Kit for joint detection of markers A beta 1-40 and A beta 1-42 as well as preparation method and application of kit

Abstract

The invention relates to a kit for jointly detecting markers A beta 1-40 and A beta 1-42 as well as a preparation method and application of the kit, and relates to the technical field of immunodetection. The kit comprises a first reagent and a second reagent, the first reagent comprises a magnetic bead working solution and a magnetic microsphere coated with an A beta polyclonal antibody; and the second reagent comprises a labeled antibody diluent and an acridinium ester labeled anti-A beta antibody. According to the method, an anti-A beta 1-40 antibody and an anti-A beta 1-42 antibody with high specificity are adopted, and the ratio of A beta 1-40 to A beta 1-42 is calculated; a magnetic bead working solution and a labeled antibody diluent are adopted, so that the sensitivity and the stabilizer of the kit are further improved; the detection time is 8 minutes, the result can be obtained, the repeatability is within 4%, the lowest detection limit of A beta 1-40 is 1.08 pg / ml, and the lowest detection limit of A beta 1-42 is 1.08 pg / ml.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassays, and in particular to a kit for jointly detecting markers Aβ1-40 and Aβ1-42, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD), also known as senile dementia, is a severe, primary, degenerative disease of the central nervous system characterized by cognitive impairment and memory loss. It is the most common type of dementia and has a high incidence, making it one of the most common causes of intellectual impairment in the elderly. Characteristic pathological changes in AD patients are neurofibrillary tangles, senile plaques, and neuronal loss. The amount of these pathological changes is also believed to be related to the degree of dementia. Genetic evidence suggests that Aβ (β-amyloid protein) is deposited early and selectively in senile plaques in AD patients. This is not only associated with neuronal degeneration but also activates a series of pathological events, including activation of astrocytes and microglia, disruption of the blood-brain barrier, and changes in microcirculation, leading to the formation of senile plaques and neuronal apoptosis in the brain, making it a key factor in the development of AD.

[0003] Amyloid precursor protein (APP) is an integral membrane protein expressed in various tissues, secreted by cells and concentrated at neuronal synapses. APP can be broken down by α-, β-, and γ-proteinases; the sequential action of β- and γ-proteinases results in the breakdown of APP to produce Aβ. Aβ is hydrolyzed by β- and γ-proteinases into a 4-kDa protein fragment consisting of 39-43 amino acids. Accumulating in the cellular matrix, Aβ exhibits potent neurotoxicity, consisting of microtubules and diffuse plaques. Aβ exists in two forms: a 40-amino acid fragment, designated Aβ1-40, which is present in the brains of both healthy elderly individuals and AD patients. The 42-amino acid fragment, designated Aβ1-42, is primarily found in the brains of AD patients, with relatively low levels in the cerebrospinal fluid. Aβ circulates in the blood, cerebrospinal fluid, and interstitial fluid, mostly bound to chaperone protein molecules, with a small amount existing in a free state. In human cerebrospinal fluid and blood, the content levels of Aβ1-40 are 10 times and 1.5 times that of Aβ1-42, respectively. Aβ1-42 is more toxic and more likely to aggregate, thus forming the core of Aβ precipitation and triggering neurotoxic effects.

[0004] The Aβ1-42 / Aβ1-40 ratio in humans reflects brain Aβ pathology and can be used to early assess the risk of Alzheimer's dementia and mild cognitive impairment. Currently, cerebrospinal fluid analysis and positron emission tomography (PET) are commonly used to measure Aβ in the brain. However, CSF testing is invasive and has low patient acceptance, while the high cost of PET limits its application. Therefore, studying Aβ in peripheral fluids has attracted widespread attention. Aβ monomers can be cleared from the brain to the blood via various pathways, such as clearance via the blood-brain barrier and absorption and clearance from the CSF circulation. However, due to the very low concentrations of Aβ1-40 and Aβ1-42 in plasma, the varying sensitivity of different detection techniques can lead to significant discrepancies in results.

[0005] Currently available testing products, such as colloidal gold immunochromatography, suffer from large batch-to-batch variability and low sensitivity. They utilize physical adsorption, which can easily cause antigen / antibody detachment from the gold particle surface and label instability, resulting in only qualitative or semi-quantitative results. Enzyme-linked immunosorbent assays (ELISAs) are complex and time-consuming, requiring specialized personnel. Furthermore, numerous operational factors can influence the process, leading to false-positive and false-negative results. Chemiluminescence methods offer high sensitivity, good specificity, excellent stability, and are environmentally and human-safe. However, currently available products utilize a single sample type and can only measure marker concentrations in plasma samples. This single assay is time-consuming and produces poor reproducibility. Rapid and accurate results for Alzheimer's disease diagnosis and treatment, as well as accurate results to guide subsequent treatment, are pressing challenges in clinical diagnostic research. The development of a chemiluminescence detection kit for the combined detection of Aβ1-40 / Aβ1-42 with high sensitivity, short reaction time, simple operation, and high interference resistance is highly desirable. In view of this, the present invention provides a kit for jointly detecting markers Aβ1-40 and Aβ1-42, as well as a preparation method and application thereof. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a kit for the combined detection of the markers Aβ1-40 and Aβ1-42, as well as its preparation method and application. The purpose is to provide a chemiluminescent detection kit for the combined detection of Aβ1-40 / Aβ1-42 with high sensitivity, short reaction time, simple operation, and high interference resistance.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] In a first aspect, a kit for jointly detecting the markers Aβ1-40 and Aβ1-42 is provided, the kit comprising a first reagent and a second reagent; the first reagent comprising a magnetic bead working solution and magnetic microspheres coated with Aβ polyclonal antibodies, the magnetic microspheres coated with Aβ polyclonal antibodies comprising magnetic microspheres coated with Aβ1-40 polyclonal antibodies and magnetic microspheres coated with Aβ1-42 polyclonal antibodies; the second reagent comprising a labeled antibody diluent and an acridinium ester-labeled anti-Aβ antibody, the acridinium ester-labeled anti-Aβ antibody comprising an acridinium ester-labeled anti-Aβ1-40 antibody and an acridinium ester-labeled anti-Aβ1-42 antibody.

[0009] The principle of the present invention is as follows: a chemiluminescent immunosandwich method is used to detect the relative concentration of Aβ1-40 and Aβ1-42, a highly specific anti-Aβ polyclonal antibody is coupled to a magnetic microsphere, and acridinium ester is used to label the highly specific anti-Aβ1-40 antibody and anti-Aβ1-42 antibody respectively. The sample and the magnetic microspheres coated with Aβ1-40 polyclonal antibody, the magnetic microspheres coated with Aβ-42 polyclonal antibody, and the acridinium ester-labeled anti-Aβ1-40 antibody and the acridinium ester-labeled anti-Aβ1-42 antibody are added to the reaction tube respectively. After incubation, the Aβ1-40 and Aβ1-42 proteins in the sample and the Aβ1-40 and Aβ1-42 polyclonal antibodies coated on the magnetic beads are detected. The cloned antibody binds to the acridinium ester-labeled anti-Aβ1-40 antibody and anti-Aβ1-42 antibody simultaneously, forming an antibody-antigen-antibody sandwich complex. After the reaction is complete, the magnetic field attracts the magnetic beads and washes away unbound substances. Chemiluminescent substrates (pre-excitation solution / chemiluminescent substrate A solution, excitation solution / chemiluminescent substrate B solution) are added to the reaction tubes to generate chemiluminescence. The number of photons produced by the reaction is measured by a photomultiplier tube, and the number of photons produced is proportional to the analyte concentration in the sample. The amount of analyte in the sample is determined by a calibration curve, and the instrument automatically calculates the Aβ1-40 / Aβ1-42 protein ratio based on the concentration in the detected sample.

[0010] The beneficial effects of the present invention are as follows: the use of highly specific anti-Aβ1-40 antibodies and Aβ1-42 antibodies can simultaneously detect the values of Aβ1-40 and Aβ1-42 proteins in human serum, plasma or urine samples in one sampling, and calculate the ratio of Aβ1-40 / Aβ1-42; and the use of magnetic bead working solution and labeled antibody diluent further improves the sensitivity and stabilizer of the kit; the detection result is available within 8 minutes, the repeatability is within 4%, the minimum detection limit of Aβ1-40 is 1.08pg / ml, and the minimum detection limit of Aβ1-42 is 1.08pg / ml; at the same time, the kit of the present invention is more stable during transportation and storage, and can be kept stable for 2 years at 2-8°C for a long time. It can be kept stable for 13 days after aging test at 37°C. The calibrators and quality control products matched with the reagents do not need to be freeze-dried, and are in liquid form and ready for use. They can be kept stable for 2 years at 2-8°C for a long time.

[0011] On the basis of the above technical solution, the present invention can also be improved as follows.

[0012] Furthermore, the mass ratio of the Aβ1-40 polyclonal antibody to the magnetic microspheres coated with the Aβ1-40 polyclonal antibody is 25-50:50-100;

[0013] The mass ratio of the Aβ1-42 polyclonal antibody to the magnetic microspheres in the magnetic microspheres coated with the Aβ1-42 polyclonal antibody is 25-50:50-100.

[0014] Furthermore, the mass ratio of acridinium ester to anti-Aβ1-40 antibody in the acridinium ester-labeled anti-Aβ1-40 antibody is 5-9:30-50;

[0015] The mass ratio of acridinium ester to anti-Aβ1-42 antibody in the acridinium ester-labeled anti-Aβ1-42 antibody is 5-9:30-50.

[0016] Furthermore, each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH=7.4.

[0017] Furthermore, each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

[0018] Furthermore, the kit also includes a calibrator, a quality control product and a chemiluminescent substrate; the chemiluminescent substrate is a pre-excitation solution / chemiluminescent substrate A solution and an excitation solution / chemiluminescent substrate B solution.

[0019] In a second aspect, a method for preparing a kit for the combined detection of markers Aβ1-40 and Aβ1-42 comprises the following steps:

[0020] Preparation of the first reagent:

[0021] activating the magnetic microspheres in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding Aβ1-40 polyclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with Aβ1-40 polyclonal antibodies; and storing the magnetic microspheres coated with Aβ1-40 polyclonal antibodies in a magnetic bead working solution;

[0022] and / or, activating the magnetic microspheres in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding Aβ1-42 polyclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with Aβ1-42 polyclonal antibodies; and storing the magnetic microspheres coated with Aβ1-42 polyclonal antibodies in a magnetic bead working solution;

[0023] Preparation of the second reagent:

[0024] carrying out a labeling reaction between an anti-Aβ1-40 antibody and acridine in a luminescent labeling buffer to obtain an antibody labeling mixture; ultrafiltration of the antibody labeling mixture to obtain an acridinium ester-labeled anti-Aβ1-40 antibody; and storing the acridinium ester-labeled anti-Aβ1-40 antibody in a labeled antibody diluent;

[0025] And / or, the anti-Aβ1-42 antibody is labeled with acridine in a luminescent labeling buffer to obtain an antibody-labeled mixture; the antibody-labeled mixture is ultrafiltered to obtain an acridinium ester-labeled anti-Aβ1-42 antibody, and the acridinium ester-labeled anti-Aβ1-42 antibody is stored in a labeled antibody diluent.

[0026] Furthermore, the activators are all EDC; the mass ratio of the activators to the magnetic beads is 10:0.5-2;

[0027] The activation parameters are: room temperature, 20 min to 40 min;

[0028] The coupling reaction parameters are: room temperature, 3h to 8h.

[0029] The ratio of the magnetic microspheres coated with Aβ1-40 polyclonal antibodies to the magnetic bead working solution can be any ratio as long as it meets the requirements. The ratio of the magnetic microspheres coated with Aβ1-42 polyclonal antibodies to the magnetic bead working solution can be any ratio as long as it meets the requirements.

[0030] Furthermore, the parameters of the labeling reaction are: 37°C, 20 min to 40 min;

[0031] The ultrafiltration parameters are: 14000xg, 5-15min.

[0032] The dosage ratio of the acridinium ester-labeled anti-Aβ1-40 antibody to the labeled antibody diluent can be any ratio as long as it meets the requirements. The dosage ratio of the acridinium ester-labeled anti-Aβ1-42 antibody to the labeled antibody diluent can be any ratio as long as it meets the requirements.

[0033] In a third aspect, a kit for the combined detection of markers Aβ1-40 and Aβ1-42 is used to prepare a detection product for the Alzheimer's disease markers Aβ1-40 and Aβ1-42. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a calibration curve diagram of the reagent Aβ1-40 of the present invention;

[0035] Figure 2 This is a calibration curve diagram of the reagent Aβ1-42 of the present invention;

[0036] Figure 3 This is the linear correlation diagram of the reagent Aβ1-40 of the present invention;

[0037] Figure 4 This is a linear correlation diagram of the reagent Aβ1-42 of the present invention. DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0039] Example

[0040] 1. Materials and reagents.

[0041] (1) Antigen and Antibody:

[0042] Aβ1-40 polyclonal magnetic microsphere-coated antibody, Aβ1-42 polyclonal magnetic microsphere-coated antibody, anti-Aβ1-40 labeled antibody, and anti-Aβ1-42 labeled antibody were all purchased from Beijing Baixinyi Biotechnology Co., Ltd. at a concentration of 10 mg / mL; Aβ1-40 antigen and Aβ1-42 antigen were purchased from Beijing Aibosheng Biotechnology Co., Ltd. at a concentration of 5 mg / mL.

[0043] (2) Other reagents:

[0044] Acridinium ester (AE) was purchased from Helison (Xiamen) Biotechnology Co., Ltd., carboxyl magnetic beads were purchased from Nanjing Ruibeixi Biotechnology Co., Ltd., with a concentration of 10 mg / mL; pre-excitation solution / excitation solution (pre-excitation solution / chemiluminescent substrate A solution, excitation solution / chemiluminescent substrate B solution) were purchased from Guangzhou Hongrunkang Technology Development Co., Ltd.; disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, HEPES (4-hydroxyethylpiperazineethanesulfonic acid), sodium chloride, sucrose, 3-morpholinepropanesulfonic acid (MOPS), N-tris(hydroxymethyl)methylglycine (Tricine), dextran Sugar, sodium caseinate, hydrazine yellow, fruit green, lactose, Tween-20, Triton-100, glycerol, tris(hydroxymethyl)aminomethane hydrochloride (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), ethylene glycol, hydrochloric acid, sodium periodate, sodium borohydride and other conventional chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), bovine serum albumin (BSA), Proclin-300, polyethylene glycol 6000 and others were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0045] (3) Instruments:

[0046] SMART 500S fully automatic chemiluminescence immunoassay analyzer produced by Chongqing Cosmay Biotechnology Co., Ltd. (Yu Medical Device Registration No. 20192220077).

[0047] 2. Experimental method.

[0048] 2.1 Reagent preparation:

[0049] (1) Magnetic bead coating buffer:

[0050] Add 9.762 g of MES, 0.1 mL of Triton-100, and 5.00 g of disodium alkyl polyglycoside sulfosuccinate, and dilute to 1 L with purified water.

[0051] (2) Magnetic bead working solution:

[0052] HEPES (4-hydroxyethylpiperazineethanesulfonic acid) 11.91 g, sodium chloride 9.00 g, BSA 25.00 g, trehalose 100.00 g, glucose 80.00 g, Tween-20 1 mL, Proclin 300 1 mL, polyethylene glycol 20000 80.00 g; alkyl polyglycoside glycerol ether 7.2 g, adjust the pH to 7.4, and dilute to 1 L with purified water.

[0053] (3) Luminescent labeling buffer:

[0054] 13.435 g of disodium hydrogen phosphate dodecahydrate, 0.8265 g of sodium dihydrogen phosphate dihydrate, 2.3 g of α-sulfo fatty acid methyl ester (mono) sodium salt, 0.6 g of fatty alcohol sulfate (ester), and dilute to 1 L with purified water.

[0055] (4) Luminescent blocking buffer:

[0056] 12.1 g of tris(hydroxymethyl)aminomethane, 9.0 g of sodium chloride, 10.0 g of lysine, 100 g of BSA, 1.5 g of lauryl citrate monoester, 5.0 g of sorbitan polyoxyethylene ether tetraoleate, and dilute to 1 L with purified water.

[0057] (5) Acridinium ester labeled antibody dilution solution:

[0058] 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate, and 0.01 g of amaranth were diluted to 1 L with purified water.

[0059] (6) Calibrator diluent:

[0060] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5 g of cocamidopropylhydroxysulfobetaine, 1 mL of Proclin 300, and dilute to 1 L with purified water.

[0061] (7) Quality control diluent:

[0062] 8.19 g of 3-morpholinepropanesulfonic acid (MOPS), 9.00 g of sodium chloride, 26.00 g of sorbitol, 5.00 g of sodium caseinate, 5.0 g of glycine, 40 mL of β-mercaptoethanol, 1.45 g of magnesium chloride, 0.58 g of zinc chloride, 200 mL of glycerol, 5.2 g of cocamidopropylhydroxysulfobetaine, and 1 mL of Proclin 300 were added to make up to 1 L with purified water.

[0063] (8) Working cleaning fluid:

[0064] N-Tris(hydroxymethyl)methylglycine (Tricine) 9.76g, hydrochloric acid 0.70mL, sodium chloride 9.00g, methyl glucoside polyethylene glycol-20 ether sesquistearate 7.9g, Triton 1002.5mL, surfactant sorbitan sesquioleate 40g, Proclin 300 1mL, dilute to 1L with purified water.

[0065] 2.2 Preparation of magnetic microspheres:

[0066] (1) Antibody ultrafiltration:

[0067] Rinse: Pipette 500 μL of magnetic bead activation solution into the sample reservoir, close the lid, and centrifuge at 10,000 x g for 10 minutes. Aspirate the liquid from the filtrate receiver. Repeat the rinse cycle.

[0068] Ultrafiltration: Add 50 μg of antibody to the sample pool, cover it, and centrifuge at 10,000 x g for 10 minutes to concentrate the antibody in the sample pool;

[0069] Collection: Add 100 μL of magnetic bead activation solution (to make the antibody concentration 0.5 mg / mL) to the sample pool, pipette and re-dissolve (pipe and pipette more than 30 times to avoid bubbles), and transfer to a 1.5 ml centrifuge tube to obtain the dialyzed antibody for magnetic bead coating in the next step.

[0070] (2) Preparation of magnetic microspheres:

[0071] Oscillate the magnetic beads to resuspend them, add 1 mg of magnetic beads to a 1.5 mL centrifuge tube, add 200 μL of magnetic bead activation solution, and oscillate; place the centrifuge tube in a magnetic rack, allow the magnetic beads to adsorb to the side close to the magnet, and remove the solution; add 200 μL of magnetic microsphere activation solution and oscillate; wash the magnetic beads twice; remove the solution; add 60 μL of magnetic microsphere activation solution and oscillate;

[0072] Add 20 μL each of EDC (50 mg / mL) and NHS (50 mg / mL), and activate in a 37°C incubator with shaking for 30 minutes; aspirate the solution; add 200 μL of magnetic microsphere activation solution, wash twice; aspirate the solution;

[0073] Add 100 μL of activation solution containing 50 μg of antibody (the volume here increases with the amount of antibody), and shake the reaction in a 37°C incubator for 4 hours; place the centrifuge tube in a magnetic rack and aspirate the solution;

[0074] Add 200 μL of magnetic microsphere blocking solution, shake to reconstitute, and shake in a 37°C incubator for 2 hours. Place the centrifuge tube in a magnetic rack and aspirate the solution. Add 200 μL of magnetic microsphere diluent to wash and aspirate the solution. Repeat this step once. Add 100 μL of magnetic microsphere diluent and mix thoroughly. The antibodies mentioned above include Aβ1-40 polyclonal antibodies and Aβ1-42 polyclonal antibodies. Magnetic microspheres coated with Aβ1-40 polyclonal antibodies and magnetic microspheres coated with Aβ1-42 polyclonal antibodies were prepared, respectively.

[0075] Take magnetic microspheres and magnetic microsphere diluent, dilute them at a ratio of 1:19 between magnetic microspheres and magnetic bead working solution, and obtain the first reagent.

[0076] 2.3 Preparation of luminescent markers:

[0077] (1) Antibody labeling:

[0078] Take 1uL of acridinium ester mother solution (5mM) and add 9uL of DMF (N,N-dimethylformamide) to prepare the acridinium ester working solution (acridinium ester mother solution: anhydrous dimethylformamide = 1:9).

[0079] Add 50 μg of anti-Aβ1-40 labeled antibody to a centrifuge tube, add luminescent labeling buffer to 300 μL (the volume here increases with the amount of antibody), add 30 μL of acridine working solution, and shake in a 37°C incubator for 30 minutes;

[0080] Add 100 μL of luminescent blocking solution and shake in a 37° C. incubator for 30 minutes.

[0081] (2) Ultrafiltration of antibody after labeling:

[0082] Rinse: Pipette 500 μL of luminescent labeling buffer into the sample reservoir, close the lid, and centrifuge at 10,000 x g for 10 minutes. Aspirate the liquid from the filtrate receiver. Repeat the rinse cycle.

[0083] Ultrafiltration: Add labeled antibodies to the sample pool, cover the lid, and centrifuge at 14,000 x g for 10 minutes to concentrate the labeled antibodies in the sample pool;

[0084] Washing: Add 200 μL of acridinium ester labeled antibody diluent to the sample pool, centrifuge at 14,000 x g for 20 minutes, and concentrate the labeled antibody in the sample pool after washing;

[0085] Collection: Add 100 μL of acridinium ester-labeled antibody diluent to the sample pool, pipette and reconstitute (pipetting and reconstitute at least 30 times to avoid creating bubbles), transfer to a 1.5 ml centrifuge tube, add acridinium ester-labeled antibody diluent to 1 ml, and store at -20°C. (Luminescent marker storage solution, concentration: 0.05 mg / ml); this includes acridinium ester-labeled anti-Aβ1-40 antibody and acridinium ester-labeled anti-Aβ1-42 antibody, i.e., the second reagent.

[0086] (3) Preparation of luminescent marker working solution:

[0087] Take 5 mL of the luminescent marker stock solution stored at -20°C in the dark, add 495 mL of acridinium ester labeled antibody diluent, mix at 30 rpm / min, 2-8°C in the dark for 30 minutes, and then store at 2-8°C in the dark.

[0088] 2.4 Preparation of calibrators:

[0089] Take 0.5 μL of each of the Aβ1-40 and Aβ1-42 antigens and add them to 999 μL of Calibrator Diluent. Mix thoroughly at room temperature to prepare a 2.5 μg / mL stock solution. Then, use Calibrator Diluent to serially dilute the stock solution to six concentration points: 1500 ng / mL, 750 ng / mL, 250 ng / mL, 50 ng / mL, 25 ng / mL, and 0 ng / mL. Store at 2-8°C.

[0090] 2.5 Preparation of quality control products:

[0091] Take 0.5 μL of each of the Aβ1-40 and Aβ1-42 antigens and add them to 999 μL of Calibrator Diluent. Mix at room temperature to prepare a 2.5 μg / mL stock solution. Then, dilute the stock solutions to 200 ng / mL for Control Level 1 and 600 ng / mL for Control Level 2 using Control Diluent. Store at 2-8°C.

[0092] 2.6 Detection method:

[0093] (1) Testing procedures:

[0094] When loading the reagent bottle onto the machine for the first time, it should be gently turned over 30 times before opening, and the reagent bottle should be observed to ensure that the magnetic beads are completely suspended. If the magnetic beads are still attached to the bottom of the reagent bottle, continue to turn the reagent bottle until they are completely suspended. If they still cannot be suspended, the bottle of reagent cannot be used.

[0095] The system requires a sample volume of 10 μL per measurement. Before applying for a test, you should prepare all the materials required for the test and carefully read the chemiluminescence immunoassay user manual to obtain information on system operating procedures, sample management, usage precautions, maintenance, and other information. The main steps for applying for a test are as follows:

[0096] ① Enter the sample application interface and enter the sample number, sample rack number, position number, sample type, remarks and other information according to the test requirements;

[0097] ② Select the test item, select the sample type in the item options, enter the number of repetitions and other information;

[0098] ③ After preparing the test sample and placing it correctly, click the start button to start the test. The chemiluminescence immunoassay analyzer will perform the following operations in sequence:

[0099] a. The sample processing system transfers the sample rack to the sample suction position;

[0100] b. The cuvette loading and scheduling system loads the cuvette from the loading area to the sample loading position;

[0101] c. The sample injection system completes the sample injection and cleaning of the sample needle;

[0102] The reagent processing system provides the reagents required for the test, mixes each reagent, and sends it to the reagent aspiration position for aspiration; the sample injection system completes the reagent dispensing and cleaning of the reagent needle;

[0103] d. Mix the reaction mixture with the counter-liquid mixing system and incubate in the reaction plate;

[0104] e. After the incubation is completed, the magnetic separation system uses a cleaning solution to clean and separate the reaction mixture;

[0105] f. The substrate system injects the preheated substrate solution into the reaction cup after magnetic separation, mixes, incubates, and waits for light detection;

[0106] g. The optical reaction system sends each reaction cup to the signal collection position to collect signals for calculating the luminescence value;

[0107] h. Calculate the amount of analyte in the sample.

[0108] (2) Calibration:

[0109] Perform a calibration test using the included kit and calibrator. Before starting the kit calibration, scan the master curve in the kit's 2D barcode and import it into the system.

[0110] The chemiluminescence immunoassay analyzer uses the results of calibrator tests to adjust the master curve and generate the calibration curve for the current system. The instrument operating software includes a calibration check function that automatically verifies the validity of the calibration curve. If calibration fails, consider the instrument settings, status, location of the calibrators (place low-value calibrators first, then high-value calibrators), and expiration dates.

[0111] A valid calibration curve is required before all tests. Recalibration is required in the following situations:

[0112] ① Use a new batch of test kit;

[0113] ② The same batch of test kits has been used on the analyzer for more than 30 days;

[0114] ③After instrument maintenance;

[0115] ④The quality control value exceeds the specified range.

[0116] (3) Quality Control:

[0117] To ensure the reliability of test results, it is recommended that both high and low level quality control products be tested once every 24 hours. In addition, quality control testing is also recommended after each calibration test, reagent batch change, maintenance and troubleshooting.

[0118] The test values of quality control samples should be within the specified range. If they are outside the specified range, the user should check the detection system, such as the location, expiration date, storage method, calibration process, instrument performance and status, and recalibrate if necessary. If the results are still outside the range after retesting, please contact customer service.

[0119] (4) Calculation:

[0120] Using stored calibration data, the system software automatically determines the sample test results using a weighted four-parameter logarithmic curve (4PLC, Y-weighted) mathematical method. Results are given in ng / mL.

[0121] 2.7 Comparison reagents:

[0122] (1) Aβ1-40: Human Amyloid beta (aa1-40) Quantikine ELISA Kit produced by R&D Systems, USA;

[0123] (2) Aβ1-42: Human Amyloid beta (aa1-42) Quantikine ELISA Kit produced by R&D Systems, USA was selected.

[0124] 3 Experimental results.

[0125] 3.1 Establishment of calibration curve:

[0126] The establishment of the calibration curve is shown in Table 1 and Table 2. Figure 1 and Figure 2 .

[0127] Table 1 Aβ1-40 calibration results of the reagent of the present invention

[0128]

[0129]

[0130] Table 2 Calibration results of the reagent Aβ1-42 of the present invention

[0131]

[0132] 3.2 Minimum detection limit:

[0133] Using PBS buffer as a blank sample, the detection kit prepared by the present invention and the comparison reagent were simultaneously measured 20 times. The detection kit prepared by the present invention and the comparison value plus twice the standard deviation were respectively substituted into the fitting curve for regression. The results showed that the detection kit prepared by the present invention had a minimum detection limit of 1.08 pg / ml for Aβ1-40 and 1.08 pg / ml for Aβ1-42. The minimum detection limit of the comparison reagent for Aβ1-40 was 9.74 pg / ml, and the minimum detection limit of the comparison reagent for Aβ1-42 was 6.45 pg / ml. It can be seen that the method for preparing the test strips prepared by the present invention has high sensitivity. The results are shown in Table 3.

[0134] Table 3 Results of minimum detection limits of reagents

[0135]

[0136]

[0137] 3.3 Repeatability:

[0138] The kit of the present invention and the comparison reagent were tested 10 times at two levels of quality control, and the CVs were calculated. The results showed that the Aβ1-40 quality control level of the kit of the present invention was 2.847% at the 1CV level and 2.49% at the 2CV level; the Aβ1-40 quality control level of the comparison reagent was 11.19% at the 1CV level and 12.91% at the 2CV level; the Aβ1-40 quality control level of the kit of the present invention was 3.807% at the 1CV level and 2.87% at the 2CV level; and the Aβ1-40 quality control level of the comparison reagent was 13.69% at the 1CV level and 10.79% at the 2CV level. The method for preparing the test strips of the present invention has good reproducibility. The results are shown in Tables 4 and 5.

[0139] Table 4 Aβ1-40 repeatability results

[0140]

[0141]

[0142] Table 5 Aβ1-42 repeatability results

[0143]

[0144] 3.4 Linearity:

[0145] A high-dose sample containing Aβ1-40 at a concentration of 1250 pg / ml and Aβ1-42 at a concentration of 625 pg / ml was diluted with PBS buffer and mixed into dilution ratios of 1 / 512, 1 / 256, 1 / 128, 1 / 64, 1 / 32, 1 / 16, 1 / 8, 1 / 4, 1 / 2, and 1 / 10 (xi). The samples were tested using the test strips and comparison reagents prepared by the present invention. The test was repeated three times for each dilution, and the mean of the results was calculated (yi). The linear regression equation and linear regression coefficient were obtained with the dilution concentration (xi) as the independent variable and the mean value of the measurement result (yi) as the dependent variable. The results showed that for the reagent of the present invention, the linear correlation coefficient (r) of Aβ1-40 was not less than 0.9990 in the range of [10.0-125] pg / ml, and the absolute deviation was within ±0.5 ng / mL in the linear range of [3.0-40] ng / ml, and the relative deviation was within 1% in the linear range of [40-120] ng / ml. It can be seen that the preparation method of the test strip prepared by the present invention has good linearity. The results are shown in Tables 6 and 7. Figure 2 and 3 .

[0146] Table 6 Results of linearity test of the inventive reagent Aβ1-40

[0147]

[0148] Table 7 Linearity test results of the inventive reagent Aβ1-42

[0149]

[0150]

[0151] 3.5 Accuracy:

[0152] The high concentration reference substance (liquid A) was added to the low concentration reference substance (liquid B), and the volume ratio between the high concentration reference substance (liquid A) and the low concentration reference substance (liquid B) was not greater than 1:9. Each sample was measured 3 times and the average value was taken. According to the calculation results of formula (1), it can be seen that the recovery rates of Aβ1-40 and Aβ1-42 in the detection preparation method prepared by the present invention are respectively , and the accuracy is good. The results are shown in FIG.

[0153] Tables 8 and 9.

[0154]

[0155] Where: R is the recovery rate; V is the volume of solution A added; V0 is the volume of serum sample B; c is the detection concentration of serum sample after adding solution A; c0 is the detection concentration of serum sample B; c s is the concentration of solution A.

[0156] Table 8 Accuracy test results of the invented reagent Aβ1-40

[0157]

[0158] Table 9 Accuracy test results of the invented reagent Aβ1-42

[0159]

[0160]

[0161] 3.6 Stability:

[0162] 3.6.1 Long-term stability at 2-8°C:

[0163] (1) Three batches of the chemiluminescent immunoassay kit for the high-sensitivity combined detection of Alzheimer's disease markers Aβ1-40 and Aβ1-42 were stored in a finished product warehouse at 2-8°C in a dry environment. The temperature of the finished product warehouse was monitored using a thermometer and required to be between 2-8°C to ensure compliance with the product storage conditions during the stability study.

[0164] (2) Three batches of the reagent of the present invention stored in a finished product warehouse at 2-8°C were randomly sampled for testing at month 0, month 6, month 12, month 18, month 24, and month 25. The test items were: appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target value of quality control product, and uniformity of quality control product.

[0165] (3) Judgment of test content and results.

[0166] ①3.1 Appearance

[0167] a. All components of the test kit should be complete and intact, with no leakage of liquid; the Chinese packaging label should be clear and not worn;

[0168] b. The magnetic bead reagent component is a liquid containing brown solid particles. The magnetic beads are not caking, and the liquid is free of flocs and foreign matter.

[0169] c. Other reagent components are clear and uniform liquids without precipitation or flocs.

[0170] ② Accuracy: The recovery rate should be within the range of 85% to 115%;

[0171] ③Detection limit: no more than 1pg / mL;

[0172] ④ Linearity: The detection range of this kit is Aβ1-40 [2-1250] pg / mL, and the correlation coefficient R of its linear interval should be no less than 0.9900; Aβ1-42 [1-625] pg / mL, and the correlation coefficient R of its linear interval should be no less than 0.9900;

[0173] ⑤ Repeatability: coefficient of variation CV is not greater than 5%;

[0174] ⑥ Inter-batch difference: inter-batch coefficient of variation CV is not greater than 8%;

[0175] ⑦Accuracy of calibrator value assignment: The relative deviation of the results of the calibration sample detected by the chemiluminescence immunoassay analyzer calibrated with the calibrator is ±8%;

[0176] ⑧ Homogeneity of calibration material: homogeneity ≤ 8%

[0177] ⑨ Target value validity of quality control products: When quality control products are tested using a chemiluminescence immunoassay analyzer calibrated with the calibrator, the results should be within the target value range;

[0178] ⑩ Homogeneity of quality control products: Homogeneity ≤ 8%.

[0179] (4) Three batches of the kits of the present invention were stored at 2-8°C for 25 months, and all performance characteristics met the requirements. This indicates that the kits are stable when stored at 2-8°C for 25 months. Therefore, the kits of the present invention are stored at 2-8°C and have a shelf life of 24 months. The results are detailed in Tables 10-12.

[0180] Table 10 Real-time stability test results (first batch)

[0181]

[0182]

[0183] Table 11 Real-time stability test results (second batch)

[0184]

[0185]

[0186] Table 12 Real-time stability test results (third batch)

[0187]

[0188]

[0189] 3.6.2 37℃ aging stability:

[0190] (1) The main raw material of the detection kit of the present invention is the coated antibody. Since temperature has a great influence on the activity of the antibody, the higher the temperature, the more serious the attenuation of the antibody activity. The accelerated stability test design adopts the destruction test at 37°C. The stability study is carried out by examining the appearance, detection limit, accuracy, linearity, repeatability, batch difference, calibration value assignment accuracy, calibration uniformity, target value validity of the quality control product, and quality control product uniformity performance indicators, which are required to be no less than the product design requirements.

[0191] (2) Three batches of test kits, 4 boxes each, totaling 12 boxes, were placed in a 37°C incubator according to the commercial packaging for accelerated testing. The full performance (appearance, detection limit, accuracy, linearity, repeatability, inter-batch variation, accuracy of calibrator value assignment, uniformity of calibrator, validity of target values of quality control products, and uniformity of quality control products) of the test kits were tested on day 0, day 5, day 10, and day 13, respectively.

[0192] (3) Judgment of test content and results: consistent with the long-term stability at 2-8℃.

[0193] (4) After accelerating the test kit at 37°C for 13 days, all performances of the three batches of the test kit of the present invention met the requirements, indicating that the performance of the test kit is stable within 13 days of accelerating the test kit at 37°C. The results are detailed in Tables 13 to 15.

[0194] Table 13 Accelerated stability test results (first batch)

[0195]

[0196] Table 14 Accelerated stability test results (second batch)

[0197]

[0198] Table 15 Accelerated stability test results (third batch)

[0199]

[0200]

[0201] In summary, the kit of the present invention adopts the magnetic microparticle chemiluminescence-double antibody sandwich method. Compared with other methodologies such as enzyme-linked immunosorbent assay and fluorescent immunochromatography, it is simple to operate and can be tested on the machine directly after adding the sample. It can also simultaneously detect two indicators, Aβ1-40 and Aβ1-42. In addition, through the independently developed acridinium ester working solution, magnetic bead working solution and working cleaning solution, after adding the sample, the incubation time of the magnetic beads and acridinium ester with the sample is greatly shortened. It only takes 3 minutes to reach the optimal reaction time. The subsequent cleaning process only requires 3 cleanings, each lasting 30 seconds, to remove unreacted impurities. The entire test only takes 360 seconds. Most similar chemiluminescence product detection reagents on the market require about 900 seconds. Therefore, the detection time is greatly shortened, achieving the purpose of real-time detection, fully meeting the diagnosis and treatment needs of time-sensitive diseases, and providing patients with accurate test results. At the same time, the kit of the present invention is more stable during transportation and storage. It can be kept stable for 2 years at 2-8°C for a long time. It can be kept stable for 13 days in an aging test at 37°C. The calibrators and quality control products that come with the reagents do not need to be freeze-dried and are ready for use in liquid form. They can be kept stable for 2 years at 2-8°C for a long time.

[0202] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for the combined detection of markers Aβ1-40 and Aβ1-42, characterized in that: The kit includes a first reagent and a second reagent; the first reagent includes a magnetic bead working solution and magnetic microspheres coated with Aβ polyclonal antibodies, and the magnetic microspheres coated with Aβ polyclonal antibodies include magnetic microspheres coated with Aβ1-40 polyclonal antibodies and magnetic microspheres coated with Aβ1-42 polyclonal antibodies; the second reagent includes a labeled antibody diluent and an acridinium ester-labeled anti-Aβ antibody, and the acridinium ester-labeled anti-Aβ antibody includes an acridinium ester-labeled anti-Aβ1-40 antibody and an acridinium ester-labeled anti-Aβ1-42 antibody.

2. A kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 1, characterized in that: The mass ratio of the Aβ1-40 polyclonal antibody to the magnetic microspheres coated with the Aβ1-40 polyclonal antibody is 25-50:50-100; The mass ratio of the Aβ1-42 polyclonal antibody to the magnetic microspheres in the magnetic microspheres coated with the Aβ1-42 polyclonal antibody is 25-50:50-100.

3. A kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 1, characterized in that: The mass ratio of acridinium ester to anti-Aβ1-40 antibody in the acridinium ester-labeled anti-Aβ1-40 antibody is 5-9:30-50; The mass ratio of acridinium ester to anti-Aβ1-42 antibody in the acridinium ester-labeled anti-Aβ1-42 antibody is 5-9:30-50.

4. A kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 1, characterized in that: Each 1L of the magnetic bead working solution includes the following components: HEPES 11.91g, sodium chloride 9.00g, BSA 25.00g, trehalose 100.00g, glucose 80.00g, Tween-20 1mL, Proclin300 1mL, polyethylene glycol-20000 80.00g, alkyl polyglycoside glycerol ether 7.2g, pH=7.

4.

5. A kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 1, characterized in that: Each 1 L of the labeled antibody dilution solution includes the following components: 2.42 g of tris(hydroxymethyl)aminomethane, 1.41 mL of hydrochloric acid, 9.00 g of sodium chloride, 10.00 g of BSA, 0.5 mL of Proclin 300, 4.5 g of dextran, 7.4 g of isodecyl citrate monoester, and 0.01 g of amaranth.

6. A kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 1, characterized in that: The kit further comprises a calibrator, a quality control product and a chemiluminescent substrate; the chemiluminescent substrate is a pre-excitation solution / chemiluminescent substrate A solution and an excitation solution / chemiluminescent substrate B solution.

7. A method for preparing a kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to any one of claims 1 to 6, characterized in that: The steps include: Preparation of the first reagent: activating the magnetic microspheres in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding Aβ1-40 polyclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with Aβ1-40 polyclonal antibodies; and storing the magnetic microspheres coated with Aβ1-40 polyclonal antibodies in a magnetic bead working solution; and / or, activating the magnetic microspheres in a magnetic bead coating buffer using an activator to obtain activated magnetic beads; adding Aβ1-42 polyclonal antibodies to the activated magnetic beads for coupling reaction to obtain magnetic microspheres coated with Aβ1-42 polyclonal antibodies; and storing the magnetic microspheres coated with Aβ1-42 polyclonal antibodies in a magnetic bead working solution; Preparation of the second reagent: carrying out a labeling reaction between an anti-Aβ1-40 antibody and acridine in a luminescent labeling buffer to obtain an antibody labeling mixture; ultrafiltration of the antibody labeling mixture to obtain an acridinium ester-labeled anti-Aβ1-40 antibody; and storing the acridinium ester-labeled anti-Aβ1-40 antibody in a labeled antibody diluent; And / or, the anti-Aβ1-42 antibody is labeled with acridine in a luminescent labeling buffer to obtain an antibody-labeled mixture; the antibody-labeled mixture is ultrafiltered to obtain an acridinium ester-labeled anti-Aβ1-42 antibody, and the acridinium ester-labeled anti-Aβ1-42 antibody is stored in a labeled antibody diluent.

8. The method for preparing a kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 7, characterized in that: The activators are all EDC; the mass ratio of the activators to the magnetic beads is 10:0.5-2; The activation parameters are: room temperature, 20 min to 40 min; The coupling reaction parameters are: room temperature, 3h to 8h.

9. The method for preparing a kit for the combined detection of markers Aβ1-40 and Aβ1-42 according to claim 7, characterized in that: The parameters of the labeling reaction are: 37°C, 20min-40min; The ultrafiltration parameters are: 14000xg, 5-15min.

10. Use of a kit for the combined detection of markers Aβ1-40 and Aβ1-42, characterized in that: The kit for jointly detecting the markers Aβ1-40 and Aβ1-42 according to any one of claims 1 to 6 is used to prepare a detection product for the Alzheimer's disease markers Aβ1-40 and Aβ1-42.

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