A Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry

Abstract

This invention discloses a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, belonging to the field of clinical immunology testing. It includes solutions R1 and R2. Solution R1 is a dispersion of double-coupled latex microspheres coated with natural Mycoplasma pneumoniae antigens, and solution R2 is a sample diluent containing an enhancer. The test samples can be serum, plasma, or whole blood samples, and it is applicable to biochemical analyzers, protein analyzers, or combined blood cell and immunoturbidimetric analyzers. The kit prepared by this invention can be used on various instruments, has a wide range of detection targets, and can be coupled with other instruments to detect various clinically valuable blood indicators, further shortening the testing time. It solves the problems of poor accuracy, low efficiency, and limited application scenarios caused by the subjective interpretation of results, long processing time, and limited applicability of blood samples in existing technologies. It can effectively simplify the testing process and improve diagnostic efficiency.

Description

Technical Field

[0001] This invention relates to the field of clinical immunology testing technology, specifically to a kit for detecting Mycoplasma pneumoniae antibodies based on immunoturbidimetry. Background Technology

[0002] Mycoplasma pneumoniae (MP) is a common human pathogen causing respiratory infections, accounting for 15-20% of all pneumonia cases. It is primarily transmitted through respiratory droplets, and cases are usually sporadic. While it can occur year-round, the peak season is autumn, winter, and early spring, with regional epidemics occurring every 3-7 years. Infants and school-aged children are the most affected. The incubation period for MP infection is 10-21 days. The symptoms are varied, with most patients presenting with low-grade fever and fatigue. Dry cough is the most prominent respiratory symptom. Lower respiratory tract infection can lead to bronchitis and pneumonia. Extra-respiratory symptoms commonly include earache, measles-like or scarlet fever-like rashes. A small number of patients may experience gastroenteritis, pericarditis, myocarditis, meningoencephalitis, myelitis, hemolytic anemia, disseminated intravascular coagulation, arthritis, and hepatitis. Some patients may experience sudden high fever accompanied by significant headache, muscle pain, nausea, and other systemic toxic symptoms. Laboratory testing for Mycoplasma pneumoniae can help diagnose acute Mycoplasma pneumoniae infection. Commonly used detection methods include isolation and culture, serological testing, and nucleic acid amplification technology. Currently, serological antibody testing is the main clinical method used.

[0003] The "Chinese Expert Consensus on Laboratory Diagnosis of Mycoplasma pneumoniae Respiratory Infections in Children" published by the Chinese Medical Association in 2019 mentions the following methods for detecting Mycoplasma pneumoniae serological antibodies: ELISA, latex particle agglutination, colloidal gold method, chemiluminescence, and immunofluorescence. Among these, latex particle agglutination and immunofluorescence rely on manual observation of results, making interpretation highly subjective and requiring over 2 hours. ELISA and chemiluminescence are suitable for batch testing, with chemiluminescence requiring specialized equipment and taking over 1 hour. These methods are only applicable to serum or plasma samples and are primarily used in central laboratories or for inpatients in large hospitals. The colloidal gold method is a point-of-care testing (POCT) rapid detection method, typically providing results in 15 minutes, and is suitable for whole blood and serum or plasma samples, making it ideal for outpatient and primary care hospitals. However, Mycoplasma pneumoniae is a pathogen that causes respiratory infections. During peak seasons for respiratory infections, it is difficult to distinguish the specific pathogen and medication based solely on symptoms. Therefore, hospitals typically combine infection markers such as CRP / SAA, complete blood counts, and pathogen marker testing for comprehensive treatment and medication. In outpatient clinics and primary care hospitals, biochemical and blood cell analyzers are widely available. Collecting blood from a child's fingertip allows for simultaneous analysis of inflammatory markers and complete blood counts, aiding in diagnosis and medication. Children are the primary population affected by Mycoplasma pneumoniae, and their medication differs from that for bacteria and viruses. Therefore, collecting fingertip blood from suspected infected children for colloidal gold antibody testing is also essential. If a patient requires simultaneous testing for inflammatory markers CRP / SAA, complete blood counts, and Mycoplasma pneumoniae, at least one additional capillary blood sample is needed for colloidal gold Mycoplasma pneumoniae antibody testing. This increases the difficulty for children, whose blood collection is already challenging, and the colloidal gold antibody test is a manual, subjective assessment, which is inconvenient.

[0004] Therefore, there is an urgent need for an instrumental detection method for Mycoplasma pneumoniae that can simultaneously detect multiple indicators in whole blood and serum to solve the above problems. Summary of the Invention

[0005] To address this, the present invention provides a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, thereby solving the problems of poor detection accuracy, low efficiency, and limited application scenarios caused by the subjective nature of result interpretation, long processing time, and limited applicability of blood samples in existing technologies.

[0006] According to a first aspect of the present invention, a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry is provided, comprising R1 solution and R2 solution, wherein R1 solution is a dispersion of dual-coupled latex microspheres coated with natural Mycoplasma pneumoniae antigen, and R2 solution is a sample diluent containing an enhancer; the test sample of the kit is serum, plasma or whole blood sample, and the applicable instrument is a biochemical analyzer, a protein analyzer or a blood cell and immunoturbidimetric analyzer;

[0007] The protein analyzer is a specific protein analyzer, specifically an instrument that uses immunoturbidimetry to detect C-reactive protein in serum / plasma / whole blood samples.

[0008] Furthermore, the R2 solution contains PEG6000 and an enhancer to enhance the antigen-antibody reaction signal, as well as one or more of SDS, Triton X100, and ammonium salts to lyse red blood cells.

[0009] Furthermore, the method for preparing the reinforcing agent includes the following steps:

[0010] S1. Dissolve PMS in anhydrous ethanol to 10 mM, add ANS to 20 mM, stir at 100 rpm for 3 h at room temperature in the dark, remove ethanol by rotary evaporation, and dry to obtain ANS-PMS conjugate.

[0011] S2. Dissolve NBT in anhydrous ethanol to a concentration of 10 mg / mL to obtain an NBT solution;

[0012] S3. Dissolve the ANS-PMS conjugate in ethanol to 5 mM, dilute with Tris-HCl buffer to 1 mM, add NBT solution to a final NBT concentration of 0.03 mM, stir well, and then add trehalose and preservative P300 to a mass fraction of 0.5% and 0.05% respectively to obtain the enhancer.

[0013] Furthermore, the Mycoplasma pneumoniae natural antigen-coated dual-coupled latex microsphere dispersion in the R1 solution includes antigen-latex microsphere A, antigen-latex microsphere B, and a preservation solution. The concentrations of antigen-latex microsphere A and antigen-latex microsphere B in the preservation solution are both 0.5-1 mg / mL, and their mass ratio is (1-3):1.

[0014] Furthermore, both antigen-latex microspheres A and B have a particle size of 50-500 nm and are made of polystyrene. The surface of the latex microspheres of antigen-latex microsphere A is modified with carboxyl groups, and the surface of the latex microspheres of antigen-latex microsphere B is modified with maleimide groups. The natural antigen of Mycoplasma pneumoniae is a high-concentration Mycoplasma pneumoniae glycolipid and membrane protein complex extracted with a surfactant. The surfactant is one or more of SDS, Triton X100, and ammonium salts.

[0015] Further, the preservation solution is prepared by dissolving glycerol, sucrose, polyvinyl alcohol, preservative P300 and preservative BND in 0.05M, pH 7.4 Tris-HCl buffer until the volume fraction of glycerol is 1-10%, and the mass fractions of sucrose, polyvinyl alcohol, preservative P300 and preservative BND are 1-10%, 0.1-1%, 0.1-0.5% and 0.01-0.05% respectively, and dispersing them evenly.

[0016] According to a second aspect of the present invention, a method for preparing a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry is provided, for preparing the above-mentioned Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, specifically including the following steps:

[0017] S1. Preparation of natural antigens of Mycoplasma pneumoniae:

[0018] Mycoplasma pneumoniae FH strain was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. The bacterial cells were resuspended in phosphate buffer containing 0.2% Tween 80, and then ether was added and mixed thoroughly. The mass-to-volume ratio of bacterial cells to phosphate buffer and ether was 1g:10mL:10mL. The mixture was shaken at 2-8℃ for 30 min, centrifuged at 12000rpm for 10 min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was freeze-dried to obtain the natural antigen of Mycoplasma pneumoniae.

[0019] S2. Preparation of antigen-latex microspheres A:

[0020] Carboxylated latex microspheres were washed with coupling buffer and resuspended, then mixed with EDC / NHS solution. After reaction, the supernatant was removed by centrifugation. The precipitate was resuspended with coupling buffer, and mycoplasma pneumoniae natural antigen was added. After reaction, the supernatant was removed by centrifugation, the precipitate was resuspended, and then rapidly aged by heat to obtain antigen-latex microspheres A.

[0021] S3. Preparation of antigen-latex microspheres B:

[0022] The surface-modified maleimide latex microspheres were washed with phosphate buffer and resuspended, then mycoplasma pneumoniae natural antigen was added. After reaction, the supernatant was removed by centrifugation, and the precipitate was resuspended to obtain antigen-latex microspheres B.

[0023] S4. Preparation of a dispersion of dual-coupled latex microspheres coated with natural antigens of Mycoplasma pneumoniae:

[0024] Antigen-latex microspheres A and antigen-latex microspheres B were mixed at a mass ratio of (1-3):1 and dispersed evenly to obtain a dispersion of latex microspheres coated with natural antigen of Mycoplasma pneumoniae.

[0025] S5. Preparation of reinforcing agent:

[0026] PMS and ANS were dissolved in ethanol, stirred and mixed, and dried. The mixture was then mixed with an ethanol solution of NBT in Tris-HCl, and trehalose and preservative P300 were added to obtain the reinforcing agent.

[0027] S6. Preparation of sample diluent containing enhancer:

[0028] Dissolve BSA, casein, PEG6000, enhancer, SDS, preservative P300, and preservative BND in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.1-1%, 0.01-0.3%, 3-5%, 0.5-2%, 0.1-2%, 0.1-0.5%, and 0.01-0.05%.

[0029] Furthermore, step S2 specifically includes the following steps:

[0030] a. Wash 50 mg / mL carboxylated latex microspheres 2-3 times with 9 times the volume of coupling buffer, and then resuspend the carboxylated latex microspheres with the same volume of coupling buffer to obtain latex microsphere resuspension A.

[0031] b. Dissolve EDC and NHS separately in coupling buffer to a final concentration of 20 mg / mL, mix in equal volumes to obtain microsphere activation solution;

[0032] c. Mix latex microsphere resuspension A and microsphere activation solution at a volume ratio of 18:1, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend in an equal volume of coupling buffer to obtain activated latex microspheres.

[0033] d. Add 10 mg / mL of Mycoplasma pneumoniae natural antigen dispersion to make the mass ratio of activated latex microspheres to Mycoplasma pneumoniae natural antigen 5:1. After shaking and reacting at room temperature for 120 min, add 20% of the volume of Mycoplasma pneumoniae natural antigen dispersion in 10% BSA. After shaking and reacting at room temperature for 60 min, centrifuge to remove the supernatant.

[0034] e. After resuspending the precipitate to 1 mg / mL in the preservation solution, disperse it by sonication, accelerate aging at 37°C for 3 days, and replace with a new preservation solution to obtain antigen-latex microspheres A.

[0035] Further, the coupling buffer in step S1 is a 0.1M 2-morpholine ethanesulfonic acid solution with a pH of 5.0.

[0036] Furthermore, step S3 specifically includes the following steps:

[0037] a. Wash the 50 mg / mL maleimide-modified latex microspheres 2-3 times with 9 times the volume of phosphate buffer. After washing, resuspend the latex microspheres with the same volume of phosphate buffer to obtain latex microsphere resuspension B.

[0038] b. Add 10 mg / mL of Mycoplasma pneumoniae natural antigen dispersion to make the mass ratio of surface-modified maleimide latex microspheres to Mycoplasma pneumoniae natural antigen 5:1. After shaking and reacting at room temperature for 120 min, add 20% of the volume of Mycoplasma pneumoniae natural antigen dispersion in 10% BSA. Shake and react at room temperature for 60 min. After centrifugation to remove the supernatant, resuspend the precipitate in preservation solution to 1 mg / mL to obtain antigen-latex microspheres B.

[0039] The present invention has the following advantages:

[0040] 1. This invention provides a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, primarily for rapid clinical detection of Mycoplasma pneumoniae antibodies. It is suitable for whole blood samples, such as finger-prick blood samples from children, requiring only a small amount of whole blood for immediate testing, making it patient-friendly for children. The detection of Mycoplasma pneumoniae antibodies can be performed on a standard biochemical analyzer in outpatient clinics, emergency rooms, and central laboratories; it can also be simultaneously detected with inflammatory markers CRP / SAA and complete blood count (CBC) on a specific model of hematology analyzer. A single finger-prick blood collection can detect multiple clinically valuable diagnostic results, including CBC, CRP / SAA, and Mycoplasma pneumoniae antibodies, further shortening the testing time, especially during respiratory epidemic seasons in pediatric outpatient clinics, significantly improving testing efficiency.

[0041] 2. This invention uses a dispersion of dual-coupled latex microspheres coated with Mycoplasma pneumoniae natural antigen as the reaction solution. The Mycoplasma pneumoniae natural antigen is a high-concentration Mycoplasma pneumoniae glycolipid and membrane protein complex extracted with a surfactant. The two types of coupled latex microspheres are carboxylated latex microspheres and surface-modified maleimide latex microspheres, respectively coupled to the amino and thiol groups of the antigen. The amino groups are abundant on the antigen surface, ensuring coupling efficiency and detection sensitivity. However, this binding is disordered, which may lead to the antigen-antibody binding sites being encapsulated internally, potentially affecting the spatial orientation of the antigen-antibody reaction and reducing binding efficiency and detection precision. The thiol groups are only present in small amounts at specific sites on the antigen surface. While their binding alone cannot guarantee coupling efficiency, their binding to the latex microspheres ensures that a large number of antigen-antibody binding sites are exposed, improving detection precision. Therefore, this invention prepares dual-coupled latex microspheres coated with Mycoplasma pneumoniae natural antigen in a specific ratio, optimizing the spatial conformation of the antigen and thus significantly improving detection precision.

[0042] 3. This invention adds substances that enhance antigen-antibody reactions and substances that lyse red blood cells to the sample diluent to enhance the binding between antigens and antibodies and improve detection sensitivity. Furthermore, by lysing red blood cells, background interference from red blood cells in whole blood samples is effectively eliminated, improving the accuracy and sensitivity of whole blood sample detection.

[0043] 4. This invention uses the ANS-NBT complex as an enhancer. ANS, as a hydrophobic molecule, can specifically bind to the antigen-antibody hydrophobic microenvironment, anchoring NBT molecules to the immune complex, causing them to aggregate in the hydrophobic microenvironment to form a blue-purple formazan precipitate, thus enhancing the turbidity signal. A small amount can improve the sensitivity and precision of detecting Mycoplasma pneumoniae antibodies. Attached Figure Description

[0044] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0045] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0046] Figure 1 This is a standard curve for detecting Mycoplasma pneumoniae antibody standard samples using an immunoturbidimetric assay kit provided in Embodiment 1 of the present invention.

[0047] Figure 2 This is a standard curve for detecting Mycoplasma pneumoniae antibody standard samples using an immunoturbidimetric assay kit provided in Embodiment 2 of the present invention.

[0048] Figure 3 The standard curve for detecting Mycoplasma pneumoniae antibody standard samples using a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, provided in Comparative Example 1 of this invention;

[0049] Figure 4 The standard curve for detecting Mycoplasma pneumoniae antibody standard samples using a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, provided in Comparative Example 2 of this invention;

[0050] Figure 5 This is a standard curve for detecting Mycoplasma pneumoniae antibody standard samples using an immunoturbidimetric assay kit provided in Comparative Example 3 of the present invention. Detailed Implementation

[0051] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The abbreviations for the components in this invention specifically include:

[0053] 5-Methylphenazinium methyl sulfate (PMS), 1-Anilinonaphthalene-8-sulfonic acid (ANS), Nitro Blue Tetrazolium (NBT), Sodium Dodecyl Sulfate (SDS), Octylphenol ethoxylate (Triton X-100), Polyethylene Glycol 6000 (PEG6000), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-Hydroxysuccinimide (NHS), and Bovine Serum Albumin (BSA).

[0054] According to a first aspect of the present invention, a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry is provided, comprising R1 solution and R2 solution, wherein R1 solution is a dispersion of dual-coupled latex microspheres coated with natural Mycoplasma pneumoniae antigen, and R2 solution is a sample diluent containing an enhancer; the test sample of the kit is serum, plasma or whole blood sample, and the applicable instrument is a biochemical analyzer, a protein analyzer or a blood cell and immunoturbidimetric analyzer;

[0055] Among them, the protein analyzer is a specific protein analyzer, specifically an instrument that uses immunoturbidimetry to detect C-reactive protein in serum / plasma / whole blood samples.

[0056] R2 solution contains PEG6000 and an enhancer to enhance the antigen-antibody reaction signal, as well as one or more of SDS, Triton X100, and ammonium salts to lyse red blood cells.

[0057] The preparation method of the reinforcing agent includes the following steps:

[0058] S1. Dissolve PMS in anhydrous ethanol to 10 mM, add ANS to 20 mM, stir at 100 rpm for 3 h at room temperature in the dark, remove ethanol by rotary evaporation, and dry to obtain ANS-PMS conjugate.

[0059] S2. Dissolve NBT in anhydrous ethanol to a concentration of 10 mg / mL to obtain an NBT solution;

[0060] S3. Dissolve the ANS-PMS conjugate in ethanol to 5 mM, dilute with Tris-HCl buffer to 1 mM, add NBT solution to a final NBT concentration of 0.03 mM, stir well, and then add trehalose and preservative P300 to a mass fraction of 0.5% and 0.05% respectively to obtain the enhancer.

[0061] Among them, the Mycoplasma pneumoniae natural antigen-coated dual-coupled latex microsphere dispersion in R1 solution includes antigen-latex microsphere A, antigen-latex microsphere B and preservation solution. The concentrations of antigen-latex microsphere A and antigen-latex microsphere B in the preservation solution are both 0.5-1 mg / mL, and the mass ratio of the two is (1-3):1.

[0062] Among them, the particle size of antigen-latex microspheres A and antigen-latex microspheres B is 50-500nm, and the material is polystyrene. The surface of the latex microspheres of antigen-latex microspheres A is modified with carboxyl groups, and the surface of the latex microspheres of antigen-latex microspheres B is modified with maleimide groups. The natural antigen of Mycoplasma pneumoniae is a high-concentration Mycoplasma pneumoniae glycolipid and membrane protein complex extracted with surfactants. The surfactants are one or more of SDS, Triton X100, and ammonium salts.

[0063] The preservation solution is prepared by dissolving glycerol, sucrose, polyvinyl alcohol, preservative P300, and preservative BND in 0.05M, pH 7.4 Tris-HCl buffer until the volume fraction of glycerol is 1-10%, and the mass fractions of sucrose, polyvinyl alcohol, preservative P300, and preservative BND are 1-10%, 0.1-1%, 0.1-0.5%, and 0.01-0.05%, respectively, and dispersing them evenly.

[0064] According to a second aspect of the present invention, a method for preparing a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry is provided, for preparing the above-mentioned Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, specifically including the following steps:

[0065] S1. Preparation of natural antigens of Mycoplasma pneumoniae:

[0066] Mycoplasma pneumoniae FH strain was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. The bacterial cells were resuspended in phosphate buffer containing 0.2% Tween 80, and then ether was added and mixed thoroughly. The mass-to-volume ratio of bacterial cells to phosphate buffer and ether was 1g:10mL:10mL. The mixture was shaken at 2-8℃ for 30 min, centrifuged at 12000rpm for 10 min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was freeze-dried to obtain the natural antigen of Mycoplasma pneumoniae.

[0067] S2. Preparation of antigen-latex microspheres A:

[0068] Carboxylated latex microspheres were washed with coupling buffer and resuspended, then mixed with EDC / NHS solution. After reaction, the supernatant was removed by centrifugation. The precipitate was resuspended with coupling buffer, and mycoplasma pneumoniae natural antigen was added. After reaction, the supernatant was removed by centrifugation, the precipitate was resuspended, and then rapidly aged by heat to obtain antigen-latex microspheres A.

[0069] S3. Preparation of antigen-latex microspheres B:

[0070] The surface-modified maleimide latex microspheres were washed with phosphate buffer and resuspended, then mycoplasma pneumoniae natural antigen was added. After reaction, the supernatant was removed by centrifugation, and the precipitate was resuspended to obtain antigen-latex microspheres B.

[0071] S4. Preparation of a dispersion of dual-coupled latex microspheres coated with natural antigens of Mycoplasma pneumoniae:

[0072] Antigen-latex microspheres A and antigen-latex microspheres B were mixed at a mass ratio of (1-3):1 and dispersed evenly to obtain a dispersion of latex microspheres coated with natural antigen of Mycoplasma pneumoniae.

[0073] S5. Preparation of reinforcing agent:

[0074] PMS and ANS were dissolved in ethanol, stirred and mixed, and dried. The mixture was then mixed with an ethanol solution of NBT in Tris-HCl, and trehalose and preservative P300 were added to obtain the reinforcing agent.

[0075] S6. Preparation of sample diluent containing enhancer:

[0076] Dissolve BSA, casein, PEG6000, enhancer, SDS, preservative P300, and preservative BND in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.1-1%, 0.01-0.3%, 3-5%, 0.5-2%, 0.1-2%, 0.1-0.5%, and 0.01-0.05%.

[0077] Step S2 specifically includes the following steps:

[0078] a. Wash 50 mg / mL carboxylated latex microspheres 2-3 times with 9 times the volume of coupling buffer, and then resuspend the carboxylated latex microspheres with the same volume of coupling buffer to obtain latex microsphere resuspension A.

[0079] b. Dissolve EDC and NHS separately in coupling buffer to a final concentration of 20 mg / mL, mix in equal volumes to obtain microsphere activation solution;

[0080] c. Mix latex microsphere resuspension A and microsphere activation solution at a volume ratio of 18:1, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend in an equal volume of coupling buffer to obtain activated latex microspheres.

[0081] d. Add 10 mg / mL of Mycoplasma pneumoniae natural antigen dispersion to make the mass ratio of activated latex microspheres to Mycoplasma pneumoniae natural antigen 5:1. After shaking and reacting at room temperature for 120 min, add 20% of the volume of Mycoplasma pneumoniae natural antigen dispersion in 10% BSA. After shaking and reacting at room temperature for 60 min, centrifuge to remove the supernatant.

[0082] e. After resuspending the precipitate to 1 mg / mL in the preservation solution, disperse it by sonication, accelerate aging at 37°C for 3 days, and replace with a new preservation solution to obtain antigen-latex microspheres A.

[0083] In step S1, the coupling buffer is a 0.1M 2-morpholine ethanesulfonic acid solution with a pH of 5.0.

[0084] Step S3 specifically includes the following steps:

[0085] a. Wash the 50 mg / mL maleimide-modified latex microspheres 2-3 times with 9 times the volume of phosphate buffer. After washing, resuspend the latex microspheres with the same volume of phosphate buffer to obtain latex microsphere resuspension B.

[0086] b. Add 10 mg / mL of Mycoplasma pneumoniae natural antigen dispersion to make the mass ratio of surface-modified maleimide latex microspheres to Mycoplasma pneumoniae natural antigen 5:1. After shaking and reacting at room temperature for 120 min, add 20% of the volume of Mycoplasma pneumoniae natural antigen dispersion in 10% BSA. Shake and react at room temperature for 60 min. After centrifugation to remove the supernatant, resuspend the precipitate in preservation solution to 1 mg / mL to obtain antigen-latex microspheres B.

[0087] Example 1

[0088] S1. Preparation of natural antigens of Mycoplasma pneumoniae:

[0089] Mycoplasma pneumoniae FH strain (purchased from Zhengzhou Minyou Biotechnology Co., Ltd., catalog number MY01A001, cultured in our laboratory) was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. 1g of bacterial cells was resuspended in 10mL of phosphate buffer containing 0.2% Tween 80, and then 10mL of ether was added and mixed thoroughly. The mixture was shaken at 2-8℃ for 30min, centrifuged at 12000rpm for 10min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was lyophilized to obtain the natural antigen of Mycoplasma pneumoniae.

[0090] S2. Preparation of preservation solution:

[0091] Dissolve 0.25 mL of glycerol, 0.25 g of sucrose, 0.025 g of polyvinyl alcohol, 0.006 g of preservative P300 and 0.001 g of preservative BND in 5 mL of 0.05 M, pH 7.4 Tris-HCl buffer solution and disperse evenly to obtain the preservation solution.

[0092] S3. Preparation of antigen-latex microspheres A:

[0093] a. Add 100 μL of 50 mg / mL latex microspheres (JSR IMMUTEX) TM P0323 (purchased from JSR Corporation of Japan) was washed twice with 900 μL of 0.1 M 2-morpholine ethanesulfonic acid solution at pH 5.0. After washing, the latex microspheres were resuspended in 900 μL of the same 2-morpholine ethanesulfonic acid solution to obtain a latex microsphere resuspension.

[0094] b. Dissolve EDC and NHS in 0.1M 2-morpholine ethanesulfonic acid solution (MES) at pH 5.0 to a final concentration of 20 mg / mL, respectively, and mix them in equal volumes to obtain microsphere activation solution;

[0095] c. Mix 900 μL of latex microsphere resuspension with 100 μL of microsphere activation solution, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend in 900 μL of 0.1 M 2-morpholine ethanesulfonic acid solution (pH 5.0) to obtain activated latex microspheres.

[0096] d. Add 100 μL of 10 mg / mL Mycoplasma pneumoniae natural antigen dispersion, shake at room temperature for 120 min, then add 20 μL of 10% BSA, shake at room temperature for 60 min, and centrifuge to remove the supernatant.

[0097] e. After resuspending the precipitate to 1 mg / mL in the preservation solution, it was ultrasonically dispersed, and then aged at 37°C for 3 days. The preservation solution was then replaced to obtain antigen-latex microspheres A.

[0098] S4. Preparation of antigen-latex microspheres B:

[0099] a. Wash 100 μL of 50 mg / mL maleimide-modified latex microspheres (purchased from Xi'an Qiyue Biotechnology Co., Ltd.) three times with 900 μL of phosphate buffer. After washing, resuspend the latex microspheres in 900 μL of the same phosphate buffer to obtain latex microsphere resuspension B.

[0100] b. Add 100 μL of 10 mg / mL Mycoplasma pneumoniae natural antigen dispersion, shake at room temperature for 120 min, then add 20 μL of 10% BSA, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend the precipitate in preservation solution to 1 mg / mL to obtain antigen-latex microspheres B.

[0101] S5. Preparation of a dispersion of dual-coupled latex microspheres coated with natural antigens of Mycoplasma pneumoniae:

[0102] Antigen-latex microspheres A and antigen-latex microspheres B were mixed at a mass ratio of 3:1 and dispersed evenly to obtain a dispersion of latex microspheres coated with natural antigen of Mycoplasma pneumoniae.

[0103] S6. Preparation of reinforcing agent:

[0104] a. Dissolve 18.3 mg of phenazine methyl sulfate (PMS, purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd.) in anhydrous ethanol, add 35.1 mg of 8-phenylamino-1-naphthalenesulfonic acid (ANS), stir at 100 rpm for 3 h at room temperature in the dark, remove ethanol by rotary evaporation, and dry to obtain ANS-PMS conjugate.

[0105] b. Dissolve 40 mg of nitrotetrazole blue (NBT, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) in 4 mL of anhydrous ethanol to obtain an NBT solution;

[0106] c. Dissolve the ANS-PMS conjugate in 10 mL of ethanol, dilute to 1 mM with Tris-HCl buffer, add 0.15 mL of NBT solution, stir well, and then add 0.3 g of trehalose and 0.03 g of preservative P300 to obtain the enhancer.

[0107] S7. Preparation of sample diluent containing enhancer:

[0108] BSA, casein, PEG6000, SDS, enhancer, preservative P300 and preservative BND were dissolved in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.5%, 0.1%, 4%, 1%, 1%, 0.1% and 0.02%, respectively.

[0109] Example 2

[0110] This embodiment is based on Example 1, except that the mass ratio of antigen-latex microspheres A to antigen-latex microspheres B is 1:1, and the other specific parameters are the same as in Example 1.

[0111] Comparative Example 1

[0112] S1. Preparation of natural antigens of Mycoplasma pneumoniae:

[0113] Mycoplasma pneumoniae FH strain was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. 1g of bacterial cells was resuspended in 10mL of phosphate buffer containing 0.2% Tween 80, and then 10mL of ether was added and mixed thoroughly. The mixture was shaken at 2-8℃ for 30min, centrifuged at 12000rpm for 10min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was freeze-dried to obtain the natural antigen of Mycoplasma pneumoniae.

[0114] S2. Preparation of preservation solution:

[0115] Dissolve 0.25 mL of glycerol, 0.25 g of sucrose, 0.025 g of polyvinyl alcohol, 0.006 g of preservative P300 and 0.001 g of preservative BND in 5 mL of 0.05 M, pH 7.4 Tris-HCl buffer solution and disperse evenly to obtain the preservation solution.

[0116] S3. Preparation of antigen-latex microspheres A:

[0117] a. Add 100 μL of 50 mg / mL latex microspheres (JSR IMMUTEX) TM P0323 (purchased from JSR Corporation of Japan) was washed twice with 900 μL of 0.1 M 2-morpholine ethanesulfonic acid solution at pH 5.0. After washing, the latex microspheres were resuspended in 900 μL of the same 2-morpholine ethanesulfonic acid solution to obtain a latex microsphere resuspension.

[0118] b. Dissolve EDC and NHS in 0.1M 2-morpholine ethanesulfonic acid solution (MES) at pH 5.0 to a final concentration of 20 mg / mL, respectively, and mix them in equal volumes to obtain microsphere activation solution;

[0119] c. Mix 900 μL of latex microsphere resuspension with 100 μL of microsphere activation solution, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend in 900 μL of 0.1 M 2-morpholine ethanesulfonic acid solution (pH 5.0) to obtain activated latex microspheres.

[0120] d. Add 100 μL of 10 mg / mL Mycoplasma pneumoniae natural antigen dispersion, shake at room temperature for 120 min, then add 20 μL of 10% BSA, shake at room temperature for 60 min, and centrifuge to remove the supernatant.

[0121] e. After resuspending the precipitate to 1 mg / mL in the preservation solution, it was ultrasonically dispersed, and then aged at 37°C for 3 days. The preservation solution was then replaced to obtain antigen-latex microspheres A.

[0122] S4. Preparation of reinforcing agent:

[0123] a. Dissolve 18.3 mg of PMS (purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd.) in anhydrous ethanol, add 35.1 mg of ANS, stir at 100 rpm for 3 h at room temperature in the dark, remove ethanol by rotary evaporation, and dry to obtain ANS-PMS conjugate.

[0124] b. Dissolve 40 mg of NBT (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) in 4 mL of anhydrous ethanol to obtain an NBT solution;

[0125] c. Dissolve the ANS-PMS conjugate in 10 mL of ethanol, dilute to 1 mM with Tris-HCl buffer, add 0.15 mL of NBT solution, stir well, and then add 0.3 g of trehalose and 0.03 g of preservative P300 to obtain the enhancer.

[0126] S5. Preparation of sample diluent containing enhancer:

[0127] BSA, casein, PEG6000, enhancer, SDS, preservative P300 and preservative BND were dissolved in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.5%, 0.1%, 4%, 1%, 1%, 0.1% and 0.02%, respectively.

[0128] This comparative example is based on Example 1, except that antigen-latex microspheres B are not added, while the other specific parameters are the same as in Example 1.

[0129] Comparative Example 2

[0130] S1. Preparation of natural antigens of Mycoplasma pneumoniae:

[0131] Mycoplasma pneumoniae FH strain was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. 1g of bacterial cells was resuspended in 10mL of phosphate buffer containing 0.2% Tween 80, and then 10mL of ether was added and mixed thoroughly. The mixture was shaken at 2-8℃ for 30min, centrifuged at 12000rpm for 10min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was freeze-dried to obtain the natural antigen of Mycoplasma pneumoniae.

[0132] S2. Preparation of preservation solution:

[0133] Dissolve 0.25 mL of glycerol, 0.25 g of sucrose, 0.025 g of polyvinyl alcohol, 0.006 g of preservative P300 and 0.001 g of preservative BND in 5 mL of 0.05 M, pH 7.4 Tris-HCl buffer solution and disperse evenly to obtain the preservation solution.

[0134] S3. Preparation of antigen-latex microspheres B:

[0135] a. Wash 100 μL of 50 mg / mL maleimide-modified latex microspheres (purchased from Xi'an Qiyue Biotechnology Co., Ltd.) three times with 900 μL of phosphate buffer. After washing, resuspend the latex microspheres in 900 μL of the same phosphate buffer to obtain latex microsphere resuspension B.

[0136] b. Add 100 μL of 10 mg / mL Mycoplasma pneumoniae natural antigen dispersion, shake at room temperature for 120 min, then add 20 μL of 10% BSA, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend the precipitate in preservation solution to 1 mg / mL to obtain antigen-latex microspheres B.

[0137] S4. Preparation of reinforcing agent:

[0138] a. Dissolve 18.3 mg of PMS (purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd.) in anhydrous ethanol, add 35.1 mg of ANS, stir at 100 rpm for 3 h at room temperature in the dark, remove ethanol by rotary evaporation, and dry to obtain ANS-PMS conjugate.

[0139] b. Dissolve 40 mg of NBT (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) in 4 mL of anhydrous ethanol to obtain an NBT solution;

[0140] c. Dissolve the ANS-PMS conjugate in 10 mL of ethanol, dilute to 1 mM with Tris-HCl buffer, add 0.15 mL of NBT solution, stir well, and then add 0.3 g of trehalose and 0.03 g of preservative P300 to obtain the enhancer.

[0141] S5. Preparation of sample diluent containing enhancer:

[0142] BSA, casein, PEG6000, SDS, preservative P300 and preservative BND were dissolved in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.5%, 0.1%, 4%, 1%, 1%, 0.1% and 0.02%, respectively.

[0143] This comparative example is based on Example 1, except that antigen-latex microspheres A are not added, while the other specific parameters are the same as in Example 1.

[0144] Comparative Example 3

[0145] S1. Preparation of natural antigens of Mycoplasma pneumoniae:

[0146] Mycoplasma pneumoniae FH strain was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. 1g of bacterial cells was resuspended in 10mL of phosphate buffer containing 0.2% Tween 80, and then 10mL of ether was added and mixed thoroughly. The mixture was shaken at 2-8℃ for 30min, centrifuged at 12000rpm for 10min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was freeze-dried to obtain the natural antigen of Mycoplasma pneumoniae.

[0147] S2. Preparation of preservation solution:

[0148] Dissolve 0.25 mL of glycerol, 0.25 g of sucrose, 0.025 g of polyvinyl alcohol, 0.006 g of preservative P300 and 0.001 g of preservative BND in 5 mL of 0.05 M, pH 7.4 Tris-HCl buffer solution and disperse evenly to obtain the preservation solution.

[0149] S3. Preparation of antigen-latex microspheres A:

[0150] a. Add 100 μL of 50 mg / mL latex microspheres (JSR IMMUTEX) TM P0323 (purchased from JSR Corporation of Japan) was washed twice with 900 μL of 0.1 M 2-morpholine ethanesulfonic acid solution at pH 5.0. After washing, the latex microspheres were resuspended in 900 μL of the same 2-morpholine ethanesulfonic acid solution to obtain a latex microsphere resuspension.

[0151] b. Dissolve EDC and NHS in 0.1M 2-morpholine ethanesulfonic acid solution (MES) at pH 5.0 to a final concentration of 20 mg / mL, respectively, and mix them in equal volumes to obtain microsphere activation solution;

[0152] c. Mix 900 μL of latex microsphere resuspension with 100 μL of microsphere activation solution, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend in 900 μL of 0.1 M 2-morpholine ethanesulfonic acid solution (pH 5.0) to obtain activated latex microspheres.

[0153] d. Add 100 μL of 10 mg / mL Mycoplasma pneumoniae natural antigen dispersion, shake at room temperature for 120 min, then add 20 μL of 10% BSA, shake at room temperature for 60 min, and centrifuge to remove the supernatant.

[0154] e. After resuspending the precipitate to 1 mg / mL in the preservation solution, it was ultrasonically dispersed, and then aged at 37°C for 3 days. The preservation solution was then replaced to obtain antigen-latex microspheres A.

[0155] S4. Preparation of antigen-latex microspheres B:

[0156] a. Wash 100 μL of 50 mg / mL maleimide-modified latex microspheres (purchased from Xi'an Qiyue Biotechnology Co., Ltd.) three times with 900 μL of phosphate buffer. After washing, resuspend the latex microspheres in 900 μL of the same phosphate buffer to obtain latex microsphere resuspension B.

[0157] b. Add 100 μL of 10 mg / mL Mycoplasma pneumoniae natural antigen dispersion, shake at room temperature for 120 min, then add 20 μL of 10% BSA, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend the precipitate in preservation solution to 1 mg / mL to obtain antigen-latex microspheres B.

[0158] S5. Preparation of a dispersion of dual-coupled latex microspheres coated with natural antigens of Mycoplasma pneumoniae:

[0159] Antigen-latex microspheres A and antigen-latex microspheres B were mixed at a mass ratio of 3:1 and dispersed evenly to obtain a dispersion of latex microspheres coated with natural antigen of Mycoplasma pneumoniae.

[0160] S6. Preparation of sample diluent:

[0161] BSA, casein, PEG6000, SDS, preservative P300 and preservative BND were dissolved in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.5%, 0.1%, 4%, 1%, 0.1% and 0.02%, respectively.

[0162] This comparative example is based on Example 1, except that no enhancer is added to the sample dilution solution, while the other specific parameters are the same as in Example 1.

[0163] Test Example 1

[0164] To verify the detection performance of the kit prepared in this invention for Mycoplasma pneumoniae antigen, standard curves for Mycoplasma pneumoniae antibodies were determined for the kits prepared in each example and comparative example. The specific steps included:

[0165] S1. The Mycoplasma pneumoniae antibody standard (purchased from Thermo Fisher Scientific) was diluted with phosphate buffer to concentrations of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL, respectively.

[0166] S2. Load the R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry into a Siemens Attellica IM 1300 biochemical analyzer. The sample loading volume is 10 μL of standard sample, 100 μL of R2 solution, and 25 μL of R1 solution (for Comparative Example 3, replace the enhancer with an equal volume of Tris-HCl buffer). Set up 3 replicates for each concentration of Mycoplasma pneumoniae antibody standard sample. After reacting for 10 min, the results are automatically interpreted, and a standard curve is plotted.

[0167] The standard curves for Mycoplasma pneumoniae antibodies prepared in the various examples and comparative examples are provided by [the following]. Figures 1-5 As shown, all examples and comparative examples exhibit good linearity, indicating a stable linear relationship between signal value and antibody concentration. Example 1 shows the largest signal range, with an average signal value of 2618 (mg / mL antibody standard). Figure 1 The average signal value of the 5 mg / mL antibody standard in Example 2 was 2053 (). Figure 2 The average signal value for the 5 mg / mL antibody standard in Comparative Example 1 was 1024 ( Figure 3 The average signal value of the 5 mg / mL antibody standard in Comparative Example 2 was 853 ( Figure 4 The average signal value for the 5 mg / mL antibody standard in Comparative Example 3 was 1369 ( Figure 5 Compared to Example 1, the average signal value of the 5 mg / mL antibody standard in Example 2 decreased by 23.6%, and the average signal values ​​of the 5 mg / mL antibody standards in Comparative Examples 1-3 decreased by 60.9%, 67.4%, and 47.7%, respectively. This indicates that the complete set of examples exhibited higher detection sensitivity for Mycoplasma pneumoniae antibodies. However, insufficient carboxylated latex microspheres led to a decrease in the antigen loading of the latex microspheres. Simultaneously, the absence of antigen-latex microsphere A (Comparative Example 2) weakened the antigen-binding ability of the latex, resulting in the smallest detection range. The absence of antigen-latex microsphere B led to excessive disordered coupling between the antigen and the latex microspheres, obscuring the antigen-antibody binding sites and preventing effective binding, thus reducing detection sensitivity. The enhancer acts as an amplifier for the turbidity signal in the kit prepared in this invention. Comparative Example 3, lacking the enhancer, exhibited the largest detection range among the comparative examples, but significantly lower than the example groups, confirming the auxiliary effect of the enhancer. Furthermore, the detection range of the example group was significantly larger than that of each comparative group, indicating that the components such as antigen-latex microspheres A, antigen-latex microspheres B, and enhancers in the kit prepared by the present invention have a synergistic effect on enhancing the turbidity signal.

[0168] Test Example 2

[0169] This invention uses a biochemical analyzer to detect Mycoplasma pneumoniae antibody standard samples to verify the detection precision of the kit prepared by this invention. Specifically, it includes the following steps:

[0170] The R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kits based on immunoturbidimetry prepared in each example and comparative example were loaded into a Siemens Attellica IM 1300 biochemical analyzer. The sample loading volume was 10 μL of 3 mg / mL Mycoplasma pneumoniae antibody standard sample, 100 μL of R2 solution, and 25 μL of R1 solution (for comparative example 3, an equal volume of Tris-HCl buffer was used instead of the enhancer). Ten replicates were set up for each sample, and the reaction was automatically interpreted after 10 min. The results are recorded in Table 1, and repeatability analysis was performed.

[0171] It can be seen that the kit prepared in this invention exhibits good reproducibility for detecting 3 mg / mL Mycoplasma pneumoniae antibody standard samples. The coefficients of variation for Examples 1, 2, and Comparative Examples 1-3 are 4.99%, 5.37%, 7.23%, 7.38%, and 6.95%, respectively. Generally, a coefficient of variation <10% is considered to indicate good reproducibility, and a coefficient of variation <5% is considered to indicate excellent reproducibility. This shows that the kit prepared in this invention demonstrates good or better precision in detecting Mycoplasma pneumoniae antibody standard samples, good intra-batch stability, and stable and reliable results.

[0172] Compared to Example 1, the coefficients of variation for Example 2 and Comparative Examples 1-3 increased by 7.6%, 44.9%, 47.9%, and 39.3%, respectively. It can be seen that the kits prepared in the complete example groups exhibit good precision in detecting Mycoplasma pneumoniae antibodies, with Example 1 showing the lowest coefficient of variation, indicating the best detection precision. This may be because the absence of maleimide-modified latex microspheres leads to increased disorder in the coupling between antigen and latex microspheres, with antigen-antibody binding sites encapsulated internally. This results in greater differences in the number of effective antigen-coated microspheres between different batches, leading to an increased coefficient of variation. However, the antigen has fewer and unevenly distributed thiol sites, and excessive maleimide-modified latex microspheres result in low antigen loading and large intra-batch variability, leading to decreased detection precision. Comparative Example 3, which lacks signal amplification and stabilization capabilities and relies solely on the aggregation ability of the antibody-antigen latex microspheres for turbidity detection, shows a significant decrease in signal value and relatively large background noise, resulting in large intra-group fluctuations.

[0173] Based on the combined results of Test Example 1 and Test Example 2, the Mycoplasma pneumoniae antibody detection kit prepared in this invention, using immunoturbidimetry, exhibits good and stable detection precision and repeatability for Mycoplasma pneumoniae antibody samples, meeting clinical needs. Furthermore, Example 1 demonstrates the best sensitivity and precision in detecting Mycoplasma pneumoniae antibodies.

[0174] Due to time and cost considerations, Example 1 was selected to conduct the experiments of Examples 3-8, and the Zhuhai Lizhu colloidal gold method reagent kit was used as the control group for the control experiment.

[0175] Test Example 3

[0176] This invention verifies the performance of the kit prepared in Example 1 of this invention in detecting Mycoplasma pneumoniae in serum samples by comparing it with a commercially available colloidal gold kit. The specific steps include:

[0177] S1. Zhuhai Lizhu Colloidal Gold Reagent Kit (purchased from Zhuhai Lizhu Reagent Co., Ltd.) was operated according to the kit instructions: 5uL of sample and 50uL of sample diluent were added to the sample wells of the reagent card, and the results were observed after 15 minutes. A color development ≥G2 was interpreted as positive.

[0178] S2. The R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 were loaded onto a Siemens Attellica IM 1300 biochemical analyzer. The sample loading volume was 10 μL of serum sample, 100 μL of R2 solution, and 25 μL of R1 solution. The reaction was allowed to proceed for 10 min, and the results were automatically interpreted. The data are recorded in Table 2.

[0179] Note: "-" indicates not detected. In the table, P group samples represent positive samples, N group samples represent negative samples, and L group samples represent borderline or weakly positive samples.

[0180] Table 2 shows that the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 of this invention has good distinguishability between the detection signal values ​​of positive and negative samples detected by the commercial colloidal gold method kit. The highest signal value of the negative sample in the kit of Example 1 is <60, and the lowest signal value of the positive sample is >100. Therefore, a signal value of 100 is used as the cutoff value for positive and negative. In addition, the kit prepared in Example 1 of this invention has a dynamic detection range of over 5000, which can distinguish different antibody titer levels, while the colloidal gold method kit can only provide colorimetric grade and cannot quantitatively provide antibody titer levels.

[0181] Test Example 4

[0182] This invention verifies the performance of the kit prepared in Example 1 of this invention in detecting Mycoplasma pneumoniae in whole blood by comparing it with a commercially available colloidal gold kit. The specific steps include:

[0183] Whole blood samples were collected using two capillary tubes. One tube was tested using the Zhuhai Lizhu colloidal gold method reagent kit according to the kit instructions. The other tube was tested using R1 and R2 solutions from Example 1 on a Shenzhen Dimai DP-H12 POCT integrated machine. The sample loading volume was 20uL of whole blood sample, 100uL of R2 solution, and 25uL of R1 solution. The results were automatically interpreted after 10 minutes and recorded in Table 3.

[0184] Note: "-" indicates not detected.

[0185] As shown in Table 3, the lowest signal value corresponding to the reagent kit of this invention for whole blood samples that tested positive by the colloidal gold method was 92, while the highest signal value corresponding to samples that tested negative by the colloidal gold method was 101. After excluding interference from critical data, the present invention can still use a signal value of 100 as the critical value for detecting positive and negative results for whole blood samples. Therefore, among the 30 whole blood samples, one positive sample (sample 9) and one negative sample (sample 25) were inconsistent with the commercial colloidal gold detection method, with an overall concordance rate of 93.3%. This indicates that the reagent kit prepared in Example 1 of this invention has good adaptability, is suitable for rapid detection of whole blood samples, and has high detection accuracy. The reason why the reagent kit prepared in this invention can use the same critical value for whole blood and serum samples is that a substance that can lyse red blood cells (SDS) is added to the sample diluent. This releases the components in the red blood cells, promotes the dissolution or denaturation of these components, thereby forming a homogeneous liquid environment similar to serum and eliminating background interference from red blood cells in whole blood samples. Based on the results of test examples 3 and 4, the kit prepared in Example 1 of this invention showed good detection accuracy for both whole blood and serum samples, and the same threshold value could be used, simplifying clinical operation and enabling large-scale clinical detection of Mycoplasma pneumoniae antibodies.

[0186] Test Example 5

[0187] This invention verifies the performance of the kit prepared in this invention in detecting Mycoplasma pneumoniae in expanded samples by comparing it with a commercially available colloidal gold kit. The specific steps include:

[0188] S1. Zhuhai Lizhu Colloidal Gold Reagent Kit (purchased from Zhuhai Lizhu Reagent Co., Ltd.) was operated according to the kit instructions: 5uL of sample and 50uL of sample diluent were added to the sample wells of the reagent card, and the results were observed after 15 minutes. A color development ≥G2 was interpreted as positive.

[0189] S2. The R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 of this invention were loaded onto a Siemens Attellica IM 1300 biochemical analyzer. The sample loading volume was 10 μL of serum sample, 100 μL of R2 solution, and 25 μL of R1 solution. The reaction was allowed to proceed for 10 min and the results were automatically interpreted. The serum samples of 200 patients with respiratory symptoms were tested in parallel, and the results are recorded in Table 4.

[0190] The results in Table 4 show that the positive concordance rate of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 of this invention was 91.1%, the negative concordance rate was 95.5%, and the overall concordance rate was 94.5% compared with the commercial colloidal gold method kit. This indicates that the kit prepared in Example 1 of this invention has good sensitivity and specificity for detecting Mycoplasma pneumoniae antibodies, which is helpful for clinical diagnosis and screening.

[0191] Test Example 6

[0192] This invention uses a biochemical analyzer to detect serum samples to verify the detection precision of the kit prepared by this invention, specifically including the following steps:

[0193] The R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 were loaded into a Siemens Attellica IM 1300 biochemical analyzer. The sample loading volume was 10 μL of serum sample, 100 μL of R2 solution, and 25 μL of R1 solution. Ten replicates of one positive serum sample were detected. The reaction was carried out for 10 min and the results were automatically interpreted. The results are recorded in Table 5 and repeatability analysis was performed.

[0194] As can be seen, the kit prepared in Example 1 of this invention exhibits good reproducibility for detecting Mycoplasma pneumoniae antibodies in serum samples. For a sample with an average value of 477.4, the standard deviation is only 24.7, and the coefficient of variation is only 5.2%. Generally, a coefficient of variation <10% is considered good reproducibility, and a coefficient of variation <5% is considered excellent reproducibility. The kit prepared in this invention shows a coefficient of variation close to excellent for detecting Mycoplasma pneumoniae antibodies in serum samples, indicating good intra-batch stability and stable and reliable results.

[0195] Test Example 7

[0196] This invention uses a biochemical analyzer to test whole blood samples to verify the detection precision of the kit prepared by this invention, specifically including the following steps:

[0197] The R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 were loaded onto the Shenzhen Dimai DP-H12 POCT integrated machine. The sample loading volume was 20uL of whole blood sample, 100uL of R2 solution, and 25uL of R1 solution. Ten replicates of one positive whole blood sample were detected. The reaction was carried out for 10 minutes and the results were automatically interpreted. The results are recorded in Table 6 and repeatability analysis was performed.

[0198] As can be seen, the kit prepared in Example 1 of this invention exhibits good reproducibility for detecting Mycoplasma pneumoniae antibodies in whole blood samples. For a sample with an average value of 360.2, the standard deviation is only 21.4, and the coefficient of variation is only 5.9%. The coefficient of variation of the kit prepared in this invention for detecting Mycoplasma pneumoniae antibodies in whole blood samples is close to the excellent level, indicating good intra-assay stability and stable and reliable results. Combining the results of Test Examples 6 and 7, the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in this invention shows good and stable detection precision and reproducibility for both whole blood and serum samples, which can meet clinical needs.

[0199] Test Example 8

[0200] The R1 and R2 solutions of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry prepared in Example 1 were placed at 4°C and 37°C for 7 days and 10 days, respectively. These solutions were then loaded onto a Siemens Attellica IM 1300 biochemical analyzer to detect positive and negative serum samples. The signal value reduction was expressed by the formula: The result of the calculation is multiplied by 100% and recorded in Table 7.

[0201] Note: In the table, P group samples represent positive samples, N group samples represent negative samples, and L group samples represent borderline or weakly positive samples.

[0202] Stability is a key indicator for the commercialization and clinical application of in vitro diagnostic reagents. As shown in Table 7, after 7 days of accelerated heat treatment at 37°C, the signal value reduction of the reagent kit prepared in Example 1 of this invention was controlled within 8%-10% for positive samples, and 9%-25% for borderline samples. For negative samples, due to their inherently lower signal values, the percentage reduction fluctuated more significantly, but the absolute signal value reduction was minimal, and its actual impact on the determination of positive or negative results was negligible. After 10 days of accelerated heat treatment at 37°C, the reduction in positive samples remained controlled within 9%-13%, demonstrating overall stability.

[0203] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A kit for detecting Mycoplasma pneumoniae antibodies based on immunoturbidimetry, characterized in that, The kit includes R1 solution and R2 solution, wherein R1 solution is a dispersion of double-coupled latex microspheres coated with natural antigen of Mycoplasma pneumoniae, and R2 solution is a sample diluent containing an enhancer; the test sample of the kit is serum, plasma or whole blood sample, and the applicable instruments are biochemical analyzers, protein analyzers or blood cell and immunoturbidimetric analyzers; The Mycoplasma pneumoniae natural antigen-coated dual-coupled latex microsphere dispersion in the R1 solution includes antigen-latex microsphere A, antigen-latex microsphere B, and a preservation solution. The particle size of antigen-latex microsphere A and antigen-latex microsphere B are both 50-500 nm, and both are made of polystyrene. The surface of the latex microspheres of antigen-latex microsphere A is modified with carboxyl groups, and the surface of the latex microspheres of antigen-latex microsphere B is modified with maleimide groups.

2. The Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 1, characterized in that, The R2 solution contains PEG6000 and an enhancer to enhance the antigen-antibody reaction signal, as well as one or more of SDS, Triton X100, and ammonium salts to lyse red blood cells.

3. The Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 2, characterized in that, The method for preparing the reinforcing agent includes the following steps: S1. Dissolve PMS in anhydrous ethanol to 10 mM, add ANS to 20 mM, stir at 100 rpm for 3 h at room temperature in the dark, remove ethanol by rotary evaporation, and dry to obtain ANS-PMS conjugate. S2. Dissolve NBT in anhydrous ethanol to a concentration of 10 mg / mL to obtain an NBT solution; S3. Dissolve the ANS-PMS conjugate in ethanol to 5 mM, dilute with Tris-HCl buffer to 1 mM, add NBT solution to a final NBT concentration of 0.03 mM, stir well, and then add trehalose and preservative P300 to a mass fraction of 0.5% and 0.05% respectively to obtain the enhancer.

4. The Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 1, characterized in that, The concentrations of antigen-latex microspheres A and antigen-latex microspheres B in the preservation solution are both 0.5-1 mg / mL, and their mass ratio is (1-3):

1.

5. The Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 1, characterized in that, The natural antigen of Mycoplasma pneumoniae is a high-concentration Mycoplasma pneumoniae glycolipid and membrane protein complex extracted with a surfactant, wherein the surfactant is one or more of SDS, Triton X100, and ammonium salts.

6. The Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 1, characterized in that, The preservation solution is prepared by dissolving glycerol, sucrose, polyvinyl alcohol, preservative P300, and preservative BND in 0.05M, pH 7.4 Tris-HCl buffer until the volume fraction of glycerol is 1-10%, and the mass fractions of sucrose, polyvinyl alcohol, preservative P300, and preservative BND are 1-10%, 0.1-1%, 0.1-0.5%, and 0.01-0.05%, respectively, and dispersing them evenly.

7. A method for preparing a Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry, used to prepare the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in any one of claims 1-6, characterized in that, Specifically, the following steps are included: S1. Preparation of natural antigens of Mycoplasma pneumoniae: Mycoplasma pneumoniae FH strain was cultured in broth medium for 7-10 days. The bacterial cells were collected by high-speed centrifugation. The bacterial cells were resuspended in phosphate buffer containing 0.2% Tween 80, and then ether was added and mixed thoroughly. The mass-to-volume ratio of bacterial cells to phosphate buffer and ether was 1g:10mL:10mL. The mixture was shaken at 2-8℃ for 30 min, centrifuged at 12000rpm for 10 min, and the supernatant was collected. The ether was removed under negative pressure, and the remaining liquid was freeze-dried to obtain the natural antigen of Mycoplasma pneumoniae. S2. Preparation of antigen-latex microspheres A: Carboxylated latex microspheres were washed with coupling buffer and resuspended, then mixed with EDC / NHS solution. After reaction, the supernatant was removed by centrifugation. The precipitate was resuspended with coupling buffer, and mycoplasma pneumoniae natural antigen was added. After reaction, the supernatant was removed by centrifugation, the precipitate was resuspended, and then rapidly aged by heat to obtain antigen-latex microspheres A. S3. Preparation of antigen-latex microspheres B: The surface-modified maleimide latex microspheres were washed with phosphate buffer and resuspended, then mycoplasma pneumoniae natural antigen was added. After reaction, the supernatant was removed by centrifugation, and the precipitate was resuspended to obtain antigen-latex microspheres B. S4. Preparation of a dispersion of dual-coupled latex microspheres coated with natural antigens of Mycoplasma pneumoniae: Antigen-latex microspheres A and antigen-latex microspheres B were mixed at a mass ratio of (1-3):1 and dispersed evenly to obtain a dispersion of latex microspheres coated with natural antigen of Mycoplasma pneumoniae. S5. Preparation of reinforcing agent: PMS and ANS were dissolved in ethanol, stirred and mixed, and dried. The mixture was then mixed with an ethanol solution of NBT in Tris-HCl, and trehalose and preservative P300 were added to obtain the reinforcing agent. S6. Preparation of sample diluent containing enhancer: Dissolve BSA, casein, PEG6000, enhancer, SDS, preservative P300, and preservative BND in 0.05M, pH 7.4 Tris-HCl buffer to a mass fraction of 0.1-1%, 0.01-0.3%, 3-5%, 0.5-2%, 0.1-2%, 0.1-0.5%, and 0.01-0.05%.

8. The preparation method of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 7, characterized in that, Step S2 specifically includes the following steps: a. Wash 50 mg / mL carboxylated latex microspheres 2-3 times with 9 times the volume of coupling buffer, and then resuspend the carboxylated latex microspheres with the same volume of coupling buffer to obtain latex microsphere resuspension A. b. Dissolve EDC and NHS separately in coupling buffer to a final concentration of 20 mg / mL, mix in equal volumes to obtain microsphere activation solution; c. Mix latex microsphere resuspension A and microsphere activation solution at a volume ratio of 18:1, shake at room temperature for 60 min, centrifuge to remove supernatant, and resuspend in an equal volume of coupling buffer to obtain activated latex microspheres. d. Add 10 mg / mL of Mycoplasma pneumoniae natural antigen dispersion to make the mass ratio of activated latex microspheres to Mycoplasma pneumoniae natural antigen 5:

1. After shaking and reacting at room temperature for 120 min, add 20% of the volume of Mycoplasma pneumoniae natural antigen dispersion in 10% BSA. After shaking and reacting at room temperature for 60 min, centrifuge to remove the supernatant. e. After resuspending the precipitate to 1 mg / mL in the preservation solution, disperse it by sonication, accelerate aging at 37°C for 3 days, and replace with a new preservation solution to obtain antigen-latex microspheres A.

9. The preparation method of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 7, characterized in that, The coupling buffer in step S1 is a 0.1M 2-morpholine ethanesulfonic acid solution with a pH of 5.

0.

10. The preparation method of the Mycoplasma pneumoniae antibody detection kit based on immunoturbidimetry as described in claim 7, characterized in that, Step S3 specifically includes the following steps: a. Wash the 50 mg / mL maleimide-modified latex microspheres 2-3 times with 9 times the volume of phosphate buffer. After washing, resuspend the latex microspheres with the same volume of phosphate buffer to obtain latex microsphere resuspension B. b. Add 10 mg / mL of Mycoplasma pneumoniae natural antigen dispersion to make the mass ratio of surface-modified maleimide latex microspheres to Mycoplasma pneumoniae natural antigen 5:

1. After shaking and reacting at room temperature for 120 min, add 20% of the volume of Mycoplasma pneumoniae natural antigen dispersion in 10% BSA. Shake and react at room temperature for 60 min. After centrifugation to remove the supernatant, resuspend the precipitate in preservation solution to 1 mg / mL to obtain antigen-latex microspheres B.

Images