Kit for detecting mycoplasma pneumoniae IgM antibody

Abstract

The invention relates to the technical field of biology, in particular to a kit for detecting a mycoplasma pneumoniae IgM antibody. The invention provides a mycoplasma pneumoniae antigen, a preparation method thereof and a kit for detecting a mycoplasma pneumoniae IgM antibody. The kit provided by the invention not only has good inter-batch stability, specificity and repeatability, but also can obviously reduce the false positive rate and improve the accuracy of the kit.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a kit for detecting Mycoplasma pneumoniae IgM antibodies. Background Technology

[0002] Mycoplasma pneumoniae (MP) is a common pathogen that primarily infects the human respiratory system, causing pneumonia. The clinical manifestations of MP infection are diverse, with patients experiencing symptoms such as cough, sputum production, fever, and fatigue. These symptoms are similar to other respiratory infections and are therefore easily overlooked or confused with other diseases; therefore, early detection of MP is crucial.

[0003] IgM antibodies are an early immune response in the body to a new infection, and their production increases rapidly within a few days after infection. By detecting the level of IgM antibodies in a patient's blood, it is possible to determine whether MP infection is present.

[0004] Magnetic particle chemiluminescence immunoassay is a highly sensitive and specific immunological detection method widely used in clinical diagnostics. It offers unique principles and advantages in detecting Mycoplasma pneumoniae IgM antibodies. Specifically, it utilizes magnetic particles as a solid-phase carrier to immobilize specific antigens or antibodies on the particle surface. When specific Mycoplasma pneumoniae IgM antibodies are present in the sample, they specifically bind to the antigens on the solid-phase carrier. Subsequently, a chemiluminescent reagent is introduced, and the reaction between the Mycoplasma pneumoniae IgM antibodies bound to the antigens on the solid-phase carrier and the resulting chemiluminescent signal is emitted.

[0005] The current reagent kit uses a passive adsorption process for magnetic bead coating, resulting in significant data variations across different instrument models, with an average variation of 54%. Furthermore, after washing with a small amount of surfactant (Tween 80 or Tritium X-100), significant protein detachment occurs, leading to a decrease in luminescence value of up to 97%. In addition, the current reagent kit exhibits some false positives when evaluating clinical samples.

[0006] In summary, existing Mycoplasma pneumoniae IgM antibody detection kits (magnetic microparticle chemiluminescence method) have been found to have batch-to-batch and inter-unit variability and a high false positive rate in clinical applications. Summary of the Invention

[0007] In view of this, the present invention provides a kit for detecting Mycoplasma pneumoniae IgM antibodies. The present invention provides Mycoplasma pneumoniae antigen and its preparation method, as well as a kit for detecting Mycoplasma pneumoniae IgM antibodies. Experiments have shown that the kit, after chemical cross-linking and coating protein modification, not only exhibits batch-to-batch stability and good specificity and repeatability, but also significantly reduces the false positive rate and improves the accuracy of the kit.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] This invention provides a method for preparing Mycoplasma pneumoniae antigen, comprising the following steps:

[0010] Step S1: Take Mycoplasma pneumoniae cells, sonicate them in buffer solution, centrifuge and take the supernatant;

[0011] Step S2: The supernatant is precipitated using 40-55 wt% ammonium sulfate for fractional precipitation. After centrifugation, the supernatant is collected, filtered, and the Mycoplasma pneumoniae antigen is obtained.

[0012] The buffer solution in step S1 comprises 0.5 wt% to 1 wt% Chaps.

[0013] In some specific embodiments of the present invention, the buffer solution includes one or more of sodium phosphate buffer, Tris buffer, HEPES buffer, or carbonate buffer.

[0014] In some specific embodiments of the present invention, the buffer solution comprises a sodium phosphate buffer containing 0.5 wt% to 1 wt% Chaps, NaCl, EDTA, 2-mercaptoethanol and PMSF.

[0015] In some specific embodiments of the present invention, the buffer solution comprises a sodium phosphate buffer containing 1 wt% Chaps, 400 mmol / L NaCl, 1 mmol / L EDTA, 5 mmol / L 2-mercaptoethanol and 0.6 mmol / L PMSF.

[0016] In some specific embodiments of the present invention, step S2 includes: after precipitation with 40wt% and 55wt% ammonium sulfate, the precipitates are collected and combined, reconstituted with salt solution, centrifuged to obtain the supernatant, filtered, and the filtrate is collected to obtain the Mycoplasma pneumoniae antigen.

[0017] In some specific embodiments of the present invention, the filter membrane used for filtration has a pore size of 0.22 μm.

[0018] The present invention also provides mycoplasma pneumoniae antigen prepared by the aforementioned preparation method.

[0019] The present invention also provides the application of the aforementioned Mycoplasma pneumoniae antigen in the preparation of a Mycoplasma pneumoniae IgM antibody detection kit.

[0020] The present invention also provides a kit for detecting Mycoplasma pneumoniae IgM antibodies, including the Mycoplasma pneumoniae antigen.

[0021] In some specific embodiments of the present invention, the Mycoplasma pneumoniae antigen is chemically cross-linked and coated with magnetic beads.

[0022] In some specific embodiments of the present invention, the method for chemically cross-linking magnetic beads with Mycoplasma pneumoniae antigen includes the following steps:

[0023] Step S1: Wash the carboxyl magnetic beads in magnetic bead buffer solution A to obtain the washed magnetic beads;

[0024] Step S2: Take the washed magnetic beads, mix them with the cross-linking agent, activate the magnetic beads, mix them with magnetic bead buffer solution B, centrifuge and discard the supernatant to obtain the activated magnetic beads;

[0025] Step S3: Take the activated magnetic beads and the Mycoplasma pneumoniae, mix them, chemically cross-link them, centrifuge and discard the supernatant to obtain the coated magnetic beads;

[0026] Step S4: Take the coated magnetic beads and mix them with the sealing and preservation solution to obtain the chemically cross-linked magnetic beads of Mycoplasma pneumoniae antigen.

[0027] In some specific embodiments of the present invention, the magnetic bead buffer solution A includes sodium phosphate buffer, sodium chloride, BSA, and a preservative;

[0028] The magnetic bead buffer solution B includes sodium acetate and a preservative;

[0029] The sealed preservation solution includes sodium phosphate buffer, sodium chloride, and preservatives.

[0030] In some specific embodiments of the present invention, the crosslinking agent comprises EDC and NHS. Preferably, the magnetic beads:EDC:NHS ratio is 3:4:4 (v:v:v).

[0031] In some specific embodiments of the present invention, the volume ratio of the activated magnetic beads to the Mycoplasma pneumoniae includes 2:1.

[0032] Based on a 1L system, the magnetic bead buffer solution A comprises 5.8g disodium hydrogen phosphate, 0.59g sodium dihydrogen phosphate, 8.766g sodium chloride, 1mL Proclin 300, and pH 7.4;

[0033] The magnetic bead buffer solution B comprises 6.15g sodium acetate, 1mL Proclin 300, and has a pH of 4.7.

[0034] The sealing and preservation solution comprises 5.8 g of disodium hydrogen phosphate, 0.59 g of sodium dihydrogen phosphate, 8.766 g of sodium chloride, and 1 mL of Proclin 300, with a pH of 7.4.

[0035] This invention provides the following beneficial effects:

[0036] 1) Chemically cross-linked magnetic beads are used to solve the antigen shedding caused by cleaning and improve batch-to-batch and inter-unit differences;

[0037] 2) Using the antigen that has been precipitated with ammonium sulfate and then had glycolipids removed as the coating antigen reduces the false positive rate. Detailed Implementation

[0038] This invention discloses a kit for detecting Mycoplasma pneumoniae IgM antibodies. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0039] This invention provides a Mycoplasma pneumoniae IgM antibody detection kit, comprising: magnetic microparticle suspension (containing natural Mycoplasma pneumoniae antigen), enzyme conjugate (containing horseradish peroxidase-labeled mouse anti-human IgM monoclonal antibody), sample diluent, and calibrator (containing Mycoplasma pneumoniae IgM positive serum or plasma).

[0040] The formula for the diluent of the magnetic microparticles described in this invention is as follows: 5.8g disodium hydrogen phosphate, 0.59g sodium dihydrogen phosphate, 8.766g sodium chloride, 30g BSA, 50mL glycerol, 1mL Proclin 300, and water to a final volume of 1L, with a pH of 7.4.

[0041] The diluent for the enzyme conjugate described in this invention is formulated as follows: 5.8g disodium hydrogen phosphate, 0.59g sodium dihydrogen phosphate, 8.766g sodium chloride, 30g BSA, 50mL glycerol, 1mL Proclin 300, and water to a final volume of 1L, with a pH of 7.4.

[0042] The sample diluent of this invention is formulated as follows: 5.8g disodium hydrogen phosphate, 0.59g sodium dihydrogen phosphate, 8.766g sodium chloride, 50g BSA, 1mL Proclin 300, water to a final volume of 1L, pH 7.4.

[0043] The formulation of magnetic bead buffer solution A of the present invention is as follows: 5.8g disodium hydrogen phosphate, 0.59g sodium dihydrogen phosphate, 8.766g sodium chloride, 1mL Proclin 300, water to make up to 1L, pH 7.4.

[0044] The formulation of magnetic bead buffer solution B of the present invention is as follows: 6.15g sodium acetate, 1mL Proclin 300, water to a final volume of 1L, pH 4.7.

[0045] The formula of the magnetic bead sealing and preservation solution of the present invention is as follows: 5.8g disodium hydrogen phosphate, 0.59g sodium dihydrogen phosphate, 8.766g sodium chloride, 1mL Proclin 300, water to make up to 1L, pH 7.4.

[0046] The present invention also provides the following technical solutions:

[0047] 1) Chemical cross-linking coated magnetic bead process; Chemical cross-linking coated magnetic beads are used to solve the antigen shedding caused by cleaning and improve batch-to-batch and inter-unit differences;

[0048] 2) Ammonium sulfate precipitation to remove glycolipid coating antigens for purification; using the antigens after ammonium sulfate precipitation to remove glycolipids as coating antigens reduces the false positive rate.

[0049] The reagent kit provided by this invention, which has undergone chemical cross-linking and coating protein modification, is not only stable between batches, but also has good specificity and reproducibility. At the same time, it can significantly reduce the false positive rate and improve the accuracy of the reagent kit.

[0050] Unless otherwise specified, all raw materials and reagents used in the kit for detecting Mycoplasma pneumoniae IgM antibodies provided by this invention can be purchased commercially.

[0051] The present invention will be further illustrated below with reference to the embodiments:

[0052] Example: Preparation of Mycoplasma pneumoniae IgM antibody detection kit (magnetic microparticle chemiluminescence method)

[0053] In clinical applications, the Mycoplasma pneumoniae IgM antibody detection kit (magnetic microparticle chemiluminescence method) has been found to have batch-to-batch and inter-unit variability and a high false positive rate.

[0054] A method for preparing natural antigens of Mycoplasma pneumoniae in humans includes the following steps:

[0055] Step 1) MP cell culture: Add the strain seed to the MP medium at a ratio of 1:10 (v / v) and shake to mix. Incubate at 37°C on a shaker. When the culture medium changes from red to orange, it can be harvested. Under aseptic conditions, add β-propiolactone at a ratio of 1 / 4000 (v / v) (β-propiolactone / bacterial suspension) and mix well. After incubating overnight at 4°C, transfer to a 37°C water bath and hydrolyze for 2 hours to achieve complete inactivation.

[0056] Step 2) Cell washing: Resuspend MP cells in 10 mmol / L PBS at pH 7.2, centrifuge at 15000 rpm for 30 min, wash a total of 4 times, and collect the precipitate;

[0057] Step 3) Protein enrichment: The precipitate was resuspended in 1% Chaps + 400 mmol / L NaCl + 1 mmol / L EDTA + 5 mmol / L 2-mercaptoethanol + 0.6 mmol / L PMSF sodium phosphate buffer at pH 6.75, then ultrasonically cleaned for 20 min, centrifuged at 15000 rpm for 30 min, the supernatant was collected, and the precipitate was collected.

[0058] Step 4) 1% Chaps supernatant ammonium sulfate precipitation: Take the supernatant from step 3) centrifugation and carry out the entire process at 4℃. Perform fractional precipitation of the supernatant with 40% + 55% ammonium sulfate. Combine the 40% + 55% ammonium sulfate precipitates and redissolve them with 75 mmol / L PB + 400 mmol / L NaCl. Centrifuge at 15000 rpm for 30 min. Take the supernatant and filter it through a 0.22 μm filter membrane to obtain the MP natural antigen.

[0059] The method for chemically modifying MP natural antigens and coating magnetic beads includes the following steps:

[0060] Step (1) Magnetic bead washing: Take an appropriate amount of carboxyl magnetic beads (Merck magnetic beads, magnetic bead concentration is 100mg / mL), magnetic beads: magnetic bead buffer A solution = 1:20 (v:v) Add magnetic bead buffer A solution to a centrifuge tube, mix slowly, place on a shaker and mix for 5min, place the reaction vessel on a magnetic rack to separate until the supernatant is clear, discard the supernatant, wash a total of 5 times;

[0061] Step (2) Activation of magnetic beads: Prepare a 20 mg / mL stock solution of EDC and NHS. Add 100 μL of the washed magnetic beads (magnetic beads: EDC: NHS = 3:4:4 (v:v:v)) to each of them. Mix and activate for 1 h. After activation, add magnetic bead buffer solution B at a ratio of 1:10 (v:v). Mix slowly and place on a shaker to mix for 5 min. Activate twice in total. Place the reaction vessel on a magnetic rack to separate until the supernatant is clear. Discard the supernatant to obtain the activated magnetic beads.

[0062] Step (3) Coating of magnetic beads:

[0063] Take the activated magnetic beads from step (2) and add MP natural antigen at a ratio of 2:1 (20 μg / T of activated magnetic beads and 40 μg / T of MP natural antigen) as the chemical cross-linking conditions;

[0064] Mix on a shaker for 2 hours, then place the reaction vessel on a magnetic rack to separate until the supernatant is clear. Discard the supernatant to obtain the coated magnetic beads.

[0065] Step (4) Magnetic bead sealing: Add magnetic bead sealing solution according to the ratio of coated magnetic beads: sealing solution = 1:10 (v:v), place on a shaker and mix for 10 minutes. Repeat the sealing process 5 times to obtain chemically cross-linked magnetic beads.

[0066] Take the prepared chemically cross-linked magnetic beads (containing natural antigen of Mycoplasma pneumoniae), enzyme conjugate (containing horseradish peroxidase-labeled mouse anti-human IgM monoclonal antibody), sample diluent and calibrator (containing Mycoplasma pneumoniae IgM positive serum or plasma) to obtain the Mycoplasma pneumoniae IgM antibody detection kit (magnetic particle chemiluminescence method).

[0067] Comparative Example 1

[0068] A kit for detecting Mycoplasma pneumoniae IgM antibodies (magnetic microparticle chemiluminescence method) was prepared using purchased antigens.

[0069] Comparative Example 2

[0070] The magnetic bead coating steps are as follows: Take the magnetic beads (1) washed and not activated (2) in the example and add them to the MP natural antigen prepared in the example at a ratio of 1:2 (v:v) as a passive adsorption condition; mix on a shaker for 2 hours, place the reaction container on a magnetic rack to separate until the supernatant is clear, and discard the supernatant.

[0071] The remaining operations are the same as in the example.

[0072] Comparative Example 3

[0073] In Example 3), the precipitate after centrifugation was resuspended in 2% octyl glucoside + 400 mmol / L NaCl + 1 mmol / L EDTA + 5 mmol / L 2-mercaptoethanol + 0.6 mmol / L PMSF sodium phosphate buffer at pH 6.75, vortexed at room temperature for 30 min, and centrifuged at 15000 rpm for 30 min. The supernatant was collected. The supernatant was subjected to fractional precipitation with 40% + 55% ammonium sulfate. The 40% + 55% ammonium sulfate precipitates were combined and reconstituted with 75 mmol / L PB + 400 mmol / L NaCl. The mixture was centrifuged at 15000 rpm for 30 min. The supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the MP natural antigen.

[0074] The remaining operations are the same as in the example.

[0075] Example of effect 1

[0076] To determine the appropriate proportion of Chaps in this invention, the concentration of Chaps in the examples was varied, and the following verification experiments were conducted: verification was performed using 0.5%, 1%, and 1.5% Chaps, respectively. The experimental results are as follows:

[0077] Table 1. Detection of MP after antigen enrichment with different concentrations of Chaps

[0078]

[0079] Three false positives were found in the purchased antigen, three false positives were found in 0.5% Chaps, but the overall reactivity was consistent with that of the purchased antigen. Two false positives were found in 1% Chaps, which was consistent with the reactivity of the purchased antigen. Seven false positives were found in 1.5% Chaps, and some samples were inconsistent with the reactivity of the purchased antigen. Based on this verification experiment, we finally selected 0.5%~1% Chaps as the extraction process range, with 1% Chaps being the preferred option.

[0080] Example 2: Stability testing of different magnetic beads

[0081] The Mycoplasma pneumoniae IgM antibody detection kit (magnetic microparticle chemiluminescence method) prepared in Comparative Example 2 was used to detect Mycoplasma pneumoniae IgM antibodies using different models. The results showed that the existing magnetic bead coating process uses passive adsorption (Comparative Example 2), and there are significant differences in data on different models, with an average difference of 54%, as shown in Table 2.

[0082] Table 2 Evaluation data of MP across different models

[0083]

[0084] The Mycoplasma pneumoniae IgM antibody detection kits (magnetic microparticle chemiluminescence method) prepared in Examples 1 and 2 were used. A small amount of surfactant (Tween 80 or Tritium X-100) was added to wash the chemically cross-linked magnetic beads and passively adsorbed magnetic beads. Specifically, cross-linked magnetic beads or passively adsorbed magnetic beads were added to a solution of magnetic bead buffer A at a ratio of 1:20 (v:v, with 1% Tritium X-100 added to magnetic bead buffer A). The solution was placed in a centrifuge tube, slowly mixed, and then stirred on a shaker for 5 minutes. The reaction vessel was then placed on a magnetic rack to separate until the supernatant became clear. The supernatant was discarded, and the mixture was washed 5 times. Then, Mycoplasma pneumoniae IgM antibody detection was performed. The results are shown in Table 3.

[0085] Table 3. Surfactant cleaning data for passive adsorption and chemical cross-linking coating processes.

[0086]

[0087] Existing coating processes use passive adsorption. After washing with a small amount of surfactant (Tween 80 or Tritium X-100), a significant amount of protein is shed, resulting in a decrease in luminescence value of up to 97%.

[0088] This invention employs a chemical cross-linking coating process for magnetic beads. Even after multiple washes with 1% Trition X-100, the protein remains intact, and the luminescence value remains at a high level with a reduction of less than 15%. The chemical cross-linking coating process removes non-specific background signals while also reducing protein shedding, significantly improving batch-to-batch and inter-unit differences.

[0089] Example 3

[0090] The Mycoplasma pneumoniae IgM antibody detection kits (magnetic microparticle chemiluminescence method) prepared in Examples 1 and 3 were used to detect Mycoplasma pneumoniae IgM antibodies.

[0091] The results showed that the present invention removes most of the glycolipid components through ammonium sulfate fractionation precipitation. It was also found that compared with 2% octyl glucoside supernatant ammonium sulfate precipitation, the component coating of 1% Chaps supernatant ammonium sulfate precipitation can significantly reduce the false positive rate and improve the accuracy of the kit, as shown in Table 4.

[0092] Table 4. Luminescence values ​​of purchased antigens coated with different ammonium sulfate precipitates

[0093]

[0094]

[0095] In the existing process, 1% Chaps buffer is used to enrich P1 in the precipitate, and 2% octyl glucoside buffer is used to solubilize P1 protein into the supernatant. Then, a 40+55% ammonium sulfate precipitation process is used to remove most of the glycolipids and other substances. The purchased antigen, 1% Chaps buffer ammonium sulfate precipitation and 2% octyl glucoside ammonium sulfate precipitation are coated. The data shows that the 2% octyl glucoside component has a high false positive rate, the purchased antigen has a small number of false positives, and the 1% Chaps component has a low background and can significantly reduce the false positive rate and improve the accuracy of the kit. This indicates that in addition to P1, other proteins play a role in IgM detection. The 1% Chaps centrifugation supernatant has a low proportion of P1 and an increased proportion of other proteins, which is conducive to the binding of IgM antibodies.

[0096] Currently, two batches of the process, 20240810 and 20240815, are being replicated. The evaluation results show that the positive and negative reactivity is consistent between different batches, the repeatability is good, and the differences between batches are small.

[0097] Overall, the kits, after optimization of the coating protein and chemical cross-linking coating process, are not only stable between batches, but also have good specificity and reproducibility. They can be used for clinical sample testing and provide technical support for timely measures to be taken for infected patients.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Mycoplasma pneumoniae antigen, characterized in that, Includes the following steps: Step S1: Take Mycoplasma pneumoniae cells, sonicate them in buffer solution, centrifuge and take the supernatant; Step S2: The supernatant is precipitated using 40-55 wt% ammonium sulfate for fractional precipitation. After centrifugation, the supernatant is collected, filtered, and the Mycoplasma pneumoniae antigen is obtained. The buffer solution in step S1 comprises 0.5 wt% to 1 wt% Chaps.

2. The preparation method according to claim 1, characterized in that, The buffer solution includes one or more of sodium phosphate buffer, Tris buffer, HEPES buffer, or carbonate buffer.

3. The preparation method according to claim 1 or 2, characterized in that, The buffer solution comprises a sodium phosphate buffer containing 0.5 wt% to 1 wt% Chaps, NaCl, EDTA, 2-mercaptoethanol and PMSF.

4. The preparation method according to any one of claims 1 to 3, characterized in that, Step S2 includes: precipitating the mycoplasma pneumoniae antigen successively with 40wt% and 55wt% ammonium sulfate, combining the precipitates, redissolving them with salt solution, centrifuging to obtain the supernatant, filtering, collecting the filtrate, and obtaining the mycoplasma pneumoniae antigen.

5. Mycoplasma pneumoniae antigen prepared by the preparation method according to any one of claims 1 to 4.

6. The use of the Mycoplasma pneumoniae antigen as described in claim 5 in the preparation of a Mycoplasma pneumoniae IgM antibody detection kit.

7. A kit for detecting Mycoplasma pneumoniae IgM antibodies, characterized in that, Including the Mycoplasma pneumoniae antigen as described in claim 5.

8. The kit according to claim 7, characterized in that, The Mycoplasma pneumoniae antigen is chemically cross-linked and coated with magnetic beads.

9. The reagent kit as described in claim 8, characterized in that, The method for chemically cross-linking magnetic beads with Mycoplasma pneumoniae antigen includes the following steps: Step S1: Wash the carboxyl magnetic beads in magnetic bead buffer solution A to obtain the washed magnetic beads; Step S2: Take the washed magnetic beads, mix them with the cross-linking agent, activate the magnetic beads, mix them with magnetic bead buffer solution B, centrifuge and discard the supernatant to obtain the activated magnetic beads; Step S3: Take the activated magnetic beads and the Mycoplasma pneumoniae, mix them, chemically cross-link them, centrifuge and discard the supernatant to obtain the coated magnetic beads; Step S4: Take the coated magnetic beads and mix them with the sealing and preservation solution to obtain the chemically cross-linked magnetic beads of Mycoplasma pneumoniae antigen.

10. The kit according to claim 9, characterized in that, The magnetic bead buffer solution A includes sodium phosphate buffer, sodium chloride, BSA, and preservatives; The magnetic bead buffer solution B includes sodium acetate and a preservative; The sealed preservation solution includes sodium phosphate buffer, sodium chloride, and preservatives.

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