. Theoretically, similar to antibody 4H84, these two antibodies can recognize seven HLA-G isoforms (HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7), but they also cannot distinguish the expression of specific HLA-G isoforms. Antibodies 2A12 and 5A6G7 were obtained by immunizing mice with the 22 amino acid residues located at the C-terminus 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 non-HLA-G6 molecules with molecular weights in the range of 36-175 kDa. In immunohistochemical applications, cross-reactivity may occur, leading to false positives and preventing the correct expression of HLA-G5 and HLA-G6 molecules. Monoclonal antibodies 87G and MEM-G / 9 are used for flow cytometry detection of HLA-G molecules, but they can only detect HLA-G1 and HLA-G5 molecules.

[0007] Therefore, in the context of targeted precision medicine, existing antibodies for detecting HLA-G molecules are not entirely satisfactory, and there is an urgent need to develop more monoclonal antibodies targeting different HLA-G isoform molecules. Summary of the Invention

[0008] In view of the above-mentioned shortcomings of existing HLA-G antibodies, the purpose of this invention is to provide a specific monoclonal antibody (YWHG-4) against HLA-G1, HLA-G4 and HLA-G5 isoform molecules, a nucleic acid molecule and amino acid sequence encoding the antibody (YWHG-4) of this invention; and the use of the YWHG-4 antibody for HLA-G1, HLA-G4 and HLA-G5 immunohistochemistry, immunoblotting and flow cytometry detection.

[0009] This invention uses the antigenic peptide at amino acid positions 106-126 of the HLA-G molecule heavy chain as the immunogen, the amino acid sequence of which is shown in SEQ ID No. 17 (RGYYNQSEASSHTLQWMIGC). The RGYYNQSEASSHTLQWMIGC peptide is located in the α1 and α2 hinge regions commonly found in HLA-G1, HLA-G4, and HLA-G5 isoforms, and based on this, a specific monoclonal antibody against HLA-G1, HLA-G4, and HLA-G5 isoforms (YWHG-4) was prepared. Figure 1 ).

[0010] Based on the inventor's research findings, this invention provides a monoclonal antibody (YWHG-4) against HLA-G isoform molecules HLA-G1, HLA-G4 and HLA-G5, comprising at least one or more of the heavy chain hypervariable regions CDR1, CDR2 and CDR3, and / or one or more of the light chain hypervariable regions CDR1, CDR2 and CDR3.

[0011] The amino acid sequence of the monoclonal antibody (YWHG-4) light chain is as shown in SEQ ID No. 1, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 1 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR1 of the antibody light chain is the sequence QSIVHSNGNTY shown in SEQ ID No. 2, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 2 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR2 of the light chain is the sequence KVS shown in SEQ ID No. 3, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 3 formed by substitution, deletion, or addition of one or more amino acids; and the amino acid sequence of the hypervariable region CDR3 of the light chain is the sequence FQASHVPYT shown in SEQ ID No. 4, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 4 formed by substitution, deletion, or addition of one or more amino acids.

[0012] The nucleotide sequence of the heavy chain of the monoclonal antibody (YWHG-4) is as shown in SEQ ID No. 5, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 5 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR1 of the antibody heavy chain is the sequence GYASTNYL shown in SEQ ID No. 6, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 6 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of the hypervariable region CDR2 of the heavy chain is the sequence INPGSGRI shown in SEQ ID No. 7, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 7 formed by substitution, deletion, or addition of one or more amino acids; and the amino acid sequence of the hypervariable region CDR3 of the heavy chain is the sequence ARHYGNSAWFAY shown in SEQ ID No. 8, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 8 formed by substitution, deletion, or addition of one or more amino acids.

[0013] Furthermore, the monoclonal antibody (YWHG-4) further includes a light chain framework region (FR) and a heavy chain framework region; wherein, the light chain framework region includes one or more of light chains FR1, FR2, and FR3, the amino acid sequence of light chain FR1 is the sequence shown in SEQ ID No. 9, GDIVITQDELSLPVSLGDQAAISCRS, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 9 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of light chain FR2 is the sequence shown in SEQ ID No. 10, LEWYLQKPGQSPKLLIY, or an amino acid sequence with equivalent function to the sequence shown in SEQ ID No. 10 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of light chain FR3 is the sequence shown in SEQ ID No. 11, NRFSGVPDRFRGSGSGTDFTLKISRVEAEDLGVYYC, or an amino acid sequence with equivalent function to 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 one or more of heavy chains FR1, FR2, and FR3, wherein: the amino acid sequence of heavy chain FR1 is the sequence VQLQQSGAEVVRPGTSVKVSCKAS shown in SEQ ID No. 12, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 12 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of heavy chain FR2 is the sequence VEWIKQRPGQGLEWIGV shown in SEQ ID No. 13, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 13 formed by substitution, deletion, or addition of one or more amino acids; the amino acid sequence of heavy chain FR3 is the sequence YYSEKFKGKTTLTADKSSSN AYMQLSSLTSDDSAVYFC shown in SEQ ID No. 14, or an amino acid sequence with the same function as the sequence shown in SEQ ID No. 14 formed by substitution, deletion, or addition of one or more amino acids.

[0014] Furthermore, the nucleotide sequence encoding the light chain variable region in the monoclonal antibody (YWHG-4) is the sequence shown in SEQ ID No. 15, or a nucleotide sequence with the same function as the sequence shown in SEQ ID No. 15 formed by replacing, deleting, or adding one or more nucleotides to the sequence; and the nucleotide sequence encoding the heavy chain variable region in the monoclonal antibody (YWHG-4) is the sequence shown in SEQ ID No. 16, or a nucleotide sequence with the same function as the sequence shown in SEQ ID No. 16 formed by replacing, deleting, or adding one or more nucleotides to the sequence.

[0015] According to one aspect of the present invention, a preferred monoclonal antibody (YWHG-4) against the HLA-G isomers HLA-G1, HLA-G4 and HLA-G5 is provided. The monoclonal antibody (YWHG-4) is produced from a hybridoma with accession number CCTCC NO:2021204, deposited at the China Center for Type Culture Collection on August 11, 2021.

[0016] The present invention also provides the use of the anti-HLA-G1, HLA-G4 and HLA-G5 isoform molecular antibody (YWHG-4) for the detection of HLA-G isoforms HLA-G1, HLA-G4 and HLA-G5 molecular immunohistochemistry, immunoblotting and flow cytometry, etc., with 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 following will further explain this application through specific embodiments 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. For those skilled in the art, several improvements and adjustments can be made without departing from the technical principles of this application, and these improvements and adjustments should also be considered to fall within the protection scope of this application.

[0018] Unless otherwise defined, all technical 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 3-letter and / or 1-letter codes used in the art to refer to the 20 commonly used amino acids.

[0019] The hypervariable region of light chain or hypervariable region of heavy chain mentioned in this invention is also called the complementarity determining region (CDR).

[0020] The term "sequence" as used in this invention can refer to amino acid sequences containing certain biologically equivalent amino acids or "conserved substitutions," while "other sequences" can refer to amino acids with non-equivalent functions or "non-conserved substitutions," which are genetically engineered to improve the properties of CDR or CDR-containing antibodies. Without substantially affecting antibody activity, those skilled in the art can manipulate the sequences in this 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. These should be considered to be included within the scope of protection of this application. For example, amino acids with similar properties can be substituted in the variable region. The sequences of the variants mentioned in this invention can have at least 95%, 96%, 97%, 98%, or 99% sequence identity with their source sequences. Sequence identity can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. Various amino acid sequences described herein are detailed in the sequence listing. Attached Figure Description

[0021] Figure 1 These are schematic diagrams of the molecular structures of seven different HLA-G isomers and the locations of their immunogenic fragments.

[0022] Figure 2 This involves the identification of the heavy and light chain subclasses of the antibody (YWHG-4).

[0023] Figure 3 The antibody (YWHG-4) was analyzed by SDS-PAGE to determine its purity.

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

[0025] Figure 5 The antibody (YWHG-4) is used for immunoblotting detection of HLA-G1, HLA-G4, and HLA-G5 molecules.

[0026] Figure 6 The antibody (YWHG-4) ELISA method is used to detect the HLA-G5 molecular concentration.

[0027] Figure 7 The antibody (YWHG-4) was used for flow cytometry detection of HLA-G1 and HLA-G4 molecules.

[0028] Figure 8 The intracellular flow cytometry detection of the antibody (YWHG-4) for HLA-G5 molecular detection.

[0029] Figure 9The antibody (YWHG-4) was used for immunohistochemical detection of HLA-G1, HLA-G4, and HLA-G5 expression in different tissues. Specifically, 6.1 shows HLA-G1, HLA-G4, and HLA-G5 expression in decidual tissue (100X); 6.2 shows HLA-G1, HLA-G4, and HLA-G5 expression in adjacent gastric tissue (100X); and 6.3 and 6.4 show HLA-G1, HLA-G4, and HLA-G5 expression in gastric cancer tissue (400×). Detailed Implementation

[0030] 1. Preparation of anti-HLA-G monoclonal antibody (YWHG-4)

[0031] ① Antigenic peptide synthesis

[0032] Synthesize the antigenic peptide of the HLA-G molecular heavy chain from amino acid position 106 to 126, SEQ No. 23RGYYNQSEASSHTLQWMIGC.

[0033] ② Mouse immunization

[0034] Four SPF-grade female BALB / c mice were initially immunized with 60 μg / mouse. A first booster immunization was given to each mouse with 30 μg. A second booster immunization was given to each mouse with 30 μg. A third booster immunization was given to each mouse with 30 μg. Blood was collected from the orbital sinus to determine serum titers. The immunized mice were coated with "SEQ No. 23RGYYNQSEASSHTLQWMIGC" and ELISA was used to determine the titers. The mice were then coated overnight at 4°C with "SEQ No. 23RGYYNQSEASSHTLQWMIGC" at 2 μg / ml; blocked with 2% milk at 37°C for 2 hours; serum was serially diluted 2-fold starting from 200-fold, with PBS as the blank control and a 200-fold dilution of negative serum as the negative control. Before fusion, mice were immunized with a shock immunization using 50 μg of the "SEQ No. 23RGYYNQSEASSHTLQWMIGC" immunogen. In the fusion experiment, mouse spleen cells and SP2 / 0 cells were fused using the PEG method. After fusion, the cells were screened and cultured in a semi-solid culture medium (containing HAT).

[0035] ③ Monoclonal cell screening

[0036] Select 10 plates × 93 cell clones and culture them in 96-well cell culture plates (pre-coated with thymocytes, 100 μL / well). Coat the plates with "SEQ No. 23RGYYNQSEASSHTLQWMIGC", dilute "SEQ No. 23RGYYNQSEASSHTLQWMIGC" with coating buffer to a final concentration of 2 μg / ml, 100 μL / well, incubate overnight at 4°C; then wash 3 times with washing buffer. Block with 2% milk blocking buffer, 200 μL / well, incubate at 37°C for 2 h; then wash 3 times with washing buffer. Add primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum PBS diluted 1000 times), 100 μL / well, incubate at 37°C for 1 h; then wash 3 times with washing buffer. Secondary antibody diluted 20,000 times with PBS, 100 μL / well, incubated at 37°C for 1 h; washed 3 times with washing buffer after incubation. For color development, 100 μL / well of development solution, development time approximately 5 min. Stop the reaction by adding 50 μL of stop solution to each well. Measure absorbance at dual wavelengths (450, 630). Selected clones were screened using ELISA for the first time to obtain positive hybridoma cell lines. The positive cell lines were then re-coated with “SEQ No. 23RGYYNQSEASSHTLQWMIGC” and screened again using ELISA for the second time to obtain positive hybridoma cell lines. After multiple screenings, a monoclonal antibody against the fragment SEQ No. 23RGYYNQSEASSHTLQWMIGC was obtained and named YWHG-4 (accession number CCTCCNO: 2021204).

[0037] ④ Identification of subclasses of anti-HLA-G monoclonal antibody (YWHG-4)

[0038] Ten positive cell lines were selected for subclass identification. The coating antibody was diluted to 0.5 μg / mL with 100 mM PBS (pH 7.4), and 0.1 mL was added to each well. The cells were incubated overnight at 4°C. After washing twice with PBS-T, 200 μL of blocking buffer was added to each well, and the cells were incubated at 37°C for 2 hours. After washing three times with PBS-T, 100 μL of hybridoma supernatant was added to each well, and the cells were incubated at 37°C for 1 hour. After washing three times with PBS-T, 0.1 mL of HRP-labeled antibody diluted 1:10000 (κ,λ) or 1:20000 (other) blocking buffer was added to each well, and the cells were incubated at 37°C for 1 hour. After washing three times with PBS-T, 50 μL of substrate solution was added to each well, and the absorbance was measured at dual wavelengths (450, 630 nm) within 10–20 minutes. The antibody subtype was again confirmed using Thermo Biotech's Pierce Rapid Isotyping Kits – Mouse (Catalog #26178) to be the subtype of the monoclonal antibody produced by this cell line: heavy chain IgG1 and light chain kappa(κ). Figure 2 ).

[0039] ⑤ Antibody purification

[0040] Sample pretreatment: Dilute 1:3 with the appropriate conjugation buffer, centrifuge at 12000 rpm for 10 min at 4℃, and filter through a 0.22 μm filter to remove fat, cell debris, and small particulate matter. Equilibration: Equilibrate the column with 10 column volumes of the appropriate conjugation buffer, maintaining a flow rate of 1 ml / min. Sample loading: Inject the sample into the top inlet of the column, collect the eluent, and maintain a flow rate of 1 ml / min. Washing: Pass the sample through the column with 5 column volumes of conjugation buffer, maintaining a flow rate of 1 ml / min. Elution: Elute the antibody with 5 column volumes of elution buffer, collect it in the EP tube mentioned above, and maintain a flow rate of 1 ml / min. Immediately adjust the pH to 7.0 with 1M pH 9.0 Tris-HCl buffer. Equilibration: Equilibrate the column back to pH 7.0 with 10 column volumes of conjugation buffer, maintaining a flow rate of 1 ml / min. Dialysis: Dialyze the antibody overnight using 0.01M PBS buffer, changing the buffer 3 times.

[0041] ⑥ SDS-PAGE assay for antibody purity

[0042] Prepare SDS-PAGE gels; the separating gel concentration is 12%. Sample preparation: After adding sample buffer, boil the sample in water for 10 min. Sample loading: 10 μL per well. Gel running: Stacking gel 80 V, 30 min; separating gel 120 V, 60 min. Stop electrophoresis when the bromophenol blue front reaches the bottom of the glass plate and remove the gel. Staining and destaining: Immerse the gel in Coomassie Brilliant Blue staining solution and gently shake on a shaker for at least 30 min (staining time needs to be adjusted appropriately according to gel thickness). Remove the gel and rinse it several times in water, then add Coomassie Brilliant Blue destaining solution and shake. Destain the gel until the bands are roughly visible (1 hour); for complete destaining, change the destaining solution 2-3 times and shake for at least 24 hours. After destaining, the gel can be scanned and recorded using an ECL gel imaging system. The purified antibody YWHG-4 has a purity >90%. Figure 3 ).

[0043] ⑦ Determination of antibody affinity constant for ELISA detection

[0044] Dilute the antigen (SEQ No. 23RGYYNQSEASSHTLQWMIGC) with coating buffer to a final concentration of 2 μg / ml, 100 μL / well, incubate overnight at 4°C; then wash twice with washing buffer. Block with blocking buffer, 200 μL / well, incubate at 37°C for 2 hours; then wash once with washing buffer. Serially dilute the purified antibody 2-fold (PBS) starting at 200-fold, with a blank control in PBS, 100 μL / well for both, incubate at 37°C for 1 hour; then wash three times with washing buffer. Add secondary antibody diluted 20000-fold in PBS, 100 μL / well, incubate at 37°C for 1 hour; then wash three times with washing buffer. Develop the colorimetric solution, 100 μL / well, for 5-15 minutes. Add 50 μL of stop solution to each well to stop the reaction. Measure the absorbance at dual wavelengths (450, 630 nm) and plot for analysis. According to the Logistic regression equation, the affinity constant is approximately 150000 × A / antibody concentration (where A is the antibody dilution factor corresponding to 1 / 2 OD value; dilution factor = 197388.03; YWHG-4 antibody concentration = 3.0 mg / mL), and the affinity constant of antibody YWHG-4 is 9.87 × 10⁻⁶. 9 L / mol ( Figure 4 ).

[0045] II. Application Examples

[0046] Example 2.1. Detection of HLA-G1, HLA-G4, and HLA-G5 molecules using the antibody (YWHG-4) immunoblotting.

[0047] HLA-G isoform standard proteins HLA-G1, HLA-G2, HLA-G3, HLA-G4, HLA-G5, HLA-G6, and HLA-G7 were denaturing PAGE electrophoresis. After semi-dry electrotransfer, the membranes were blocked with 5% skim milk powder at room temperature for 4 h, and washed with 0.2% TBS (Teween-20 PBS). The antibody (YWHG-4) was added, and its recognition specificity was tested. The membranes were incubated overnight at 4°C, and then washed. HRP-labeled rabbit anti-mouse IgG antibody was added, and the membranes were incubated at room temperature for 30 min. After washing, the membranes were irradiated with Dako REAL. TM EnVision TM The detection system (DAKO) was incubated for 1-3 minutes. Results showed that the antibody (YWHG-4) specifically recognizes HLA-G1, HLA-G4, and HLA-G5 standard proteins, and does not cross-react with other HLA-G isoforms. Figure 5 ).

[0048] Example 2.2. Detection of HLA-G5 molecular concentration using the antibody (YWHG-4) ELISA method

[0049] HLA-G5 protein was serially diluted two-fold with coating buffer (0.1M, pH 9.6, NaHCO3) to final concentrations of 4 μg / ml, 2 μg / ml, 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, and 0.0625 μg / ml, with the coating buffer serving as a blank control. 100 μL / well was coated and incubated overnight at 4°C; followed by washing twice with washing buffer. 200 μL / well was blocked with 1% BSA and incubated at 37°C for 2 hours; followed by washing three times with washing buffer.

[0050] After washing, add purified antibody (YWHG-4) at a final concentration of 1.0 ug / ml, 100 μl / well, incubate at 37°C for 1 h; then wash 3 times with washing buffer. Add HRP-labeled anti-mouse IgG secondary antibody diluted 20000 times with PBS, 100 μl / well, incubate at 37°C for 1 h; after removal, wash 3 times with washing buffer. For color development, use 100 μl / well of TMB chromogenic solution, and the color development time is 5-15 min. Add 50 μl of stop solution to each well to stop the reaction. Measure the absorbance at wavelength (450 nm) and plot for analysis. OD values ​​of 4 ug / ml, 2 ug / ml, 1 ug / ml, 0.5 ug / ml, 0.25 ug / ml, 0.125 ug / ml, 0.0625 ug / ml and the blank control of the coating solution. 450The concentrations were 1.855, 1.699, 1.318, 1.213, 0.982, 0.644, 0.424, and 0.06, respectively. The results showed that the antibody (YWHG-4) specifically recognized HLA-G5, and the concentration was significantly correlated with HLA-G5 concentration [Y (concentration) = 1.772 x (OD)]. 2 -1.479x (OD); R 2 =0.914]( Figure 6 ).

[0051] Example 2.3. Flow cytometry detection of HLA-G1, HLA-G4 and HLA-G5 molecules using the antibody (YWHG-4).

[0052] Standard proteins of HLA-G1, HLA-G4, and HLA-G5 molecules, K562-HLA-G1, K562-HLA-G4, and K562-HLA-G5, were collected from cell cultures expressing HLA-G1, HLA-G4, and HLA-G5 molecules during the logarithmic growth phase. HLA-G1 and HLA-G4 are cell membrane surface-expressed molecules, while HLA-G5 is a soluble molecule, and intracellular flow cytometry analysis was performed.

[0053] Flow cytometry detection of HLA-G1 and HLA-G4 molecular expression on cell membrane surface: K562-HLA-G1 and K562-HLA-G4 cell cultures were washed twice (250g) by centrifugation with PBS / BSA, and then prepared into 2×10⁻⁶ cells / mL solution with PBS / BSA. 6 Cell suspension. Take 200 μL of cell suspension and add 2 μL (1.0 mg / mL) of FITC-labeled purified antibody (YWHG-4). Incubate at 4°C for 1 hour. (1×BD Perm / Wash) TM Centrifuge and wash 3 times (250g); then rinse the cells with 1×BD Perm / Wash. TM Resuspend the cells in 200 μL suspension and perform flow cytometry analysis. Figure 7 ).

[0054] Intracellular HLA-G5 molecule expression flow cytometry detection: 50 μL BD Perm / Wash TM K562-HLA-G5 cells were resuspended, and purified antibody (YWHG-4) containing 2 μL (1.0 mg / mL) of FITC-labeled solution was added. The cells were incubated at 4°C for 30 minutes. Then, 1 mL of 1×BDPerm / Wash solution was added. TM Centrifuge and wash 3 times (250g); then rinse the cells with 1×BD Perm / Wash. TM Resuspend the cells in 200 μL suspension and perform flow cytometry analysis. Figure 8 ).

[0055] Example 2.4. The antibody (YWHG-4) was used for immunohistochemical detection of HLA-G1, HLA-G4 and HLA-G5 molecular expression in different tissues.

[0056] Tissue samples were fixed in 10%-12% neutral formalin and embedded in paraffin. Tissue sections underwent standard slide preparation procedures including baking, dewaxing, hydration, and antigen retrieval. A suitable amount of 1% BSA was added to the tissue, covering the tissue and its edges by 2 mm, and incubated at room temperature for 10 min for blocking. Anti-HLA-G1, HLA-G4, and HLA-G5 isoform antibodies (YWHG-4) (1 mg / mL, 1:500 dilution) were added, and the slides were incubated overnight (16-20 h) at 4°C in a humidified chamber. After washing with TBS buffer, secondary antibody (TBS diluted antibody at a goat anti-mouse ratio of 1:300) was added, and the slides were incubated at 37°C for 30 min. After further washing with TBS buffer, DAB working solution was added as the chromogenic agent. Once the tissue was fully stained, the slides were rinsed in running water for 5 min and then soaked in distilled water for 5 min. After HE counterstaining, dehydration, clearing, and mounting, the tissue sections were observed under a light microscope in each field of view. The total number of cells and the number of brown-stained cells in each field of view were counted. Brown-stained cells were positive for HLA-G1, HLA-G4, and HLA-G5 molecules. The intensity of HLA-G1, HLA-G4, and HLA-G5 positivity was determined based on the depth of brown staining. Figure 9 ).

Claims

1. A monoclonal antibody YWHG-4 against HLA-G isoform molecules HLA-G1, HLA-G4 and HLA-G5, wherein the monoclonal antibody YWHG-4 is produced from a hybridoma with accession number CCTCC NO: C2021204.

2. A monoclonal antibody YWHG-4 against the HLA-G isoform molecules HLA-G1, HLA-G4, and HLA-G5, characterized in that, The monoclonal antibody YWHG- includes 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 variable region of the light chain of the monoclonal antibody YWHG-4 is shown in SEQ ID No. 1; the amino acid sequence of the hypervariable region CDR1 of the antibody light chain is shown in SEQ ID No. 2; the amino acid sequence of the hypervariable region CDR2 of the light chain is shown in SEQ ID No. 3; and the amino acid sequence of the hypervariable region CDR3 of the light chain is shown in SEQ ID No.

4. The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody YWHG-4 is shown in SEQ ID No. 5; the amino acid sequence of the hypervariable region CDR1 of the antibody heavy chain is shown in SEQ ID No. 6; the amino acid sequence of the hypervariable region CDR2 of the heavy chain is shown in SEQ ID No. 7; and the amino acid sequence of the hypervariable region CDR3 of the heavy chain is shown in SEQ ID No.

8.

3. The monoclonal antibody YWHG-4 against the HLA-G isoform molecules HLA-G1, HLA-G4 and HLA-G5 according to claim 2, characterized in that, The monoclonal antibody YWHG-4 includes a light chain framework region and a heavy chain framework region; wherein, the light chain framework region includes light chains FR1, FR2, and FR3, the amino acid sequence of light chain FR1 is the sequence shown in SEQ ID No. 9; the amino acid sequence of light chain FR2 is the sequence shown in SEQ ID No. 10; and the amino acid sequence of light chain FR3 is the sequence shown in SEQ ID No. 11; the heavy chain framework region includes heavy chains FR1, FR2, and FR3, wherein: the amino acid sequence of heavy chain FR1 is the sequence shown in SEQ ID No. 12; the amino acid sequence of heavy chain FR2 is the sequence shown in SEQ ID No. 13; and the amino acid sequence of heavy chain FR3 is the sequence shown in SEQ ID No.

14.

4. The monoclonal antibody YWHG-4 against the HLA-G isoform molecules HLA-G1, HLA-G4 and HLA-G5 according to claim 2, characterized in that, The nucleotide sequence encoding the light chain variable region of the monoclonal antibody YWHG-4 is the sequence shown in SEQ ID No. 15, and the nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody YWHG-4 is the sequence shown in SEQ ID No. 16.

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