Chlamydia pneumoniae igm antibody detection kit with high stability and preparation method

Abstract

The application discloses a high-stability chlamydia pneumoniae IgM antibody detection kit and a preparation method, and belongs to the technical field of in-vitro detection. The kit comprises a sample pad, a gold mark pad, a reaction film coated with a detection line and a quality control line and a water absorption pad fixed on a PVC plate. The gold mark pad is loaded with colloidal gold labeled mouse anti-human IgM monoclonal antibody. The colloidal gold labeled mouse anti-human IgM monoclonal antibody is obtained by labeling the antibody after modifying the surface of colloidal gold particles with cysteamine and performing targeted stabilization treatment with boron-functionalized polyamide-amine dendrimers. The sample pad is fixed with composite functional microspheres. The composite functional microspheres are polystyrene microspheres fixed with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer. The application has made significant progress in detection performance, stability and anti-interference ability through systematic optimization and innovative design.

Description

Technical Field

[0001] This invention belongs to the field of in vitro detection technology, and relates to a highly stable Chlamydia pneumoniae IgM antibody detection kit and its preparation method. Background Technology

[0002] Chlamydia pneumoniae ( Chlamydia pneumoniae Chlamydia pneumoniae is a common pathogen that can cause respiratory infections in humans. It is a significant cause of community-acquired pneumonia, bronchitis, sinusitis, and pharyngitis, and is prevalent globally, capable of widespread transmission among the population. Therefore, achieving rapid and accurate diagnosis of Chlamydia pneumoniae infection is of great significance for clinical treatment and public health prevention and control.

[0003] In clinical laboratory testing, the diagnosis of acute Chlamydia pneumoniae infection mainly relies on the detection of specific IgM antibodies in serum. Traditional enzyme-linked immunosorbent assay (ELISA) is cumbersome, time-consuming, and instrument-dependent, making it difficult to meet the needs of rapid diagnosis in outpatient and emergency departments. In contrast, colloidal gold immunochromatography (CIGC) offers advantages such as ease of operation, rapid detection, and intuitive result interpretation, making it more suitable for primary healthcare institutions and rapid on-site screening.

[0004] However, commercially available Chlamydia pneumoniae IgM antibody colloidal gold detection kits currently have problems in practical applications. Firstly, there is the issue of kit stability; the colloidal gold-labeled antibody complex, which serves as the core signal element, exhibits poor stability during storage. On one hand, colloidal gold particles are prone to aggregation; on the other hand, the biological activity of the labeled antibody decreases with time and rising ambient temperature. These factors result in some commercially available products having either short shelf lives or requiring stringent storage conditions. Improper transportation or storage can easily lead to weakened detection line signals and decreased sensitivity. A more critical problem is their insufficient resistance to interference. When existing kits encounter complex clinical samples (such as whole blood), high concentrations of endogenous interfering substances such as bilirubin, blood lipids, and hemoglobin can easily cause non-specific color development, leading to false positive or false negative results, severely affecting the specificity and accuracy of the test.

[0005] Therefore, there is an urgent need in this field for a highly stable and reliable Chlamydia pneumoniae IgM antibody detection kit to simultaneously address the two major pain points mentioned above. On the one hand, it can significantly improve the stability of colloidal gold-labeled complexes during long-term storage to extend the product's shelf life; on the other hand, it can effectively overcome matrix interference in clinical samples, thereby ensuring that the detection results always maintain high sensitivity and specificity. Summary of the Invention

[0006] The application aims to provide a high-stability Chlamydia pneumoniae IgM antibody detection kit and a preparation method, so as to realize stable storage and accurate detection of complex clinical samples at room temperature, solve the technical bottlenecks of the existing technology, such as easy inactivation of the labeled antibody, easy aggregation of the colloidal gold particles, and false positive caused by endogenous substances, and improve the storage stability, detection specificity and reliability of the kit in clinical application.

[0007] The technical solution adopted by the application provides the high-stability Chlamydia pneumoniae IgM antibody detection kit, and the key lies in that the kit comprises a sample pad, a gold label pad, a reaction membrane coated with a detection line and a quality control line, and a water absorption pad fixed on a PVC plate; the gold label pad is loaded with colloidal gold-labeled mouse anti-human IgM monoclonal antibody, the colloidal gold-labeled mouse anti-human IgM monoclonal antibody is obtained by labeling the antibody after modifying the surface of colloidal gold particles with cysteamine and performing targeted stabilization treatment with boron-functionalized polyamide-amine dendrimers; the sample pad is fixed with composite functional microspheres, the composite functional microspheres are polystyrene microspheres fixed with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer; and the boron-functionalized polyamide-amine dendrimers are obtained by coupling 3-aminobenzene boronic acid and polyamide-amine dendrimers under the action of a coupling agent.

[0008] Specifically, in the colloidal gold-labeled mouse anti-human IgM monoclonal antibody, the modification concentration of cysteamine is 0.9 mM to 1.1 mM.

[0009] More specifically, the coupling agent is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1:1.

[0010] Preferably, in the composite functional microspheres, the polystyrene microspheres are amino-modified polystyrene microspheres; the theoretical loading amount of bilirubin oxidase on each milligram of the polystyrene microspheres is 15 μg to 25 μg of enzyme protein; and the theoretical loading amount is calculated based on the ratio of the total mass of added bilirubin oxidase to the total mass of the polystyrene microspheres.

[0011] The preparation method of the high-stability Chlamydia pneumoniae IgM antibody detection kit is used to prepare the kit, and the key lies in comprising the following steps:

[0012] S1, preparation of the gold label pad:

[0013] The concentrated colloidal gold solution is obtained by synthesizing colloidal gold and concentrating, the colloidal gold surface is modified by cysteamine, the colloidal gold-antibody complex is obtained after labeling the mouse anti-human IgM monoclonal antibody, the high-stable colloidal gold labeled antibody concentrate is obtained after blocking, purifying, and concentrating by using the boron acid functionalized polyamide-amine dendrimers for targeted stabilization treatment, and the finished product gold label pad is obtained after dilution, spraying on the gold label pad substrate, and drying.

[0014] S2, sample pad preparation: the amino-modified polystyrene microspheres are co-immobilized with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer to obtain composite functional microspheres, and the stable composite functional microspheres suspension is obtained after stabilization by a protective agent, and the finished product sample pad is obtained after dilution, spraying on the sample pad substrate, and drying.

[0015] S3, reaction membrane coating: the recombinant Chlamydia pneumoniae antigen solution and the goat anti-mouse IgG antibody solution are sprayed on the nitrocellulose membrane at a spraying amount of 0.08 μL / mm to 0.12 μL / mm to form a detection line and a quality control line, and the finished product reaction membrane is obtained after drying.

[0016] S4, reagent strip assembly: the finished product sample pad, the finished product gold label pad, the finished product reaction membrane, and the water absorption pad are sequentially pasted on the PVC bottom plate, and the above-mentioned kit is obtained after cutting, casing, and packaging.

[0017] Further, in the above-mentioned S1 step, the colloidal gold particles are prepared by the method of reducing tetrachloroauric acid with trisodium citrate, and the specific process is as follows: 800 μL to 1200 μL of 1% tetrachloroauric acid solution is added to 100 mL of ultrapure water, heated to boiling, 1.6 mL to 2.0 mL of 1% trisodium citrate solution is added under stirring at 300 r / min to 500 r / min, boiling is maintained for 5 min to 10 min, heating is stopped, and the concentrated colloidal gold solution is obtained after cooling, membrane filtration, centrifugation, and concentration.

[0018] Further, in the above-mentioned S1 step, the reaction conditions for the cysteamine modification are as follows: the cysteamine is prepared into a stock solution and added to the concentrated colloidal gold solution, the cysteamine modification concentration is controlled at 0.9 mM to 1.1 mM, and the reaction is carried out at room temperature for 4 h to 6 h in the dark.

[0019] Further, the boronic acid functionalized polyamidoamine dendrimer is used for targeted and stable treatment in the above S1 step, and the boronic acid functionalized polyamidoamine dendrimer solution is mixed with the above colloidal gold-antibody complex, and incubated in the dark at 50 r / min-100 r / min for 1 h-2 h; wherein, the preparation process of the boronic acid functionalized polyamidoamine dendrimer solution is: 10 mg of polyamidoamine dendrimer is dissolved in a borate buffer, 5 mg-10 mg of 3-aminophenylboronic acid and 6 mg-12 mg of a coupling agent are added, and after reaction for 2 h-4 h, the product is purified by dialysis, and then diluted with a borate buffer to 1 mL to obtain the boronic acid functionalized polyamidoamine dendrimer solution.

[0020] Preferably, the S2 step is specifically: 100 mg-200 mg of polystyrene microspheres is weighed, washed with a phosphate buffer, and then resuspended to prepare a microsphere stock suspension of 20 mg / mL; 10 mL of the above microsphere stock suspension is taken, 300 μL-500 μL of a bilirubin oxidase solution with a concentration of 1 mg / mL is added, and 100 μL-300 μL of a polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer aqueous solution with a mass concentration of 10% is added; the pH of the system is adjusted to 8.0-8.5 with a lye, and the system is incubated in the dark at 30℃-35℃ and 150 r / min-250 r / min for 1.5 h-2 h to obtain a functionalized microsphere suspension; a protective agent is added to the above functionalized microsphere suspension, and mixed at 4℃ and 100 r / min-200 r / min for 40 min-60 min to obtain a stable composite functional microsphere suspension.

[0021] Preferably, the protective agent comprises a trehalose solution with a mass concentration of 20%, a bovine serum albumin solution with a mass concentration of 10%, and a sorbitol solution with a mass concentration of 20% in a volume ratio of 3:2:1.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] The high-stability chlamydia pneumoniae IgM antibody detection kit (colloidal gold method) provided by the present application has made significant progress in detection performance, stability and anti-interference ability through systematic optimization and innovative design. The specific technical effects include:

[0024] Firstly, the present application establishes a double-stable mechanism of chemical anchoring-targeting cross-linking for gold label pad, fundamentally improves the stability of the labeled probe from the molecular level. The present application constructs an active amine group layer on the surface of colloidal gold by cysteamine modification, and then realizes the directional anchoring and spatial protection of antibody molecules by specific and reversible binding of boron-functionalized polyamidoamine dendrimers with antibody Fc segment glycosyl. The design introduces specific functional materials, which can effectively protect the active structure of the antibody and significantly inhibit the inactivation of the kit under storage and environmental stress, so as to improve the shelf life and batch consistency of the product. The present application reduces the product scrap and quality risk caused by probe failure from the source, and the comprehensive benefits are significant.

[0025] Secondly, the present application constructs a new sample pretreatment strategy of enzyme digestion and dispersion integration for sample pad, and realizes the synchronous removal of key interferents at the starting end of chromatography. The present application fixes the bilirubin oxidase and the lipid dispersant Pluronic F-127 in the composite functional microspheres, and modifies the sample pad into a high-efficiency sample pretreatment center. The design realizes precise component compatibility and process, which can simultaneously degrade the color interference of bilirubin and disperse the steric hindrance of lipids or proteins after sample loading, reduces the risk of false positive and false negative caused by hemoglobin and blood lipids, greatly improves the accuracy and success rate of detection, and effectively avoids the subsequent cost caused by misdiagnosis.

[0026] Thirdly, the present application completes the whole chain design from key material screening to system integration optimization, realizes the best balance of performance, cost and robustness. The core material polyamidoamine dendrimer selected by the present application reflects the perfect balance between molecular size and functional group density in "providing sufficient stability" and "maintaining efficient reaction". The kit system constructed thereby not only shows a wider operation window and environmental tolerance, but also reduces the dependence on the operating environment due to its stable performance, so that the kit of the present application shows excellent practical value and competitive advantage in the point-of-care testing scene.

[0027] In summary, the innovative design of the present application significantly improves the reliability, detection accuracy and user experience of the kit product. The product finally realizes the comprehensive performance of high sensitivity, high specificity, long life and strong anti-interference, and provides a complete solution with significant competitive advantage for solving the core pain points of traditional colloidal gold kit. DETAILED DESCRIPTION

[0028] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Unless otherwise specified in the examples, conventional conditions may be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products, including recombinant Chlamydia pneumoniae antigen, mouse anti-human IgM monoclonal antibody, and goat anti-mouse IgG antibody. The polyamide-amine dendritic macromolecule used is a type of dendritic polyamide-amine copolymer. The commercial name of this substance used in this invention is PAMAM G4-NH2 dendritic polyamide-amine fourth-generation copolymer, which can be abbreviated as PAMAM G4. The polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer used in this invention is commercially available as Pluronic F127 or Pluronic F-127. The amino-modified polystyrene microspheres are commercially available functionalized materials with amino groups introduced onto the surface of polystyrene microspheres through chemical modification. The microspheres are regularly spherical, and their particle size can be controlled between 20 nm and 200 μm. The bilirubin oxidase specification is 100 U.

[0034] The coupling agent used in the embodiment of the application is composed of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide with a mass ratio of 1:1, which is referred to as EDC / NHS coupling agent; and bovine serum albumin is referred to as BSA.

[0035] The room temperature in the embodiment refers to a laboratory temperature in the range of 22℃ to 26℃. Embodiment I

[0036] The chlamydia pneumoniae IgM antibody detection kit provided in the embodiment has high stability, and includes a sample pad, a gold label pad, a reaction membrane coated with a detection line and a quality control line, and a water absorption pad. The gold label pad is loaded with colloidal gold-labeled mouse anti-human IgM monoclonal antibody. The colloidal gold-labeled mouse anti-human IgM monoclonal antibody is obtained by labeling the antibody after modifying the surface of colloidal gold particles with cysteamine and performing targeted stabilization treatment with boron-functionalized polyamidoamine dendrimers. The sample pad is fixed with composite functional microspheres. The composite functional microspheres are polystyrene microspheres fixed with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer.

[0037] The specific preparation process is as follows:

[0038] S1, preparation of the gold label pad:

[0039] S11, synthesis and preliminary purification of colloidal gold particles:

[0040] 100mL of ultrapure water was taken, 1000μL of 1% tetrachlorogold acid solution was added, and heated to vigorous boiling;

[0041] Under vigorous stirring at a speed of 400r / min, 1.8mL of 1% trisodium citrate solution was added;

[0042] Boil for 8min until the solution turns bright red, stop heating, cool under stirring at 24℃, filter through a 0.22μm water phase filter membrane, use an ultrafiltration centrifuge tube, centrifuge at 8000×g for 25min at 4℃, concentrate to about 1 / 5 of the original volume, and obtain a concentrated colloidal gold solution with a volume of 20mL.

[0043] S12, stability modification of the surface of colloidal gold particles:

[0044] Cysteamine was used as a linking molecule, and the cysteamine was dissolved in ultrapure water to prepare a 10mM cysteamine stock solution;

[0045] Under continuous stirring at a speed of 400r / min, 200μL of the cysteamine stock solution was added dropwise to the 20mL concentrated colloidal gold solution to make the final concentration of cysteamine 1.0mM, and the continuous reaction was carried out at 24℃ in the dark for 5h.

[0046] Again use ultrafiltration centrifuge tube, wash and centrifuge 2 times with 10 mM borate buffer solution of pH 8.5, centrifugal condition is 4℃, 8000xg centrifugal 25min; discard the supernatant, resuspended in 10 mL of the borate buffer solution, to obtain the surface amine modified colloidal gold solution.

[0047] S13, labeling of antibody and preparation of boronic acid functionalized polymer:

[0048] S131, antibody labeling:

[0049] The pH of the obtained surface amine modified colloidal gold solution is adjusted to 8.5 with 0.1M K2CO3 solution, 150μL of 1mg / mL mouse anti-human IgM monoclonal antibody is added, and mixed at 300r / min for 35min to obtain a colloidal gold-antibody complex.

[0050] S132, preparation of boronic acid functionalized polyamidoamine dendrimers:

[0051] Take 10mg of PAMAM G4, dissolve in 1mL of 10mM borate buffer solution of pH 8.5;

[0052] Add 7.5mg of 3-aminobenzoic acid and 9mg of EDC / NHS coupling agent, react at 24℃ for 3h;

[0053] Purify by dialysis to obtain boronic acid functionalized polyamidoamine dendrimers, and dilute to 1mL with the borate buffer solution to obtain a boronic acid functionalized polyamidoamine dendrimer solution.

[0054] S14, target stabilization treatment and post-treatment:

[0055] S141, target stabilization reaction:

[0056] Slowly add 1mL of boronic acid functionalized polyamidoamine dendrimer solution to the above colloidal gold-antibody complex;

[0057] Incubate at 24℃, 75r / min shaking in the dark for 1.5h to obtain a target stabilized colloidal gold-antibody complex.

[0058] S142, blocking and stabilization:

[0059] Add 1.1mL of 10% BSA solution to the target stabilized colloidal gold-antibody complex, and continue to react for 15min to block non-specific sites;

[0060] Then, 1.1 mL of 50% trehalose solution was added to obtain a stable colloidal gold-labeled antibody solution.

[0061] S143, purification and concentration:

[0062] The obtained stable colloidal gold-labeled antibody solution was centrifuged at 4℃ and 12000×g for 40 min;

[0063] The supernatant was discarded, and the precipitated and stabilized colloidal gold-antibody complex was resuspended in 1 mL of gold label pad treatment solution to obtain a high-stability colloidal gold-labeled antibody concentrate solution.

[0064] The gold label pad treatment solution is 50 mM PBS buffer containing 1.0% BSA, 5.0% trehalose, and 0.5% Tween-20, and the pH is 7.4.

[0065] S15, spraying and drying of the gold label pad:

[0066] 1 mL of high-stability colloidal gold-labeled antibody concentrate solution was mixed with 5 mL of gold label pad treatment solution to obtain a working solution, and the glass fiber membrane was treated with a uniform spraying amount of 1.5 mL / strip.

[0067] After the sprayed gold label pad was dried at 37℃ for 3 h, it was placed in a high-strength water and oxygen isolation aluminum foil bag and sealed with a desiccant to obtain a finished gold label pad.

[0068] S2, preparation of sample pad:

[0069] S21, pretreatment of composite functional microspheres

[0070] 150 mg of amino-modified polystyrene microspheres with a particle size of 150 nm were weighed and placed in a 50 mL centrifuge tube.

[0071] 20 mL of pH 7.4, 50 mM PBS buffer was added, and the vortex oscillator was used for full suspension for 1.5 min;

[0072] Centrifugation was performed at 4℃ and 12000×g for 18 min, the supernatant was discarded, and the washing step was repeated twice to remove possible impurities during transportation and storage.

[0073] The washed microspheres were resuspended with the PBS buffer to prepare a 20 mg / mL microsphere stock suspension, which was stored at 4℃ for standby use.

[0074] S22, functional loading

[0075] 10 mL of the microsphere stock suspension was taken and placed in a 50 mL reaction bottle.

[0076] Add 400 μL of 1 mg / mL bilirubin oxidase solution, and calculate the expected value according to the total amount of enzyme added and the total mass of the microspheres, that is, the theoretical loading of the enzyme is 20 μg of enzyme protein per milligram of microspheres; the theoretical loading is calculated based on the ratio of the total mass of bilirubin oxidase added to the total mass of polystyrene microspheres;

[0077] Add 200 μL of a 10% polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer aqueous solution to make the final concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer 0.2% by mass concentration;

[0078] Adjust the pH of the mixed system to 8.3 with a NaOH solution, and incubate the system at 32°C and 200 r / min for 1.75 h in the dark to obtain a functionalized microsphere suspension.

[0079] S23, adding a protective agent stabilizing system

[0080] Add a protective agent to the obtained functionalized microsphere suspension, and the protective agent is prepared from 1.5 mL of a 20% trehalose solution, 1.0 mL of a 10% BSA solution, and 0.5 mL of a 20% sorbitol solution;

[0081] Continue mixing at 4°C and 150 r / min for 50 min to allow the protective agent to fully bind, and obtain a stable composite functional microsphere suspension.

[0082] S24, preparation and spraying of sample pad working solution

[0083] Mix and dilute the obtained stable composite functional microsphere suspension with the sample pad treatment solution at a volume ratio of 1:2 to obtain a sample pad working solution;

[0084] The sample pad treatment solution is a 50 mM PBS buffer containing 0.75% BSA, 4% trehalose, and 0.45% Tween-20 by mass concentration, and the pH is 7.4;

[0085] Spray the working solution onto the glass fiber membrane at a uniform spraying amount of 1.5 mL per strip;

[0086] After drying the sprayed sample pad at 37°C for 3 h, place it in a high-strength water- and oxygen-proof aluminum foil bag, add a desiccant, and seal and store it to obtain a finished sample pad.

[0087] S3, coating of the reaction membrane:

[0088] S31, preparation and coating of the test line (T line) coating solution

[0089] Dissolve the recombinant Chlamydia pneumoniae antigen in 10 mL of T-line coating buffer to prepare a T-line coating working solution with a concentration of 1.0 mg / mL;

[0090] The T-line coating buffer is 10 mM PBS buffer containing 5.0% sucrose by mass concentration, pH 7.3;

[0091] Use the membrane scribing and gold spraying instrument to accurately spray the T-line coating working solution on the set position of the nitrocellulose membrane at a spraying amount of 0.10 μL / mm to form the T-line.

[0092] S32, preparation and coating of quality control line (C-line) coating solution:

[0093] Dissolve the goat anti-mouse IgG antibody in 10 mL of C-line coating buffer to prepare a C-line coating working solution with a concentration of 2.0 mg / mL of goat anti-mouse IgG antibody;

[0094] The C-line coating buffer is 10 mM PBS buffer containing 5.0% sucrose by mass concentration, pH 7.3;

[0095] Use the membrane scribing and gold spraying instrument to accurately spray the C-line coating working solution on the set position above the T-line on the same nitrocellulose membrane at a spraying amount of 0.10 μL / mm to form the C-line.

[0096] S33, drying and preservation of the reaction membrane:

[0097] Place the reaction membrane with coated T-line and C-line in a 37°C constant temperature incubator and dry for 3.0 h;

[0098] After drying, place the reaction membrane in a high-strength water and oxygen isolation aluminum foil bag and seal it for preservation to obtain the finished reaction membrane.

[0099] S4, assembly of the reagent strip:

[0100] Paste the finished sample pad, finished gold-labeled pad, finished reaction membrane, and water absorption pad on the PVC bottom plate in sequence, and cut them into long strips in a clean environment using a numerical control cutting machine;

[0101] In the order of sample flow direction, sequentially overlap and paste the following components on the PVC bottom plate: finished sample pad, finished gold-labeled pad, finished reaction membrane, and water absorption pad, with an overlap of 1.2 mm between each component. Use the roller to press three times to ensure that each component is firmly bonded to the bottom plate without air bubbles;

[0102] Cut the assembled large plate into individual detection reagent strips;

[0103] The cut test reagent strip is loaded into a plastic card shell, and a cover is covered, pressed tightly, and a complete detection card is obtained; the complete detection card is loaded into a high-strength water-oxygen isolation aluminum foil bag, a drying agent is put into each bag, and a heat sealing machine is used for sealing, and a Chlamydia pneumoniae IgM antibody test kit (colloidal gold method) finished product is obtained, which is recorded as kit sample 1. Example two

[0104] The Chlamydia pneumoniae IgM antibody test kit provided in the embodiment has high stability, and includes a sample pad fixed on a PVC plate, a gold label pad, a reaction membrane coated with a detection line and a quality control line, and a water absorption pad; the gold label pad is loaded with colloidal gold-labeled mouse anti-human IgM monoclonal antibody, the colloidal gold-labeled mouse anti-human IgM monoclonal antibody is obtained by labeling the antibody after modifying the surface of colloidal gold particles with cysteamine, and is obtained by targeted stabilization treatment with boron-functionalized polyamidoamine dendrimers; the sample pad is fixed with composite functional microspheres, and the composite functional microspheres are polystyrene microspheres fixed with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer.

[0105] The specific preparation process is as follows:

[0106] S1, preparation of the gold label pad:

[0107] S11, synthesis and preliminary purification of colloidal gold particles:

[0108] Take 100 mL of ultrapure water, add 800 μL of 1% tetrachlorogold acid solution, and heat to vigorous boiling;

[0109] Under the condition of 300 r / min of stirring speed, 2.0 mL of 1% trisodium citrate solution is added;

[0110] Boil for 10 min until the solution turns bright red, stop heating, cool under stirring at 22°C, filter through a 0.22 μm water phase filter membrane, use an ultrafiltration centrifuge tube, centrifuge at 8000 x g for 20 min at 4°C, concentrate to about 1 / 5 of the original volume, and obtain a concentrated colloidal gold solution with a volume of 20 mL.

[0111] S12, stability modification of the surface of colloidal gold particles:

[0112] Use cysteamine as a linking molecule, dissolve the cysteamine in ultrapure water, and prepare a 10 mM cysteamine stock solution;

[0113] Under the condition of 300 r / min of stirring speed, 180 μL of the cysteamine stock solution is added dropwise to the 20 mL concentrated colloidal gold solution, so that the final concentration of cysteamine is 0.9 mM, and the continuous reaction is carried out at 22°C for 6 h in the dark;

[0114] The surface amine-modified colloidal gold solution was washed and centrifuged three times using an ultrafiltration centrifuge tube and 10 mM borate buffer solution at pH 8.3, and the centrifugation condition was 8000 x g at 4°C for 20 min; the supernatant was discarded, and the surface amine-modified colloidal gold solution was resuspended in 10 mL of the borate buffer solution.

[0115] S13, labeling of the antibody and preparation of the boronate-functionalized polymer:

[0116] S131, labeling of the antibody:

[0117] The pH of the obtained surface amine-modified colloidal gold solution was adjusted to 8.7 using a 0.1 M K2CO3 solution, 150 μL of mouse anti-human IgM monoclonal antibody with a concentration of 1 mg / mL was added, and the mixture was mixed at a rotation speed of 200 r / min for 40 min to obtain a colloidal gold-antibody complex.

[0118] S132, preparation of boronate-functionalized polyamidoamine dendrimers:

[0119] 10 mg of PAMAM G4 was dissolved in 1 mL of 10 mM borate buffer solution at pH 8.3;

[0120] 5 mg of 3-aminophenylboronic acid and 12 mg of EDC / NHS coupling agent were added, and the reaction was carried out at 22°C for 2 h;

[0121] The boronate-functionalized polyamidoamine dendrimers were purified by dialysis, and the volume was adjusted to 1 mL with the borate buffer solution to obtain a boronate-functionalized polyamidoamine dendrimer solution.

[0122] S14, targeted stabilization treatment and post-treatment:

[0123] S141, targeted stabilization reaction:

[0124] 1 mL of the boronate-functionalized polyamidoamine dendrimer solution was slowly added to the colloidal gold-antibody complex;

[0125] The mixture was incubated at 22°C and 100 r / min under light shielding for 1 h to obtain a colloidal gold-antibody complex with targeted stabilization.

[0126] S142, blocking and stabilization:

[0127] 1.0 mL of a BSA solution with a mass concentration of 10% was added to the colloidal gold-antibody complex with targeted stabilization, and the reaction was continued for 18 min to block non-specific sites;

[0128] 1.0 mL of a trehalose solution with a mass concentration of 50% was further added to obtain a stable colloidal gold-labeled antibody solution.

[0129] S143, purification and concentration:

[0130] Centrifuge the obtained stable colloidal gold-labeled antibody solution at 4℃, 12000xg for 35min;

[0131] Discard the supernatant, and resuspend the precipitated and stabilized colloidal gold-antibody complex in 1mL of gold label pad treatment solution to obtain a high-stability colloidal gold-labeled antibody concentrate solution;

[0132] The gold label pad treatment solution is 45mM PBS buffer containing 0.8% BSA, 4.5% trehalose and 0.4% Tween-20 by mass concentration, and the pH is 7.3.

[0133] S15, spraying and drying of gold label pad:

[0134] Mix 1mL of high-stability colloidal gold-labeled antibody concentrate solution with 4mL of gold label pad treatment solution to obtain a working solution, and treat the glass fiber membrane with a uniform spraying amount of 1.6mL / strip;

[0135] After the sprayed gold label pad is dried at 37℃ for 3h, it is placed in a high-strength water and oxygen isolation aluminum foil bag and sealed for storage with a desiccant, to obtain a finished gold label pad.

[0136] S2, preparation of sample pad:

[0137] S21, pretreatment of composite functional microspheres

[0138] Weigh 100mg of amino-modified polystyrene microspheres with a particle size of 200nm and place them in a 50mL centrifuge tube;

[0139] Add 20mL of pH 7.3, 50mM PBS buffer, and use a vortex oscillator to fully suspend for 1min;

[0140] Centrifuge at 4℃, 12000xg for 15min, discard the supernatant, and repeat the washing step 3 times to remove possible impurities during transportation and storage;

[0141] Resuspend the washed microspheres with the PBS buffer to prepare a 20mg / mL microsphere stock suspension, and store it at 4℃ for later use.

[0142] S22, functional loading

[0143] Take 10mL of the microsphere stock suspension and place it in a 50mL reaction bottle;

[0144] Add 300 μL of 1 mg / mL bilirubin oxidase solution, and calculate the expected value according to the total amount of added enzyme and the total mass of the microspheres, that is, the theoretical loading of the enzyme is 15 μg of enzyme protein per milligram of microspheres; the theoretical loading is calculated based on the ratio of the total mass of added bilirubin oxidase to the total mass of polystyrene microspheres;

[0145] Add 300 μL of a 10% mass concentration of a polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer aqueous solution, so that the final concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer is 0.3% by mass concentration;

[0146] Adjust the pH of the mixed system to 8.0 with a NaOH solution, and incubate the system at 35°C and a rotation speed of 150 r / min for 2 h in the dark to obtain a functionalized microsphere suspension.

[0147] S23, adding a protective agent to stabilize the system

[0148] Add a protective agent to the obtained functionalized microsphere suspension, and the protective agent is prepared from 1.5 mL of a 20% mass concentration trehalose solution, 1.0 mL of a 10% mass concentration BSA solution, and 0.5 mL of a 20% mass concentration sorbitol solution;

[0149] Continue mixing at 4°C and a rotation speed of 100 r / min for 60 min to allow the protective agent to fully bind, and obtain a stable composite functional microsphere suspension.

[0150] S24, preparation and spraying of sample pad working solution

[0151] Mix and dilute the obtained stable composite functional microsphere suspension with the sample pad treatment solution at a volume ratio of 1:1 to obtain a sample pad working solution;

[0152] The sample pad treatment solution is a 45 mM PBS buffer containing 0.5% BSA, 3% trehalose, and 0.6% Tween-20 by mass concentration, and the pH is 7.6;

[0153] Spray the working solution onto the glass fiber membrane at a uniform spraying amount of 1.6 mL per strip;

[0154] After drying the sprayed sample pad at 37°C for 3 h, place it in a high-strength water- and oxygen-proof aluminum foil bag, add a desiccant, and seal and store it to obtain a finished sample pad.

[0155] S3, coating of the reaction membrane:

[0156] S31, preparation and coating of the test line (T line) coating solution

[0157] Dissolve the recombinant Chlamydia pneumoniae antigen in 10 mL of T-line coating buffer to prepare a T-line coating working solution with a concentration of 0.8 mg / mL;

[0158] The T-line coating buffer is 8 mM PBS buffer containing 4.5% sucrose by mass concentration, pH 7.2;

[0159] Using a membrane scribing and gold spraying instrument, accurately spray the T-line coating working solution on the set position of the nitrocellulose membrane at a spraying amount of 0.12 μL / mm to form the T-line.

[0160] S32, preparation and coating of quality control line (C-line) coating solution:

[0161] Dissolve the goat anti-mouse IgG antibody in 10 mL of C-line coating buffer to prepare a C-line coating working solution with a concentration of 1.8 mg / mL of goat anti-mouse IgG antibody;

[0162] The C-line coating buffer is 8 mM PBS buffer containing 4.5% sucrose by mass concentration, pH 7.2;

[0163] Using a membrane scribing and gold spraying instrument, accurately spray the C-line coating working solution on the set position above the T-line on the same nitrocellulose membrane at a spraying amount of 0.08 μL / mm to form the C-line.

[0164] S33, drying and preservation of the reaction membrane:

[0165] Place the reaction membrane with coated T-line and C-line in a 38°C constant temperature incubator and dry for 2.5 h;

[0166] After drying, place the reaction membrane in a high-strength water and oxygen isolation aluminum foil bag and seal it for preservation, obtaining the finished reaction membrane.

[0167] S4, assembly of reagent strips:

[0168] Paste the finished sample pad, finished gold-labeled pad, finished reaction membrane, and water absorption pad on the PVC base plate in sequence, and cut them into long strips in a clean environment using a numerical control cutting machine;

[0169] In the order of sample flow direction, sequentially overlap and paste the following components on the PVC base plate: finished sample pad, finished gold-labeled pad, finished reaction membrane, and water absorption pad, with an overlap of 1.0 mm between each component. Use a roller to press twice to ensure that each component is firmly bonded to the base plate without air bubbles;

[0170] Cut the assembled large plate into individual test reagent strips;

[0171] The cut test reagent strip is loaded into a plastic card shell, and a cover is covered, pressed tightly, and a complete detection card is obtained; the complete detection card is loaded into a high-strength water-oxygen isolation aluminum foil bag, a drying agent is put into each bag, and a heat sealing machine is used for sealing, and a Chlamydia pneumoniae IgM antibody test kit (colloidal gold method) finished product is obtained, which is recorded as kit sample 2. Example three

[0172] The Chlamydia pneumoniae IgM antibody test kit provided in the embodiment has high stability, and includes a sample pad fixed on a PVC plate, a gold label pad, a reaction membrane coated with a detection line and a quality control line, and a water absorption pad; the gold label pad is loaded with colloidal gold-labeled mouse anti-human IgM monoclonal antibody, the colloidal gold-labeled mouse anti-human IgM monoclonal antibody is obtained by labeling the antibody after modifying the surface of colloidal gold particles with cysteamine, and is obtained by targeted stabilization treatment with boron-functionalized polyamidoamine dendrimers; the sample pad is fixed with composite functional microspheres, and the composite functional microspheres are polystyrene microspheres fixed with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer.

[0173] The specific preparation process is as follows:

[0174] S1, preparation of the gold label pad:

[0175] S11, synthesis and preliminary purification of colloidal gold particles:

[0176] Take 100 mL of ultrapure water, add 1200 μL of 1% tetrachlorogold acid solution, and heat to vigorous boiling;

[0177] Under the condition of 500 r / min stirring, 1.6 mL of 1% trisodium citrate solution is added;

[0178] Boil for 5 min until the solution turns bright red, stop heating, cool under stirring at 26℃, filter through a 0.22 μm water phase filter membrane, use an ultrafiltration centrifuge tube, centrifuge at 8000 x g for 30 min at 4℃, concentrate to about 1 / 5 of the original volume, and obtain a concentrated colloidal gold solution with a volume of 20 mL.

[0179] S12, stability modification of the surface of colloidal gold particles:

[0180] Use cysteamine as a linking molecule, dissolve the cysteamine in ultrapure water, and prepare a 10 mM cysteamine stock solution;

[0181] Under the condition of 500 r / min continuous stirring, 220 μL of the cysteamine stock solution is added dropwise to the 20 mL concentrated colloidal gold solution, so that the final concentration of cysteamine is 1.1 mM, and the continuous reaction is carried out at 26℃ for 4 h in the dark;

[0182] The surface amine-modified colloidal gold solution was washed and centrifuged twice using an ultrafiltration centrifuge tube with 10 mM borate buffer at pH 8.7, and the centrifugation condition was 8000 x g for 30 min at 4°C. The supernatant was discarded, and the surface amine-modified colloidal gold solution was resuspended in 10 mL of the borate buffer.

[0183] S13, Labeling of the antibody and preparation of the boronate-functionalized polymer:

[0184] S131, Labeling of the antibody:

[0185] The pH of the obtained surface amine-modified colloidal gold solution was adjusted to 8.3 using a 0.1 M K2CO3 solution, and 150 μL of mouse anti-human IgM monoclonal antibody with a concentration of 1 mg / mL was added, and mixed at a speed of 400 r / min for 30 min to obtain a colloidal gold-antibody complex.

[0186] S132, Preparation of boronate-functionalized polyamidoamine dendrimers:

[0187] 10 mg of PAMAM G4 was dissolved in 1 mL of 10 mM borate buffer at pH 8.7;

[0188] 10 mg of 3-aminophenylboronic acid and 6 mg of EDC / NHS coupling agent were added, and the reaction was carried out at 26°C for 4 h;

[0189] The boronate-functionalized polyamidoamine dendrimers were purified by dialysis, and the volume was adjusted to 1 mL with the borate buffer to obtain a boronate-functionalized polyamidoamine dendrimer solution.

[0190] S14, Targeting and stabilization treatment and post-treatment:

[0191] S141, Targeting and stabilization reaction:

[0192] 1 mL of the boronate-functionalized polyamidoamine dendrimer solution was slowly added to the colloidal gold-antibody complex described above;

[0193] The resulting colloidal gold-antibody complex was incubated at 26°C with shaking at 50 r / min in the dark for 2 h.

[0194] S142, Blocking and stabilization:

[0195] 1.2 mL of a BSA solution with a mass concentration of 10% was added to the targeting and stabilized colloidal gold-antibody complex, and the reaction was continued for 12 min to block non-specific sites;

[0196] 1.2 mL of a trehalose solution with a mass concentration of 50% was further added to obtain a stabilized colloidal gold-labeled antibody solution.

[0197] S143, purification and concentration:

[0198] Centrifuge the obtained stable colloidal gold-labeled antibody solution at 4℃, 12000xg for 45min;

[0199] Discard the supernatant, and resuspend the precipitated and stabilized colloidal gold-antibody complex in 1mL of gold label pad treatment solution to obtain a high-stability colloidal gold-labeled antibody concentrate solution;

[0200] The gold label pad treatment solution is 55mM PBS buffer containing 1.2% BSA, 5.5% trehalose and 0.6% Tween-20 by mass concentration, and the pH is 7.5.

[0201] S15, spraying and drying of gold label pad:

[0202] Mix 1mL of high-stability colloidal gold-labeled antibody concentrate solution with 6mL of gold label pad treatment solution to obtain a working solution, and treat the glass fiber membrane with a uniform spraying amount of 1.4mL / strip;

[0203] After the sprayed gold label pad is dried at 37℃ for 3h, it is placed in a high-strength water and oxygen isolation aluminum foil bag and sealed for storage with a desiccant, to obtain a finished gold label pad.

[0204] S2, preparation of sample pad:

[0205] S21, pretreatment of composite functional microspheres

[0206] Weigh 200mg of amino-modified polystyrene microspheres with a particle size of 100nm and place them in a 50mL centrifuge tube;

[0207] Add 20mL of pH 7.5, 50mM PBS buffer, and use a vortex oscillator to fully suspend for 2min;

[0208] Centrifuge at 4℃, 12000xg for 20min, discard the supernatant, and repeat the washing step 2 times to remove possible impurities during transportation and storage;

[0209] Resuspend the washed microspheres with the PBS buffer to prepare a 20mg / mL microsphere stock suspension, and store it at 4℃ for later use.

[0210] S22, functionalization loading

[0211] Take 10mL of the microsphere stock suspension and place it in a 50mL reaction bottle;

[0212] Add 500 μL of 1 mg / mL bilirubin oxidase solution, and calculate the expected value according to the total amount of added enzyme and the total mass of the microspheres, that is, the theoretical loading of the enzyme is 25 μg of enzyme protein per milligram of microspheres; the theoretical loading is calculated based on the ratio of the total mass of added bilirubin oxidase to the total mass of polystyrene microspheres;

[0213] Add 100 μL of a polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer aqueous solution with a mass concentration of 10%, so that the final concentration of the polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer is 0.1% by mass concentration;

[0214] Adjust the pH of the mixed system to 8.5 with a NaOH solution, and incubate the system at 30°C and a rotation speed of 250 r / min for 1.5 h in the dark to obtain a functionalized microsphere suspension.

[0215] S23, adding a protective agent stabilizing system

[0216] Add a protective agent to the obtained functionalized microsphere suspension, and the protective agent is prepared from 1.5 mL of a trehalose solution with a mass concentration of 20%, 1.0 mL of a BSA solution with a mass concentration of 10%, and 0.5 mL of a sorbitol solution with a mass concentration of 20%;

[0217] Continue mixing at 4°C and a rotation speed of 200 r / min for 40 min to allow the protective agent to fully bind, and obtain a stable composite functional microsphere suspension.

[0218] S24, preparation and spraying of sample pad working solution

[0219] Mix and dilute the obtained stable composite functional microsphere suspension with a sample pad treatment solution at a volume ratio of 1:3 to obtain a sample pad working solution;

[0220] The sample pad treatment solution is a 55 mM PBS buffer containing 1% BSA, 5% trehalose, and 0.3% Tween-20 by mass concentration, and the pH is 7.2;

[0221] Spray the working solution onto the glass fiber membrane at a uniform spraying amount of 1.4 mL per strip;

[0222] After drying the sprayed sample pad at 37°C for 3 h, place it in a high-strength water- and oxygen-proof aluminum foil bag, add a desiccant, and seal and store it to obtain a finished sample pad.

[0223] S3, coating of the reaction membrane:

[0224] S31, preparation and coating of the test line (T line) coating solution

[0225] The recombinant Chlamydia pneumoniae antigen was dissolved in 10 mL of T-line coating buffer to prepare a T-line coating working solution with a concentration of 1.2 mg / mL;

[0226] The T-line coating buffer was 12 mM PBS buffer containing 5.5% sucrose by mass concentration, pH 7.4;

[0227] The T-line coating working solution was accurately sprayed on the set position of the nitrocellulose membrane using a membrane scribing and gold spraying instrument at a spraying amount of 0.08 μL / mm to form a T-line.

[0228] S32, preparation and coating of quality control line (C-line) coating solution:

[0229] The goat anti-mouse IgG antibody was dissolved in 10 mL of C-line coating buffer to prepare a C-line coating working solution with a goat anti-mouse IgG antibody concentration of 2.2 mg / mL;

[0230] The C-line coating buffer was 12 mM PBS buffer containing 5.5% sucrose by mass concentration, pH 7.4;

[0231] The C-line coating working solution was accurately sprayed on the same nitrocellulose membrane at a set position above the T-line at a spraying amount of 0.12 μL / mm to form a C-line.

[0232] S33, drying and preservation of the reaction membrane:

[0233] The reaction membrane with coated T-line and C-line was placed in a 36°C constant temperature oven for drying for 3.5 h;

[0234] After drying, the reaction membrane was placed in a high-strength water and oxygen isolation aluminum foil bag and sealed for preservation to obtain a finished reaction membrane.

[0235] S4, assembly of the reagent strip:

[0236] The finished sample pad, finished gold-labeled pad, finished reaction membrane, and water absorption pad were sequentially pasted on the PVC bottom plate, and were cut into strips in a clean environment using a numerical control cutting machine;

[0237] The following components were sequentially overlapped on the PVC bottom plate in the order of the sample flow direction, with an overlap of 1.4 mm between each component: the finished sample pad, the finished gold-labeled pad, the finished reaction membrane, and the water absorption pad. The roller was pressed three times to ensure that each component was firmly bonded to the bottom plate without air bubbles.

[0238] The assembled large plate was cut into individual detection reagent strips;

[0239] The cut test reagent strip is loaded into a plastic card shell, and a cover is covered, pressed tightly, and a complete detection card is obtained; the complete detection card is loaded into a high-strength water-oxygen isolation aluminum foil bag, a drying agent is put into each bag, and a heat sealing machine is used for sealing, and a Chlamydia pneumoniae IgM antibody test kit (colloidal gold method) finished product is obtained, which is recorded as kit sample 3. Comparative Example One

[0240] This comparative example provides a Chlamydia pneumoniae IgM antibody test kit, and the specific implementation is the same as that of Example One, except that the targeting and stabilizing step of the boron acid functional polymer is omitted in the preparation of the gold label pad, which is recorded as kit control 1.

[0241] The preparation process of the kit control 1 of the present comparative example is as follows:

[0242] S1, preparation of the gold label pad:

[0243] S11, synthesis and preliminary purification of colloidal gold particles: the same as S11 of Example One.

[0244] S12, stability modification of the surface of colloidal gold particles: the same as S12 of Example One, to obtain a colloidal gold solution with amine group modification on the surface.

[0245] S13, antibody labeling:

[0246] The pH of the obtained colloidal gold solution with amine group modification on the surface is adjusted to 8.5 with 0.1M K2CO3 solution, 150μL of mouse anti-human IgM monoclonal antibody with a concentration of 1mg / mL is added, and mixed at a speed of 300r / min for 35min to obtain a colloidal gold-antibody complex. Thereafter, the targeting and stabilizing step is omitted, and the blocking treatment is directly performed

[0247] S14, blocking and stabilization:

[0248] 1.1mL of BSA solution with a mass concentration of 10% is added to the above colloidal gold-antibody complex, and the reaction is continued for 15min to block the non-specific sites;

[0249] 1.1mL of trehalose solution with a mass concentration of 50% is further added to obtain a stable colloidal gold-labeled antibody solution.

[0250] S15, purification and concentration:

[0251] The obtained stable colloidal gold-labeled antibody solution is centrifuged at 4℃, 12000xg for 40min;

[0252] The supernatant is discarded, and the precipitated colloidal gold-antibody complex is resuspended in 1mL of gold label pad treatment solution to obtain a colloidal gold-labeled antibody concentrate;

[0253] The gold label pad treatment solution is 50 mM PBS buffer solution containing 1.0% BSA, 5.0% trehalose and 0.5% Tween-20 by mass concentration, and the pH is 7.4.

[0254] S16, spraying and drying of the gold label pad:

[0255] 1 mL of the colloidal gold labeled antibody concentrate was mixed with 5 mL of the gold label pad treatment solution to obtain a working solution, and the glass fiber membrane was treated with a uniform spraying amount of 1.5 mL per strip.

[0256] After the sprayed gold label pad was dried at 37°C for 3 h, it was sealed and stored to obtain the finished gold label pad.

[0257] S2, preparation of the sample pad: same as step S2 of Example One.

[0258] S3, coating of the reaction membrane: same as step S3 of Example One.

[0259] S4, assembly of the reagent strip: same as step S4 of Example One, to obtain a Chlamydia pneumoniae IgM antibody detection kit, which is referred to as Kit Control 1. Comparative Example Two

[0260] This comparative example provides a Chlamydia pneumoniae IgM antibody detection kit, and the specific implementation is the same as that of Example One, except that polyvinylpyrrolidone is used for colloidal gold stabilization in the preparation of the gold label pad instead of cysteamine modification, which is referred to as Kit Control 2.

[0261] The preparation process of Kit Control 2 of this comparative example is as follows:

[0262] S1, preparation of the gold label pad:

[0263] S11, synthesis and preliminary purification of colloidal gold particles: same as step S11 of Example One, to obtain a concentrated colloidal gold solution.

[0264] S12, conventional stabilization treatment of the surface of the colloidal gold particles:

[0265] Under continuous stirring at a speed of 400 r / min, 200 μL of a 1% polyvinylpyrrolidone aqueous solution was added to 20 mL of the concentrated colloidal gold solution, and the reaction was continuously carried out at 24°C in the dark for 30 min.

[0266] The ultrafiltration centrifuge tube was washed and centrifuged twice with 10 mM borate buffer solution at pH 8.5, and the centrifugation conditions were 4°C, 8000 x g for 25 min; the supernatant was discarded, and the colloidal gold solution stabilized by PVP was obtained by resuspending in 10 mL of the borate buffer solution.

[0267] S13, Labeling of antibody and preparation of boronic acid functionalized polymer:

[0268] S131, Labeling of antibody: same as step S131 of example one, but after finishing this step, directly proceed to blocking, purification step, completely delete the preparation of boronic acid functionalized polymer and the treatment of target stabilization of S132 and S141 of example one.

[0269] S14, Blocking, purification and concentration:

[0270] S141, Blocking and stabilization:

[0271] To the above PVP stabilized gold colloid-antibody complex, 1.1 mL of 10% BSA solution by mass concentration was added, and the reaction was continued for 15 min to block the non-specific sites;

[0272] Then 1.1 mL of 50% trehalose solution by mass concentration was added to obtain the PVP stabilized gold colloid labeled antibody solution.

[0273] S142, Purification and concentration:

[0274] The obtained PVP stabilized gold colloid labeled antibody solution was centrifuged at 4℃, 12000xg for 40 min;

[0275] The supernatant was discarded, and the precipitated gold colloid-antibody complex was resuspended in 1 mL of gold label pad treatment solution to obtain the PVP stabilized gold colloid labeled antibody concentrate; wherein the gold label pad treatment solution is the same as example one.

[0276] S15, Spraying and drying of gold label pad: same as step S15 of example one to obtain the finished gold label pad.

[0277] S2, Preparation of sample pad: same as step S2 of example one.

[0278] S3, Coating of reaction membrane: same as step S3 of example one.

[0279] S4, Assembly of reagent strip: same as step S4 of example one to obtain the Chlamydia pneumoniae IgM antibody detection kit, which is referred to as kit control 2. Comparative example three

[0280] This comparative example provides a Chlamydia pneumoniae IgM antibody detection kit, and the specific implementation is the same as example one, except that the composite functional microspheres in the sample pad only load bilirubin oxidase, and no polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer is added, which is referred to as kit control 3.

[0281] The preparation process of kit control 3 of this comparative example is as follows:

[0282] S1, Preparation of gold label pad: same as step S1 of Example One.

[0283] S2, Preparation of sample pad:

[0284] S21, Pretreatment of composite functional microspheres: same as step S21 of Example One.

[0285] S22, Functionalization loading:

[0286] Take 10 mL of microsphere stock suspension and place it in a 50 mL reaction bottle;

[0287] Add 400 μL of 1 mg / mL bilirubin oxidase solution;

[0288] Adjust the pH of the mixed system to 8.3 with NaOH solution, and incubate at 32°C with stirring at 200 r / min in the dark for 1.75 h to obtain a functionalized microsphere suspension.

[0289] S23, Add protective agent to stabilize the system: same as step S23 of Example One.

[0290] S24, Preparation and spraying of sample pad working solution: same as step S24 of Example One to obtain the finished sample pad.

[0291] S3, Coating of reaction film: same as step S3 of Example One.

[0292] S4, Assembly of reagent strip: same as step S4 of Example One to obtain a Chlamydia pneumoniae IgM antibody test kit, designated as Kit Control 3. Comparative Example Four

[0293] A Chlamydia pneumoniae IgM antibody test kit is provided in the comparative example, and the specific implementation is the same as Example One, except that PAMAM G8-NH2 dendrimer polyamide amine 8 (referred to as PAMAM G8) is used instead of PAMAM G4 in the preparation of the gold label pad, designated as Kit Control 4. The main difference between PAMAM G8 and PAMAM G4 is that PAMAM G8 has a higher molecular generation, a larger molecular size, and a more compact three-dimensional spherical structure, with about 16 times the number of surface functional groups (-NH2) of PAMAM G4.

[0294] The preparation process of Kit Control 4 of the present comparative example is as follows:

[0295] S1, Preparation of gold label pad:

[0296] S11, Synthesis and preliminary purification of colloidal gold particles: same as step S11 of Example One.

[0297] S12, modification of the stability of the surface of the colloidal gold particles: same as step S12 of Example 1.

[0298] S13, preparation of the antibody labeled with the boronic acid functionalized polymer:

[0299] S131, antibody labeling: same as S131 of Example 1, to obtain a colloidal gold-antibody complex.

[0300] S132, preparation of the boronic acid functionalized polyamidoamine dendrimer:

[0301] Take 10 mg of PAMAM G8 and dissolve in 1 mL of 10 mM borate buffer at pH 8.5;

[0302] Add 16 mg of 3-aminophenylboronic acid and 18 mg of EDC / NHS coupling agent, and react at 24°C for 3 h. The amount of reactants is increased in this step to match the significantly increased surface functional groups of the 8th generation.

[0303] Purify by dialysis to obtain the boronic acid functionalized polyamidoamine dendrimer, and dilute to 1 mL with the borate buffer to obtain a boronic acid functionalized polyamidoamine dendrimer solution.

[0304] S14, target stabilization treatment and post-treatment:

[0305] S141, target stabilization reaction: same as step S141 of Example 1.

[0306] S142, blocking and stabilization: same as step S142 of Example 1.

[0307] S143, purification and concentration: same as step S143 of Example 1.

[0308] S15, spraying and drying of the gold label pad: same as step S15 of Example 1, to obtain the finished gold label pad.

[0309] S2, preparation of the sample pad: same as step S2 of Example 1.

[0310] S3, coating of the reaction membrane: same as step S3 of Example 1.

[0311] S4, assembly of the reagent strip: same as step S4 of Example 1, to obtain a Chlamydia pneumoniae IgM antibody test kit, which is designated as kit control 4.

[0312] Analysis and testing

[0313] In the present application, "-" represents negative; "- / +" represents T line if any; "+" represents weakly positive; "++" represents positive; and "+++" represents strong positive.

[0314] I. Sensitivity test

[0315] To evaluate the core detection ability of the kit of the present application, the detection limit is used to evaluate the sensitivity, and the precision is used to evaluate the reliability.

[0316] 1. Detection limit determination:

[0317] By comparing the detection end points of the same dilution series of samples under different methods, the relative sensitivity of the kit of the present application relative to the ELISA method is evaluated. Specifically, a clinically diagnosed positive mixed serum of Chlamydia pneumoniae IgM antibody is taken as a starting sample; the negative human serum is used to dilute the sample by a series of ratios (1:2, 1:4, 1:8, 1:16, 1:32); the commercially available Chlamydia pneumoniae IgM antibody enzyme-linked immunosorbent assay (ELISA) kit is used to detect all dilution samples, and the OD 450 values and the positive / negative judgment of the color of the well plate of each dilution gradient sample are recorded, and the results are shown in Table 1.

[0318] The kit samples 1-3 and the kit control samples 1-4 are used to test the samples with gradient concentrations, and the results are recorded in Table 1.

[0319] Table 1: Detection limit determination results

[0320]

[0321] As can be seen from Table 1, the kit samples 1-3 prepared in the embodiments of the present application all exhibit significantly better detection sensitivity than the comparative examples 1, 2 and 4.

[0322] Specifically, the kit control sample 1 has a greatly reduced sensitivity due to the missing of the target stable layer, which damages the activity of the antibody; the kit control sample 2 has the worst performance because its basic stability is replaced by polyvinylpyrrolidone, which cannot effectively perform subsequent functionalization; the kit control sample 3 has an initial detection sensitivity comparable to the embodiments of the present application because its gold label pad is prepared completely, and only the sample pad lacks a lipid dispersing agent; however, its anti-lipid interference ability (see Table 3) and stability (see Table 4) are significantly reduced due to the defect; the kit control sample 4 has a greatly reduced sensitivity and the highest detection limit due to the steric hindance effect of G8 generation PAMAM, which seriously hinders the immune reaction.

[0323] 2. Precision determination:

[0324] The kit samples 1-3 prepared by the same method and different batches are used to repeatedly test the weak positive sample (1:4 dilution) and the negative sample (1:16 dilution) near the critical value for 20 times each, and the positive coincidence rate and the negative coincidence rate are calculated, and the results are shown in Table 2.

[0325] Table 2: Precision determination results

[0326]

[0327] From the results of Table 2, all the embodiments of the present application show more than 95% positive coincidence rate and 100% negative coincidence rate in terms of batch precision, which shows that the kit of the present application has high stability and strong reliability.

[0328] II. Analysis of specificity and anti-interference ability test

[0329] In order to evaluate the reliability of the kit in the complex real sample environment, the specificity of the kit to common endogenous interferents and cross-reactive antigens was investigated.

[0330] Specificity evaluation: rheumatoid factor (rheumatoid factor concentration of 480 IU / mL), anti-nuclear antibody (1:320 titer), high bilirubin (bilirubin concentration of 80 mmol / L and 160 mmol / L), hemolysis (hemoglobin concentration of 10 g / L and 20 g / L), and high blood lipids (blood lipid concentration of 6 mM and 9 mM) were detected in negative serum samples, respectively, and each serum sample was tested 10 times to investigate the false positive results, and the results are shown in Table 3.

[0331] Table 3: Specificity evaluation results

[0332]

[0333] The results of Table 3 show that the kits prepared in the embodiments of the present application (samples 1-3) do not have false positive results for all potential interfering substances tested, and in samples with bilirubin as high as 160 mmol / L, hemolysis as high as 20 g / L, and high blood lipids as high as 9 mM, the results are still negative, showing excellent analytical specificity, which shows that the technical scheme of the present application is effective in resisting complex sample matrix interference.

[0334] In comparison, the detection results of each comparative example have different degrees of specificity problems due to their own process defects. First, the double stabilization strategy of the gold label pad is the key design to resist non-specific binding. The kit control 1 lacks the targeting stabilization step of the boron acid functional polymer, and the labeled antibody is exposed to a complex environment, resulting in insufficient structural stability. The experimental results show that there is a clear false positive in the high blood lipid sample, which shows that lipid molecules can induce conformational changes or non-specific aggregation of unstabilized antibodies, and the targeting stabilization treatment of the present application can effectively lock the active conformation of the antibody, avoiding such risks.

[0335] Secondly, the cysteamine of the present application has irreplaceable function as a connecting molecule. The kit control 2 uses polyvinylpyrrolidone, a traditional steric stabilizer, to replace cysteamine. Although it can maintain the colloidal stability of colloidal gold, it cannot provide the active amine group necessary for subsequent targeting and stabilization, resulting in false positives in hemolyzed samples. It can be seen that the steric protection provided by polyvinylpyrrolidone is easily destroyed in the face of substances such as hemoglobin and cell fragments, while cysteamine forms Au-S bonds and mediates subsequent polymer stabilization, building a more effective defense system.

[0336] Furthermore, the complete composition of the composite functional microspheres in the sample pad of the present application is the key to achieving synergistic interference removal. The microspheres of kit control 3 only load bilirubin oxidase and lack the lipid dispersant Pluronic F-127, resulting in false positives in high lipid samples. This proves that Pluronic F-127 plays an independent and crucial role in dispersing lipids, preventing them from blocking membrane pores and nonspecific adsorption, and that it complements the function of bilirubin oxidase, both of which are indispensable.

[0337] It is worth noting that although kit control 4 is negative in all specific tests, this is not because of its superior performance, but rather because of the strong steric hindrance caused by its large molecular size and dense structure. This hindrance not only blocks the nonspecific contact of interferents, but also severely hinders the effective binding of antibodies to target antigens, resulting in a significant decrease in the sensitivity of the kit. This result confirms that the PAMAM G4 used in the present application can provide an optimal balance between effective stabilization and maintenance of high reaction activity.

[0338] III. Accelerated stability test

[0339] To evaluate the shelf stability of the kit, an accelerated stability test was performed.

[0340] Test method: Place the kits to be tested in a 37℃ constant temperature incubator for 7 days, 14 days, 21 days and 28 days respectively. At each time point, take out the samples and compare the color intensity of the test line (T line) before and after acceleration by detecting weak positive samples near the critical value (determine the dilution ratio corresponding to each kit sample or control according to "I. Sensitivity test"). If the color of the T line is still clear and visible after acceleration (i.e. judged as "+"), it is considered that the sensitivity is maintained. The test results are shown in Table 4.

[0341] Table 4: Sensitivity determination results during acceleration

[0342]

[0343] Note: "N / A" in Table 4 means that the sample is completely disabled.

[0344] The results in Table 4 show that the kit samples 1-3 prepared by the embodiments of the present application all exhibit excellent accelerated stability. At 37°C, the detection lines of the kit sample 1 and sample 2 for the weak positive sample still present clear positive (“+”) at the 28th day, showing excellent long-term stability; although the detection line of the kit sample 3 is weakened to “- / +” at the 28th day, it remains stable positive (“+”) in the test before the 21st day, and its stability is still significantly better than all the comparative examples.

[0345] In contrast, the stability defects of each comparative example are clearly reflected from their performance decay trajectories: the detection line of the kit control 1 has decayed to “- / +” at the 14th day and completely turned to negative at the 21st day, indicating that its antibody activity decays rapidly due to the absence of the targeted stabilization layer; the kit control 2 has completely failed at the 14th day, proving that its labeling structure is the most fragile; the detection line of the kit control 3 has decayed to “- / +” at the 14th day, indicating that the absence of the lipid dispersing agent does not affect its initial labeling efficiency, but significantly reduces its long-term stability; the kit control 4 remains positive at the 7th day, but its detection line continuously presents a weak positive state of “- / +” during the entire aging process, and finally fails at the 28th day, which is consistent with the steric hindrance effect it exhibits in the sensitivity test, further proving that it has a certain stability, but sacrifices the efficiency of the detection reaction.

[0346] IV. Opened stability (humidity influence) test

[0347] To evaluate the sensitivity of the kit to humidity in the actual use environment, the stability after opening is simulated.

[0348] Test method: After opening the aluminum foil bag of the kit to be tested, place the kit in a constant temperature and humidity chamber with relative humidity of 25%, 45% and 65% (temperature is 25°C). Take out the sample at 0, 2, 4, 6, 8 hours after opening, and test it using the weak positive sample corresponding to the detection limit. Taking the inability of the detection line (T line) to produce a clear color visible to the naked eye (judged as “-”) as the criterion for the failure of the kit. Record the time for each sample to maintain effective performance under different humidity conditions, and the test results are shown in Table 5.

[0349] Table 5: Opened stability test results

[0350]

[0351] As can be seen from the results of Table 5, the kit prepared in the embodiment of the present application exhibits good opening stability. In a conventional humidity environment (25% and 45%), the performance of kit sample 1 and kit sample 2 does not attenuate within 8h; in a high humidity environment of 65%, the effective detection window can also be maintained for more than 4h. This proves that the reagent core components have strong moisture resistance through the optimized components, stable structure and thorough drying process. The excellent moisture resistance of the kit of the present application is mainly due to its unique structural design: the cysteamine in the gold label pad and the constructed PAMAM G4 targeting stable system provide hydrophobic spatial protection for the antibody marker, effectively blocking the invasion of water molecules; at the same time, the Pluronic F-127 in the sample pad cooperatively fixes the composite functional microspheres for hydrophobic modification. Both of them cooperatively construct a stable moisture-resistant barrier at the molecular level.

[0352] In contrast, each of the comparative examples again shows its technical defects. The kit control 1 lacks a targeting stable layer, and the antibody is easily deactivated by moisture, so that the performance retention time is sharply shortened to 3h under a humidity of 45%. The basic stable layer of the kit control 2 is replaced by polyvinylpyrrolidone, and its structure is fragile, so that the performance attenuates rapidly in a high humidity environment. The kit control 3 lacks a lipid dispersing agent in the sample pad, causing the chromatographic system to be more sensitive to humidity, and the performance retention time is shorter than the embodiment under high humidity. The kit control 4 may be affected by the stronger hydrophilicity of the G8 generation polymer, which is easily absorbed by moisture, thereby affecting its stability in a high humidity environment.

[0353] The above-described embodiments are only preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A highly stable Chlamydia pneumoniae IgM antibody detection kit, characterized in that, The kit includes a sample pad fixed on a PVC plate, a gold-labeled pad, a reaction membrane coated with detection lines and control lines, and an absorbent pad. The gold-labeled pad carries a colloidal gold-labeled mouse anti-human IgM monoclonal antibody. This colloidal gold-labeled mouse anti-human IgM monoclonal antibody is obtained by modifying the surface of colloidal gold particles with cysteine, labeling the antibody, and then performing targeted stabilization treatment with boric acid-functionalized polyamide-amine dendritic macromolecules. The sample pad is immobilized with composite functional microspheres, which are polystyrene microspheres immobilized with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer. The boric acid-functionalized polyamide-amine dendritic macromolecules are obtained by coupling 3-aminophenylboronic acid with polyamide-amine dendritic macromolecules under the action of a coupling agent.

2. The reagent kit according to claim 1, characterized in that, In the colloidal gold-labeled mouse anti-human IgM monoclonal antibody, the cysteine ​​modification concentration is 0.9 mM to 1.1 mM.

3. The reagent kit according to claim 2, characterized in that, The coupling agent consists of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 1:

1.

4. The reagent kit according to claim 1, characterized in that, In the composite functional microspheres, the polystyrene microspheres are amino-modified polystyrene microspheres; the theoretical loading capacity of bilirubin oxidase on each milligram of polystyrene microsphere is 15 μg to 25 μg of enzyme protein.

5. A method for preparing a highly stable Chlamydia pneumoniae IgM antibody detection kit, used to prepare the kit as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of the gold-labeled pad: Colloidal gold was synthesized and concentrated to obtain a concentrated colloidal gold solution. The surface of the colloidal gold was modified with cysteine ​​and labeled with mouse anti-human IgM monoclonal antibody to obtain a colloidal gold-antibody complex. The complex was then targeted and stabilized using boric acid-functionalized polyamide-amine dendritic macromolecules. After blocking, purification, and concentration, a highly stable colloidal gold-labeled antibody concentrate was obtained. After dilution, the concentrate was sprayed onto a gold-labeled pad substrate and dried to obtain the finished gold-labeled pad. S2. Preparation of sample pad: Amino-modified polystyrene microspheres are co-immobilized with bilirubin oxidase and polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer to obtain composite functional microspheres. After stabilization with a protective agent, a stable composite functional microsphere suspension is obtained. The suspension is diluted and sprayed onto the sample pad substrate. After drying, the finished sample pad is obtained. S3. Coating of the reaction membrane: The recombinant Chlamydia pneumoniae antigen solution and the goat anti-mouse IgG antibody solution were sprayed onto the nitrocellulose membrane at a spray volume of 0.08 μL / mm to 0.12 μL / mm to form the detection line and the control line, respectively. After drying, the finished reaction membrane was obtained. S4. Assembly of reagent strips: The finished sample pad, finished gold label pad, finished reaction membrane and absorbent pad are sequentially pasted onto the PVC base plate. After cutting, shelling and packaging, the reagent kit is obtained.

6. The preparation method according to claim 5, characterized in that, The colloidal gold synthesized in step S1 is prepared by reducing tetrachloroauric acid with trisodium citrate. The specific process is as follows: 800 μL to 1200 μL of 1% tetrachloroauric acid solution is added to 100 mL of ultrapure water and heated to boiling. Then, 1.6 mL to 2.0 mL of 1% trisodium citrate solution is added while stirring at 300 r / min to 500 r / min. The mixture is kept boiling for 5 min to 10 min, then heating is stopped. After cooling, membrane filtration, centrifugation, and concentration, a concentrated colloidal gold solution is obtained.

7. The preparation method according to claim 5, characterized in that, The reaction conditions for cysteamine modification in step S1 are as follows: cysteamine is prepared into a stock solution and added to a concentrated colloidal gold solution. The concentration of cysteamine modification is controlled at 0.9 mM to 1.1 mM, and the reaction is carried out at room temperature in the dark for 4 to 6 hours.

8. The preparation method according to claim 5, characterized in that, In step S1, the boric acid-functionalized polyamide-amine dendritic macromolecules are used for targeted stabilization. This involves mixing the boric acid-functionalized polyamide-amine dendritic macromolecule solution with the colloidal gold-antibody complex and incubating it in the dark at 50 r / min to 100 r / min for 1 h to 2 h. The preparation process of the boric acid-functionalized polyamide-amine dendritic macromolecule solution is as follows: 10 mg of polyamide-amine dendritic macromolecules are dissolved in borate buffer, 5 mg to 10 mg of 3-aminophenylboronic acid and 6 mg to 12 mg of coupling agent are added, and after reacting for 2 h to 4 h, the solution is purified by dialysis and then brought to a final volume of 1 mL with borate buffer to obtain the boric acid-functionalized polyamide-amine dendritic macromolecule solution.

9. The preparation method according to claim 5, characterized in that, The S2 step specifically involves: weighing 100 mg to 200 mg of polystyrene microspheres, washing them with phosphate buffer, and resuspending them to prepare a 20 mg / mL microsphere stock suspension; taking 10 mL of the microsphere stock suspension, adding 300 μL to 500 μL of 1 mg / mL bilirubin oxidase solution and 100 μL to 300 μL of 10% (w / w) polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer aqueous solution; adjusting the pH of the system to 8.0 to 8.5 with alkali solution, and incubating under light-protected stirring at 30°C to 35°C and 150 r / min to 250 r / min for 1.5 h to 2 h to obtain a functionalized microsphere suspension; adding a protective agent to the functionalized microsphere suspension, and mixing at 4°C and 100 r / min to 200 r / min for 40 min to 60 min to obtain a stable composite functionalized microsphere suspension.

10. The preparation method according to claim 9, characterized in that, The protective agent comprises a trehalose solution with a mass concentration of 20% in a volume ratio of 3:2:1, a bovine serum albumin solution with a mass concentration of 10% and a sorbitol solution with a mass concentration of 20%.

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