CN117106079A

CN117106079A - Anti SDcatcher, GVopti murine monoclonal antibody, preparation method and application thereof

Info

Publication number: CN117106079A
Application number: CN202311128307.5A
Authority: CN
Inventors: 张辉; 邹帆; 刘炳峰; 涂洛扬
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-09-04
Filing date: 2023-09-04
Publication date: 2023-11-24

Abstract

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

Description

Anti SDcatcher, GVopti murine monoclonal antibody, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a SDcatcher, GVopti murine monoclonal antibody, a preparation method and application thereof.

Background

Cell function depends on a number of reversible non-covalent protein-protein interactions, while the precise arrangement of proteins in the complex affects and determines their function. However, non-covalent interactions tend to be fragile and are often interrupted over long periods of time or in the presence of mechanical forces, etc. Thus, designing covalent protein-protein interactions can bring a new set of opportunities for basic research, synthetic biology and biotechnology.

Proteins capable of spontaneously forming isopeptidic linkages have been used to develop peptide tags and their corresponding binding polypeptides (i.e., conjugates of two peptide fragments) that rely on mutual covalent binding between isopeptidic linkages to provide irreversible interactions. In this case, a protein capable of spontaneously forming an isopeptide bond may be expressed as two fragments, a peptide tag and a polypeptide binding partner. Among these, proteins capable of possessing extremely strong interactions are present in some gram-positive bacteria.

In the earlier studies, the SDcatcher polypeptide is taken as the 486-591 amino acid sequence in CnaB2 of streptococcus agalactiae (Streptococcus dysgalactiae), the GVtag peptide tag is taken as the 97-111 amino acid sequence in CnaB2 of gardnerella vaginalis (Gardnerella vaginalis), and the corresponding amino acid modification optimization is carried out at the N-terminal of the GVtag peptide tag, so that an SD-Gvopti connecting system (doi: 10.1038/s 41423-021-00736-2) of the combination of the polypeptide capable of spontaneously forming the isopeptide bond and the peptide tag is established. The connection system provides a simple, specific and genetically encodable method for the creation of various biological materials, and has great application value in various fields, such as biological materials, next generation sequencing, enzyme stabilization, vaccine development and the like. Therefore, the development of the murine monoclonal antibody aiming at SDcatcher, GVtag can be used for qualitative and quantitative detection, tracing and the like of the fusion peptide and the fusion protein containing the tag, and can be used for enrichment, extraction or purification and the like of the fusion peptide and the fusion protein containing the tag.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides an anti-SDcatcher, GVopti murine monoclonal antibody, wherein one antibody (SD-Mab-1/2) is used for specifically recognizing a polypeptide (SDcatcher) derived from streptococcus agalactiae (Streptococcus dysgalactiae), and the other antibody (GV-Mab-1/2) is used for specifically recognizing a polypeptide (Gvopti) with an optimized 97-111 amino acid sequence in an adhesin domain (Cna B2) of fibronectin binding protein (Fba B) derived from gardnerella vaginalis (Gardnerella vaginalis), so that the antibody can be used for qualitative, quantitative and tracing related detection work of fusion protein containing SDcatcher, GVopti peptide, and has wide application prospect.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

the first aspect of the present invention provides a murine monoclonal antibody directed against a fusion protein comprising a SDcatcher, GVopti peptide, said murine monoclonal antibody being selected from the group consisting of an anti-SDcatcher monoclonal antibody selected from SD-Mab-1 and/or SD-Mab-2 and/or an anti-GV-monoclonal antibody selected from GV-Mab-1 and/or GV-Mab-2; the SD-Mab-1, SD-Mab-2, GV-Mab-1 and GV-Mab-2 are composed of a heavy chain and a light chain (the heavy chain and the light chain are connected through disulfide bonds); the heavy chain of the SD-Mab-1 has an amino acid sequence shown as SEQ ID NO.9, and the light chain has an amino acid sequence shown as SEQ ID NO. 10; the heavy chain of the SD-Mab-2 has an amino acid sequence shown as SEQ ID NO.11, and the light chain has an amino acid sequence shown as SEQ ID NO. 12; the heavy chain of the GV-Mab-1 has an amino acid sequence shown as SEQ ID NO.13, and the light chain has an amino acid sequence shown as SEQ ID NO. 14; the heavy chain of GV-Mab-2 has an amino acid sequence shown as SEQ ID NO.15, and the light chain has an amino acid sequence shown as SEQ ID NO. 16.

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

In a third aspect, the 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, and the GV-Mab-1 and the GV-Mab-2 are composed of a heavy chain and a light chain; the heavy chain of the GV-Mab-1 has an amino acid sequence shown as SEQ ID NO.13, and the light chain has an amino acid sequence shown as SEQ ID NO. 14; the heavy chain of GV-Mab-2 has an amino acid sequence shown as SEQ ID NO.15, and the light chain has an amino acid sequence shown as SEQ ID NO. 16.

The amino acid sequence of the heavy chain variable region has at least 80% sequence homology with the amino acid sequence of the light chain variable region, and the amino acid sequence of the heavy chain hypervariable region also has at least 80% sequence homology with the amino acid sequence of the light chain hypervariable region (the variable region comprises a hypervariable region in which the immunoglobulin light chain and the heavy chain are greatly changed near the N-terminal amino acid sequence, and the hypervariable region comprises a specific part in which the immunoglobulin light chain and the heavy chain are most severely changed). 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: ARHSNSYFDY 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 the SD-Mab-2 is CDR1: GYTFTTAG, CDR2: IKTHSGVT and CDR3: ARSGPLNWYYPMDY 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 the SD-Mab-1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the SD-Mab-2 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

The amino acid sequence of the heavy chain variable region has at least 80% sequence homology with the amino acid sequence of the light chain variable region, and the amino acid sequence of the heavy chain hypervariable region also has at least 80% sequence homology with the amino acid sequence of the light chain hypervariable region (the variable region comprises a hypervariable region in which the immunoglobulin light chain and the heavy chain are greatly changed near the N-terminal amino acid sequence, and the hypervariable region comprises a specific part in which the immunoglobulin light chain and the heavy chain are most severely changed). Preferably, in the first and third aspects, the heavy chain hypervariable region amino acid sequence of the GV-Mab-1 is CDR1: GFTFSGYI, CDR2: issgsyt and CDR3: LVADLDFDV 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 the GV-Mab-2 is CDR1: GFTFSSYT, CDR2: INSTGTYT and CDR3: LVADLDFD, light chain hypervariable region amino acid sequence CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT; the amino acid sequence of the heavy chain variable region of the GV-Mab-1 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region of the GV-Mab-2 is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

In a fourth aspect, the invention provides a nucleic acid molecule encoding a monoclonal antibody of murine origin as described in the second or third aspect.

Preferably, the nucleotide sequence of the heavy chain of the SD-Mab-1 is shown as SEQ ID NO.17, and the nucleotide sequence of the light chain of the SD-Mab-1 is shown as SEQ ID NO. 18; the nucleotide sequence of the heavy chain of the coded SD-Mab-2 is shown as SEQ ID NO.19, and the nucleotide sequence of the light chain of the coded SD-Mab-2 is shown as SEQ ID NO. 20.

Preferably, the nucleotide sequence of the heavy chain of GV-Mab-1 is shown as SEQ ID NO.21, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 22; the nucleotide sequence of the heavy chain of the GV-Mab-2 is shown as SEQ ID NO.23, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 24.

In a fifth aspect, the invention provides the use of a murine monoclonal antibody according to the first to third aspects or a nucleic acid molecule according to the fourth aspect for qualitative, quantitative, or tracer purposes of fusion proteins comprising an SDcatcher and/or GVGti.

In a sixth aspect, the present invention provides the use of a murine monoclonal antibody according to the first to third aspects or a nucleic acid molecule according to the fourth aspect for the enrichment, extraction and purification of fusion proteins comprising sdcatchers and/or GVopti.

The anti-SDcatcher murine monoclonal antibody and the anti-GVGpati murine monoclonal antibody disclosed by the invention are composed of a heavy chain and a light chain, wherein the anti-SDcatcher monoclonal antibody is SD-Mab-1 (shown as SEQ ID NO.9 and 10) and SD-Mab-2 (shown as SEQ ID NO.11 and 12), and the anti-GVGpati antibody is GV-Mab-1 (shown as SEQ ID NO.13 and 14) and GV-Mab-2 (shown as SEQ ID NO.15 and 16). The antibody can be used for the related works of qualitative, quantitative, tracing, enrichment, extraction, purification and the like of fusion proteins containing SDcatcher, GVopti peptide, and has great potential application value.

The seventh aspect of the present invention provides a method for preparing the murine monoclonal antibody of the first to third aspects, comprising designing amino acid sequences of humanized or other animal-derived antibodies by computer assistance, splicing the heavy chain and light chain variable region genes synthesized by whole genes with human or other animal-derived immunoglobulin heavy and light chain constant region genes respectively through gene recombination, cloning into expression vectors, constructing light and heavy chain expression vectors of the humanized or other animal-derived antibodies respectively, co-transfecting eukaryotic cell expression systems with the light and heavy chain expression vectors by a liposome method, and then screening, culturing and purifying.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses an anti-SDcatcher, GVopti murine monoclonal antibody, which comprises an anti-SDcatcher murine monoclonal antibody and an anti-Gvopti murine monoclonal antibody, and consists of a heavy chain and a light chain. The anti-SDcatcher monoclonal antibodies are SD-Mab-1 (shown as SEQ ID NO.9 and 10) and SD-Mab-2 (shown as SEQ ID NO.11 and 12), and the anti-Gvopti antibodies are GV-Mab-1 (shown as SEQ ID NO.13 and 14) and GV-Mab-2 (shown as SEQ ID NO.15 and 16). Wherein the amino acid sequence of the heavy chain hypervariable region of the first anti-SDcatcher monoclonal antibody (SD-Mab-1) is CDR1: GFTFTSYT, CDR2: ISNGGSTT and CDR3: ARHSNSYFDY the amino acid sequence of the light chain hypervariable region 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 the amino acid sequence of the light chain hypervariable region is CDR1: QNVGTN; CDR2: SAS and CDR3: QQYNSYPLT. The amino acid sequence of the hypervariable region of the heavy chain of the first anti-GVGpati antibody (GV-Mab-1) is CDR1: GFTFSGYI, CDR2: issgsyt and CDR3: LVADLDFDV the amino acid sequence of the light chain hypervariable region is CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT; the heavy chain hypervariable region amino acid sequence of the second anti-GVGpati antibody (GV-Mab-2) is CDR1: GFTFSSYT, CDR2: INSTGTYT and CDR3: LVADLDFD, light chain hypervariable region amino acid sequence CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT. The antibody disclosed by the invention can be used for qualitative, quantitative and tracing related detection work of fusion protein containing SDcatcher, GVopti peptide, and can also be used for enrichment, extraction and purification of fusion protein containing SDcatcher, GVopti peptide, thereby having great potential application value.

Drawings

FIG. 1 is a gel electrophoresis-Coomassie brilliant blue staining chart of a murine antibody expressed by anti-SDcatcher hybridoma cells (SD-mab-1/2) and a murine antibody expressed by anti-GVGpati hybridoma cells (GV-mab-1/2) after purification;

FIG. 2 shows the Western-blot detection results of murine antibodies (SD-mab-1/2) against SDcatcher hybridoma cell expression for a plurality of fusion proteins containing SDcatcher tags [ 2.75, XBB.1.5, BA.5, BQ.1.1, delta, CH.1.1 ] representing fusion proteins containing RBD protein dimers of each strain of the novel coronavirus, respectively, with positive controls of RBD protein dimer-SD fusion proteins of 2.75, XBB.1.5 of the novel coronavirus (i.e., SD-2.75-XBB.1.5) ];

FIG. 3 shows the Western-blot detection results of murine antibodies (GV-mab-1/2) expressed by anti-GVGti hybridoma cells for GVGti-tag containing fusion proteins (GV-Delta-RBD);

FIG. 4 shows ELISA detection results of a murine monoclonal antibody (SD-Mab-1) against SDcatche hybridoma cell expression for a fusion protein containing an SDcatche tag (SD-Ferritin);

FIG. 5 shows ELISA detection results of murine monoclonal antibody (SD-Mab-2) against SDcatche hybridoma cell expression for fusion proteins with SDcatche tag (SD-Ferritin);

FIG. 6 shows the results of ELISA assays of murine monoclonal antibody (GV-Mab-1) expressed by anti-GVGti hybridoma cells for GVGti-tag-containing fusion proteins (GV-Ferritin);

FIG. 7 shows the results of ELISA assays of murine monoclonal antibody (GV-Mab-2) expressed by anti-GVGti hybridoma cells for GVGti-tag-containing fusion proteins (GV-Ferritin);

FIG. 8 shows ELISA detection results of fusion proteins (RBD-Ferritin) containing SDcatcher tag and GVGpti tag using an SDcatcher peptide fragment antibody (SD-Mab-1) as a wrapper and an HPR-conjugated anti-GVGpti peptide fragment antibody (GV-Mab-1) as a detection index.

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

EXAMPLE 1 acquisition of anti-SDcatcher, GVopti murine monoclonal antibody

1. Immunizing animals with SDcatcher peptide fragment and Gvopti peptide fragment respectively, and inducing animals to generate polyclonal antibody B cells of the SDcatcher peptide fragment and the Gvopti peptide fragment respectively

(1) Synthesis of SDcatcher peptide and Gvopti peptide

The SDcatche peptide fragment (amino acid sequence: SGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYL MPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI) and Gvopeti peptide fragment (amino acid sequence: KVGNTIVMVDKLKEVPTP) were synthesized by the company of Biotechnology, inc. of mountain, asia, commission.

(2) Immunized Balb/c mice and serum titer detection

(1) The SDcatcher peptide is diluted to 500 mug/mL as antigen, freund's complete adjuvant is adopted in the first immunization, wherein the volume ratio of the immunization antigen (peptide) to the immunization adjuvant (Freund's complete adjuvant) is 1:1, the antigen and the adjuvant are only required to be gently mixed before immunization (note that the antigen and the adjuvant are put on ice before immunization), the immunization is carried out by adopting an abdominal subcutaneous multipoint injection mode, and 5 female BALB/c mice with the age of 6-8 weeks are immunized at intervals of 14 days for 4 times (four points are selected subcutaneously for each immunization, and the immunization time is 0 day, 14 day, 28 day and 42 day). The first immunization adopts Freund's complete adjuvant, and 50 mug of antigen is injected; two weeks apart followed by 3 consecutive boosts (two weeks apart) at this stage with Freund's incomplete adjuvant, 30. Mu.g per spot.

(2) In addition, gvopti peptide is diluted to 500 mug/mL to be used as an antigen, freund's complete adjuvant is adopted in the first immunization, the volume ratio of an immunization antigen (peptide) to an immunization adjuvant (Freund's complete adjuvant) is 1:1, the antigen and the adjuvant are only required to be gently mixed before immunization (note that the antigen and the adjuvant are put on ice before immunization), and an abdomen subcutaneous multipoint injection mode is adopted in the immunization, and 50 mug is injected at each point; boost was performed 3 consecutive times (two weeks apart) after two weeks apart: this phase used Freund's incomplete adjuvant, injected 30. Mu.g per spot.

(3) Collecting blood with potency: at week 8, the mice were subjected to orbital bleeding, allowed to stand for a period of time until serum was separated out, centrifuged at 2800rpm at 4℃for 15 minutes to obtain mouse serum, and then subjected to antibody titer detection by ELISA.

ELISA coating liquid (manufacturer: soilebao; product number C1050-100 mL) is used for diluting the SDcatcher peptide fragment and Gvopti peptide fragment antigen respectively, 1 mug antigen is coated on each hole, 50 mug antigen is added into the enzyme-labeled plate hole in the amount of 50 mug/hole, and the mixture is placed at 4 ℃ overnight or coated for 2 hours at 37 ℃; afterwards, the liquid in the hole is discarded, and simultaneously, 200 mu L of PBST is used for washing 3 times, and the mixture is beaten dry; mu.L of blocking solution (5% nonfat milk powder+PBS) was added to each well and blocked at 4℃overnight or at 37℃for 2 hours.

Washing with 200 μl of PBST for 3 times, and storing the coated plate at-20deg.C or 4deg.C; add 50. Mu.L primary antibody (mouse serum to be tested) to each well, and set up negative control wells (non-immunized mouse serum); after incubation at 37℃for 1h, 200. Mu.L of the wash was washed 5 times and patted dry.

Adding enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP; manufacturer: invitrogen), incubating for 1h at 37 ℃ with 50 μl of each well, washing with 200 μl of washing solution for 5 times, and drying; adding 50 mu L of substrate solution TMB, and developing at 37 ℃ for 2-15 min; stopping the reaction by using a stopping solution (manufacturer: soy Bao, product number: C1058-500 mL), and reading an OD value at 450nm by using an enzyme-labeled instrument; as a result, cell fusion was observed when the antibody titer (OD value) was more than 3 times 10.

(4) Sprint immunity

The sprint immunization is carried out at the 10 th week, at this time, no adjuvant is added, only antigen is used for carrying out intraperitoneal immunization, 50 mug is injected into each point, and the subsequent cell fusion can be carried out after 3 days of sprint immunization.

2. Fusing spleen cells of the anti-SDcatcher peptide fragment and the anti-Gvopati peptide fragment B cells with SP20 myeloma cells to obtain two hybridoma cell pools, and screening monoclonal hybridoma cell strains

(1) Acquisition of hybridoma cells

(1) After sprint immunization, removing one side eyeball for exsanguination, and then killing a mouse by matching with spinal dislocation, and placing the mouse in 70% alcohol; the skin inside the left hind limb femoral canal of the mouse is held up with forceps, cut horizontally with scissors, then cut vertically, or torn with hands to expose the tie film. If there are lymph nodes in the femoral canal, the femoral canal can be removed and immersed in 10mL of RMPI 1640 medium (in a 9cm dish). Carefully picking spleen, immersing the spleen in the culture medium, carefully shearing off excessive white fat or connective tissue, immersing the spleen in the fresh culture medium again, and shaking and washing the spleen for 1-2 minutes to obtain immune cells;

(2) the washed spleen and lymph nodes were transferred into a cell sieve (the cell sieve was immersed in fresh 10-20ml RMPI 1640 medium in advance), and the spleen was ground in the cell sieve by pushing a syringe, so that the spleen cells were individually filtered through a 200 mesh sterile filter. The medium (containing cells) was then transferred to a clean centrifuge tube; slowly adding 10-20mL of culture medium into the cell sieve for flushing, transferring the filtered cells into a centrifuge tube, lightly blowing liquid in the centrifuge tube, and mixing uniformly; centrifuging at 1000rpm for 5min, and discarding supernatant; adding 40mL of culture medium, resuspending the cells, centrifuging again, discarding the supernatant, and washing the obtained immune cells with GKN buffer solution three times;

(3) Add 5mL red blood cell lysate (Sigma, R7757-100 ML) to the immune cells in the tube for resuspension, carefully blow for 1-2min; adding 40mL of culture medium to terminate the cleavage reaction; centrifuging at 1000rpm for 5min, and discarding supernatant; adding 10mL of culture medium to resuspend the cells;

(4) mu.L of the cell suspension was transferred into a clean EP tube and diluted with 90. Mu.L of medium, 10. Mu.L of this dilution was taken up into a fresh EP tube and 10. Mu.L of 4% trypan blue was added. Cells were counted under an inverted microscope and cell viability was calculated (viability = 100% -stained cells/total cell count x 100%);

(5) the majority of the cells remaining in step (3) were fixed to 30mL with medium, centrifuged, and the supernatant discarded (at this time, a water bath was set at 40℃to preheat PEG solution (Sigma 1450)); cells were then resuspended in 10mL of medium according to Sp2/0 tumor cells (manufacturer: punuocele; cat.: CL-0445): cell suspension = 1:3 (Sp 2/0 was collected in advance, washed, resuspended to medium and the density was about 1X 10) ⁷ /mL). The ratio of Sp2/0 to the number of splenocytes is controlled between 1:9 and 1:4. Then the culture medium is used for fixing the volume to 20mL, the centrifugation is carried out, and the supernatant is discarded; pouring distilled water at 40 ℃ into a beaker, and placing a centrifuge tube filled with cells into the beaker for water bath for about 2min; then slowly dripping 1mL of preheated PEG into the tube within 60 seconds, mixing uniformly while adding, and keeping water bath; sucking the cell suspension, slowly dripping the cell suspension back into the tube, maintaining the water bath, slowly adding 1mL of culture medium in 60s, mixing the culture medium while adding the culture medium, and maintaining the water bath; slowly adding 10mL of culture medium, continuously and uniformly mixing and carrying out water bath; slowly adding the culture medium, fixing the volume to 20mL, mixing uniformly, and carrying out water bath for 10min. Slowly cooling the water temperature to room temperature Centrifuging, and discarding the supernatant; a hybridoma cell mixture was obtained.

(2) Screening of monoclonal hybridoma cells

(1) Monocloning: taking 1×10 ⁴ The individual hybridoma cell mixtures were resuspended using complete medium (HAT containing 20% fbs), individual hybridoma cells were inoculated into individual 96-well cell culture wells by limiting dilution (hybridoma cell suspensions to be cloned were prepared, diluted with HAT medium containing 20% serum to a dilution of 5 cells per ml, mixed cells, cell culture medium was inoculated at a volume of 100 μl per well, detected under a 30min postmicroscope after inoculation was completed, and monoclonal cell wells were labeled).

(2) Monoclonal culture was performed using complete medium (HAT with 20% fbs): placing at 37deg.C, 5% CO ₂ Culturing in an incubator for 7 days.

(3) And (3) detection: 50 mu L of supernatant was harvested from each of the monoclonal cell wells, two multiplex wells were set, and the levels of the anti-SDcatcher antibody and the anti-GVGti antibody in the supernatant were detected by ELISA, and the cell well expressing the anti-SDcatcher antibody and the anti-GVGti antibody, which is the best and normal cell morphology, was labeled.

(4) The 5 wells with the highest positive values (the highest OD450 detection) were transferred to about 1mL of complete medium (RPMI with 20% FBS) and cultured in 24 well plates for 2-3 days followed by expansion.

(5) Expansion culture (12 well plate): cells were transferred to appropriate cell culture plates with complete medium (RPMI with 20% fbs), placed at 37 ℃,5% CO ₂ Culturing in an incubator for 48 hours.

(6) Counting: when the density reaches 2-3×10 ⁶ At the time of/mL, carrying out expansion culture again; such as density<2×10 ⁶ Per mL, the culture was continued for 24 hours and then counted again.

(7) Re-expansion culture (6 well plate): cell density was adjusted to 1X 10 with complete medium (RPMI with 20% FBS) ⁶ Per mL, resuspended cells were transferred to appropriate cell culture plates and placed at 37℃with 5% CO ₂ Culturing in an incubator for 48 hours.

(8) Counting: when the density reaches 2-3×10 ⁶ At the time of/mL, carrying out expansion culture again; such asDensity of<2×10 ⁶ Per mL, the culture was continued for 24 hours and then counted again.

(9) Re-expansion culture (T25 flask): cell density was adjusted to 1X 10 with complete medium (RPMI with 20% FBS) ⁶ Per mL, cells were resuspended in T25 flasks and placed at 37℃with 5% CO ₂ Culturing in an incubator for 48 hours.

Counting: the cell suspension is evenly resuspended before sampling, and the cell suspension is taken out with the amount controlled between 1 and 2 drops. When the cell density is>2×10 ⁶ the/mL is put into the next step of expansion culture.

Re-expansion culture (T75 flask): cell density was adjusted to 1X 10 with complete medium (RPMI with 20% FBS) ⁶ Per mL, cells were resuspended in T75 flask, placed at 37℃in 5% CO ₂ Culturing in an incubator for 48 hours.

Counting: when the density reaches 2-3×10 ⁶ Harvesting culture solution for titer detection; such as density<2×10 ⁶ Culturing for 24 hr, and counting again until the density reaches 2-3×10 ⁶ And (3) harvesting culture solution for titer detection.

(3) Titer detection of hybridoma cells

ELISA screening is carried out on the fused hybridoma cells to determine positive cell strains, wherein the titer detection steps of the anti-SDcatcher mouse-derived monoclonal hybridoma cells are as follows:

(1) SD protein was diluted with ELISA coating solution (manufacturer: soy treasure; cat. No. C1050-100 mL) to a final concentration of 0.1. Mu.g/mL and 1. Mu.g/mL, 100. Mu.L/well overnight at 4 ℃;

(2) the supernatant was discarded and washed 3 times with PBST: 5% skim milk blocking solution, 200 μl/well, incubation at 37deg.C for 2 hr;

(3) removing the supernatant, washing 3 times with washing liquid, adding primary antibody (namely cell culture supernatant obtained by screening the monoclonal hybridoma cells in the step (2)), negative control (HAT medium containing 20% FBS) and blank control (PBS), and incubating at 37 ℃ for 1 hour;

(4) the supernatant was discarded, washed 3 times, and secondary antibody (Rabbit Anti-Mouse IgG F (ab) 2/HRP-0.09. Mu.g/mL), 100. Mu.L/well, was added and incubated at 37℃for 1 hour.

(5) Washing the supernatant with washing solution for 3 times, adding TMB color development solution (manufacturer: soilebao; product number: PR 1200-100) 100 μl/hole, and developing for about 5 minutes;

(6) each well was terminated by adding 50. Mu.L of a stop solution (manufacturer: soy Bao, cat. No. C1058-500 mL), OD450 was measured at 450nm, and Blank was used as a Blank. The data were recorded and saved, and the dilution corresponding to the minimum OD450 reading with titer greater than the maximum OD/2 was finally screened to obtain two anti-SDcatcher monoclonal hybridoma cells (SD-mab-1/2) with the highest positive values, as shown in Table 1.

In addition, the titer detection of the anti-GVGti murine monoclonal hybridoma cells was consistent with the titer detection procedure of the anti-SDcatche murine monoclonal hybridoma cells except that the coating solution was used to dilute the GV protein to a final concentration of 0.1. Mu.g/mL and 1. Mu.g/mL. Finally, two anti-Gvopti monoclonal hybridoma cells (GV-mab-1/2) with highest positive values are obtained through screening, and are shown in table 2.

TABLE 1 antibody levels for each positive well of anti-SDcatcher monoclonal hybridoma cell screening plates

Sample(1ug/mL)	OD-450-1	OD-450-2	Average	OD450-Blank	OD-450-1	OD-450-2	Average	OD450-Blank
									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

TABLE 2 anti-Gvopti monoclonal hybridoma cell screening plates for Positive well antibody levels

(4) Cryopreservation of monoclonal hybridoma cells

(1) Collecting each hybridoma in a 50mL centrifuge tube, and centrifuging at room temperature (18-26 ℃ C., speed: 500g, speed: 5 g) for 5min;

(2) Discarding the supernatant, adding 30mL sodium chloride injection to resuspend for cell counting, and centrifuging at room temperature (18-26 ℃ C., speed: 500g, speed: 5 g) for 5min;

(3) the supernatant was discarded at 1X 10 ⁶ Adding serum-free cell freezing solution (manufacturer: biyun day, product number: C0210B-200 mL) at cell/mL density, and gently beating with a Pasteur pipette;

(4) 1 mL/branch is split into cell freezing tubes, the cells are placed into a gradient cooling box, the cells are placed into a refrigerator at the temperature of minus 80 ℃ for freezing overnight, and the cell freezing tubes are transferred to liquid nitrogen for preservation the next day.

(5) Determination of monoclonal hybridoma cell antibody sequences

The two anti-SDcatcher monoclonal hybridoma cells (SD-mab-1/2) with highest positive values and two anti-GVGpti monoclonal hybridoma cells (GV-mab-1/2) were subjected to antibody sequence determination by the client Guangzhou mountain biosciences, and analyzed by using antibody sequence analysis software (VASE 2) to further determine the CDR sequence, variable region and constant region of the antibody.

In this example, the mouse was immunized with the SDcatcher peptide fragment and the GVGti peptide fragment, respectively, a plurality of times, and the mouse was induced to produce polyclonal antibody B cells against the SDcatcher peptide fragment and the rabbit anti-GVGti peptide fragment. Then, 3 days after the immunization of the mice, the mice were sacrificed, spleen cells containing the anti-SDcatcher peptide fragment and the anti-GVopti peptide fragment B cells were obtained from the spleen, and pools of anti-SDcatcher peptide fragment and rabbit anti-GVopti peptide fragment hybridoma cells were obtained after fusion with SP20 myeloma cells, respectively. Obtaining monoclonal hybridomas through a single cell sorting scheme, respectively detecting the titers of anti-SDcatcher antibodies and anti-Gvopti antibodies generated by the hybridoma monoclonal cells after the hybridoma cells are amplified to a certain number, and determining antibody sequences of two anti-SDcatcher monoclonal hybridoma cells (SD-mab-1/2) with the highest positive value and two anti-SDcatcher monoclonal hybridoma cells (GV-mab-1/2).

The measurement result shows that the anti-SDcatcher monoclonal hybridoma cell (SD-mab-1/2) and the anti-GVGti monoclonal hybridoma cell (GV-mab-1/2) are both composed of a heavy chain and a light chain (the heavy chain and the light chain are connected through disulfide bonds). The amino acid sequence of the heavy chain hypervariable region of the first anti-SDcatcher monoclonal antibody (SD-Mab-1) is CDR1: GFTFTSYT, CDR2: ISNGGSTT and CDR3: ARHSNSYFDY the amino acid sequence of the light chain hypervariable region 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 the amino acid sequence of the light chain hypervariable region is CDR1: QNVGTN; CDR2: SAS and CDR3: QQYNSYPLT. Wherein the amino acid sequence of the heavy chain variable region of SD-Mab-1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of SD-Mab-2 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4. The heavy chain amino acid sequence of SD-Mab-1 is shown as SEQ ID NO.9, and the light chain amino acid sequence is shown as SEQ ID NO. 10; the heavy chain amino acid sequence of SD-Mab-2 is shown as SEQ ID NO.11, and the light chain amino acid sequence is shown as SEQ ID NO. 12. The nucleotide sequence of the heavy chain of the encoding SD-Mab-1 is shown as SEQ ID NO.17, and the nucleotide sequence of the light chain of the encoding SD-Mab-1 is shown as SEQ ID NO. 18; the nucleotide sequence of the heavy chain of the coded SD-Mab-2 is shown as SEQ ID NO.19, and the nucleotide sequence of the light chain of the coded SD-Mab-2 is shown as SEQ ID NO. 20.

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

SEQ ID NO. 1 (anti-SDcatcher murine monoclonal antibody SD-Mab-1, heavy chain variable region amino acid sequence):

MNFGLSLVFLVLVLKGVLCEVKLVESGGGLVQPGGSLKLSCAASGFTFTSYTMSWVR QTPEKNLEWVAYISNGGSTTYYPDTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCAR HSNSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKG (hypervariable region underlined, corresponding to CDR1: GFTFSGYI, CDR2: ISSGGSYT and CDR3: LVADLDFDV in this order).

SEQ ID NO. 2 (anti-SDcatcher murine monoclonal antibody SD-Mab-1, light chain variable region amino acid sequence):

MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQ KQGKSPQVLVYNAKTLVEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGNPPTFG AGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (hypervariable regions underlined, corresponding in order to CDR1: ENIYSY; CDR2: NAK and CDR3:QHHYGNPPT)。

SEQ ID NO. 3 (anti-SDcatcher murine monoclonal antibody SD-Mab-2, heavy chain variable region amino acid sequence):

MEWLWNLLFLMAAAQSIQAQIQLVQSGPELKKPGETVRISCKASGYTFTTAGMQWVQKIPGKGLKWIGWIKTHSGVTKYAEDFKGRFAFSLETSASSAYLQISNLKNEDTATYFCARSGPLNWYYPMDYWGQGTSVTVSVAKTTPPSVYPLAPGSAAQTNSMVTLGC (hypervariable region underlined, corresponding in sequence to CDR1: GYTFTTAG, CDR2: IKTHSGGT and CDR3: ARSGPLNWYYPMDY).

SEQ ID NO. 4 (anti-SDcatcher murine monoclonal antibody SD-Mab-2, light chain variable region amino acid sequence):

MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVNVTCKASQNVGTNVAWYQQKPGQSPKALIYSASFRYSGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFCQQYNSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (hypervariable regions underlined, corresponding in sequence to CDR1: QNVGTN; CDR2: SAS and CDR3: QQYNSYPLT).

SEQ ID NO. 5 (anti-GVGti murine monoclonal antibody GV-Mab-1, heavy chain variable region amino acid sequence):

MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAASGFTFSGYIMSWVRQNSEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMRSLKSEDTAMYYCLVADLDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGY (hypervariable region underlined, corresponding to CDR1: GFTFSGYI, CDR2: ISSGGSYT and CDR3: LVADLDFDV in this order).

SEQ ID NO. 6 (anti-GVGti murine monoclonal antibody GV-Mab-1, light chain variable region amino acid sequence):

MESHTQAFVFAFLWLSGVDGDIVMTQSQKFMSTSIGGRVSITCKASQNVGTAVAWCQQKPGQSPKVLIHSASNRYTGVPDRFTGSGSGTDFTLTISNIQSEDLADYFCQQYSSYPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (hypervariable regions underlined, corresponding in sequence to CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT).

SEQ ID NO. 7 (anti-GVGti murine monoclonal antibody GV-Mab-2, heavy chain variable region amino acid sequence):

MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATINSGVTYTYYPDSVKGRFTISRDNAKNILYLQMSSLKSEDTAMYYCLVADLDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGY (hypervariable region underlined, corresponding in sequence to CDR1: GFTFSSYT, CDR2: INSTGHTYT and CDR3: LVADLDFD).

SEQ ID NO. 8 (anti-GVGti murine monoclonal antibody GV-Mab-2, light chain variable region amino acid sequence):

MESHTQAFVFAFLWLSGIDGDIVMTQSQKFMSTSVGDRVSITCKASQNVGTAIAWCQQKPGQSPKVLIHSASNRYTGVPDRFTGSGSGTDFTLTIRNVQSDDLADYFCQQYSSYPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFY (hypervariable regions underlined, corresponding in sequence to CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT).

SEQ ID NO. 9 (anti-SDcatcher murine monoclonal antibody SD-Mab-1, heavy chain amino acid sequence):

MNFGLSLVFLVLVLKGVLCEVKLVESGGGLVQPGGSLKLSCAASGFTFTSYTMSWVRQTPEKNLEWVAYISNGGSTTYYPDTVKGRFTISRDNARNTLYLQMSSLKSEDTAMYYCARHSNSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

SEQ ID NO. 10 (anti-SDcatcher murine monoclonal antibody SD-Mab-1, light chain amino acid sequence):

MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQVLVYNAKTLVEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGNPPTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

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

MEWLWNLLFLMAAAQSIQAQIQLVQSGPELKKPGETVRISCKASGYTFTTAGMQWVQKIPGKGLKWIGWIKTHSGVTKYAEDFKGRFAFSLETSASSAYLQISNLKNEDTATYFCARSGPLNWYYPMDYWGQGTSVTVSVAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

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

MESQTQVFVYMLLWLSGVDGDIVMTQSQKFMSTSVGDRVNVTCKASQNVGTNVAWYQQKPGQSPKALIYSASFRYSGVPDRFTGSGSGTDFTLTNSNVQSEDLADYFCQQYNSYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

SEQ ID NO. 13 (anti-GGVGti murine monoclonal antibody GV-Mab-1, heavy chain amino acid sequence):

MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAASGFTFSGYIMSWVRQNSEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMRSLKSEDTAMYYCLVADLDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

SEQ ID NO. 14 (anti-GGVGti murine monoclonal antibody GV-Mab-1, light chain amino acid sequence):

MESHTQAFVFAFLWLSGVDGDIVMTQSQKFMSTSIGGRVSITCKASQNVGTAVAWCQQKPGQSPKVLIHSASNRYTGVPDRFTGSGSGTDFTLTISNIQSEDLADYFCQQYSSYPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

SEQ ID NO. 15 (anti-GGVGti murine monoclonal antibody GV-Mab-2, heavy chain amino acid sequence):

MNFGLRLIFLVLTLKGVQCDVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVATINSGVTYTYYPDSVKGRFTISRDNAKNILYLQMSSLKSEDTAMYYCLVADLDFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

SEQ ID NO. 16 (anti-GVGpti murine monoclonal antibody GV-Mab-2, light chain amino acid sequence):

MESHTQAFVFAFLWLSGIDGDIVMTQSQKFMSTSVGDRVSITCKASQNVGTAIAWCQQKPGQSPKVLIHSASNRYTGVPDRFTGSGSGTDFTLTIRNVQSDDLADYFCQQYSSYPLTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

SEQ ID NO. 17 (anti-SDcatcher murine monoclonal antibody SD-Mab-1, heavy chain nucleotide sequence):

ATGAATTTCGGGCTCAGCTTGGTTTTCCTTGTCCTTGTTTTAAAAGGTGTCCTGTGTGAAGTTAAGTTGGTGGAGTCGGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCACTAGCTATACCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAACCTGGAGTGGGTCGCATACATTAGTAATGGTGGTAGTACCACCTACTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACGGCCATGTATTACTGTGCAAGACATTCTAACTCCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATAA。

SEQ ID NO. 18 (anti-SDcatcher murine monoclonal antibody SD-Mab-1, light chain nucleotide sequence):

ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCGAGTGAAAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGGTCCTGGTCTATAATGCAAAAACCTTAGTAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTAATCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

SEQ ID NO. 19 (anti-SDcatcher murine monoclonal antibody SD-Mab-2, heavy chain nucleotide sequence):

ATGGAATGGCTGTGGAACTTGCTCTTTCTCATGGCAGCAGCTCAAAGTATCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAGGATCTCCTGCAAGGCTTCTGGGTACACCTTCACAACTGCTGGAATGCAGTGGGTTCAAAAGATACCAGGAAAGGGTTTGAAGTGGATTGGCTGGATAAAAACCCACTCTGGAGTGACAAAATATGCTGAAGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCAGTGCATATTTACAGATAAGCAATCTCAAAAATGAGGACACGGCTACGTATTTCTGTGCGAGATCGGGTCCCCTTAACTGGTACTATCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCGTAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATAA。

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

ATGGAGTCACAGACTCAGGTCTTTGTATACATGTTGCTGTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAACGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTATTCGGCATCCTTCCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAACAGCAATGTGCAGTCTGAAGACTTGGCAGATTATTTCTGTCAGCAATATAACAGCTATCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

SEQ ID NO. 21 (anti-GGVGti murine monoclonal antibody GV-Mab-1, heavy chain nucleotide sequence):

ATGAACTTCGGGCTCAGATTGATTTTCCTTGTCCTTACTTTAAAAGGTGTCCAGTGTGACGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGGCTATATTATGTCTTGGGTTCGCCAGAATTCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGAAGTTACACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGAAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTTTAGTAGCGGACTTGGACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATAA。

SEQ ID NO. 22 (anti-GGVGti murine monoclonal antibody GV-Mab-1, light chain nucleotide sequence):

ATGGAGTCTCATACTCAGGCCTTTGTATTCGCGTTTCTTTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAATAGGAGGCAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTGTAGCCTGGTGTCAACAGAAACCAGGACAATCTCCTAAAGTTCTGATTCATTCGGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGTAATATACAGTCTGAAGACCTGGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCTCTGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

SEQ ID NO. 23 (anti-GGVGti murine monoclonal antibody GV-Mab-2, heavy chain nucleotide sequence):

ATGAACTTCGGGCTCAGATTGATTTTCCTTGTCCTTACTTTAAAAGGTGTCCAGTGTGACGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATACCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAATAGTGGTGTTACTTACACCTACTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCCGAGACAATGCCAAGAACATCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTTTAGTAGCGGACTTGGACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATAA。

SEQ ID NO. 24 (anti-GGVGti murine monoclonal antibody GV-Mab-2, light chain nucleotide sequence):

ATGGAGTCTCATACTCAGGCCTTTGTATTCGCGTTTCTCTGGTTGTCTGGTATTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTGGGTACTGCTATAGCCTGGTGTCAACAGAAACCAGGTCAATCTCCTAAAGTACTGATTCACTCGGCATCCAATCGGTACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGGAATGTGCAGTCTGACGACCTGGCAGATTATTTCTGCCAGCAATATAGCAGCTATCCTCTGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTAA。

EXAMPLE 2 expression and purification of antibodies of interest (anti-SDcatcher peptide antibody and anti-GVGti peptide antibody) Using anti-SDcatcher peptide and anti-GVGti peptide hybridoma cells

(1) Recovery of hybridoma cells

(1) Hybridoma cells were first placed in RPMI complete medium containing 20% fetal bovine serum, and dishes were placed in 5% CO ₂ Culturing in a cell culture incubator at 37 ℃, completing one passage after the hybridoma cells are recovered, and counting cells every day in the culture process (3 days);

(2) growing the cell density to 1X 10 ⁶ The culture medium was transferred to a 15mL centrifuge tube at about 4℃at 1500rpm for 5min when the color of the culture medium became orange, and the supernatant was removed from the centrifuge tube.

(3) After washing the cells with PBS solution, the cells were resuspended in RPMI complete medium containing 20% fetal bovine serum and the cell resuspension was transferred to T25 cell culture flasks.

(4) The complete culture medium of RPMI containing 20% of fetal bovine serum is used for fixing the volume to 120mL; t25 cell culture flasks were placed in 5% CO ₂ Culturing in a cell culture incubator at 37 ℃ and counting cells at intervals in the culture process (3 days);

(2) Amplified culture of hybridoma cells

The hybridoma cells were transferred to RPMI complete medium containing 10% fetal bovine serum for expansion culture, as follows:

(1) to grow to 1×10 cell density ⁶ When the color of the culture solution is about one mL or the color of the culture solution is changed to orange, 120mL of the culture solution in the T25 cell culture flask is transferred into a 50mL centrifuge tube for multiple times, the culture solution is centrifuged at 1500rpm for 5min at 4 ℃, and the supernatant in the centrifuge tube is removed.

(2) After washing the cells with PBS solution, the cells were resuspended in RPMI complete medium containing 10% fetal bovine serum; and the cell resuspension was transferred to T75 cell culture flasks.

(3) The complete culture medium of RPMI containing 10% of fetal bovine serum is used for fixing the volume to 540mL; t75 cell culture flasks were placed in 5% CO ₂ The cells were cultured in a 37℃incubator at intervals according to the cell expansion (color change of the medium) during the culture (5 days).

(3) Expression and purification of anti-SDcatcher peptide fragment and anti-Gvopati peptide fragment hybridoma monoclonal antibody

To grow to 1×10 cell density ⁶ Transferring the culture solution in T75 cell culture flask into 50mL centrifuge tube for 5min at 4deg.C and 1500rpm when the color of culture solution is orange, removing cell supernatant, washing with PBS solution, re-suspending cells in T75 cell culture flask containing 540mL serum-free CD-hybrid oma culture medium, and placing T75 cell culture flask in 5% CO ₂ Culturing in a 37 ℃ cell incubator, and producing monoclonal antibodies; the antibody production cycle was 10 days, and the culture broth was collected on day 11 for the next antibody purification (affinity chromatography using AKTA equipment from general company and a column of Mabselect Protein A to purify antibodies, supra).

(4) Antibody electrophoresis

5 volumes of purified antibody and 1 volume of SDS-PAGE loading buffer (manufacturer: bejingrui Biotechnology Co., ltd.; product No. RB 005-001) were mixed, boiled for 5 minutes, and centrifuged to obtain a supernatant, wherein 10. Mu.L of the sample was loaded, and the protein Marker was 5. Mu.L.

Electrophoresis conditions: the voltage is 95V, the current is about 75mA, electrophoresis is carried out for 0.5 hour, after electrophoresis is finished, the electrophoresis gel is taken out, the electrophoresis gel is put into Super SDS-PAGE staining solution (manufacturer: bejingui Biotechnology Co., ltd.; product number: RB 010-500) for 1 hour, a shaking table is slowly shaken, after the staining is completed, the electrophoresis gel is taken out and put into dehydration solution (formula: 40% ethanol, 10% acetic acid and 50% distilled water), the shaking table is slowly shaken for 1-2 hours, and after the decoloration is completed, the observation result is taken out.

FIG. 1 shows the gel electrophoresis-Coomassie Brilliant blue staining patterns of purified antibodies SD-mab-1, SD-mab-2, GV-mab-1, GV-mab-2, respectively, with two bands (the heavy chain band of the antibody with the larger molecular weight and the light chain band of the antibody with the smaller molecular weight) developed after electrophoresis of all antibody sequences. It was demonstrated that purified antibodies SD-mab-1, SD-mab-2, GV-mab-1, GV-mab-2 could be successfully extracted by the method of this example.

Example 3 qualitative detection of anti-SDcatcher peptide antibodies and anti-Gvopti peptide antibodies

The anti-Gvopti peptide antibody and the fusion protein containing the SDcatche peptide are subjected to chromogenic detection through a Western blot experiment, and the anti-Gvopti peptide antibody and the fusion protein containing the Gvopti peptide are subjected to chromogenic detection, wherein the detection is specifically as follows:

1. western blot detection of fusion protein containing SDcatcher tag

(1) Dilution of test samples: a plurality of fusion proteins expressing a plurality of SDcatcher tags [ fusion proteins containing RBD protein dimers of each strain of the novel coronavirus (2.75, XBB.1.5, BA.5, BQ.1.1, delta, CH.1.1) ], were independently constructed for the previous team study, and the expression vectors were used for the present study, see patent CN116375884A for details. Firstly, dissolving frozen CHO cells expressing various proteins in a water bath kettle at the room temperature of 18-26 ℃ and the speed of: 500g, rise speed: 5g, speed reduction: 5g, centrifuging for 5min, and removing the frozen stock solution; 1mL of S4 CHO medium was added to resuspend cells using a Pasteur pipette, the cell suspension was transferred to a 125mL triangle flask, and S4 CHO medium [ 1% Glutamax ] ^TM -I(100×)]To 10mL, then placed in a 125mL triangle flask at 37deg.C, 8% CO ₂ Culturing on a high-capacity overlapped constant-temperature shaker at 100rpm for 2-3 days, and collecting culture supernatant. The culture supernatant was diluted to 100ng/mL with PBS, while introducing positive control (purified high purity SD-2.75-XBB.1.5 protein, novel coronavirus 2.75, RBD protein dimer-SD fusion protein of XBB.1.5, team pre-study autonomously constructs expression vector), negative control (GVO-XBB.1.5, no SDcatcher tag, team pre-study autonomously constructs expression vector). The control supernatant was obtained in the same manner as the test sample supernatant.

(2) Purified antibody (SD-Mab-1/2, 10-200. Mu.g/mL) was mixed with 1 volume of 5 XSDS-PAGE loading buffer (manufacturer: bosch & Biotechnology Co., beijing Rui; product number: RB 005-001) and boiled for 5 minutes, and the supernatant was collected after centrifugation, wherein the sample was 10. Mu.L and the protein Marker was 5. Mu.L.

(3) Electrophoresis conditions: and (3) the voltage is 95V, the current is about 75mA, the electrophoresis is carried out for 0.5 to 1 hour, and when bromophenol blue runs to the bottom surface of the separation gel, the electrophoresis is ended, and the power supply is turned off.

(4) Protein transfer: after electrophoresis, cutting an NC film with the size close to the required gel, taking out a pair of thick filter papers and a pair of thin sponges, and immersing the NC film in a 1X film transfer buffer solution to fully soak the NC film. A layer of sponge and a layer of filter paper are paved on the film transferring clamp in sequence, and a layer of filter paper is paved on each of two sides. Then, an NC film is paved on a surface close to a white clamp, glue is paved on a surface close to a black clamp, bubbles between the NC film and filter paper are removed at the same time, and the NC film is made into a sandwich of 'white surface-sponge-filter paper-NC film-gel-filter paper-sponge-black surface', clamped by the clamp and placed into a rotating film electrophoresis tank. Turning on the transfer device, placing the electrophoresis device on ice, transferring film with constant current of 200mA for 100min, and turning off the power supply after the film transfer is completed.

(5) Closing: after the transfer, a TBS solution containing 5% skimmed milk powder was prepared. The membrane was removed from the electrophoresis tank, put into a TBS solution containing 5% nonfat dry milk for blocking, and placed on a horizontal shaking table with slow shaking at room temperature for 1h.

(6) Incubation of primary antibody (SD-Mab-1): after blocking, a primary antibody incubation (i.e., the purified antibody harvested and purified in example 2) was prepared, the blocking solution was decanted, and the primary antibody incubation (SD-Mab-1, 1:3000 dilution) was added and slowly shaken on a room temperature horizontal shaker for 1h. After the completion, the primary antibody was collected, and the membrane was washed with 1 XTBE solution 3-4 times for 8min each.

(7) Incubating a secondary antibody: after the membrane washing is finished, TBST membrane washing liquid is removed, and an incubation liquid mixed with a fluorescent secondary antibody (FITC anti-Mouse antibody, manufacturer: biolegend, product number: 16059) is added, and the membrane is slowly shaken on a horizontal shaking table at room temperature and in a dark place for 1h.

(8) Color development: after completion, the secondary antibodies were collected and the membrane was washed with 1 XTBE solution 4 times for 8min each. After the film washing was completed, the film was scanned using a two-color infrared laser imaging system (Li-COR Odyssey) and the obtained result was analyzed.

SD-Mab-2 primary antibodies were tested by the same experimental means and anti-SD rabbit polyclonal antibodies (prepared from pre-team autoimmune rabbits) were introduced as control primary antibodies and the above experiment was repeated.

Wherein, the preparation process of the rabbit polyclonal antibody is as follows: the SDcatcher peptide is diluted to 500 mug/mL as antigen, freund's complete adjuvant is adopted in the first immunization, wherein the volume ratio of the immunization antigen (peptide) to the immunization adjuvant (Freund's complete adjuvant) is 1:1, the antigen and the adjuvant are only required to be gently mixed before immunization (note that the antigen and the adjuvant are put on ice before immunization), 2 rabbits are immunized 4 times at intervals of 14 days by adopting an abdominal subcutaneous multipoint injection mode (four points are selected subcutaneously for each immunization, and the immunization time is 0 day, 14 days, 28 days and 42 days). The first immunization adopts Freund's complete adjuvant, and 50 mug of antigen is injected; two weeks apart followed by 3 consecutive boosts (two weeks apart) at this stage with Freund's incomplete adjuvant, 30. Mu.g per spot. At week 8, the mice were subjected to orbital bleeding, allowed to stand for a period of time until serum was separated out, centrifuged at 2800rpm at 4 ℃ for 15 minutes to obtain mouse serum, and purified using SD polypeptides to obtain anti-SD rabbit polyclonal serum. FIG. 2 shows the results of the analysis after incubation with SD-mab-1 antibody (left panel), SD-mab-2 antibody (middle panel), SD-Pab rabbit polyclonal antibody (right panel, test control) as primary antibody, respectively. The results showed that the color development effect of the SD-mab-1 antibody and the SD-mab-2 antibody on various proteins containing SDcatche tag was significantly better than that of the SD-Pab rabbit polyclonal antibody as primary antibody, and furthermore, the color development results of the negative control containing no SDcatche tag showed that the SD-Pab rabbit polyclonal antibody as primary antibody showed nonspecific color development bands, while the color development specificity of the SD-mab-1 antibody and the SD-mab-2 antibody as primary antibody was better, confirming the feasibility of the SD-mab-1 antibody and the SD-mab-2 antibody for qualitative detection of fusion proteins containing SDcatche tag.

2. Western blot detection of fusion protein containing GVGti tag

(1) Dilution of test samples: GVGti-tagged fusion proteins [ GV-Delta-RBD, team earlier studies were obtained from expression and purification of autonomously constructed vectors, namely Gv-B.1.617.2_RBD protein as described in the literature (DOI: 10.1002/advs.202105378) ].

(2) The frozen CHO cells expressing the protein are dissolved in a water bath kettle at the room temperature of 18-26 ℃ and the speed of: 500g, rise speed: 5g, speed reduction: centrifuging at 5deg.C for 5min, and removing the frozen stock solution; cells were resuspended by adding 1mL of S4 CHO medium using a Pasteur pipette and transferredThe cell suspension was transferred to a 125mL triangular flask and S4 CHO medium [ 1% Glutamax ] ^TM -I(100×)]To 10mL; placed in a 125mL triangular flask at 37deg.C with 8% CO ₂ Culturing on a high-capacity overlapped constant-temperature shaker at 100rpm for 2-3 days, harvesting culture supernatant, and diluting the culture supernatant to 100ng/mL.

(3) Purified antibody (GV-Mab-1/2, 10-200. Mu.g/mL) was mixed with 1 volume of 5 XSDS-PAGE loading buffer (manufacturer: bosch & Biotechnology Co., beijing Rui; product number: RB 005-001) and boiled for 5 minutes, and the supernatant was collected after centrifugation, wherein the sample was 10. Mu.L and the protein Marker was 5. Mu.L.

(4) Electrophoresis conditions: and (3) the voltage is 95V, the current is about 75mA, the electrophoresis is carried out for 0.5 to 1 hour, and when bromophenol blue runs to the bottom surface of the separation gel, the electrophoresis is ended, and the power supply is turned off.

(3) Protein transfer: after electrophoresis, cutting an NC film with the size close to the required gel, taking out a pair of thick filter papers and a pair of thin sponges, and immersing the NC film in a 1X film transfer buffer solution to fully soak the NC film. A layer of sponge and a layer of filter paper are paved on the film transferring clamp in sequence, and a layer of filter paper is paved on each of two sides. Then, an NC film is paved on a surface close to a white clamp, glue is paved on a surface close to a black clamp, bubbles between the NC film and filter paper are removed at the same time, and the NC film is made into a sandwich of 'white surface-sponge-filter paper-NC film-gel-filter paper-sponge-black surface', clamped by the clamp and placed into a rotating film electrophoresis tank. Turning on the transfer device, placing the electrophoresis device on ice, transferring film with constant current of 200mA for 100min, and turning off the power supply after the film transfer is completed.

(4) Closing: after the transfer, a TBS solution containing 5% skimmed milk powder was prepared. The membrane was removed from the electrophoresis tank, put into a TBS solution containing 5% nonfat dry milk for blocking, and placed on a horizontal shaking table with slow shaking at room temperature for 1h.

(5) Incubation of primary antibody (GV-Mab-1): after blocking, a primary antibody incubation (i.e., the purified antibody harvested and purified in example 2) was prepared, the blocking solution was decanted, and the primary antibody incubation (GV-Mab-1, 1:3000 dilution) was added and slowly shaken on a room temperature horizontal shaker for 1h. After the completion, the primary antibody was collected, and the membrane was washed with 1 XTBE solution 3-4 times for 8min each.

(6) Incubating a secondary antibody: after the membrane washing is finished, TBST membrane washing liquid is removed, and an incubation liquid (FITC anti-Mouse anti-body, manufacturer: biolegend, product number: 16059) mixed with a fluorescent secondary antibody is added, and the membrane is slowly shaken on a horizontal shaking table for 1h at room temperature and in a dark place.

(7) Color development: after completion, the secondary antibodies were collected and the membrane was washed with 1 XTBE solution 4 times for 8min each. After the film washing was completed, the film was scanned using a two-color infrared laser imaging system (Li-COR Odyssey) and the obtained result was analyzed.

The GV-Mab-2 primary antibody was tested by the same experimental means and the above experiment was repeated.

The results are shown in FIG. 3, which shows the results of the analysis after incubation with GV-mab-1 antibody (left panel) and GV-mab-2 antibody (right panel) as primary antibodies, respectively. The results showed that GV-mab-1 antibody and GV-mab-2 antibody were used as primary antibodies, and GV-Delta-RBD protein containing GV opti tag was specifically developed, confirming the feasibility of GV-mab-1 antibody and GV-mab-2 antibody for qualitative detection of fusion protein containing GV opti tag.

As can be seen, in this example, the anti-SDcatcher murine monoclonal antibody and the anti-GV opti murine monoclonal antibody were used as the binding antibodies for the western blot experiment, and the results indicate that the anti-SDcatcher murine monoclonal antibody can well specifically bind to a plurality of fusion proteins containing SDcatcher tags [ fusion proteins containing RBD protein dimers of each strain (2.75, xbb.1.5, ba.5, bq.1.1, delta, ch.1.1) of the novel coronavirus (SD-2.75-xbb.1.5) ] and that the positive control was RBD protein dimer-SD fusion protein of the novel coronavirus 2.75, xbb.1.5 ], and that the anti-GV opti murine monoclonal antibody can well specifically bind to the fusion protein (GV-Delta-RBD) containing the gvalve tag, and that an accurate and specific indicator band appears after color development.

Example 4 quantitative detection of anti-SDcatcher peptide antibody and anti-Gvopti peptide antibody

The quantitative detection of the anti-SDcatche peptide antibody and the target protein containing the SDcatche peptide fragment is carried out through a Western blot experiment, and the quantitative detection of the fusion protein of the anti-Gvopti peptide antibody and the Gvopti peptide fragment is carried out:

1. SD-Ferritin content detection of fusion protein containing SDcatcher tag

The method comprises the steps of adopting a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA), pre-coating an anti-SDcatcher peptide antibody (SD-mab-1/2), sequentially adding fusion proteins (SD-Ferritin) with different concentrations and an HRP-labeled anti-mouse SD-IgG antibody (I.S. provide anti-mouse SD-IgG antibodies SD-mab-1 and SD-mab-2, and provide HRP labeling service by the technology Co., gmbH of Beijing Yinqiao), incubating and thoroughly washing, developing with a substrate TMB, converting TMB into blue under the catalysis of peroxidase, and converting TMB into final yellow under the action of acid. The shade of the color and the SD-Ferritin protein content in the sample are positively correlated. The concentration of the sample and the reference were calculated by measuring absorbance (OD value) at a wavelength of 450nm using an enzyme-labeled instrument, and then the antigen content was measured. Wherein, the reference dilution is: the fusion proteins SD-Ferritin containing the SDcatcher tag were diluted to 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0ng/mL with PBS. The specific method comprises the following steps:

(1) Sample incubation: each reference (team earlier study autonomously constructs expression vector and expresses purified fusion protein SD-Ferritin protein containing SDcatcher tag, see DOI:10.1016/j. Cellrep. 2021.110256) was spotted in an amount of 100. Mu.L/well, two duplicate wells were each set, and 2 well blank control (PBS diluent) was added and incubated at 37℃for 1.5h;

(2) washing the plate: taking out the high adsorption 96-well plate, discarding the liquid, adding 300 mu L of Wash buffer (PBS-T) into each well, tapping, oscillating and throwing out the washing liquid, repeatedly washing the plate for 3 times, and finally drying the plate on the absorbent paper;

(3) antibody incubation: HRP-labeled anti-mouse SD-IgG antibody diluted with PBS (1:3000 dilution) was added at 100 μl/well and incubated for 1h at 37 ℃;

(4) washing the plate: taking out the high adsorption 96-well plate, discarding the liquid, adding 300 mu L of Wash buffer (PBS-T) into each well, tapping, oscillating and throwing out the washing liquid, repeatedly washing the plate for 3 times, and finally drying the plate on the absorbent paper;

(5) color development: 50 mu L of TMB chromogenic substrate (manufacturer: invitrogen; cat. No. 00-4201-56) was added to each well, and incubated at room temperature for 15min in a dark place;

(6) termination and read values: 50. Mu.L of stop solution (manufacturer: solarbio Life Sciences, cat# C1058) was added to each well, and the OD value of each well was measured at a wavelength of 450 nm;

(7) And (3) calculating: in an Excel worksheet, linear regression analysis is adopted, the concentration value of a reference is taken as the X axis, the OD value of the reference is taken as the Y axis, a standard curve is drawn, and a linear equation is obtained: y=bx+a, and gives a, b and R ² Values. Wherein b is the slope, a is the intercept with the Y axis, R ² For determining the coefficient (R ² The fitting goodness is the fitting degree of the regression line to the observed value. R is R ² The closer to 1, the better the fitting effect, the linear equation and R in this experiment ² Generated by the formula in Excel).

FIGS. 4 and 5 show the results of ELISA reactions of 96-well high adsorption flat bottom plates pre-coated with SD-mab-1 antibody and SD-mab-2 antibody for quantitative detection of SD-Ferritin proteins at different concentrations, and linear analysis of OD values after development of SD-Ferritin at different concentrations, respectively. The results show that the standard curve R2 drawn by ELISA kits for preparing the two antibodies is more than 0.99, and the feasibility of the SD-mab-1 antibody and the SD-mab-2 antibody for quantitative detection of fusion proteins containing SDcatcher labels is confirmed.

2. GV-Delta-RBD content detection of fusion proteins containing Gvopti tag

The preparation method comprises the steps of adopting a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA), pre-coating anti-Gvopti peptide antibody, sequentially adding fusion proteins with different concentrations and HRP-labeled anti-mouse Gvopti-IgG antibodies (GV-mab-1 and GV-mab-2 provided by I.S. and provided by HRP labeling service provided by Beijing Yiqiao Shenzhou sciences and technologies Co., ltd.), incubating and thoroughly washing, developing with a substrate TMB, converting TMB into blue under the catalysis of peroxidase, and converting TMB into final yellow under the action of acid. The color shade and the content of the stock GV-Delta-RBD protein in the sample are positively correlated. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme-labeled instrument, and the concentrations of the sample and the reference were calculated to determine the antigen content. Wherein, reference dilution: GV-Delta-RBD containing GVGti tag fusion proteins were diluted to 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0ng/mL with PBS. The specific method comprises the following steps:

(1) Sample incubation: each reference [ team earlier study autonomously constructs expression vector and expresses purified GV-Delta-RBD protein containing Gvopti tag, namely Gv-B.1.617.2_RBD protein described in literature (DOI: 10.1002/advs.202105378), spotted in an amount of 100. Mu.L/well, two wells were each placed while adding 2 well blank (PBS diluent), and incubated for 1h at 37 ℃;

(3) antibody incubation: HRP-labeled anti-mouse GVGti-IgG antibody diluted in PBS (1:3000 dilution) was added in an amount of 100. Mu.L/well and incubated at 37℃for 1h;

(4) washing the plate: taking out the high adsorption 96-well plate, discarding the liquid, adding 300 mu L Wash buffer (PBS-T) r into each well, tapping, oscillating and throwing out the washing liquid, repeatedly washing the plate for 3 times, and finally drying the plate on the water absorption paper;

(5) color development: 50 mu L of TMB chromogenic substrate (manufacturer: invitrogen; cat. No. 00-4201-56) is added to each well, and incubated at room temperature for 12min in a dark place;

(6) termination and read values: 50. Mu.L of stop solution (manufacturer: solarbio Life Sciences, cat#: C1058) was added to each well, and the OD value of each well was measured at a wavelength of 450 nm;

(7) And (3) calculating: in an Excel worksheet, linear regression analysis is adopted, the concentration value of a reference is taken as the X axis, the OD value of the reference is taken as the Y axis, a standard curve is drawn, and a linear equation is obtained: y=bx+a, yielding a, b and R ² Values. Wherein b is the slope, a is the intercept with the Y axis, R ² Is a decision coefficient.

FIGS. 6 and 7 show the results of ELISA reactions of 96-well high adsorption flat plates pre-coated with GV-mab-1 antibody (FIG. 6) and GV-mab-2 antibody (FIG. 7) for quantitative detection of GV-Delta-RBD proteins at different concentrations, and the linear analysis of OD values after development of GV-Delta-RBD at different concentrations, respectively. The results show that the standard curve R drawn by ELISA kits for preparing two antibodies ² All > 0.99, confirm GThe feasibility of the V-mab-1 antibody and the GV-mab-2 antibody for quantitative detection of fusion proteins containing GVGti tags.

It can be seen that ELISA kits prepared by using anti-SDcatcher murine monoclonal antibodies and anti-GVGti murine monoclonal antibodies as binding antibodies can accurately quantify the fusion proteins containing SDcatcher tags (SD-Ferritin) and GVGti tags (GV-Delta-RBD), respectively.

Example 5 Combined quantitative test of anti-SDcatcher Peptidomimetic antibody with anti-Gvopti Peptidomimetic antibody

The fusion protein (RBD-Ferritin) containing the SDcatche peptide fragment and the Gvopti peptide fragment is analyzed by Western blot experiment.

The double antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA) is adopted, the anti-SDcatche peptide antibody (SD-Mab-1) protein is diluted to 2.5 mug/mL by using a coating liquid (manufacturer: soilebao; product number C1050-100 mL), 50 mug of each hole is added into a high-adsorption 96-well plate, a sealing plate film is used for sealing the reaction hole, the reaction hole is incubated for 4 hours or overnight at 4 ℃, and the anti-SDcatche peptide antibody (SD-Mab-1) is pre-coated. Then sequentially adding reference (RBD-Ferritin containing double-tag fusion protein, independently constructing an expression vector by early research of a team, expressing and purifying the double-tag fusion protein, and further referring to document DOI: 10.1002/advs.202105378), an HRP-labeled anti-GVGti peptide-HRP antibody (namely GV-Mab-1, provided by GYP technology Co., GYP.) and developing by using a substrate TMB after incubation and thorough washing, wherein TMB is converted into blue under the catalysis of peroxidase and is converted into final yellow under the action of acid. The shade of the color and the RBD-Ferritin content in the sample are positively correlated. The absorbance (OD value) was measured at a wavelength of 450nm using an enzyme-labeled instrument, and the concentrations of the sample and the reference were calculated to determine the antigen content. Wherein, the dilution of the reference is: the fusion protein RBD-Ferritin containing the ditag was diluted to 500, 250, 125, 62.5, 31.25, 15.625,7.8125,0ng/mL with PBS. The method comprises the following steps:

(1) Sample incubation: taking out the plate pre-coated with antibody, spotting the reference sample according to the amount of 100 mu L/hole, respectively arranging two compound holes, simultaneously adding 2-hole blank control (PBS diluent), and incubating for 1h at 37 ℃;

(2) washing the plate: taking out the high adsorption 96-well plate, discarding the liquid, adding 300 mu L of Wash buffer into each well, tapping, oscillating and throwing out the washing liquid, repeatedly washing the plate for 3 times, and finally drying the plate on the water absorption paper;

(3) antibody incubation: anti-Gvopti peptide-HRP antibody diluted with PBS (1:3000 dilution) was added at 100. Mu.L/well and incubated at 37℃for 1h;

(4) washing the plate: taking out the high adsorption 96-well plate, discarding the liquid, adding 300 mu L of Wash buffer into each well, tapping, oscillating and throwing out the washing liquid, repeatedly washing the plate for 3 times, and finally drying the plate on the water absorption paper;

(5) color development: adding 50 mu L of TMB chromogenic substrate into each hole, standing at room temperature in a dark place, and incubating for 20min;

(6) termination and read values: adding 50 mu L of stop solution into each hole, and measuring the OD value of each hole at the wavelength of 450 nm;

FIG. 8 shows the results of an ELISA reaction for quantitative detection of RBD-Ferritin proteins (containing SDcatcher, GVopti ditag) at different concentrations and a linear analysis of OD values after development of the RBD-Ferritin at different concentrations, using a 96-well high adsorption flat bottom plate pre-coated with SD-Mab-1 antibody and an HRP-labeled anti-Gvopti peptide-HRP antibody (GV-Mab-1) as an antibody for incubation and development. The results show that the ELISA kit prepared by the scheme draws a standard curve R ² > 0.99, the feasibility of the GV-mab-1 antibodies, GV-mab-1 antibodies in combination for quantitative detection of SDcatcher, GVopti ditag-containing fusion proteins was demonstrated.

It can be seen that the ELISA kit prepared by using the anti-SDcatcher murine monoclonal antibody and the anti-GVGti murine monoclonal antibody as the binding antibodies can accurately quantify the fusion protein (RBD-Ferritin) containing the SDcatcher tag and the GVGti tag.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Claims

1. A murine monoclonal antibody directed against a fusion protein comprising a SDcatcher, GVopti peptide, wherein the murine monoclonal antibody is selected from the group consisting of an anti-SDcatcher monoclonal antibody selected from SD-Mab-1 and/or SD-Mab-2 and/or an anti-GV opti monoclonal antibody selected from GV-Mab-1 and/or GV-Mab-2; the SD-Mab-1, SD-Mab-2, GV-Mab-1 and GV-Mab-2 are composed of a heavy chain and a light chain; the heavy chain of the SD-Mab-1 has an amino acid sequence shown as SEQ ID NO.9, and the light chain has an amino acid sequence shown as SEQ ID NO. 10; the heavy chain of the SD-Mab-2 has an amino acid sequence shown as SEQ ID NO.11, and the light chain has an amino acid sequence shown as SEQ ID NO. 12; the heavy chain of the GV-Mab-1 has an amino acid sequence shown as SEQ ID NO.13, and the light chain has an amino acid sequence shown as SEQ ID NO. 14; the heavy chain of GV-Mab-2 has an amino acid sequence shown as SEQ ID NO.15, and the light chain has an amino acid sequence shown as SEQ ID NO. 16.

2. An anti-SDcatcher murine monoclonal antibody, wherein the anti-SDcatcher murine monoclonal antibody is selected from SD-Mab-1 and/or SD-Mab-2, and both SD-Mab-1 and SD-Mab-2 are composed of a heavy chain and a light chain; the heavy chain of the SD-Mab-1 has an amino acid sequence shown as SEQ ID NO.9, and the light chain has an amino acid sequence shown as SEQ ID NO. 10; the heavy chain of the SD-Mab-2 has an amino acid sequence shown as SEQ ID NO.11, and the light chain has an amino acid sequence shown as SEQ ID NO. 12.

3. An anti-Gvopti murine monoclonal antibody, wherein the anti-Gvopti murine monoclonal antibody is selected from Gv-Mab-1 and/or Gv-Mab-2, and wherein the Gv-Mab-1 and Gv-Mab-2 are composed of two parts, namely a heavy chain and a light chain; the heavy chain of the GV-Mab-1 has an amino acid sequence shown as SEQ ID NO.13, and the light chain has an amino acid sequence shown as SEQ ID NO. 14; the heavy chain of GV-Mab-2 has an amino acid sequence shown as SEQ ID NO.15, and the light chain has an amino acid sequence shown as SEQ ID NO. 16.

4. A murine monoclonal antibody directed against a fusion protein comprising a SDcatcher, GVopti peptide according to claim 1 or an anti-SDcatcher murine monoclonal antibody according to claim 2, wherein the heavy chain hypervariable region amino acid sequence of SD-Mab-1 is CDR1: GFTFTSYT, CDR2: ISNGGSTT and CDR3: ARHSNSYFDY 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 the SD-Mab-2 is CDR1: GYTFTTAG, CDR2: IKTHSGVT and CDR3: ARSGPLNWYYPMDY 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 the SD-Mab-1 is shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the SD-Mab-2 is shown as SEQ ID NO.3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

5. A murine monoclonal antibody directed against a fusion protein comprising a SDcatcher, GVopti peptide according to claim 1 or an anti-GVopti murine monoclonal antibody according to claim 2, wherein the heavy chain hypervariable region amino acid sequence of GV-Mab-1 is CDR1: GFTFSGYI, CDR2: issgsyt and CDR3: LVADLDFDV 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 the GV-Mab-2 is CDR1: GFTFSSYT, CDR2: INSTGTYT and CDR3: LVADLDFD, light chain hypervariable region amino acid sequence CDR1: QNVGTA; CDR2: SAS and CDR3: QQYSSYPLT; the amino acid sequence of the heavy chain variable region of the GV-Mab-1 is shown as SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region of the GV-Mab-2 is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8.

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

7. The nucleic acid molecule of claim 6, wherein the nucleotide sequence encoding the heavy chain of SD-Mab-1 is shown in SEQ ID No.17 and the nucleotide sequence encoding the light chain is shown in SEQ ID No. 18; the nucleotide sequence of the heavy chain of the coded SD-Mab-2 is shown as SEQ ID NO.19, and the nucleotide sequence of the light chain of the coded SD-Mab-2 is shown as SEQ ID NO. 20.

8. The nucleic acid molecule of claim 6, wherein the nucleotide sequence encoding the heavy chain of GV-Mab-1 is set forth in SEQ ID NO.21 and the nucleotide sequence encoding the light chain is set forth in SEQ ID NO. 22; the nucleotide sequence of the heavy chain of the GV-Mab-2 is shown as SEQ ID NO.23, and the nucleotide sequence of the light chain is shown as SEQ ID NO. 24.

9. Use of a monoclonal antibody of murine origin according to any one of claims 1 to 5 or of a nucleic acid molecule according to any one of claims 6 to 8 for qualitative, quantitative, or tracer of fusion proteins comprising sdcatchers and/or GVopti.

10. Use of a murine monoclonal antibody according to any one of claims 1-5 or a nucleic acid molecule according to any one of claims 6-8 for the enrichment, extraction, purification of fusion proteins comprising sdcatchers and/or GVopti.