A streptococcus pneumoniae urine antigen elisa kit and a preparation method thereof

Abstract

The present application relates to the technical field of biology, in particular to a streptococcus pneumoniae urine antigen ELISA kit and a preparation method thereof.The solid-phase coating material of the kit is CRP, which is easy to prepare and obtain and low in cost;the reaction system only needs one antibody, greatly reducing the workload and labor cost of screening and preparation of paired antibodies;the antigen detection is fully automatic, greatly increasing the detection throughput and reducing the labor cost.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a pneumococcal urinary antigen ELISA kit and its preparation method. Background Technology

[0002] Pneumonia is a common childhood illness and a leading cause of death in children under five years old, with Streptococcus pneumoniae infection accounting for 95% of pneumonia cases (particularly in low- and middle-income countries). Streptococcus pneumoniae is a leading cause of community-acquired pneumonia and may be a significant causative agent of community-acquired pneumonia of unknown origin. In 2005, the WHO estimated that pneumococcal disease (PDs) caused 1.6 million deaths globally annually, including 700,000 to 1 million children under five years old, with the majority occurring in developing countries and a higher mortality rate among children under two years old. In 2015, approximately 5.83 million children under five years old died globally, with an estimated 294,000 of these deaths attributed to Streptococcus pneumoniae infection. In 2016, lower respiratory tract infections caused approximately 653,000 deaths in children under five years old globally, and Streptococcus pneumoniae remains the leading cause of high morbidity and mortality from lower respiratory tract infections across all age groups worldwide. Streptococcus pneumoniae infection can lead to serious illnesses such as bacteremia and meningitis. Pneumococcal meningitis often leads to irreversible brain damage or brain death. The disease progresses rapidly, and can progress from mild to coma within hours, making immediate diagnosis and treatment crucial.

[0003] Bacterial isolation and culture remains the "gold standard" for confirming Streptococcus pneumoniae (SP) infection, but its positive rate is low and it is time-consuming, making it unsuitable for rapid detection. Streptococcus pneumoniae antigen detection kits are simple and rapid to use, providing timely and highly accurate diagnoses to support early clinical treatment.

[0004] However, existing pneumococcal antigen detection kits use a double-antibody sandwich method, requiring at least two monoclonal antibodies (polyclonal antibodies are difficult to apply in large industrial-scale production, requiring multiple immunizations and preparations, and making it difficult to control batch-to-batch variations in live material preparation). Although monoclonal antibody preparation technology is now common in the industry, the cost and workload of antibody preparation and screening remain high. Furthermore, industry professionals are aware that due to differences in antibody properties and limitations in the kit's reaction system, preparing two specific antibodies for immunofluorescence (IF) or immunohistochemistry (IHC) assays is not equivalent to preparing two specific antibodies for double-antibody sandwich ELISA assays; the latter is far more difficult and labor-intensive to screen. Summary of the Invention

[0005] In view of this, the kit and its preparation method provided by the present invention, which combine C-reactive protein and a single antibody in an ELISA kit, solve the problem of large screening quantity and difficulty in screening monoclonal antibody pairing during the establishment of a double antibody sandwich detection kit.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of C-reactive protein in serological detection;

[0008] The C-reactive protein is directly immobilized or adsorbed onto a solid support via chemical bonds to capture the target to be tested.

[0009] The serological tests include one or more of radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, time-resolved fluorescence immunoassay, or chemiluminescent immunoassay.

[0010] In some specific embodiments of the present invention, the above applications are as follows:

[0011] The target to be tested includes Streptococcus pneumoniae cell wall polysaccharides; and / or

[0012] The serological tests include enzyme-linked immunosorbent assay (ELISA) or magnetic particle chemiluminescence immunoassay.

[0013] In some specific embodiments of the present invention, the above applications are as follows:

[0014] The enzyme-linked immunosorbent assay (ELISA) is an ELISA assay for Streptococcus pneumoniae cell wall polysaccharides; and / or

[0015] The magnetic microparticle chemiluminescent immunoassay is a magnetic microparticle chemiluminescent immunoassay of Streptococcus pneumoniae cell wall polysaccharides.

[0016] The present invention also provides the application of any of the following in the preparation of a kit for detecting Streptococcus pneumoniae cell wall polysaccharides or a Streptococcus pneumoniae diagnostic kit:

[0017] (i) C-reactive protein; or

[0018] (ii) Monoclonal antibodies against C-reactive protein and anti-pneumococcal cell wall polysaccharides.

[0019] The present invention also provides a detection kit for preparing Streptococcus pneumoniae antigen using C-reactive protein and monoclonal antibody, comprising component A and component B, wherein component A is a solid-phase carrier coated with C-reactive protein, and component B is a monoclonal antibody specific to Streptococcus pneumoniae cell wall polysaccharide labeled with a tracer.

[0020] In some specific embodiments of the present invention, the C-reactive protein coating method of the above-mentioned kit includes direct coating or indirect coating.

[0021] In some specific embodiments of the present invention, the tracer markers described in the above kit include enzymes and / or luminescent markers;

[0022] The enzymes include horseradish peroxidase and / or alkaline phosphatase;

[0023] The luminescent marker includes at least one of luminol and its derivatives, isoluminol and its derivatives, and acridine esters.

[0024] In some specific embodiments of the present invention, the above-mentioned kit further includes a blocking buffer containing 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM calcium chloride.

[0025] The blocking buffer solution is mixed with component A.

[0026] In some specific embodiments of the present invention, the solid support of the above-mentioned kit includes magnetic microparticles.

[0027] The present invention also provides a method for detecting pneumococcal antigen, comprising the above-mentioned kit, wherein pneumococcal antigen in the sample to be tested is qualitatively detected by chemiluminescent immunoassay.

[0028] The sample to be tested is a urine sample.

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

[0030] Step (1): Mix component A and component B of the kit with the sample to be tested, incubate, wash, separate magnetically, add luminescent substrate to the precipitate, detect the light signal value, and obtain the light signal value of the sample to be tested;

[0031] Step (2): Measure the light signal values ​​of the negative control and the positive control, and obtain the light signal value of the CutOff value of the kit. Compare the light signal value of the sample to be tested with the light signal value of the CutOff value to obtain the qualitative detection result of the pneumococcal antigen in the sample to be tested.

[0032] The present invention also provides magnetic microparticles coated with C-reactive protein.

[0033] In some specific embodiments of the present invention, the method of coating the above-mentioned magnetic microparticles includes direct coating or indirect coating;

[0034] The indirect coating includes: linking avidinized magnetic beads to biotinylated antibodies;

[0035] The indirect coating further includes: linking tagged recombinant CRP to tagged antibodies coated on magnetic microparticles.

[0036] In some specific embodiments of the present invention, the method for preparing the above-mentioned magnetic microparticles includes the following steps:

[0037] Step (1): Wash the magnetic microparticles containing carboxyl groups on the surface three times; the washing process is as follows: take 200 μl of the magnetic microparticles, remove the supernatant, add 2 ml of 0.02 M PBS (pH 8.0), and resuspend.

[0038] Step (2): Activate the magnetic microparticles washed in step (1); the activation is as follows: dissolve EDC and NHS in 0.1M MES (pH 5.0) buffer solution to a concentration of 20mg / ml, then take 1ml of each and mix with the magnetic microparticles washed in step (1), and shake for 30 minutes.

[0039] Step (3): Wash the activated magnetic microparticles from step (2) twice; the washing process is as follows: remove the supernatant, mix with 2 ml of 0.1 M MES (pH 5.0) buffer, and resuspend.

[0040] Step (4): Take 2 μg, 4 μg, 6 μg, 8 μg, 10 μg, 12 μg, 14 μg, 16 μg, 18 μg or 20 μg of coating material CRP and add it to the magnetic microparticles washed in step (3); mix with 0.1M MES (pH 5.0) buffer, then add 10mM to 50mM calcium chloride to a final volume of 1 ml, shake for 60 minutes, remove the supernatant, wash 3 times with 2 ml of 0.02M PBS (pH 8.0), and add 20 ml of blocking buffer;

[0041] The blocking buffer formulation is: 0.02M PBS buffer, calcium chloride, and 1% blocking protein BSA, wherein the final concentration of calcium chloride is 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50mM.

[0042] The present invention also provides a magnetic microparticle suspension comprising the above-mentioned magnetic microparticles, and acceptable excipients or additives.

[0043] In some specific embodiments of the present invention, the above-mentioned magnetic microparticle suspension contains 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM of calcium chloride.

[0044] The present invention also provides a kit comprising the above-described magnetic microparticles or the above-described magnetic microparticle suspension, and acceptable excipients or additives.

[0045] In some specific embodiments of the present invention, the kit further includes a monoclonal antibody against Streptococcus pneumoniae cell wall polysaccharide; the monoclonal antibody is labeled with horseradish peroxidase.

[0046] In some specific embodiments of the present invention, the test sample for the above-mentioned reagent kit is a urine sample or an upper respiratory tract sample.

[0047] In some specific embodiments of the present invention, the monoclonal antibody described in the above application or the above kit further includes one or more of recombinant antibodies, single-domain antibodies, Fv, ScFv, Fab, or nanobodies.

[0048] In some specific embodiments of the present invention, the above-described kit does not include other antibodies.

[0049] In some specific embodiments of the present invention, the above-mentioned kit includes a magnetic microparticle suspension, an enzyme conjugate, a negative control, and a positive control;

[0050] The magnetic microparticle suspension is coated with C-reactive protein;

[0051] The enzyme conjugate is a horseradish peroxidase-labeled Streptococcus pneumoniae-specific antibody.

[0052] The negative control was a diluted PBS solution containing surfactants and preservatives;

[0053] The positive control was a diluted PBS solution containing Streptococcus pneumoniae C polysaccharide, surfactant, and preservative.

[0054] The present invention also provides a detection method, comprising detecting a sample to be tested based on the above-mentioned reagent kit to obtain detection results;

[0055] The samples to be tested include urine samples or upper respiratory tract samples.

[0056] The reagent kit and its preparation method of the present invention have the following effects:

[0057] 1. The solid-phase coating material is CRP, which is easy to prepare and obtain and has low cost;

[0058] 2. The reaction system requires only one antibody at most, which greatly reduces the workload and manpower costs of screening and preparing paired antibodies;

[0059] 3. Fully automated antigen detection greatly increases testing throughput and reduces labor costs. Attached Figure Description

[0060] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0061] Figure 1 This illustrates the principle of the reagent kit of the present invention. Detailed Implementation

[0062] This invention discloses a reagent kit and its preparation method. 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.

[0063] Existing detection kits are based on the traditional double-antibody sandwich method for detecting Streptococcus pneumoniae cell wall polysaccharide (C-polysaccharide) antigen. This invention utilizes C-reactive protein (CRP), a protein specifically bound to C-polysaccharide and a common inflammatory marker following bacterial infection. This invention uses a combination of CRP and a specific antibody to construct an ELISA kit, requiring only one specific antibody to detect C-polysaccharide antigen in urine samples after Streptococcus pneumoniae infection. The combination of C-reactive protein and a single antibody in the ELISA solution solves the problem of large monoclonal antibody pairing and screening difficulties encountered in the development of double-antibody sandwich detection kits (the principle is as follows). Figure 1 (As shown).

[0064] This invention is based on an optimized design of a fully automated chemiluminescence platform; the sample type is urine, which is easy to obtain; a single specific antibody is used to detect urine antigens after Streptococcus pneumoniae infection; CRP is used as the target material for capture; 10-50 mM calcium chloride is added during CRP coating and storage to ensure the effective activity of CRP.

[0065] Specifically:

[0066] 1. CRP coating: A 96-well microplate or magnetic microspheres can be used as the coating solid phase. Magnetic microspheres can be prepared according to the following steps:

[0067] Take 200 μl of carboxyl-containing magnetic microparticles and place them in a glass bottle. Use a magnet to attract the magnetic microparticles to the bottom of the bottle and remove the supernatant. Add 2 ml of 0.02 M PBS (pH 8.0) and repeat the above operation 3 times. Dissolve EDC and NHS separately in 0.1 M MES (pH 5.0) buffer to a concentration of 20 mg / ml, and then add 1 ml of each to the magnetic microparticles. Gently shake the mixture at room temperature for 30 minutes. Use a magnet to attract the magnetic microparticles to the bottom and remove the supernatant. Add another 2 ml of 0.1 M MES (pH 5.0) buffer to resuspend the magnetic microparticles. Repeat the above operation 2 times. Add 20 μg of coating material CRP to the activated magnetic microparticles; then add 0.1 M M ES (pH 5.0) buffer, specifically adding 10 mM to 50 mM calcium chloride to the coating solution to ensure the C-polysaccharide binding activity of CRP, to a final volume of 1 ml; gently shake at room temperature for 60 minutes; use a magnet to adsorb the magnetic microparticles to the bottom, remove the supernatant, and wash three times with 2 ml of 0.02 M PBS (pH 8.0); add 20 ml of blocking buffer (blocking buffer formula: 0.02 M PBS buffer, 10 mM calcium chloride, BSA as the blocking protein, 1% ratio), and store at 2–8 °C.

[0068] 2. Preparation of specific antibodies: Monoclonal antibodies can be prepared by immunizing animals with Streptococcus pneumoniae. The preparation process of monoclonal antibodies is a mature technology in the industry. Hybridoma technology, monoclonal B cell technology, phage platform technology, etc. can all be used to obtain specific monoclonal antibodies. Recombinant antibodies, single-domain antibodies, Fv, ScFv, Fab, and nanobodies can also be further prepared.

[0069] 3. Labeling with specific antibodies: Horseradish peroxidase (HRP) was activated using a conventional modified sodium periodate method. Specific antibodies were added, and the reaction was incubated overnight at 2–8°C. Sodium borohydride reductase conjugate was added, and unreacted reagents were removed by dialysis. 50% volumetric glycerol was added, and the mixture was stored at -20°C. When using, it was stored in preservation solution 1 at a ratio of 1:5000. Preservation solution 1 was formulated as follows: Bis-Tris buffer containing 20% ​​fetal bovine serum, 0.1% P300 preservative, and 10%–30% polyol protein protectant, and stored at 2–8°C.

[0070] 4. Preparation of negative control: 0.02M PBS buffer, 0.1% poloxamer, with P300 added as a preservative.

[0071] 5. Preparation of positive control: The obtained Streptococcus pneumoniae culture was resuspended in PBS until the OD value was greater than 2.0-3.0. The supernatant after centrifugation at 7000-10000 rpm for 5-10 minutes was used as positive additive and added to the negative control dilution at a ratio of 1 / 10k as positive control.

[0072] 6. Sample processing: Urine samples are usually clear liquids. If the sample contains insoluble substances, it should be processed by centrifugation at 12,000 rpm for 5 to 10 minutes or by filtration. A 1μm to 10μm filter can be selected for filtration.

[0073] Kit components:

[0074] Magnetic microparticle suspension coated with CRP protein, 100 tests per bottle;

[0075] Enzyme conjugate, HRP-labeled Streptococcus pneumoniae-specific antibody;

[0076] Negative control: PBS dilution containing surfactants and preservatives;

[0077] Positive control, PBS dilution kit containing Streptococcus pneumoniae C polysaccharide, surfactant and preservative: Instructions:

[0078] Place the sample container in the instrument's sample holder; load the sample holder and enter the sample information in the instrument's software interface; select "Run" to start the test, and the instrument will perform the following operations:

[0079] 1) Transfer the sample rack to the sample aspiration position and load the reaction vessel into the sample loading position.

[0080] 2) Complete the dispensing of 100 μL sample or 100 μL calibrator, 20 μL magnetic microparticle suspension, and 100 μL enzyme conjugate.

[0081] 3) Mix the reaction solution thoroughly and incubate at 37°C for 15 minutes.

[0082] 4) After the incubation is complete, the reaction solution is cleaned and separated using a cleaning solution.

[0083] 5) Complete the dispensing of 50 μL of substrate solution and 50 μL of enhancement solution.

[0084] 6) Mix the reaction solution thoroughly and test the luminescence intensity.

[0085] 7) The instrument automatically reports the test results.

[0086] The coating method can be direct coating or indirect coating. Indirect coating can be, for example, selecting avidin magnetic beads to link with biotinylated antibodies, or adding a tag to recombinant CRP, coating the tagged antibody with magnetic microparticles and then linking it to CRP.

[0087] Labeling methods can include direct labeling to conjugate antibodies to HRP, or indirect methods such as linking biotinylated antibodies to HRP-labeled avidin, or linking FITC-labeled antibodies to HRP-labeled anti-FITC antibodies.

[0088] The magnetic microspheres described in this invention are magnetic microparticles.

[0089] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0090] The present invention will be further illustrated below with reference to the embodiments:

[0091] Preparation Example 1

[0092] 1. CRP Coating: Magnetic microspheres were selected as the coating solid phase. The magnetic microspheres were prepared according to the following steps (the CRP protein was purchased from Aibotek Biotechnology Co., Ltd., catalog number: RP01019):

[0093] Take 200 μl of carboxyl-containing magnetic microparticles and place them in a glass bottle. Use a magnet to attract the magnetic microparticles to the bottom of the bottle and remove the supernatant. Add 2 ml of 0.02 M PBS (pH 8.0) and repeat the above operation 3 times. Dissolve EDC and NHS separately in 0.1 M MES (pH 5.0) buffer to a concentration of 20 mg / ml, and then add 1 ml of each to the magnetic microparticles. Gently shake the mixture at room temperature for 30 minutes. Use a magnet to attract the magnetic microparticles to the bottom and remove the supernatant. Add another 2 ml of 0.1 M MES (pH 5.0) buffer to resuspend the magnetic microparticles. Repeat the above operation 2 times. Add 20 μg of coating material CRP to the activated magnetic microparticles; then add 0.1 M M ES (pH 5.0) buffer, specifically adding 10 mM calcium chloride to the coating solution to ensure the C-polysaccharide binding activity of CRP, to a final volume of 1 ml; gently shake at room temperature for 60 minutes; use a magnet to adsorb the magnetic microparticles to the bottom, remove the supernatant, and wash three times with 2 ml of 0.02 M PBS (pH 8.0); add 20 ml of blocking buffer (blocking buffer formula: 0.02 M PBS buffer, 10 mM calcium chloride, BSA as the blocking protein, 1% ratio), and store at 2–8 °C.

[0094] The specific antibody was a rabbit monoclonal antibody, purchased from Meridian (catalog number C01758R).

[0095] 3. Labeling with specific antibodies: Horseradish peroxidase (HRP) was activated using a conventional modified sodium periodate method. Specific antibodies were added, and the reaction was incubated overnight at 2–8°C. Sodium borohydride reductase conjugate was added, and unreacted reagents were removed by dialysis. 50% volumetric glycerol was added, and the mixture was stored at -20°C. When using, it was stored in preservation solution 1 at a concentration of 0.1 mg / L. Preservation solution 1 was formulated as follows: Bis-Tris buffer containing 20% ​​fetal bovine serum, 0.1% P300 preservative, and 10%–30% polyol protein protectant, and stored at 2–8°C.

[0096] 4. Preparation of negative control: 0.02M PBS buffer, 0.1% poloxamer, with P300 added as a preservative.

[0097] 5. Preparation of positive control: The obtained Streptococcus pneumoniae culture was resuspended in PBS until the OD value was greater than 2.0-3.0. The supernatant after centrifugation at 7000-10000 rpm for 5-10 minutes was used as positive additive and added to the negative control dilution at a ratio of 1 / 10k as positive control.

[0098] 6. Sample processing: Urine samples are usually clear liquids. If the sample contains insoluble substances, it should be processed by centrifugation at 12,000 rpm for 5 to 10 minutes or by filtration. A 1μm to 10μm filter can be selected for filtration.

[0099] Kit components:

[0100] Magnetic microparticle suspension coated with CRP protein, 100 tests per bottle;

[0101] Enzyme conjugate, HRP-labeled Streptococcus pneumoniae-specific antibody;

[0102] Negative control: PBS dilution containing surfactants and preservatives;

[0103] Positive control, PBS dilution kit containing Streptococcus pneumoniae C polysaccharide, surfactant and preservative: Instructions:

[0104] Place the sample container in the instrument's sample holder; load the sample holder and enter the sample information in the instrument's software interface; select "Run" to start the test, and the instrument will perform the following operations:

[0105] 1) Transfer the sample rack to the sample aspiration position and load the reaction vessel into the sample loading position.

[0106] 2) Complete the dispensing of 100 μL sample or 100 μL calibrator, 20 μL magnetic microparticle suspension, and 100 μL enzyme conjugate.

[0107] 3) Mix the reaction solution thoroughly and incubate at 37°C for 15 minutes.

[0108] 4) After the incubation is complete, the reaction solution is cleaned and separated using a cleaning solution.

[0109] 5) Complete the dispensing of 50 μL of substrate solution and 50 μL of enhancement solution.

[0110] 6) Mix the reaction solution thoroughly and test the luminescence intensity.

[0111] 7) The instrument automatically reports the test results.

[0112] Preparation Example 2

[0113] 1. CRP Coating: Magnetic microspheres were selected as the coating solid phase. The magnetic microspheres were prepared according to the following steps (the CRP protein was purchased from Aibotek Biotechnology Co., Ltd., catalog number: RP01019):

[0114] Take 200 μl of carboxyl-containing magnetic microparticles and place them in a glass bottle. Use a magnet to attract the magnetic microparticles to the bottom of the bottle and remove the supernatant. Add 2 ml of 0.02 M PBS (pH 8.0) and repeat the above operation 3 times. Dissolve EDC and NHS separately in 0.1 M MES (pH 5.0) buffer to a concentration of 20 mg / ml, and then add 1 ml of each to the magnetic microparticles. Gently shake the mixture at room temperature for 30 minutes. Use a magnet to attract the magnetic microparticles to the bottom and remove the supernatant. Add another 2 ml of 0.1 M MES (pH 5.0) buffer to resuspend the magnetic microparticles. Repeat the above operation 2 times. Add 20 μg of coating material CRP to the activated magnetic microparticles; then add 0.1 M M ES (pH 5.0) buffer, specifically adding 10 mM calcium chloride to the coating solution to ensure the C-polysaccharide binding activity of CRP, to a final volume of 1 ml; gently shake at room temperature for 60 minutes; use a magnet to adsorb the magnetic microparticles to the bottom, remove the supernatant, and wash three times with 2 ml of 0.02 M PBS (pH 8.0); add 20 ml of blocking buffer (blocking buffer formula: 0.02 M PBS buffer, 30 mM calcium chloride, BSA as the blocking protein, 1% ratio), and store at 2–8 °C.

[0115] The subsequent preparation process of the kit is the same as that in Preparation Example 1.

[0116] Preparation Example 3

[0117] 1. CRP Coating: Magnetic microspheres were selected as the coating solid phase. The magnetic microspheres were prepared according to the following steps (the CRP protein was purchased from Aibotek Biotechnology Co., Ltd., catalog number: RP01019):

[0118] Take 200 μl of carboxyl-containing magnetic microparticles and place them in a glass bottle. Use a magnet to attract the magnetic microparticles to the bottom of the bottle and remove the supernatant. Add 2 ml of 0.02 M PBS (pH 8.0) and repeat the above operation 3 times. Dissolve EDC and NHS separately in 0.1 M MES (pH 5.0) buffer to a concentration of 20 mg / ml, and then add 1 ml of each to the magnetic microparticles. Gently shake the mixture at room temperature for 30 minutes. Use a magnet to attract the magnetic microparticles to the bottom and remove the supernatant. Add another 2 ml of 0.1 M MES (pH 5.0) buffer to resuspend the magnetic microparticles. Repeat the above operation 2 times. Add 20 μg of coating material CRP to the activated magnetic microparticles; then add 0.1 M M ES (pH 5.0) buffer, specifically adding 10 mM calcium chloride to the coating solution to ensure the C-polysaccharide binding activity of CRP, to a final volume of 1 ml; gently shake at room temperature for 60 minutes; use a magnet to adsorb the magnetic microparticles to the bottom, remove the supernatant, and wash three times with 2 ml of 0.02 M PBS (pH 8.0); add 20 ml of blocking buffer (blocking buffer formula: 0.02 M PBS buffer, 50 mM calcium chloride, BSA as the blocking protein, 1% ratio), and store at 2–8 °C.

[0119] The subsequent preparation process of the kit is the same as that in Preparation Example 1.

[0120] Example 1: Sensitivity Verification

[0121] Samples identified positive by Streptococcus pneumoniae strain (ATCC-49619) and the imported colloidal gold reagent kit were diluted with physiological saline. The diluted samples were then tested using the kit of this invention (preparation method and operation steps as described in Preparation Example 1). The results are shown in Table 1.

[0122] Table 1

[0123]

[0124] The results showed that the kit of the present invention had higher sensitivity than the control kit in detecting Streptococcus pneumoniae strains and clinical urine samples.

[0125] Example 2: Serological Inclusivity Verification

[0126] Six common serotypes of Streptococcus pneumoniae in China: 19F, 19A, 6A, 14, 6B, and 23F (all bacterial concentrations were 10). 9 The sample was diluted with physiological saline (approximately CFU / ml), and the diluted sample was tested using the kit of this invention (preparation method and operation steps as described in Preparation Example 1). The results are shown in Table 2.

[0127] Table 2

[0128]

[0129] The results show that this invention can effectively detect a variety of common Streptococcus pneumoniae.

[0130] Example 3: Specificity Verification

[0131] The kit of this invention (preparation method and operation steps as described in Preparation Example 2) was used to detect common urogenital tract microorganisms and pneumonia-related microorganisms, including: Streptococcus agalactiae, Bordetella pertussis, Escherichia coli, Corynebacterium diphtheriae, Staphylococcus aureus, Moraxella catarrhalis, Lactobacillus acidophilus, Acinetobacter bacillus, Haemophilus influenzae, Pseudomonas aeruginosa, Candida albicans, Pseudomonas aeruginosa, and Salmonella enteritidis. The kit was tested at a high concentration of 10... 8 The test results at CFU / ml concentrations were all negative, indicating that the kit has good specificity.

[0132] Example 4: Clinical Sample Validation

[0133] A retrospective study of clinical samples was conducted using the kit of this invention. Urine samples from 15 patients with blood culture-positive Streptococcus pneumoniae pneumonia were simultaneously tested using the kits prepared in Preparation Examples 1, 2, and 3. The detection performance of this kit in clinically confirmed samples was evaluated, and all results were statistically expressed as S / CO. The results show that all 15 urine samples were detected, and there was no significant difference in the detection results of the kit with 10, 30, and 50 mM calcium chloride.

[0134] Table 3

[0135] Critical value: S / CO = 1 Preparation Example 1 Preparation Example 2 Preparation Example 3 1 1,108.41 1,043.32 1,069.26 2 98.33 184.10 197.98 3 22.12 16.67 17.95 4 448.27 452.21 463.39 5 68.92 53.32 55.65 6 163.39 189.10 201.02 7 15.90 17.69 15.85 8 1.16 1.16 1.08 9 90.98 101.01 50.57 10 11.93 14.68 12.74 11 0.95 0.95 0.95 12 770.44 762.78 885.44 13 107.95 127.27 113.09 14 8.49 10.17 9.04 15 202.54 283.54 222.21

[0136] 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 detection kit for preparing Streptococcus pneumoniae antigen using C-reactive protein and monoclonal antibody, comprising component A and component B, wherein component A is a solid-phase carrier coated with C-reactive protein, and component B is a monoclonal antibody specific to Streptococcus pneumoniae cell wall polysaccharide labeled with a tracer marker; The test kit also includes a blocking buffer containing 10mM~50mM calcium chloride; The monoclonal antibody was a rabbit monoclonal antibody, purchased from Meridian, catalog number C01758R.

2. The test kit according to claim 1, characterized in that, The C-reactive protein coating method includes direct coating or indirect coating.

3. The test kit according to claim 1 or 2, characterized in that, The tracer markers include enzymes and / or luminescent markers; The enzymes include horseradish peroxidase and / or alkaline phosphatase; The luminescent marker includes at least one of luminol and its derivatives, isoluminol and its derivatives, and acridine esters.

4. The test kit according to claim 1, characterized in that, The blocking buffer solution is mixed with component A.

5. The test kit according to claim 1, characterized in that, The solid support includes magnetic microparticles.

6. A method for detecting Streptococcus pneumoniae antigen for non-diagnostic purposes, comprising using a detection kit as described in any one of claims 1 to 5 to qualitatively detect Streptococcus pneumoniae antigen in a sample to be tested by chemiluminescent immunoassay; The sample to be tested is a urine sample.

7. The method of claim 6, wherein, Includes the following steps: Step (1): Mix component A and component B of the test kit with the sample to be tested, incubate, wash, separate magnetically, add luminescent substrate to the precipitate, detect the light signal value, and obtain the light signal value of the sample to be tested; Step (2): Measure the light signal values ​​of the negative control and the positive control, and obtain the light signal value of the CutOff value of the kit. Compare the light signal value of the sample to be tested with the light signal value of the CutOff value to obtain the qualitative detection result of the pneumococcal antigen in the sample to be tested.

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