CN117434273A

CN117434273A - Joint inspection kit for auxiliary diagnosis of Alzheimer disease

Info

Publication number: CN117434273A
Application number: CN202311472474.1A
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: 2023-11-07
Filing date: 2023-11-07
Publication date: 2024-01-23

Abstract

The invention discloses a joint inspection kit for diagnosing Alzheimer's Disease (AD), and particularly discloses application of a detection reagent for beta amyloid protein, phosphorylated Tau protein and alpha-synuclein in preparation of a kit for assisting in diagnosing Alzheimer's disease. The kit can take blood (whole blood, plasma, serum and the like), saliva, tear and other non-cerebrospinal fluid as samples, realizes early auxiliary diagnosis of AD, is suitable for a full-automatic single-molecule immunity analyzer, and can be well applied to AD screening.

Description

Joint inspection kit for auxiliary diagnosis of Alzheimer disease

Technical Field

The invention relates to the technical field of biological detection, in particular to a joint inspection kit for assisting early diagnosis of Alzheimer's disease based on a single-molecule immunodetection technology characterized by single-molecule counting.

Background

Alzheimer's disease (Alzheimer disease, AD) is a chronic neurodegenerative disease of unknown cause, the pathological changes of which are mainly manifested by senile plaques caused by the deposition of betA-Amyloid, neurofibrillary tangles caused by abnormal phosphorylation of Tau protein, loss of neurons and synapses, and clinical manifestations of progressive cognitive decline and non-cognitive neuropsychiatric symptoms. The main diagnostic means of AD at present are neuropsychological scales, imaging examinations, biomarker detection, etc. The neuropsychological scale evaluates the cognitive function of the subject, and the simple mental state scale, the teryle cognitive evaluation scale, the Changguchun dementia scale and the like are mainly adopted in the prior art, but the test result is greatly floated due to different education degrees and individual understanding abilities of the subject; the neuroimaging detection mainly comprises Magnetic Resonance Imaging (MRI), positron Emission (PET), computer Tomography (CT) and the like, which are strong evidence of AD diagnosis, and the CT can directly display the brain atrophy phenomenon of an AD patient, but has limited value due to poor resolution of soft tissues, and the MRI and the PET are expensive and are not suitable for crowd screening.

Accurate and early diagnosis of AD facilitates early medical intervention by patients, thereby providing the possibility to delay the onset or progression of AD. Both cerebrospinal fluid aβ42/aβ40 (or brain ridge aβ2) and aβpet are associated with brain aβ pathology, and are considered the earliest biomarkers for AD. However, cerebrospinal fluid analysis is generally not a necessary option during diagnosis because of the need for invasive lumbar puncture. Thus, in china there is an urgent need to diagnose AD using a method that is non-invasive, readily available and cost-effective.

Currently, there are few Alzheimer's diagnosis kits marketed in China, among which there are Shenzhen An Qun organism (AD 7C-NTP), beta amyloid 1-42 (Abeta 1-42), phosphorylated tau-181 protein) 3 diagnosis kit products based on the traditional ELISA method, nanjinoman related neurofilament 1 diagnosis products based on the chemiluminescence method, hunan Qiankang urine beta amyloid 1 detection kit based on the colloidal gold immunochromatography method.

In terms of literature report, patent literature 1 reports a biomarker for diagnosis of cognitive disorders, which uses the Fatty Acid Binding Protein (FABP) family as an index for diagnosis of neurodegenerative diseases. Patent document 2 reports a joint inspection kit for diagnosing alzheimer's disease, which uses at least two of phosphorylated Tau protein, blood protein or DNA molecular markers for the detection of alzheimer's disease, and performs joint inspection by both dimensions of immunodetection and molecular detection. Patent document 3 reports a kit for early diagnosis of Alzheimer's disease, which detects T-Tau, p-Tau-181, p-Tau-217, p-Tau-231 in serum and plasma based on a chemiluminescence method. Patent document 4 relates to a biomarker for diagnosing alzheimer's disease and a device in which a body fluid biomarker is selected from one or more of the group consisting of cerebrospinal fluid P-tau, cerebrospinal fluid aβ42/aβ40 and plasma P-tau, the device comprising: the input module is used for inputting clinical detection indexes of a subject, wherein the clinical detection indexes comprise plasma biomarkers, key physiological indexes and nuclear magnetic resonance imaging measurement; a machine learning module for screening an optimal model, wherein the machine learning utilizes stepwise backward regression and red pool information criteria to screen an optimal model index; a model determination module for quantifying an area under ROC curve (AUC) and determining a difference in AUC using the dilong statistics.

Single molecule immunodetection (SMD) is a method that is built on the basis of analysis of single molecules, which has been of great interest in life sciences research to date. In recent years, the method has been applied to cell imaging, research of protein interactions, and quantitative detection of proteins and nucleic acids. In the above mentioned applications, the quantitative detection is achieved by counting target molecules one after the other, which means of quantitative detection represent the final limit of detection. Traditional averaging methods such as immunochromatography, chemiluminescence, enzyme-linked immunosorbent assay and the like are quantified in terms of the relationship between signal intensity and target concentration. The higher the signal intensity, the higher the concentration of the object to be measured. Unlike the average measurement method, in the SMD quantification method, the molecules capable of generating signals are counted, so that the method has visibility and digital property, and high reproducibility and high detection sensitivity are ensured. Since the SMD quantitative detection method has an advantage incomparable with the average measurement, there has been work to apply SMD to quantitative analysis at present (see patent document 5).

Specifically, patent document 5 is a prior application of the inventor of the present application, and relates to labeling a molecule to be detected by using in-situ signal enhanced nanoparticles with a specific particle size (180-400 nm), so that a single molecule signal can be captured and identified by an optical imaging device, and ultrahigh-sensitivity quantitative detection of the molecule to be detected is realized. Wherein, in-situ signal enhanced nano particles and magnetic beads are used, and based on a double antibody sandwich method, single-molecule immunodetection is carried out on cTnI protein, IL-6 protein, DNA and the like, so that a lower detection lower limit is obtained.

Prior art literature

Patent document 1: CN115667927a;

patent document 2: CN114324890a;

patent document 3: CN114034872a;

patent document 4: CN115201495a;

patent document 5: CN111771126B.

Disclosure of Invention

Problems to be solved by the invention

As described above, a single or combined test kit for diagnosing Alzheimer's disease is reported in the prior art, but basically, the kit is based on the conventional immunoassay technology, such as a radioactive method, an enzyme-linked immunoassay method, a fluorescent immunoassay method, a flow-type fluorescent method, a latex turbidimetry method, a biochemical method, an immunochromatography method, a chemiluminescent method, etc., which results in insufficient sensitivity. Given the low concentration of some biomarkers in the blood (e.g., serum, plasma) for alzheimer's disease, the above conventional methods are not satisfactory and tend to result in reduced diagnostic accuracy. In addition, although the above patent documents 1 to 4 relate to the application of a combination of a plurality of different biomarkers in the detection of alzheimer's disease, there is room for improvement in the accuracy.

In view of the above-mentioned state of the art, it is an object of the present application to provide a joint test kit and related applications that enable an assisted diagnosis of alzheimer's disease with a high specificity and sensitivity.

Means for solving the problems

The applicant conducted intensive studies on a joint test kit that can be well applied to the auxiliary diagnosis of alzheimer's disease, found that a marker combination composed of beta amyloid, phosphorylated Tau protein and alpha-synuclein can be used for the auxiliary diagnosis of alzheimer's disease with high accuracy, and also found that a kit comprising an antibody against beta amyloid, an antibody against phosphorylated Tau protein and an antibody against alpha-synuclein based on a single-molecule immunodetection technique featuring single-molecule counting can be used for the auxiliary diagnosis of alzheimer's disease with high sensitivity and specificity. Although the prior art relates to the use of β -amyloid and phosphorylated Tau protein alone as biomarkers in a diagnostic aid kit for alzheimer's disease, the use of combinations of the above three markers in the diagnostic aid of AD has not been reported, and no kit comprising detection reagents for the above three markers based on a single-molecule immunodetection technique featuring single-molecule counting has been mentioned.

One technical scheme of the application is as follows.

Kit based on a single-molecule immunodetection technique featuring single-molecule counting, for aiding in the diagnosis of alzheimer's disease, characterized in that it comprises an antibody against beta amyloid, an antibody against phosphorylated Tau protein and an antibody against alpha-synuclein.

It is known that depending detection techniques need to be noted at the time of registration and reporting of a kit, because the detection technique on which the kit is based directly determines its composition, for example, an enzyme or the like is necessarily contained in an ELISA-based AD kit, and a chemiluminescent reagent is necessarily contained in an ELISA-based AD kit. That is, the feature of "a single molecule immunoassay technique based on a single molecule count" in the above-described scheme means that the kit of the present invention does not contain a reagent specific to other immunoassay methods such as an enzyme and a chemiluminescent agent, and the like, because the technology on which the kit is based has a limiting effect on the scope of protection.

Preferably, the amyloid beta is Abeta-40 and Abeta-42.

Preferably, the phosphorylated Tau proteins are p-Tau 181 and p-Tau 217.

Preferably, it further comprises in situ signal enhancing particles comprising fluorescent material and carrier and having a particle size of 180-300 nm.

Preferably, the carrier is polyethylene glycol, silicon dioxide, polyacrylamide or polystyrene, and the fluorescent material is a fluorescein luminescent material, a rhodamine luminescent material, an aggregation-induced emission material, a quantum dot luminescent material or a semiconductor polymer fluorescent material.

Preferably, the antibody comprises an antibody against beta amyloid, an antibody against phosphorylated Tau protein and an antibody against alpha-synuclein, and the kit further comprises a buffer system, wherein the pH of the buffer solution is 7.0-7.6, and each 100mL of the buffer solution comprises 0.5-2.0 g of 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 0.5-2.0 g of surfactant, 0.5-2.0 g of fish skin gelatin, 2.5-4.0 g of inorganic metal salt, 0.02-0.08 mL of preservative and 0.3 mL-1.0 mL of blocking agent.

The invention also relates to the use of a detection reagent for a biomarker in the preparation of a kit for aiding in the diagnosis of Alzheimer's disease, characterized in that the biomarker is a combination of beta amyloid, phosphorylated Tau protein and alpha-synuclein.

Preferably, the beta amyloid is Abeta-40 and Abeta-42 and the phosphorylated Tau proteins are p-Tau 181 and p-Tau 217.

The invention also relates to a single-molecule immunity detection system for auxiliary diagnosis of Alzheimer's disease, which comprises the kit and an optical imaging device, wherein 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 spot 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.

Particle size in this application particles were observed using a Scanning Electron Microscope (SEM) at a magnification at which about 100 in-situ signal enhancing particles were observed in 1 field of view, and the longest diameter of the 50 randomly selected in-situ signal enhancing particles containing fluorescent probes and carrier was measured with a caliper, and the number average of the values was determined as the average particle size.

The kit has the beneficial effects that the specificity and sensitivity of early diagnosis and screening of Alzheimer's disease are improved by adopting a joint detection mode of three markers, namely beta amyloid, phosphorylated Tau protein and alpha-synuclein; and a single-molecule immunodetection technology featuring counting is adopted, so that higher detection sensitivity and stability are realized. Further, by selecting aβ -40 and aβ -42 as β amyloid, p-Tau 181 and p-Tau 217 as phosphorylated Tau protein, and α -synuclein as an index, better detection accuracy can be obtained, and the applicant has unexpectedly found that these five biomarkers can use substantially the same buffer system and signal amplification system, which greatly reduces the difficulty of preparation of the kit and reduces the kinds of reagents, and improves the usability of the kit, which is an advantage not possessed by any of patent documents 1 to 4. Further, by using in-situ signal enhancing particles comprising a fluorescent material and a carrier of a specific particle size as a signal amplifying material in a kit, it is possible to particularly suitably use for detection of the above-mentioned specific marker combinations while achieving single molecule immunodetection of these markers.

Detailed Description

In order that those skilled in the art will better understand the present application, a technical solution in the embodiments of the present application will be clearly and completely described in the following in connection with examples, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments herein without making any inventive effort, shall fall within the scope of the present application.

In the present specification, the "biological sample" of a subject is not particularly limited to a biological sample derived from a subject, but is preferably a biological sample less traumatic to a subject, and examples thereof include biological samples that are easily collected from a living body, such as blood, plasma, serum, saliva, urine, tears, and sweat, biological samples that are relatively easily collected, such as marrow fluid, bone marrow fluid, pleural effusion, peritoneal effusion, joint effusion, aqueous humor, vitreous humor, and lymph, and tissue samples such as a tissue biopsy. In some embodiments, the biological sample is comprised of a body fluid selected from the group consisting of blood (whole blood), plasma, serum, saliva, urine, medullary fluid, pleural fluid, peritoneal fluid, joint fluid, tears, sweat, aqueous humor, vitreous humor, and lymphatic fluid. When blood and serum are used, a sample of a subject is collected according to a usual method, and the sample is prepared by analyzing the sample directly or after separating the liquid components without pretreatment. The present invention is capable of separating and removing a high molecular weight protein component in advance by using a protein as a detection target, if necessary, an antibody column, an adsorbent column, a spin column or the like. It is noted that the samples of the present invention may be used without enrichment or extraction of vesicles (including exosomes, microparticles/microvesicles, apoptotic bodies, tumor vesicles, and other various EV sub-populations).

In the present specification, amyloid beta (amyloid) is a 39-43 amino acid polypeptide produced by proteolytic action of betA-And gamma-secretase, which causes toxic reactions in and out of nerve cells, leading to degeneration and death of neurons. The most common subtypes of Abeta in humans are Abeta 1-40 and Abeta 1-42.

In the present specification, phosphorylated Tau protein is a Tau protein having at least one amino acid residue modified by a phosphate group. Tau is generally considered to comprise up to 85 amino acid residues compatible with phosphorylation. Typically, the phosphate group is post-translational and may be bound on a side chain of the amino acid residue. The phosphorylated amino acid residue may be, for example, a serine (S), threonine (T), or tyrosine (Y) residue, or a combination thereof. For example, p-tau 181 is a protein of tau protein phosphorylated at threonine position 181, p-tau 231 is a protein of tau protein phosphorylated at threonine position 231, p-tau 217 is a protein of tau protein phosphorylated at threonine position 217, and p-tau 199 is a protein of tau protein phosphorylated at threonine position 199. The phosphorylated tau protein herein may be deposited or may be undeposited and may be soluble. Phosphorylated tau protein may be a triple-repeat tau, a quadruple-repeat tau or a combination thereof. In some embodiments, phosphorylated tau protein may comprise a mixture of tri-and tetra-repeat tau. Phosphorylated tau proteins may be fibrillar, for example as neurofibrillary tangles (NFT). NFT can be found in the dendritic chamber of neurons. The phosphorylated tau protein of the present invention may be at least one of p-tau 181, p-tau 231, p-tau 217, p-tau 199, p-tau 202, p-tau 404 or p-tau 205.

In this specification, α -synuclein (α -syn) is a very conserved acidic small protein of 140 amino acids, with a molecular weight of 19kDa, encoded by the SNCA gene located on chromosome 4. It is located in high concentrations at the presynaptic nerve terminals of the central nervous system where it acts on synaptic vesicle biology. Synucleinopathies are a group of neurodegenerative disorders associated with the deposition of fibrous aggregates of a-syn in selective populations of neurons and glia. These deposits can be found in the neurites of neurons bodies such as Lewy Bodies (LB) or diseases such as Parkinson's Disease (PD) or lewy body Dementia (DLB); or Multiple System Atrophy (MSA) in glial cytoplasmic inclusion bodies.

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

Monoclonal antibodies, antibodies produced by methods other than those produced by normal germ cells), multispecific antibodies, humanized antibodies (fully or partially humanized antibodies), animal antibodies (e.g., but not limited to: birds (e.g., ducks or geese), sharks, whales and mammals, including non-primates (e.g., cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.), or non-human primates (e.g., monkeys, chimpanzees, etc.), recombinant antibodies, chimeric antibodies, single chain Fv ("scFv"), single chain antibodies, single domain antibodies, fab fragments, F (ab') 2 fragments, etc., preferably monoclonal/polyclonal murine/rabbit antibodies. In addition, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an analyte binding site. Immunoglobulin molecules may be of any type (e.g., igG, igE, igM, igD, igA and IgY), of any class (e.g., igG1, igG2, igG3, igG4, igA1 and IgA 2), or of any subclass of protein molecule, preferably IgG.

Examples of monoclonal and polyclonal antibodies derived from mammals include: antibodies produced in animal blood, antibodies produced by hybridomas, and antibodies produced by a host transfected with an expression vector containing an antibody gene obtained by genetic engineering techniques, antibodies produced in large quantities in CHO cell factories using the gene of the optimal antibody, or human antibodies produced directly using an anti-gene mouse producing a human antibody, and the like. Monoclonal and polyclonal antibodies can be produced by methods known to those skilled in the art, and commercially available products can be purchased.

The in situ signal enhancing particles of the present invention are materials that enhance the fluorescent signal in situ (in-situ) to a level that can be detected by conventional optical imaging equipment, and which contain both fluorescent materials and carriers.

In the in-situ signal enhancement particles, the carrier plays a very important role, for example, more fluorescent materials can be combined, so that the 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 immunodetection by a conventional fluorescence microscope, and cannot realize single-molecule immunodetection without a carrier. The carrier is classified according to materials and can be one or more of polyethylene glycol, silicon dioxide, polystyrene or polyacrylamide. 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, polyethylene glycol is preferable as the carrier from the viewpoint of uniformly distributing the fluorescent material and making the fluorescent material high in brightness.

The fluorescent material in the in situ signal enhancing particles is also necessary to achieve single molecule immunodetection. The fluorescent material can be one or more of fluorescent dye molecules, rare earth elements, rare earth chelates, fluorescent proteins, quantum dots, aggregation-induced emission materials, semiconductor polymer fluorescent materials 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 in-situ signal enhancement particles are preferably fluorescent particles (commercially available) formed by coating quantum dots with polyethylene glycol, fluorescent particles formed by coating fluorescent dye molecules (such as fluorescein) with silicon dioxide, fluorescent particles formed by coating fluorescent dye molecules (such as fluorescein) with polyacrylamide, fluorescent particles formed by coating quantum dots with polystyrene, fluorescent particles formed by coating rare earth elements or rare earth chelate with polystyrene, and the like.

In the invention, the particle size of the in-situ signal enhancement particles needs to be controlled within 180-300 nm, such as 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 290nm and 300nm. If the particle diameter of the in-situ signal enhancing particles is less than 180nm, for example, 150nm, no signal can be detected in the conventional optical imaging apparatus, and if the particle diameter is more than 300nm, the detection sensitivity is not excellent enough, 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 particle size of the in-situ signal enhancement particles can be controlled by adjusting parameters such as the mass ratio of the fluorescent material to the carrier, the reaction temperature, the reaction time, the stirring rotating speed, the solvent dosage and the like.

In this application, the surface of the in situ signal enhancing particle is modified with reactive functional groups capable of covalent coupling with antibodies, including one or more of hydroxyl, carboxyl, amino, sulfhydryl, alkenyl, alkynyl, succinimidyl ester groups and derivatives thereof.

In this application, the surface of the in situ signal enhancing particle may be modified with a length of linker arms, including multi-carbon straight chains, multi-carbon branched chains, polymer chains, peptide chains, proteins. The length of the connecting arm is preferably 180-350 nm, more preferably 2-20 nm, and most preferably 5-10 nm.

The term "single molecule count" as used herein refers to a measurement of the concentration of a protein to be detected labeled with an in-situ signal-enhancing particle by counting the single molecule, not by measuring the fluorescence intensity of the whole solution.

The kit of the invention is a kit based on a double antibody sandwich method, wherein the buffer solution can be a buffer solution for a detection antibody marked with in-situ signal enhancement particles, or a buffer solution for a capture antibody combined with a solid carrier such as magnetic beads, and is preferably a buffer solution for a detection antibody marked with in-situ signal enhancement particles. Specifically, the buffer solution is sucked up and added to a centrifuge tube containing the washed marking fluid to disperse the marking fluid, and then stored for use. The buffer may also be referred to as a labeling dispersion. The applicant analyzed that by dispersing a specific labeling working fluid (i.e., a liquid containing a detection antibody labeled with specific in-situ signal enhancing particles) with the above-described specific composition and concentration of buffer, the in-situ signal enhancing particles of a specific particle size used in the present invention can be made to exist stably without excessive aggregation, and the generation of fluorescence quenching can be avoided, thereby enabling higher sensitivity to be achieved. In addition, it is presumed that the buffer solution with the specific composition and concentration has good compatibility with antibodies against Abeta-40, abeta-42, p-tau 181, p-tau 217 and alpha-synuclein, ensures the stability of the system, and further contributes to the improvement of detection sensitivity and preservation stability.

As described above, another significant advantage of the present invention is the versatility of the buffer system, and by selecting β amyloid, phosphorylated Tau protein and α -synuclein, particularly aβ -40, aβ -42, p-Tau 181, p-Tau 217, and α -synuclein as diagnostic markers for alzheimer's disease, a set of buffer systems can be shared, which can reduce the difficulty of preparation of the kit and the variety of reagents, and improve the ease of use of the kit, which is an advantage not possessed by patent documents 1 to 4.

In the buffer solution, the surfactant is surfactant S9, and the mass of the surfactant is 0.8-1.5 g in every 100mL of the buffer solution. Preferably, the inorganic alkali metal salt is sodium chloride or potassium chloride, preferably sodium chloride, and the mass of the inorganic alkali metal salt is 3.0-3.5 g per 100mL of the buffer solution. Preferably, the preservative is PC-300, and the content of the preservative is 0.02-0.08 mL in every 100mL of the buffer solution. Preferably, the blocking agent comprises 0.1-0.4 mL of Mak33 and 0.2-0.6 mL of TRU Block 2. The buffer of the present invention does not contain Tris base, urea, tris, cellulose salt, cellulose derivative, sodium azide or glycerol, which are often contained in the conventional buffer system. The buffer solution does not contain an enzyme stabilizer, zinc chloride, sodium caseinate, mannitol, or the like. Still more preferably, the buffer in the present invention consists of 4-hydroxyethylpiperazine ethanesulfonic acid, surfactant S9, inorganic alkali metal salt, fish skin gelatin, mak33, TRU Block 2, and sterile distilled water.

In the present invention, an 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 special detection system is adopted, so that the requirement on the optical imaging equipment is low, and the optical imaging equipment is conventional optical imaging equipment (namely, the optical imaging equipment which does not break through the optical diffraction limit) without expensive imaging equipment which breaks 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.

In the present application, the calculation mode of each protein concentration includes two modes, namely a single molecule counting mode and a fluorescence intensity integration mode. And the number of the bright spots formed by the in-situ signal enhancement nano particles in the generated image is directly analyzed and counted, and the number of the bright spots is directly or indirectly converted into concentration information of the protein in the sample. The term "directly converted to concentration information of a protein in a sample" means absolute quantification, that is, conversion to concentration information is performed without standard curve correction. The term "indirectly converted into concentration information of a protein in a sample" means that the concentration information is converted into concentration information by the number of bright spots and a standard curve (or correction parameter). In the fluorescence intensity integration mode, the area of the bright spots formed by the in-situ signal enhancement nanoparticles in the generated image is counted and integrated, and the integration result is divided by a specific parameter, such as an average bright spot area formed by each in-situ signal nanoparticle or a bright spot area related variable (such as a power square, an evolution square, a polynomial, and the like), so as to obtain the approximate number of the in-situ signal enhancement nanoparticles in a conversion mode, and the value is converted into concentration information of the protein in the sample. Wherein the average speckle area is obtained by counting and averaging the speckle area of individual molecules at a lower concentration. From the viewpoint of obtaining a larger detection dynamic range, it is important to use a single molecule count mode in a low concentration section and a fluorescence intensity integration mode in a high concentration section, and then combine the standard curves plotted in these two modes, thereby drawing a complete standard curve. The boundary between the low concentration and the high concentration is generally the concentration when more than one molecule to be measured is bound to one surface of the magnetic bead, and may be preferably the concentration when 0.5 molecules to be measured are bound to one surface of the magnetic bead or the concentration when 2 molecules to be measured are bound on average according to the standard curve fitting result.

In the present invention, experiments are performed using an AST-Dx90, AST-Sc-Ulti ultrasensitive single-molecule immunodetector, an AST-Sc-Lite single-molecule immunodetector, or an AST-Sc-Semi ultrasensitive single-molecule immunodetector developed by the present applicant, and preferably using an AST-Sc-Ulti ultrasensitive single-molecule immunodetector.

Examples

Hereinafter, the present application will be described in further detail with reference to examples, but the present application is not limited thereto.

1. Method for detecting protein

(1) Determination of particle size and coefficient of variation of in situ Signal enhancing nanoparticles

Taking polyethylene glycol-supported fluorescent material, styrene- (meth) acrylic acid copolymer-supported fluorescent particles, and silica-supported fluorescent particles as examples, each in-situ signal enhancement particle was diluted 1000-fold with water, and then 100. Mu.L was dropped on the surface of a clean silicon wafer, air-dried, and platinum was deposited on the surface of the silicon wafer by sputtering with a small-sized sputtering apparatus at 5nm, and image analysis was performed by using SEM (SU 3900 manufactured by Hitachi, ltd.) to obtain particle diameters.

The coefficient of variation of the particle diameter is calculated by the following formula.

Coefficient of variation of particle diameter (CV value) =standard deviation of particle diameter/average particle diameter

Taking polyacrylamide-supported fluorescent particles as an example, the obtained polyacrylamide fluorescent nanoparticles were diluted 1000-fold with pure water, and the particle diameter of the particles was measured using a malvern particle size analyzer (Zetasizer Nano S90).

(2) Single molecule imaging

Single molecule imaging is performed using a conventional fluorescence microscope such as the Nikon Eclipse Ti-U fluorescence microscope, and in addition, other fluorescence microscopes of the Nikon Eclipse Ti series, lycra DMi8 fluorescence microscope, and the like may also be employed.

(3) Standard curve drawing method

In the application, a single molecule counting mode and a fluorescence intensity integrating mode are used in combination, and the specific implementation method is as follows:

when the concentration of the molecules to be detected is lower, the number of the magnetic beads is more than that of the molecules to be detected combined with the magnetic beads, so that standard curve drawing is carried out on the samples of the molecules to be detected with different concentrations by using a single-molecule counting mode;

when the concentration of the molecules to be detected exceeds a certain threshold, more than 1 molecules to be detected can be combined on the surface of one magnetic bead, single-molecule signals are easy to overlap, and the detection result is deviated, so that the fluorescent intensity integration mode is more suitable to be used.

Specifically, when the number of single molecules in one imaging picture does not exceed a set threshold value, drawing a standard curve by using a single molecule counting mode; when the number of single molecules in one imaging picture exceeds a set threshold, a fluorescence intensity integration mode is used, and the total fluorescence intensity area is divided by the average fluorescence intensity area of each molecule and converted into an approximate number of single molecules, so that standard curve drawing is performed.

And finally, combining the standard curve obtained by using the single molecule counting mode with the standard curve obtained by using the fluorescence intensity integration mode, performing curve fitting by using a fitting formula, and drawing a complete standard curve.

(4) Preparation of in situ Signal enhancing particles

The preparation of the in-situ signal enhancement particles using polyethylene glycol as a carrier is exemplified by the following steps: the method comprises the steps of placing polyethylene glycol and fluorescent material in an organic solvent according to a specified proportion, stirring and slightly heating, dripping the liquid into an aqueous solution containing a surfactant such as Pluronic F-127, performing ultrasonic treatment to form emulsion, mixing with the aqueous solution containing the surfactant again, performing magnetic stirring, evaporating to remove the organic solvent, centrifuging, and washing to obtain the in-situ signal enhancement particles with the polyethylene glycol as a carrier and embedded with the fluorescent material. Commercial products such as PEG-coated quantum dot fluorescent materials of specific particle sizes biospecifically tailored to sieanziue may also be used.

When the carrier is silica or polyacrylamide, the method for producing the in-situ signal enhancing particles can be described in the example of patent document 1. When the support is styrene, the in situ signal enhancing particles may be synthesized by methods common in the art (see, for example, CN102676157 a).

2. Data sets and statistical methods.

2.1 data set of study

Subjects (n=40, 19 AD patients, 21 healthy controls) were enrolled from Jiangsu area for analysis of protein differences.

The diagnosis standard of AD is based on the American national aging institute (NIA-AA) diagnosis standard of 2011. In addition, AD and normal controls were determined based on the ratio of cerebrospinal fluid P-tau/Abeta 42 (cut-off taken to 0.14), which was calculated based on data published by the teaching task group of Beijing Xuan Wu Hospital Gu Longfei, consistent with other research reports (see, jia L et al Concordance between the assessment of Abeta42, T-tau, and P-T181-tau in peripheral blood neuronal-derived exosomes and cerebrospinal fluid. Alzheimer's de. 2019; 15:1071-80). According to the ATN framework, low levels of cerebrospinal fluid aβ42 are a critical pathological change in AD. Thus, the already reported threshold value 500pg/ml of cerebral spinal fluid Abeta42 was used as another criterion for determining AD and normal controls. All subjects or their legal guardians had fully informed and signed written consent. The study was approved by the institutional review board.

2.2 participant characteristics

The participant characteristics are shown below and table 1 below lists the characteristics of the study subjects.

Table 1 participant characteristics

Features (e.g. a character)	Control (n=21)	AD（n=19）
			Age, average (SD)	66.2（2.7）	68.2（3）
Educational years, average (SD)	9.1（2.6）	9.5（2.3）
			Male, quantity (%)	11（52%）	9（47%）
ApoE ε 4 positive (%)	5（24%）	6（32%）
			MMSE Score (SD)	26.5（0.6）	16.8（1.3）
Aβ42 (pg/mL), average (SD)	816.2（98.5）	326.1（104.3）
			P-tau (pg/mL), average (SD)	60.3（25.0）	112（29.8）
P-tau/Abeta, average (SD)	0.074（0.03）	0.34（0.12）

Note that: age, age-to-education, and MMSE values are expressed as average values (standard deviation). Abbreviations: apoE epsilon 4, apolipoprotein epsilon 4; MMSE, simple mental state examination; SD, standard deviation; * P < 0.05 compared to the control group.

As shown in the table above, in the data set, no significant differences in age and sex were observed for AD and control groups, whereas there were significant differences in the proportion of persons carrying ApoE ε 4, MMSE score, cerebrospinal fluid Aβ42 and P-tau (P < 0.05).

3. Protein extraction and analysis.

3.1 cerebrospinal fluid collection and detection.

Cerebrospinal fluid specimens were collected according to international guidelines, and briefly, subjects were placed in a left lateral recumbent position in the early morning on an empty stomach (12 hours fasted) with the L3-L5 intervertebral space selected as the puncture site. 15ml of cerebrospinal fluid was collected with an atraumatic 20 gauge needle and centrifuged at 2,000Xg for 10min at room temperature, and Aβ42 and P-tau were tested according to the standards already proposed. Here, the cerebrospinal fluid collection is to confirm whether the patient is an AD patient by the above-described index.

3.2 plasma collection.

All subjects were collected with 20ml venous blood in the early morning on an empty stomach (12 hours fasted) and plasma was extracted and taken as a sample for further protein level detection.

3.3 Protein level detection.

The concentrations of aβ40, aβ42, p-tau 181, p-tau 217, and α -synuclein were determined using a single-molecule immunoassay kit developed by the applicant, all on the same AST-Sc-Ulti ultrasensitive single-molecule immunoassay instrument. The kit comprises capturing antibodies and detection antibodies for Abeta 40, abeta42, p-tau 181, p-tau 217 and alpha-synuclein, in-situ signal enhancement particles (ZnO quantum dots coated by polyethylene glycol and with the particle size of 230 nm), a single buffer solution (see the specification of the application), a matched specification, a standard substance and a quality control substance.

3.4 Statistical analysis

According to the method of the document CN114324890A, the concentrations of the related markers are subjected to natural logarithmic transformation, a regression equation is obtained through Logistic regression analysis (carried out by Medcalc software), and corresponding AUC values, specificities and sensitivities are obtained through the regression equation and the analysis. The results are shown in Table 2.

TABLE 2

Marker name	Alzheimer's disease and health comparison AUC	Specificity (%)	Sensitivity (%)
				Aβ40	0.85	90	82
Aβ42	0.86	90	81
				p-tau 181	0.78	90	68
p-tau 217	0.80	90	73
				Alpha-synuclein	0.63	73	65
The five combined inspection combinations	0.96	96	95

As shown in the above table, the combined auc=0.96, the specificity was 96%, and the sensitivity was 95% for the above five proteins, which were significantly better than the combination of the single non-combined assays.

4. Correlation of the protein with MMSE score

To further investigate the relationship of protein levels to AD cognitive impairment, a linear correlation analysis was performed on the MMSE score of AD patients with 5 protein levels. The levels of the five protein combinations in AD patients correlated significantly positively with the MMSE score (corrected r2=0.983, P < 0.001), whereas the individual proteins correlated less strongly with the MMSE score than their combinations (aβ40, aβ42, P-tau 181, P-tau 217, and R2 of α -synuclein were 0.642, 0.653, 0.542, 0.513, and 0.426, respectively, with P <0.001, respectively), suggesting that the 5 protein combinations have the potential to predict cognitive impairment.

Claims

1. A joint test kit based on a single molecule immunoassay featuring single molecule counting for aiding in the diagnosis of alzheimer's disease, characterized in that it comprises antibodies against beta amyloid, antibodies against phosphorylated Tau protein and antibodies against alpha synuclein.

2. The joint test kit of claim 1, wherein the beta amyloid is aβ -40 and aβ -42.

3. The joint detection kit of claim 1, wherein the phosphorylated Tau proteins are p-Tau 181 and p-Tau 217.

4. The joint inspection kit according to any one of claims 1 to 3, further comprising in-situ signal enhancing particles containing a fluorescent material and a carrier and having a particle size of 180 to 300nm.

5. The joint detection kit according to claim 4, wherein the carrier is polyethylene glycol, silicon dioxide, polyacrylamide or polystyrene, and the fluorescent material is a fluorescein-based luminescent material, a rhodamine-based luminescent material, an aggregation-induced luminescent material, a quantum dot-based luminescent material, or a semiconductor polymer fluorescent material.

6. The joint test kit of claim 1, comprising an antibody to beta amyloid, an antibody to phosphorylated Tau protein, and an antibody to alpha synuclein, further comprising a buffer solution having a pH of 7.0-7.6, comprising 0.5-2.0 g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 0.5-2.0 g of surfactant, 0.5-2.0 g of fish skin gelatin, 2.5-4.0 g of inorganic metal salt, 0.02-0.08 mL of preservative, and 0.3 mL-1.0 mL of blocking agent per 100mL of buffer solution.

7. Use of a detection reagent for a biomarker in the preparation of a joint detection kit for aiding in the diagnosis of alzheimer's disease, characterized in that the biomarker is a combination of beta amyloid, phosphorylated Tau protein and alpha-synuclein.

8. The use of claim 7, wherein the beta amyloid is aβ -40 and aβ -42 and the phosphorylated Tau proteins are p-Tau 181 and p-Tau 217.

9. A single molecule immunoassay system for aiding in the diagnosis of alzheimer's disease comprising the joint inspection kit of any one of claims 1-6, and an optical imaging device comprising a light source and an optical signal acquisition unit, and the detection system does not include a total internal reflection microscope, a near field microscope, and an airy disk focus detection device, nor a micro-reaction chamber of volume nanoliter, picoliter, or flying-scale.