An immunochromatographic test kit for apolipoprotein E4 detection, preparation method and application

Abstract

This invention discloses an immunochromatographic assay kit, preparation method, and application for the detection of apolipoprotein E4. The kit includes a test card and sample diluent. The test card comprises a test strip, which includes a PVC base, absorbent paper, a glass fiber pad, and a nitrocellulose membrane. The glass fiber pad is loaded with polystyrene latex microspheres for physical adsorption. The surface of the polystyrene latex microspheres is modified with sulfonyl or hydroxyl groups, and the zeta potential ranges from -15V to -20V. After the polystyrene latex microspheres are uniformly coated on a glass slide and dried, the contact angle between them and water droplets is greater than 90°. The nitrocellulose membrane is loaded with anti-APOE4 antibody, coating solution, and protein A. The sample diluent includes PBS, TWEEN-20, Tetronic 1307, and CHAPS. This invention eliminates the need for monoclonal antibody-labeled latex microspheres, reducing false positives and reagent batch-to-batch variation, and achieving higher sensitivity.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, and specifically to an immunochromatographic detection kit, preparation method, and application for the detection of apolipoprotein E4. Background Technology

[0002] Alzheimer's disease (AD) is a common, multifactorial, primary degenerative brain disease, and the most common type. The World Health Organization classifies AD into early-onset Alzheimer's disease (EOAD) and late-onset Alzheimer's disease (LOAD), with LOAD accounting for over 95% of all AD cases and considered a complex result of gene-environment interactions. Risk factors for LOAD include advanced age, a history of cardiovascular disease, depression, and the apolipoprotein E4 gene. Apolipoprotein E4 (APOE4) is considered the strongest genetic risk factor for AD.

[0003] Currently, there are kits for detecting apolipoproteins based on latex turbidimetry. However, since apolipoproteins have multiple protein subtypes, such as APOE2, APOE3, and APOE4, latex turbidimetry cannot distinguish between these three subtypes. Reagents that can directly detect APOE4 often employ immunochromatographic techniques based on a double-antibody sandwich method. Specifically, monoclonal antibody 1 is labeled onto latex microspheres, and the carboxyl groups of the microspheres are activated using EDC and NHS salts. The activated carboxyl groups are then linked to the amino groups of the antibody, followed by blocking with 1% BSA and preservation. Monoclonal antibody 2 is coated onto a nitrocellulose membrane. When the sample to be tested binds to the latex microsphere label of monoclonal antibody 1, it migrates forward on the chromatographic membrane through capillary action, triggering a specific immunoreaction at the site coated with monoclonal antibody 2. The concentration of the sample to be tested is directly proportional to the deposition intensity of latex at that site.

[0004] However, the above immunochromatographic techniques based on the double-antibody sandwich method have the following technical drawbacks:

[0005] First, monoclonal antibody 1 requires labeling latex microspheres, which is a cumbersome and time-consuming process (generally taking 1 to 2 days). The entire labeling process is very complex and difficult to control, resulting in large batch-to-batch variations and high costs.

[0006] Second, ideally, antibody-labeled latex microspheres are covalently coupled to the microspheres through the amino group at their Fc end, meaning the antibody Fab faces outward and the Fc end faces inward. However, in reality, the Fab and Fc ends are often randomly facing outward. The non-directional nature of the antibody causes non-specific adsorption at the Fc end between monoclonal antibody 1 and monoclonal antibody 2, leading to false positives.

[0007] Third, the sensitivity is sufficient when using venous blood to measure APOE4, but the low sensitivity can lead to false negatives when using small samples (such as finger prick blood). This is because the distribution of monoclonal antibody 1 on the microspheres is affected by its own volume and labeling efficiency, resulting in a limited number of APOE4 proteins carried on a single latex microsphere.

[0008] Therefore, this patent application is filed. Summary of the Invention

[0009] To address the above issues, this invention provides an immunochromatographic assay kit for the detection of apolipoprotein E4, along with its preparation method and applications. This invention eliminates the step of labeling latex microspheres with monoclonal antibody 1. Utilizing the property that APOE4 in the test sample can be stably and physically adsorbed onto polystyrene latex microspheres, untreated latex microspheres can be used directly. Since monoclonal antibody 1 is not used, false positives caused by non-specific adsorption at the Fc end between the two antibodies are directly eliminated, thereby reducing false positives. It also allows for a greater quantity of APOE4 protein carried on a single latex microsphere, increasing the probability of capture by protein A on the T-line and improving sensitivity. Simultaneously, it significantly reduces batch-to-batch variability.

[0010] The present invention is implemented using the following technical solutions:

[0011] The first objective of this invention is to provide an immunochromatographic assay kit for the detection of apolipoprotein E4, comprising a test card and a sample diluent. The test card comprises a test strip, and the test strip comprises a PVC base plate, absorbent paper, a fiberglass pad, and a nitrocellulose membrane. The absorbent paper, the fiberglass pad, and the nitrocellulose membrane are all adhered to the PVC base plate.

[0012] The glass fiber pad is loaded with polystyrene latex microspheres for physical adsorption. The surface of the polystyrene latex microspheres for physical adsorption is modified with sulfonyl or hydroxyl groups. The Zeta potential range is -15V to -20V. After the polystyrene latex microspheres for physical adsorption are uniformly coated on the glass slide and dried, the contact angle formed with water droplets is greater than 90°.

[0013] The nitrocellulose membrane is loaded with anti-APOE4 antibody, coating solution and protein A;

[0014] The sample diluents include PBS, TWEEN-20, Tetronic 1307, and CHAPS.

[0015] This invention employs polystyrene latex microspheres with surface-modified sulfonyl or hydroxyl groups for physical adsorption, instead of conventional chemically coupled latex microspheres with surface-modified carboxyl groups (such as silica microspheres). Furthermore, the polystyrene latex microspheres of this invention have a lower absolute Zeta potential and a larger contact angle than chemically coupled microspheres. Utilizing the adsorption characteristics between APOE4 and these latex microspheres, the C-terminus of the APOE4 protein is rich in hydrophobic amino acids such as Val, Leu, and Phe, which can insert into the hydrophobic region of polystyrene. This allows for stable and efficient physical adsorption of the APOE4 protein onto the latex microspheres, maximizing the adsorption capacity of the microspheres for APOE4 protein. This eliminates the need for monoclonal antibody 1 labeling of the latex microspheres, solving not only the problems of false positives and batch-to-batch variation but also increasing the number of APOE4 proteins carried on a single microsphere, resulting in a higher probability of capture by protein A on the T-line, higher sensitivity, and stronger specificity. It also significantly shortens reagent preparation time and reduces production costs. Meanwhile, the components in the sample diluent include PBS, TWEEN-20, Tetronic 1307, and CHAPS, which work synergistically with the polystyrene latex microspheres with the above properties to exert specific adsorption capabilities and produce accurate latex deposition intensity.

[0016] As a preferred technical solution, the physical adsorption polystyrene latex microspheres adsorb protein up to 20~90 μg protein / mg microspheres.

[0017] As a preferred technical solution, the polystyrene latex microspheres used for physical adsorption are latex microspheres from MF manufacturer.

[0018] As a preferred technical solution, the proportions of the components in the sample diluent are as follows:

[0019] PBS: molar concentration 10~50mM, pH=6.4~7.6; where pH can be 6.4, 6.8, 7.2, 7.6, and molar concentration can be 10mM, 20mM, 50mM;

[0020] TWEEN-20: 0.025~0.1%; can be 0.025%, 0.05%, or 0.1%.

[0021] Tetronic 1307: 0.1~0.4%; can be 0.1%, 0.2%, or 0.4%.

[0022] CHAPS: 0.2~0.6%, which can be 0.2%, 0.4%, or 0.6%.

[0023] This invention incorporates Tween-20, a polyoxyethylene sorbitan monolaurate, into the sample diluent. Tween-20's molecule comprises a hydrophilic polyoxyethylene chain and a hydrophobic laurate chain, making it an oil-in-water emulsifier. Being uncharged, it has minimal impact on protein activity and does not cause protein denaturation like ionic detergents (such as SDS). Tween-20 effectively reduces non-specifically adsorbed biomolecules on solid-phase surfaces (such as microplate pore walls, NC membranes, and polystyrene microspheres), ensuring that only specifically bound molecules remain. The target substance, APOE4 protein, has a C-terminus rich in hydrophobic amino acids such as Val, Leu, and Phe, which can insert into the hydrophobic region of the latex microsphere surface. By adjusting the Tween-20 content in the sample diluent, APOE4 protein can be stably adsorbed onto the latex microsphere surface, reducing the adsorption of other proteins such as albumin, thus minimizing the occupancy effect of these proteins on the latex microsphere surface. Meanwhile, Tween-20 can act as a dispersant and stabilizer, helping the latex microspheres on the binding pad to regain their dispersed state after drying, and can also prevent the highly hydrophobic latex microspheres in this invention from aggregating during chromatography.

[0024] Tetronic 1307, chemically known as a propylene oxide-ethylene oxide-vinyl diamine copolymer, possesses excellent interfacial activity and combines the advantages of both anionic and cationic surfactants. It also exhibits good emulsifying, dispersing, and antistatic properties. Compared to other surfactants, Tetronic 1307 does not form a hydrophobic surface regardless of whether it adsorbs onto a positively or negatively charged interface. Tetronic 1307 has fixed hydrophilic and lipophilic groups, allowing it to align oriented on the surface of a solution. Adding a small amount of Tetronic 1307 to a solution system significantly alters its interfacial state, reducing the absorption of the NC membrane by the analyte and latex microspheres. Since this invention uses bare microspheres with physical adsorption properties, the microspheres themselves may bind to the NC membrane through hydrophobic interactions, leading to a darker background and non-specific signals. The amphoteric properties of Tetronic 1307 effectively mitigate these problems. Furthermore, adding an appropriate amount of Tetronic 1307 can also improve sensitivity, i.e., it has a color enhancement effect (increased line intensity).

[0025] CHAPS is the inner salt of 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonic acid, containing a steroid backbone (similar to bile acids), which gives it a strong ability to dissolve lipids and membrane proteins (effectively disrupting HDL / VLDL particles). It also possesses a zwitterionic head group (quaternary ammonium cation + sulfonic acid anion), making it electrically neutral in solution. Unlike ionic surfactants (such as SDS), CHAPS does not disrupt the secondary and tertiary structures of proteins. Regarding the detection of APOE4: the main difference between APOE4 and APOE3 lies in the domain interaction caused by the 112th amino acid (Arg vs Cys), a difference in spatial conformation. If a strong denaturing agent (such as SDS) is used, APOE4 will unfold, losing its specific conformational epitope, causing specific antibodies to fail to recognize it or incorrectly recognize APOE3. CHAPS can unwrap the lipoprotein shell, exposing APOE, without denaturing APOE itself, thus protecting the conformational epitopes required to distinguish APOE4. CHAPS has a high CMC (approximately 6-10 mM), and on the chromatographic strip, the CHAPS concentration rapidly decreases below the CMC as the sample solution flows and is diluted, reducing its potential interference with subsequent antigen-antibody binding. Furthermore, as a zwitterionic reagent, CHAPS exhibits extremely low nonspecific binding to proteins and nitrocellulose membranes, helping to reduce background noise in immunofluorescence detection and improve the signal-to-noise ratio.

[0026] As a preferred technical solution, the proportions of the components in the sample diluent are as follows:

[0027] PBS: 50 mM, pH 7.2;

[0028] TWEEN-20: 0.05%;

[0029] Tetronic 1307: 0.2%;

[0030] CHAPS: 0.4%.

[0031] As a preferred technical solution, the fiberglass pad is coated with a treatment liquid, the formula of which is:

[0032] The composition consisted of 10% blocking agent, 2% sucrose, 0.04% Tween-20, 1% anti-erythrocyte antibody, 50 mmol / L PBS solution, and 1% Evans blue, with the pH of the PBS solution being 7.2.

[0033] As a preferred technical solution, the blocking agent is a mixture of active blocking agent and passive blocking agent.

[0034] As a preferred technical solution, the coating solution includes PBS and sucrose, wherein the concentration of PBS is 50 mmol / L and the mass concentration of sucrose is 2%.

[0035] A second objective of this invention is to provide a method for preparing an immunochromatographic assay kit for detecting apolipoprotein E4 as described in any of the preceding claims, wherein the preparation process of the test strip includes:

[0036] Preparation of nitrocellulose membrane: Anti-APOE4 antibody was diluted with coating buffer and streaked on one side of the cellulose membrane as the T line, and protein A was diluted with coating buffer and streaked on the cellulose membrane as the C line. The membrane was then dried.

[0037] Preparation of glass fiber mat: After cutting the glass fiber, the treatment solution was sprayed onto one side of the glass fiber. Polystyrene latex microspheres were diluted with sucrose in PBS solution and sprayed onto the other side of the glass fiber. The mixture was then dried.

[0038] Attach absorbent paper, fiberglass pad, and nitrocellulose membrane to a PVC base plate, cut them to obtain test strips, pack the test strips into a card and seal them in an aluminum foil bag.

[0039] A third objective of this invention is to provide the application of the immunochromatographic assay kit described in any of the above claims in the detection of apolipoprotein E4.

[0040] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0041] This invention utilizes the unique properties of the APOE4 protein, whose C-terminus is rich in hydrophobic amino acids such as Val, Leu, and Phe, allowing it to insert into the hydrophobic region of polystyrene. Combined with a specific sample diluent and latex microspheres possessing physical adsorption properties, the APOE4 protein can be stably and efficiently physically adsorbed onto the latex microspheres. This property eliminates the need for monoclonal antibody 1 labeling of latex microspheres, making kit preparation simpler, more economical, faster, and avoiding false positives. It also improves the sensitivity and specificity of the kit while maintaining good batch-to-batch consistency. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0043] Example 1:

[0044] This embodiment presents an immunochromatographic assay kit for the detection of apolipoprotein E4, comprising a test card and a sample diluent. The test card includes a test strip, which consists of a PVC base, absorbent paper, a fiberglass pad, and a nitrocellulose membrane. The absorbent paper, fiberglass pad, and nitrocellulose membrane are all adhered to the PVC base, with the adhered portions overlapping by 2 mm. After assembly and cutting, the strips are made into 4 mm wide strips, which are then placed in a card case and sealed in an aluminum foil bag. The specific preparation process of the test strip is as follows:

[0045] 1. Preparation of nitrocellulose membrane: Anti-APOE4 antibody was diluted to 1 mg / mL with coating buffer (2% sucrose, 50 mmol / L PBS) and streaked on one side of Sartorius CN95 cellulose membrane as the detection line (T line); Protein A was diluted to 0.2 mg / mL with coating buffer (2% sucrose, 50 mmol / L PBS) and streaked on the cellulose membrane as the control line (C line); the streaking volume was 1 μL / cm, and the membrane was dried at 45℃ for 16 h.

[0046] 2. Preparation of Fiberglass Pads: Fiberglass was cut into 19mm*300mm pieces. Using a gold sprayer, a treatment solution (10% blocking agent, 2% sucrose, 0.04% Tween-20, 1% anti-erythrocyte antibody, 50mmol / L, pH 7.2 PBS, and 1% Evans Blue) was sprayed onto one side of the fiberglass at a rate of 4ul / cm. The blocking agent was a mixture of active and passive blocking agents. MF latex microspheres (diluted with PBS and sucrose) were sprayed onto the other side of the fiberglass at a rate of 4ul / cm. The sheets were then dried at 45℃ for 16 hours.

[0047] 3. Attach absorbent paper, the prepared fiberglass pad, and the nitrocellulose membrane to the PVC base plate, cut them into 4mm wide test strips, put them into the cartridge, and seal them into an aluminum foil bag.

[0048] The components of the sample diluent are:

[0049] PBS: 50 mM, pH 7.2;

[0050] TWEEN-20: 0.05%;

[0051] Tetronic 1307: 0.2%;

[0052] CHAPS: 0.4%.

[0053] Among them, the latex microspheres manufactured by MF are produced by Shenzhen Maifu New Materials Co., Ltd. They are modified with sulfonyl groups, have a potential value of -20V, a contact angle of 110°, and an adsorption capacity of 50ug protein / mg microsphere.

[0054] The process of determining the latex microsphere potential (Zeta potential) (usually using electrophoretic light scattering method):

[0055] Sample preparation: Dilute the latex microspheres with a suitable solvent (usually a buffer solution) to an appropriate concentration (e.g., 0.1-1 mg / mL) to ensure that the particles are fully dispersed and to avoid multiple scattering affecting the measurement.

[0056] Instrument preparation: Turn on the Zeta potential analyzer, preheat it, and calibrate it using standard samples as specified.

[0057] Parameter settings: Enter or confirm the measurement parameters in the instrument software. The most critical parameters are temperature (usually set to 25°C), solvent pH, and ionic strength, as these factors will significantly affect the results of the Zeta potential.

[0058] Sample loading and measurement: The prepared sample is injected into a clean measurement cell and placed in the instrument. An electric field is applied, and the instrument measures the electrophoretic mobility of the particles using techniques such as laser Doppler electrophoresis and automatically calculates the Zeta potential value.

[0059] Data analysis: Record the measurement results, typically including zeta potential values, particle size distribution, etc. To ensure the reliability of the results, it is recommended to perform multiple repeated measurements.

[0060] Procedure for determining the contact angle of latex microspheres (usually using the seated drop method):

[0061] Sample preparation: The dried latex microsphere powder is pressed into smooth and flat discs in a mold using a tablet press.

[0062] Instrument calibration: Turn on the contact angle measuring instrument, adjust the light source and camera to ensure that the droplet outline can be clearly captured.

[0063] Droplet deposition: Place the sample sheet horizontally on the sample stage and use a microsyringe to drop a drop of liquid with known surface tension (usually ultrapure water) onto the sample surface.

[0064] Image acquisition and analysis: Static images of the droplet on the solid surface are captured using the instrument's optical system. The software automatically or manually fits the droplet profile and calculates the contact angle θ2 based on Young's Equation.

[0065] Results recording: Usually, multiple measurements are taken at different locations on the sample piece, and the average value is taken as the final result to reduce errors.

[0066] The procedure for determining protein adsorption capacity (taking the BCA method as an example):

[0067] Establish an adsorption system:

[0068] Take a certain amount of latex microsphere suspension of known concentration.

[0069] Add a protein solution (such as an antibody solution) of known concentration and volume, and incubate under specific conditions (such as specific pH, temperature, and time) to allow the protein to fully adsorb onto the surface of the microspheres.

[0070] Microsphere separation: After incubation, the protein-microsphere complex was precipitated by high-speed centrifugation.

[0071] Collect the supernatant: Carefully aspirate the supernatant, which contains free proteins that were not adsorbed by the microspheres.

[0072] Determine the protein concentration in the supernatant: Use a protein quantification kit (such as the BCA method or the Bradford method) to determine the concentration of free protein in the supernatant.

[0073] Creating a standard curve: Use a standard protein of known concentration (such as BSA) to create a standard curve.

[0074] Absorbance measurement: React the supernatant with BCA working solution and measure the absorbance value at a specific wavelength (e.g., 562 nm).

[0075] Calculate the concentration: Calculate the protein concentration in the supernatant based on the standard curve.

[0076] Calculate the adsorption capacity:

[0077] Total adsorbed protein = (Initial protein concentration × Initial volume) - (Supernatant protein concentration × Supernatant volume)

[0078] Protein adsorption capacity per unit microsphere = Total adsorbed protein / Total mass (or total surface area) of microspheres.

[0079] The above measurement process uses existing detection methods.

[0080] Comparative Example 1:

[0081] Compared with Example 1, the only difference in Comparative Example 1 is that it uses latex microspheres from MF manufacturer labeled with monoclonal antibody 1; otherwise, they are the same as in Example 1.

[0082] Comparative Example 2:

[0083] The difference between Comparative Example 2 and Example 1 is that:

[0084] The latex microspheres used are latex microspheres from MK manufacturer; MK latex microspheres are produced by Merck Technologies in Germany, and their surface is modified with carboxyl groups. Their zeta potential ranges from -25 to -30, their contact angle is less than 50°, and their protein adsorption capacity is 10~30 micrograms of protein / mg of microspheres.

[0085] Sample diluent components:

[0086] TWEEN-20 (0.1%), S9 (0.1%), TX-100 (0.1%), sucrose, and 50 mmol / L PBS (pH 7.2).

[0087] Comparative Example 3:

[0088] Compared with Example 1, the only difference in Comparative Example 3 is that it uses latex microspheres from MK manufacturer, whose surface is modified with carboxyl groups; otherwise, it is the same as Example 1.

[0089] Comparative Example 4:

[0090] Compared with Example 1, the only difference in Comparative Example 4 is that:

[0091] Sample diluent components:

[0092] PBS: 50 mM, pH 7.2;

[0093] TWEEN-20: 0.10%;

[0094] Tetronic 1307: 0.4%;

[0095] CHAPS: 0.4%.

[0096] Detection:

[0097] The kits obtained in Example 1 and the comparative examples were used for apolipoprotein E4 genotyping detection. The specific procedures were as follows:

[0098] 1. Serum, plasma, and whole blood samples: Open the sample diluent tube, use a pipette to take 4 μL of serum / plasma or 6 μL of venous whole blood into the sample diluent, insert the quantitative blood collection device into the provided sample diluent tube, tighten it downwards, and gently shake it up and down for about 30 seconds to fully mix the sample, which is then used as the sample to be tested.

[0099] 2. Finger tip blood sample: Insert the collected sample (about 6 microliters) into the provided sample diluent tube, tighten it downwards, and gently shake it up and down for about 30 seconds to mix the sample thoroughly. This is the sample to be tested.

[0100] 3. With the end of the sample diluent tube containing the quantitative blood collection device facing down, gently press the sample diluent tube and vertically add 2 drops of the treated sample into the sample well of the test strip. Do not move the test strip after adding the sample.

[0101] 4. Observe the results within 12 to 15 minutes; results after 15 minutes are invalid.

[0102] Taking the fingertip capillary blood sample as an example, the test results are as follows:

[0103] 1. Eight APOE4 samples with known different genotypes were collected. According to the current AD diagnostic criteria, they were identified as ε2 / ε2, ε2 / ε3, ε3 / ε3, ε3 / ε4 heterozygous, ε2 / ε4 heterozygous, ε3 / ε4 heterozygous, ε4 / ε4 homozygous, and ε4 / ε4 homozygous. Expression of ε4 is considered a risk factor for AD.

[0104] The results of the color intensity of the reaction bands at the detection line (T line) for the above 8 samples were tested using the kit obtained in Example 1, and are shown in Table 1.

[0105] Table 1

[0106] The results in the table above show that the visual characterization results of Example 1 of the present invention are consistent with current genotyping methods.

[0107] 2. The above 8 samples were tested using the kit of Comparative Example 1 (traditional POCT double antibody sandwich method). The results of the color intensity of the reaction bands at the detection line (T line) are shown in Table 2. A comparison was made with Example 1. The results show that Comparative Example 1 resulted in false positives and false negatives.

[0108] Table 2. Detection results of the kit in Comparative Example 1

[0109]

[0110] 3. The apolipoprotein E4 (APOE4) levels in the eight blood samples of different genotypes mentioned above were compared between Example 1 and Comparative Examples 2-4. The results of the color intensity of the reaction bands on the detection line (T line) are shown in Table 3. The results show that Comparative Example 2 is prone to false positives and false negatives, Comparative Example 3 causes false positives, and Comparative Example 4 causes false negatives. The results indicate that neither using latex microspheres with suitable properties nor sample diluents with suitable physicochemical properties alone can accurately reflect the concentration of apolipoprotein E4 (APOE4) in blood samples. Both properties need to be suitable simultaneously to achieve adequate specific adsorption capacity and produce accurate latex deposition intensity.

[0111] Table 3

[0112]

[0113] 4. Stability Testing After Opening: The stability of the reagent kit obtained in Example 1 of this invention was tested in both the opened state and under normal sealed storage before testing. Specifically, the degree of conformity (negative / positive compliance rate) with the company's negative / positive reference samples was tested within 3 hours of opening. Testing was conducted every 0.5 hours after opening, using the reference sample compliance rate as a reference. The test results under normal sealed storage are shown in Table 4, and the test results under opened state are shown in Table 5. The results show that the reagent kit of Example 1 of this invention conforms to the reference sample results within 3 hours of opening, indicating that the reagent is stable within 3 hours of opening and meets the testing requirements.

[0114] Table 4

[0115]

[0116] Table 5

[0117]

[0118] 5. Heat Treatment Stability Test: The stability of the kit obtained in Example 1 was tested under heat treatment at 45℃ for 4 days and under normal storage conditions. Specifically, the degree of conformity (negative / positive compliance rate) with the company's negative / positive reference samples was tested during the 4-day heat treatment at 45℃. Tests were conducted once per day, using the reference sample compliance rate as a reference. The test results are shown in Tables 6 and 7. The results show that the kit in this example conforms to the reference sample results during the 4-day heat treatment at 45℃, indicating that the reagent is stable during this period and meets the testing requirements.

[0119] Table 6

[0120]

[0121] Table 7

[0122]

[0123] 6. Inter-batch difference test: Inter-batch difference tests were conducted on three batches of reagent kits from Example 1 and Comparative Example 1, respectively. The company's negative / positive reference samples were tested, and the degree of agreement between the inter-batch results and the qualitative reference samples (reference negative / positive concordance rate) was observed. The test results are shown in Tables 8 and 9. The results show that the inter-batch difference of the reagent kit from Example 1 of this invention is significantly smaller than that of Comparative Example 1.

[0124] Table 8. Batch-to-batch variability test of the kit in Example 1

[0125]

[0126] Table 9. Batch-to-batch variability test of reagent kits in Comparative Example 1

[0127]

[0128] In summary, this invention utilizes the stable and efficient physical adsorption of APOE4 protein onto latex microspheres, eliminating the need for monoclonal antibody 1 labeling of latex microspheres. This results in a simpler, more economical, and faster kit preparation, improving the kit's sensitivity and specificity while maintaining good batch-to-batch consistency. Furthermore, this label-free process reduces reagent preparation time by two days, decreases the use of expensive raw materials, and significantly lowers production costs.

Claims

1. An immunochromatographic assay kit for the detection of apolipoprotein E4, characterized in that, The test includes a test card and a sample diluent. The test card includes a test strip, and the test strip includes a PVC base plate, absorbent paper, a fiberglass pad, and a nitrocellulose membrane. The absorbent paper, fiberglass pad, and nitrocellulose membrane are all adhered to the PVC base plate. The glass fiber pad is loaded with polystyrene latex microspheres for physical adsorption. The surface of the polystyrene latex microspheres for physical adsorption is modified with sulfonyl or hydroxyl groups. The Zeta potential range is -15V to -20V. After the polystyrene latex microspheres for physical adsorption are uniformly coated on the glass slide and dried, the contact angle formed with water droplets is greater than 90°. The nitrocellulose membrane is loaded with anti-APOE4 antibody, coating solution and protein A; The sample diluents include PBS, TWEEN-20, Tetronic 1307, and CHAPS.

2. The immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 1, characterized in that, The physical adsorption capacity of the polystyrene latex microspheres for protein adsorption is 20~90 μg protein / mg microsphere.

3. The immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 1, characterized in that, The polystyrene latex microspheres used for physical adsorption are latex microspheres from MF manufacturer.

4. The immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 1, characterized in that, The proportions of each component in the sample diluent are as follows: PBS: 10-50 mM molar concentration, pH 6.4-7.6; TWEEN-20: 0.025~0.1%; Tetronic 1307: 0.1~0.4%; CHAPS: 0.2~0.6%.

5. An immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 1, characterized in that, The proportions of each component in the sample diluent are as follows: PBS: 50 mM, pH 7.2; TWEEN-20: 0.05%; Tetronic 1307: 0.2%; CHAPS: 0.4%.

6. The immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 1, characterized in that, The fiberglass pad is coated with a treatment solution, the formula of which is: The composition consisted of 10% blocking agent, 2% sucrose, 0.04% Tween-20, 1% anti-erythrocyte antibody, 50 mmol / L PBS solution, and 1% Evans blue, with the pH of the PBS solution being 7.

2.

7. An immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 6, characterized in that, The blocking agent is a mixture of active and passive blocking agents.

8. The immunochromatographic assay kit for detecting apolipoprotein E4 according to claim 1, characterized in that, The coating solution includes PBS and sucrose, wherein the concentration of PBS is 50 mmol / L and the mass concentration of sucrose is 2%.

9. A method for preparing an immunochromatographic assay kit for detecting apolipoprotein E4 according to any one of claims 1 to 8, characterized in that, The preparation process of the test strip includes: Preparation of nitrocellulose membrane: Anti-APOE4 antibody was diluted with coating buffer and streaked on one side of the cellulose membrane as the T line, and protein A was diluted with coating buffer and streaked on the cellulose membrane as the C line. The membrane was then dried. Preparation of glass fiber mat: After cutting the glass fiber, the treatment solution was sprayed onto one side of the glass fiber. Polystyrene latex microspheres were diluted with sucrose in PBS solution and sprayed onto the other side of the glass fiber. The mixture was then dried. Attach absorbent paper, fiberglass pad, and nitrocellulose membrane to a PVC base plate, cut them to obtain test strips, pack the test strips into a card and seal them in an aluminum foil bag.

10. The use of the immunochromatographic assay kit according to any one of claims 1 to 8 in the detection of apolipoprotein E4.

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