Monoclonal antibodies to group b streptococcal surface immunogenic proteins and uses thereof

Abstract

The application discloses a monoclonal antibody or antigen-binding fragment thereof with high affinity to recognize group B streptococcus SIP protein. The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 5. The minimum detection limit of a double antibody sandwich enzyme-linked immunosorbent method for the SIP protein established by using the monoclonal antibody or antigen-binding fragment thereof as a capture antibody is 0.32 ng / mL. The minimum detection limit of a double antibody sandwich fluorescence immunochromatography method for the SIP protein established by using the monoclonal antibody or antigen-binding fragment thereof as a capture antibody is 0.08 ng / mL, and the minimum detection limit of a group B streptococcus liquid reaches 1×10 3 CFU / mL, which is significantly higher than the minimum detection limit of existing detection reagents and reported in the literature, and can be used for qualitative and quantitative detection of group B streptococcus SIP protein.

Description

Technical Field

[0001] This invention relates to the field of medical immunoassay technology, specifically to a monoclonal antibody that recognizes immunogenic proteins on the surface of group B streptococci and its application in immunoassay. Background Technology

[0002] Group B Streptococcus (GBS), also known as agalactiae, is a hemolytic, aerobic, Gram-positive streptococcus. Serological classification is based on the specificity of the capsular polysaccharide of each strain. According to literature reports, more than ten serotypes have been detected, namely Ia, Ib, Ic, II, III, IV, V, VI, VII, VIII, and IX, with serotype III being the most virulent. GBS can colonize the vagina, intestines, and urethra of pregnant women. Newborns can be infected directly from the mother or through ascending infection from maternal reproductive tract parasites during delivery. GBS is an important pathogen causing reproductive tract infections in pregnant and postpartum women. It can lead to urinary tract infections, chorioamnionitis, puerperal infections, and maternal sepsis. It is associated with premature birth, premature rupture of membranes, neonatal sepsis, and meningitis. In particular, neonatal infection and the resulting neonatal sepsis and death are characterized by high morbidity, mortality, and disability rates, seriously endangering the health of pregnant women and newborns.

[0003] Currently, the main methods for detecting GBS infection include microbiological detection, molecular biological detection, and immunological detection. Direct GBS culture is the gold standard for confirming GBS infection. The principle is that specific enzyme substrates are added to the basal culture medium, and the enzymatic digestion of GBS releases chromogens, causing the bacteria to exhibit different colors, which are then used for analysis and identification of GBS. However, bacterial culture requires at least 18 to 24 hours to observe results, and it is difficult to detect when the bacterial count is low. Furthermore, the sampling process is easily affected by vaginal and perianal bacteria, leading to false negatives and false positives. Molecular biological detection methods have high sensitivity and specificity, but they also require sophisticated equipment and personnel, and are cumbersome to operate, making them unsuitable for widespread application in primary healthcare institutions. Clinically used immunological detection methods mainly include latex agglutination and immunochromatography. The experimental principle of both methods is to use specific antibodies to detect GBS capsular polysaccharide antigen. However, due to the poor immunogenicity of capsular polysaccharide, it is difficult to prepare high-affinity antibodies. Furthermore, there are differences in antigenic epitopes between different serotypes, making it difficult to prepare monoclonal antibodies that can cover multiple serotypes. As a result, the sensitivity of the reagents cannot meet clinical needs.

[0004] The discovery of surface immunogenic protein (SIP) overcomes the complexity caused by serotype differences. SIP is a GBS surface protein discovered through multi-strain genomic analysis, and it has four advantages compared to GBS capsular polysaccharide antigens: First, SIP is expressed in various serotypes of GBS strains, making it a ubiquitous antigen; second, SIP is highly conserved, with over 98% homology in its amino acid sequences across different serotypes of GBS strains; third, SIP protein has strong immunogenicity and good antigenicity, making it easy to prepare high-affinity antibodies; and fourth, SIP is a protein located on the surface of GBS, making it easy to detect. However, there is currently a lack of GBS detection reagents targeting SIP that have been approved for clinical use. Therefore, this invention aims to improve the sensitivity of existing GBS infection detection reagents and promote the clinical application of SIP-targeted GBS infection detection reagents by using SIP protein as an immunogen and combining it with rapid reaction screening technology to prepare monoclonal antibodies with high affinity for SIP protein. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a monoclonal antibody with high affinity for Group B Streptococcus SIP protein prepared using mouse hybridoma technology. The antibody can be used for qualitative and quantitative detection of Group B Streptococcus SIP protein, as well as for preparing detection reagents for detecting Group B Streptococcus infection.

[0006] Therefore, a first aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof with high affinity for Group B Streptococcus SIP protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, wherein...

[0007] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2;

[0008] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3;

[0009] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4;

[0010] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6;

[0011] The amino acid sequence of the light chain CDR2 is DTS;

[0012] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.7.

[0013] Furthermore, the present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.5.

[0014] Furthermore, the present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a single-chain antibody or a humanized antibody, which, because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain, are able to recognize Group B Streptococcus SIP protein with high affinity.

[0015] A second aspect of the present invention relates to a nucleic acid molecule comprising a nucleic acid encoding the above-described monoclonal antibody or an antigen-binding fragment thereof.

[0016] A third aspect of the present invention relates to an expression vector comprising the above-described nucleic acid molecules, said expression vector being capable of expressing the above-described monoclonal antibody or its antigen-binding fragment.

[0017] The fourth aspect of the present invention relates to a recombinant comprising the above-mentioned nucleic acid molecule or the above-mentioned expression vector, which can produce the above-mentioned monoclonal antibody or its antigen-binding fragment, and further, it can be a mammalian cell recombinant, a bacterial recombinant or a yeast recombinant.

[0018] The fifth aspect of this invention relates to a monoclonal antibody mouse hybridoma cell line that secretes the aforementioned monoclonal antibody with high affinity for the SIP protein of Group B Streptococcus. Further, this invention relates to a mouse hybridoma cell line that secretes a monoclonal antibody with high affinity for the SIP protein of Group B Streptococcus, specifically the monoclonal antibody mouse hybridoma cell line 3H617, with accession number CGMCC No. 46596.

[0019] The sixth aspect of this invention relates to the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in the preparation of a kit for detecting Group B Streptococcus SIP protein.

[0020] A seventh aspect of this invention relates to a kit for detecting Group B Streptococcus SIP protein, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment for high-affinity binding to Group B Streptococcus SIP protein. Furthermore, the kit utilizes a double-antibody sandwich detection principle, wherein the monoclonal antibody or its antigen-binding fragment serves as a capture antibody.

[0021] Instructions for the Preservation of Biological Materials

[0022] The monoclonal antibody mouse hybridoma cell line 3H617 of this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with the collection number CGMCC No. 46596, deposit date of September 18, 2025, and classified as: Monoclonal Antibody Mouse Hybridoma Cell Line. The address of the China General Microbiological Culture Collection Center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. Attached Figure Description

[0023] Figure 1 This is an SDS-PAGE electrophoresis image showing prokaryotic expression of group B streptococcal SIP protein, where M is the molecular weight standard.

[0024] Figure 2 This is a graph showing the limit of detection results for the SIP protein double antibody sandwich enzyme-linked immunosorbent assay (ELISA).

[0025] Figure 3 This is a diagram showing the identification results of the 3H617 subtype of the monoclonal antibody against Group B Streptococcus SIP protein. Detailed Implementation

[0026] The purpose of this invention is to provide a mouse hybridoma cell line capable of secreting a monoclonal antibody with high affinity for Group B Streptococcus SIP protein, prepared using mouse hybridoma technology. The specific preparation process involves first expressing the commonly used serotype GBS III SIP protein in prokaryotic cells of *E. coli* Rosetta (DE3) and BL21 (DE3), respectively, resulting in E-SIP and G-SIP proteins. The expressed G-SIP protein is used as an immunogen to immunize female BALB / c mice, generating a monoclonal antibody. Then, using the E-SIP protein as a screening antigen, combined with a rapid high-affinity monoclonal antibody screening technique, a monoclonal antibody capable of recognizing Group B Streptococcus SIP protein with high affinity is obtained. The mouse hybridoma cell line secreting this monoclonal antibody is named 3H617. This monoclonal antibody mouse hybridoma cell line was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 18, 2025, with accession number CGMCC No. 46596.

[0027] Simultaneously, a polyclonal antibody against group B streptococcal SIP protein in rabbits was prepared using the aforementioned E-SIP protein as an immunogen. Using the aforementioned G-SIP protein as a coating antigen, the titer of the prepared polyclonal antibody against group B streptococcal SIP protein in rabbits was determined to be 1:1024000 by indirect enzyme-linked immunosorbent assay (ELISA).

[0028] The inventors amplified and sequenced the gene sequence of the monoclonal antibody secreted by the mouse hybridoma cell line CGMCC No. 46596. Then, using public software from the National Center for Biotechnology Information (NCBI) website, they analyzed the immunoglobulin domain sequence of this monoclonal antibody and found that its heavy chain variable region has 116 amino acids, specifically: EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGHGLEWIGYINPYNDGTKYNENFKGKATLSSDKSSNTAYMELSSLTSEDSAVYYCARTVTTVPDYWGQGTTLTVS (SEQ ID NO. 1). Specifically, CDR1 is located at 26-33 aa, with the amino acid sequence GYTFTSYV (SEQ ID NO. 2); CDR2 is located at 51-58 aa, with the amino acid sequence INPYNDGT (SEQ ID NO. 3); and CDR3 is located at 97-106 aa, with the amino acid sequence ARTVTTVPDY (SEQ ID NO. 1). (SEQ ID NO.4). The light chain variable region has 105 amino acids, with the specific sequence as follows: IVLTQSPAIMSASPGEKVTMTCSASSSIGHIHWYQQRPGTSPKRWIFDTSNLASGVPARFSGSGSGTSYSLIISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK (SEQ ID NO.5), where CDR1 is located at 26-30 aa with the amino acid sequence SSIGH (SEQ ID NO.6); CDR2 is located at 48-50 aa with the amino acid sequence DTS; and CDR3 is located at 87-95 aa with the amino acid sequence HQRSSYPWT (SEQ ID NO.7).

[0029] The inventors have established enzyme-linked immunosorbent assay (ELISA) and fluorescence immunochromatography (FIC) techniques for detecting Group B Streptococcus (GBS) SIP protein based on the double-antibody sandwich principle using the monoclonal and polyclonal antibodies of this invention. Detection of SIP protein and GBS strains showed that the limit of detection (LOD) for SIP protein using the ELISA method was 0.32 ng / mL, and the LOD for SIP protein using the FIC was 0.08 ng / mL. The LOD for GBS bacterial culture reached 1 × 10⁻⁶. 3 The CFU / mL level was significantly higher than the limits of detection for SIP protein (0.5-1.0 ng / mL) and GBS bacterial suspension (10 ng / mL) reported in existing detection reagents and literature. 5 -10 6 The limit of detection for CFU / mL.

[0030] As is well known in the art, although the CDR regions of the antibody heavy chain and light chain are important amino acid sequence regions for recognizing and binding to corresponding antigens, conserved amino acid substitution is a biotechnological means in protein engineering to maintain the functional properties of proteins by replacing amino acid residues of the same family with similar physicochemical properties. This method mainly involves the directional substitution between amino acids of the same family, thereby ensuring that the binding affinity and specificity of the protein do not change significantly after substitution. In this patent application, the conserved amino acid substitution includes the substitution between aromatic amino acids Phe, Trp, and Tyr; the substitution between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; the substitution between polar amino acids Gln and Asn; the substitution between basic amino acids Lys, Arg, and His; the substitution between acidic amino acids Asp and Glu; and the substitution between hydroxyl amino acids Ser and Thr. The conserved substitution of a single amino acid in the amino acid sequences of the heavy chain CDR region and the light chain CDR region should not change the structure of the protein. Therefore, the conserved substitution of a single amino acid in the above-mentioned regions may still have the property of binding to the corresponding antigen. Therefore, monoclonal antibodies or their antigen-binding fragments obtained by making a conserved substitution of one amino acid in heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 and / or light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 can still recognize Group B Streptococcus SIP protein.

[0031] Those skilled in the art can also use existing techniques to prepare various antibody fragments, i.e., antigen-binding fragments, capable of recognizing Group B Streptococcus SIP proteins, from the monoclonal antibodies of the present invention described above, such as, but not limited to, Fab, Fab', and F(ab')2. The Fab fragment is a region in the antibody structure that can bind to the antigen, consisting of a complete light chain and a variable region VH and a constant region CH1 domain (Fd segment) of the heavy chain. Both the light and heavy chains have a constant region and a variable region, and disulfide bonds link the light and heavy chains. The antigen-binding fragments can be prepared as follows: for example, after enzymatic digestion with papain, antibody IgG is degraded into two Fab fragments and one Fc fragment. Under the action of pepsin, antibody IgG is degraded into one F(ab')2 fragment and one Fc fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. Because the above antigen-binding fragments can still bind the corresponding antigens, they can be used to prepare products for detecting Group B Streptococcus SIP proteins.

[0032] Those skilled in the art can also prepare single-chain antibodies (scFv) from the monoclonal antibodies of the present invention using existing techniques. A single-chain antibody is an antibody composed of a heavy chain variable region and a light chain variable region linked by a short peptide linker of several amino acids; it has only one chain and is a synthetically produced antibody. A single-chain antibody may also contain only the heavy chain variable region. The length and amino acid composition of the short peptide linker are well known in the art, and usable short peptide linkers for the monoclonal antibodies of the present invention can be determined through simple, repeatable experiments. The single-chain antibody can be expressed, for example, in *Escherichia coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the characteristic of recognizing Group B Streptococcus SIP protein and can be applied to the detection of Group B Streptococcus SIP protein.

[0033] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable region of monoclonal antibodies that recognize Group B Streptococcus SIP proteins based on the aforementioned high-affinity recognition. They can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors that express monoclonal antibodies or antigen-binding fragments of the high-affinity recognition of Group B Streptococcus SIP proteins. Furthermore, those skilled in the art can introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as mammalian cells, bacteria, or yeast to obtain mammalian cell recombinants, bacterial recombinants, or yeast recombinants, and then express these recombinants to produce the antibodies or antigen-binding fragments of the present invention. The antibodies or antigen-binding fragments expressed in this way can recognize Group B Streptococcus SIP proteins; therefore, the aforementioned nucleic acid molecules, expression vectors, and mammalian cell recombinants, bacterial recombinants, or yeast recombinants are within the scope of protection of the claims of this invention. Moreover, the above-described techniques are all well-known in the art and can be carried out by those skilled in the art without inventive effort.

[0034] As described above, the antibody or its antigen-binding fragment of the present invention can recognize Group B Streptococcus SIP protein with high affinity, and therefore can be used to prepare a kit for detecting Group B Streptococcus SIP protein. The kit can be any kit that utilizes the antibody or its antigen-binding fragment of the present invention to react with Group B Streptococcus SIP protein, such as, but not limited to, kits using enzyme-linked immunosorbent assay (ELISA), chemiluminescence, fluorescence immunochromatography, colloidal gold immunochromatography, Western blotting, and immunohistochemistry.

[0035] To explain in detail the technical content, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments.

[0036] Example 1: Prokaryotic expression of Group B Streptococcus SIP protein

[0037]

[0038] The pET-SIP recombinant expression plasmid with the correct inserted sequence, verified by sequencing, was transformed into E. coli Rosetta (DE3) competent cells. Single colonies were picked and cultured in 5 mL of LB liquid medium containing ampicillin sodium at 37°C with shaking overnight. The next day, the cells were inoculated into 250 mL of fresh LB liquid medium and cultured until the logarithmic growth phase. The temperature was then adjusted to 16°C, and after 30 minutes, 150 μl of 1 mol / L IPTG induction solution was added. The cells were induced at 16°C for 12-14 h. The induced bacterial cells were collected by centrifugation, resuspended in 25 mmol / L Tris-HCl (pH 8.5), and sonicated. The cells were then centrifuged at 20,000 g for 30 min at 4°C. The supernatant was collected for Ni column purification. First, the Ni column was equilibrated with equilibration buffer (25 mmol / L TE, 6 mol / L urea, pH 8.5). The supernatant was added to the Ni column, and after complete sample incorporation, the target protein was collected by elution with wash buffers containing 25 mmol / L and 250 mmol / L imidazole, respectively. The 250 mmol / L imidazole wash buffer eluted most of the protein. SDS-PAGE gel electrophoresis was performed, and the results are shown below. Figure 1 As shown, the SIP protein expressed by the recombinant expression plasmid pET-SIP is soluble, with a molecular weight of approximately 50 kDa, and is labeled E-SIP. Similarly, the pGEX-SIP recombinant expression plasmid, verified by sequencing to have the correct inserted sequence, was transformed into BL21(DE3) competent cells, and the induction conditions were the same as above. The induced bacterial cells were collected by centrifugation, resuspended in PBS (pH 7.4), and sonicated. The supernatant was collected by centrifugation at 20000g for 30 min at 4℃ and purified using a glutathione agarose gel 4B column. The target protein was eluted with 10 mmol / L reduced glutathione and collected for SDS-PAGE gel electrophoresis. The results are shown below. Figure 1 As shown, the SIP protein expressed by the recombinant expression plasmid pGEX-SIP is also soluble, with a molecular weight of approximately 75 kDa, and is labeled as G-SIP.

[0039] Example 2: Screening and preparation of high-affinity monoclonal antibodies against Group B Streptococcus SIP protein

[0040] Using the G-SIP protein prepared in Example 1 as the immunogen, 6-8 week old female BALB / c mice were immunized with 100 µg of antigen per mouse plus an equal volume of Freund's complete adjuvant. After thorough emulsification with a stirrer, the mice were immunized subcutaneously in the back and intraperitoneally, with 3 mice immunized in total. A second immunization was performed 4 weeks later, and a third immunization was performed 8 weeks later. For each third immunization, 50 µg of antigen per mouse plus incomplete Freund's adjuvant was emulsified thoroughly with a stirrer and then injected subcutaneously in the back and intraperitoneally. One week after the third immunization, blood was collected from the tail vein of the mice to detect the titer of the immune serum. Mice with the highest titer were selected for a booster immunization via intraperitoneal injection (50 µg per mouse). Spleen cells were harvested 3 days later for fusion. SP20 myeloma cells were resuscitated and cultured until they entered the logarithmic growth phase. Spleen cells were prepared from the spleens of the immunized BALB / c mice. Spleen cells and myeloma cells were mixed at a ratio of 9:1 in serum-free DMEM medium, centrifuged at 1500 rpm for 5 minutes, the supernatant was aspirated, and the cells were gently shaken to disperse them. The cells were then fused in a 37°C water bath. 1 mL of preheated 50% PEG fusion cells was added within 1 minute, while gently shaking to mix. After the addition was complete, the cells were allowed to stand for 90 seconds, and serum-free DMEM medium was added to terminate the fusion. The cells were then incubated at 37°C for 10 minutes, centrifuged at 1500 rpm for 5 minutes, and the pellet was resuspended in HAT medium. The pellet was then aliquoted into 96-well cell culture plates containing feeder cells and cultured in a 37°C, 5% CO2 cell culture incubator for 5 days. The medium was changed once with HAT medium. The medium was changed again on day 10. When the fused cells covered about 60% of the bottom of the wells, the cell culture supernatant was collected. A rapid screening method was used to screen for high-affinity positive clones. The earlier a significant color change occurred during the reaction, the higher the affinity of the monoclonal antibody secreted by the positive clone for the SIP protein. The specific method is as follows: Dilute the E-SIP protein prepared in Example 1 with carbonate coating buffer to a concentration of 2.0 μg / ml, coat 150 μl per well, and incubate overnight at 4°C; wash the plate twice with washing buffer; add 200 μl / well blocking buffer and block at room temperature for 6 hours; wash the plate 5 times with washing buffer. After adding 100 μl of sample dilution buffer to each well, add 10 μl of cell culture supernatant, and incubate with shaking at room temperature for 15 min, then discard the supernatant. Wash the plate 5 times, invert the washed ELISA plate on absorbent paper to dry, add 100 μl / well of HRP-labeled goat anti-mouse IgG antibody, and incubate with shaking at room temperature for 15 min. Wash the plate 5 times. Add 50 μl each of TMB chromogenic solution A and B to each well, and develop the color at room temperature in the dark. Continuously observe during this time, and select the positive clone that first shows a significant color change to determine the positive clone secreting high-affinity monoclonal antibody. Name the cell line secreting high-affinity monoclonal antibody 3H617.

[0041] Hybridoma cells from 3H617 mice were cultured in 1640 medium containing 10% fetal bovine serum. Each male BALB / c mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. After 10 days, cells were collected and resuspended in 10 mL of physiological saline at a cell density of 1 × 10⁻⁶ cells / mL. 7 The antibody concentration was 0.5 mL / mL, and each mouse was injected intraperitoneally. Two weeks later, ascites fluid was collected and the antibody was purified using the Melon Gel Monoclonal IgG Purification Kit (PIERCE, catalog number 45214). The purified antibody was aliquoted into 1 mg vials and stored at -20°C.

[0042] Example 3: Preparation of rabbit polyclonal antibody against group B streptococcus SIP protein

[0043] Polyclonal antibodies against Group B Streptococcus SIP protein were prepared using the E-SIP protein prepared in Example 1 as an immunogen. The specific preparation process is as follows: One healthy New Zealand white rabbit was selected, and 1.0 mg of the E-SIP protein prepared in Example 1 was mixed with 1.0 mL of Freund's complete adjuvant. After thorough emulsification with a stirrer, 0.2 mL was injected subcutaneously at both sides of the rabbit's spine. Four weeks later, 1.0 mg of E-SIP protein and 1.0 mL of Freund's incomplete adjuvant were mixed and thoroughly emulsified with a stirrer, and a second immunization was performed at different points on the above sites. A third booster immunization was performed four weeks later to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After the blood clots clumped and contracted, the blood was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored at -20°C for later use. The indirect ELISA method for determining the titer of polyclonal antibodies is performed as follows: The enzyme-linked plate is coated with G-SIP protein prepared in Example 1 at a concentration of 2.0 μg / ml, with 150 μL coated per well, and incubated overnight at 4°C; the plate is washed twice with washing buffer; 200 μL / well blocking buffer is added and the plate is blocked at room temperature for 6 hours; the plate is washed 5 times with washing buffer. Rabbit serum was diluted with PBS at dilution ratios of 1:2000, 1:8000, 1:32000, 1:128000, 1:512000, 1:1024000, 1:2048000, and 1:4096000, with 100 μL added to each well. The plate was incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer, 200 μL per well. HRP-labeled goat anti-rabbit secondary antibody was added and incubated at 37°C for 45 min. The plate was washed 5 times with washing buffer, 200 μL per well. Freshly prepared substrate solution was added, 100 μL per well, and the plate was incubated at 37°C for 10 min. The reaction was terminated by adding 50 μL of 2 M H2SO4 to each well. The absorbance of each well was measured using a microplate reader at a wavelength of 450 nm, and the readings were taken within 10 minutes after termination. The results are shown in Table 1. Using pre-immunization rabbit serum (1:2000 dilution) as a negative control, the titer of the prepared rabbit anti-Group B Streptococcus SIP protein polyclonal antibody reached 1:1024000.

[0044] Table 1. Results of rabbit anti-Group B Streptococcus SIP protein polyclonal antibody titer determination (OD450nm)

[0045] Dilution ratio OD450nm 1:2000 3.761 1:8000 3.004 1:32000 2.173 1:128000 0. 924 1:512000 0.338 1:1024000 0.159 1:2048000 0.081 1:4096000 0.047 negative control 0.056

[0046] Example 4: Establishment of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for Group B Streptococcus SIP protein.

[0047] Using the high-affinity monoclonal antibody 3H617 against Group B Streptococcus SIP protein prepared in this invention as the capture antibody, and the rabbit polyclonal antibody against Group B Streptococcus SIP protein prepared in Example 3 as the detection antibody, a protein-linked immunosorbent assay (ELISA) method for detecting Group B Streptococcus SIP protein based on the double-antibody sandwich principle was established. The specific steps were as follows: The ELISA plate was coated with monoclonal antibody 3H617 at a concentration of 2.0 μg / mL, 100 μL per well, incubated overnight at 4°C, and washed twice with washing buffer; 120 μL / well blocking buffer was added and the plate was blocked at room temperature for 6 hours, followed by washing five times with washing buffer; the E-SIP protein expressed in prokaryotes in Example 1 was serially diluted with double-distilled water to concentrations of 1000, 200, 40, 8.0, 1.6, 0.32, 0.064, and 0 ng / mL. 100 μL of each diluted protein was added to the wells, incubated at 37°C for 45 min, and the diluted protein was discarded. Wash the plate 5 times with washing buffer. Add 100 μL of horseradish peroxidase-labeled rabbit anti-Group B Streptococcus SIP protein polyclonal antibody to each well and incubate at 37°C for 30 min. Wash the plate 5 times, blot dry, and add 50 μL each of TMB chromogenic solutions A and B to each well. Incubate at room temperature in the dark for 15 min. Add 50 μL of 2 M H₂SO₄ stop solution per well to stop the reaction. Measure the OD value of each well at a wavelength of 450 nm using a microplate reader, and read the value within 10 minutes after termination. Each concentration of sample was tested 5 times. Results are as follows. Figure 2 The figure shows the limit of detection (LOD) of the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for SIP protein. Using the negative control average + 3SD as the cutoff value (Cutoff = 0.070), the LOD of the double-antibody sandwich ELISA established using the high-affinity monoclonal antibody 3H617 against Group B Streptococcus SIP protein prepared in this invention as the capture antibody can reach 0.32 ng / mL, significantly higher than the 1.0 ng / mL reported in the literature (Shiliang Cheng, Jiae Han, Yidan Huang, et al. The correlation between expression of sip protein in different serotypes of groupb streptococcus and diagnosis. Heliyon 5 (2019) e01899. https: / / doi.org / 10.1016 / j.heliyon.2019.e01899.).

[0048] Example 5: Establishment of a double-antibody sandwich fluorescence immunochromatographic assay for Group B streptococcal SIP protein

[0049] Based on the double-antibody sandwich principle, fluorescent microspheres were labeled with the high-affinity monoclonal antibody 3H617 against Group B Streptococcus SIP protein of this invention, and a nitrocellulose membrane was coated with the rabbit anti-Group B Streptococcus SIP protein polyclonal antibody prepared in this invention. A fluorescence immunochromatographic assay was established to detect the content of SIP protein and evaluate the limit of detection. Specifically, 100 µL of serially diluted E-SIP protein expressed in prokaryotes in Example 1 (1000, 200, 40, 8.0, 1.6, 0.32, 0.16, 0.08, 0.04, 0.02 ng / mL) was vertically added to the sample loading point of the detection card and allowed to react at room temperature for 15 min. The SIP protein in the sample binds to the fluorescent microsphere-labeled high-affinity monoclonal antibody 3H617 against group B streptococcus SIP protein to form an antigen-antibody immune complex. The immune complex and the fluorescent microsphere-labeled chicken IgY antibody move forward along the nitrocellulose membrane during chromatography. When they reach the detection zone (T line), the reaction complex is captured by the rabbit anti-group B streptococcus SIP protein polyclonal antibody on the detection line of the nitrocellulose membrane. The fluorescent microsphere-labeled chicken IgY antibody is captured in the control zone (C line) by the goat anti-chicken IgY antibody coated on the nitrocellulose membrane. The AFS-1000 dry fluorescence immunoassay analyzer was used for testing. After the sample was added to the various wells of the reagent card, it migrated to the chromatography zone by capillary action. At the test and control lines, fluorescent microsphere-labeled particles accumulated due to antigen-antibody reactions, forming complexes and either forming or not forming fluorescent microsphere reaction bands. Under the excitation light source, the fluorescent material in the microspheres emitted fluorescence signals of specific wavelengths. The fluorescence immunoassay analyzer captured these signals and calculated the ratio of the T-line fluorescence value to the C-line fluorescence value (T / C) through signal conversion. The amount of SIP protein in the sample is positively correlated with the signal intensity of the fluorescent antibody. The cutoff value was calculated as three times the T / C value of the sample dilution. The ratio of the sample's T / C value to the cutoff value, i.e., the S / CO value, was then used to detect SIP protein. An S / CO value ≥ 1 indicates a positive result; an S / CO value < 1 indicates a negative result. Each concentration of sample was tested five times.The 3H617 monoclonal antibody of this invention was used as the capture antibody. The limit of detection for SIP protein using a double-antibody sandwich fluorescence immunochromatographic assay was 0.08 ng / mL, which is significantly higher than the 1.0 ng / mL reported in the literature (Shiliang Cheng, JiaeHan, Yidan Huang, et al. The correlation between expression of sip protein indifferent serotypes of group b streptococcus and diagnosis. Heliyon 5 (2019)e01899. https: / / doi.org / 10.1016 / j.heliyon.2019.e01899.). The results are shown in Table 2.

[0050] Table 2. Limit of Detection for SIP Protein Double Antibody Sandwich Fluorescent Immunochromatographic Assay

[0051] SIP concentration (ng / mL) S / CO value 1000 19.013±2.345 200 12.254±2.296 40 8.653±1.112 8.0 6.970±1.107 1.6 4.605±0.349 0.32 2.038±0.227 0.16 1.762±0.114 0.08 1.294±0.091 0.04 0.743±0.057 0.02 0.619±0.063

[0052] SIP protein in bacterial culture was detected using the fluorescence immunochromatographic assay described above. First, group III Streptococcus B (ATCC 12403™) was serially diluted with PBS to a concentration of 1×10⁻⁶. 7 CFU / mL, 1×10 6 CFU / mL, 1×10 5 CFU / mL, 1×10 4 CFU / mL, 1×10 3 CFU / mL, 1×10 2 CFU / mL, 1×10 1CFU / mL. Sample processing steps are as follows: 1) Add 250 μL of extraction solution A (0.5 M NaOH, 0.02% Triton X-100) to the sample extraction tube. 2) Dip a vaginal swab into the bacterial dilution solution and place it into extraction solution A, incubate at room temperature for 3 minutes. 3) Add 250 μL of extraction solution B (0.6 M TAPS, 0.15 M HCl, 0.02% Triton X-100), and mix thoroughly. 4) Shake the swab for a few seconds and repeatedly squeeze and rotate it against the tube wall; remove the swab after squeezing out the liquid, and the extract in the tube is ready for detection. Vertically add 100 μL of extract into the sample well, incubate at room temperature for 15 min, and then perform the detection. An S / CO value ≥ 1 indicates a positive result; an S / CO value < 1 indicates a negative result. The results are shown in Table 3. Using the 3H617 monoclonal antibody of this invention as the capture antibody, the limit of detection for Group B Streptococcus bacterial suspensions in the double-antibody sandwich fluorescence immunochromatographic assay was 1 × 10⁻⁶. 3 CFU / mL, significantly higher than the reported 1×10⁻⁶ in the literature. 5 CFU / mL (Shiliang Cheng, Jiae Han, Yidan Huang, et al. The correlationbetween expression of sip protein in different serotypes ofgroup bstreptococcus and diagnosis. Heliyon 5 (2019) e01899. https: / / doi.org / 10.1016 / j.heliyon.2019.e01899.).

[0053] Table 3. Limit of Detection for Group B Streptococcus Double Antibody Sandwich Fluorescent Immunochromatographic Assay

[0054] Bacterial concentration (CFU / mL) <![CDATA[1×10 7 ]]> <![CDATA[1×10 6 ]]> <![CDATA[1×10 5 ]]> <![CDATA[1×10 4 ]]> <![CDATA[1×10 3 ]]> <![CDATA[1×10 2 ]]> <![CDATA[1×10 1 ]]> S / CO value 11.247 4. 079 2.169 1.798 1.332 0.751 0.445

[0055] Example 6: Isotype analysis of monoclonal antibody 3H617 against group B streptococcus SIP protein

[0056] The heavy and light chain isotypes of mouse antibodies were identified using the rapid mouse antibody subtype detection card (catalog number THJ-ISO-M8a, batch number 052725) from Antaiji (Beijing) Biotechnology Co., Ltd. 80 μL of the supernatant from the culture of mouse hybridoma cell line 3H617 was added to the sample wells of the rapid mouse antibody subtype detection card. After standing for 5-10 min, the results were observed and recorded. The results are as follows: Figure 3 As shown, the anti-Group B Streptococcus SIP protein monoclonal antibody 3H617 is mouse IgG1 subtype, and the antibody light chain is Igκ subtype.

[0057] Example 7: Determination of the amino acid sequence of the variable region of the anti-Group B Streptococcus SIP protein monoclonal antibody 3H617

[0058] Mouse hybridoma cell line 3H617 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. After reverse transcription of cDNA, PCR amplification was performed using primers for the Fab fragment of mouse monoclonal antibodies synthesized by Beijing Qingke Biotechnology Co., Ltd. The cells were preheated at 95℃ for 2 min, followed by 30 cycles of 95℃ for 30 seconds, 58℃ for 30 seconds, and 72℃ for 30 seconds, with a final extension at 72℃ for 5 min. The resulting cells were ligated into the pMD18-T vector and transformed into *E. coli* JM109. Positive clones were selected for sequencing. The sequenced data was compared with the mouse-derived monoclonal antibody CDR region sequence using IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) in the NCBI website's BLAST module.

[0059] Sequence analysis revealed that the heavy chain variable region consists of 116 amino acids, with the following sequence: EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGHGLEWIGYINPYNDGTKYNENFKGKATLSSDKSSNTAYMELSSLTSEDSAVYYCARTVTTVPDYWGQGTTLTVS (SEQ ID NO.1). Among these, CDR1 is located at 26-33 aa, with the amino acid sequence GYTFTSYV (SEQ ID NO.2); CDR2 is located at 51-58 aa, with the amino acid sequence INPYNDGT (SEQ ID NO.3); and CDR3 is located at 97-106 aa, with the amino acid sequence ARTVTTVPDY (SEQ ID NO.4). The light chain variable region has an amino acid sequence of 105 amino acids, as follows: IVLTQSPAIMSASPGEKVTMTCSASSSIGHIHWYQQRPGTSPKRWIFDTSNLASGVPARFSGSGSGTSYSLIISSMEAEDAATYYCHQRSSYPWTFGGGTKLEIK (SEQ ID NO.5), where CDR1 is located at 26-30 aa and has the amino acid sequence SSIGH (SEQ ID NO.6); CDR2 is located at 48-50 aa and has the amino acid sequence DTS; and CDR3 is located at 87-95 aa and has the amino acid sequence HQRSSYPWT (SEQ ID NO.7).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof with high affinity for recognizing Group B Streptococcus SIP protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, and the light chain variable region comprises light chain CDR1, light chain CDR2, and light chain CDR3, characterized in that, The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.4; The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the light chain CDR2 is DTS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO.

5.

3. The monoclonal antibody according to claim 2, characterized in that, It is secreted by the monoclonal antibody mouse hybridoma cell line 3H617 with accession number CGMCC No.46596.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or antigen-binding fragment is a Fab fragment, Fab' fragment, F(ab')2 fragment, single-chain antibody, or humanized antibody.

5. A nucleic acid molecule, characterized in that, It comprises a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A recombinant, characterized in that, It comprises the nucleic acid molecule of claim 5 or the expression vector of claim 6.

8. The recombinant according to claim 7, characterized in that, It is a mammalian cell recombinant, a bacterial recombinant, or a yeast recombinant.

9. A mouse hybridoma cell line that secretes a monoclonal antibody with high affinity for recognizing Group B Streptococcus SIP protein, characterized in that, It is the monoclonal antibody mouse hybridoma cell line 3H617 with accession number CGMCC No.46596.

10. The use of the high-affinity monoclonal antibody or antigen-binding fragment thereof for recognizing Group B Streptococcus SIP protein as described in any one of claims 1 to 4 in the preparation of a kit for detecting Group B Streptococcus SIP protein.

11. A kit for detecting SIP protein in Group B Streptococcus, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

12. The kit according to claim 11, characterized in that, It is a detection kit using a double-antibody sandwich method, wherein the monoclonal antibody or its antigen-binding fragment serves as the capture antibody.

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