Glial Fibrillary Acidic Protein Detection Kit

Abstract

Glial fibrillary acid protein detection kit. The present invention relates to a kit for diagnosing markers of neurodegenerative diseases (particularly Alzheimer's disease), and in particular to a kit for detecting glial fibrillary acid protein (GFAP), wherein the kit detects GFAP based on single molecule detection technology. In addition, it also relates to a single molecule detection system for detecting GFAP. The present invention can achieve extremely high sensitivity while performing rapid detection, and the kit has high stability, a simple system, and low cost.

Description

Technical Field

[0001] The present invention relates to the field of immunoassay, in particular to a kit for detecting glial fibrillary acid protein (GFP) based on single molecule counting, and a single molecule detection system for detecting glial fibrillary acid protein. Background Art

[0002] Glial fibrillary acidic protein (GFAP) is a specific acidic protein in the cytoplasm of astrocytes (AS) in the central nervous system. In recent years, many studies have found that serum and cerebrospinal fluid GFAP levels have important guiding significance in the diagnosis, treatment and prevention of traumatic brain injury or primary brain injury, especially neurodegenerative diseases (such as Alzheimer's disease, cerebrovascular dementia, Parkinson's disease, Huntington's disease, etc.), and have attracted more and more attention.

[0003] Currently known GFAP detection methods mainly include colloidal gold method (see patent document 1), enzyme-linked immunosorbent assay (see patent documents 2-3), immunochromatography (see patent documents 4-6), fluorescent immunoassay (see patent document 7), chemiluminescence method (see patent documents 8-12), etc.

[0004] Specifically (one example for each type of method), Patent Document 1 discloses a colloidal gold test strip for semi-quantitative detection of GFAP concentration in human serum, wherein a colloidal gold-labeled anti-GFAP antibody is coated on a colloidal gold-binding membrane, a detection line and a quality control line are provided on the detection membrane, an anti-GFAP capture antibody is fixed on the detection line, and rabbit anti-mouse IgG is fixed on the quality control line; the test strip is composed of a sample addition site, a colloidal gold-binding membrane, a detection line, a quality control line, and a blotting paper from bottom to top. Although this method is simple to operate and low in cost, the detection limit can only reach the ng / mL level, and the sensitivity is poor.

[0005] Patent document 2 discloses a kit and in vitro detection equipment for luminescent enzyme-linked immunosorbent assay (ELISA), the kit comprising: an aqueous solution for stabilizing body fluid samples, the body fluid samples containing a protein component that is a marker of nerve damage, the solutes in the aqueous solution for stabilizing body fluid samples include human serum, animal serum albumin, an inorganic alkali metal salt, Tris base, a protein denaturant, and a nonionic surfactant, the pH value of the aqueous solution is 6.7 to 7.6; and, optionally, antibodies against a protein component that is a marker of nerve damage. It uses a luminescent ELISA method, which has a higher sensitivity than the conventional ELISA method, but the operation steps are still cumbersome and time-consuming (1 to 2 hours, determined by the characteristics of the ELISA method itself), and the sensitivity is only about 20pg / mL.

[0006] Patent document 4 discloses a fluorescent immunochromatographic test strip for rapid detection of GFAP, wherein a fluorescent probe of a GFAP monoclonal antibody labeled with fluorescent microspheres is fixed on a conjugate release pad, the detection line is coated with a GFAP antigen paired with the microsphere labeled antibody, and the quality control line is coated with a goat anti-mouse IgG antibody. Although this method can achieve rapid detection, it has a low sensitivity and cannot meet the detection needs of extremely small amounts of GFAP.

[0007] Patent document 7 discloses a double antibody sandwich quantitative determination of GFAP in serum based on carbon dots as fluorescent markers, but the stability of carbon dots is weak, the practicality is insufficient, and the detection limit can only reach the ng / mL level, with poor sensitivity.

[0008] Patent document 9 discloses a magnetic particle separation chemiluminescent immunoassay for detecting glial fibrillary acidic protein (GFAP), wherein the kit comprises: a calibrator, a quality control reagent A, a reagent B, a cleaning fluid concentrate, and a luminescent substrate solution, wherein the calibrator is a GFAP antigen containing a series of concentrations, used to establish a standard curve; the quality control is prepared with a buffer solution containing a certain concentration of GFAP antigen; the reagent A is a GFAP antibody solution labeled with a certain concentration of magnetic particles; the reagent B is a GFAP antibody solution labeled with a certain concentration of alkaline phosphatase; the cleaning fluid concentrate is used to prepare the cleaning fluid; and the luminescent substrate solution is a luminescent substrate solution catalyzed by ALP. This invention improves the signal strength and sensitivity of the immune response, allowing low-content substances to produce strong chemiluminescent signals during immune binding, providing a more accurate, precise, convenient, quick and simple method for detecting human GFAP. However, the GFAP antibody labeled with magnetic particles used in this invention is prone to aggregation and precipitation, has poor fluidity, and is prone to wall residues when used, thereby reducing the number of particles that play a role in the detection process, resulting in poor accuracy and stability of the test results, and the sensitivity can only reach about 0.05ng / mL.

[0009] The single molecule detection technology characterized by counting has a breakthrough in principle. Compared with the existing enzyme-linked immunosorbent assay and chemiluminescence method based on overall light intensity, the sensitivity has been greatly improved. Therefore, the application of single molecule detection technology to quantitatively detect biomarkers with low content is being studied. The inventors of this application proposed a unique single molecule detection method (see patent document 13), in which in situ signal enhancement nanoparticles and magnetic beads were used to perform single molecule detection of cTnI protein, IL-6 protein, DNA, etc. based on the double antibody sandwich method, and a lower detection limit was obtained, but no mention was made on how to detect glial fibrillary acidic protein, nor was attention paid to the influence of the buffer system on the detection of glial fibrillary acidic protein.

[0010] Prior art literature

[0011] Patent document 1: CN102353787A;

[0012] Patent document 2: CN109696552A;

[0013] Patent document 3: CN102411056A;

[0014] Patent document 4: CN109633171A;

[0015] Patent document 5: CN110488007A;

[0016] Patent document 6: CN104142404A;

[0017] Patent document 7: CN107102146A;

[0018] Patent document 8: CN108051586A;

[0019] Patent document 9: CN109521004A;

[0020] Patent document 10: CN113607958A;

[0021] Patent document 11: CN114563577A;

[0022] Patent document 12: CN114624448A;

[0023] Patent Document 13: CN111771126B Summary of the invention

[0024] Problems to be solved by the invention

[0025] It is well known that when the detection is performed with a longer detection time (especially incubation time), the detection sensitivity can be improved. There is a certain trade-off relationship between shortening the detection time and improving the detection sensitivity, and it is difficult to achieve both shortening the detection time and improving the sensitivity at the same time. Patent document 13 does not mention how to detect glial fibrillary acidic protein, nor does it focus on the influence of the buffer system on the detection of glial fibrillary acidic protein. The inventors of the present application attempted to use the single molecule detection method of patent document 13 for the development of a glial fibrillary acidic protein kit, but because glial fibrillary acidic protein is completely different from the cTnI antigen, IL-6 antigen, DNA, etc. recorded in the document, when the detection system of cTnI antigen, IL-6 antigen, DNA, etc. is directly applied to the detection of glial fibrillary acidic protein, the sensitivity is not satisfactory.

[0026] In order to solve the above problems, the applicant has repeatedly optimized the types of capture antibodies, detection antibodies, fluorescent dyes, and groups of magnetic beads, but the sensitivity is still unsatisfactory. Regarding the buffer system, the inventors of the present application tried to use the buffer in the existing kit for GFAP during the initial optimization process, such as the buffer described in Patent Document 11, Patent Document 12, CN114994332A, and CN115290898A, but found that when these buffer systems are directly applied to the single-molecule detection method characterized by counting, even if the incubation time is extended, the optimal sensitivity is still unsatisfactory, and the detection requirements for extremely small amounts of GFAP cannot be met, and these buffer systems have many components and high preparation costs. This may be because the buffers in the existing kits for GFAP are all used in conjunction with their respective methods, and when these buffer systems are directly used in the single-molecule detection method characterized by counting, there may be adverse interactions with other components in the method (such as in situ signal enhancement particles).

[0027] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a glial fibrillary acid protein kit that can achieve high sensitivity and good stability while achieving rapid detection.

[0028] Means for solving problems

[0029] In order to solve the above-mentioned problems, the inventors of the present application have conducted intensive studies and, as a result, have found a glial fibrillary acid protein kit that can achieve high sensitivity and good stability while achieving rapid detection.

[0030] A technical solution of the present application is as follows.

[0031] A kit for detecting glial fibrillary acidic protein based on single molecule counting, characterized in that it comprises magnetic beads coated with a capture antibody, a detection antibody labeled with a fluorescent dye, and a buffer for the detection antibody labeled with a fluorescent dye, wherein the capture antibody and the detection antibody can bind to different sites of the glial fibrillary acidic protein, respectively.

[0032] The fluorescent dye contains a fluorescent material and a carrier, and has a particle size of 180 to 450 nm.

[0033] The pH of the aforementioned buffer solution is 7.0 to 7.6, and each 100 mL of the buffer solution contains 10 mM to 40 mM PBS, 0.05 mL to 0.30 mL of a nonionic surfactant, 0.4 g to 1.0 g of an inorganic alkali metal salt, 0.2 g to 0.8 g of animal serum albumin, 0.03 mL to 0.06 mL of a biological preservative, and sterile distilled water, and does not contain Tris base, urea, trishydroxymethylaminomethane, cellulose salts, cellulose derivatives, sodium azide or glycerol. In addition, the buffer solution also does not contain enzyme stabilizers, gelatin, zinc chloride, 4-hydroxyethylpiperazineethanesulfonic acid, sodium caseinate and mannitol. It should also be noted that the aforementioned inorganic alkali metal salt does not include the corresponding salt contained in PBS, but refers to the inorganic alkali metal salt added in addition to PBS.

[0034] Preferably, the nonionic surfactant is Tween-20, Tween-40, Tween-60, Tween-80 or a mixture of at least two of them, preferably Tween-20, and the nonionic surfactant is 0.08 mL to 0.20 mL in every 100 mL of the buffer.

[0035] Preferably, the inorganic alkali metal salt is sodium chloride or potassium chloride, preferably sodium chloride, and the inorganic alkali metal salt is 0.5 g to 0.8 g per 100 mL of the buffer solution.

[0036] Preferably, the animal serum albumin is bovine serum albumin, and its mass is 0.4 g to 0.6 g in every 100 mL of the buffer.

[0037] Preferably, the aforementioned biological preservative is proclin-300, and its volume is 0.04 mL to 0.06 mL in every 100 mL of buffer.

[0038] Preferably, the fluorescent material is an aggregation-induced emission material, a fluorescein-based luminescent material, a rhodamine-based luminescent material, or a quantum dot-based luminescent material, and the carrier is silicon dioxide, polyacrylamide, or polystyrene.

[0039] Preferably, each 100 mL of the aforementioned buffer further comprises 2 g to 10 g of trehalose, preferably 4 g to 6 g of trehalose.

[0040] Another technical solution of the present application is as follows.

[0041] A single molecule detection system for detecting glial fibrillary acidic protein, comprising the above-mentioned kit and an optical imaging device,

[0042] The aforementioned optical imaging device includes a light source and an optical signal acquisition unit, and the aforementioned detection system does not include a total internal reflection microscope, a near-field microscope and an Airy disk focusing detection device, nor does it include a micro-reaction chamber with a volume of nanoliter level, picoliter level or femtoliter level.

[0043] Effects of the Invention

[0044] The kit for detecting glial fibrillary acid protein of the present invention can achieve high sensitivity and good stability while performing rapid detection. The inventors of the present application have found that when used for the detection of glial fibrillary acid protein (GFAP), in some embodiments, the sensitivity for GFAP can reach 0.20 pg / mL, and in some embodiments, the sensitivity for GFAP can reach 0.15 pg / mL, or even 0.10 pg / mL. In addition, the present application can detect GFAP in a shorter time on the basis of ensuring high sensitivity. Specifically, the incubation time can be compressed to about 5 minutes (about 5 minutes and 40 seconds from injection to result output), thereby greatly shortening the clinical detection time. In addition, the kit has high stability, and the relative error (Relative Error; RE) and coefficient of variation (Coefficient of variation; CV) of the test results within one year are both less than 10%. In addition, the present invention can detect glial fibrillary acid protein using whole blood samples, serum samples or plasma samples as samples, without using samples taken from cerebrospinal fluid, which causes little damage to patients and greatly reduces the cost of detection.

[0045] Compared with the existing test kits based on colloidal gold method, enzyme-linked immunosorbent assay, immunochromatography, fluorescent immunoassay, chemiluminescence method, etc. (such as patent documents 1 to 12), the GFAP detection kit based on single molecule counting of the present invention can significantly shorten the detection time while improving the sensitivity, and the stability is also roughly equivalent, and the dynamic range is wide. Since the results can be obtained more widely when using the detection kit of the present invention, the amount of signal generated by the measurement can be controlled, thereby reducing the supersaturation phenomenon of the signal, and the hook effect is lower. In addition, in the present application, the buffer system for the detection antibody labeled with a fluorescent dye is simple, with fewer components, and the components used are inexpensive and easy to obtain, so the cost is also low. Based on the above reasons, the kit of the present invention has broad commercial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the standard curve obtained in Example 1, wherein the ordinate is the number of single molecule signals; DETAILED DESCRIPTION

[0047] The present invention is further described below by the description of specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention. Unless otherwise specified, the meanings of all technical terms used herein are the same as those generally understood by those skilled in the art. In the event of a conflict, this specification shall prevail. The description of the numerical range in the specification explicitly includes each intermediate number with the same precision within the range. For example, for the range of 10mM to 40mM, in addition to 10mM and 40mM, 20mM and 30mM are also included, and for the range of 7.0 to 7.6, the numerical values ​​7.0, 7.1, 7.2, 7.3, 7.4, 7.5 and 7.6 are explicitly included.

[0048] 1. Test Kit

[0049] The first embodiment of the present application relates to a kit for detecting glial fibrillary acidic protein based on single molecule counting, characterized in that it comprises magnetic beads coated with a capture antibody, a detection antibody labeled with a fluorescent dye, and a buffer for the detection antibody labeled with a fluorescent dye, wherein the capture antibody and the detection antibody can bind to different sites of the glial fibrillary acidic protein, respectively.

[0050] The fluorescent dye contains a fluorescent material and a carrier, and has a particle size of 180 to 450 nm.

[0051] The pH of the aforementioned buffer is 7.0 to 7.6, and each 100 mL of the buffer contains 10 mM to 40 mM PBS, 0.05 mL to 0.30 mL of a nonionic surfactant, 0.4 g to 1.0 g of an inorganic alkali metal salt, 0.2 g to 0.8 g of animal serum albumin, 0.03 mL to 0.06 mL of a biological preservative, and sterile distilled water, and does not contain Tris base, urea, trishydroxymethylaminomethane, cellulose salts, cellulose derivatives, sodium azide or glycerol. In addition, the buffer also does not contain enzyme stabilizers, gelatin, zinc chloride, 4-hydroxyethylpiperazineethanesulfonic acid, sodium caseinate and mannitol.

[0052] The single molecule counting used in this specification refers to measuring the concentration of GFAP by counting individual molecules of GFAP labeled with a fluorescent dye instead of measuring the fluorescence intensity of the entire solution.

[0053] Glial fibrillary acid protein (GFAP) refers to a 50kDa intracytoplasmic filamentous protein that constitutes part of the cytoskeleton in astrocytes and has been shown to be the most specific marker in the astrocyte family. GFAP protein is encoded by the human GFAP gene. GFAP is the main intermediate filament of mature astrocytes. In the central rod-shaped region of the molecule, GFAP shares considerable structural homology with other intermediate filaments. GFAP participates in the movement and shaping of astrocytes by providing structural stability for astrocyte processes.

[0054] The kit refers to a box for containing chemical reagents for detecting chemical components, drug residues, virus types, etc. In this manual, it specifically refers to a box containing multiple reagents for detecting GFAP concentration in a sample. In addition, the kit also includes a matching instruction manual, reaction cup, waste cup, etc., which are omitted here for detailed description.

[0055] The term "antibody" as used herein refers to a monoclonal antibody, a monospecific antibody (for example, a monoclonal antibody or an antibody produced by methods other than ordinary germ cell production), a multispecific antibody, a human antibody, a humanized antibody (a fully or partially humanized antibody), an animal antibody (for example, but not limited to, birds (such as ducks or geese), sharks, whales, and mammals, including non-primates (such as cattle, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, etc.), or non-human primates (such as monkeys, chimpanzees, etc.), a recombinant antibody, a chimeric antibody, a single-chain Fv ("scFv" ), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, F(ab')2 fragments, etc., preferably monoclonal / polyclonal mouse / rabbit antibodies. In addition, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing analyte binding sites. Immunoglobulin molecules can be protein molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass, preferably IgG. For simplicity, antibodies against GFAP in this specification are generally referred to as GFAP antibodies.

[0056] The present invention is also a kit based on the double antibody sandwich method, in which the capture antibody and the detection antibody are combined with different epitopes of the antigen (GFAP). Ideally, the binding of the capture antibody to the GFAP epitope will not interfere with the binding of the detection antibody to the GFAP epitope. Both monoclonal and polyclonal antibodies can be used as capture antibodies and detection antibodies in sandwich immunoassays. The capture antibody is classified according to the antibody specificity, and can be one or both of polyclonal antibodies and monoclonal antibodies. According to the source classification, it can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies, and alpaca antibodies. The detection antibody is classified according to the antibody specificity, and can be one or both of polyclonal antibodies and monoclonal antibodies. According to the source classification, it can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies, and alpaca antibodies.

[0057] The magnetic beads can be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluid. Typical ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (FeO·Fe2O3). The magnetic beads can be a magnetic solid core surrounded by one or more non-magnetic layers. The magnetic part can also be a layer surrounding the non-magnetic core. The surface of the magnetic beads is modified with active functional groups that can be covalently coupled to antibodies, such as one or more of hydroxyl, carboxyl, amino, succinimidyl ester, sulfonyl (such as toluenesulfonyl) and their derivative groups.

[0058] The so-called "capture antibody-coated magnetic beads" refer to a complex obtained by combining the capture antibody with magnetic beads whose surface is modified with active functional groups that can be covalently coupled to the capture antibody. The preparation process may include steps such as magnetic bead washing, activation treatment, coupling of magnetic beads and capture antibodies, post-coupling washing and sealing treatment. Regarding the activator in the activation treatment, for example, when the modification group on the surface of the magnetic beads is a carboxyl group, the activator can be one or more of N-hydroxysuccinimide (NHS), SulfoNHS, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), etc., and when the modification group is an amino group, the activator can be succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Sulfo-SMCC, etc.

[0059] The so-called "detection antibody labeled with a fluorescent dye" refers to a complex obtained by direct or indirect binding of a fluorescent dye to a detection antibody, wherein direct binding to the detection antibody means that the detection antibody is directly adsorbed or coupled to the fluorescent dye by physical adsorption or chemical modification, and indirect binding to the detection antibody means that the fluorescent dye is specifically labeled on the detection antibody through binding via an anti-detection antibody (i.e., a secondary antibody) or a biotin-streptavidin system. The preparation process of the detection antibody labeled with a fluorescent dye may include the following steps:

[0060] (1) Preparation of dilution solution of fluorescent dye

[0061] Take buffer a (such as carbonate buffer, phosphate buffer PBS or borate buffer) and dilute the fluorescent dye to a specified concentration;

[0062] (2) Preparation of labeling incubation solution

[0063] The activator is dissolved in PBS buffer to prepare a buffer solution containing the activator, the solution is added to the diluted fluorescent dye, mixed and centrifuged, and the buffer solution is added to prepare a labeling incubation solution for use.

[0064] (3) Preparation of labeling working solution

[0065] Add the detection antibody stock solution to the above-mentioned labeled incubation solution, mix well and incubate for 1 hour.

[0066] (4) Seal the marking working fluid

[0067] Add the blocking solution to the above-mentioned labeling working solution, mix well and incubate for 1 hour.

[0068] (5) Washing Mark Working Solution

[0069] (6) Preservation of the labeling working solution

[0070] Wherein, in step (6), the above-mentioned "buffer for the detection antibody labeled with a fluorescent dye" is used. Specifically, the buffer is drawn into a centrifuge tube containing the washed labeling working solution to disperse the labeling working solution, and then stored for standby use. Therefore, the buffer can also be called a labeling dispersion. The applicant analyzed that by using the buffer of the specific composition and concentration of the present invention to disperse the specific labeling working solution (that is, the liquid containing the detection antibody labeled with a specific fluorescent dye), the fluorescent dye of the specific particle size used in the present invention can be stably present without excessive aggregation, avoiding the generation of fluorescence quenching, thereby achieving higher sensitivity. In addition, it is also speculated that the buffer of the specific composition and concentration of the present invention has good compatibility with the detection antibody for GFAP, which ensures the stability of the system, and further contributes to the improvement of detection sensitivity and storage stability.

[0071] As used in this specification, the sensitivity of the test, also known as the limit of detection (LoD), refers to the lowest concentration of the measured variable (i.e., the number of planned measurements) that can be detected at a specific confidence level. The confidence level is usually 95%, and the probability of a false negative measurement is 5%. Sensitivity is the lowest analyte concentration that can be reliably distinguished from the blank limit (LoB) and can be detected.

[0072] The fluorescent dye of the present invention is a material that enhances the fluorescent signal in situ to a level that can be detected by conventional optical imaging equipment, and contains two parts: a fluorescent material and a carrier.

[0073] In the fluorescent dye, the carrier plays a very important role. For example, it can combine more fluorescent materials to make the luminescent signal stronger; it provides sites for functional modification, can combine a large number of antibodies, and improves the reaction activity; it provides the possibility of single molecule detection by conventional fluorescence microscope. If there is no carrier, single molecule detection cannot be achieved. The carrier can be classified according to the material, and can be one or more of silica, polystyrene, polyacrylamide, poly(methyl)acrylate, dextran, agarose and inorganic metal compounds. The carrier can be classified according to the 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 making the fluorescent material evenly distributed and making the brightness of the fluorescent material high, the carrier is preferably silica, polyacrylamide, polystyrene and dextran.

[0074] The fluorescent material in the fluorescent dye is also necessary for realizing 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, aggregation-induced luminescence materials and up-conversion nanoparticles. The fluorescent material is preferably fluorescein (such as fluorescein isothiocyanate), rhodamine (such as rhodamine green, rhodamine B, etc.), coumarin, quantum dots (such as CdS, CdSe, CdTe, ZnSe), rare earth elements (such as Eu, Ce) and their complexes, etc. The fluorescent material is adsorbed or wrapped on the surface or inside of the carrier by one or more of covalent modification, chelation, spatial encapsulation, hydrophobic effect and electrostatic adsorption. It should be noted that from the viewpoint of being conducive to optical imaging recognition and improving sensitivity, it is preferred that the fluorescent material is uniformly wrapped inside the carrier.

[0075] In the present application, the fluorescent dye is preferably a fluorescent particle formed by fluorescent dye molecules (such as fluorescein) wrapped in silica, a fluorescent particle formed by fluorescent dye molecules (such as fluorescein) wrapped in polyacrylamide, a fluorescent particle formed by quantum dots wrapped in polystyrene, a fluorescent particle formed by rare earth elements or rare earth chelates wrapped in polystyrene, a fluorescent particle formed by fluorescent protein wrapped in dextran, and a fluorescent particle formed by quantum dots wrapped in cross-linked agarose, etc.

[0076] In the present invention, the particle size of the fluorescent dye needs to be controlled within the range of 180 to 450 nm, for example, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm. If the particle size of the fluorescent dye is less than 180 nm, for example, 150 nm, no signal can be detected in conventional optical imaging equipment. If the particle size is greater than 450 nm, for example, 460 nm, the detection sensitivity is low and it is difficult to achieve the sensitivity required by clinical practice. It should be noted that the particle size can 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 agglomerate.

[0077] After repeated exploration, the inventors of the present application found that in order to solve the technical problems described above, the pH of the above-mentioned buffer in the present invention must be 7.0-7.6, and PBS, nonionic surfactant, inorganic alkali metal salt, animal serum albumin, biological preservative, and sterile distilled water are essential components, and the concentration of each of the above components must also be within the range defined by the present invention. Among them, the nonionic surfactant is preferably Tween-20, and the concentration is preferably 0.08mL-0.20mL (per 100mL buffer). The inorganic alkali metal salt is preferably sodium chloride, and the concentration is preferably 0.5g-0.8g. Animal serum albumin is preferably bovine serum albumin, and its mass is 0.4g-0.6g in every 100mL of the buffer. The biological preservative is preferably proclin-300, and its volume is 0.03mL-0.06mL in every 100mL buffer. In the present invention, each 100 mL of the above buffer preferably also contains 2g to 10g of trehalose, and more preferably contains 4g to 6g of trehalose. It should also be noted that the above buffer in the present invention does not contain Tris base, urea, trishydroxymethylaminomethane, cellulose salts, cellulose derivatives, sodium azide or glycerol, which are often contained in the existing buffer system for GFAP detection. In addition, the buffer also does not contain enzyme stabilizers, gelatin, zinc chloride, 4-hydroxyethylpiperazineethanesulfonic acid, sodium caseinate and mannitol. More preferably, the "buffer for the detection antibody labeled with a fluorescent dye" in the present invention is composed of PBS, nonionic surfactants, inorganic alkali metal salts, animal serum albumin, biological preservatives, and sterile distilled water, or is composed of PBS, nonionic surfactants, inorganic alkali metal salts, animal serum albumin, biological preservatives, trehalose and sterile distilled water.

[0078] In addition, items required but not provided in the kit include reaction cups, wash solutions, waste cup boxes, and circulation pool care solutions.

[0079] The kit of the present invention can be used for rapid diagnosis of traumatic brain injury or primary brain injury, and is particularly suitable for rapid diagnosis of neurodegenerative diseases (Alzheimer's disease, cerebrovascular dementia, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.).

[0080] 2. Single-molecule detection system for detecting glial fibrillary acidic protein

[0081] The detection system includes the above-mentioned test kit and an optical imaging device, wherein the optical imaging device includes 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 focusing detection device, nor does it include a micro-reaction chamber with a volume of nanoliter level, picoliter level, or femtoliter level, and does not require expensive detection equipment based on nanopores and nanowells as used in document CN110249226A.

[0082] In the present invention, the optical imaging device mainly includes the following components: an excitation light source, an objective lens, a filter, a photosensitive element, a data acquisition module, a data processing module and a dichroic mirror (if it is an upright microscope, there may be no dichroic mirror). Among them, the excitation light source is an optical emitting device used to excite the reacted sample to emit an optical signal. The objective lens is used for signal acquisition and amplification of the sample to be tested. The dichroic mirror is used for reflection of the excitation light path and acquisition of sample optical signals. The filter is used for filtering the excitation light band and filtering the sample emission light signal. The photosensitive element is used for acquisition of sample optical signals. The data acquisition module is configured to receive the optical signal captured by the photosensitive element and convert it into a digital signal. The data processing module is configured for conversion of digital signals and formation and processing of optical images.

[0083] In some embodiments of the device, the excitation light source includes one or more of a gas laser, a solid laser, a semiconductor laser, a liquid laser, and a free electron laser. In some embodiments of the device, the objective lens is classified according to the magnification, including one or more of 1X, 2X, 4X, 5X, 10X, 20X, 40X, 50X, and 100X; the objective lens is classified according to the field curvature correction, including a flat objective lens or a curved objective lens. In some embodiments of the device, the photosensitive element includes one or both of CCD (Charge Coupled Device) or CMOS (Complementary Metal-Oxide Semiconductor).

[0084] The optical imaging device of the present invention can be a conventional optical imaging device (i.e., an optical imaging device that has not broken the optical diffraction limit), and there is no need for expensive imaging devices that have broken the optical diffraction limit, such as a total internal reflection fluorescence microscope, an epifluorescence microscope, a scanning near-field optical microscope, or a confocal fluorescence microscope.

[0085] Example

[0086] The present invention is further described in detail below with reference to Examples and Comparative Examples, but the present invention is not limited thereto. Regarding the determination of the particle size of the fluorescent dye, single molecule imaging and standard curve drawing method, see the Examples section of Patent Document 13 applied by the present applicant, which is hereby incorporated herein.

[0087] 1. Preparation of buffer solution Example 1 : (Preparation of buffer 1)

[0088] Prepare 50 mL of sterile distilled water, add PBS (purchased from Thermo Fisher Scientific) with a final concentration of 20 mM, 0.2 g of bovine serum albumin (purchased from Sigma-Aldrich), 0.8 g of sodium chloride (purchased from Beijing Inokai Technology), 0.04 mL of proclin-300 (purchased from Sigma-Aldrich, hereinafter referred to as PC300), and 0.2 mL of Tween-20 (purchased from Sigma-Aldrich), and finally add the remaining amount of sterile distilled water, mix well and make up to 100 mL.

[0089] 2. Preparation of buffer solutions Examples 2 to 8 (Preparation of buffer solutions 2 to 8)

[0090] Buffer solutions 2 to 8 were obtained in the same manner as in Preparation Example 1 except that the amounts of the above components were changed and trehalose (purchased from Shanghai Yuanju Biotechnology) was added. The contents of the components in these buffer solutions are shown in Table 1 below.

[0091] Table 1

[0092] PBS BSA NaCl PC300 Tween-20 Trehalose Buffer 2 10mM 0.2g 1.0g 0.06mL 0.08mL 2g Buffer 3 10mM 0.8g 0.4g 0.03mL 0.30mL 10g Buffer 4 30mM 0.5g 0.65g 0.05mL 0.10mL 5g Buffer 5 20mM 0.2g 0.8g 0.04mL 0.20mL 5g Buffer 6 20mM 0.6g 0.8g 0.06mL 0.05mL 6g Buffer 7 40mM 0.5g 0.5g 0.03mL 0.20mL 4g Buffer 8 40mM 0.4g 0.8g 0.05mL 0.10mL 4g

[0093] 3. Comparative Preparation Examples 1 to 10 of Buffer Solutions (Preparation of Comparative Buffers 1 to 10)

[0094] Comparative buffer solutions 1 to 10 were obtained in the same manner as in Preparation Example 1 except that the amounts of the above-mentioned components were changed. The contents of the components in these buffer solutions are shown in Table 2 below.

[0095] Table 2

[0096]

[0097] 4. Example of preparation of the kit

[0098] (1) Experimental components

[0099] Carboxyl activated magnetic beads (purchased from Merck), GFAP capture antibody (self-developed), GFAP detection antibody (self-developed), 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 diluent, PBS washing solution, labeling blocking solution (0.01% NaCl, 0.5% BSA dissolved in 10mM PBS, pH=7.4), labeling dispersion (buffer obtained in the above preparation examples 1 to 8 and comparative preparation examples 1 to 10), labeling preservation solution (0.0088% NaCl, 0.1% BSA dissolved in 10mM PBS, pH = 7.4), magnetic bead cleaning solution (PBST solution), cross-linking agent dissolving solution (DMSO), magnetic bead coupling solution (TRIS buffer), magnetic bead blocking solution (0.01% NaCl, 0.2% BSA and 0.02% PC300 dissolved in 10mM PBS, pH = 7.4) and magnetic bead storage solution (0.5% NaCl, 0.5% BSA and 0.02% PC300 dissolved in 10mM PBS, pH = 7.4).

[0100] (2) Preparation of magnetic bead solution coated with GFAP capture antibody

[0101] a. Take 100 μL of carboxyl-modified magnetic beads (purchased from Merck), wash them 5 times with 18 mM PBS buffer, and remove the buffer.

[0102] b. Take 80 μg of GFAP capture antibody and add it to the magnetic beads in (1), mix well, and incubate on a roller incubator at 20° C. for 2 h at a speed of 80 rpm / min.

[0103] c. Add magnetic bead cleaning solution for cleaning, mix well and place on a magnetic rack for magnetic separation.

[0104] d. Add magnetic bead blocking solution to the coated tube and incubate on a roller incubator for 50 min at a speed of 80 rpm / min. Remove the supernatant after magnetic separation.

[0105] e. Take the magnetic bead storage solution and add it into the centrifuge tube. Mix well to obtain the magnetic bead solution coated with GFAP capture antibody.

[0106] (3) Preparation of GFAP detection antibody solution labeled with fluorescent dye

[0107] a. Prepare dilution of fluorescent dye

[0108] Take 30 μL of buffer PBS to dilute 20 μL of fluorescent dye (silica microspheres coated with FITC, particle size 250 nm);

[0109] b. Preparation of labeling incubation solution

[0110] 0.004 g of activator EDC was dissolved in 25 μL of PBS buffer to prepare a buffer solution containing the activator, and the solution was added to the diluted fluorescent dye, mixed and centrifuged, and the buffer solution was added to prepare a labeling incubation solution for use.

[0111] c. Preparation of labeling working solution

[0112] 30 μg of GFAP detection antibody stock solution was added to the above-mentioned labeling incubation solution, mixed and incubated for 1.5 hours.

[0113] d. Seal the marking working fluid

[0114] Take 25 μL of labeling blocking solution and add it to the above labeling working solution, mix well and incubate for 1 hour.

[0115] e. Washing mark working solution

[0116] f. Save the labeling working solution

[0117] Take the labeled dispersion (ie, the buffer solution obtained in the above-mentioned Preparation Examples 1 to 8 and Comparative Preparation Examples 1 to 10) and add it to a centrifuge tube and mix it evenly. After mixing, store it in a refrigerator at 2 to 8°C.

[0118] (4) The magnetic bead solution coated with GFAP capture antibody, the GFAP detection antibody solution labeled with fluorescent dye, and the GFAP quality control and calibration products prepared in the above steps are combined to form kits 1 to 8 (using the buffer solutions obtained in Preparation Examples 1 to 8, respectively) and comparative kits 1 to 10 (using the buffer solutions obtained in Comparative Preparation Examples 1 to 10, respectively).

[0119] Example 1 :Use kit 1 to measure the concentration of GFAP based on single molecule detection technology

[0120] (1) The concentrations of GFAP were diluted to 0, 0.30, 0.91, 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 pg / mL, respectively.

[0121] (2) According to the requirements of the AST-Dx90 fully automatic fluorescent immunoassay analyzer (equipment developed by Suzhou Yuce Biotechnology), the samples and reagents are loaded into the designated positions in order. When ready, the test is started. The equipment automatically sends the samples to the loading position and loads the reaction cup into the incubation plate. The sampling needle draws 25 μL of the sample of each concentration obtained in (1) from the sample tube and adds it to the reaction cup. The reagent needle draws 25 μL of the magnetic bead solution (reagent 1) coated with GFAP capture antibody from the reagent kit 1 and adds it to the reaction cup. The mixture is mixed and incubated for 3 minutes. The magnetic beads modified with surface-specific antibodies in reagent 1 can recognize and capture target molecules with extremely low content in the sample.

[0122] (3) The reagent needle draws 10 μL of GFAP detection antibody (reagent 2) labeled with fluorescent dye from the reagent kit 1 and adds it to the reaction cup, mixes and incubates for 2 minutes. Reagent 2 contains a single molecule signal marker that modifies the detection antibody and can convert the target molecule into a single molecule signal.

[0123] (4) The detection needle transfers the reaction system to the flow cell, uses magnetic separation to attract the magnetic beads to the bottom of the flow cell and spreads them on the surface of the detection hole, washes to remove other components, uses a common fluorescence microscope (purchased from Olympus) for single-molecule imaging, and uses the single-molecule counting mode and fluorescence intensity integration mode together to complete subsequent single-molecule counting statistics and analysis. It should be noted that the incubation time is 5 minutes, and the time from injection to output of the results is 5 minutes and 40 seconds.

[0124] (5) Complete a series of concentration tests, repeat each concentration point 6 times, and draw a standard curve based on the test results.

[0125] Test results such as Figure 1 As shown in FIG. 1 , it can be seen that in this embodiment, the detection range of GFAP is 0.20 pg / mL to 1000 pg / mL. In this range, the number of single-molecule signals and the sample concentration show a good linear relationship (R 2 Close to 1), the detection limit can reach 0.20pg / mL, and the time from injection to output of results is only 5 minutes and 40 seconds. In addition, the CV value is also less than 10%.

[0126] In addition, the results of six independent experiments conducted two months apart show that the GFAP kit of the present invention has highly stable detection results. Specifically, the relative errors calculated based on the results of the six independent experiments are all less than 10%.

[0127] Examples 2 to 8, Comparative Examples 1 to 10 (using the above kits 2 to 8, Comparative kits 1 to 10, respectively)

[0128] The above kits 2 to 8 and comparative kits 1 to 10 were operated in the same manner as in Example 1 (the incubation time was 5 minutes, and the time from injection to output of results was 5 minutes and 40 seconds), and the detection limits were obtained. The results of the detection limits are shown in Tables 3 and 4, respectively.

[0129] In addition, the applicant also conducted experiments on the cases where the fluorescent dyes were particles made of polyacrylamide encapsulated fluorescein, fluorescent particles made of polystyrene encapsulated rare earth elements (europium), fluorescent particles made of dextran encapsulated fluorescent protein (GFP), and fluorescent particles made of cross-linked agarose encapsulated quantum dots (cadmium sulfide). Specifically, in Example 1, the fluorescent dye made of silicon dioxide encapsulated fluorescein was replaced with the above-mentioned fluorescent dyes, and the results showed that the sensitivity was also relatively excellent (the detection limit of some dyes can also reach about 0.15pg / mL, especially the particles made of polyacrylamide encapsulated fluorescein).

[0130] Table 3

[0131]

[0132] Table 4

[0133]

Claims

1. A kit for detecting glial fibrillary acidic protein based on single molecule counting, characterized in that: The invention comprises magnetic beads coated with a capture antibody, a detection antibody labeled with a fluorescent dye, and a buffer for the detection antibody labeled with the fluorescent dye, wherein the capture antibody and the detection antibody can bind to different sites of glial fibrillary acid protein respectively, the fluorescent dye contains a fluorescent material and a carrier, and has a particle size of 180 to 450 nm, the fluorescent material is a fluorescein-type luminescent material, a rhodamine-type luminescent material, an aggregation-induced luminescent material or a quantum dot-type luminescent material, the carrier is silicon dioxide, polyacrylamide or polystyrene, the pH of the buffer is 7.0 to 7.6, and each 100 mL of the buffer consists of 10 mM to 40 mM PBS, 0.2 g to 0.8 g of bovine serum albumin, 0.05 mL to 0.30 mL of Tween-20, 0.4 g to 1.0 g of sodium chloride, 0.03 mL to 0.06 mL of proclin-300, 2 g to 10 g of trehalose and sterile distilled water.

2. The kit for detecting glial fibrillary acid protein according to claim 1, wherein: The kit also includes a glial fibrillary acid protein calibrator and a glial fibrillary acid protein control.

3. A single molecule detection system for detecting glial fibrillary acidic protein, comprising the kit according to claim 1 or 2 and an optical imaging device, The optical imaging device includes 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 focusing detection device, nor does it include a micro-reaction chamber with a volume of nanoliter level, picoliter level, or femtoliter level.

Images