An apolipoprotein e4 detection kit and application thereof

Abstract

The application relates to the technical field of in-vitro diagnostic kits, and discloses an apolipoprotein E4 detection kit and application thereof. An immunochromatographic test paper comprises a detection immunochromatographic test paper, the detection immunochromatographic test paper comprises a bottom plate, a sample pad, a binding pad, an NC membrane and a water absorption pad, one side of the NC membrane is sequentially provided with the binding pad and the sample pad, and the other side of the NC membrane is provided with the water absorption pad; gold-manganese oxide nano color developing particles and apolipoprotein APOE4 monoclonal antibodies are marked on the binding pad. Through the technical scheme, the problems of complex operation, high cost, low detection sensitivity and weak specificity of apolipoprotein E4 detection in the related art are solved.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent kit technology, specifically to a detection kit for apolipoprotein E4 and its application. Background Technology

[0002] Alzheimer's disease (AD) is a common, multifactorial, primary degenerative neurodegenerative disease, and the most common type. Data published on the World Health Organization (WHO) website shows that AD may cause 60%–70% of Alzheimer's cases. The primary symptom of AD is irreversible progressive decline in intelligence, memory, sensory orientation, judgment, and language and thinking abilities, often accompanied by personality changes, severely impacting the quality of life of the elderly. Clinically, AD is often divided into early-onset Alzheimer's disease (EOAD) and late-onset Alzheimer's disease (LOAD) with age 65 as the dividing line. LOAD accounts for more than 95% of AD cases and is considered to be a complex result of the interaction between genes and the environment. Besides advanced age, risk factors for LOAD include a history of cardiovascular disease, low education level, depression, and the apolipoprotein E4 gene. Apolipoprotein E4 (APOE4) is considered the strongest genetic risk factor for AD.

[0003] Apolipoprotein E (APOE) is an important component of plasma lipoproteins, participating in the transport and metabolism of plasma cholesterol and triglycerides. It plays a key role in lipid metabolism and maintaining cholesterol balance, and is a determinant of lipid metabolism and cardiovascular disease. APOE also participates in activating enzymes that hydrolyze fat, and has functions such as immune regulation, participation in the regeneration of nerve tissue, and inhibition of platelet aggregation.

[0004] The APOE gene is located on the long arm of human chromosome 19 and is an autosomal dominant gene with significant genetic polymorphism. It has three common alleles: E2 (Cys112 / Cys158), E3 (Cys112 / Arg158), and E4 (Arg112 / Arg158), representing three isoforms of the APOE protein: E2, E3, and E4. These alleles differ only at amino acid positions 112 and 158, but play distinctly different immunomodulatory roles. Studies have shown that APOE gene polymorphism is closely related to the development and progression of Alzheimer's disease. The incidence of Alzheimer's disease (AD) is significantly higher in APOE ε4 carriers than in those with other alleles, and the ε4 / ε4 genotype is a high-risk factor for AD. The ε2 allele is not associated with AD development. Studies by Strittmatter et al. show that carrying one ε4 allele increases the risk of late-onset AD by 2-3 times, while carrying two ε4 alleles increases the risk by more than 8 times. Sando et al. reported that the prevalence of AD in APOE ε4 carriers is significantly higher than in the ε3 / ε3 genotype. The risks of AD in the ε4 / ε4, ε2 / ε4, and ε3 / ε4 genotypes are 12.9, 3.2, and 4.2 times higher than in the ε3 / ε3 genotype, respectively. Furthermore, the age of AD onset is 3.1 years earlier in carriers of one ε4 allele compared to those with the ε3 / ε3 genotype, and 5.5 years earlier in carriers of two ε4 alleles.

[0005] Apolipoprotein E (APOE) genotyping is performed on APOE, a very important protein in the human body. It is composed of polypeptides encoded by three alleles (E2, E3, and E4) at one gene locus, resulting in six genotypes: three homozygous types (APOE2 / 2, E3 / 3, and E4 / 4) and three heterozygous types (APOE2 / 3, E2 / 4, and E3 / 4), which are expressed in three protein forms: E2, E3, and E4.

[0006] APOE3: Wild type; accounting for approximately 78% of the natural population, it plays a crucial role in the normal physiological functions of the body.

[0007] APOE2: Variant; It belongs to the longevity gene and has a protective effect against Alzheimer's disease, but it is associated with hyperglyceridemia.

[0008] APOE4: Variant; associated with a high incidence of Alzheimer's disease and cardiovascular disease, and has a negative impact on the recovery of function from various brain injuries.

[0009] The longevity gene E2 corresponds to the genotypes ε2 / ε2 and ε2 / ε3, accounting for 7% of humans. Individuals with this genotype are less likely to suffer from diseases such as Alzheimer's disease, coronary heart disease, and cerebral infarction, but are more prone to macular degeneration. Their blood lipid profile is characterized by high triglycerides, low LDH-C, and uncertain HDL.

[0010] The wild-type gene E3 corresponds to the genotypes ε3 / ε3 and ε2 / ε4, accounting for 78% of human cases. These genotypes are common genes.

[0011] The genotype E4, which corresponds to the ε3 / ε4 genotype, accounts for 15% of all human cases. Individuals with this genotype are more susceptible to diseases such as Alzheimer's disease, coronary heart disease, cerebral infarction, and retinitis pigmentosa. Their blood lipid profile is characterized by normal triglycerides, high LDH-C, and low HDL.

[0012] APOE genotypes have lifelong effects on a person's health and lifestyle. They guide an individual's eating habits, help maintain good health, prevent a range of major diseases, and, once a disease develops, determine unique, individualized treatment.

[0013] Currently, the main methods for detecting APOE gene polymorphism on the market include PCR-chip hybridization, fluorescence PCR, and PCR-melting curve methods. Laboratory methods primarily include direct sequencing, high-performance liquid chromatography (DHPLC), restriction fragment length polymorphism detection (PCR-RFLP), time-of-flight mass spectrometry, and various PCR methods. While these methods offer good specificity and high sensitivity, their complexity, time-consuming nature, and high cost make them unsuitable for initial screening. Clinically, the main method for testing apolipoprotein E is immunoturbidimetry, which targets APOE and cannot distinguish APOE4, thus failing to meet the requirements for APOE4 initial screening. Therefore, designing and developing a simple, low-cost, highly sensitive, and highly specific apolipoprotein E4 (APOE4) detection kit would have excellent usability. Summary of the Invention

[0014] This invention proposes a detection kit for apolipoprotein E4 and its application, which solves the problems of complex operation, high cost, low detection sensitivity and weak specificity in related technologies for apolipoprotein E4 detection.

[0015] The technical solution of the present invention is as follows: The present invention proposes an immunochromatographic test strip, comprising a detection immunochromatographic test strip, wherein the detection immunochromatographic test strip comprises a base plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. The conjugate pad and the sample pad are sequentially disposed on one side of the NC membrane, and the absorbent pad is disposed on the other side of the NC membrane. The conjugate pad is labeled with gold manganese oxide nanocolor particles and apolipoprotein APOE4 monoclonal antibody.

[0016] As a further technical solution, the base plate is made of PVC.

[0017] As a further technical solution, the particle size of the gold manganese oxide nanocolorizing particles can be any size, preferably 200~700nm, and more preferably 408.7nm.

[0018] As a further technical solution, the preparation method of the gold manganese oxide nanocolor particles includes the following steps: manganese chloride and chloroauric acid undergo a reduction reaction in an alkaline solution to obtain gold manganese oxide nanocolor particles.

[0019] As a further technical solution, the manganese chloride is added in the form of a manganese chloride solution for the reduction reaction, and the concentration of the manganese chloride solution is 0.1 mol / L; the alkaline solution is a sodium hydroxide solution with a concentration of 0.1 mol / L; the chloroauric acid is added in the form of a tetrachloroauric acid trihydrate solution for the reduction reaction, and the concentration of the tetrachloroauric acid trihydrate solution is 0.1 wt%.

[0020] As a further technical solution, water is added during the preparation of gold manganese oxide nanocolor particles, and the volume ratio of the manganese chloride solution, the chloroauric acid solution, the water and the sodium hydroxide solution is 0.05~0.125:0.5:0.34:20.

[0021] As a further technical solution, the preparation method of the gold manganese oxide nanocolor particles includes the following steps: mixing the manganese chloride solution, the chloroauric acid solution and the water, stirring and then adding sodium hydroxide solution, stirring again and then centrifuging to obtain the gold manganese oxide nanocolor particles.

[0022] As a further technical solution, the stirring time is 2-4 minutes, preferably 3 minutes, the re-stirring time is 10-15 minutes, preferably 10 minutes, the centrifugation rate is 8000-10000 rpm, preferably 10000 rpm, and the time is 5-8 minutes, preferably 5 minutes.

[0023] As a further technical solution, the conjugate pad is a conjugate pad treated with a conjugate pad treatment solution, which includes gold manganese oxide nanocoloring particles, blocking solution, and apolipoprotein APOE4 monoclonal antibody.

[0024] As a further technical solution, the sealant is a composite sealant, which is composed of the following components in weight percentage: 5% casein, 0.5%~1.5% polyacrylic acid, 0.1%~0.5% polyvinylpyrrolidone, and the balance being water.

[0025] In this invention, the composite blocking solution used during labeling can increase the stability of macromolecular chromogenic nanoparticles, reduce the sedimentation of macromolecular particles, and ensure that mouse anti-human apolipoprotein E4 monoclonal antibody is stably dispersed in the solution, thereby increasing sensitivity and stability.

[0026] As a further technical solution, the method for preparing the conjugate pad includes the following steps:

[0027] S1. Disperse gold manganese oxide nanocoloring particles in water, adjust the pH to 7.0~9.0, add apolipoprotein APOE4 monoclonal antibody and react, add blocking solution for blocking, centrifuge to remove supernatant, collect precipitate, resuspend the precipitate with resuspension to obtain conjugation pad treatment solution;

[0028] S2. Coat the bonding pad treatment solution evenly onto the glass fiber paper, 10cm. 3 240±50uL of conjugate pad treatment solution was wrapped in glass fiber paper and dried to obtain the conjugate pad.

[0029] As a further technical solution, in step S1, the solution used to adjust the pH is a potassium carbonate aqueous solution with a concentration of 0.2 mol / L.

[0030] As a further technical solution, in step S1, the reaction time is 1 hour.

[0031] As a further technical solution, in step S1, the concentration of the apolipoprotein APOE4 monoclonal antibody is 1.0 mg / mL.

[0032] As a further technical solution, in step S1, the sealing time is 1 hour and the temperature is room temperature.

[0033] As a further technical solution, in step S1, the resuspension is composed of the following raw materials: Tris buffer, sucrose, bovine serum albumin, casein, PVP-30, PEG8000, Tween, and the remainder is water; the final concentration of the Tris buffer in the resuspension is 0.1 mol / L, the pH is 8, the final mass concentration of the sucrose is 5%, the final mass concentration of the bovine serum albumin is 2.5%, the final mass concentration of the casein is 0.1%, the final volume concentration of the PVP-30 is 0.025%, the final mass concentration of the PEG8000 is 0.025%, and the final mass concentration of the Tween is 0.7%.

[0034] As a further technical solution, the 10cm 3 240uL of bonding pad treatment solution was wrapped in glass fiber paper.

[0035] As a further technical solution, the preparation method of the sample pad includes the following steps: cutting non-woven fabric into 300mm×280mm pieces, immersing it in the sample pad treatment solution for 3~5 minutes, taking it out and placing it horizontally for drying, drying at 37℃ for 2~3 hours, and after drying, the sample pad is obtained.

[0036] As a further technical solution, the sample pad treatment solution is composed of the following raw materials: Tris buffer, casein, sucrose, Tween, NaCl, anti-erythrocyte antibody, and the balance is water; the final concentration of Tris buffer in the sample pad treatment solution is 0.1 mol / L, the pH is 9, the final mass concentration of casein is 0.5%, the final mass concentration of sucrose is 0.5%, the final mass concentration of Tween is 0.7%, the final mass concentration of NaCl is 0.9%, and the final concentration of anti-erythrocyte antibody is 0.25 mg / mL.

[0037] As a further technical solution, the NC membrane is coated with goat anti-human apolipoprotein E polyclonal antibody and goat anti-mouse IgG antibody.

[0038] As a further technical solution, the preparation method of the detection immunochromatographic test strip includes the following steps: the sample pad, the conjugation pad, the NC membrane and the absorbent pad are sequentially placed on the base plate, and after cutting, the detection immunochromatographic test strip is obtained.

[0039] In this invention, gold manganese oxide nanoparticles are used as the colorimetric system. Labeling APOE4 monoclonal antibody can significantly improve detection sensitivity (0.5 μg / mL), accelerate antigen-antibody reaction, improve detection efficiency, and produce results rapidly in 5-10 minutes.

[0040] The present invention also proposes a detection kit for apolipoprotein E4, comprising the aforementioned detection immunochromatographic strip.

[0041] The present invention also proposes the application of the immunochromatographic test strip or the apolipoprotein E4 detection kit in the detection of apolipoprotein E4.

[0042] In this invention, immunochromatography is employed, utilizing the double-antibody sandwich principle, to qualitatively detect apolipoprotein E4 in human serum, plasma, and whole blood. A mouse anti-human apolipoprotein E4 monoclonal antibody is pre-coated onto glass fibers. Goat anti-human apolipoprotein E polyclonal antibody and goat anti-mouse IgG polyclonal antibody are coated onto the test line and control line of a nitrocellulose membrane, respectively. When the sample contains an appropriate concentration of apolipoprotein E4, it forms an antigen-antibody complex with the labeled APOE4 monoclonal antibody on the glass fibers. Due to chromatographic action, this complex moves forward along the paper strip until it reaches the test line, where it reacts with the pre-coated APOE polyclonal antibody, forming a double-antibody sandwich complex that aggregates and develops color, indicating a positive result. The free gold-labeled APOE4 monoclonal antibody continues to move forward, reacting with the pre-coated goat anti-mouse IgG polyclonal antibody at the control line, forming a complex that aggregates and develops color. Negative samples only show color at the control line.

[0043] The working principle and beneficial effects of this invention are as follows:

[0044] 1. This invention provides a detection kit for detecting the allele of apolipoprotein E4 in human serum, plasma, and whole blood. The kit uses gold manganese oxide nanoparticles as the colorimetric system, which can promote highly sensitive immunochromatographic analysis. The gold manganese oxide nanoparticles have excellent colorimetric signal brightness, enhanced antibody conjugation efficiency, and unrestricted immunogenic affinity.

[0045] 2. The immunochromatographic detection of APOE4 provided by this invention can help the general public identify their own risk of AD, and also help clinicians to quickly screen and analyze patients' APOE genotypes. It can provide early reference for the causes of related diseases such as Alzheimer's disease, hyperlipidemia, and coronary heart disease.

[0046] 3. The immunochromatographic method for ApoE4 detection provided by this invention is convenient and low-cost, suitable for early large-scale ApoE4 screening, allowing for early assessment of disease risk and enabling early prevention. Combined with subsequent PCR genotyping, it not only facilitates early screening, diagnosis, and treatment of Alzheimer's disease (AD), but also helps conserve social medical resources. Attached Figure Description

[0047] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0048] Figure 1 This is a particle size diagram of the gold manganese oxide nanocoloring particles in Example 1 of the present invention;

[0049] Figure 2 This is a particle size diagram of the gold manganese oxide nanocoloring particles in Example 5 of the present invention;

[0050] Figure 3This is a particle size diagram of the gold manganese oxide nanocoloring particles in Example 6 of the present invention. Detailed Implementation

[0051] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0052] In the following examples and comparative examples:

[0053] Polyacrylic acid: weight average molecular weight 2000;

[0054] Polyvinylpyrrolidone: weight average molecular weight 40,000;

[0055] Mouse anti-human APOE4 monoclonal antibody: purchased from Hainan Shengyang Biotechnology Co., Ltd.;

[0056] Goat anti-human apolipoprotein E polyclonal antibody: purchased from Shanghai Bolesi Biotechnology Co., Ltd.;

[0057] Goat anti-mouse IgG antibody: purchased from Luoyang Baitong Experimental Materials Center;

[0058] Casein: 99 wt% of active ingredients;

[0059] The resuspension consisted of the following ingredients: Tris buffer, sucrose, bovine serum albumin, casein, PVP-30, PEG8000, Tween, and the remainder being water. The final concentration of Tris buffer in the resuspension was 0.1 mol / L, the pH was 8, the final mass concentration of sucrose was 5%, the final mass concentration of bovine serum albumin was 2.5%, the final mass concentration of casein was 0.1%, the final volume concentration of PVP-30 was 0.025%, the final mass concentration of PEG8000 was 0.025%, and the final mass concentration of Tween was 0.7%.

[0060] The coating buffer consisted of phosphate buffer, bovine serum albumin, and Tween. The final concentration of the phosphate buffer was 0.1 mol / L, the final mass concentration of the bovine serum albumin was 0.1%, and the final volume concentration of the Tween was 1%.

[0061] The sample pad treatment solution consists of the following ingredients: Tris buffer, casein, sucrose, Tween, NaCl, anti-erythrocyte antibody, and the balance being water. The final concentration of Tris buffer in the sample pad treatment solution is 0.1 mol / L, pH is 9, the final mass concentration of casein is 0.5%, the final mass concentration of sucrose is 0.5%, the final mass concentration of Tween is 0.7%, the final mass concentration of NaCl is 0.9%, and the final concentration of anti-erythrocyte antibody is 0.25 mg / mL.

[0062] Example 1

[0063] S1. Preparation of gold manganese oxide nanoparticles for color development:

[0064] The gold manganese oxide nanoparticles were prepared using a one-step reduction method. 125 μL of 0.1 M MnCl2 and 340 μL of 1 wt% HAuCl4•3H2O were added to 20 mL of purified water and stirred at 500 rpm for three minutes. Then, 500 μL of 0.1 M NaOH solution was added to create an alkaline environment, and the mixture was stirred for 10 minutes. The mixture was then centrifuged (10000 rpm, 5 minutes) to separate the silver ear-shaped gold manganese oxide (Au-MnOX). The gold manganese oxide nanoparticles were then dispersed in purified water.

[0065] S2, Labeled Antibody:

[0066] Take 1 mL of the gold manganese oxide nanoparticles prepared in step S1, add 0.2 mol / L K2CO3 aqueous solution, and adjust the pH of the system to 7.0; then add 10 μg of 1.0 mg / mL mouse anti-human apolipoprotein E4 monoclonal antibody, and mix and react at room temperature for 1 h.

[0067] S3, Closed:

[0068] Blocking solution was added to the mouse anti-human apolipoprotein E4 monoclonal antibody labeled with gold manganese oxide nanoparticles in step S2. The blocking agent was a composite blocking agent, which consisted of the following components by weight percentage: casein 5%, polyacrylic acid 1%, polyvinylpyrrolidone 0.2%, and the balance being water. After adding the composite blocking solution, the mixture was stirred at room temperature and reacted at room temperature for 1 hour to obtain a labeled gold solution with a volume concentration of 1.0%.

[0069] S4, Heavy Suspension:

[0070] The blocked gold manganese oxide nanoparticles labeled with antibodies were centrifuged at 3500 r / min for 10 min, the supernatant was discarded, and the precipitate was resuspended in resuspension.

[0071] S5. Prepare the sample pad:

[0072] Cut the non-woven fabric into 300mm×280mm pieces, soak them in the sample pad treatment solution for 3 minutes, take them out and place them horizontally to dry at 37℃ for 2 hours. After drying, the sample pad is obtained and placed in a self-sealing bag for later use.

[0073] S6. Preparation of the binding pad:

[0074] The mouse anti-human apolipoprotein E4 monoclonal antibody labeled with gold manganese oxide nanoparticles in step S2 is evenly spread on an 8975 glass fiber membrane, dried, and the gold manganese oxide nanoparticles labeled with the antibody are solidified on the glass fiber membrane to obtain the binding pad. It is then removed, placed in a sealed bag, desiccant is added, and it is sealed and stored.

[0075] S7. Preparation of the reaction pad:

[0076] Nitrocellulose membranes were attached to PVC plates, and then goat anti-human apolipoprotein E4 polyclonal antibody was diluted to a concentration of 1 mg / mL with coating buffer and used as the detection line coating. Goat anti-mouse IgG was diluted to 2 mg / mL and used as the control line C coating. The membrane was then drawn with a gold-spraying membrane scribing instrument at a concentration of 1 μL / cm and dried at 40℃ for 6 hours to obtain the reaction pad. The pad was then removed and placed in a sealed bag for later use.

[0077] S8. Reagent kit assembly:

[0078] The sample pad, conjugate pad, NC membrane, and absorbent pad are sequentially overlapped and pasted onto a PVC base plate. After cutting, the detection immunochromatographic test strip for apolipoprotein E4 is obtained.

[0079] Figure 1 This is a particle size diagram of the gold manganese oxide nanocoloring particles in Example 1 of the present invention.

[0080] Example 2

[0081] S1. Preparation of gold manganese oxide nanoparticles for color development:

[0082] The gold manganese oxide nanoparticles were prepared using a one-step reduction method. 125 μL of 0.1 M MnCl2 and 340 μL of 1 wt% HAuCl4•3H2O were added to 20 mL of purified water and stirred at 500 rpm for three minutes. Then, 500 μL of 0.1 M NaOH solution was added to create an alkaline environment, and the mixture was stirred for 10 minutes. The mixture was then centrifuged (10000 rpm, 5 minutes) to separate the silver ear-shaped gold manganese oxide (Au-MnOX). The gold manganese oxide nanoparticles were then dispersed in purified water.

[0083] S2, Labeled Antibody:

[0084] Take 1 mL of the gold manganese oxide nanoparticles prepared in step S1, add 0.2 mol / L K2CO3 aqueous solution, and adjust the pH of the system to 8.0; then add 10 μg of 1.0 mg / mL mouse anti-human apolipoprotein E4 monoclonal antibody, and mix and react at room temperature for 1 h.

[0085] S3, Closed:

[0086] Blocking solution was added to the mouse anti-human apolipoprotein E4 monoclonal antibody labeled with gold manganese oxide nanoparticles in step S2. The blocking agent was a composite blocking agent, which consisted of the following components by weight percentage: casein 5%, polyacrylic acid 1%, polyvinylpyrrolidone 0.3%, and the balance being water. After adding the composite blocking solution, the mixture was stirred at room temperature and reacted at room temperature for 1 hour to obtain a labeled gold solution with a volume concentration of 1.0%.

[0087] S4, Heavy Suspension:

[0088] The blocked gold manganese oxide nanoparticles labeled with antibodies were centrifuged at 4200 r / min for 10 min, the supernatant was discarded, and the precipitate was resuspended in resuspension.

[0089] S5. Prepare the sample pad:

[0090] Cut the non-woven fabric into 300mm×280mm pieces, soak them in the sample pad treatment solution for 4 minutes, take them out and place them horizontally to dry at 37℃ for 2.5 hours. After drying, the sample pad is obtained and placed in a self-sealing bag for later use.

[0091] S6. Preparation of the binding pad:

[0092] The mouse anti-human apolipoprotein E4 monoclonal antibody labeled with gold manganese oxide nanoparticles in step S2 is evenly spread on an 8975 glass fiber membrane, dried, and the gold manganese oxide nanoparticles labeled with the antibody are solidified on the glass fiber membrane to obtain the binding pad. It is then removed, placed in a sealed bag, desiccant is added, and it is sealed and stored.

[0093] S7. Preparation of the reaction pad:

[0094] Nitrocellulose membranes were attached to PVC plates, and then goat anti-human apolipoprotein E4 polyclonal antibody was diluted to a concentration of 1 mg / mL with coating buffer and used as the detection line coating. Goat anti-mouse IgG was diluted to 2 mg / mL and used as the control line C coating. The membrane was then drawn with a gold-spraying membrane scribing instrument at a concentration of 1 μL / cm and dried at 40℃ for 6 hours to obtain the reaction pad. The pad was then removed and placed in a sealed bag for later use.

[0095] S8. Reagent kit assembly:

[0096] The sample pad, conjugate pad, NC membrane, and absorbent pad are sequentially overlapped and pasted onto a PVC base plate. After cutting, the detection immunochromatographic test strip for apolipoprotein E4 is obtained.

[0097] Example 3

[0098] S1. Preparation of gold manganese oxide nanoparticles for color development:

[0099] The gold manganese oxide nanoparticles were prepared using a one-step reduction method. 125 μL of 0.1 M MnCl2 and 340 μL of 1 wt% HAuCl4•3H2O were added to 20 mL of purified water and stirred at 500 rpm for three minutes. Then, 500 μL of 0.1 M NaOH solution was added to create an alkaline environment, and the mixture was stirred for 10 minutes. The mixture was then centrifuged (10000 rpm, 5 minutes) to separate the silver ear-shaped gold manganese oxide (Au-MnOX). The gold manganese oxide nanoparticles were then dispersed in purified water.

[0100] S2, Labeled Antibody:

[0101] Take 1 mL of the gold manganese oxide nanoparticles prepared in step S1, add 0.2 mol / L K2CO3 aqueous solution, and adjust the pH of the system to 9.0; then add 10 μg of 1.0 mg / mL mouse anti-human apolipoprotein E4 monoclonal antibody, and mix and react at room temperature for 1 h.

[0102] S3, Closed:

[0103] Blocking solution was added to the mouse anti-human apolipoprotein E4 monoclonal antibody labeled with gold manganese oxide nanoparticles in step S2. The blocking agent was a composite blocking agent, which consisted of the following components by weight percentage: casein 5%, polyacrylic acid 1.5%, polyvinylpyrrolidone 0.5%, and the balance being water. After adding the composite blocking solution, the mixture was mixed at room temperature and reacted at room temperature for 1 hour to obtain a labeled gold solution with a volume concentration of 1.0%.

[0104] S4, Heavy Suspension:

[0105] The blocked gold manganese oxide nanoparticles labeled with antibodies were centrifuged at 5000 r / min for 10 min, the supernatant was discarded, and the precipitate was resuspended in resuspension.

[0106] S5. Prepare the sample pad:

[0107] Cut the non-woven fabric into 300mm×280mm pieces, soak them in the sample pad treatment solution for 5 minutes, take them out and place them horizontally to dry at 37℃ for 3 hours. After drying, the sample pad is obtained and placed in a self-sealing bag for later use.

[0108] S6. Preparation of the binding pad:

[0109] The mouse anti-human apolipoprotein E4 monoclonal antibody labeled with gold manganese oxide nanoparticles in step S2 is evenly spread on an 8975 glass fiber membrane, dried, and the gold manganese oxide nanoparticles labeled with the antibody are solidified on the glass fiber membrane to obtain the binding pad. It is then removed, placed in a sealed bag, desiccant is added, and it is sealed and stored.

[0110] S7. Preparation of the reaction pad:

[0111] Nitrocellulose membranes were attached to PVC plates, and then goat anti-human apolipoprotein E4 polyclonal antibody was diluted to a concentration of 1 mg / mL with coating buffer and used as the detection line coating. Goat anti-mouse IgG was diluted to 2 mg / mL and used as the control line C coating. The membrane was then drawn with a gold-spraying membrane scribing instrument at a concentration of 1 μL / cm and dried at 40℃ for 6 hours to obtain the reaction pad. The pad was then removed and placed in a sealed bag for later use.

[0112] S8. Reagent kit assembly:

[0113] The sample pad, conjugate pad, NC membrane, and absorbent pad are sequentially overlapped and pasted onto a PVC base plate. After cutting, the detection immunochromatographic test strip for apolipoprotein E4 is obtained.

[0114] Example 4

[0115] The difference between Example 4 and Example 1 is that the sealing agent consists of the following components by weight percentage: 5% casein and the balance being water.

[0116] Example 5

[0117] The difference between Example 1 and Example 5 is that the amount of manganese chloride solution added in step S1 is 50 μL.

[0118] Figure 2 This is a particle size diagram of the gold manganese oxide nanocoloring particles in Example 5 of the present invention.

[0119] Example 6

[0120] The difference between Example 1 and Example 5 is that the amount of manganese chloride solution added in step S1 is 80 μL.

[0121] Figure 3 This is a particle size diagram of the gold manganese oxide nanocoloring particles in Example 6 of the present invention.

[0122] Example 7

[0123] The difference between Example 7 and Example 1 is that the sealing agent consists of the following components by weight percentage: 5% casein, 1% polyacrylic acid, and the balance being water.

[0124] Example 8

[0125] The difference between Example 1 and Example 8 is that the sealing agent consists of the following components by weight percentage: 5% casein, 0.2% polyvinylpyrrolidone, and the balance being water.

[0126] Example 9

[0127] The difference between Example 1 and Example 9 is that the sealant is composed of the following components by weight percentage: 1% polyacrylic acid, 0.2% polyvinylpyrrolidone, and the balance being water.

[0128] Comparative Example 1

[0129] The only difference from Example 1 is that the gold manganese oxide nanoparticles are replaced with colloidal gold solution;

[0130] The colloidal gold solution was prepared by the following method:

[0131] Colloidal gold was prepared using chloroauric acid and sodium citrate as raw materials. 99 mL of distilled water and 1 mL of 1% chloroauric acid were added to a glass reagent bottle under stirring and heating. The mixture was boiled for 5 minutes, and then 3 mL of 1% sodium citrate was added. The mixture was stirred and heated for another 10 minutes until the solution turned from purple to red. The solution was then stirred and cooled to room temperature to obtain a colloidal gold solution containing colloidal gold particles with a diameter of 30-40 nm.

[0132] Experimental Example 1

[0133] The sensitivity of the detection kits for apolipoprotein E4 prepared in Examples 1-3 and Comparative Example 1 was tested. The recombinant APOE4 protein was diluted with serum and plasma of genotype E3 / E3 to a concentration of 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0 μg / mL to test the detection limits of Examples 1-3 and Comparative Example 1.

[0134] The test results are shown in Table 1:

[0135] Table 1 Performance test results of the apolipoprotein E4 detection kits prepared in Examples 1-3 and Comparative Example 1

[0136]

[0137] Note: Positive: +; Negative: -.

[0138] As shown in Table 1, the detection limits of Examples 1-3 are 0.5 μg / mL, while the detection limit of Comparative Example 1 is 2 μg / mL. The detection limits of Examples 1-3 are lower than those of Comparative Example 1, indicating that the kit has higher sensitivity when gold manganese oxide nanoparticles are added as the colorimetric system.

[0139] Experiment Example 2

[0140] The stability of the apolipoprotein E4 detection kits prepared in Examples 1, 4, and 7-9 was tested. The recombinant APOE4 protein was diluted with serum and plasma of genotype E3 / E3 to prepare reference standards: L1: 4 μg / mL; L2: 2 μg / mL; L3: 0 μg / mL. The apolipoprotein E4 detection kits prepared in Examples 1 and 4 were placed in an incubator at 55°C for accelerated stability testing. The kits were placed for 0, 10, 20, 30, 40, 50, and 62 days, respectively. Three samples were tested each time, and the color intensity of the detection kits was recorded.

[0141] The test results are shown in Table 2:

[0142] Table 2 Performance test results of the apolipoprotein E4 detection kits prepared in Examples 1, 4 and 7-9

[0143]

[0144] Continued from Table 2

[0145]

[0146] As shown in Table 2, Examples 4 and 7-9 showed poor color development and reproducibility on day 40, while Example 1 maintained good color development intensity on day 62. This indicates that the addition of a blocking agent composed of casein, polyacrylic acid, polyvinylpyrrolidone and water helps to improve the stability of the apolipoprotein E4 detection kit.

[0147] Experimental Example 3

[0148] The sensitivity of the detection kits for apolipoprotein E4 prepared in Examples 1, 5, and 6 was tested. The recombinant APOE4 protein was diluted with serum and plasma of genotype E3 / E3, and the detection limits of Examples 1, 5, and 6 were then tested.

[0149] The test results are shown in Table 3:

[0150] Table 3 Performance test results of the apolipoprotein E4 detection kits prepared in Examples 1, 5, and 6

[0151]

[0152] As shown in Table 3, when the particle size of gold manganese oxide nanoparticles is 659.13 nm, the detection limit is relatively low but non-specific results may occur. When the particle size of gold manganese oxide nanoparticles is 408.70 nm, the detection limit is relatively low and no non-specific results may occur.

[0153] Experiment Example 4

[0154] The apolipoprotein E4 detection kit prepared in Example 1 was used to detect 296 venous whole blood samples. At the same time, some samples were sent to Zhengzhou Aividi Medical Laboratory Co., Ltd. for APOE genotyping detection using the human APOE genotyping kit (fluorescent PCR-enzyme digestion method) produced by Guangzhou Baochuang Biotechnology Co., Ltd. The results are shown in Tables 4 and 5.

[0155] Table 4 Performance test results of the apolipoprotein E4 detection kit prepared in Example 1

[0156]

[0157] Positive concordance rate = 149 / 149 × 100% = 100.00% (95% CI: 93.79%, 100.00%).

[0158] Negative compliance rate = 147 / 147 × 100% = 100% (95% CI: 94.34%, 100.00%).

[0159] Overall compliance rate = (149 + 147) / 296 × 100% = 100% (95% CI: 96.95%, 100.00%).

[0160] Kappa=1;

[0161] The Kappa coefficient of the consistency analysis was 1, indicating that the test results of the reagent to be evaluated and the comparison reagent were highly consistent (Kappa≥0.75).

[0162] Table 5 Performance test results of the apolipoprotein E4 detection kit prepared in Example 1

[0163]

[0164] Note: Positive: +; Negative: -.

[0165] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An immunochromatographic test strip, characterized by, The detection immunochromatography test paper comprises a base plate, a sample pad, a conjugate pad, an NC membrane and a water absorption pad, one side of the NC membrane is sequentially provided with the conjugate pad and the sample pad, and the other side of the NC membrane is provided with the water absorption pad; the conjugate pad is marked with gold-manganese oxide nano chromogenic particles and apolipoprotein APOE4 monoclonal antibody; The preparation method of the gold-manganese oxide nano chromogenic particles comprises the following steps: reducing manganese chloride and chloroauric acid in an alkaline solution to obtain gold-manganese oxide nano chromogenic particles; the gold-manganese oxide nano chromogenic particles are prepared by using a one-step reduction method, 125 μL of 0.1M MnCl2 and 340 μL of 1wt% HAuCl4·3H2O are added into 20 mL of purified water, stirring is performed at 500 rpm for three minutes, then 500 μL of 0.1M NaOH solution is added into the solution to form an alkaline environment, stirring is performed for 10 min, centrifugation is performed at 10000 rpm for 5 min, the silver ear-shaped gold-manganese oxide is separated, and the gold-manganese oxide nano chromogenic particles are dispersed in purified water; The conjugate pad is a conjugate pad treated by a conjugate pad treatment solution, and the conjugate pad treatment solution comprises gold-manganese oxide nano chromogenic particles, a blocking solution and apolipoprotein APOE4 monoclonal antibody; The blocking solution is a composite blocking solution, and the composite blocking solution is composed of the following components by weight percentage: casein 5%, polyacrylic acid 0.5%-1.5%, polyvinylpyrrolidone 0.1%-0.5%, and the balance is water.

2. The immunochromatographic test strip according to claim 1, characterized in that, The particle size of the gold-manganese oxide nano chromogenic particles is 200-700 nm.

3. The immunochromatographic test strip according to claim 1, wherein The manganese chloride is added in the form of a manganese chloride solution for the reduction reaction, the concentration of the manganese chloride solution is 0.1 mol / L; the alkaline solution is a sodium hydroxide solution with a concentration of 0.1 mol / L; the chloroauric acid is added in the form of a tetrachloroauric acid trihydrate solution for the reduction reaction, and the concentration of the tetrachloroauric acid trihydrate solution is 0.1wt%.

4. The immunochromatographic test strip according to claim 1, wherein The preparation method of the conjugate pad comprises the following steps: S1, dispersing the gold-manganese oxide nano chromogenic particles in water, adjusting the pH to 7.0-9.0, then adding apolipoprotein APOE4 monoclonal antibody to react, adding a blocking solution for blocking, removing the supernatant by centrifugation, collecting the precipitate, resuspending the precipitate with a resuspension solution, and obtaining a conjugate pad treatment solution; S2, coat the binding pad treatment liquid uniformly on the glass fiber paper, 10 cm 3 Coat 240±50 uL of the binding pad treatment liquid on the glass fiber paper, dry, and obtain the binding pad.

5. The immunochromatographic test strip according to claim 1, wherein The NC membrane is coated with goat anti-human apolipoprotein E polyclonal antibody and goat anti-mouse IgG antibody.

6. An assay kit for apolipoprotein E4, characterized in that, The detection immunochromatography test paper comprises the detection immunochromatography test paper according to any one of claims 1-5.

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