Mycobacterium abscessus marker and application thereof

Abstract

The invention discloses a biomarker for identifying and detecting mycobacterium abscessus, the biomarker is MAB2734c signal peptide removal protein, and the amino acid sequence of the biomarker is as shown in SEQ ID NO. 2. By identifying the molecular marker, the mycobacterium abscessus can be accurately identified and detected, the process is simpler, quicker and more accurate, the cost is lower, and a quick and cheap mycobacterium abscessus immunological diagnosis technology suitable for mycobacterium positive culture supernatant is established; the detection method disclosed by the invention can be widely applied to auxiliary diagnosis, epidemiological monitoring and the like of the lung diseases caused by the mycobacterium abscessus, the cure rate is increased, and the prognosis is improved.

Description

Technical Field

[0001] This invention belongs to the field of rapid disease diagnosis and detection, specifically involving biomarkers for Mycobacterium abscessus and their applications. Background Technology

[0002] Nontuberculous mycobacteria (NTM) refer to mycobacteria other than Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium leprae. Nontuberculous mycobacterial disease refers to lesions in related tissues or organs caused by NTM infection in humans. In recent years, the global prevalence of nontuberculous mycobacterial lung disease has been on the rise, becoming a common disease and posing a threat to human health (Li-Ping Cheng et al. 2022). Due to the inherent resistance of NTM to antibiotics and anti-tuberculosis drugs, diagnosis relies on laboratory identification of the pathogen, which must be down to the species level (Uyan ZS et al. 2010; Tang Shenjie et al. 2012). However, NTM lung disease lacks specific clinical manifestations and signs, making it difficult to differentiate from other diseases (YeGu et al. 2019). Many clinicians often overlook the existence of NTM lung disease, frequently leading to misdiagnosis or missed diagnosis. Apart from Mycobacterium tuberculosis complex, there are currently no immunodiagnostic products available domestically or internationally that can rapidly identify other NTM species, nor are there any species identification technologies that can be directly used for patient body fluid specimens.

[0003] Among the rapidly growing nontuberculous mycobacterial pathogens (NTMs) that infect humans, the most common is the Mycobacterium abscessus complex, with Mycobacterium abscessus (Mab) being the most prevalent. It primarily causes lung infections but can also lead to lesions in the skin, soft tissues, and bones (Cara D Varley et al. 2021), especially in immunocompromised patients, where it can cause systemic infections such as sepsis. Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterium, often referred to as an "incurable nightmare" for antibiotics, as it is resistant to almost all anti-tuberculosis drugs. Therefore, Mab lung disease is the most difficult to cure among all mycobacterial lung diseases, with an initial treatment success rate of only 34% and a retreatment success rate of only 20%, making it a significant cause of death in patients with underlying chronic lung diseases. Because the clinical symptoms and imaging findings of Mab lung disease are similar to those of tuberculosis, and both diseases show granulomatous changes and positive acid-fast staining, it is easily misdiagnosed as tuberculosis. When Mab disease is misdiagnosed as tuberculosis, it not only delays the treatment of mycobacterial abscess and leads to some avoidable complications, but also causes patients to suffer from the adverse reactions of anti-tuberculosis drugs, further reducing the cure rate.

[0004] Studies show that approximately 50% of Mab patients in clinical practice are pathogen-negative, making it impossible to identify Mab infection using mycobacterial identification markers. These pathogen-negative Mab patients are often misdiagnosed with tuberculosis. Currently, clinical identification of abscess mycobacteria mainly relies on the sequence comparison of mycobacterial identification markers (16S rRNA, hsp65, rpoB, 16-23S rRNA), utilizing techniques such as DNA sequencing, gene chips, linear probes, and multicolor fluorescence melting curves. However, these methods all have specific equipment and technical requirements. Sequencing technology requires expensive instruments, is complex and time-consuming, making it unsuitable for routine implementation, and the result reporting time is 1-2 days, limiting its widespread application in primary care laboratories. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention discovered that Mycobacterium abscessis can specifically secrete MAB_2734c signal peptide protein, and based on this, the invention was completed.

[0006] The technical solution of this invention is as follows:

[0007] In a first aspect, a biomarker for Mycobacterium abscessus is provided, the biomarker being MAB_2734c signal peptide protein with its amino acid sequence shown in SEQ ID NO: 2, and the biomarker being used for the detection of Mycobacterium abscessus.

[0008] Secondly, this invention provides a monoclonal antibody mouse hybridoma cell line HHR2734 with MAB_2734c depleted of signal peptide protein, with accession number CGMCC NO.45846 and deposit address: China General Microbiological Culture Collection Center.

[0009] Thirdly, this invention provides the application of MAB_2734c signal peptide protein removal in the preparation of a kit for the differential diagnosis of Mycobacterium abscessis infection.

[0010] Furthermore, the amino acid sequence of the MAB_2734c signal peptide protein is shown in SEQ ID NO: 2.

[0011] Furthermore, if the MAB_2734c signal peptide protein can be detected in the patient's biological sample, it is determined that the patient is infected with Mycobacterium abscessus.

[0012] Furthermore, the patient's biological samples were selected from blood, pleural fluid, or sputum.

[0013] More preferably, the patient's biological sample is at least one of peripheral blood, plasma, and serum.

[0014] Fourthly, the present invention provides a kit for the differential diagnosis of Mycobacterium abscessus infection, the kit containing a reagent for detecting the MAB_2734c signal peptide protein removal.

[0015] Furthermore, the amino acid sequence of the MAB_2734c signal peptide protein is shown in SEQ ID NO: 2.

[0016] Furthermore, the kit can be used to prepare ELISE detection kits and / or colloidal gold detection kits.

[0017] Furthermore, the diagnostic methods of the kit include direct methods, indirect methods, double-antibody sandwich methods, and / or competitive methods.

[0018] Beneficial effects:

[0019] This invention identifies a specific MAB_2734c signal peptide protein, screened from *Mycobacterium abscessus* strains, as a biomarker for diagnosing *Mycobacterium abscessus*, ensuring the method's specificity and avoiding the non-specificity and false positives of bacterial common genes such as rpoB and 16SRNA. A rapid and inexpensive immunological diagnostic technique for *Mycobacterium abscessus* is established for mycobacterial-positive culture supernatants. ELISA and immunogold assays are used to differentiate between tuberculosis and *Mycobacterium abscessus* infection in a "bacterial-negative" state. The detection method based on this invention can be widely applied in the auxiliary diagnosis and epidemiological monitoring of *Mycobacterium abscessus* lung disease, improving cure rates and prognosis. Attached Figure Description

[0020] Figure 1 Venn diagram of Mab and secreted proteins of common pathogenic mycobacteria

[0021] Figure 2 SDS-PAGE electrophoresis image of rabbit polyclonal antibody against anti-MAB_2734c signal peptide protein removal.

[0022] Note: 1. marker; 2. Anti-MAB_2734c signal peptide removal rabbit polyclonal antibody-1; 3. Anti-MAB_2734c signal peptide removal rabbit polyclonal antibody-2

[0023] Figure 3 The titer and localization of anti-MAB_2734c signal peptide-removing rabbit polyclonal antibody in Mab.

[0024] Note: 1. MAB_2734c antigen 0.1 μg / mL; 2. MAB_2734c antigen 1 μg / mL; 3. MAB_2734c antigen 5 μg / mL; 4. MAB_2734c antigen 10 μg / mL; 5. Mab standard strain supernatant

[0025] Figure 4 .MAB_2734c removes the specificity of the signal peptide protein in the standard strain of mycobacteria.

[0026] Note: 1. Mycobacterium smegmatis; 2. Recombinant Mycobacterium smegmatis (pMV261-2734c); 3. Mycobacterium kansasii; 4. Intracellular Mycobacterium; 5. Occasional Mycobacterium; 6. Mycobacterium avium; 7. Mycobacterium bufotae; 8. Standard strain of Mycobacterium abscessus; 9. Clinical strain of Mab; 10. Escherichia coli;

[0027] Figure 5 The MAB_2734c signal peptide protein is widely present in clinical Mab strains.

[0028] Note: 1-10 Mab clinical strains

[0029] Figure 6 Western blot screening of antigen-binding positive hybridoma cell supernatant

[0030] Note: 1. Marker; 2. 0.1 μg / ml protein; 3. 1 μg / ml protein; 4. Mab supernatant

[0031] Figure 7 The titer and specificity of the anti-MAB_2734c signal peptide-removing monoclonal antibody in the culture filtrate of mycobacterial standard strains.

[0032] Note: 1. MAB_2734c antigen 0.1 μg / mL; 2. MAB_2734c antigen 1 μg / mL; 3. MAB_2734c antigen 5 μg / mL; 4. MAB_2734c antigen 10 μg / mL; 5. Mab standard strain; 6. Mab knockout strain; 7. Mab complement strain; 8. Mycobacterium smegmatis; 9. Mycobacterium kansasii; 10. Intracellular mycobacteria; 11. Occasional mycobacteria; 12. Mycobacterium bufo; 13. Recombinant Escherichia coli; 14. Escherichia coli

[0033] Figure 8 Detection of Mab clinical strains with anti-MAB_2734c signal peptide removal monoclonal antibody.

[0034] Note: 1-24 Mab clinical strains; 25-27 Intracellular mycobacterial clinical strains; 28-31 Kansas mycobacterial clinical strains; 32-34 Occasional mycobacterial clinical strains

[0035] Figure 9 Schematic diagram of immunogold detection based on Mab-specific secreted proteins.

[0036] Figure 10 ELISA detection of culture filtrates of different NTM clinical strains Detailed Implementation

[0037] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0038] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0039] the term:

[0040] As used in this article, the term "sample" or "sample" refers to material specifically associated with the person being tested, from which specific information relating to the person being tested can be determined or inferred. A sample may consist wholly or partially of biological material from the person being tested.

[0041] In this invention, the term "specific combination" has a general immunological meaning, such as the binding between antigens and antibodies.

[0042] Biomarkers are generally indicators that can be objectively measured and evaluated, reflecting physiological or pathological processes and the biological effects of therapeutic interventions. Biomarkers are typically derived from human tissues or fluids and can encompass changes at the physiological, biochemical, immune, cellular, and molecular levels.

[0043] Sputum-negative pulmonary tuberculosis refers to active pulmonary tuberculosis that tests negative for both acid-fast bacilli (AFB) in sputum smears and mycobacterium in sputum cultures. Sputum-negative pulmonary tuberculosis includes both smear-negative and culture-negative pulmonary tuberculosis, accounting for approximately 40%-70% of all pulmonary tuberculosis cases. This type of pulmonary tuberculosis patients also possess a certain degree of transmissibility and pathogenicity, making them a major group contributing to the prevalence of pulmonary tuberculosis.

[0044] Enzyme-linked immunosorbent assay (ELISA): ELISA was established in the 1970s by Cliquet et al. Currently, the enzyme immunoassay methods used worldwide for detecting rabies virus antibodies are primarily indirect ELISA. The antibodies detected by ELISA vary depending on the purity and composition of the adsorbed antigen. This method is now available as a kit (Serelisa) and is mainly used for large-scale screening of seroconversion in vaccinated pets.

[0045] Gold-immunochromatography assay (GICA) is a diagnostic technique that combines chromatography with immunogold technology, integrating immune reaction and chromatographic analysis. This method can achieve antigen concentration and has high sensitivity.

[0046] Label-free proteomics is a label-free method for protein quantification that uses mass spectrometry to quantitatively analyze proteins in a sample. The principle of label-free proteomics is based on mass spectrometry and mainly includes two steps: sample digestion and mass spectrometry analysis. First, the sample to be tested is digested to break down proteins into peptides. Then, the digested sample is analyzed using a mass spectrometer, and protein quantification is achieved by detecting the mass and abundance of peptides in the sample. Compared with traditional labeling methods, label-free proteomics has advantages such as ease of operation, high throughput, and high sensitivity.

[0047] UniProt Database: UniProt (Universal Protein) is a protein database that contains protein sequences, functional information, and research paper indexes. It integrates resources from three major databases: EBI (European Bioinformatics Institute), SIB (the Swiss Institute of Bioinformatics), and PIR (Protein Information Resource).

[0048] Peptides are precise protein fragments. A compound with two amino acids linked by peptide bonds is called a dipeptide; a compound with three amino acids linked by peptide bonds is called a tripeptide, and so on. Only molecules with a molecular weight between 5000 and 180 can be called peptides. Molecular weights between 5000 and 10000 are called macropeptides. Molecular weights between 180 and 480 are called small peptides, oligopeptides, or low-molecular-weight peptides, also known as small-molecule bioactive polypeptides. Biologists refer to peptides as "amino acid chains," and collectively call small-molecule bioactive polypeptides "bioactive peptides."

[0049] Protein: Amino acids form peptides, and peptides form proteins. A peptide is a biochemical substance between amino acids and proteins; it is smaller in molecular weight than a protein but larger than an amino acid, and is a fragment of a protein. That is, a peptide is formed by the aggregation of two or more, up to dozens, of amino acids linked by peptide bonds, and multiple peptides are then linked by side chains to form a protein. Proteins are the most abundant and functionally diverse macromolecules in cells, playing a crucial role in various life functions.

[0050] Example 1

[0051] Screening and identification of specific secreted proteins in culture filtrate of Mycobacterium abscessus

[0052] 1.1 Experimental Methods

[0053] Mycobacterium abscessus and standard strains of common pathogenic mycobacteria (including Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansas, Mycobacterium guildrums, and Mycobacterium occulta) were cultured in Soton medium to the logarithmic growth phase (OD). 600 =0.6-0.8), the culture filtrate was collected by centrifugation and concentrated to a concentration greater than 0.5 μg / mL, with a total amount greater than 50 μg. Label-free proteomics was used to identify peptides, obtaining peptide profiles of *Mycobacterium abscessus* and common mycobacteria. Peptides identified by mass spectrometry in the Mab filtrate supernatant were searched in the UniProt database, and protein signal peptides were predicted using the SignaIP server. Using a protein homology <30% as the cut-off value, three *Mycobacterium abscessus*-specific secretory proteins were further screened, with MAB_2734c being one of them. Figure 1 Since the signal peptide guides the protein to the cell membrane, the portion ultimately secreted extracellularly is the protein with the signal peptide removed. Therefore, MAB_2734c with the signal peptide removed was selected to prepare polyclonal and monoclonal antibodies.

[0054] 1.2 Selection Criteria for Specific Proteins

[0055] (1) The abundance of peptides in the filtrate ranked among the top 30 of all detected peptides;

[0056] (2) The proteins corresponding to the peptides are commonly found in different clinical samples of Mycobacterium abscessus;

[0057] (3) The protein was not found in the culture filtrate of other common mycobacterial species;

[0058] (4) Based on bioinformatics analysis, no homologous proteins were found in other mycobacteria and other bacteria (homology <30%).

[0059] 1.3 Amino acid sequence of MAB_2734c protein and MAB_2734c signal peptide protein (without signal peptide)

[0060] Table 1. Amino acid sequences of MAB_2734c protein and MAB_2734c signal peptide removed protein

[0061]

[0062] Example 2: Preparation of rabbit polyclonal antibody against MAB_2734c signal peptide protein removal.

[0063] 2.1 Animal Immunization

[0064] (1) Two Japanese white rabbits were immunized with 500 μg of protein. For the first immunization, the same volume of complete Freund's adjuvant was prepared as an emulsion and administered via multiple subcutaneous injections on the back.

[0065] (2) Every 2-3 weeks, take an equal volume of immunogen and incomplete Freund's adjuvant to make an emulsion. The immunization route is the same. After four immunizations, the serum titer is measured.

[0066] (3) If the serum titer is qualified, kill the rabbit and take its blood to purify the antibody for later use; if the titer is unqualified, immunize again, then measure the serum titer, kill the rabbit and take its blood for later use.

[0067] 2.2 Blood collection and titer testing

[0068] (1) One week after the last immunization, 3-4 mL of blood was taken from the marginal ear vein, refrigerated overnight, and then centrifuged at 1500g for 15 minutes to separate the upper serum layer.

[0069] (2) Then take a portion of the protein needed for detection and dilute it to 0.1 μg / mL, 1 μg / mL and 5 μg / mL (diluted with coating buffer). Add 100 μL to each well of the 96-well plate, shake the plate to mix the sample, seal it and place it at 4°C overnight.

[0070] (3) Wash the microplate with 300 μL / well of washing solution, and then pat the microplate dry;

[0071] (4) Add 300 μL of sealing solution to each well, seal the plate, and seal for 1 hour;

[0072] (5) Wash the microplate with 300 μL / well washing buffer, dilute the sample serially, then add 100 μL of sample diluent to each well, and add an equal amount of detection antibody at the same time, and incubate for two hours.

[0073] (6) Wash the microplate with 300 μL / well washing buffer, add 200 μL of colorimetric solution (A solution: B solution volume ratio is 1:1) to each well, and then place at room temperature for 15 minutes;

[0074] (7) After adding 50 μL of stop solution to each well, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the enzyme at a wavelength of 450 nm.

[0075] 2.3 Purification of rabbit polyclonal antibody against anti-MAB_2734c signal peptide protein removal

[0076] 2.3.1 Protein Affinity Purification

[0077] (1) Protein A affinity chromatography column, then add equilibration buffer for equilibration;

[0078] (2) Load rabbit serum onto an affinity chromatography column and then wash with equilibration buffer.

[0079] (3) Use elution buffer to elute the sample, collect the elution peaks and then neutralize them with Tris buffer.

[0080] 2.3.2 Antigen affinity purification

[0081] (1) Rinse the antigen affinity chromatography column with ultrapure water and then equilibrate it with equilibration buffer;

[0082] (2) Load the purified protein sample from the previous part onto an antigen affinity chromatography column, and then wash with equilibration buffer after loading.

[0083] (3) Elute the sample with elution buffer, collect the elution peaks and neutralize them with Tris buffer;

[0084] (4) Samples eluted with PBS at pH 7.4.

[0085] 2.4 Purity of rabbit polyclonal antibody against anti-MAB_2734c with signal peptide removed

[0086] Rabbit polyclonal antibody against MAB_2734c signal peptide protein was purified from rabbit serum using a Protein A affinity chromatography column and an antigen affinity chromatography column. The purification results are shown in the figure. The results show that, in the non-reducing state, the target band corresponding to the antibody appeared at 150 kDa. Figure 2 A); Under reducing conditions, bands corresponding to the antibody heavy chain (55kDa) and light chain (25kDa) appear, with no other extraneous bands. Figure 2 B) Upon testing, the purity of both antibodies was above 95%, meeting the purity requirements for subsequent experiments. UV 280 analysis showed that the purified rabbit polyclonal antibody concentration was approximately 1.0 mg / mL, and each rabbit yielded over 5 mg of purified polyclonal antibody, meeting the concentration requirements for subsequent experiments.

[0087] 2.5 ELISA method for detecting the binding activity of anti-MAB_2734c signal peptide-removing polyclonal antibody to antigen.

[0088] The antigen was detected by ELISA using a 0.1 μg / mL anti-MAB_2734c signal peptide polyclonal antibody, typically with OD... 450 A Blank value greater than 1 indicates that the antibody has bound to the antigen. The OD values ​​of two anti-MAB_2734c signal peptide-removing polyclonal antibodies were measured at 0.1 μg / mL of antigen. 450The lowest Blank value was 2.9, indicating that the antibody and antigen were tightly bound, which met the requirements for subsequent experiments (Table 2).

[0089] Table 2. ELISA method for detecting the binding activity of anti-MAB_2734c signal peptide removal polyclonal antibody to antigen.

[0090]

[0091] 2.6 Western blot method for detecting polyclonal antibody titer and localization in mab

[0092] This study used different concentrations of MAB_2734c protein as the antigen, with anti-MAB_2734c signal peptide-removing polyclonal antibody as the primary antibody and HRP-labeled Goat Anti-Rabbit IgG as the secondary antibody. Western blot was used to detect the titer and localization of the anti-MAB_2734c signal peptide-removing rabbit polyclonal antibody in mabs. The results showed that at a concentration of 1 μg / ml, the anti-MAB_2734c signal peptide-removing polyclonal antibody produced a clear band, which could be detected in the mab supernatant. Figure 3 ).

[0093] 2.7 Western blot method for detecting the specificity of polyclonal antibodies in culture filtrate

[0094] As previously demonstrated, MAB_2734c protein is a secreted protein present in mab supernatant. To verify the specificity of MAB_2734c protein in mab, we used 1 μg / ml of anti-MAB_2734c signal peptide-removed polyclonal antibody as the primary antibody and HRP-labeled GoatAnti-Rabbit IgG as the secondary antibody to detect nontuberculous mycobacterial standard strains and clinical strain culture supernatant filtrates. The results showed that MAB_2734c protein is specific in Mycobacterium abscessus standard strains. Figure 4 ), which is commonly found in clinical Mab strains. Figure 5 ).

[0095] Example 3: Preparation of mouse monoclonal antibody against MAB_2734c signal peptide protein removal

[0096] 3.1 Animal Immunization

[0097] (1) Five Balb / c mice aged 6-8 weeks were prepared for immunization, with an immunization dose of 50 μg protein per mouse. For the first immunization, the immunogen and an equal volume of complete Freund's adjuvant were prepared into an emulsion and administered via multiple subcutaneous injections into the abdomen. Before the first immunization, blood was collected by tail amputation to extract serum, which was stored at -80℃ for later use.

[0098] (2) Two to three weeks after the first immunization, blood was collected by cutting off the tail, and the serum was separated and stored at -80℃ for later use.

[0099] (3) 50 μg of protein immunogen was mixed with the same volume of incomplete Freund's adjuvant to form an emulsion, which was then injected subcutaneously into the abdomen at multiple points for a second immunization. The same method was used for three immunizations, followed by tail amputation to collect blood for serum, which was stored at -80°C for later use.

[0100] (4) 100 μg of protein was injected into the peritoneal cavity of mice with qualified titers for booster immunization. Three days later, the spleen of the mice was removed for hybridoma fusion.

[0101] 3.2 Blood collection and titer testing

[0102] (1) Seven days after each immunization, blood was collected from the orbital venous plexus of mice, about 50-60 μL, and refrigerated overnight. The next day, the blood was centrifuged until the upper serum was separated.

[0103] (2) Coating: Dilute the detection protein to 5 μg / mL, then add 100 μL / well to a 96-well plate, gently shake the plate to mix the sample, seal the plate with a sealing film, and refrigerate at 4°C overnight.

[0104] (3) Wash the microplate with 300 μL / well of washing solution and pat the microplate dry;

[0105] (4) Add 300 μL of blocking solution to each well, seal the plate, and seal at room temperature for 1 hour;

[0106] (5) Wash the microplate with 300 μL / well of washing solution;

[0107] (6) Wash the plate 5 times with 300 μL / well of washing buffer, and add 100 μL / well of chromogenic solution and let it stand at room temperature for 15 min. Dilute the sample serially, and then add 100 μL of sample diluent to each well, along with an equal amount of detection antibody. Incubate at room temperature for two hours. (7) Add 50 μL / well of stop solution. After the reaction is terminated, measure the absorbance at 450 nm wavelength using an ELISA reader.

[0108] 3.3 Hybridoma cell fusion and screening

[0109] 3.3.1 Preparation of feeder layer cells

[0110] (1) The blank mice were euthanized by removing their eyeballs and bleeding them to death the day before fusion, and then soaked in 75% alcohol for 10 minutes.

[0111] (2) Cell Acquisition: The mice were then transferred to a biosafety cabinet with their abdomens facing upwards. The skin was then cut open with sterile ophthalmic scissors to fully expose the peritoneum. A small incision was made in the peritoneum using sterile forceps and scissors. Pre-cooled HAT medium was injected into the mouse's peritoneal cavity using a sterile pipette, and the medium was gently pipetted repeatedly. The medium was then aspirated from the peritoneal cavity and injected into a 50 mL sterile centrifuge tube. This process was repeated 3-5 times. The cells were centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in HAT medium.

[0112] (3) Plate culture: Dilute with HAT medium to a cell concentration of approximately 5 × 10⁻⁶. 5 Add 100 μL / well to a 96-well cell culture plate and incubate overnight in a cell culture incubator;

[0113] (4) Observe the cell status the next day and pay attention to whether there is any contamination. If the cell growth status is good, it can be used for subsequent experiments.

[0114] 3.3.2 Preparation of mouse spleen lymphocytes.

[0115] (1) Preparation of positive serum: After the mice were immunized, the level of immune antibodies was measured. The mice with higher antibody levels were excised by removing the eyeballs and blood was collected. The whole blood of the mice was centrifuged at 4°C and the serum was collected and stored for later use.

[0116] (2) Preparation of cell suspension: After sacrifice, the mice were immersed in 75% alcohol for 10 minutes and placed in a biosafety cabinet to expose the left abdominal wall and peritoneum of the mice. The operation must be performed in a sterile environment.

[0117] (3) Spleen isolation: expose the mouse spleen, remove the connective tissue on the surface of the spleen, place it in a sterile petri dish, cut the spleen into pieces, grind it to make it into single cells, let it stand, and then aspirate the upper cell suspension to prepare a single cell suspension. Centrifuge at 1000 rpm for 10 min, discard the supernatant and set aside for later use.

[0118] 3.3.3 Cell Fusion

[0119] (1) SP2 / 0 myeloma cell preparation: Collect SP2 / 0 cells in the logarithmic growth phase and in good growth condition. Before fusion, place them in a 50mL sterile centrifuge tube and centrifuge horizontally at 1000rpm for 5min. Then add 10% FBS RPMI 1640 medium to the centrifuge tube, resuspend the cells, mix by pipetting, repeat the operation, and then add 10% FBS RPMI 1640 medium to resuspend them as single lymphocytes.

[0120] (2) Remove all spleen lymphocytes from the immunized mice and place them in a sterile centrifuge tube. Mix them with mouse myeloma cells at a ratio of 1:1 and centrifuge at 1000 rpm for 10 min.

[0121] (3) Remove the supernatant, gently shake the centrifuge tube until the mouse myeloma cells and spleen lymphocytes are evenly mixed and become a loose paste, and then place it in a 37°C water bath.

[0122] (4) Add 1 mL of PEG preheated at 37°C to the mixed cell pellet. Note that when adding, add along the side of the centrifuge tube while slowly rotating the centrifuge tube.

[0123] (5) Shake the centrifuge tube gently for 1 minute, then let it stand for 90 seconds;

[0124] (6) Termination of fusion: Add 1 mL of DMEM medium to the centrifuge tube within 1 min, and add it slowly and evenly along the wall of the centrifuge tube; then add 4 mL of DMEM medium; then add 10 mL of DMEM medium within the same time period, let stand at room temperature, and centrifuge at 1000 rpm for 5 min after 5 min.

[0125] (7) Cell seeding: Discard the supernatant and add 6 mL of HAT medium to resuspend the cells; then add another 5 mL of HAT medium, mix by pipetting, and then add another 20 mL of HAT medium to mix the cells evenly. Add the above cells to a 96-well cell culture plate containing feeder cells, 100 μL per well, and place in a cell culture incubator for culture;

[0126] (8) Carefully observe the growth status of the fused cells. Three days after fusion, replace the cells with HAT medium in a half-replacement manner.

[0127] 3.3.4 Screening of positive hybridoma cells

[0128] (1) When the fused cell clones have grown to 1 / 4-1 / 3 of the bottom of the well, the cell supernatant is first screened by Western blot, and then the screened cell supernatant is detected by the established indirect ELISA method.

[0129] (2) The cell supernatant was tested every two days for a total of 3 times. The positive cell clones were transferred to 24-well cell culture plates for expansion culture.

[0130] 3.3.5 Subcloning of positive hybridoma cells

[0131] (1) When the number of cells in the 24-well plate reaches about 25-50% of the bottom area of ​​a single well, select the positive wells with good growth and about 100 cells, and continue to add HI medium to make the total volume 10 mL. Add 100 μL to each well of the 96-well cell culture plate at densities of 0.5 cells / 100 μL, 1 cell / 100 μL, and 2 cells / 100 μL, and place it in a cell culture incubator (37℃, 5% CO2) for culture.

[0132] (2) Observe the growth of cells every day and check for contamination. After 5 days, select cell wells containing only one cell clone, repeat subcloning, and then perform ELISA detection. After selecting positive wells, continue subcloning 3 times until a stable positive hybridoma cell line is obtained. Then expand the culture and freeze according to the above freezing method.

[0133] 3.4 Purification of mouse monoclonal antibody against MAB_2734c with signal peptide removed

[0134] Rinse the Protein A affinity chromatography column with ultrapure water and equilibrate with equilibration buffer. After processing the hybridoma cell supernatant, load the column with the affinity chromatography solution, followed by rinsing with equilibration buffer. Elute with elution buffer, collect the elution peak, and neutralize with Tris buffer. Desalt using PBS at pH 7.4.

[0135] 3.5 ELISA method for detecting the binding activity of anti-MAB_2734c signal peptide monoclonal / polyclonal antibodies to antigen.

[0136] (1) Coating: MAB_2734c was coated one day in advance to remove the signal peptide protein. The concentration was set at 0.1 μg / mL, 1 μg / mL, 100 μL / well, and placed overnight at 4℃.

[0137] (2) Sealing: Pat the liquid in the plate dry, add 3% BSA, 300 μL / well, seal, and incubate at room temperature for 1 h;

[0138] (3) Washing the plate: Add 300 μL of washing solution to each well, wash the plate 3 times, and pat dry the plate after the last wash;

[0139] (4) Antibody dilution: Dilute the antibody to a concentration of 0.1 μg / mL, add 100 μL to each well of a 96-well plate, mix well, and incubate at room temperature for 2 hours;

[0140] (5) Washing the plate: Add 300 μL of washing solution to each well and wash the plate 3 times. Be sure to pat it dry during the third wash.

[0141] (6) Add secondary antibody: Dilute GoatAnti-Rabbit / mouse IgG Fc / HRP secondary antibody to the required concentration, add 100 μL to each well of a 96-well plate, mix well, incubate at room temperature for 1 h, add 300 μL of washing buffer to each well, wash the plate 3 times, and pat dry.

[0142] (7) Color development: Mix equal amounts of solution A and solution B, 200 μL / well, and incubate at room temperature for 20 min in the dark.

[0143] (8) Termination: Prepare the termination solution (2% H2SO4) in advance, add 50 μL to each well to terminate the reaction, and then measure the OD value at a wavelength of 450 nm.

[0144] 3.6 Western blot method for detecting the specificity of monoclonal / polyclonal antibodies

[0145] (1) Preparation of protein from culture filtrate of different nontuberculous mycobacterial standard strains: Different nontuberculous mycobacterial standard strains were grown to the logarithmic growth phase, centrifuged at 4℃, 10000rpm for 30min, and then the culture supernatant was filtered through a 0.22μm filter membrane. The culture supernatant was concentrated through an ultrafiltration tube with a molecular weight cutoff of 10kDa. After being concentrated to 100 times, 5× protein loading buffer was added, and the mixture was heated in a 100℃ metal bath. After 5min, the mixture was centrifuged and loaded at 20μL / well.

[0146] (2) MAB_2734c signal peptide protein with different concentrations: The MAB_2734c signal peptide protein purified in Part II was adjusted to 0.1 μg / mL, 1 μg / mL, 5 μg / mL and 10 μg / mL respectively. 5× protein loading buffer was added and the mixture was heated in a 100℃ metal bath. After 5 min, it was centrifuged and loaded at 20 μL / well.

[0147] (3) Protein from the supernatant of different clinical strains of nontuberculous mycobacteria: Different NTM clinical strains were grown to the logarithmic growth phase, centrifuged at 4℃, 10000rpm for 30min, and then filtered through a 0.22μm filter membrane. The culture supernatant was concentrated using an ultrafiltration tube with a molecular weight cutoff of 3kDa. After concentration according to the above concentration factor, 5× protein loading buffer was added, and the mixture was placed in a 100℃ metal bath, heated for 5min, and then centrifuged and loaded at 20μL / well.

[0148] (4) Specificity was detected by Western blot: Proteins from the culture supernatant of different nontuberculous mycobacterial standard strains and clinical strains and different concentrations of MAB_2734c signal peptide protein were tested using purified anti-MAB_2734c signal peptide protein monoclonal / rabbit polyclonal antibody as primary antibody and HRP-labeled GoatAnti-Rabbit / mouse IgG as secondary antibody.

[0149] 3.7 Western blot and ELISA screening of monoclonal antibody-positive hybridoma cells

[0150] After obtaining hybridoma cells during the preparation of monoclonal antibodies, the presence and concentration of antibodies in the positive supernatant need to be detected by Western blotting. We used the positive supernatant diluted 100-fold as the primary antibody and HRP-labeled Goat Anti-mouse IgG as the secondary antibody. We detected different concentrations of MAB_2734c signal peptide protein removed and Mab standard strain supernatant. The expression level and quality of the antibodies were determined by the imaging results. The results showed that bands 11E12, 13E10, 6E9, 7G6, and 8A6 were relatively clear, with 11E12 showing a distinct band in the Mab supernatant. Figure 6 Subsequently, the signal peptide protein was removed using 1 μg / ml MAB_2734c and used as the antigen. The supernatant of the above 5 cell lines was detected by ELISA. Combined with the results of Western blot, the final cell line 11E12 was determined for subsequent monoclonal antibody purification (Table 3).

[0151] Table 3. Supernatant of ELISA-selected positive hybridoma cells

[0152]

[0153]

[0154] 3.8 ELISA method for detecting the binding activity of anti-MAB_2734c signal peptide-removed mouse monoclonal antibody to antigen.

[0155] Different concentrations of anti-MAB_2734c mouse monoclonal antibody to remove signal peptide protein were used to detect 1 μg / ml antigen by ELISA. The results showed that the antibody bound tightly to the antigen when the concentration was greater than 7.81 ng / ml, and the antibody quality met the requirements for subsequent experiments (Table 4).

[0156] Table 4. ELISA detection of antigen-antigen binding activity of anti-MAB_2734c signal peptide removal monoclonal antibody.

[0157]

[0158] 3.9 Western blot method for detecting monoclonal antibody titer and specificity in culture filtrate

[0159] This study used different concentrations of MAB_2734c signal peptide-removed protein and non-tuberculous mycobacterial standard and clinical strains as antigens. Anti-MAB_2734c signal peptide-removed monoclonal antibody was used as the primary antibody, and HRP-labeled Goat Anti-Mouse IgG as the secondary antibody. Western blot was used to detect the titer and localization of the anti-MAB_2734c signal peptide-removed mouse monoclonal antibody in mabs. Figure 7 The results showed that the anti-MAB_2734c signal peptide-removed mouse monoclonal antibody could be detected as a band in Mab supernatant. The MAB_2734c signal peptide-removed protein was specific in both standard and clinical strains, and was prevalent in clinical Mab strains. The MAB_2734c signal peptide-removed protein was not detected in other non-tuberculous mycobacterial clinical strains. Figure 8 ).

[0160] Example 4: Detection of NTM Culture Filtrate Specificity by Double Antibody Sandwich ELISA

[0161] 4.1 Establish an immunoassay technique for specific secreted proteins of Mycobacterium abscessus

[0162] A double-antibody sandwich ELISA method was initially established using monoclonal antibodies and HRP-labeled polyclonal antibodies to detect Mab-specific secreted proteins in test samples, enabling rapid and inexpensive bacterial identification. A colloidal gold-labeled monoclonal antibody against Mab secreted proteins was placed at the conjugation pad. If Mab-specific secreted proteins were present in the sample, they formed an immune complex with the colloidal gold-labeled antibody. This complex migrated on the nitrocellulose membrane due to capillary chromatography and was captured by the Mab secreted protein polyclonal antibody immobilized at the detection line, forming a purple-red band. Figure 9 ).

[0163] 4.2 Double Antibody Sandwich ELISA Method

[0164] (1) Coating: Dilute the anti-MAB_2734c signal peptide-removed mouse monoclonal antibody to 1 μg / mL, add 100 μL to each well of the microplate, seal and coat at 4°C overnight.

[0165] (2) The next day, take out the microplate, wash the microplate with 1×PBST, 300μL / well, and pat dry for the last time.

[0166] (3) Then add 3% BSA blocking solution, 200 μL / well, place at room temperature and shake horizontally for 1 h;

[0167] (4) Add 300 μL of 1×PBST to each well and wash, then blot dry for the last time;

[0168] (5) Sample addition: Add 100 μL of concentrated filtrate of different nontuberculous mycobacterial clinical strains to each well. Set up multiple replicates for each sample and set up a positive control (i.e., 0.1 μg / mL immunogen), a negative control (3% BSA blocking solution) and a blank control. Place on a horizontal shaker and incubate for 2 h.

[0169] (6) Add 300 μL of 1×PBST to each well and wash 3 times, then pat dry the last time;

[0170] (7) Add 100 μL of diluted MAB_2734c signal peptide removal rabbit polyclonal antibody (final concentration 0.1 μg / mL) to each well, place at room temperature on a horizontal shaker, and block for 1 h;

[0171] (8) Add 300 μL of 1×PBST to each well for washing, pat dry, add 100 μL of goat anti-rabbit IgG-HRP secondary antibody to each well, and incubate at room temperature for 1 h;

[0172] (9) Prepare TMB colorimetric solution in advance, wash and dry with 1×PBST, add 100μL to each well of the plate, protect from light, and incubate at room temperature for 15-20min.

[0173] (10) Terminate the reaction with 50 μL of 2% H2SO4 per well and measure OD. 450nm Absorbance value at point;

[0174] (11) ROC curves for OD values ​​were generated using SPSS to calculate the sensitivity and specificity at the optimal diagnostic cutoff point. 4.3 Specificity analysis of culture filtrates of different nontuberculous mycobacterial clinical strains detected by the double-antibody sandwich method.

[0175] A double-antibody sandwich ELISA was established using purified mouse monoclonal antibody and rabbit polyclonal antibody against the removal of the MAB_2734c signal peptide protein. The assay consisted of "anti-MAB_2734c signal peptide protein removal mouse monoclonal antibody - detection of different NTM clinical strain culture filtrates - anti-MAB_2734c signal peptide protein removal rabbit polyclonal antibody - HRP-labeled secondary antibody" to detect the specificity of proteins in the culture filtrates of different NTM clinical strains. The assay included 45 Mab clinical strains and 9 other non-tuberculous mycobacterial clinical strains. The negative control was 3% BSA, and the positive control was 0.1 μg / mL MAB_2734c antigen. Results showed good reaction with proteins in the Mab clinical strain culture filtrate, with high specificity of the antigen-antibody reaction. However, the binding signal was weaker with proteins in the culture filtrate of other NTM clinical strains. Figure 10 A). By using the ROC curve, it was found that when OD 450At a value of 0.7805, the Youden index is largest, and the area under the ROC curve is 0.916 (P < 0.001). Therefore, 0.7805 can be used as the optimal reference value for detecting Mab. If OD is used... 450 A value greater than 0.7805 is considered a positive result for the detection of Mab. Therefore, the sensitivity of this diagnostic method is 84.4%, and the specificity is 100%. Figure 10 B).

Claims

1. A biomarker for Mycobacterium abscessus, said biomarker being a protein of MAB_2734c with its signal peptide removed, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. Application of MAB_2734c signal peptide-removed protein in the preparation of a kit for the differential diagnosis of Mycobacterium abscessis infection. The amino acid sequence of the MAB_2734c signal peptide-removed protein is shown in SEQ ID NO:

2. When the MAB_2734c signal peptide-removed protein can be detected in the patient's biological sample, the patient is judged to be infected with Mycobacterium abscessis.

3. The use of the MAB_2734c protein with the signal peptide removed as described in claim 2 in the preparation of a kit for the differential diagnosis of Mycobacterium abscessis infection, wherein the patient's biological sample is selected from blood, pleural fluid, or sputum.

4. A kit for the differential diagnosis of Mycobacterium abscessis infection, the kit containing a reagent for detecting a protein of MAB_2734c with its signal peptide removed, the amino acid sequence of the MAB_2734c protein being shown in SEQ ID NO:

2.

5. The kit for differential diagnosis of Mycobacterium abscessis infection as described in claim 4, wherein the kit can be used to prepare an ELISE detection kit and / or a colloidal gold detection kit.

6. A monoclonal antibody-based mouse hybridoma cell line, HHR2734, for screening MAB_2734c with the removal of the signal peptide protein, with accession number CGMCC NO.45846, wherein... The amino acid sequence of the protein with the signal peptide removed from MAB_2734c is shown in SEQ ID NO: 2.

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