Magnetic bead coating process and detection reagent for detecting chlamydia pneumoniae IgM antibody

Abstract

The invention relates to the technical field of biology, in particular to a magnetic bead coating process and a detection reagent for detecting a chlamydia pneumoniae IgM antibody. The invention provides a process for coating a chlamydia pneumoniae natural antigen with magnetic beads. The performance of a chlamydia pneumoniae IgM antibody detection kit can be improved. According to the process, a coating antigen is a chlamydia pneumoniae natural antigen and is coated on a magnetic bead by adopting a passive adsorption method, a traditional method is that a high-concentration surfactant is added into a reaction system to elute non-specific adsorption IgM, but the passive adsorption antigen falls off and the reactivity is reduced; according to the invention, latex particles with a certain particle size and a certain concentration are added into the magnetic particle preserving fluid, and experiments prove that false positive samples can be remarkably eliminated, and meanwhile, the reactivity of positive samples is not influenced, so that the specificity is improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a magnetic bead coating process and a detection reagent for detecting Chlamydia pneumoniae IgM antibodies. Background Art

[0002] Chlamydia pneumoniae (CP) is a new species of Chlamydia officially named in 1989. The morphology of the Chlamydia pneumoniae is diverse. Under the electron microscope, it is a typical pear-shaped body with a long axis of 0.44 μm, a short axis of 0.31 μm, and an average diameter of 0.38 μm. The nuclear region is round and located in the center of the cell, with an average diameter of 0.24 μm. There is a wide protoplasmic region between the nuclear region and the cell membrane. The inclusion bodies of Chlamydia pneumoniae do not contain glycogen and are iodine-negative. Their morphology in Hela cells is very similar to that of Chlamydia psittaci. After Giemsa staining, they are oval with deep density. The inclusion bodies in HEP-2 cells have a variety of density and morphology, including dense, osmanthus-like scattered, extracellular tumor-like, and round inclusion bodies with less intracellular staining.

[0003] Chlamydia pneumoniae cannot be cultured in vitro and can only parasitize within cells. Chicken embryos are not sensitive to it, so it is generally not passaged using chicken embryos but cell culture. The cell lines sensitive to Chlamydia pneumoniae are HEP-2 or H-292.

[0004] Chlamydia pneumoniae has genus-specific antigens and species-specific antigens. The genus-specific antigens mainly have two antigenic determinant epitopes, one is the third KDO residue of the LPS core polysaccharide of Chlamydia pneumoniae, and the other is the major outer membrane protein (MOMP) with a molecular weight of 39.5 KD. Both antigenic determinants are sensitive to methanol and have cross-reactions with other Chlamydia species. The species-specific antigen of Chlamydia pneumoniae is 98 KD MOMP, which is sensitive to acetone and does not cross-react with Chlamydia trachomatis and Chlamydia psittaci antisera. These outer membrane proteins are rich in cysteine, and disulfide bonds are easily formed between proteins.

[0005] In order to indirectly detect Chlamydia pneumoniae, the existing technology provides a method for detecting Chlamydia pneumoniae-specific IgM antibodies based on magnetic particle chemiluminescence. During the detection process, a high concentration of surfactant needs to be added to the reaction system to elute the non-specifically adsorbed IgM, but this will also cause the passively adsorbed antigen to fall off, thereby reducing the reactivity. Summary of the invention

[0006] In view of this, the present invention provides a magnetic bead coating process and detection reagent for detecting Chlamydia pneumoniae IgM antibodies. The present invention adds latex particles of a certain particle size and a certain concentration to the magnetic particle preservation solution, which can significantly eliminate false positive samples without affecting the reactivity of positive samples, thereby improving specificity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a magnetic particle preservation solution, comprising latex microspheres with a particle size of less than 145 nm and a content of more than 9.09 mg / mL.

[0009] In some specific embodiments of the present invention, the particle size of the latex microspheres in the magnetic particle preservation solution is less than 107 nm.

[0010] In some specific embodiments of the present invention, the particle size of the latex microspheres in the magnetic particle preservation solution is less than 95 nm.

[0011] In some specific embodiments of the present invention, the particle size of the latex microspheres in the magnetic particle preservation solution is less than 80 nm.

[0012] In some specific embodiments of the present invention, the particle size of the latex microspheres in the magnetic particle preservation solution is 80-95 nm, 80-107 nm or 80-145 nm.

[0013] In some specific embodiments of the present invention, the particle size of the latex microspheres in the magnetic particle preservation solution is 80 nm or 95 nm.

[0014] In some specific embodiments of the present invention, the magnetic particle preservation solution comprises latex microspheres of different particle sizes, and the particle sizes of the latex microspheres are all below 145 nm.

[0015] In some specific embodiments of the present invention, the magnetic particle preservation solution comprises latex microspheres with different particle sizes, and the particle sizes are all 80-95 nm, 80-107 nm or 80-145 nm.

[0016] In some specific embodiments of the present invention, the content of the latex microspheres in the magnetic particle preservation solution is 4.76 mg / mL or more.

[0017] In some specific embodiments of the present invention, the content of the latex microspheres in the magnetic particle preservation solution is above 9.09 mg / mL.

[0018] The present invention also provides a magnetic particle suspension, comprising magnetic particles and the magnetic particle preservation solution.

[0019] In some specific embodiments of the present invention, the magnetic particles of the magnetic particle suspension are coated with Chlamydia pneumoniae antigens.

[0020] In some specific embodiments of the present invention, the magnetic particle content of the magnetic particle suspension is 0.5-2 mg / mL.

[0021] In some specific embodiments of the present invention, the particle size of the magnetic particles in the magnetic particle suspension is 1 μm.

[0022] The present invention also provides a method for preparing a magnetic particle suspension, comprising mixing magnetic particles with a magnetic particle protection liquid to obtain the magnetic particle suspension;

[0023] The magnetic particle protection liquid is the magnetic particle preservation liquid mentioned above.

[0024] The present invention also provides an anti-Chlamydia pneumoniae antibody detection kit or device, comprising the magnetic particle preservation solution or the magnetic particle suspension.

[0025] In some specific embodiments of the present invention, the anti-Chlamydia pneumoniae antibody detection kit or device further comprises an enzyme conjugate, and the enzyme conjugate may be an anti-human IgM antibody labeled with horseradish peroxidase.

[0026] The present invention also provides a method for detecting anti-Chlamydia pneumoniae antibodies based on magnetic particle chemiluminescence, comprising: mixing a sample to be tested with the magnetic particle suspension, removing the supernatant and washing, then mixing with an enzyme conjugate, removing the supernatant and washing, then mixing with a substrate, detecting a signal and obtaining a detection result;

[0027] Wherein, the step of eluting the magnetic particles in the magnetic particle suspension by using a high concentration surfactant is not included, and the high concentration surfactant is Triton X-100 with a content of ≥0.5%;

[0028] The enzyme conjugate can be an anti-human IgM antibody labeled with horseradish peroxidase.

[0029] In some specific embodiments of the present invention, the above method comprises the following steps:

[0030] S1, mixing the diluted sample to be tested, magnetic beads and sample diluent, reacting, removing the supernatant and washing;

[0031] S2, mixing with enzyme conjugate, reacting, removing supernatant and washing;

[0032] S3, mixing with the substrate, reacting, detecting the signal, and obtaining the test result;

[0033] The step of eluting the magnetic beads with a high concentration of surfactant is not included.

[0034] The magnetic bead coating process and detection reagent of the present invention have the following effects:

[0035] The present invention adds latex particles of a certain particle size and a certain concentration to the magnetic particle preservation solution, which can significantly eliminate false positive samples without affecting the reactivity of positive samples, thereby improving specificity. Specifically, experiments show that adding latex particles with a particle size of 80 nm, 95 nm, 107 nm or 145 nm to the magnetic particle preservation solution has the effect of eliminating false positives, and the smaller the latex particles, the better the effect of eliminating false positives. After further experiments, it was unexpectedly found that adding latex particles with a particle size of less than or equal to 95 nm to the magnetic particle preservation solution and the concentration reached 9.09 mg / mL can more significantly reduce false positive samples. DETAILED DESCRIPTION

[0036] The present invention discloses a magnetic bead coating process and a detection reagent for detecting Chlamydia pneumoniae IgM antibodies. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0037] The present invention adopts the indirect method principle for detection and is applicable to a Chlamydia pneumoniae IgM antibody detection kit (magnetic particle chemiluminescence method). Magnetic particles are coated with Chlamydia pneumoniae antigens, and anti-human IgM antibodies are labeled with horseradish peroxidase to prepare enzyme conjugates. An antigen-specific IgM antibody-enzyme-labeled secondary antibody complex is formed through immune reaction. The complex catalyzes the luminescent substrate to emit photons, and the luminescence intensity is proportional to the content of CP IgM antibodies. In the first step, 50 µL of sample diluted with the universal PBS buffer diluent provided by the fully automatic instrument, 20 µL of magnetic particle suspension and 50 µL of sample diluent (0.05 MTris-HCl + 0.5 M NaCl + 1% EDTA + 0.1% P300 + 0.01% NaN3 + 0.2% CHAPS + 100,000 units of urokinase + 1% BSA + 0.25% human IgG adsorbent + 0.1% Tween, where all percentages represent g / 100 mL) were added in sequence, and the reaction was carried out at 37°C for 15 min. In the second step, 100 µL of enzyme conjugate was added, and the reaction was carried out at 37°C for 17 min.

[0038] In some embodiments of the present invention, latex particles (polystyrene) are added to the antigen storage solution coated in the polystyrene magnetic bead passive adsorption process.

[0039] In some embodiments of the present invention, latex particles of 95 nm or less are added to the magnetic bead storage solution.

[0040] In some embodiments of the present invention, the concentration of latex particles added to the magnetic bead storage solution is not less than 9.09 mg / mL.

[0041] In some embodiments of the present invention, adding a certain concentration of latex particles to the magnetic bead storage solution can significantly eliminate false positives caused by nonspecific IgM adsorption in serum or plasma.

[0042] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0043] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0044] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0045] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0046] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0047] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0048] The present invention will be further described below in conjunction with the embodiments.

[0049] Example

[0050] 1. Preparation of magnetic beads (taking the preparation of 480 mL magnetic microparticle suspension as an example)

[0051] 1.1 Washing before coating: Place 4.8 mL of magnetic microparticle working solution (carboxyl magnetic beads, manufacturer Merck, product number LSKMAG1CBX, 100 mg / mL, particle size 1 μm) on a magnetic rack and separate until the supernatant is clear, then discard the supernatant; use a measuring cylinder to measure 20-100 mL of buffer A (0.02 M PBS buffer) and add it to the reaction vessel, slowly shake the magnetic microparticles in the reaction vessel by hand, place on a shaker at 100-300 r / min for 3-10 min, and the washing is complete. Repeat the washing 3-6 times, and finally place the reaction vessel on a magnetic rack and separate until the supernatant is clear, then discard the supernatant.

[0052] 1.2 Coating: First add buffer A, shake the magnetic particles in the reaction container slowly by hand, and observe the state of the magnetic particles. If there is agglutination, perform ultrasonic treatment for 15-30 seconds. If there is no agglutination, proceed to the next step. If there is agglutination, continue ultrasonic treatment until there is no visible agglutination of magnetic particles, and then proceed to the next step to make the magnetic particles in the reaction container evenly dispersed. Then add a specified amount of Chlamydia pneumoniae natural antigen (magnetic particle CP-IgM coating antigen, manufacturer Imeno, coating concentration: no more than 0.5 μg / person), and the total liquid volume is 16 mL. Cover the bottle cap / container sealer tightly and place on a shaker at 100-300 r / min for 1-5 hours.

[0053] 1.3 Blocking: After the reaction is completed, place the reaction container on a magnetic stand to separate until the supernatant is clear and discard the supernatant. Use a measuring cylinder to measure 20-100 mL of blocking solution and add it to the reaction container; manually shake the magnetic particles in the reaction container slowly and place it on a shaker at 100-300 r / min for 5-20 min. This completes the first blocking. Repeat the blocking 2-5 times, and finally place the reaction container on a magnetic stand to separate until the supernatant is clear and discard the supernatant.

[0054] 1.4 Storage after preparation: Accurately measure 480 mL of the control or test magnetic particle preservation solution (0.04 mol / L PB + 0.01% NaN3 + 0.1% P300 + 5% glycerol + 5% sucrose + 1% BSA, where the percentages represent g / 100 mL), and use the magnetic particle preservation solution in small amounts and multiple times to transfer the residual magnetic beads in the reaction container to the storage container so that the content of the magnetic beads is 1 mg / mL, then add the latex microspheres, place on a shaker at 100~300 r / min and mix for 3~10 min, cover the bottle cap / container seal, affix a label, and then store at 2~8℃. The magnetic particle suspension is prepared.

[0055] 2. Experimental Data

[0056] 2.1 Adding latex particles during storage after preparation to eliminate false positives

[0057] Table 1

[0058]

[0059] Note: The content of magnetic beads is 1 mg / mL; the solid content of latex microspheres is 10% w / v, i.e. 0.1 g / mL.

[0060] Conclusion: Latex particles of different sizes were added to the preservation solution, among which 95 nm latex microspheres had the best effect, reducing the number of positive samples by 16%, the number of false positive samples by 46%, and further widening the positive-negative contrast.

[0061] Table 2

[0062]

[0063] Note: The content of magnetic beads is 1 mg / mL; the solid content of latex microspheres is 10% w / v, i.e. 0.1 g / mL; CO is equal to the mean of PC1 multiplied by 0.4.

[0064] Conclusion: When the amount of 95 nm latex particles added to the preservation solution was increased to 300 μL, the number of positive samples decreased by 2%, the number of false positive samples decreased by 45%, the false positive samples decreased to negative, and the positive samples did not change much.

[0065] Table 3

[0066]

[0067] Note: The content of magnetic beads is 1 mg / mL; the solid content of latex microspheres is 10% w / v, i.e. 0.1 g / mL.

[0068] Conclusion: Adding latex particles with a particle size of 80 nm, 95 nm, 107 nm or 145 nm to the preservation solution has the effect of eliminating false positives, and the smaller the latex particles, the better the effect of eliminating false positives. The above data are summarized in Table 4.

[0069] Table 4

[0070]

[0071] Note: 9.09 mg / mL, that is, (100 mg / mL*0.3 mL) / 3.3 mL; 4.76 mg / mL, that is, (100 mg / mL*0.15 mL) / 3.15 mL.

[0072] Based on the above results, adding latex particles with a diameter of less than or equal to 95 nm and a concentration of 9.09 mg / mL to the preservation solution can significantly reduce the number of false positive samples.

[0073] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A magnetic particle storage solution, characterized in that: It includes latex microspheres with a particle size of less than 145 nm and a content of more than 9.09 mg / mL.

2. The magnetic particle storage solution according to claim 1, characterized in that The particle size of the latex microspheres is 95 nm.

3. The magnetic particle storage solution according to claim 1, characterized in that The particle size of the latex microspheres is 80 nm.

4. The magnetic particle storage solution according to any one of claims 1 to 3, characterized in that The content of the latex microspheres is greater than 4.76 mg / mL.

5. The magnetic particle storage solution according to any one of claims 1 to 4, characterized in that: The content of the latex microspheres is above 9.09 mg / mL.

6. A magnetic particle suspension, characterized in that: Comprising magnetic particles and the magnetic particle storage solution according to any one of claims 1 to 5.

7. The magnetic particle suspension according to claim 6, characterized in that: The magnetic particles are coated with Chlamydia pneumoniae antigens.

8. A method for preparing a magnetic particle suspension, characterized in that: include: Mixing the magnetic particles with the magnetic particle protection solution to obtain the magnetic particle suspension; The magnetic particle protection solution is the magnetic particle preservation solution according to any one of claims 1 to 5.

9. An anti-Chlamydia pneumoniae antibody detection kit or device, characterized in that: It comprises the magnetic particle preservation solution according to any one of claims 1 to 5 or the magnetic particle suspension according to claim 6 or 7.

10. A method for detecting anti-Chlamydia pneumoniae antibodies based on magnetic particle chemiluminescence method for purposes other than disease diagnosis and treatment, characterized in that: include: The sample to be tested is mixed with the magnetic particle suspension of claim 6 or 7, the supernatant is removed and washed, and then mixed with the enzyme conjugate, the supernatant is removed and washed, and then mixed with the substrate, and the signal is detected to obtain the test result; The method does not include the step of eluting the magnetic particles in the magnetic particle suspension by using a high concentration surfactant.

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