Mycobacterium tuberculosis secretory protein 64 monoclonal antibody and application thereof

Abstract

The invention discloses a monoclonal antibody capable of simultaneously identifying mycobacterium tuberculosis secretory protein 64 of a wild type (W-MPT64) and a 63bp deletion mutant type (D-MPT64). The amino acid sequences of variable regions of a heavy chain and a light chain of the antibody are shown as SEQ ID NO.1 and SEQ ID NO.5. The lowest detection limits of W-MPT64 and D-MPT64 by a mycobacterium tuberculosis MPT64 double-antibody sandwich enzyme-linked immunosorbent assay and a fluorescence immunochromatography assay established by using the monoclonal antibody both reach 1.5625 pg / mL, which are significantly higher than the lowest detection limit in the prior art, and the monoclonal antibody can be used for qualitative and quantitative detection of mycobacterium tuberculosis secretory protein 64.

Description

Technical Field

[0001] This invention relates to the field of medical immunoassay technology, specifically to a dual-recognition monoclonal antibody that simultaneously recognizes wild-type Mycobacterium tuberculosis secretory protein 64 (W-MPT64) and 63bp deletion mutant Mycobacterium tuberculosis secretory protein 64 (D-MPT64) and its application in immunoassay. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). The internationally recognized gold standard for TB diagnosis is a positive MTB culture in sputum, bronchoalveolar lavage fluid, or other body fluids. However, MTB cultures take at least 3-4 weeks to produce results and have a low positive rate. Acid-fast staining can identify mycobacteria, but it cannot differentiate between non-tuberculous mycobacteria (NTM) infections with similar clinical symptoms. While the tuberculin skin test is simple to perform, its sensitivity and specificity are low, and it cannot distinguish between BCG vaccination and MTB or NTM infection. Nucleic acid molecular detection methods have high sensitivity and specificity, but they are complex to perform and require specialized equipment, making them unsuitable for widespread use in primary healthcare institutions. There are three types of tuberculosis diagnostic methods based on immunological principles. The first type uses antibodies produced by humoral immunity as the detection target, but the positive rate of existing reagents cannot meet clinical needs and cannot distinguish between current and past infections. The second type is the gamma-interferon release assay based on cellular immunity, which also cannot distinguish between current and past infections. The third type uses MTB-specific antigens as the detection target. After the body is infected with MTB, MTB-specific antigens appear earlier than antibodies, which is direct evidence of active MTB infection. At the same time, it can avoid false negatives in humoral or cellular immunity tests due to low immune response in patients.

[0003] Current MTB antigen detection reagents primarily target Mycobacterium tuberculosis secreted protein 64 (MPT64). MPT64, also known as MPB64, is encoded by the Rv1980c (mpt64) gene in the differential coding region RD2. It has a full length of 228 amino acids and a molecular weight of approximately 24 kDa. The advantages of MPT64 as an MTB antigen detection target are mainly threefold: First, MPT64 is one of the main proteins secreted by MTB during its growth and reproduction phase, accounting for 8% of the total secreted proteins, making it relatively easy to detect; second, MPT64 is present in actively replicating bacterial cells and is secreted in large quantities in the early stages of culture, making it a specific marker for early and active MTB infection; third, MPT64 is secreted only in MTB and not in NTM, making it a specific marker for MTB. However, in clinical testing, some culture-positive samples still show negative results for MPT64 antigen, leading to missed detection and delayed treatment. The reason for false negatives in MPT64 antigen testing lies in the polymorphism of the MPT64 protein, including nucleotide deletion mutations at positions 197-259 of the MPT64 protein gene leading to the deletion of 21 amino acids from positions 66-86 (63 bp deletion), single nucleotide mutations resulting in single amino acid sense or nonsense mutations, frameshift mutations resulting from single amino acid insertions or deletions, and amino acid sequence changes caused by the insertion of the IS6110 fragment. According to literature reports, the 63 bp deletion mutation accounts for over 90% of all mutations and is the main cause of negative MPT64 antigen tests. This 63 bp deletion mutation alters the tertiary structure of the MPT64 protein, causing α-helix defects and increased coiling of adjacent β-fragments, thus preventing recognition by monoclonal antibodies that specifically bind to wild-type MPT64 protein, ultimately leading to false negative results in existing MPT64 antigen tests.

[0004] To address the above shortcomings, this invention prepares and screens dual-recognition monoclonal antibodies capable of simultaneously recognizing wild-type MPT64 (W-MPT64) and 63bp-deleted MPT64 (D-MPT64), enabling simultaneous detection of two MPT64 proteins, avoiding missed detections, reducing the false negative rate of existing reagents, and improving the detection rate. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a monoclonal antibody prepared using mouse hybridoma technology that can simultaneously recognize wild-type MPT64 (W-MPT64) and 63 bp deletion mutant MPT64 (D-MPT64) (in this patent application, the antibody may also be referred to as W-MPT64 and D-MPT64 dual-recognition monoclonal antibody or dual-recognition monoclonal antibody). The monoclonal antibody can be used for qualitative and quantitative detection of Mycobacterium tuberculosis MPT64 protein, as well as for the preparation of detection reagents for detecting Mycobacterium tuberculosis infection.

[0006] Therefore, a first aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that simultaneously recognizes wild-type MPT64 (W-MPT64) and 63 bp deletion mutant MPT64 (D-MPT64), 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 LVS;

[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 can simultaneously recognize W-MPT64 and D-MPT64 because they retain the variable regions of the light chain and the heavy chain, or only retain the variable region of the heavy chain.

[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 a dual-recognition monoclonal antibody that simultaneously recognizes wild-type MPT64 (W-MPT64) and 63 bp deletion mutant MPT64 (D-MPT64). Further, the monoclonal antibody mouse hybridoma cell line is monoclonal antibody mouse hybridoma cell line 6112, with accession number CGMCC No. 46599.

[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 Mycobacterium tuberculosis MPT64 protein.

[0020] A seventh aspect of this invention relates to a kit for detecting MPT64 protein in Mycobacterium tuberculosis, the kit comprising the aforementioned monoclonal antibody or its antigen-binding fragment for simultaneously recognizing W-MPT64 and D-MPT64. Further, the kit is a double-antibody sandwich type kit, wherein the monoclonal antibody or its antigen-binding fragment is used as a capture antibody or a detection antibody. Further still, the kit is an enzyme-linked immunosorbent assay (ELISA) kit or a fluorescence immunochromatographic assay kit.

[0021] Instructions for the Preservation of Biological Materials

[0022] The monoclonal antibody mouse hybridoma cell line 6112 of this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC), with the collection number CGMCC No. 46599, deposit date of October 15, 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 the W-MPT64 and D-MPT64 proteins expressed in prokaryotic Mycobacterium tuberculosis, where M is the molecular weight standard; 1 is the W-MPT64 protein expressed by the recombinant expression vector pBV-W-MPT64; and 2 is the D-MPT64 protein expressed by the recombinant expression vector pGEX-D-MPT64.

[0024] Figure 2This is a graph showing the limit of detection (LOD) results of a sandwich enzyme-linked immunosorbent assay (ELISA) for MPT64 protein in Mycobacterium tuberculosis. Figure A shows the antibody group using the anti-W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 as the capture antibody and the HRP-labeled rabbit anti-MPT64 protein polyclonal antibody as the detection antibody. Figure B shows the antibody group using the rabbit anti-MPT64 protein polyclonal antibody as the capture antibody and the HRP-labeled 6112 monoclonal antibody as the detection antibody.

[0025] Figure 3 This is a diagram showing the identification results of the W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 subtype. Detailed Implementation

[0026] The purpose of this invention is to provide a mouse hybridoma cell line prepared using mouse hybridoma technology that can secrete dual-recognition monoclonal antibodies that simultaneously recognize wild-type Mycobacterium tuberculosis secretory protein 64 (W-MPT64) and 63bp deletion mutant Mycobacterium tuberculosis secretory protein 64 (D-MPT64). Specifically, the preparation process involves first expressing W-MPT64 and D-MPT64 in prokaryotic cells of *Escherichia coli*. Then, BALB / c female mice were cross-immunized with W-MPT64 and D-MPT64 as immunogens to generate monoclonal antibodies. A dual-recognition monoclonal antibody capable of simultaneously recognizing both W-MPT64 and D-MPT64 with high affinity was obtained through screening. This antibody is also referred to in this patent application as monoclonal antibody 6112 that simultaneously recognizes W-MPT64 and D-MPT64, or dual-recognition monoclonal antibody 6112, or monoclonal antibody 6112. The mouse hybridoma cell line secreting this monoclonal antibody is named 6112 or monoclonal antibody mouse hybridoma cell line 6112 in this patent application. The inventors deposited this monoclonal antibody mouse hybridoma cell line at the China General Microbiological Culture Collection Center (CGMCC) on October 15, 2025, with accession number CGMCC No. 46599, and classified it as a monoclonal antibody mouse hybridoma cell line.

[0027] Meanwhile, using the aforementioned prokaryotically expressed W-MPT64 and D-MPT64 as mixed immunogens, a rabbit polyclonal antibody against Mycobacterium tuberculosis MPT64 protein was prepared. The detection titer of this polyclonal antibody against both W-MPT64 and D-MPT64 reached 1:2048000.

[0028] Next, using the prepared dual-recognition monoclonal antibody 6112 for W-MPT64 and D-MPT64 and a rabbit anti-MPT64 polyclonal antibody, the inventors established enzyme-linked immunosorbent assay (ELISA) and fluorescence immunochromatography (FIC) techniques for detecting Mycobacterium tuberculosis MPT64 protein based on the double-antibody sandwich principle. Both detection methods achieved a limit of detection (LOD) of 1.5625 pg / mL for prokaryotically expressed W-MPT64 and D-MPT64 proteins. The LOD of the Mycobacterium tuberculosis MPT64 double-antibody sandwich FIC assay for both W-MPT64 and D-MPT64 expressed Mycobacterium tuberculosis cultures reached 1×10⁻⁶ pg / mL. 3 The CFU / mL level is significantly higher than the limit of detection reported by existing test reagents and literature. The positive detection time of Mycobacterium tuberculosis culture medium is 7 days, which is earlier than the positive detection time reported by existing test reagents and literature. It can be used for the qualitative and quantitative detection of Mycobacterium tuberculosis secretory protein 64.

[0029] The inventors amplified and sequenced the gene sequence of monoclonal antibody 6112 secreted by the mouse hybridoma cell line CGMCC No. 46599. 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 115 amino acids, specifically: EVKVVESGGGLVQPGGSMKLSCAAS GFTFSDAW MDWVRQSPEKGLEWIAE IRNKANDHAT YYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYC TTVPFDC WGQGTTLTVS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 26-33 amino acids, with the amino acid sequence GFTFSDAW (SEQ ID NO.2); CDR2 is located at 51-60 amino acids, with the amino acid sequence IRNKANDHAT (SEQ ID NO.3); and CDR3 is located at 99-105 amino acids, with the amino acid sequence TTVPFDC (SEQ ID NO.4). The light chain variable region has an amino acid sequence of 109 amino acids, as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVSTSG YSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTRSEGAPSWKS (SEQ ID NO.5) has three CDR regions marked with underscores. CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO.7).

[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 W-MPT64 and D-MPT64 simultaneously.

[0031] Those skilled in the art can also use existing techniques to prepare various antibody fragments capable of simultaneously recognizing W-MPT64 and D-MPT64 from the monoclonal antibodies of the present invention, i.e., antigen-binding fragments, 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 kits for detecting Mycobacterium tuberculosis MPT64 protein.

[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 *E. coli* using genetic engineering techniques. The single-chain antibody of the present invention prepared in this way has the characteristic of simultaneously recognizing W-MPT64 and D-MPT64, and therefore can be used to prepare kits for detecting the MPT64 protein in *Mycobacterium tuberculosis*.

[0033] Those skilled in the art can design and synthesize nucleic acid molecules encoding the variable regions of monoclonal antibodies that simultaneously recognize W-MPT64 and D-MPT64 based on the aforementioned amino acid sequences. They can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors capable of expressing monoclonal antibodies or their antigen-binding fragments that simultaneously recognize W-MPT64 and D-MPT64. 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 their antigen-binding fragments of the present invention. Since the expressed antibodies or their antigen-binding fragments can simultaneously recognize W-MPT64 and D-MPT64, 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 performed 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 simultaneously recognize W-MPT64 and D-MPT64, and therefore can be used to prepare a kit for detecting Mycobacterium tuberculosis MPT64 protein. The kit can be any kit that utilizes the antibody or its antigen-binding fragment of the present invention to react with Mycobacterium tuberculosis MPT64 protein, such as, but not limited to, double-antibody sandwich kits. Specific kits include, but are 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 W-MPT64 and D-MPT64 proteins in Mycobacterium tuberculosis

[0037] Because the wild-type W-MPT64 protein undergoes a 63bp deletion mutation, its tertiary structure is altered, resulting in α-helix defects and increased coiling of adjacent β-fragments, thus preventing it from being recognized by monoclonal antibodies that specifically bind to wild-type MPT64 protein. Therefore, this invention prepares prokaryotic expression of wild-type W-MPT64 protein and 63bp deletion mutant D-MPT64 protein, respectively.

[0038] The W-MPT64 protein of Mycobacterium tuberculosis H37Rv strain (GenBank: CAA53143.1) was searched in the GenBank database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ). It is 228 amino acids in length and its sequence is MRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACLIQMSDPAYNTNISLPSYYPDQKSLENYIAQTRDKFLSAATSSTPREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVHRSAIDSMLA (SEQ ID NO. 8). The 63bp deletion mutant D-MPT64 deletes amino acids 66-86, resulting in a full-length sequence of 207 amino acids. Its sequence is: MRIKIFMLVTAVVLLCCSGVATAAPKTYCEELKGTDTGQACQIQMSDPAYNINISLPSYYPDQKSREAPYELNITSATYQSAIPPRGTQAVVLKVYQNAGGTHPTTTYKAFDWDQAYRKPITYDTLWQADTDPLPVVFPIVQGELSKQTGQQVSIAPNAGLDPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVPRSAIDSMLA (SEQ ID NO).9). Based on the genetic code preferences of *E. coli*, the optimized nucleotide sequences suitable for expression in *E. coli* expression systems were derived. The optimized nucleotide sequence of W-MPT64 is (SEQ ID NO).10); The optimized nucleotide sequence of D-MPT64 is (SEQ ID NO. 11). The optimized nucleotide sequences of W-MPT64 and D-MPT64 were synthesized by Beijing Qingke Biotechnology Co., Ltd. For the expression of W-MPT64, the pBVIL expression vector was used, and the fusion protein was interleukin IL1, with a molecular weight of approximately 10 kDa. The upstream primer used for amplification (named W-MPT64-F) has the sequence GC. CTCGAG ATGCGTATCAAGATCTTC (SEQ ID NO.12) contains an XhoⅠ restriction site (indicated by an underline); the downstream primer (named W-MPT64-R) has the sequence GC. TCTAGAAGCGAGCATGCTATCGAT (SEQ ID NO.13) contains an XbaⅠ restriction site (indicated by an underline). For D-MPT64 expression, the pGEX-4T-2 expression vector was used, and the fusion protein was the glutathione S-transferase GST, with a molecular weight of approximately 26 kDa. The upstream primer used for amplification (named D-MPT64-F) had the sequence GC... GGATCC ATGCGTATCAAGATCTTC (SEQ ID NO.14) contains a BamH I restriction site (indicated by an underline); the downstream primer (named D-MPT64-R) has the sequence GC. GAATTC CGCAAGCATAGAGTCAAT (SEQ ID NO.15) contains an EcoR I restriction site (indicated by an underline). Recombinant expression vectors were constructed using techniques known in the field; the constructed recombinant expression vectors are named pBV-W-MPT64 and pGEX-D-MPT64, respectively.

[0039] The correctly sequenced pBV-W-MPT64 recombinant expression plasmid was transformed into E. coli HB101. The identified positive clones were cultured overnight at 37°C, then transferred to fresh LB medium and cultured at 37°C for 2-3 hours until the OD600nm value reached 0.4-0.6. The culture was then incubated in a 42°C water bath for at least 4 hours to induce expression. Bacterial cells were collected, inclusion bodies were extracted, and the protein was purified. The purified protein was identified by SDS-PAGE electrophoresis. The correctly sequenced pGEX-D-MPT64 recombinant expression plasmid was transformed into E. coli BL21. The identified positive clones were cultured overnight at 37°C with shaking. The next day, they were inoculated into 250 mL of fresh LB liquid medium and cultured to the logarithmic growth phase. The temperature was adjusted to 16°C, and after 30 minutes, 150 μl of 1 mol / L IPTG induction medium was added. Induction was performed at 16°C for 12-14 hours. Bacterial cells were collected, inclusion bodies were extracted, and the protein was purified. The purified protein was identified by SDS-PAGE electrophoresis. The results are as follows Figure 1 As shown, 1 is the W-MPT64 protein expressed by the recombinant expression vector pBV-W-MPT64, with a molecular weight of approximately 35 kDa; 2 is the D-MPT64 protein expressed by the recombinant expression vector pGEX-D-MPT64, with a molecular weight of approximately 48 kDa.

[0040] Example 2: Preparation and screening of W-MPT64 and D-MPT64 dual-recognition monoclonal antibodies

[0041] To obtain a dual-recognition monoclonal antibody capable of recognizing both W-MPT64 and D-MPT64, 6-8 week old female BALB / c mice were used. For the first immunization, W-MPT64 was used as the immunogen, with 100 µg of antigen per mouse plus an equal volume of complete Freund's adjuvant, emulsified and injected subcutaneously and intraperitoneally into the back of the mice. A second immunization was performed 4 weeks later, using D-MPT64 as the immunogen, with 60 µg of antigen per mouse plus an equal volume of incomplete Freund's adjuvant. Eight weeks later, a third immunization was performed, with 30 µg of W-MPT64 and 30 µg of D-MPT64 emulsified with incomplete Freund's adjuvant, respectively, and then immunized into the mice. One week after the third immunization, mice with the highest serum titers were selected for a booster immunization via intraperitoneal injection at the same dose as the third immunization. Three days later, spleen cells were harvested and fused with SP20 myeloma cells according to standard procedures. When the confluent cells covered approximately 60% of the bottom of the wells, the cell culture supernatant was collected, and an indirect enzyme-linked immunosorbent assay (ELISA) was used to screen for dual-recognition monoclonal antibodies that could simultaneously recognize W-MPT64 and D-MPT64. The specific method was as follows: W-MPT64 and D-MPT64 were diluted with carbonate coating buffer to a concentration of 2.0 μg / ml, and 100 μl of either W-MPT64 or D-MPT64 was coated per well and incubated overnight at 4°C. The plates were washed twice with washing buffer; 150 μl / well of blocking buffer was added and the plates were blocked at room temperature for 6 hours; the plates were washed 5 times with washing buffer. After adding 100 μl of sample dilution buffer to each well, 10 μl of cell culture supernatant was added, and the plates were incubated with shaking at room temperature for 15 min, then the supernatant was discarded; the plates were washed 5 times with washing buffer, and 100 μl / well of HRP-labeled goat anti-mouse IgG antibody was added, and the plates were incubated with shaking at room temperature for 15 min. The plates were washed 5 times. Add 50 μL each of TMB chromogenic solutions A and B to each well and incubate at room temperature in the dark for 15 minutes. Stop the reaction by adding 50 μL of 2 M H₂SO₄ to each well. Measure the absorbance of each well using a microplate reader at 450 nm, reading the value within 10 minutes after termination. Select positive clones that show strong positive reactions with both W-MPT64-coated and D-MPT64-coated wells as positive clones for the dual-recognition monoclonal antibodies against W-MPT64 and D-MPT64, obtaining three positive clones: 1134, 6112, and 6261. Culture the three positive clone mouse hybridoma cell lines in 1640 medium containing 10% fetal bovine serum. After 10 days, collect the cells, resuspend them in physiological saline, and inject each mouse intraperitoneally with 1 × 10⁻⁶ cells. 6 Ascites fluid was prepared from hybridoma cells. After 2 weeks, the ascites fluid was collected and antibody was purified using the Montage Antibody Purification Kit with PROSEP-G (Millipore, catalog number LSK2ABG 20). The purified antibody was aliquoted into 1 mg vials and stored at -20°C.

[0042] The titers of three dual-recognition monoclonal antibodies against W-MPT64 and D-MPT64 were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Specifically, 50 μl of each of W-MPT64 and D-MPT64 was coated into each well, with the remaining steps following the method described above. The three monoclonal antibodies were diluted to a concentration of 1 mg / mL with PBS and then serially diluted to 1:50, 1:200, 1:800, 1:3200, 1:12800, 1:51200, and 1:204800. The results are shown in Table 1. The dual-recognition monoclonal antibody 6112 exhibited the highest antibody titer, reaching 1:204800, and was selected for subsequent studies.

[0043] Table 1. Results of titer assays for W-MPT64 and D-MPT64 dual-recognition monoclonal antibodies (OD450nm)

[0044]

[0045] Example 3: Preparation of rabbit anti-MPT64 protein polyclonal antibody

[0046] Polyclonal antibodies against MPT64 protein were prepared using W-MPT64 and D-MPT64 prepared in Example 1 as immunogens. The specific steps are as follows: One healthy male white rabbit was selected, and 2 mL of blood was collected from the marginal ear vein. The serum was separated as a negative control. 0.5 mg each of W-MPT64 and D-MPT64 prepared in Example 1 were mixed and then mixed with 1.0 mL of Freund's complete adjuvant. The mixture was thoroughly emulsified into a milky white, viscous water-in-oil emulsion. Multiple subcutaneous injections were performed on both sides of the rabbit's spine, with each injection containing at least 0.1 mL. Four weeks later, 1.0 mg of an equal volume of the mixed W-MPT64 and D-MPT64 protein was mixed with 1.0 mL of Freund's incomplete adjuvant and thoroughly emulsified before a second immunization. A third booster immunization was performed four weeks later, and blood was collected from the heart one week later. After the blood clots and contracted, the mixture was centrifuged at 5000 rpm for 15 minutes, and the serum was collected for titer determination using an indirect ELISA method. Each well was coated with 100 μl of either W-MPT64 or D-MPT64, using HRP-labeled goat anti-rabbit secondary antibody. The remaining steps were the same as described above. The results are shown in Table 2. Using pre-immunization rabbit serum (1:2000 dilution) as a negative control, the prepared rabbit anti-MPT64 polyclonal antibody achieved a detection titer of 1:2048000 against both W-MPT64 and D-MPT64.

[0047] Table 2. Results of rabbit anti-MPT64 protein polyclonal antibody titer determination (OD450nm)

[0048]

[0049] Example 4: Mycobacterium tuberculosis MPT64 protein double antibody sandwich enzyme-linked immunosorbent assay

[0050] Using the anti-W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 and rabbit anti-MPT64 protein polyclonal antibody prepared in this invention, a proteinase-linked immunosorbent assay (ELISA) method for detecting MPT64 in Mycobacterium tuberculosis based on the principle of double antibody sandwich was established.

[0051] First, horseradish peroxidase (HRP) was labeled with monoclonal and polyclonal antibodies, respectively. The specific procedure was as follows: 5 mg of HRP was dissolved in 0.5 mL of deionized water, and 1 mL of 0.06 mol / L NaIO4 was added. The mixture was stirred gently at room temperature in the dark for 30 min. 1 mL of 0.16 mol / L ethylene glycol was added, and the mixture was stirred gently at room temperature for 1 hour to terminate the oxidation reaction. The mixture was then placed in a dialysis bag and dialyzed overnight at 4°C with 1000 mL of 0.01 mol / L carbonate buffer (pH 9.5), changing the buffer 3 times to obtain aldehyde-modified HRP. 1 mL of carbonate buffer containing 5 mg of antibody was added to 3 mL of the aldehyde-modified HRP solution, and the mixture was incubated at room temperature in the dark with gentle stirring for 2-3 hours. 5 mg of NaHB4 was added, and the mixture was incubated overnight at 4°C. The mixture was then placed in a dialysis bag and dialyzed for 24 hours at 4°C with 0.01 mol / L PBS (pH 7.2), changing the buffer 3 times. Centrifuge at 3000 r / min for 30 min, remove the precipitate, add an equal amount of glycerol to the supernatant to obtain HRP-labeled antibody, aliquot and store at low temperature.

[0052] The experiment was divided into two groups, A and B. Group A used anti-W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 as the capture antibody and HRP-labeled rabbit anti-MPT64 protein polyclonal antibody as the detection antibody. Group B used rabbit anti-MPT64 protein polyclonal antibody as the capture antibody and HRP-labeled 6112 monoclonal antibody as the detection antibody. ELISA plates were coated with either anti-W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 or rabbit anti-MPT64 protein polyclonal antibody as the capture antibody, and the corresponding HRP-labeled polyclonal antibody or HRP-labeled monoclonal antibody was used as the detection antibody. The specific steps were as follows: antibody was coated onto the ELISA plate at a concentration of 2.5 μg / mL, 100 μL per well, incubated overnight at 4°C, and washed twice with washing buffer. 110 μL / well blocking buffer was added, incubated overnight at 4°C, and the buffer was discarded and the plate was air-dried. The prokaryotically expressed W-MPT64 and D-MPT64 from Example 1 were serially diluted with double-distilled water to concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, and 0 pg / mL, respectively. 100 μL of each was added to each well and incubated at 37°C for 60 min, then discarded. The plate was washed 5 times, and 100 μL of the corresponding HRP-labeled antibody was added to each well, incubating at 37°C for 45 min. After washing 5 times and patting dry, 50 μL each of TMB chromogenic solutions A and B were added to each well, and the plate was incubated at room temperature in the dark for 15 min. 50 μL of 2 M H₂SO₄ stop solution was added to each well to terminate the reaction. The OD450 nm value was measured using a microplate reader within 10 minutes. Each concentration was tested three times. The results are as follows: Figure 2 As shown, the limits of detection for both W-MPT64 and D-MPT64 in group A reached 1.5625 pg / mL, which is higher than the 6.25 pg / mL in group B. Therefore, the monoclonal antibody 6112, which recognizes both W-MPT64 and D-MPT64, was selected as the capture antibody, and the HRP-labeled rabbit anti-MPT64 protein polyclonal antibody was selected as the detection antibody.

[0053] Example 5: Double-antibody sandwich fluorescence immunochromatographic assay for MPT64 protein in Mycobacterium tuberculosis

[0054] To establish a sensitive, simple, and rapid method for detecting MPT64 protein in Mycobacterium tuberculosis, a double-antibody sandwich fluorescent immunochromatographic assay was developed. This invention uses the anti-W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 as the capture antibody-labeled fluorescent microspheres, and a rabbit anti-MPT64 protein polyclonal antibody as the detection antibody, coated onto a nitrocellulose membrane. For detection, 100 µL of serially diluted W-MPT64 and D-MPT64 protein as described in Example 4 was vertically added to the sample loading area of ​​the test card, and the reaction was allowed to proceed at room temperature for 15 min. The test was performed using an AFS-1000 dry fluorescence immunoassay analyzer. After the sample was added to each well of the reagent card, it migrated to the chromatography zone via capillary action. At the test and control lines, fluorescent microsphere-labeled particles accumulated due to antigen-antibody reaction, 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 MPT64 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 MPT64 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. This invention utilizes a double-antibody sandwich fluorescence immunochromatographic assay with W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112 as the capture antibody and rabbit anti-MPT64 protein polyclonal antibody as the detection antibody. The limits of detection for both W-MPT64 and D-MPT64 reach 1.5625 pg / mL. The detection time is only 15-20 min, significantly shorter than that of enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 3.

[0055] Table 3. Limit of detection (S / CO value) for MPT64 protein fluorescence immunochromatographic assay in Mycobacterium tuberculosis.

[0056]

[0057] Example 6: Limit of detection of MPT64 protein in Mycobacterium tuberculosis by fluorescence immunochromatography

[0058] MPT64 protein in Mycobacterium tuberculosis cultures was detected using the fluorescence immunochromatographic assay described above. Cultures of the standard Mycobacterium tuberculosis strain (H37Rv) and the clinical 63bp deletion strain AH03009 were placed in physiological saline and shaken to prepare a physiological saline bacterial suspension with a turbidity of 1 McFarland unit; the bacterial count was 3 × 10⁻⁶. 8 CFU / mL, then serially diluted with physiological saline to a bacterial 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 1 CFU / mL. Take 50 μL of bacterial suspension and add it to 50 μL of sample processing solution, mix well, and vertically drop it onto the sample application point of the test card. Incubate at room temperature for 15 min, and then detect using an AFS-1000 dry fluorescence immunoassay analyzer. The results are shown in Table 4. The detection limit of the double-antibody sandwich fluorescence immunochromatographic assay for Mycobacterium tuberculosis MPT64 protein of this invention reached 1 × 10⁻⁶ CFU / mL. 3 The CFU / mL was significantly higher than the 3-5 × 10⁻⁶ reported in the literature. 3 CFU / mL (Zhang Yuanliang, Cai Xingshan, Tan Yaoju. Application of secretory proteins in the identification of Mycobacterium tuberculosis complexes in sputum and culture medium. Modern Hospital, 2011, 11(2): 17-19).

[0059] Table 4. Limit of detection (S / CO value) for Mycobacterium tuberculosis

[0060]

[0061] Example 7: Earliest detection time of MPT64 protein in Mycobacterium tuberculosis culture by fluorescence immunochromatography

[0062] MPT64 protein in Mycobacterium tuberculosis cultures was detected using the fluorescence immunochromatographic assay described above to determine the earliest detection time of Mycobacterium tuberculosis. Three sputum samples from patients with clinically active tuberculosis were cultured using the BACTEC MGIT 960 rapid culture system, and the experimental procedures were strictly performed according to the system operation guidelines. Samples were taken on days 3, 5, 7, 10, 12, and 15 of culture for testing, following the same procedures as described above. The results are shown in Table 5. Samples 1 and 2 were positive for MPT64 protein on day 7, and sample 3 was positive on day 10, earlier than the average detection time of 12 days reported in the literature (Zhang Yuanliang, Cai Xingshan, Tan Yaoju. Application of secretory proteins in the identification of Mycobacterium tuberculosis complexes in sputum and culture medium. Modern Hospital, 2011, 11(2): 17-19).

[0063] Table 5. Earliest detection time of Mycobacterium tuberculosis (S / CO value)

[0064]

[0065] Example 8: Identification of the 6112 subtype of the W-MPT64 and D-MPT64 dual-recognition monoclonal antibody

[0066] The heavy and light chain subtypes 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. 100 μL of supernatant from mouse hybridoma cell line 6112 was added to the sample wells of the rapid mouse antibody subtype detection card, and the results were observed and recorded after standing for 5-10 min. The results are as follows: Figure 3 As shown, the dual recognition monoclonal antibody 6112 for Mycobacterium tuberculosis W-MPT64 and D-MPT64 is mouse IgG1 subtype, and the antibody light chain is Igκ subtype.

[0067] Example 9: Determination of the amino acid sequence of the variable region of W-MPT64 and D-MPT64 dual-recognition monoclonal antibody 6112

[0068] Mouse hybridoma cell line 6112 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 primer sequences are available in *Recombinant Antibodies* (Science Press, 2005), edited by Shen Beifen. Amplification conditions were as follows: preheating 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 PCR product was 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.

[0069] Sequence analysis revealed that the heavy chain variable region consists of 115 amino acids, with the following sequence: EVKVVESGGGLVQPGGSMKLSCAAS GFTFSDAW MDWVRQSPEKGLEWIAE IRNKANDHAT YYAESVKGRFTISRDDSKSSVYLQMNSLRAEDTGIYYC TTVPFDCWGQGTTLTVS (SEQ ID NO.1) has three CDR regions indicated by underscores: CDR1 is located at 26-33 amino acids, with the amino acid sequence GFTFSDAW (SEQ ID NO.2); CDR2 is located at 51-60 amino acids, with the amino acid sequence IRNKANDHAT (SEQ ID NO.3); and CDR3 is located at 99-105 amino acids, with the amino acid sequence TTVPFDC (SEQ ID NO.4). The light chain variable region has an amino acid sequence of 109 amino acids, as follows: DIVLTQSPASLAVSLGQRATISYRAS KSVST SGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGAPSWKS (SEQ ID NO.5) has three CDR regions marked with underscores. CDR1 is located at 27-36 aa and has the amino acid sequence KSVSTSGYSY (SEQ ID NO.6); CDR2 is located at 54-56 aa and has the amino acid sequence LVS; and CDR3 is located at 93-100 aa and has the amino acid sequence QHIRELTR (SEQ ID NO.7).

Claims

1. A monoclonal antibody or its antigen-binding fragment that simultaneously recognizes secretory protein 64 of wild-type Mycobacterium tuberculosis and secretory protein 64 of 63bp deletion mutant Mycobacterium tuberculosis, 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 LVS; 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 6112 with accession number CGMCC No.46599.

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 monoclonal antibodies that simultaneously recognize wild-type Mycobacterium tuberculosis secretory protein 64 and 63bp-deleted mutant Mycobacterium tuberculosis secretory protein 64, characterized in that, It is the monoclonal antibody mouse hybridoma cell line 6112 with accession number CGMCC No.46599.

10. The use of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4 in the preparation of a kit for detecting Mycobacterium tuberculosis secretory protein 64.

11. A kit for detecting Mycobacterium tuberculosis secretory protein 64, 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, The kit is a double-antibody sandwich kit, in which a monoclonal antibody or its antigen-binding fragment is used as a capture antibody or a detection antibody.

13. The kit according to claim 12, characterized in that, The kit is an enzyme-linked immunosorbent assay kit or a fluorescence immunochromatographic assay kit.

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