CN114814240B

CN114814240B - Beta amyloid protein detection kit

Info

Publication number: CN114814240B
Application number: CN202210598463.7A
Authority: CN
Inventors: 汤健; 安源; 官志超
Original assignee: Suzhou Yuce Biotechnology Co ltd
Current assignee: Suzhou Yuce Biotechnology Co ltd
Priority date: 2022-05-30
Filing date: 2022-05-30
Publication date: 2023-04-28
Anticipated expiration: 2042-05-30
Also published as: CN114814240A

Abstract

The invention relates to a kit for diagnosing Alzheimer disease markers, in particular to a kit for detecting beta-amyloid, and further relates to application of fluorescent dye and specific magnetic beads in preparation of the kit for detecting beta-amyloid. The kit is used for detecting the beta-amyloid based on a single-molecule detection technology and a double-antibody sandwich method, and also relates to a single-molecule detection system for detecting the beta-amyloid, which can realize high sensitivity while rapidly detecting, has small CV value, and has low cost and can accurately determine the concentration of the beta-amyloid.

Description

Beta amyloid protein detection kit

Technical Field

The invention relates to the field of immunodetection, in particular to a kit for detecting beta-amyloid, application of fluorescent dye and magnetic beads in preparation of the kit for detecting beta-amyloid and a single-molecule detection system for detecting beta-amyloid.

Background

Alzheimer's Disease (AD) is a common form of Alzheimer's disease, and no effective therapy exists at present. The patient is painful to the patient, and can have a negative effect on the family and even the whole society. The exact mechanism of the onset of AD has not been fully understood so far. However, a great deal of research supports that amyloid 1-Peptide (A1) produced by amyloid precursor protein plays an important role in the pathology of AD. Abnormal accumulation of amyloid beta in the brain of a patient often results in the formation of brain fibrous tissue tangles (neurofibrillary tangle, NFT) and Senile Plaques (SP) outside the brain cells. Therefore, NFT and SP are also considered as a basis for pathological diagnosis of AD. Beta Amyloid (beta-Amyloid) is a polypeptide formed from the precursor substance beta Amyloid precursor protein (aβ) by enzymatic hydrolysis to 40 to 43 amino acids. Normally, aβ is expressed only in very small amounts, with low concentrations of aβ having a trophic effect on undifferentiated, immature neurons and high concentrations of aβ having a toxic effect on differentiated mature neurons.

Early accumulation of amyloid beta inside brain nerve cells was studied and found that Alzheimer's disease has occurred when amyloid beta accumulation has not occurred outside brain nerve cells. This finding may be useful in the early diagnosis and treatment of Alzheimer's disease, thereby minimizing the delay of progression. It is important to develop a human amyloid beta detection reagent or kit, particularly a kit capable of realizing high sensitivity in a short detection time and having a small CV value, and capable of accurately measuring the concentration of amyloid beta (see patent document 1).

In healthy normal humans, the concentration of Abeta 1-40 in blood ranges from 200pg/ml to 400pg/ml and the concentration of Abeta 1-42 in plasma ranges from 15pg/ml to 30pg/ml. However, in the case of AD, Aβ1-42 levels decrease and are often undetectable by currently available detection techniques. Furthermore, an interventional strategy based on depleting the formation of aβ1-42 requires a method to determine the decline of aβ1-42. Thus, there is a need for accurate and precise quantification of low concentrations of amyloid in blood.

Compared with the existing ELISA and chemiluminescence methods, the single-molecule detection technology has extremely high sensitivity due to the breakthrough of the principle, so that the single-molecule technology is applied to the research to detect the biomarker. Patent document 2 discloses a method and a composition for high sensitivity detection of single molecules, and specifically discloses an aβ1-40 and aβ1-42 (amyloid beta 40 and 42) test in example 22 thereof, with detection lower limits of 0.2pg/mL and 0.1pg/mL, respectively. However, this method requires the use of specific single-molecule detectors including an electromagnetic radiation source, an inspection space and an electromagnetic radiation detector, and in particular, it is necessary to use a total internal reflection microscope, a near field microscope and an airy disk focus detection device that can realize a single-molecule fluorescence response, which is costly. Moreover, the detection time of the method is as long as 4 hours, and the requirement of clinical rapid detection cannot be met. In addition, the kit does not contain detection antibodies marked with specific fluorescent dyes.

The inventors of the present application proposed a single molecule detection method (see patent document 3) different from the principle of patent document 2 in that single molecule detection was performed on cTnI antigen, IL-6 antigen, DNA, etc. based on the double antibody sandwich method using in-situ signal enhanced nanoparticles and magnetic beads, but how to detect β amyloid was not mentioned, nor was the influence of the diameter, concentration, etc. of magnetic beads on the detection of β amyloid focused.

Among the mainstream immunological platforms available in vitro diagnosis, electrochemiluminescence is the detection platform with highest sensitivity, and the lower limit of detection of the kit for detecting Abeta 42 in cerebrospinal fluid (Cerebrospinal Fluid, CSF) is 120pg/mL (see China medical network med.china.com.cn).

Prior art literature

Patent document 1: CN102192985a;

patent document 2: CN102016552a;

patent document 3: CN111771126A

Disclosure of Invention

Problems to be solved by the invention

Patent document 1 discloses a beta amyloid kit comprising an antibody-coated panel, a calibrator, a concentrated biotinylated antibody, etc., which is low in sensitivity (up to 7 pg/mL) based on a conventional ELISA method. Patent document 2 mentions that aβ1-40 and aβ1-42 are measured by a single-molecule detection technique, but it requires detection using optical, electric and magnetic detection devices (such as a total internal reflection microscope, a near field microscope, an airy focal detection device, etc.) with extremely high precision, which is costly and takes too long. It is known that when detection is performed for a long detection time (particularly, incubation time), the detection sensitivity can be improved, and a certain trade-off relationship exists between shortening the detection time and improving the detection sensitivity, so that it is difficult to achieve both the shortening of the detection time and the improvement of the sensitivity. Patent document 3 does not mention how to detect amyloid β, nor does it concern the influence of the diameter, concentration, etc. of magnetic beads on amyloid β detection. The inventors of the present application tried to use the single molecule detection method of patent document 3 for development of a beta amyloid kit, but since beta amyloid is completely different from cTnI antigen, IL-6 antigen, DNA, etc. described in the document, there is a low sensitivity in the case of applying a detection system of cTnI antigen, IL-6 antigen, DNA, etc. to detection of beta amyloid, the lower limit of detection is only 100pg/mL.

In order to solve the above problems, the applicant repeatedly carried out optimization of the type of capture antibody, the type of detection antibody, the type of fluorescent dye, the pH of the system, the type of group of magnetic beads, etc., but the sensitivity was still low and the lower detection limit could only reach 80pg/mL. Regarding the working concentration of the magnetic beads, in the field of immunodetection, a concentration of 0.5mg/mL or more is generally selected in order to improve the capturing efficiency. Therefore, the inventors of the present application have similarly set the working concentration of the magnetic beads to 0.5mg/mL or more in the initial optimization process (this working concentration is also used in patent document 3). However, as described above, the inventors of the present application have found that when a kit is constructed and the concentration of amyloid β is measured using a conventional common system, the lower detection limit can be only 80pg/mL, and the detection requirements for Alzheimer's disease cannot be satisfied.

The present invention has been made in view of the above-described problems, and an object thereof is to provide a beta amyloid kit capable of realizing high sensitivity and small CV value while rapidly detecting.

Means for solving the problems

The inventors of the present application have repeatedly conducted intensive studies to solve the above-mentioned problems, and as a result, have found a beta amyloid kit which can realize rapid detection and high sensitivity and a small CV value.

One technical scheme of the application is as follows.

A kit for detecting amyloid beta, comprising magnetic beads coated with a capture antibody and a detection antibody labeled with a fluorescent dye,

wherein the capture antibody and the detection antibody are capable of binding to different sites of amyloid beta respectively,

the fluorescent dye contains fluorescent material and carrier, and has particle size of 180-450 nm,

the diameter of the magnetic beads is 0.3-5 mu m, and the working concentration of the magnetic beads is more than 0.01mg/mL and less than 0.5mg/mL.

Preferably, the magnetic beads have a diameter of 0.5 to 2.2. Mu.m, preferably 0.8 to 2. Mu.m, more preferably 1 to 1.5. Mu.m.

Preferably, the working concentration of the magnetic beads is 0.05-0.25 mg/mL, preferably 0.08-0.22 mg/mL, preferably 0.1-0.2 mg/mL.

Preferably, the surface of the magnetic beads is modified with one or more of carboxyl groups, amino groups or tosyl groups, preferably with carboxyl groups, wherein the carboxyl group concentration is 15 to 30 microequivalents per gram (μeq/g) when the surface of the magnetic beads is modified with carboxyl groups.

Preferably, the kit further comprises a beta amyloid calibrator and a beta amyloid control, the beta amyloid being aβ1-40, aβ1-42.

Preferably, the fluorescent material is fluorescein, rhodamine, coumarin or quantum dot, and the carrier is silicon dioxide, polyacrylamide or polystyrene.

Another technical solution of the present application is as follows.

The application of fluorescent dye and magnetic beads in preparing a kit for detecting beta amyloid, wherein the fluorescent dye contains fluorescent material and carrier, has the particle size of 180-450 nm,

Still another embodiment of the present invention is as follows.

A single molecule detection system for detecting amyloid beta comprising the above-described kit, and an optical imaging device,

the optical imaging device comprises a light source and an optical signal acquisition unit, and the detection system does not comprise a total internal reflection microscope, a near-field microscope and an airy disk focusing detection device, and does not comprise a micro-reaction cavity with the volume of nano-liter level, pico-liter level or flying-liter level.

Effects of the invention

The kit for detecting the beta-amyloid can realize high sensitivity and small CV value while rapidly detecting the beta-amyloid. The inventors of the present application have found that when used for detection of amyloid beta, in some embodiments, the sensitivity to aβ1-42 may reach 0.2pg/mL, and in some embodiments, the sensitivity to aβ1-42 may reach 0.05pg/mL. In some embodiments, the sensitivity to Abeta 1-40 may be up to 0.2pg/mL, in some embodiments, the sensitivity to Abeta 1-40 may be up to 0.15pg/mL, and in other embodiments, the sensitivity to Abeta 1-40 may be up to 0.07pg/mL. In addition, the detection of the beta amyloid can be realized in a short time (namely, only about 5 minutes of incubation time is needed, and about 5 minutes and 40 seconds are needed from sample injection to result output) on the basis of ensuring higher sensitivity, so that the clinical detection time is greatly shortened. In addition, the CV values are all less than 10%, and in some embodiments, the detection CV values for Abeta 1-40 are 7 or less, 6 or less, or even 2 or less, and the detection CV values for Abeta 1-42 are 7 or less, 6 or less, or even 2 or less. In addition, the invention can detect amyloid beta by taking serum and the like as samples, and does not need to use samples taken from cerebrospinal fluid, so that the damage to patients is small.

The sensitivity was significantly improved as compared with the conventional ELISA method-based kit of patent document 1. Compared with the detection system based on a specific microscope of patent document 2, the detection system of the present invention achieves equivalent or more excellent detection sensitivity without requiring use with a specific microscope such as a total internal reflection microscope, a near field microscope, and an airy disk focus detection device at a significantly reduced cost, and particularly for aβ1-40, the method of the present invention can achieve a detection sensitivity of at least 0.07 pg/mL. The technique of patent document 2 requires a detection time of up to 4 hours, and the method of the present invention can significantly shorten the detection time, specifically, the incubation time is only about 5 minutes, and the time from sample injection to result output (i.e., detection time) is only 5 minutes and 40 seconds. Compared with patent document 3, the present invention breaks through the general knowledge of the conventional single-molecule detection technique, and can perform single-molecule detection of amyloid β with remarkably excellent sensitivity by adjusting the diameter and working concentration of the magnetic beads.

Drawings

FIG. 1 is a standard curve obtained in example 1, wherein the ordinate is the number of single molecule signals;

FIG. 2 is a standard curve obtained in example 18.

Detailed Description

< kit >

A first embodiment of the present application relates to a kit for detecting amyloid-beta, characterized by comprising magnetic beads coated with a capture antibody, and a detection antibody labeled with a fluorescent dye (the kit may further comprise a amyloid-beta calibrator and an amyloid-beta controller),

In the present invention, it is important that the magnetic beads have a diameter of 0.3 to 5. Mu.m, and a working concentration of 0.01mg/mL or more and less than 0.5mg/mL. The diameter is required to be strictly controlled within a range of 0.3 to 5. Mu.m, for example, 0.4. Mu.m, 0.5. Mu.m, 0.6. Mu.m, 0.7. Mu.m, 0.8. Mu.m, 0.9. Mu.m, 1. Mu.m, 1.1. Mu.m, 1.2. Mu.m, 1.3. Mu.m, 1.4. Mu.m, 1.5. Mu.m, 1.6. Mu.m, 1.7. Mu.m, 1.8. Mu.m, 1.9. Mu.m, 2.0. Mu.m, 2.1. Mu.m, 3.5. Mu.m, 4. Mu.m, 4.5. Mu.m, preferably 0.5 to 2.2. Mu.m, more preferably 0.8 to 2. Mu.m, still more preferably 1 to 1.5. Mu.m. The working concentration is required to be strictly controlled within a range of 0.01mg/mL or more and less than 0.5mg/mL, for example, 0.02mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL, 0.09mg/mL, 0.10mg/mL, 0.11mg/mL, 0.12mg/mL, 0.13mg/mL, 0.14mg/mL, 0.15mg/mL, 0.16mg/mL, 0.17mg/mL, 0.18mg/mL, 0.19mg/mL, 0.20mg/mL, 0.21mg/mL, 0.22mg/mL, 0.23mg/mL, 0.24mg/mL, 0.3mg/mL, 0.35mg/mL, 0.40mg/mL, 0.45mg/mL, 0.48mg/mL, preferably 0.05 to 0.25mg/mL, more preferably 0.08 to 0.22mg/mL, still more preferably 0.22mg to 0.22 mg/mL.

The magnetic beads with proper size can be obtained by purchasing commercial products, and proper working concentration of the magnetic beads can be obtained by adjusting the addition volume of buffer solution, magnetic bead coupling solution, magnetic bead sealing solution, magnetic bead preserving solution and the like.

The magnetic beads are used as solid phase carriers in immunodetection for separation and cleaning of detection samples and reagents, and in the prior art, antibody types, fluorescent dyes and the like are generally considered to have great influence on detection sensitivity, but the size and working concentration of the magnetic beads cannot have very remarkable influence on the sensitivity. However, the inventors of the present application found that in the kit system for detecting amyloid β, a remarkably excellent technical effect can be obtained when the size and working concentration of the magnetic beads are controlled within the specific range of the present invention.

The surface of the magnetic beads is modified with reactive functional groups capable of covalent coupling with the antibody, such as one or more of hydroxyl, carboxyl, amino, succinimidyl ester, sulfonyl (e.g., tosyl) and derivatives thereof, preferably carboxyl, amino, tosyl, more preferably carboxyl. When the surface of the magnetic beads is modified with carboxyl groups, the carboxyl group concentration is 15 to 30 microequivalents/g (μeq/g), preferably 20 to 25 microequivalents/g, more preferably 22 to 24 microequivalents/g. The carboxyl group concentration can be obtained by immersing the beads in a solvent or the like and then titrating with an alkaline solution such as a sodium hydroxide solution.

The magnetic beads are washed and activated before being coupled with the capture antibodies, and washed and blocked after being coupled with the capture antibodies.

The capture antibodies are classified according to antibody specificity characteristics, can be one or two of polyclonal antibodies and monoclonal antibodies, are classified according to sources, and can be one or more of murine antibodies, rabbit antibodies, sheep antibodies and alpaca antibodies.

The detection antibody is classified according to the specificity of the antibody, can be one or two of polyclonal antibody and monoclonal antibody, is classified according to the source, and can be one or more of murine antibody, rabbit antibody, sheep antibody and alpaca antibody.

The fluorescent dye of the present invention is a material that enhances the fluorescent signal in situ (in-situ) to a level that can be detected by conventional optical imaging equipment, and contains both a fluorescent material and a carrier.

In the fluorescent dye, the carrier plays a very important role, for example, more fluorescent materials can be combined, so that a luminous signal is stronger; providing sites for functional modification, being capable of combining a large amount of antibodies and improving the reactivity; the method provides possibility for realizing single-molecule detection by a conventional fluorescence microscope, and single-molecule detection cannot be realized without a carrier. The carrier is classified according to materials and may be one or more of silica, polystyrene, polyacrylamide, poly (methyl (meth) acrylate), dextran, agarose, and an inorganic metal compound. The carrier is classified according to structure and can be one or more of a hollow structure, a core-shell structure, a porous structure, an alloy structure and a hydrogel structure. Among them, from the viewpoint of uniformly distributing the fluorescent material and making the fluorescent material high in brightness, the carrier is preferably silica, polyacrylamide, polystyrene, and dextran.

The fluorescent material in the fluorescent dye is also necessary to achieve single molecule detection. The fluorescent material can be one or more of fluorescent dye molecules, rare earth elements, rare earth chelates, fluorescent proteins, quantum dots and up-conversion nanoparticles. The fluorescent material is preferably fluorescein (such as fluorescein isothiocyanate), rhodamine (such as rhodamine green and rhodamine B), coumarin, quantum dots (such as CdS, cdSe, cdTe, znSe), rare earth elements (such as Eu and Ce), complex thereof and the like. The fluorescent material is adsorbed or coated on the surface or inside of the carrier through one or more of covalent modification, chelation, space coating, hydrophobic effect and electrostatic adsorption effect. From the viewpoint of facilitating optical imaging recognition and improving sensitivity, it is preferable that the fluorescent material is uniformly wrapped inside the carrier.

In the present application, the fluorescent dye is preferably a fluorescent particle formed by coating a fluorescent dye molecule (such as fluorescein) with silicon dioxide, a fluorescent particle formed by coating a fluorescent dye molecule (such as fluorescein) with polyacrylamide, a fluorescent particle formed by coating a quantum dot with polystyrene, a fluorescent particle formed by coating a rare earth element or a rare earth chelate with polystyrene, a fluorescent particle formed by coating a fluorescent protein with dextran, a fluorescent particle formed by coating a quantum dot with cross-linked agarose, or the like.

In the present invention, the particle size of the fluorescent dye is controlled to be within a range of 180 to 450nm, for example, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm. If the particle size of the fluorescent dye is less than 180nm, for example 150nm, no signal can be detected in the conventional optical imaging device, and if the particle size is more than 450nm, for example 460nm, the detection sensitivity is low, and it is difficult to achieve clinically required sensitivity. The particle size may be a primary particle size or a secondary particle size. The secondary particle size refers to the particle size formed after the primary particles and the secondary particles are agglomerated.

The detection antibody labeled with the fluorescent dye can be prepared by the following steps.

(1) Preparation of a dilution of a fluorescent dye

Diluting the fluorescent dye to a specified concentration by using a buffer solution a (such as carbonate buffer solution, phosphate buffer solution PBS or borate buffer solution);

(2) Preparation of the labeling incubation

The activator was dissolved in PBS buffer to prepare a buffer solution in which the activator was dissolved. Adding the solution into diluted fluorescent dye, mixing uniformly, centrifuging, and adding buffer solution to prepare a labeling incubation liquid for later use.

(3) Preparation of marking fluid

And adding the detection antibody stock solution into the labeling incubation liquid, uniformly mixing and incubating for 1 hour.

(4) Sealing the marking working fluid

Adding the sealing liquid into the marking working liquid, uniformly mixing and incubating for 1 hour.

(5) Washing marking working fluid

(6) Preserving marking working fluid

Adding the marked dispersion liquid into a centrifuge tube, uniformly mixing, sucking the marked preservation liquid according to the total volume requirement of production, adding the marked preservation liquid into the centrifuge tube, uniformly mixing, and placing the mixture into a refrigerator for preservation at 2-8 ℃.

The working concentration is the concentration at the time of actual use. Many solutions are prepared into mother solutions with high concentration for preservation, and the mother solutions are diluted according to the need for practical use.

In the case where the modifying group on the surface of the magnetic bead is a carboxyl group, the activator may be one or more of N-hydroxysuccinimide (NHS), sulfoNHS, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), and the like, and in the case where the modifying group is an amino group, the activator may be succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), sulfoo-SMCC, and the like.

Taking a kit for detecting Abeta 1-40 as an example, the quality control product can comprise Abeta 1-40 quality control product level 1 (25+/-2.5 pg/mL) and Abeta 1-40 quality control product level 2 (500+/-50 pg/mL), and the calibrator can comprise Abeta 1-40 calibrator level C0 (0 pg/mL), abeta 1-40 calibrator level C1 (50+/-5 pg/mL) and Abeta 1-40 calibrator level C2 (1000+/-100 pg/mL). Taking a kit for detecting Abeta 1-42 as an example, the quality control product can comprise Abeta 1-42 quality control product level 1 (20+/-2 pg/mL) and Abeta 1-42 quality control product level 2 (100+/-10 pg/mL), and the calibration product can comprise Abeta 1-42 calibration product level C0 (0 pg/mL), abeta 1-42 calibration product level C1 (10+/-1 pg/mL) and Abeta 1-42 calibration product level C2 (1000+/-100 pg/mL)

In addition, in the kit, the necessary but not provided articles include a reaction cup, a washing liquid, a waste cup box, a flow cell care solution, and the like.

< use of fluorescent dye and magnetic bead in preparation of kit for detecting amyloid beta >

The invention also relates to the use of a fluorescent dye and magnetic beads for the preparation of a kit for detecting amyloid beta, wherein the details of the fluorescent dye and magnetic beads are as described in column < kit >. The inventor of the application applies specific fluorescent dye and magnetic beads to single-molecule immunodetection of beta amyloid for the first time, and can realize high sensitivity and variation Coefficient (CV) lower than 10% based on a single-molecule detection technology and a double-antibody sandwich technology at the same time of rapid detection.

< Single molecule detection System for detecting amyloid beta >

The detection system comprises the kit and optical imaging equipment, wherein the optical imaging equipment comprises a light source and an optical signal acquisition unit, and the detection system does not comprise a total internal reflection microscope, a near-field microscope and an airy disk focusing detection equipment, and does not comprise a micro-reaction cavity with the volume of nanoliter level, picoliter level or flying level.

In the present invention, the optical imaging apparatus mainly includes the following components: an excitation light source, an objective lens, an optical filter, a photosensitive element, a data acquisition module, a data processing module, and a dichroic mirror (if a microscope is arranged, the dichroic mirror may be omitted). Wherein the excitation light source is an optical emission device for exciting the reacted sample to emit an optical signal. The objective lens is used for signal acquisition and amplification of a sample to be detected. The dichroic mirror is used for reflection of the excitation light path and collection of the sample optical signal. The filter is used for filtering the excitation light wave band and filtering the sample emission light signal. The photosensitive element is used for collecting optical signals of a sample. The data acquisition module is configured to receive the optical signal captured by the photosensitive element and convert the optical signal into a digital signal. The data processing module is configured for conversion of digital signals and formation and processing of optical images.

In some embodiments of the apparatus, the excitation light source comprises one or more of a gas laser, a solid state laser, a semiconductor laser, a liquid laser, and a free electron laser. In some embodiments of the apparatus, the objective lens is classified by magnification, including one or more of 1X, 2X, 4X, 5X, 10X, 20X, 40X, 50X, and 100X; the objective lens is classified according to field curvature correction and comprises a plane objective lens or a curved objective lens. In some embodiments of the apparatus, the photosensitive element comprises one or both of a CCD (Charge Coupled Device ) or CMOS (Complementary Metal-Oxide Semiconductor, complementary metal oxide semiconductor).

The optical imaging device provided by the invention is a conventional optical imaging device (namely, an optical imaging device which does not break through the optical diffraction limit), and expensive imaging devices which break through the optical diffraction limit, such as a total internal reflection fluorescence microscope, an epifluorescence microscope, a scanning near-field optical microscope, a confocal fluorescence microscope and the like, are not needed.

Examples

The present invention will be described in further detail with reference to examples and comparative examples, but the present invention is not limited thereto. For the measurement of the particle diameter of the fluorescent dye, single-molecule imaging, and standard curve drawing method, see the examples section of patent document 3 filed by the applicant.

1. Measurement of particle size of magnetic beads

The particle size of the magnetic beads is determined by microscopy or a nanosize meter (e.g., accuSizer 780A7000 SIS).

2. Determination of carboxyl concentration of magnetic beads

The beads were placed in a solvent and titrated with sodium hydroxide solution to determine the unit of microequivalents per gram (μeq/g).

3. Main components of the kit

Taking 50 human parts/box of beta amyloid 1-40 (Abeta 1-40) as an example, the main composition is as follows.

Reagent bottle (two cavities)

Cavity 1: 1.4mL of a magnetic bead solution coated with the Abeta 1-40 capture antibody;

cavity position 2: aβ1-40 detection antibody solution labeled with fluorescent dye, 0.7mL.

Quality control product:

aβ1-40 quality control level 1 (25.+ -. 2.5 pg/mL). Times.3

Aβ1-40 quality control level 2 (500.+ -.50 pg/mL). Times.3

Calibration material:

aβ1-40 calibrator level C0 (0 pg/mL). Times.1

Aβ1-40 calibrator level C1 (50.+ -. 5 pg/mL). Times.1

Aβ1-40 calibrator level C2 (1000.+ -. 100 pg/mL). Times.1

Taking 50 human parts/box of beta amyloid 1-42 (Abeta 1-42) as an example, the main composition is as follows.

Reagent bottle (two cavities)

Cavity 1: 1.4mL of a magnetic bead solution coated with the Abeta 1-42 capture antibody;

cavity position 2: aβ1-42 detection antibody solution labeled with fluorescent dye, 0.7mL.

Quality control product:

aβ1-42 quality control level 1 (20.+ -. 2 pg/mL). Times.3

Aβ1-42 quality control level 2 (100.+ -. 10 pg/mL). Times.3

Calibration material:

aβ1-42 calibrator level C0 (0 pg/mL). Times.1

Aβ1-42 calibrator level C1 (10.+ -. 1 pg/mL). Times.1

Aβ1-42 calibrator level C2 (1000.+ -. 100 pg/mL). Times.1

Preparation example 1: (preparation of kit 1 containing magnetic beads having a diameter of 1.5 μm and a working concentration of 0.15 mg/mL)

1. Experimental Components

Carboxyl activated magnetic beads (purchased from Merck), aβ1-40 capture antibody (self-made), aβ1-40 detection antibody (self-made), silane coupling Agent (APTES), silica microspheres coated with Fluorescein Isothiocyanate (FITC), polyacrylamide coated with fluorescent dye, serum sample to be tested, PBS buffer, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), microsphere preservation solution, sample dilution, PBS wash, label blocking solution (0.01% nacl, 0.5% bsa in 10mm PBS, ph=7.4), label dispersion (0.012% nacl, 0.2% bsa in 10mm PBS, ph=7.4), label preservation solution (0.0088% nacl, 0.1% bsa in 10mm PBS, ph=7.4), magnetic bead washing solution (PBST solution), cross-linker dissolution solution (DMSO), magnetic bead coupling solution (TRIS buffer), magnetic bead blocking solution (0.01% nacl, 0.2% and 0.02% pcbsa in 10mm PBS, ph=7.02% PBS, ph=7.4), and pcb in 10mm PBS, ph=7.02% PBS.

2. Preparation of magnetic bead solution coated with Abeta 1-40 Capture antibody

(1) 100. Mu.L of 5mg/mL carboxyl modified beads (from Merck, diameter 1.5 μm, carboxyl concentration 22 microequivalents/gram) were washed 5 times with 15mM PBS buffer and the buffer removed.

(2) Taking 80 mug of Abeta 1-40 capture antibody, adding the Abeta 1-40 capture antibody into the magnetic beads of the step (1), uniformly mixing, incubating the mixture on a roller incubator for 3 hours at the temperature of 25 ℃, and rotating the mixture at the speed of: 80rpm/min.

(3) Adding magnetic bead cleaning solution for cleaning, mixing, and placing on a magnetic rack for magnetic separation.

(4) Adding magnetic bead sealing liquid into the coating tube, and incubating for 1h on a roller incubator at the rotating speed: 80rpm/min, and the supernatant was removed after magnetic separation.

(5) And adding the magnetic bead preservation solution into a centrifuge tube, and uniformly mixing to obtain a magnetic bead solution coated with the Abeta 1-40 capture antibody, wherein the working concentration of the magnetic beads is 0.15mg/mL.

3. Preparation of A beta 1-40 detection antibody solution marked with fluorescent dye

(1) Preparation of a dilution of a fluorescent dye

Diluting 20 mu L of fluorescent dye (FITC-coated silica microspheres with the particle size of 250 nm) by using 40 mu L of buffer PBS;

(2) Preparation of the labeling incubation

0.005g of activator EDC was dissolved in 40. Mu.L of PBS buffer to prepare a buffer solution in which the activator was dissolved. Adding the solution into diluted fluorescent dye, mixing uniformly, centrifuging, and adding buffer solution to prepare a labeling incubation liquid for later use.

(3) Preparation of marking fluid

25. Mu.g of Abeta.1-40 detection antibody stock solution was added to the above-mentioned labeling incubation liquid, and after mixing, the incubation was carried out for 1 hour.

(4) Sealing the marking working fluid

25 mu L of the labeling sealing solution is added into the labeling working solution, and the mixture is uniformly mixed and then incubated for 1 hour.

(5) Washing marking working fluid

(6) Preserving marking working fluid

4. And (3) matching the magnetic bead solution coated with the Abeta 1-40 capture antibody, the Abeta 1-40 detection antibody solution marked with the fluorescent dye, and the quality control product and the calibrator of the Abeta 1-40, thus forming the kit 1.

Preparation example 2: (preparation of kit 2 containing magnetic beads having a diameter of 0.5 μm and a working concentration of 0.15 mg/mL)

Kit 3 was obtained in the same manner as in preparation example 1 except that a magnetic bead having a diameter of 0.5 μm (similarly available from Merck, the same applies hereinafter) was selected.

Preparation example 3: (preparation of kit 3 containing magnetic beads having a diameter of 0.8 μm and a working concentration of 0.15 mg/mL)

Kit 3 was obtained in the same manner as in preparation example 1 except that the magnetic beads having a diameter of 0.8 μm were selected.

Preparation example 4: (preparation of kit 4 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.15 mg/mL)

Kit 4 was obtained in the same manner as in preparation example 1 except that the magnetic beads having a diameter of 1.2. Mu.m.

Preparation example 5: (preparation of kit 5 containing magnetic beads having a diameter of 2.2 μm and a working concentration of 0.15 mg/mL)

Kit 5 was obtained in the same manner as in preparation example 1 except that the magnetic beads having a diameter of 2.2. Mu.m.

Preparation example 6: (preparation of kit 6 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.05 mg/mL)

Kit 6 was obtained in the same manner as in preparation example 4 except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.05 mg/mL.

Preparation example 7: (preparation of kit 7 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.1 mg/mL)

Kit 7 was obtained in the same manner as in preparation example 4, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.1 mg/mL.

Preparation example 8: (comprising a diameter of 1.2 μm and a working concentration of 0.2 m)Preparation of kit 8 of magnetic beads g/mL)

Kit 8 was obtained in the same manner as in preparation example 4, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.2 mg/mL.

Preparation example 9: (preparation of kit 9 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.25 mg/mL)

Kit 9 was obtained in the same manner as in preparation example 4 except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.25 mg/mL.

Preparation example 10: (preparation of kit 10 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 12 microequivalents/gram)

Kit 10 was obtained in the same manner as in preparation example 4 except that the carboxyl group concentration of the magnetic beads was changed to 12 microequivalents/g (from Merck, the same applies hereinafter).

PREPARATION EXAMPLE 11: (preparation of kit 11 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 32 microequivalents/g)

Kit 11 was obtained in the same manner as in preparation example 4, except that the carboxyl group concentration of the magnetic beads was set to 32 microequivalents/g.

Preparation example 12: (preparation of kit 12 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 15 microequivalents/gram)

Kit 12 was obtained in the same manner as in preparation example 4, except that the carboxyl group concentration of the magnetic beads was set to 15 microequivalents/g.

Preparation example 13: (preparation of kit 13 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 30 microequivalents/gram)

Kit 13 was obtained in the same manner as in preparation example 4 except that the carboxyl group concentration of the magnetic beads was set to 30 microequivalents/g.

PREPARATION EXAMPLE 14: (preparation of kit 14 containing magnetic beads having a diameter of 0.3 μm and a working concentration of 0.15 mg/mL)

Kit 14 was obtained in the same manner as in preparation example 1 except that the magnetic beads having a diameter of 0.3 μm were selected.

Preparation example 15: (preparation of kit 15 containing magnetic beads having a diameter of 5 μm and a working concentration of 0.15 mg/mL)

Kit 15 was obtained in the same manner as in preparation example 1 except that the magnetic beads having a diameter of 5 μm were selected.

PREPARATION EXAMPLE 16: (preparation of kit 16 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.01 mg/mL)

Kit 16 was obtained in the same manner as in preparation example 4, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.01 mg/mL.

Preparation example 17: (preparation of kit 17 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.48 mg/mL)

Kit 17 was obtained in the same manner as in preparation example 4, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.48 mg/mL.

Comparative preparation example1: (preparation of comparative kit 1 containing magnetic beads having a diameter of 0.2 μm and a working concentration of 0.15 mg/mL)

Comparative kit 1 was obtained in the same manner as in production example 1 except that a magnetic bead having a diameter of 0.2 μm (carboxyl group concentration: 22 microequivalents/g) was selected.

Comparative preparation example 2: (preparation of comparative kit 2 containing magnetic beads having a diameter of 5.1 μm and a working concentration of 0.15 mg/mL)

Comparative kit 2 was obtained in the same manner as in production example 1 except that a magnetic bead having a diameter of 5.1 μm (carboxyl group concentration: 22 microequivalents/g) was selected.

Comparative preparation example 3: (preparation of comparative kit 3 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.009 mg/mL)

Comparative kit 3 was obtained in the same manner as in production example 4, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was changed to 0.009 mg/mL.

Comparative preparation example 4: (preparation of comparative kit 4 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.5 mg/mL)

Comparative kit 4 was obtained in the same manner as in preparation example 4, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-40 capturing antibody was set to 0.5 mg/mL.

Example 1: the concentration of Abeta 1-40 was measured based on a single molecule detection technique using the kit 1 obtained in preparation example 1

(1) The concentration of Abeta 1-40 was diluted to 0, 0.1, 0.5, 1, 5, 10, 25, 50 and 100pg/mL, respectively.

(2) According to the requirements of an AST-Dx90 full-automatic fluorescence immunoassay analyzer (self-grinding device), sequentially loading samples and reagents to a designated position, starting a test after the sample is ready, automatically feeding the samples to the loading position by the device, simultaneously loading a reaction cup into an incubation plate, adding the samples with various concentrations obtained in 25 mu L (1) into the reaction cup by a sampling needle from a sample tube, adding 25 mu L of a magnetic bead solution (reagent 1) coated with Abeta 1-40 capture antibody into the reaction cup by a reagent needle from the kit 1, uniformly mixing and incubating for 3min, and identifying target molecules with extremely low content in the captured samples by the magnetic beads of the modified surface specific antibodies in the reagent 1.

(3) The reagent needle absorbs 10 mu L of Abeta 1-40 detection antibody (reagent 2) marked with fluorescent dye from the reagent kit 1, and the reagent kit is added into a reaction cup, uniformly mixed and incubated for 2min, and the reagent 2 contains a single-molecule signal marker for modifying the detection antibody, so that target molecules can be converted into single-molecule signals.

(4) The reaction system is transferred into a flow cell by a detection needle, magnetic beads are attracted to the bottom of the flow cell by magnetic separation and are spread on the surface of a detection hole, other components are removed by washing, single-molecule imaging is carried out by using a common fluorescence microscope (purchased from Olympus), and the subsequent single-molecule counting statistics and analysis are completed by using a single-molecule counting mode and a fluorescence intensity integrating mode in combination. The incubation time was 5 minutes, and the time from sample introduction to output was 5 minutes and 40 seconds.

(5) And (3) completing a series of concentration detection, repeating each concentration point for 6 times, drawing a standard curve according to the detection result, and calculating the CV% value of each point.

As shown in FIG. 1, in this example, the detection range of Abeta 1-40 was 0.14pg/mL to 100pg/mL (it is to be noted that the test is mainly for the purpose of measuring the lower detection limit, and it does not mean that the upper detection limit is only 100pg/mL, and in practice, the linearity is generally better at high concentrations), and in this interval, the number of single-molecule signals and the concentration of the sample have a good linear relationship (R ² =0.9984, close to 1), its lower detection limit can reach 0.14pg/mL, cv% value is 3%.

The respective kits obtained in preparation examples 2 to 17 and comparative preparation examples 1 to 4 (corresponding to comparative examples 1 to 4) were subjected to the same procedure as in example 1 (incubation time was 5 minutes, time from sample introduction to output of the result was 5 minutes and 40 seconds), and lower limit of detection and CV% value were obtained, and the results of lower limit of detection and CV% value are shown in tables 1 and 2, respectively.

Further, the applicant has also conducted experiments on the case where the fluorescent dye is a particle in which a fluorescein is coated with polyacrylamide, a fluorescent particle in which a rare earth element (europium) is coated with polystyrene, a fluorescent particle in which a fluorescent protein (GFP) is coated with dextran, and a fluorescent particle in which a quantum dot (cadmium sulfide) is coated with crosslinked agarose, respectively. Specifically, in example 1, the fluorescent dye obtained by coating the fluorescein with silica was replaced with the above-mentioned several fluorescent dyes, and as a result, it was found that the sensitivity was also excellent (the lower limit of detection of a part of the dye could be about 0.07pg/mL, especially particles obtained by coating the fluorescein with polyacrylamide). Further, the test was also conducted for the case where the magnetic bead particle diameter was 2.8. Mu.m, the sensitivity was about 2.5pg/mL, and the CV value was about 9 under the same conditions as in example 1.

TABLE 1

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TABLE 2

PREPARATION EXAMPLE 18: (preparation of kit 18 containing magnetic beads having a diameter of 1.5 μm and a working concentration of 0.15 mg/mL)

1. Experimental Components

The components were similar to those of preparation 1 except that the capture antibody and the detection antibody were replaced with Abeta 1-42.

2. Preparation of magnetic bead solution coated with Abeta 1-42 Capture antibody

(2) Taking 80 mug of Abeta 1-42 capture antibody, adding the Abeta 1-42 capture antibody into the magnetic beads of the step (1), uniformly mixing, incubating the mixture on a roller incubator for 3 hours at the temperature of 25 ℃, and rotating the mixture at the speed of: 80rpm/min.

(5) And adding the magnetic bead preservation solution into a centrifuge tube, and uniformly mixing to obtain a magnetic bead solution coated with the Abeta 1-42 capture antibody, wherein the working concentration of the magnetic beads is 0.15mg/mL.

3. Preparation of A beta 1-42 detection antibody solution labeled with fluorescent dye

(1) Preparation of a dilution of a fluorescent dye

(2) Preparation of the labeling incubation

(3) Preparation of marking fluid

25. Mu.g of Abeta.1-42 detection antibody stock solution was added to the above-mentioned labeling incubation liquid, and incubated for 1 hour after mixing.

(4) Sealing the marking working fluid

(5) Washing marking working fluid

(6) Preserving marking working fluid

Adding the marked dispersion liquid into a centrifuge tube, mixing uniformly, centrifuging, absorbing the marked preservation liquid according to the production total volume requirement, adding the marked preservation liquid into the centrifuge tube, mixing uniformly, and placing the mixture into a refrigerator for preservation at 2-8 ℃.

4. The magnetic bead solution coated with the Abeta 1-42 capturing antibody, the Abeta 1-42 detecting antibody solution marked with fluorescent dye, and quality control substances and calibrator in main components, which are prepared in the steps, are matched to form the kit 18.

Preparation example 19: (preparation of kit 19 containing magnetic beads having a diameter of 0.5 μm and a working concentration of 0.15 mg/mL)

A kit 19 was obtained in the same manner as in preparation example 18, except that the magnetic beads having a diameter of 0.5 μm were selected.

Preparation example 20: (preparation of kit 20 containing magnetic beads having a diameter of 0.8 μm and a working concentration of 0.15 mg/mL)

A kit 20 was obtained in the same manner as in preparation example 18 except that the magnetic beads having a diameter of 0.8 μm were selected.

Preparation example 21: (preparation of kit 21 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.15 mg/mL)

A kit 21 was obtained in the same manner as in preparation example 18, except that the magnetic beads having a diameter of 1.2 μm were selected.

PREPARATION EXAMPLE 22: (preparation of kit 22 containing magnetic beads having a diameter of 2.2 μm and a working concentration of 0.15 mg/mL)

A kit 22 was obtained in the same manner as in preparation example 18 except that the magnetic beads having a diameter of 2.2 μm were selected.

Preparation example 23: (preparation of kit 23 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.05 mg/mL)

Kit 23 was obtained in the same manner as in preparation example 21 except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.05 mg/mL.

PREPARATION EXAMPLE 24: (preparation of kit 24 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.1 mg/mL)

Kit 24 was obtained in the same manner as in preparation example 21 except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.1 mg/mL.

Preparation example 25: (preparation of kit 25 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.2 mg/mL)

Kit 25 was obtained in the same manner as in preparation example 21, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.2 mg/mL.

PREPARATION EXAMPLE 26: (preparation of kit 26 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.25 mg/mL)

Kit 26 was obtained in the same manner as in preparation example 21, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.25 mg/mL.

Preparation example 27: (preparation of kit 27 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 12 microequivalents/gram)

Kit 27 was obtained in the same manner as in preparation example 21, except that the carboxyl group concentration of the magnetic beads was changed to 12 microequivalents/g.

PREPARATION EXAMPLE 28: (preparation of kit 28 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 32 microequivalents/gram)

Kit 28 was obtained in the same manner as in preparation example 21, except that the carboxyl group concentration of the magnetic beads was changed to 32 microequivalents/g.

Preparation example 29: (preparation of kit 29 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 15 microequivalents/gram)

Kit 29 was obtained in the same manner as in preparation example 21 except that the carboxyl group concentration of the magnetic beads was changed to 15 microequivalents/g.

Preparation example 30: (preparation of kit 30 containing magnetic beads having a diameter of 1.2 μm, a working concentration of 0.15mg/mL and a carboxyl concentration of 30 microequivalents/gram)

Kit 30 was obtained in the same manner as in preparation example 21 except that the carboxyl group concentration of the magnetic beads was set to 30 microequivalents/g.

Preparation example 31: (preparation of kit 31 containing magnetic beads having a diameter of 0.3 μm and a working concentration of 0.15 mg/mL)

A kit 31 was obtained in the same manner as in preparation example 18, except that the magnetic beads having a diameter of 0.3 μm were selected.

PREPARATION EXAMPLE 32: (preparation of kit 32 containing magnetic beads having a diameter of 5 μm and a working concentration of 0.15 mg/mL)

A kit 32 was obtained in the same manner as in preparation example 18 except that the magnetic beads having a diameter of 5 μm were selected.

PREPARATION EXAMPLE 33: (preparation of kit 33 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.01 mg/mL)

Kit 33 was obtained in the same manner as in preparation example 21, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.01 mg/mL.

PREPARATION EXAMPLE 34: (preparation of kit 34 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.48 mg/mL)

Kit 34 was obtained in the same manner as in preparation example 21, except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.48 mg/mL.

Comparative preparation example 5: (preparation of comparative kit 5 containing magnetic beads having a diameter of 0.25 μm and a working concentration of 0.15 mg/mL)

Comparative kit 5 was obtained in the same manner as in production example 18, except that a magnetic bead having a diameter of 0.25 μm (carboxyl group concentration: 22 microequivalents/g) was selected.

Comparative preparation example 6: (preparation of comparative kit 6 containing magnetic beads having a diameter of 5.2 μm and a working concentration of 0.15 mg/mL)

Comparative kit 6 was obtained in the same manner as in production example 18, except that a magnetic bead having a diameter of 5.2 μm (carboxyl group concentration: 22 microequivalents/g) was selected.

Comparative preparation example 7: (preparation of comparative kit 7 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.009 mg/mL)

Comparative kit 7 was obtained in the same manner as in production example 21 except that the working concentration of magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was changed to 0.009 mg/mL.

Comparative preparation example 8: (preparation of comparative kit 8 containing magnetic beads having a diameter of 1.2 μm and a working concentration of 0.5 mg/mL)

Comparative kit 8 was obtained in the same manner as in preparation example 21 except that the working concentration of the magnetic beads in the magnetic bead solution coated with the Abeta 1-42 capturing antibody was set to 0.5 mg/mL.

Example 18: determination of Abeta 1-42 concentration Using the kit 18 obtained in preparation 18 based on Single molecule detection technique

(1) The concentration of Abeta 1-42 was diluted to 0, 0.1, 0.5, 1, 5, 10, 25, 50 and 100pg/mL, respectively.

(2) According to the requirements of an AST-Dx90 full-automatic fluorescence immunoassay analyzer, sequentially loading samples and reagents to a designated position, starting a test after the full-automatic fluorescence immunoassay analyzer is ready, automatically feeding the samples to the loading position by equipment, simultaneously loading a reaction cup into an incubation plate, sucking 25 mu L (1) of samples with various concentrations from a sample tube by a sampling needle, adding the samples into the reaction cup, sucking 25 mu L of magnetic bead solution (reagent 1) coated with Abeta 1-42 capture antibody from the reagent kit 1 by the reagent needle, adding the magnetic bead solution (reagent 1) into the reaction cup, uniformly mixing and incubating for 3min, wherein the magnetic beads of the antibodies with the specific surfaces modified in the reagent 1 can identify target molecules with extremely low content in the captured samples.

(3) The reagent needle absorbs 10 mu L of Abeta 1-42 detection antibody (reagent 2) marked with fluorescent dye from the reagent kit 1, and the reagent kit is added into a reaction cup, uniformly mixed and incubated for 2min, and the reagent 2 contains a single-molecule signal marker for modifying the detection antibody, so that target molecules can be converted into single-molecule signals.

As shown in FIG. 2, in this example, the detection range of Abeta 1-42 is 0.11pg/mL to 100pg/mL (it is to be noted that the test is mainly for the purpose of measuring the lower detection limit, and this does not mean that the upper detection limit is only 100pg/mL, and in practice, the linearity is generally better at high concentrations), and in this interval, the number of single-molecule signals and the concentration of the sample have a good linear relationship (R ² = 0.9955), the lower detection limit can reach 0.11pg/mL, and the cv% value is 5%.

The detection ranges, lower detection limits, and CV% values were obtained for the respective kits obtained in the above preparation examples 19 to 34 and comparative preparation examples (corresponding to comparative examples 5 to 8), and the results of the lower detection limits and CV% values are shown in tables 3 and 4, respectively.

Further, the applicant has also conducted experiments on the case where the fluorescent dye is a particle in which a fluorescein is coated with polyacrylamide, a fluorescent particle in which a rare earth element (europium) is coated with polystyrene, a fluorescent particle in which a fluorescent protein (GFP) is coated with dextran, and a fluorescent particle in which a quantum dot (cadmium sulfide) is coated with crosslinked agarose, respectively. Specifically, in example 18, the fluorescent dye obtained by coating fluorescein with silica was replaced with the above-mentioned several fluorescent dyes, and as a result, it was found that the sensitivity was also excellent (the lower limit of detection of a part of the dye could be about 0.05pg/mL, especially particles obtained by coating fluorescein with polyacrylamide). Further, a test was also conducted on the magnetic beads having a particle diameter of 2.8 μm (other conditions were the same as in example 18), and the sensitivity was about 3pg/mL and the CV value was about 8.

TABLE 3

TABLE 4

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Claims

1. A kit for detecting amyloid beta based on a single-molecule detection technology is characterized by comprising magnetic beads coated with a capture antibody and a detection antibody marked with fluorescent dye,

The diameter of the magnetic beads is 0.5-2.2 mu m, the working concentration is 0.05-0.25 mg/mL,

the beta amyloid is Abeta 1-40 or Abeta 1-42.

2. The kit for detecting amyloid β according to claim 1, wherein the magnetic beads have a diameter of 1 to 1.5 μm.

3. The kit for detecting amyloid beta according to claim 1 or 2, wherein the working concentration of the magnetic beads is 0.1-0.2 mg/mL.

4. The kit for detecting amyloid-beta according to claim 1 or 2, wherein the surface of the magnetic beads is modified with one or more of carboxyl groups, amino groups or tosyl groups, wherein the concentration of carboxyl groups is 15 to 30 microequivalents per gram (μeq/g) when the surface of the magnetic beads is modified with carboxyl groups.

5. The kit for detecting amyloid beta according to claim 1 or 2, wherein the kit further comprises a amyloid beta calibrator and an amyloid beta control.

6. The kit for detecting amyloid beta according to claim 1 or 2, wherein the fluorescent material is fluorescein, rhodamine, coumarin or quantum dot, and the carrier is silicon dioxide, polyacrylamide or polystyrene.

7. The application of fluorescent dye and magnetic beads in preparing a kit for detecting beta amyloid based on a single-molecule detection technology, wherein the fluorescent dye contains fluorescent materials and carriers, the particle size is 180-450 nm,

The diameter of the magnetic beads is 0.5-2.2 mu m, the working concentration of the magnetic beads is 0.05-0.25 mg/mL,

the beta amyloid is Abeta 1-40 or Abeta 1-42.

8. The use according to claim 7, wherein the magnetic beads have a diameter of 1 to 1.5 μm.

9. The use according to claim 7 or 8, wherein the working concentration of the magnetic beads is 0.1-0.2 mg/mL, the fluorescent material is fluorescein, rhodamine, coumarin or quantum dots, and the carrier is silicon dioxide, polyacrylamide or polystyrene.

10. A single molecule detection system for detecting Abeta 1-40 or Abeta 1-42, comprising the kit of any one of claims 1 to 6, and an optical imaging apparatus,

the optical imaging device comprises a light source and an optical signal acquisition unit, and the single-molecule detection system does not comprise a total internal reflection microscope, a near-field microscope and an airy disk focusing detection device, and does not comprise a micro-reaction cavity with the volume of nanoliter level, picoliter level or flying liter level.