Anti-sdcatcher, gvopti murine monoclonal antibody, its preparation method and use

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an anti-SDcatcher, GVopti mouse-derived monoclonal antibody, a preparation method and application thereof. The application discloses an anti-SDcatcher mouse-derived monoclonal antibody and an anti-GVopti mouse-derived monoclonal antibody, both of which are composed of two parts of a heavy chain and a light chain, and specifically include SD-Mab-1 (SEQ ID NO. 9, 10) and SD-Mab-2 (SEQ ID NO. 11, 12), GV-Mab-1 (SEQ ID NO. 13, 14) and GV-Mab-2 (SEQ ID NO. 15, 16). The above antibody disclosed by the application can be used for qualitative, quantitative, tracing and enrichment, extraction, purification and other related work of a fusion protein containing an SDcatcher and GVopti peptide, and has great potential application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-SDcatcher and GVopti mouse monoclonal antibody, its preparation method and uses. Background Technology

[0002] Cellular function depends on a large number of reversible non-covalent protein-protein interactions, and the precise arrangement of proteins in the complex influences and determines their function. However, non-covalent interactions are often fragile and are usually disrupted under conditions such as long cycles or the presence of mechanical forces. Therefore, designing covalent protein-protein interactions can bring a series of new opportunities to basic research, synthetic biology, and biotechnology.

[0003] Currently, proteins capable of spontaneously forming isopeptide bonds have been used to develop peptide tags and corresponding binding polypeptides (i.e., linkers of two peptide segments), relying on the covalent binding between isopeptide bonds to provide irreversible interactions. In this case, proteins capable of spontaneously forming isopeptide bonds can be expressed as two segments: a peptide tag and a polypeptide binding partner. Some Gram-positive bacteria contain proteins exhibiting extremely strong interactions.

[0004] In previous studies, amino acid sequences 486-591 from CnaB2 of *Streptococcus dysgalactiae* were used as the SDcatcher peptide, and amino acid sequences 97-111 from CnaB2 of *Gardnerella vaginalis* were used as the GVtag peptide tag. Optimization was achieved through corresponding amino acid modifications at the N-terminus of the GVtag peptide tag, establishing an SD-GVopti linker system capable of spontaneously forming heteropeptide bonds between the peptide and the peptide tag (doi:10.1038 / s41423-021-00736-2). This linker system provides a simple, specific, and genetically encoded method for creating various biomaterials, demonstrating significant application value in numerous fields, such as biomaterials, next-generation sequencing, enzyme stabilization, and vaccine development. Therefore, the development of murine monoclonal antibodies targeting SDcatcher and GVtag can be used for qualitative and quantitative detection and tracing of fusion peptides and proteins containing these tags, as well as for enrichment, extraction or purification of fusion peptides and proteins containing these tags. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, this invention proposes a mouse-derived monoclonal antibody against SDcatcher and GVopti. One antibody (SD-Mab-1 / 2) specifically recognizes a polypeptide (SDcatcher) derived from Streptococcus dysgalactiae, and the other antibody (GV-Mab-1 / 2) specifically recognizes a polypeptide (GVopti) with an optimized amino acid sequence at positions 97-111 of the adhesin domain (CnaB2) of fibronectin-binding protein (Fba B) derived from Gardnerella vaginalis. These antibodies can be used for the qualitative, quantitative, and tracing detection of fusion proteins containing SDcatcher and GVopti peptides, and have broad application prospects.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of this invention provides a murine monoclonal antibody targeting a fusion protein containing SDcatcher and GVopti peptides. The murine monoclonal antibody is selected from anti-SDcatcher monoclonal antibodies and / or anti-GVopti monoclonal antibodies. The anti-SDcatcher monoclonal antibody is selected from SD-Mab-1 and / or SD-Mab-2, and the anti-GVopti monoclonal antibody is selected from GV-Mab-1 and / or GV-Mab-2. SD-Mab-1, SD-Mab-2, GV-Mab-1, and GV-Mab-2 are each composed of a heavy chain and a light chain (linked by disulfide bonds). The heavy chain of SD-Mab-1 has the amino acid sequence shown in SEQ ID NO. 9, and the light chain has the amino acid sequence shown in SEQ ID NO. 10. The heavy chain of SD-Mab-2 has the amino acid sequence shown in SEQ ID NO. 11, and the light chain has the amino acid sequence shown in SEQ ID NO. 12. The heavy chain of GV-Mab-1 has the amino acid sequence shown in SEQ ID NO. 10. The amino acid sequence of NO.13, the light chain has the amino acid sequence as shown in SEQ ID NO.14; the heavy chain of GV-Mab-2 has the amino acid sequence as shown in SEQ ID NO.15, and the light chain has the amino acid sequence as shown in SEQ ID NO.16.

[0008] A second aspect of the present invention provides an anti-SDcatcher mouse monoclonal antibody, wherein the anti-SDcatcher mouse monoclonal antibody is selected from SD-Mab-1 and / or SD-Mab-2, wherein both SD-Mab-1 and SD-Mab-2 are composed of a heavy chain and a light chain; the heavy chain of SD-Mab-1 has the amino acid sequence shown in SEQ ID NO. 9, and the light chain has the amino acid sequence shown in SEQ ID NO. 10; the heavy chain of SD-Mab-2 has the amino acid sequence shown in SEQ ID NO. 11, and the light chain has the amino acid sequence shown in SEQ ID NO. 12.

[0009] A third aspect of this invention provides an anti-GVopti murine monoclonal antibody, wherein the anti-GVopti murine monoclonal antibody is selected from GV-Mab-1 and / or GV-Mab-2, wherein both GV-Mab-1 and GV-Mab-2 are composed of a heavy chain and a light chain; the heavy chain of GV-Mab-1 has the amino acid sequence as shown in SEQ ID NO.13, and the light chain has the amino acid sequence as shown in SEQ ID NO.14; the heavy chain of GV-Mab-2 has the amino acid sequence shown in SEQ ID NO.15, and the light chain has the amino acid sequence shown in SEQ ID NO.16.

[0010] The amino acid sequence of the variable region of the heavy chain has at least 80% sequence homology with that of the variable region of the light chain, and the amino acid sequence of the hypervariable region of the heavy chain also has at least 80% sequence homology with that of the hypervariable region of the light chain (variable region: the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes significantly, including the hypervariable region; hypervariable region: the specific site in the immunoglobulin light chain and heavy chain where the changes are most drastic). Preferably, in the first and second aspects, the amino acid sequence of the heavy chain hypervariable region of SD-Mab-1 is CDR1: GFTFTSYT, CDR2: ISNGGSTT and CDR3: ARHNSSYFDY, and the amino acid sequence of the light chain hypervariable region is CDR1: ENIYSY; CDR2: NAK and CDR3: QHHYGNPPT; the amino acid sequence of the heavy chain hypervariable region of SD-Mab-2 is CDR1: GYTFTTAG, CDR2: IKTHSGVT and CDR3: ARSGPLNWYYPMDY, and the amino acid sequence of the light chain hypervariable region is CDR1: QNVGTN; CDR2: SAS and CDR3: QQYNSYPLT; the amino acid sequence of the heavy chain variable region of SD-Mab-1 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of SD-Mab-2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0011] The amino acid sequence of the variable region of the heavy chain has at least 80% sequence homology with that of the variable region of the light chain, and the amino acid sequence of the hypervariable region of the heavy chain also has at least 80% sequence homology with that of the hypervariable region of the light chain (variable region: the region of the immunoglobulin light chain and heavy chain near the N-terminus where the amino acid sequence changes significantly, including the hypervariable region; hypervariable region: the specific site in the immunoglobulin light chain and heavy chain where the changes are most drastic). Preferably, in the first and third aspects, the amino acid sequence of the heavy chain hypervariable region of GV-Mab-1 is CDR1: GFTFSGYI, CDR2: ISSGGSYT and CDR3: LVADLDFDV, and the amino acid sequence of the light chain hypervariable region is CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT; the amino acid sequence of the heavy chain hypervariable region of GV-Mab-2 is CDR1: GFTFSSYT, CDR2: INSGVTYT and CDR3: LVADLDFD, and the amino acid sequence of the light chain hypervariable region is CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT; the amino acid sequence of the heavy chain variable region of GV-Mab-1 is as shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.6; the amino acid sequence of the heavy chain variable region of GV-Mab-2 is as shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.8.

[0012] The fourth aspect of the present invention provides a nucleic acid molecule encoding the murine monoclonal antibody described in the second or third aspect.

[0013] Preferably, the nucleotide sequence encoding the heavy chain of SD-Mab-1 is shown in SEQ ID NO.17, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.18; the nucleotide sequence encoding the heavy chain of SD-Mab-2 is shown in SEQ ID NO.19, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.20.

[0014] Preferably, the nucleotide sequence encoding the heavy chain of GV-Mab-1 is shown in SEQ ID NO.21, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.22; the nucleotide sequence encoding the heavy chain of GV-Mab-2 is shown in SEQ ID NO.23, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.24.

[0015] The fifth aspect of this invention provides the application of the murine monoclonal antibodies described in the first to third aspects or the nucleic acid molecules described in the fourth aspect in the qualitative, quantitative, and tracing of fusion proteins containing SDcatcher and / or GVopti.

[0016] The sixth aspect of this invention provides the application of the murine monoclonal antibodies described in the first to third aspects or the nucleic acid molecules described in the fourth aspect in the enrichment, extraction, and purification of fusion proteins containing SDcatcher and / or GVopti.

[0017] The anti-SDcatcher mouse monoclonal antibody and the anti-GVopti mouse monoclonal antibody disclosed in this invention are both composed of a heavy chain and a light chain. Specifically, the anti-SDcatcher monoclonal antibody is SD-Mab-1 (as shown in SEQ ID NO. 9 and 10) and SD-Mab-2 (as shown in SEQ ID NO. 11 and 12), and the anti-GVopti antibody is GV-Mab-1 (as shown in SEQ ID NO. 13 and 14) and GV-Mab-2 (as shown in SEQ ID NO. 15 and 16). These antibodies can be used for the qualitative, quantitative, tracing, enrichment, extraction, and purification of fusion proteins containing SDcatcher and GVopti peptides, and have significant potential application value.

[0018] The seventh aspect of this invention provides a method for preparing the murine monoclonal antibody described in the first to third aspects, comprising: designing the amino acid sequence of a humanized or other animal-derived antibody using computer-aided design; synthesizing the variable region genes of the heavy and light chains of a whole gene and splicing them with the constant region genes of the heavy and light chains of a human or other animal-derived immunoglobulin, respectively, cloning them into an expression vector, constructing light and heavy chain expression vectors of the humanized or other animal-derived antibody, and then co-transfecting the light and heavy chain expression vectors into a eukaryotic cell expression system using liposomes, followed by screening, culture, and purification to obtain the antibody.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] This invention discloses a mouse monoclonal antibody against SDcatcher and GVopti, comprising an anti-SDcatcher mouse monoclonal antibody and an anti-GVopti mouse monoclonal antibody, both composed of a heavy chain and a light chain. The anti-SDcatcher monoclonal antibody is SD-Mab-1 (as shown in SEQ ID NO. 9 and 10) and SD-Mab-2 (as shown in SEQ ID NO. 11 and 12), and the anti-GVopti antibody is GV-Mab-1 (as shown in SEQ ID NO. 13 and 14) and GV-Mab-2 (as shown in SEQ ID NO. 15 and 16). The first anti-SDcatcher monoclonal antibody (SD-Mab-1) has the following amino acid sequences in its heavy chain hypervariable region: CDR1: GFTFTSYT, CDR2: ISNGGSTT, and CDR3: ARHNSSYFDY; and the light chain hypervariable region amino acid sequences: CDR1: ENIYSY, CDR2: NAK, and CDR3: QHHYGNPPT. The second anti-SDcatcher monoclonal antibody (SD-Mab-2) has the following amino acid sequences in its heavy chain hypervariable region: CDR1: GYTFTTAG, CDR2: IKTHSGVT, and CDR3: ARSGPLNWYYPMDY; and the light chain hypervariable region amino acid sequences: CDR1: QNVGTN, CDR2: SAS, and CDR3: QQYNSYPLT. The first anti-GVopti antibody (GV-Mab-1) has the following amino acid sequences in its heavy chain hypervariable region: CDR1: GFTFSGYI, CDR2: ISSGGSYT, and CDR3: LVADLDFDV; and the light chain hypervariable region amino acid sequences: CDR1: QNVGTA, CDR2: SAS, and CDR3: QQYSSYPLT. The second anti-GVopti antibody (GV-Mab-2) has the following amino acid sequences in its heavy chain hypervariable region: CDR1: GFTFSSYT, CDR2: INSGVTYT, and CDR3: LVADLDFD; and the light chain hypervariable region amino acid sequences: CDR1: QNVGTA, CDR2: SAS, and CDR3: QQYSSYPLT. The antibodies disclosed in this invention can be used for the qualitative, quantitative, and tracing detection of fusion proteins containing SDcatcher and GVopti peptides. They can also be used for the enrichment, extraction, and purification of fusion proteins containing SDcatcher and GVopti peptides, demonstrating significant potential application value. Attached Figure Description

[0021] Figure 1 Gel electrophoresis-Coomas Brilliant Blue staining images of purified murine antibodies (SD-mab-1 / 2) expressed on SDcatcher hybridoma cells and GV-mab-1 / 2 expressed on GVopti hybridoma cells.

[0022] Figure 2 The results of Western blot analysis of multiple fusion proteins containing the SDcatcher tag [2.75, XBB.1.5, BA.5, BQ.1.1, Delta, CH.1.1 represent fusion proteins containing RBD protein dimers of various SARS-CoV-2 strains, respectively, and the positive control is the SARS-CoV-2 2.75-XBB.1.5 RBD protein dimer-SD fusion protein (i.e., SD-2.75-XBB.1.5)] using the murine antibody (SD-mab-1 / 2) expressed in anti-SDcatcher hybridoma cells.

[0023] Figure 3 Western blot analysis of the murine antibody (GV-mab-1 / 2) expressed in GVopti hybridoma cells for the detection of the GVopti-tagged fusion protein (GV-Delta-RBD);

[0024] Figure 4 The results of ELISA detection of the SDcatcher-tagged fusion protein (SD-Ferritin) expressed by a murine monoclonal antibody (SD-Mab-1) against SDcatcher hybridoma cells were obtained.

[0025] Figure 5 The results of ELISA detection of the SDcatcher-tagged fusion protein (SD-Ferritin) expressed by the murine monoclonal antibody (SD-Mab-2) against SDcatcher hybridoma cells were obtained.

[0026] Figure 6 The results of ELISA detection of the mouse monoclonal antibody (GV-Mab-1) expressed in GVopti hybridoma cells were obtained.

[0027] Figure 7 The results of ELISA detection of the mouse monoclonal antibody (GV-Mab-2) expressed in GVopti hybridoma cells were obtained.

[0028] Figure 8 This study aims to detect the ELISA results of a fusion protein (RBD-Ferritin) containing SDcatcher and GVopti tags using SDcatcher peptide antibody (SD-Mab-1) as the plate and HPR-conjugated anti-GVopti peptide antibody (GV-Mab-1) as the detection indicator. Detailed Implementation

[0029] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0031] Example 1: Obtaining anti-SDcatcher and GVopti mouse monoclonal antibodies

[0032] 1. Immunize animals with SDcatcher peptide and GVopti peptide respectively to induce the animals to produce polyclonal antibody B cells against SDcatcher peptide and GVopti peptide respectively.

[0033] (1) Synthesize SDcatcher peptide and GVopti peptide

[0034] Guangzhou Alexandra Biotechnology Co., Ltd. was commissioned to synthesize SDcatcher peptide (amino acid sequence: SGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYL MPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI) and GVopti peptide (amino acid sequence: KVGNTIVMVDKLKEVPTP).

[0035] (2) Immunization of Balb / c mice and serum titer detection

[0036] ① The SDcatcher peptide was diluted to 500 μg / mL as the antigen. For the first immunization, Freund's complete adjuvant was used, with a volume ratio of 1:1 between the antigen (peptide) and the adjuvant (Freund's complete adjuvant). Before immunization, the antigen and adjuvant only needed to be gently mixed (Note: Place the antigen and adjuvant on ice before immunization). Immunization was performed via subcutaneous injection at multiple sites in the abdomen, immunizing five 6-8 week old female BALB / c mice four times at 14-day intervals (four sites were selected for each immunization, immunization times: day 0, day 14, day 28, and day 42). The first immunization used Freund's complete adjuvant, injecting 50 μg of antigen; two weeks later, three consecutive booster immunizations were administered (each two-week interval), using Freund's incomplete adjuvant, injecting 30 μg at each site.

[0037] ② The GVopti peptide was diluted to 500 μg / mL as the antigen. For the first immunization, Freund's complete adjuvant was used, with a volume ratio of 1:1 between the antigen (peptide) and the adjuvant (Freund's complete adjuvant). Before immunization, the antigen and adjuvant only needed to be gently mixed (Note: the antigen and adjuvant should be placed on ice before immunization). Immunization was performed by subcutaneous injection at multiple points in the abdomen, with 50 μg injected at each point. After a two-week interval, three consecutive booster immunizations were performed (each two weeks apart): during this stage, Freund's incomplete adjuvant was used, with 30 μg injected at each point.

[0038] ③ Blood collection for titer: At week 8, blood was collected from the orbital cavity of mice. After standing for a period of time to allow the serum to separate, mouse serum was obtained by centrifugation at 4°C and 2800 rpm for 15 minutes. The antibody titer was then detected by ELISA.

[0039] Dilute the SDcatcher peptide and GVopti peptide antigens separately with ELISA coating buffer (manufacturer: Solarbio; catalog number C1050-100 mL), and coat each well with 1 μg of antigen. Add 50 μL of the antigen to each well of the ELISA plate and incubate overnight at 4°C or for 2 hours at 37°C. Then discard the liquid in the wells, wash three times with 200 μL of PBST, and blot dry. Add 200 μL of blocking buffer (5% skim milk powder + PBS) to each well and incubate overnight at 4°C or for 2 hours at 37°C.

[0040] Wash 3 times with 200 μL PBST, and store the coated plate at -20℃ or 4℃ for later use; add 50 μL of primary antibody (serum of the mouse to be tested) to each well, and set up negative control wells (serum of unimmunized mice); incubate at 37℃ for 1 h, then wash 5 times with 200 μL washing buffer and pat dry.

[0041] Add enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP; manufacturer: Invitrogen) at 50 μL per well, incubate at 37℃ for 1 h, wash 5 times with 200 μL of washing buffer, and pat dry; add substrate solution TMB at 50 μL, and develop color at 37℃ for 2–15 min; terminate the reaction with stop solution (manufacturer: Solarbio, catalog number: C1058-500 mL), and read the OD value at 450 nm using an ELISA reader; observe the results, cell fusion can only be performed when the antibody titer (OD value) reaches 10 to the power of 3 or higher.

[0042] ④ Sprint to immunize

[0043] In week 10, a sprint immunization is performed. At this time, no adjuvant is added and only the antigen is used for intraperitoneal immunization. 50μg is injected at each site. Cell fusion can be carried out 3 days after the sprint immunization.

[0044] 2. Splenic cells containing anti-SDcatcher peptide and anti-GVopti peptide B cells were fused with SP20 myeloma cells to obtain two hybridoma cell pools, and monoclonal hybridoma cell lines were screened.

[0045] (1) Obtaining hybridoma cells

[0046] ① After the initial immunization, remove one eyeball to expel blood, then euthanize the mouse by spinal dislocation and place it in 70% alcohol. Use forceps to grasp the skin on the inner side of the groin of the mouse's left hind limb, and make horizontal cuts with scissors, followed by vertical cuts, or tear it open by hand to expose the mesentery. If lymph nodes are present in the groin area, remove them and immerse them in 10 mL of RMPI 1640 medium (in a 9 cm culture dish). Then carefully remove the spleen, immerse it in the above medium, carefully remove excess white fat or connective tissue, and immerse it again in fresh medium. Shake the spleen for 1-2 minutes to obtain immune cells.

[0047] ② Transfer the washed spleen and lymph nodes into a cell strainer (the strainer should be pre-soaked in 10-20 mL of fresh RMPI 1640 medium). Use a syringe plunger to grind the spleen within the strainer, allowing individual spleen cells to pass through a 200-mesh sterile filter. Then transfer the culture medium (containing cells) to a clean centrifuge tube. Slowly add 10-20 mL of culture medium to the cell strainer to rinse, then transfer the filtered cells back into the centrifuge tube. Gently pipette the liquid in the centrifuge tube to mix it thoroughly. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant. Add 40 mL of culture medium to resuspend the cells, centrifuge again, discard the supernatant, and wash the resulting immune cells three times with GKN buffer.

[0048] ③ Add 5 mL of erythrocyte lysis buffer (Sigma, R7757-100 mL) to the immune cells in the tube and resuspend them. Gently pipette for 1-2 min. Add 40 mL of culture medium to stop the lysis reaction. Centrifuge at 1000 rpm for 5 min and discard the supernatant. Add 10 mL of culture medium to resuspend the cells.

[0049] ④ Transfer 10 μL of cell suspension into a clean EP tube and dilute with 90 μL of culture medium. Transfer 10 μL of this diluted solution to a new EP tube and add 10 μL of 4% trypan blue. Count the cells under an inverted microscope and calculate the cell viability (viability = 100% - stained cells / total cell count × 100%).

[0050] ⑤ The remaining cells from step ③ are brought to a final volume of 30 mL with culture medium, centrifuged, and the supernatant discarded (preheat the PEG solution (Sigma 1450) at 40°C in a water bath at this time); then the cells are resuspended in 10 mL of culture medium, and Sp2 / 0 is added at a volume ratio of Sp2 / 0 tumor cells (manufacturer: Pronosai; catalog number: CL-0445): cell suspension = 1:3 (Sp2 / 0 is collected in advance, washed, and resuspended in culture medium at a density of approximately 1 × 10⁻⁶). 7 The Sp2 / 0 to spleen cell ratio was maintained between 1:9 and 1:4. The volume was then adjusted to 20 mL with culture medium, centrifuged, and the supernatant was discarded. 40°C distilled water was added to a beaker, and the centrifuge tube containing the cells was placed in this beaker for approximately 2 minutes in a water bath. Then, over 60 seconds, 1 mL of preheated PEG was slowly added dropwise to the tube while mixing continuously, maintaining the water bath. The cell suspension was then aspirated and slowly added back into the tube, maintaining the water bath. Over 60 seconds, 1 mL of culture medium was slowly added while mixing continuously, maintaining the water bath. 10 mL of culture medium was slowly added, mixing continuously, and the volume was adjusted to 20 mL, mixed, and incubated for 10 minutes. After the water temperature was slowly lowered to room temperature, the mixture was centrifuged, and the supernatant was discarded; the hybridoma cell mixture was obtained.

[0051] (2) Screening of monoclonal hybridoma cells

[0052] ① Monoclonalization: Take 1×10 4 The hybridoma cell mixture was resuspended in complete culture medium (HAT containing 20% ​​FBS). Using the extreme dilution method, single hybridoma cells were seeded into single 96-well cell culture wells (preparing the hybridoma cell suspension to be cloned, diluting it with HAT medium containing 20% ​​serum to a dilution of 5 cells per milliliter, mixing the cells, and seeding the cell culture medium at a volume of 100 μL per well. After seeding, the cells were examined under a microscope 30 min later, and the wells with single cloned cells were labeled).

[0053] ② Use complete culture medium (HAT containing 20% ​​FBS) for single-clone culture: place in a 37℃, 5% CO2 incubator for 7 days.

[0054] ③ Detection: Harvest 50 μL of supernatant from each well of monoclonal cells, set up two replicates, and detect the levels of anti-SDcatcher antibody and anti-GVopti antibody in the supernatant by ELISA. Mark the wells with the best expression of anti-SDcatcher antibody and anti-GVopti antibody and normal cell morphology.

[0055] ④ Transfer the culture medium from the 5 wells with the highest positive values ​​(maximum OD450 detection value) to about 1 mL of complete culture medium (RPMI containing 20% ​​FBS) and incubate in a 24-well plate. Expand the culture after 2-3 days.

[0056] ⑤ Expanded culture (12-well plate): Transfer cells to a suitable cell culture plate with complete culture medium (RPMI containing 20% ​​FBS) and incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0057] ⑥ Counting: When the density reaches 2-3×10 6 When the density is <2×10⁶ / mL, perform another expansion culture; if the density is <2×10⁶ / mL... 6 If the count is / mL, continue culturing for 24 hours and then count again.

[0058] ⑦ Expand the culture again (6-well plate): Adjust the cell density to 1×10⁶ cells / well using complete culture medium (RPMI containing 20% ​​FBS). 6 / mL, resuspend the cells and transfer them to a suitable cell culture plate, and incubate at 37℃, 5% CO2 for 48h.

[0059] ⑧ Counting: When the density reaches 2-3×10 6 When the density is <2×10⁶ / mL, perform another expansion culture; if the density is <2×10⁶ / mL... 6 If the count is / mL, continue culturing for 24 hours and then count again.

[0060] ⑨ Further expansion culture (T25 culture flask): Adjust the cell density to 1×10⁹ cells using complete culture medium (RPMI containing 20% ​​FBS). 6 / mL, resuspend the cells in a T25 culture flask and incubate at 37℃ in a 5% CO2 incubator for 48h.

[0061] ⑩ Counting: Before sampling, the cells should be resuspended evenly. The amount of cell suspension taken should be controlled to 1-2 drops. When the cell density is >2×10⁻⁶, the counting should be performed. 6 / mL indicates the next step is to expand the culture.

[0062] Further expansion culture (T75 flasks): Adjust cell density to 1×10⁶ cells / year using complete culture medium (RPMI containing 20% ​​FBS). 6 / mL, resuspend the cells in a T75 culture flask, and incubate at 37℃ in a 5% CO2 incubator for 48h.

[0063] Counting: When the density reaches 2-3×10 6 / mL, harvest the culture medium for potency testing; if density <2×10 6 If the density reaches 2-3 × 10⁹ / mL, continue culturing for another 24 hours and count again until the density reaches 2-3 × 10⁹ / mL. 6 / mL, the culture medium was harvested for potency testing.

[0064] (3) Titer detection of hybridoma cells

[0065] The fused hybridoma cells were screened by ELISA to identify positive cell lines. The titer detection steps for anti-SDcatcher mouse monoclonal hybridoma cells are as follows:

[0066] ① Dilute SD protein with ELISA coating buffer (manufacturer: Solarbio; catalog number C1050-100 mL) to a final concentration of 0.1 μg / mL and 1 μg / mL, 100 μL / well, overnight at 4°C;

[0067] ② Discard the supernatant, wash 3 times with PBST; block with 5% skim milk blocking buffer, 200 μL / well, incubate at 37°C for 2 hours;

[0068] ③ Discard the supernatant, then wash 3 times with washing solution, and add primary antibody (i.e., cell culture supernatant harvested from the screening of monoclonal hybridoma cells in step (2), negative control (HAT medium containing 20% ​​FBS), and blank control (PBS) respectively, all at 100 μL / well, and incubate at 37°C for 1 hour;

[0069] ④ Discard the supernatant, wash 3 times with washing buffer, add secondary antibody (Rabbit Anti-Mouse IgG F(ab)2 / HRP-0.09μg / mL), 100μL / well, and incubate at 37℃ for 1 hour.

[0070] ⑤ Discard the supernatant, wash 3 times with washing solution, add 100μL / well of TMB colorimetric solution (manufacturer: Solarbio; product number: PR1200-100), and develop the color for about 5 minutes;

[0071] ⑥ Add 50 μL of stop solution (manufacturer: Solarbio, catalog number: C1058-500mL) to each well to stop the reaction. Measure the OD450 value at 450 nm. Blank is used as a blank control. Record and save the data. The titer is the dilution corresponding to the minimum OD450 reading that is greater than the maximum OD / 2. Finally, the two monoclonal hybridoma cell lines with the highest positive values ​​(SD-mab-1 / 2) were screened and obtained, as shown in Table 1.

[0072] In addition, the titer assay for anti-GVopti mouse monoclonal hybridoma cells was performed using the same procedure as for anti-SDcatcher mouse monoclonal hybridoma cells, except that the GV protein was diluted with coating buffer to a final concentration of 0.1 μg / mL and 1 μg / mL. Two anti-GVopti monoclonal hybridoma cell lines (GV-mab-1 / 2) with the highest positive values ​​were finally obtained, as shown in Table 2.

[0073] Table 1. Antibody levels in each positive well of the anti-SDcatcher monoclonal hybridoma cell screening plate.

[0074] Blank 0.06 0.06 0.06 / 0.06 0.07 0.07 / SD-Mab-1 4.37 4.33 4.35 4.29 4.27 4.11 4.19 4.12 SD-Mab-2 4.06 4.18 4.12 4.06 4.42 4.32 4.37 4.3 SD-Mab-3 3.3 3.15 3.22 3.17 3.12 4 3.06 3.01 SD-Mab-4 3.28 3.41 3.35 3.3 3.42 3.55 3.48 3.43 SD-Mab-5 3.22 3.12 3.17 3.12 3.14 3.99 3.06 3.01 SD-Mab-6 3.05 3.18 3.11 3.06 3.25 3.39 3.32 3.27

[0075] Table 2 Antibody levels in each positive well of the anti-GVopti monoclonal hybridoma cell screening plate

[0076]

[0077]

[0078] (4) Cryopreservation of monoclonal hybridoma cells

[0079] ① Collect each hybridoma into a 50mL centrifuge tube and centrifuge at room temperature (18-26℃, speed: 500g, ascending speed: 5g, descending speed: 5g) for 5min;

[0080] ② Discard the supernatant, add 30mL of sodium chloride injection to resuspend the cells for cell counting, and centrifuge at room temperature (18-26℃, speed: 500g, ascending speed: 5g, descending speed: 5g) for 5min;

[0081] ③ Discard the supernatant, and press 1×10 6 Add serum-free cell cryopreservation solution (manufacturer: Beyotime, catalog number: C0210B-200mL) at a density of cells / mL, and gently mix using a Pasteur pipette.

[0082] ④ Dispense 1 mL / vial into cell cryovials, place the cells in a gradient cooling box, freeze overnight at -80°C, and transfer the cell cryovials to liquid nitrogen for storage the next day.

[0083] (5) Determination of antibody sequences in monoclonal hybridoma cells

[0084] Guangzhou Alexandra Biotechnology Co., Ltd. was commissioned to determine the antibody sequences of the two anti-SDcatcher monoclonal hybridoma cells (SD-mab-1 / 2) and two anti-GVopti monoclonal hybridoma cells (GV-mab-1 / 2) with the highest positive values. The antibody sequence analysis software (VASE2) was used to further determine the CDR sequence, variable region, and constant region of the antibody.

[0085] In this embodiment, mice were first immunized multiple times with SDcatcher and GVopti peptides to induce the production of B cells containing polyclonal antibodies against SDcatcher and GVopti peptides, respectively. Three days after immunization, the mice were sacrificed, and spleen cells containing B cells containing both SDcatcher and GVopti peptides were collected. These cells were then fused with SP20 myeloma cells to obtain hybridoma cell pools containing both SDcatcher and GVopti peptides. Monoclonal hybridomas were obtained using a single-cell sorting method. After the hybridoma cells expanded to a certain number, the titers of anti-SDcatcher and anti-GVopti antibodies produced by the hybridoma monoclonal cells were measured. The antibody sequences of the two anti-SDcatcher monoclonal hybridoma cell lines (SD-mab-1 / 2) and two anti-SDcatcher monoclonal hybridoma cell lines (GV-mab-1 / 2) with the highest positive values ​​were determined.

[0086] The results showed that both anti-SDcatcher monoclonal hybridoma cells (SD-mab-1 / 2) and anti-GVopti monoclonal hybridoma cells (GV-mab-1 / 2) consist of two parts: a heavy chain and a light chain (linked by disulfide bonds). The heavy chain hypervariable region amino acid sequence of the first anti-SDcatcher monoclonal antibody (SD-Mab-1) is CDR1: GFTFTSYT, CDR2: ISNGGSTT, and CDR3: ARHNSSYFDY, while the light chain hypervariable region amino acid sequence is CDR1: ENIYSY, CDR2: NAK, and CDR3: QHHYGNPPT. The heavy chain hypervariable region amino acid sequence of the second anti-SDcatcher monoclonal antibody (SD-Mab-2) is CDR1: GYTFTTAG, CDR2: IKTHSGVT, and CDR3: ARSGPLNWYYPMDY, while the light chain hypervariable region amino acid sequence is CDR1: QNVGTN, CDR2: SAS, and CDR3: QQYNSYPLT. The amino acid sequence of the heavy chain variable region of SD-Mab-1 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of SD-Mab-2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4. The amino acid sequence of the heavy chain of SD-Mab-1 is shown in SEQ ID NO.9, and the amino acid sequence of the light chain is shown in SEQ ID NO.10; the amino acid sequence of the heavy chain of SD-Mab-2 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain is shown in SEQ ID NO.12. The nucleotide sequence encoding the heavy chain of SD-Mab-1 is shown in SEQ ID NO.17, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.18; the nucleotide sequence encoding the heavy chain of SD-Mab-2 is shown in SEQ ID NO.19, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.20.

[0087] The heavy chain hypervariable region amino acid sequence of the first anti-GVopti antibody (GV-Mab-1) is CDR1: GFTFSGYI, CDR2: ISSGGSYT and CDR3: LVADLDFDV, and the light chain hypervariable region amino acid sequence is CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT. The heavy chain hypervariable region amino acid sequence of the second anti-GVopti antibody (GV-Mab-2) is CDR1: GFTFSSYT, CDR2: INSGVTYT and CDR3: LVADLDFD, and the light chain hypervariable region amino acid sequence is CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT. The amino acid sequence of the heavy chain variable region of GV-Mab-1 is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region of GV-Mab-2 is shown in SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8. The amino acid sequence of the heavy chain of GV-Mab-1 is shown in SEQ ID NO. 13, and the amino acid sequence of the light chain is shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain of GV-Mab-2 is shown in SEQ ID NO. 15, and the amino acid sequence of the light chain is shown in SEQ ID NO. 16. The nucleotide sequence encoding the heavy chain of GV-Mab-1 is shown in SEQ ID NO. 21, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO. 22; the nucleotide sequence encoding the heavy chain of GV-Mab-2 is shown in SEQ ID NO. 23, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO. 24.

[0088] SEQ ID NO:1 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-1, heavy chain variable region amino acid sequence):

[0089] MNFGLSLVFLVLVLKGVLCEVKLVESGGGLVQPGGSLKLSCAAS GFTFSYT MSWVR QTPEKNLEWVAY ISNGGSTT YYPDTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYC AR HSNSYFDY WGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKG (The underlined part is the super-variable region, corresponding to CDR1:GFTFSGYI, CDR2:ISSGGSYT and CDR3:LVADLDFDV respectively).

[0090] SEQ ID NO:2 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-1, amino acid sequence of light chain variable region):

[0091] MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSSASVGETVTITCRAS BROTHER LAWYQQ KQGKSPQVLVY NAK TLVEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYC QHHYGNPPT FG AGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (The underlined area is the super-variable region, corresponding to CDR1: ENIYSY; CDR2: NAK and CDR3: QHHYGNPPT respectively).

[0092] SEQ ID NO:3 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-2, heavy chain variable region amino acid sequence):

[0093] MEWLWNLLFLLMAAAQSIQAQIQLVQSGPELKKPGETVRISCKAS GYTFTAG MQWVQKIPGKGLKWIGW ICTRST KYAEDFKGRFAFSLETSASSAYLQISNLKNEDTATYFC ARSGPLNWYYPMDY WGQGTSVTVSVAKTTPPSVYPLAPGSAAQTNSMVTLGC (The underlined part is the super-variable region, corresponding to CDR1: GYTFTTAG, CDR2: IKTHSGVT and CDR3: ARSGPLNWYYPMDY).

[0094] SEQ ID NO:4 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-2, amino acid sequence of light chain variable region):

[0095] MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVNVTCKAS QNVGTN VAWYQQKPGQSPKALIY SAS FRYSGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFC QQYNSYPLT FGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (The underlined area is the super-variable region, corresponding to CDR1: QNVGTN; CDR2: SAS and CDR3: QQYNSYPLT respectively).

[0096] SEQ ID NO:5 (Anti-GVopti mouse monoclonal antibody GV-Mab-1, heavy chain variable region amino acid sequence):

[0097] MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAAS GFTFSGYI MSWVRQNSEKRLEWVAT ISSGGSYT YYPDSVKGRFTISRDNAKNTLYLQMRSLKSEDTAMYYC LVADLDFDV WGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGY (The underlined area is the super-variable region, corresponding to CDR1:GFTFSGYI, CDR2:ISSGGSYT and CDR3:LVADLDFDV respectively).

[0098] SEQ ID NO:6 (Anti-GVopti mouse monoclonal antibody GV-Mab-1, amino acid sequence of light chain variable region):

[0099] MESHTQAFVFAFLWLSGVDGDIVMTQSQKFMSTSIGGRVSITCKAS QNVGTA VAWCQQKPGQSPKVLIH SAS NRYTGVPDRFTGSGSGTDFTLTISNIQSEDLADYFC QQYSSYPLT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (The underlined area is the super-variable region, corresponding to CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT respectively).

[0100] SEQ ID NO:7 (Anti-GVopti mouse monoclonal antibody GV-Mab-2, heavy chain variable region amino acid sequence):

[0101] MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAAS GFTFSSYT MSWVRQTPEKRLEWVAT INSTITUTION YYPDSVKGRFTISRDNAKNILYLQMSSLKSEDTAMYYC LVADLDFDV WGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGY (The underlined area is the super-variable region, corresponding to CDR1: GFTFSSYT, CDR2: INSGVTYT and CDR3: LVADLDFD respectively).

[0102] SEQ ID NO:8 (Anti-GVopti mouse monoclonal antibody GV-Mab-2, amino acid sequence of light chain variable region):

[0103] MESHTQAFVFAFLWLSGIDGDIVMTQSQKFMSTSVGDRVSITCKAS QNVGTA IAWCQQKPGQSPKVLIH SAS NRYTGVPDRFTGSGSGTDFTLTIRNVQSDDLADYFC QQYSSYPLT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (The underlined area is the super-variable region, corresponding to CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT respectively).

[0104] SEQ ID NO:9 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-1, heavy chain amino acid sequence):

[0105] MNFGLSLVFLVLVLKGVLCEVKLVESGGGLVQPGGSLKLSCAASGFTFTSYTMSWVRQTPEKNLEWVAYISNGGSTTYYPDTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYC ARHSNSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPI EKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0106] SEQ ID NO:10 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-1, light chain amino acid sequence):

[0107] MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQVLVYNAKTLVEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGNPPTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0108] SEQ ID NO: 11 (anti-SDcatcher murine monoclonal antibody SD-Mab-2, heavy chain amino acid sequence):

[0109] MEWLWNLLFLMAAAQSIQAQIQLVQSGPELKKPGETVRISCKASGYTFTTAGMQWVQKIPGKGLKWIGWIKTHSGVTKYAEDFKGRFAFSLETSASSAYLQISNLKNEDTATYFCARSGPLNWYYPMDYWGQGTSVTVSVAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0110] SEQ ID NO: 12 (anti-SDcatcher murine monoclonal antibody SD-Mab-2, light chain amino acid sequence):

[0111] MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVNVTCKASQNVGTNVAWYQQKPGQSPKALIYSASFRYSGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFCQQYNSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0112] SEQ ID NO: 13 (anti-GVopti murine monoclonal antibody GV-Mab-1, heavy chain amino acid sequence):

[0113] MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAASGFTFSGYIMSWVRQNSEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMRSLKSEDTAMYYCLVADLDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0114] SEQ ID NO: 14 (anti-GVopti murine monoclonal antibody GV-Mab-1, light chain amino acid sequence):

[0115] MESHTQAFVFAFLWLSGVDGDIVMTQSQKFMSTSIGGRVSITCKASQNVGTAVAWCQQKPGQSPKVLIHSASNRYTGVPDRFTGSGSGTDFTLTISNIQSEDLADYFCQQYSSYPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0116] SEQ ID NO: 15 (anti-GVopti murine monoclonal antibody GV-Mab-2, heavy chain amino acid sequence):

[0117] MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATINSGVTYTYYPDSVKGRFTISRDNAKNILYLQMSSLKSEDTAMYYCLVADLDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0118] SEQ ID NO: 16 (anti-GVopti murine monoclonal antibody GV-Mab-2, light chain amino acid sequence):

[0119] MESHTQAFVFAFLWLSGIDGDIVMTQSQKFMSTSVGDRVSITCKASQNVGTAIAWCQQKPGQSPKVLIHSASNRYTGVPDRFTGSGSGTDFTLTIRNVQSDDLADYFCQQYSSYPLT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0120] SEQ ID NO:17 (Anti-SDcatcher mouse monoclonal antibody SD-Mab-1, heavy chain nucleotide sequence):

[0121]

[0122] SEQ ID NO: 18 (light chain nucleotide sequence of anti-SDcatcher murine monoclonal antibody SD-Mab-1):

[0123] ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCGAGTGAAAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGGTCCTGGTCTATAATGCAAAAACCTTAGTAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTAATCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

[0124] SEQ ID NO: 19 (heavy chain nucleotide sequence of anti-SDcatcher murine monoclonal antibody SD-Mab-2):

[0125]

[0126] SEQ ID NO: 20 (anti-SDcatcher murine monoclonal antibody SD-Mab-2, light chain nucleotide sequence):

[0127] ATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAACGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTATTCGGCATCCTTCCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAACAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA.

[0128] SEQ ID NO: 21 (anti-GVopti murine monoclonal antibody GV-Mab-1, heavy chain nucleotide sequence):

[0129]

[0130] SEQ ID NO: 22 (light chain nucleotide sequence of murine anti-GVopti monoclonal antibody GV-Mab-1):

[0131] ATGGAGTCTCATACTCAGGCCTTTGTATTCGCGTTTCTTTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAATAGGAGGCAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAGCCTGGTGTCAACAGAAACCAGGACAATCTCCTAAAGTTCTGATTCATTCGGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATATACAGTCTGAAGACCTGGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCTCTGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA

[0132] SEQ ID NO: 23 (heavy chain nucleotide sequence of murine anti-GVopti monoclonal antibody GV-Mab-2):

[0133]

[0134] SEQ ID NO:24 (Anti-GVopti mouse monoclonal antibody GV-Mab-2, light chain nucleotide sequence):

[0135] .

[0136] Example 2: Expression and purification of target antibodies (anti-SDcatcher peptide antibody and anti-GVopti peptide antibody) using hybridoma cells containing anti-SDcatcher peptide and anti-GVopti peptide.

[0137] (1) Hybridoma cell resuscitation

[0138] ① First, place the hybridoma cells in RPMI complete medium containing 20% ​​fetal bovine serum, and then place the culture dish in a 5% CO2, 37℃ cell culture incubator. After the hybridoma cells are revived, complete one passage. During the culture process (3 days), count the cells every day.

[0139] ② Increase the cell density to 1×10 6 When the culture medium has reached approximately 100 cells / mL or has turned orange-yellow, transfer it to a 15mL centrifuge tube and centrifuge at 4℃ and 1500rpm for 5 minutes. Remove the supernatant from the centrifuge tube.

[0140] ③ After washing the cells with PBS solution, resuspend the cells in RPMI complete medium containing 20% ​​fetal bovine serum, and transfer the cell resuspending solution to T25 cell culture flasks.

[0141] ④ Adjust the volume to 120 mL with RPMI complete medium containing 20% ​​fetal bovine serum; place the T25 cell culture flask in a 5% CO2, 37℃ cell culture incubator for culture, and count the cells at intervals during the culture process (3 days);

[0142] (2) Scale-up culture of hybridoma cells

[0143] Hybridoma cells were transferred to RPMI complete medium containing 10% fetal bovine serum for expansion culture, as detailed below:

[0144] ① Wait until the cell density grows to 1×10 6 When the cell count is around 100 cells / mL or the culture medium turns orange-yellow, transfer 120 mL of culture medium from the T25 cell culture flask to 50 mL centrifuge tubes in multiple portions. Centrifuge at 4°C and 1500 rpm for 5 min, and remove the supernatant from the centrifuge tubes.

[0145] ② After washing the cells with PBS solution, resuspend the cells in RPMI complete medium containing 10% fetal bovine serum; and transfer the cell resuspending solution to T75 cell culture flasks.

[0146] ③ Adjust the volume to 540 mL with RPMI complete medium containing 10% fetal bovine serum; place the T75 cell culture flask in a 5% CO2, 37℃ cell culture incubator and culture. During the culture process (5 days), count the cells at intervals according to the cell expansion (based on the color change of the medium).

[0147] (3) Expression and purification of hybridoma monoclonal antibodies against SDcatcher peptide and GVopti peptide

[0148] When the cell density grows to 1×10 6When the cell density reaches approximately 10 cells / mL or the culture medium turns orange-yellow, transfer the culture medium from the T75 cell culture flask to 50mL centrifuge tubes in portions. Centrifuge at 1500rpm for 5 minutes at 4°C. Remove the supernatant from the cells, wash the cells with PBS, and resuspend the cells in a T75 cell culture flask containing 540mL of serum-free CD-hybridoma medium. Place the T75 cell culture flask in a 5% CO2, 37°C cell culture incubator for monoclonal antibody production. The antibody production cycle is 10 days. On day 11, collect the culture medium for the next step of antibody purification (using AKTA equipment from GE and a Mabselect Protein A column for affinity chromatography to purify the antibody, the same applies below).

[0149] (4) Antibody electrophoresis

[0150] Mix 5 volumes of purified antibody with 1 volume of SDS-PAGE loading buffer (manufacturer: Beijing Ruiboxingke Biotechnology Co., Ltd.; catalog number: RB005-001), boil for 5 minutes, centrifuge, and take the supernatant for loading. The loading sample is 10 μL and the protein marker is 5 μL.

[0151] Electrophoresis conditions: voltage 95V, current approximately 75mA, electrophoresis for 0.5 hours. After electrophoresis, remove the electrophoresis gel and place it in Super SDS-PAGE staining solution (manufacturer: Beijing Ruiboxingke Biotechnology Co., Ltd.; product number: RB010-500) for staining for 1 hour. Shake slowly on a shaker until staining is complete. Then, remove the gel and place it in dehydration solution (formulation: 40% ethanol, 10% acetic acid, 50% distilled water). Shake slowly on a shaker for 1-2 hours until destaining is complete. Observe the results after destaining.

[0152] Figure 1 The images show gel electrophoresis-Coomassie brilliant blue staining of the purified antibodies SD-mab-1, SD-mab-2, GV-mab-1, and GV-mab-2. All antibody sequences showed two bands after electrophoresis (the larger molecular weight band represents the antibody heavy chain, and the smaller molecular weight band represents the antibody light chain). This demonstrates that the method described in this embodiment can successfully extract and purify the antibodies SD-mab-1, SD-mab-2, GV-mab-1, and GV-mab-2.

[0153] Example 3: Qualitative detection of anti-SDcatcher peptide antibody and anti-GVopti peptide antibody

[0154] Western blot experiments were performed to detect the colorimetric reactions of anti-SDcatcher peptide antibodies and fusion proteins containing SDcatcher peptides, as well as anti-GVopti peptide antibodies and fusion proteins containing GVopti peptides. Details are as follows:

[0155] 1. Western blot detection of fusion proteins containing the SDcatcher tag

[0156] ① Sample dilution: Multiple fusion proteins expressing various SDcatcher tags [fusion proteins containing RBD protein dimers of various SARS-CoV-2 strains (2.75, XBB.1.5, BA.5, BQ.1.1, Delta, CH.1.1), the team previously constructed expression vectors independently, see invention patent CN116375884A for details] were used in this study. First, cryopreserved CHO cells expressing various proteins were thawed in a water bath at room temperature (18-26℃), centrifuged at 500g (5g increment, 5g decrement) for 5 minutes, and the cryopreservation solution was removed. The cells were resuspended in 1mL of S4 CHO medium using a Pasteur pipette, and the cell suspension was transferred to a 125mL Erlenmeyer flask, supplemented with S4 CHO medium [containing 1% GlutaMAX]. TM [I(100×)] to 10 mL, then incubate in a 125 mL Erlenmeyer flask at 37°C, 8% CO2, 100 rpm on a large-capacity stacked thermostatic shaker for 2-3 days, and harvest the culture supernatant. Dilute the culture supernatant to 100 ng / mL with PBS, and simultaneously introduce a positive control (purified high-purity SD-2.75-XBB.1.5 protein, the SARS-CoV-2 2.75 and XBB.1.5 RBD protein dimer-SD fusion protein, an expression vector independently constructed by the team in previous research), and a negative control (GVO-XBB.1.5, without the SDcatcher tag, an expression vector independently constructed by the team in previous research). The control supernatant was obtained in the same way as the test sample supernatant.

[0157] ② Mix 5 volumes of purified antibody (SD-Mab-1 / 2, 10-200 μg / mL) and 1 volume of 5×SDS-PAGE loading buffer (manufacturer: Beijing Ruiboxingke Biotechnology Co., Ltd.; catalog number: RB005-001), boil for 5 minutes, centrifuge, and take the supernatant for loading. The loading sample is 10 μL and the protein marker is 5 μL.

[0158] ③ Electrophoresis conditions: Voltage 95V, current about 75mA, electrophoresis for 0.5-1 hour. When the bromophenol blue reaches the bottom of the separating gel, stop the electrophoresis and turn off the power.

[0159] ④ Protein Transfer: After electrophoresis, cut an NC membrane close to the desired gel size. Simultaneously, take a pair of thick filter papers and a pair of thin sponges and immerse them in 1× transfer buffer, ensuring they are fully soaked. Place a layer of sponge and a layer of filter paper on each side of the transfer clamp. Then, place the NC membrane near the white clamp side and the gel near the black clamp side, removing any air bubbles between the NC membrane and filter paper to create a "white side-sponge-filter paper-NC membrane-gel-filter paper-sponge-black side" sandwich. Clamp the sandwich tightly and place it in the electrophoresis tank. Turn on the electrophoresis apparatus, place it on ice, and transfer at a constant current of 200mA for 100 minutes. After the transfer is complete, turn off the power.

[0160] ⑤ Blocking: After the transfer is complete, prepare a TBS solution containing 5% skim milk powder. Remove the membrane from the electrophoresis tank, place it in the TBS solution containing 5% skim milk powder for blocking, and place it on a horizontal shaker at room temperature for 1 hour.

[0161] ⑥ Incubation with primary antibody (SD-Mab-1): After blocking, prepare the primary antibody incubation solution (i.e., the purified antibody harvested and purified in Example 2), discard the blocking solution, add the primary antibody incubation solution (SD-Mab-1, diluted 1:3000), and slowly shake on a horizontal shaker at room temperature for 1 hour. After completion, collect the primary antibody and wash the membrane 3-4 times with 1×TBST solution, 8 minutes each time.

[0162] ⑦ Incubation with secondary antibody: After washing the membrane, remove the TBST washing solution and add the antibody (FITC anti-mouse antibody, manufacturer: Biolegend, catalog number: 16059) incubation solution mixed with fluorescent secondary antibody. Keep at room temperature and protected from light, and shake slowly on a horizontal shaker for 1 hour.

[0163] ⑧ Colorimetric Development: After the process, collect the secondary antibody and wash the membrane four times with 1×TBST solution, 8 min each time. After washing, scan the membrane using a two-color infrared laser imaging system (Li-COR Odyssey) and analyze the results.

[0164] The SD-Mab-2 primary antibody was tested using the same experimental methods, and the anti-SD rabbit polyclonal antibody (prepared by the team through previous self-immunization of rabbits) was introduced as a control primary antibody, and the above experiment was repeated.

[0165] The preparation process of the rabbit polyclonal antibody is as follows: SDcatcher peptides are diluted to 500 μg / mL as the antigen. For the first immunization, Freund's complete adjuvant is used, with a volume ratio of 1:1 between the antigen (peptide) and the adjuvant (Freund's complete adjuvant). Before immunization, the antigen and adjuvant only need to be gently mixed (note: place the antigen and adjuvant on ice before immunization). Immunization is performed via subcutaneous injection at multiple sites in the abdomen, with two rabbits immunized four times at 14-day intervals (four sites selected subcutaneously for each immunization, on days 0, 14, 28, and 42). The first immunization uses Freund's complete adjuvant, injecting 50 μg of antigen; two weeks later, three consecutive booster immunizations (each two weeks apart) are administered, using Freund's incomplete adjuvant, injecting 30 μg at each site. At week 8, blood was collected from the orbital cavity of mice. After the serum was allowed to stand for a period of time to separate, it was centrifuged at 4°C and 2800 rpm for 15 minutes to obtain mouse serum. The serum was then purified using SD peptides to obtain anti-SD rabbit polyclonal antibody rabbit serum. Figure 2 The results of incubation and color development using SD-mab-1 antibody (left), SD-mab-2 antibody (middle), and SD-Pab rabbit polyclonal antibody (right, test control) as primary antibodies are presented. The results show that using SD-mab-1 and SD-mab-2 antibodies as primary antibodies significantly improves the color development of various proteins containing the SDcatcher tag compared to using SD-Pab rabbit polyclonal antibody. Furthermore, in the color development results for the negative control without the SDcatcher tag, SD-Pab rabbit polyclonal antibody as the primary antibody exhibits non-specific color bands, while SD-mab-1 and SD-mab-2 antibodies show better color development specificity. This confirms the feasibility of using SD-mab-1 and SD-mab-2 antibodies for the qualitative detection of fusion proteins containing the SDcatcher tag.

[0166] 2. Western blot detection of fusion proteins containing the GVopti tag

[0167] ① Test sample dilution: The fusion protein expressing the GVopti tag [GV-Delta-RBD, which was expressed and purified by the team in previous studies using a vector independently constructed, i.e., the Gv-B.1.617.2_RBD protein described in the literature (DOI:10.1002 / advs.202105378)] was diluted with PBS.

[0168] ② Thaw the cryopreserved CHO cells expressing this protein in a water bath at room temperature (18-26℃), centrifuge at 500g (increase rate: 5g, decrease rate: 5℃) for 5 minutes, and remove the cryopreservation solution. Resuspend the cells in 1mL of S4 CHO medium using a Pasteur pipette, transfer the cell suspension to a 125mL Erlenmeyer flask, and replenish with S4 CHO medium (containing 1% GlutaMAX). TM -I(100×)] to 10mL; place in a 125mL Erlenmeyer flask and incubate at 37℃, 8% CO2, 100rpm on a large-capacity stacked constant temperature shaker for 2-3 days. Harvest the supernatant of the culture medium and dilute the supernatant to 100ng / mL.

[0169] ③ Mix 5 volumes of purified antibody (GV-Mab-1 / 2, 10-200 μg / mL) and 1 volume of 5×SDS-PAGE loading buffer (manufacturer: Beijing Ruiboxingke Biotechnology Co., Ltd.; catalog number: RB005-001), boil for 5 minutes, centrifuge, and take the supernatant for loading. The loading sample is 10 μL and the protein marker is 5 μL.

[0170] ④ Electrophoresis conditions: Voltage 95V, current approximately 75mA, electrophoresis for 0.5-1 hour. When bromophenol blue reaches the bottom of the separating gel, stop the electrophoresis and turn off the power.

[0171] ③ Protein Transfer: After electrophoresis, cut an NC membrane close to the desired gel size. Simultaneously, take a pair of thick filter papers and a pair of thin sponges and immerse them in 1× transfer buffer, ensuring they are fully soaked. Place a layer of sponge and a layer of filter paper on each side of the transfer clamp. Then, place the NC membrane near the white clamp side and the gel near the black clamp side, removing any air bubbles between the NC membrane and filter paper to create a "white side-sponge-filter paper-NC membrane-gel-filter paper-sponge-black side" sandwich. Clamp the sandwich tightly and place it in the electrophoresis tank. Turn on the electrophoresis apparatus and place it on ice. Transfer at a constant current of 200mA for 100 minutes. After the transfer is complete, turn off the power.

[0172] ④ Blocking: After the transfer is complete, prepare a TBS solution containing 5% skim milk powder. Remove the membrane from the electrophoresis tank and place it in the TBS solution containing 5% skim milk powder for blocking. Place the membrane on a horizontal shaker and shake it slowly at room temperature for 1 hour.

[0173] ⑤ Incubation with primary antibody (GV-Mab-1): After blocking, prepare the primary antibody incubation solution (i.e., the purified antibody harvested and purified in Example 2), discard the blocking solution, add the primary antibody incubation solution (GV-Mab-1, diluted 1:3000), and slowly shake on a horizontal shaker at room temperature for 1 hour. After completion, collect the primary antibody and wash the membrane 3-4 times with 1×TBST solution, 8 minutes each time.

[0174] ⑥ Incubation with secondary antibody: After washing the membrane, remove the TBST washing solution and add the antibody (anti-mouse antibody) incubation solution mixed with fluorescent secondary antibody (FITC anti-mouse antibody, manufacturer: Biolegend, catalog number: 16059). Incubate at room temperature in the dark and shake slowly on a horizontal shaker for 1 hour.

[0175] ⑦ Colorimetric Development: After the washing process, collect the secondary antibody and wash the membrane four times with 1×TBST solution, 8 min each time. After washing, scan the membrane using a two-color infrared laser imaging system (Li-COR Odyssey) and analyze the results.

[0176] The GV-Mab-2 primary antibody was tested using the same experimental methods, and the above experiment was repeated.

[0177] The results are as follows Figure 3 As shown, the results of incubation with GV-mab-1 antibody (left) and GV-mab-2 antibody (right) as primary antibodies, followed by color development and analysis, are presented. The results demonstrate that using GV-mab-1 and GV-mab-2 antibodies as primary antibodies can specifically detect GV-Delta-RBD proteins containing the GVopti tag, confirming the feasibility of using GV-mab-1 and GV-mab-2 antibodies for the qualitative detection of GVopti-tagged fusion proteins.

[0178] As can be seen, in this embodiment, anti-SDcatcher murine monoclonal antibody and anti-GVopti murine monoclonal antibody were used as binding antibodies for Western blot experiments. The results showed that anti-SDcatcher murine monoclonal antibody could bind well to multiple fusion proteins containing the SDcatcher tag [fusion proteins containing RBD protein dimers of various SARS-CoV-2 strains (2.75, XBB.1.5, BA.5, BQ.1.1, Delta, CH.1.1), with the positive control being the SARS-CoV-2 2.75-XBB.1.5 RBD protein dimer-SD fusion protein (i.e., SD-2.75-XBB.1.5)]. Anti-GVopti murine monoclonal antibody could bind well to the fusion protein containing the GVopti tag (GV-Delta-RBD), and accurate and specific indicator bands for each fusion protein appeared after color development.

[0179] Example 4: Quantitative detection of anti-SDcatcher peptide antibody and anti-GVopti peptide antibody

[0180] Western blot experiments were used to quantify the anti-SDcatcher peptide antibody and the target protein containing the SDcatcher peptide, and to quantify the fusion protein of the anti-GVopti peptide antibody and the GVopti peptide.

[0181] 1. Detection of SD-Ferritin content in fusion proteins containing the SDcatcher tag

[0182] A one-step sandwich enzyme-linked immunosorbent assay (ELISA) using double antibodies was employed. The sample was pre-coated with anti-SDcatcher peptide antibodies (SD-mab-1 / 2). Different concentrations of the fusion protein (SD-Ferritin) and HRP-labeled anti-mouse SD-IgG antibodies (SD-mab-1 and SD-mab-2 were provided by our company, while HRP labeling services were provided by Beijing Yiqiao Shenzhou Technology Co., Ltd.) were added sequentially. After incubation and thorough washing, the sample was developed with the substrate TMB. TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The color intensity was positively correlated with the SD-Ferritin protein content in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the concentrations of the sample and reference were calculated to determine the antigen content. The reference dilutions were as follows: the SD-Ferritin fusion protein containing the SDcatcher tag was diluted with PBS to 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 ng / mL. The specific method is as follows:

[0183] ① Sample incubation: Each reference sample (SD-Ferritin protein, a fusion protein containing the SDcatcher tag, which was independently constructed and purified by the team in previous research, see DOI:10.1016 / j.celrep.2021.110256) was spotted at a rate of 100 μL / well, with two replicates for each sample. Two blank control samples (PBS dilution) were also added. The samples were incubated at 37°C for 1.5 h.

[0184] ② Washing the plate: Take out the high-adsorption 96-well plate, discard the liquid, add 300μL of wash buffer (PBS-T) to each well, gently tap and shake to remove the washing buffer, repeat this washing process 3 times, and pat dry on absorbent paper for the last wash;

[0185] ③ Antibody incubation: Add 100 μL of HRP-labeled anti-mouse SD-IgG antibody diluted with PBS (1:3000 dilution) to each well and incubate at 37°C for 1 h;

[0186] ④ Washing the plate: Take out the high-adsorption 96-well plate, discard the liquid, add 300μL of wash buffer (PBS-T) to each well, gently tap and shake to remove the washing solution, repeat this washing process 3 times, and pat dry on absorbent paper for the last wash.

[0187] ⑤ Color development: Add 50 μL of TMB color development substrate (manufacturer: Invitrogen; product number: 00-4201-56) to each well and incubate at room temperature in the dark for 15 min;

[0188] ⑥ Termination and reading: Add 50 μL of stop solution (manufacturer: Solarbio Life Sciences, catalog number: C1058) to each well and measure the OD value of each well at a wavelength of 450 nm.

[0189] ⑦ Calculation: In the Excel worksheet, use linear regression analysis, plotting a standard curve with the concentration of the reference sample on the X-axis and its OD value on the Y-axis, to obtain the linear equation: Y = bX + a, and obtain a, b, and R. 2 Value. Where b is the slope, a is the y-intercept, and R is the value. 2 The determination coefficient (R) 2 R refers to the goodness of fit, which is how well the regression line fits the observed values. 2 The closer the value is to 1, the better the fit. The linear equation in this experiment and R0... 2 (Generated from an Excel formula).

[0190] Figure 4 , Figure 5 The results of enzyme-linked immunosorbent assays (ELISA) using 96-well high-adsorption flat-bottom plates pre-coated with SD-mab-1 and SD-mab-2 antibodies for the quantitative detection of SD-Ferritin protein at different concentrations are presented, along with linear analysis of the OD values ​​after color development of different concentrations of SD-Ferritin. The results show that the R² values ​​of the standard curves plotted for both antibody-prepared ELISA kits are >0.99, confirming the feasibility of using SD-mab-1 and SD-mab-2 antibodies for the quantitative detection of fusion proteins containing the SDcatcher tag.

[0191] 2. Detection of GV-Delta-RBD content in fusion proteins containing the GVopti tag

[0192] A one-step sandwich enzyme-linked immunosorbent assay (ELISA) using double antibodies was employed. The sample was pre-coated with anti-GVopti peptide antibodies, and then different concentrations of fusion protein and HRP-labeled anti-mouse GVopti-IgG antibodies (our company provided anti-mouse GVopti-IgG antibodies GV-mab-1 and GV-mab-2, while Beijing Yiqiao Shenzhou Technology Co., Ltd. provided HRP labeling services) were added sequentially. After incubation and thorough washing, the sample was developed with the substrate TMB. TMB was converted to blue under the catalysis of peroxidase, and then to the final yellow color under acidic conditions. The color intensity was positively correlated with the content of the original GV-Delta-RBD protein in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the concentrations of the sample and reference standard were calculated to determine the antigen content. Reference dilution: The GV-Delta-RBD fusion protein containing the GVopti tag was diluted with PBS to 2000, 1000, 500, 250, 125, 62.5, 31.25, and 0 ng / mL. The specific method is as follows:

[0193] ① Sample incubation: Each reference sample [the team independently constructed expression vector in previous research and expressed and purified the fusion protein GV-Delta-RBD containing the GVopti tag, namely the Gv-B.1.617.2_RBD protein described in the literature (DOI:10.1002 / advs.202105378)] was sampled at a rate of 100 μL / well, with two replicates for each sample. Two blank control samples (PBS dilution) were also added. The samples were incubated at 37°C for 1 h.

[0194] ② Washing the plate: Take out the high-adsorption 96-well plate, discard the liquid, add 300μL of wash buffer (PBS-T) to each well, gently tap and shake to remove the washing buffer, repeat this washing process 3 times, and pat dry on absorbent paper for the last wash;

[0195] ③ Antibody incubation: Add 100 μL of HRP-labeled anti-mouse GVopti-IgG antibody (1:3000 dilution) diluted with PBS to each well, and incubate at 37°C for 1 h;

[0196] ④ Washing the plate: Take out the high-adsorption 96-well plate, discard the liquid, add 300μL Wash buffe (PBS-T)r to each well, gently tap and shake to remove the washing solution, repeat this washing process 3 times, and pat dry on absorbent paper for the last time;

[0197] ⑤ Color development: Add 50 μL of TMB color development substrate (manufacturer: Invitrogen; product number: 00-4201-56) to each well and incubate at room temperature in the dark for 12 min;

[0198] ⑥ Termination and reading: Add 50 μL of stop solution (manufacturer: Solarbio Life Sciences, catalog number: C1058) to each well and measure the OD value of each well at a wavelength of 450 nm;

[0199] ⑦ Calculation: In the Excel worksheet, use linear regression analysis, plotting a standard curve with the concentration of the reference sample on the X-axis and its OD value on the Y-axis, to obtain the linear equation: Y = bX + a, and then obtain a, b, and R. 2 Value. Where b is the slope, a is the y-intercept, and R is the value. 2 The determination coefficient.

[0200] Figure 6 , Figure 7 They respectively demonstrated pre-coated GV-mab-1 antibody ( Figure 6 ), GV-mab-2 antibody ( Figure 7 The results of enzyme-linked immunosorbent assay (ELISA) reactions using 96-well high-adsorption flat-bottom plates for quantitative detection of different concentrations of GV-Delta-RBD protein were analyzed, along with linearity analysis of the OD values ​​after color development for different concentrations of GV-Delta-RBD. The results showed that the standard curves plotted by the two antibody-prepared ELISA kits showed consistent R-values. 2 All values ​​were >0.99, confirming the feasibility of using GV-mab-1 and GV-mab-2 antibodies for the quantitative detection of fusion proteins containing the GVopti tag.

[0201] It is evident that the ELISA kits prepared using anti-SDcatcher mouse monoclonal antibody and anti-GVopti mouse monoclonal antibody as binding antibodies can accurately quantify fusion proteins containing SDcatcher tags (SD-Ferritin) and GVopti tags (GV-Delta-RBD), respectively.

[0202] Example 5: Quantitative assay of combined anti-SDcatcher peptide antibody and anti-GVopti peptide antibody

[0203] The fusion protein (RBD-Ferritin) containing SDcatcher and GVopti peptides was analyzed by Western blot experiments.

[0204] The double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA) was used. The anti-SDcatcher peptide antibody (SD-Mab-1) protein was diluted to 2.5 μg / mL with coating buffer (manufacturer: Solarbio; catalog number C1050-100 mL). 50 μL of the solution was added to each well of a high-adsorption 96-well plate. The wells were sealed with sealing film and incubated at room temperature for 4 h or at 4 °C overnight. The plate was pre-coated with anti-SDcatcher peptide antibody (SD-Mab-1). Then, the reference sample (containing the dual-tagged fusion protein RBD-Ferritin; our team previously constructed and purified the expression vector and the dual-tagged fusion protein, see reference DOI:10.1002 / advs.202105378) and the HRP-labeled anti-GVopti peptide-HRP antibody (i.e., GV-Mab-1, with the antibody provided by our company and HRP labeling services provided by Beijing Yiqiao Shenzhou Technology Co., Ltd.) were added sequentially. After incubation and thorough washing, the sample was developed with the substrate TMB. TMB was converted to blue under the catalysis of peroxidase, and then to yellow under acidic conditions. The intensity of the color was positively correlated with the RBD-Ferritin content in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader, and the concentrations of the sample and reference sample were calculated to determine the antigen content. The reference dilutions were as follows: the RBD-Ferritin fusion protein containing the dual tag was diluted with PBS to 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 0 ng / mL. Details are as follows:

[0205] ① Sample incubation: Take out the plate pre-coated with antibody, spot the reference sample at a rate of 100 μL / well, set up two replicates for each well, and add 2 blank control wells (PBS dilution) at the same time. Incubate at 37℃ for 1 h.

[0206] ② Washing the plate: Take out the high-adsorption 96-well plate, discard the liquid, add 300μL of wash buffer to each well, gently tap and shake to remove the washing liquid, repeat this washing process 3 times, and pat dry on absorbent paper for the last time;

[0207] ③ Antibody incubation: Add 100 μL of anti-GVopti peptide-HRP antibody diluted with PBS (1:3000 dilution) to each well and incubate at 37°C for 1 h;

[0208] ④ Washing the plate: Take out the high-adsorption 96-well plate, discard the liquid, add 300μL of wash buffer to each well, gently tap and shake to remove the washing liquid, repeat this washing process 3 times, and pat dry on absorbent paper for the last time;

[0209] ⑤ Color development: Add 50 μL of TMB substrate to each well and incubate at room temperature in the dark for 20 min;

[0210] ⑥ Termination and reading: Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm;

[0211] ⑦ Calculation: In the Excel worksheet, use linear regression analysis, plotting a standard curve with the concentration of the reference sample on the X-axis and its OD value on the Y-axis, to obtain the linear equation: Y = bX + a, and then obtain a, b, and R. 2 Value. Where b is the slope, a is the y-intercept, and R is the value. 2 The determination coefficient.

[0212] Figure 8 This paper presents the results of an enzyme-linked immunosorbent assay (ELISA) protocol for the quantitative detection of different concentrations of RBD-Ferritin protein (containing SDcatcher and GVopti dual tags) using a 96-well high-absorption flat-bottom plate pre-coated with SD-mab-1 antibody and incubated with HRP-labeled anti-GVopti peptide-HRP antibody (GV-Mab-1). Linearity analysis of the OD values ​​after color development for different concentrations of RBD-Ferritin is also shown. The results indicate that the standard curve plotted using the ELISA kit prepared in this protocol yields a satisfactory result. 2 >0.99, confirming the feasibility of using GV-mab-1 antibody and GV-mab-1 antibody in combination for the quantitative detection of fusion proteins containing SDcatcher and GVopti dual tags.

[0213] It is evident that the ELISA kit prepared using anti-SDcatcher mouse monoclonal antibody and anti-GVopti mouse monoclonal antibody as binding antibodies can accurately quantify the fusion protein (RBD-Ferritin) containing SDcatcher and GVopti tags.

[0214] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A mouse monoclonal antibody against SDcatcher, characterized in that, The anti-SDcatcher mouse monoclonal antibody is selected from SD-Mab-1 or SD-Mab-2, both of which are composed of a heavy chain and a light chain. The heavy chain of SD-Mab-1 has the amino acid sequence shown in SEQ ID NO. 9, and the light chain has the amino acid sequence shown in SEQ ID NO.

10. The heavy chain of SD-Mab-2 has the amino acid sequence shown in SEQ ID NO. 11, and the light chain has the amino acid sequence shown in SEQ ID NO.

12.

2. A murine monoclonal antibody against GVopti, characterized in that, The amino acid sequence of GVopti is shown in SEQ ID NO. 26; the anti-GVopti murine monoclonal antibody is selected from GV-Mab-1 or GV-Mab-2, both of which consist of a heavy chain and a light chain; the heavy chain of GV-Mab-1 has the amino acid sequence shown in SEQ ID NO. 13, and the light chain has the amino acid sequence shown in SEQ ID NO. 14; the heavy chain of GV-Mab-2 has the amino acid sequence shown in SEQ ID NO. 15, and the light chain has the amino acid sequence shown in SEQ ID NO.

16.

3. A nucleic acid molecule encoding the murine monoclonal antibody of claim 1 or claim 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain of SD-Mab-1 is shown in SEQ ID NO.17, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.18; the nucleotide sequence encoding the heavy chain of SD-Mab-2 is shown in SEQ ID NO.19, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.

20.

5. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence encoding the heavy chain of GV-Mab-1 is shown in SEQ ID NO.21, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.22; the nucleotide sequence encoding the heavy chain of GV-Mab-2 is shown in SEQ ID NO.23, and the nucleotide sequence encoding its light chain is shown in SEQ ID NO.

24.

6. The application of the combined use of anti-SDcatcher mouse monoclonal antibody and anti-GVopti mouse monoclonal antibody in the quantification, tracing, enrichment, extraction, and purification of fusion proteins containing SDcatcher and GVopti, characterized in that, The application does not involve disease diagnosis or treatment. The amino acid sequence of GVopti is shown in SEQ ID NO.

26. The anti-SDcatcher monoclonal antibody is selected from SD-Mab-1 or SD-Mab-2, and the anti-GVopti monoclonal antibody is selected from GV-Mab-1 or GV-Mab-2. SD-Mab-1, SD-Mab-2, GV-Mab-1, and GV-Mab-2 are all composed of a heavy chain and a light chain. The heavy chain of SD-Mab-1 has the amino acid sequence shown in SEQ ID NO. 9, and the light chain has the amino acid sequence shown in SEQ ID NO.

10. The heavy chain of SD-Mab-2 has the amino acid sequence shown in SEQ ID NO. 11, and the light chain has the amino acid sequence shown in SEQ ID NO.

12. The heavy chain of GV-Mab-1 has the amino acid sequence shown in SEQ ID NO. 13, and the light chain has the amino acid sequence shown in SEQ ID NO.

14. The heavy chain of GV-Mab-2 has the amino acid sequence shown in SEQ ID NO.

14. The amino acid sequence shown in NO. 15, and the light chain having the amino acid sequence shown in SEQ ID NO.

16.

7. The application of the anti-SDcatcher mouse monoclonal antibody according to claim 1 in the qualitative, quantitative, tracing, enrichment, extraction, and purification of fusion proteins containing SDcatcher, characterized in that, The application does not involve disease diagnosis or treatment.

8. The application of the anti-GVopti mouse monoclonal antibody according to claim 2 in the qualitative, quantitative, tracing, enrichment, extraction, and purification of fusion proteins containing GVopti, characterized in that, The application does not involve disease diagnosis or treatment.

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