CN117554621A

CN117554621A - Homogeneous phase immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, preparation method, detection method and device

Info

Publication number: CN117554621A
Application number: CN202311336941.8A
Authority: CN
Inventors: 杨阳; 刘宇卉; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2024-02-13
Also published as: CN111505302A

Abstract

The invention relates to a homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, a preparation method, a detection method and a detection device, belonging to the technical field of immunoassay. The homogeneous immunoassay kit for rapidly detecting the N-terminal B-type brain natriuretic peptide prepared by the method can rapidly detect the N-terminal B-type brain natriuretic peptide in a sample to be detected, the detection time can be shortened to be within 10min, the detection sensitivity can reach 20pg/mL, and the variation coefficient is within 10%. In addition, when the kit is used for detection, the dosage of the reagent can be reduced, the raw materials can be saved, the content of the N-terminal B-type brain natriuretic peptide in a sample to be detected of a subject can be accurately and rapidly determined, and the kit has a wide application prospect.

Description

Homogeneous phase immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, preparation method, detection method and device

Technical Field

The invention belongs to the technical field of immunoassay, and particularly relates to a homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, a preparation method, a detection method and a detection device.

Background

N-terminal B-type brain natriuretic peptide, N-terminal proBNP (NT-proBNP), is the inactive N-terminal portion of proBNP (108 amino acid residues) secreted during ventricular muscle hyper-extension, which is produced in a invertase dependent reaction, consisting of 76 amino acids. The relative molecular mass of the N-terminal B-type brain natriuretic peptide is about 8.5KD, the N-terminal B-type brain natriuretic peptide is cleared by kidneys, the half-life period is 90-120 minutes, the N-terminal B-type brain natriuretic peptide is stable in vitro, and the concentration in blood is 15-20 times that of BNP. In the peripheral blood of healthy people, the content of N-terminal B-type brain natriuretic peptide is very low, when the ventricular pressure changes and the wall tension increases due to capacity load, ventricular myocytes synthesize and secrete BNP and N-terminal B-type brain natriuretic peptide, so that the increase of the concentration of N-terminal B-type brain natriuretic peptide can well reflect the changes of ventricular structure and function. Currently, N-terminal B-type brain natriuretic peptide is recognized as a sensitive and specific index during heart dysfunction, and is mainly used for auxiliary diagnosis of heart failure clinically.

For the detection method of N-terminal B-type brain natriuretic peptide, it is necessary to remove unreacted labeled antibody before detection because the amount of labeled antibody is excessive, regardless of Radioimmunoassay (RIA), enzyme Immunoassay (EIA), or chemiluminescent immunoassay. The existing heterogeneous immunoassay method all needs complicated washing steps; meanwhile, the defects of environmental pollution, short shelf life and the like of radioimmunoassay are overcome, and the defects of poor repeatability and the like of enzyme immunoassay are overcome.

In the homogeneous immunoassay technology, a separation washing step is not needed, however, in the biological detection based on homogeneous immunoassay, when the detection efficiency is further improved by shortening the detection time, various performances of the detection are seriously affected due to the fact that the reaction is not balanced, for example: the detection sensitivity may be significantly reduced. In addition, the application efficiency of the photo-excitation chemiluminescence technology is limited due to the fact that the service life of singlet oxygen ions is too short, no report of remarkably increasing the service life of the singlet oxygen ions is available at present, and the service life of the singlet oxygen ions can be increased to improve detection signals under the same conditions, so that the detection sensitivity can be increased, and the detection limit can be reduced.

Therefore, it is highly desirable to establish a rapid, accurate and sensitive homogeneous immunization method for detecting N-terminal B-type brain natriuretic peptide.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for preparing a homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, the kit obtained by the method can rapidly detect N-terminal B-type brain natriuretic peptide in a sample to be detected, the detection time can be shortened to be within 12min, the detection sensitivity can reach 5pg/mL, and the defect of poor correlation with a reference reagent is overcome.

To this end, a first aspect of the present invention provides a method for preparing a homogeneous immunoassay kit for rapid detection of an N-terminal B-type brain natriuretic peptide, comprising:

preparing component a, which is a receptor capable of reacting with active oxygen to generate a detectable signal, in combination with a primary antibody or binding fragment thereof; the first antibody or binding fragment thereof is capable of specifically binding to a first epitope of an N-terminal B-type brain natriuretic peptide;

preparing component b, wherein the component b is a second antibody or a binding fragment thereof which binds to biotin; the second antibody or binding fragment thereof is capable of specifically binding to a second epitope of an N-terminal B-type brain natriuretic peptide, and the second epitope and the first epitope do not overlap;

preparing a component c, wherein the component c is a donor which is combined with streptavidin and can generate active oxygen in an excited state.

In some embodiments of the invention, the first antibody and the second antibody are each independently selected from a monoclonal antibody, a polyclonal antibody, an artificial antibody, or a modified antibody; preferably selected from polyclonal antibodies and/or monoclonal antibodies; further preferred are monoclonal antibodies.

In some embodiments of the invention, the acceptor comprises an olefin compound and a metal chelate, which are in non-particulate form and are soluble in an aqueous medium; and/or the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide.

In other embodiments of the invention, the donor is a photoactivated or chemically activated sensitizer, which is in non-particulate form and is soluble in an aqueous medium; and/or the donor is polymer particles filled with photosensitive compounds.

In some embodiments of the invention, the first antibody or binding fragment thereof indirectly binds to a receptor; preferably, the primary antibody or binding fragment thereof binds to the receptor indirectly via the fluorescein thiocyanate-fluorescein isothiocyanate antibody system (FITC-FITC antibody system).

In other embodiments of the invention, the acceptor is an aldehyde and/or carboxyl modified acceptor; preferably, the acceptor is a carboxy-modified acceptor.

According to the invention, the method further comprises:

preparing a first composition comprising the component a and a first buffer;

preparing a second composition comprising the component b and a second buffer;

preparing a third composition comprising the component c and a third buffer.

In some embodiments of the invention, the concentration of component a in the first composition is 50 to 300 μg/mL; preferably 80-250 mug/mL; more preferably 100 to 200. Mu.g/mL; and/or the number of the groups of groups,

the concentration of component b in the second composition is 1-15 mug/mL; preferably 2 to 10. Mu.g/mL; more preferably 4 to 8. Mu.g/mL; and/or

The concentration of the component c in the third composition is 20-200 mug/mL; preferably 30 to 150. Mu.g/mL; more preferably 50 to 100. Mu.g/mL.

In some embodiments of the invention, the acceptor is a polymeric microparticle filled with a luminescent compound and a lanthanide, and the method of preparing the first composition includes:

step S1, dialyzing the first antibody or the binding fragment thereof by using a first dialysis buffer solution to obtain a dialyzed first antibody or the binding fragment thereof;

step S2, after the receptor is washed by using a crosslinking buffer solution, re-suspending the washed receptor by using the crosslinking buffer solution to obtain receptor fluid;

Step S3, adding the dialyzed primary antibody or the binding fragment thereof into the receptor liquid, uniformly mixing, and combining to obtain a receptor combined with the primary antibody or the binding fragment thereof;

step S4, after washing the receptor bound to the primary antibody or the binding fragment thereof with a washing buffer, the primary buffer is added to obtain a first composition.

In some embodiments of the invention, the mass ratio of the primary antibody or binding fragment thereof to the receptor is (0.1-0.9): 10; preferably, the mass ratio of the primary antibody or binding fragment thereof to the receptor is (0.2 to 0.8): 10; more preferably (0.3 to 0.7): 10.

In other embodiments of the invention, the first dialysis buffer and the crosslinking buffer are each 0.03 to 0.08M CB buffer having a pH of 9.5 to 10.0; the cleaning buffer solution is PBST buffer solution with the pH value of 7.0-7.5 and the concentration of 0.08-0.12M.

According to the present invention, the method for preparing the second composition comprises:

step T1, dialyzing the second antibody or the binding fragment thereof by using a labeling buffer solution to obtain a dialyzed second antibody or the binding fragment thereof;

step T2, adding a labeling buffer solution into the dialyzed secondary antibody or the binding fragment thereof, uniformly mixing, then adding a biotin solution, uniformly mixing, and rotating overnight to obtain the secondary antibody or the binding fragment thereof bound with the first marker;

And step T3, dialyzing the second antibody or the binding fragment thereof bound with the first marker by using a second dialysis buffer, and adding the second buffer after the dialysis is finished to obtain a second composition.

In some embodiments of the invention, the labeling buffer is NaHCO at a pH of from 0.08 to 0.12M at a pH of from 7.5 to 8.5 ₃ A solution; the second dialysis buffer solution is PBS buffer solution with the pH value of 7.0-7.5 and 0.08-0.12M.

In other embodiments of the invention, the mass ratio of the first label to the second antibody or binding fragment thereof is 1 (1-10); preferably, the mass ratio of the first label to the second antibody or the binding fragment thereof is 1 (1-5).

According to the invention, the first buffer solution and the second buffer solution are both Tris-HCl solutions of 0.08-0.12M with pH value of 7.0-8.0.

In some embodiments of the invention, a surfactant is also included in the third composition; preferably, the surfactant is a nonionic surfactant; further preferably, the surfactant is a linear nonionic surfactant.

In other embodiments of the invention, the concentration of the surfactant is from 0.01 to 0.04wt%; preferably 0.02 to 0.04wt%.

In a second aspect, the invention provides a homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide prepared by the method of the first aspect of the invention.

In a third aspect, the invention provides a homogeneous immunoassay for rapid detection of an N-terminal B-type brain natriuretic peptide using a kit according to the second aspect of the invention, the assay being a photoexcitation chemiluminescent assay.

According to the invention, the method comprises the following steps:

r1, mixing a V1 volume of sample to be tested with a V2 volume of first composition and a V3 volume of second composition, and then performing a first reaction to obtain a first mixture; the temperature of the first reaction is K1, and the time is T1;

step R2, mixing the first mixture with the third composition, and then performing a second reaction to obtain a second mixture; the temperature of the second reaction is K2, and the time is T2;

step R3, exciting the donor in the second mixture with energy or an active compound to generate active oxygen, the acceptor being capable of reacting with the received active oxygen to generate a detectable chemiluminescent signal; judging whether N-terminal B-type brain natriuretic peptide exists in the sample to be detected or not and/or determining the content of N-terminal B-type brain natriuretic peptide by detecting the existence and/or intensity of chemiluminescent signals;

Wherein T1 is less than or equal to 15min, T2 is less than or equal to 5min, and T1+ T2 is less than or equal to 15min.

In some embodiments of the invention, in step R1, the sample to be tested is mixed with the first composition before the second composition is added.

In other embodiments of the invention, each of K1 and K2 is independently selected from 35-45 ℃; preferably from 37 to 42 ℃.

In some embodiments of the invention, the T1 is less than or equal to 15 minutes; preferably, T1 is less than or equal to 13min; further preferably, 6 min.ltoreq.T1.ltoreq.10 min.

In other embodiments of the invention, T2 is greater than or equal to 2 minutes.

In some embodiments of the invention, the T1+T2 is less than or equal to 15 minutes; preferably, T1+T2 is less than or equal to 13min; further preferably, T1+T2 is less than or equal to 12min.

In other embodiments of the invention, T1 is 10min and T2 is 2min.

In some embodiments of the invention, 5 μL.ltoreq.V1.ltoreq.100 μL; preferably, 10. Mu.L.ltoreq.V1.ltoreq.80. Mu.L; more preferably, 20. Mu.L.ltoreq.V1.ltoreq.60. Mu.L.

In other embodiments of the invention, 5 μL.ltoreq.V2.ltoreq.50 μL; preferably, 8. Mu.L.ltoreq.V2.ltoreq.40. Mu.L; more preferably, 10. Mu.L.ltoreq.V2.ltoreq.25. Mu.L.

In some embodiments of the invention, 5 μL.ltoreq.V3.ltoreq.50 μL; preferably, 8. Mu.L.ltoreq.V3.ltoreq.40. Mu.L; more preferably, 10. Mu.L.ltoreq.V3.ltoreq.25. Mu.L.

In other embodiments of the present invention, in step R3, the second mixture is irradiated with excitation light having a wavelength of 600-700nm, the donor is excited to generate active oxygen, the acceptor reacts with the contacted active oxygen to generate light with an emission wavelength of 520-620nm, and the presence and/or intensity of the light emission signal is detected, so as to determine whether the N-terminal B-type brain natriuretic peptide is present in the sample to be detected and/or determine the content of the N-terminal B-type brain natriuretic peptide.

According to the invention, the sensitivity of the method is 5pg/mL.

In a fourth aspect, the present invention provides a homogeneous immunoassay device for rapidly detecting an N-terminal B-type brain natriuretic peptide, which employs a kit according to the second aspect of the present invention or a method according to the third aspect of the present invention to detect an N-terminal B-type brain natriuretic peptide in a sample to be detected.

According to the invention, the device comprises: the device comprises a sample adding module, a reagent module, an incubation module, a detection module and a circuit control module; the sample adding module, the reagent module, the incubation module and the detection module are all electrically connected with the circuit control module.

In some embodiments of the invention, the reagent module includes a first component for adding a first composition and a second component for adding a second composition.

In a fifth aspect the invention provides the use of a kit according to the second aspect of the invention, a method according to the third aspect of the invention or a device according to the fourth aspect of the invention in a photo-activated chemiluminescent detection technique.

In a sixth aspect, the invention provides the use of a method according to the first aspect of the invention for preparing a kit for determining the level of an N-terminal B-type brain natriuretic peptide in a test sample for detecting a subject suspected of heart failure; preferably, the sample to be tested is plasma or serum.

The beneficial effects of the invention are as follows: the homogeneous immunoassay kit for rapidly detecting the N-terminal B-type brain natriuretic peptide prepared by the method can rapidly detect the N-terminal B-type brain natriuretic peptide in a sample to be detected, the detection time can be shortened to be within 12 minutes, the detection sensitivity can reach 5pg/mL, and the variation coefficient is within 10%. In addition, when the kit is used for detection, the dosage of the reagent can be reduced, the raw materials can be saved, the content of the N-terminal B-type brain natriuretic peptide in a sample to be detected of a subject can be accurately and rapidly determined, and the kit has a wide application prospect.

Drawings

The present invention will be described in detail below with reference to the accompanying drawings.

FIG. 1 is a graph showing the relationship between the detected signals and the concentration of the kit 1 and the kit 2 in example 9.

FIG. 2 is a graph showing the correlation between the kit described in the present application and the Roche kit.

Detailed Description

In order that the invention may be more readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

I. Terminology

The term "homogeneous" as used herein is defined as "homogeneous" and refers to a detection that can be performed without separation of one or the other of the complex formed by the binding of the specific binding pair members to each other and the remaining free specific binding pair members, e.g., without separation of the bound antigen-antibody complex from the remaining free antigen or antibody.

The term "test sample" as used herein refers to a mixture that may contain an analyte, including but not limited to a protein, hormone, antibody or antigen. Typical test samples that may be used in the methods disclosed herein include body fluids, such as plasma, serum, and the like. The sample to be tested may be diluted with a diluent or buffer solution as desired prior to use. For example, in order to avoid the HOOK effect, the analyte may be diluted by a sample diluent before on-machine detection and then detected on a detection instrument, where the diluted solution that may contain the analyte is collectively referred to as a sample to be detected.

The term "antibody" as used herein is used in its broadest sense and includes antibodies of any isotype, antibody fragments that retain specific binding to an antigen, including but not limited to Fab, fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, bispecific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In any desired case, the antibody may be further conjugated to other moieties, such as specific binding pair members, e.g., biotin or streptavidin (one of the biotin-streptavidin specific binding pair members), and the like.

The term "monoclonal antibody" as used herein refers to an immunoglobulin secreted by monoclonal B lymphocytes, which can be prepared by methods well known to those skilled in the art.

The term "polyclonal antibody" as used herein refers to a collection of immunoglobulins derived from more than one B lymphocyte clone, which may be prepared by methods well known to those skilled in the art.

The term "binding" as used herein refers to the direct association between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic and/or hydrogen bonding, including but not limited to interactions such as salt and water bridges.

The term "specific binding" as used herein refers to the mutual recognition and selective binding reaction between two substances, and from a steric perspective, corresponds to the conformational correspondence between the corresponding reactants.

The term "biotin" is widely used in animal and plant tissues, and has two cyclic structures, namely an imidazolone ring and a thiophene ring, on the molecule, wherein the imidazolone ring is the main part combined with streptavidin. Activated biotin can be coupled to almost all known biomacromolecules, including proteins, nucleic acids, polysaccharides, lipids, etc., mediated by protein cross-linking agents; and "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding to a biotin. Thus, each antigen or antibody can be conjugated to multiple biotin molecules simultaneously, thereby producing a "tentacle effect" that enhances assay sensitivity. In any case where desired, any agent used in the present invention, including antigen, antibody, receptor or donor, may be conjugated to any member of the biotin-streptavidin specific binding pair member as desired.

The term "active oxygen" as used herein refers to a substance which is composed of oxygen in the body or in the natural environment, contains oxygen and is active in nature, and is mainly an excited oxygen molecule, including an electron reduction product of oxygen, superoxide anion (O ₂ Hydrogen peroxide (H), a two-electron reduction product ₂ O ₂ ) Hydroxyl radical (OH) of three-electron reduction product, nitric oxide and singlet oxygen (1O) ₂ ) Etc.

The term "donor" as used herein refers to a sensitizer that upon activation of energy or an active compound is capable of generating an active intermediate such as singlet oxygen that reacts with the acceptor. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.). In some embodiments of the invention, the donor is a photosensitizer, which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine, and chlorophyll as disclosed in U.S. Pat. No. 5709994 (which is incorporated herein by reference in its entirety), and derivatives of these compounds having 1-50 atom substituents for making these compounds more lipophilic or hydrophilic, and/or as linking groups to specific binding pair members. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, such as described in U.S. patent No. 6406913, which is incorporated herein by reference. In other embodiments of the invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Examples of other donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, and the like, and singlet oxygen is released by heating these compounds or by direct absorption of light by these compounds.

The term "acceptor" as used herein refers to a substance capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced to activate by energy or an active compound and releases singlet oxygen in a high energy state which is captured by the acceptor in close proximity, thereby transferring energy to activate the acceptor. In some embodiments of the invention, the acceptor is a substance that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that may decompose while or subsequently emit light. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan, 9-alkylidene-N-alkyl acridines, arylvinyl ethers, bisoxyethylene, dimethylthiophene, aromatic imidazoles or gloss concentrates. In other embodiments of the invention, the acceptor is an olefin capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can decompose to a ketone or carboxylic acid derivative; stable dioxetanes that can be decomposed by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides of azo compounds or azocarbonyl compounds, such as luminol, may be formed; and aromatic compounds which can form endoperoxides. A specific, non-limiting example of a receptor that can be utilized in accordance with the present disclosure and claimed invention is described in U.S. patent No. US5340716 (which is incorporated herein by reference in its entirety). In other embodiments of the present invention, the acceptor comprises an olefinic compound and a metal chelate that is non-particulated and soluble in an aqueous medium, as described in PCT/US2010/025433 (which is incorporated herein by reference in its entirety).

In the present invention, the donor may be polymer particles filled with a photosensitive compound formed by coating a functional group on a substrate, and may generate singlet oxygen under light excitation, and in this case, the donor may be called a photosensitive microsphere or a photosensitive particle, and a solution containing the photosensitive microsphere or the photosensitive particle may be called a photosensitive solution or a general solution; and/or the acceptor may be polymer particles filled with a luminescent compound and a lanthanoid element formed by coating a functional group on a substrate, which may be referred to as luminescent microspheres or luminescent particles. In this application, the system is based on the light-emitting substance coated on the surface of the substrate inducing a luminescent signal via light excitation and energy transfer, which is dependent on antigen-antibody binding resulting in the light-sensitive microspheres and the luminescent microspheres being in close proximity to each other. Thus eliminating the need for a separate process. The diameter of the nano microsphere is smaller, the suspension performance is stronger, and a three-level amplifying and light-emitting system is adopted, so that the nano microsphere has higher analysis sensitivity; the whole detection process does not need to be washed, namely the bound label and the bound label are not required to be separated, so that the reaction time is shorter; the tracer substance (photosensitizer and luminescent agent) is marked on the matrix instead of the biomolecule, which has no influence on the activity of the biomolecule, and meanwhile, the matrix has larger specific surface area, so that more tracer substance and biomolecule can be coated on the surface of the matrix, and the performance of the matrix in the aspects of effective concentration and sensitivity of the reagent, detection background and the like is better.

The "matrix" according to the invention is a microsphere or microparticle, known to the person skilled in the art, which may be of any size, preferably of nanometric size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which has any density, but preferably has a density close to that of water, preferably is floatable in water, and is composed of transparent, partly transparent or opaque material. The matrix may or may not be charged and when charged is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts, and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is generally multifunctional or capable of binding to a donor or acceptor by specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is a carboxyl modified latex particle. Details of such substrates can be found in U.S. Pat. nos. 5709994 and 5780646 (both of which are incorporated herein by reference in their entirety).

The term "epitope" as used herein refers to any protein determinant capable of specific binding to an immunoglobulin or T cell receptor. In some embodiments of the invention, an epitope is a region of an antigen surface that can be assembled specifically by an antibody. Epitope determinants may generally include chemically active surface groupings of molecules such as, but not limited to: amino acids, sugar side chains, phosphoryl and/or sulfonyl groups. In other embodiments of the invention, epitopes may be specifically specific for three-bit structural features as well as specific charge features.

Example II

As described above, in the biological detection based on homogeneous phase immunity, when the detection time is shortened to further improve the detection efficiency, various performances of the detection are seriously affected due to the fact that the reaction is not balanced. The inventor of the application finds that the reaction time can be shortened and the sensitivity and accuracy of detection can be improved by increasing the concentration of the reagent, reducing the sample adding amount of the reagent and the sample, improving the reaction temperature, indirectly combining the primary antibody or the binding fragment thereof and the receptor through a FITC-FITC antibody system and the like.

To this end, the first aspect of the present invention relates to a method for preparing a homogeneous immunoassay kit for rapid detection of an N-terminal B-brain natriuretic peptide, comprising:

In some embodiments of the invention, the first antibody or binding fragment thereof indirectly binds to a receptor; preferably, the primary antibody or binding fragment thereof binds to the receptor indirectly via the fluorescein thiocyanate-fluorescein isothiocyanate antibody system (FITC-FITC antibody system). Binding FITC antibody to the receptor, and binding FITC to the primary antibody or binding fragment thereof; the primary antibody or binding fragment thereof is then indirectly bound to the receptor by the specific binding of FITC antibody to FITC.

According to the invention, the method further comprises:

preparing a first composition comprising the component a and a first buffer;

preparing a second composition comprising the component b and a second buffer;

Preparing a third composition comprising the component c and a third buffer.

In some embodiments of the invention, the mass ratio of the primary antibody or binding fragment thereof to the receptor is 1 (15-25); preferably, the mass ratio of the primary antibody or binding fragment thereof to the receptor is 1 (18-22).

In some embodiments of the present invention, the method for preparing the first composition specifically includes:

(1) Pretreatment of

Placing the first antibody or the binding fragment thereof in a dialysis bag (with a molecular weight cut-off of 14 KD), and dialyzing with 0.03-0.08M CB buffer (first dialysis buffer) with a pH value of 9.5-10.0, which is 80-120 times of the volume, on a magnetic stirrer at 2-8 ℃; changing the dialysate at least 1 time, and dialyzing for at least 4-5 hours each time to obtain the first antibody or the binding fragment thereof after dialysis.

(2) Binding of primary antibodies or binding fragments thereof to receptors

Adding 0.03-0.08M CB buffer (cross-linking buffer) with the pH value of 9.5-10.0 into a receptor, centrifuging, discarding the supernatant, then adding 0.03-0.08M CB buffer (cross-linking buffer) with the pH value of 9.5-10.0 again, performing ultrasonic cleaning, and centrifuging to remove the supernatant; finally, 0.03-0.08M CB buffer (cross-linking buffer) with the pH value of 9.5-10.0 is used for resuspension, thus obtaining receptor solution.

Adding the dialyzed primary antibody or the binding fragment thereof into the receptor liquid, uniformly mixing, and uniformly mixing the mixture with a vertical rotary mixer at 35-40 ℃ at 25-40 rpm for overnight to obtain a mixed liquid; then cooling the mixed solution at 2-8 ℃ for 5-15 min, immediately adding NaBH ₄ Mixing the solution uniformly, and reacting for 1.5-2.5 hours at the room temperature at 25-40 rpm on a vertical rotary mixer; and finally adding Gly solution, uniformly mixing, and reacting for 0.5-1.5 hours at 25-40 rpm on a vertical rotary mixer to obtain the receptor combined with the primary antibody or the binding fragment thereof.

(3) Cleaning and compounding

The receptor bound to the primary antibody or binding fragment thereof is subjected to ultrasonic washing with 0.08-0.12M PBST buffer (washing buffer) having a pH of 7.0-7.5, and the first buffer is added to obtain a first set of compositions.

In some embodiments of the present invention, the method for preparing the second composition specifically includes:

(1) Pretreatment of

Placing the second antibody or its binding fragment in dialysis bag (molecular weight cut-off of 3 KD) and using 80-120 times volume of NaHCO with pH value of 7.5-8.5 and 0.08-0.12M ₃ The solution (marked buffer) is dialyzed on a magnetic stirrer at the temperature of 2-8 ℃; changing the dialysate at least 1 time, and dialyzing for at least 4-5 hours each time to obtain the first antibody or the binding fragment thereof after dialysis.

(2) Binding of the first marker to the second antibody or binding fragment thereof

Adding NaHCO with pH value of 7.5-8.5 and 0.08-0.12M into the dialyzed secondary antibody or binding fragment thereof ₃ The solution (labeling buffer) is mixed uniformly, then biotin solution is added and mixed uniformly rapidly, and the mixture is reacted overnight at 25 to 40rpm on a vertical rotary mixer at the temperature of 2 to 8 ℃ to obtain the second antibody or the binding fragment thereof bound with the first label.

(3) Dialysis and formulation

Placing the second antibody or the binding fragment thereof combined with biotin in a dialysis bag (with the molecular weight cut-off of 3 KD), dialyzing with a magnetic stirrer at 2-8 ℃ by using 80-120 times of PBS buffer solution (second dialysis buffer solution) with the pH value of 7.0-7.5 and 0.08-0.12M, and dialyzing for at least 4-5 hours each time; after the dialysis is completed, a second buffer is added to obtain a second composition.

In some embodiments of the invention, a surfactant is also included in the third composition; preferably, the surfactant is a nonionic surfactant; further preferably, the surfactant is a linear nonionic surfactant. In some embodiments of the invention, the surfactant isX-080。

The second aspect of the invention relates to a homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide prepared by the method according to the first aspect of the invention.

In a third aspect, the invention relates to a homogeneous immunoassay for rapid detection of N-terminal B-type brain natriuretic peptide using a kit according to the second aspect of the invention, said method being a photoexcitation chemiluminescent assay.

According to the invention, the method comprises the following steps:

In some embodiments of the invention, the T1 is less than or equal to 15 minutes; preferably, T1 is less than or equal to 13min; preferably, T1 is less than or equal to 12min; further preferably, 6 min.ltoreq.T1.ltoreq.10 min.

In other embodiments of the invention, T1 is 10min and T2 is 2min.

According to the invention, the sensitivity of the method is 5pg/mL.

In particular, the methods of the present invention are all methods for the purpose of non-disease diagnosis.

The fourth aspect of the invention relates to a homogeneous immunoassay device for rapidly detecting N-terminal type B brain natriuretic peptide, which is used for detecting N-terminal type B brain natriuretic peptide in a sample to be detected by the kit according to the second aspect of the invention or the method according to the third aspect of the invention.

A control method of a homogeneous immunoassay device for rapid detection of an N-terminal B-type brain natriuretic peptide, comprising:

step M1, controlling the sample adding module to take a sample to be detected with the V1 volume for detection; wherein, V1 is more than or equal to 5 mu L and less than or equal to 100 mu L, preferably V1 is more than or equal to 10 mu L and less than or equal to 90 mu L, and more preferably V1 is more than or equal to 20 mu L and less than or equal to 80 mu L;

step M2, controlling the reagent module to take the first composition with the volume of V2 and the second composition with the volume of V3, and mixing the first composition with the sample to be tested to obtain a first mixture; wherein, V2 is more than or equal to 5 mu L and less than or equal to 50 mu L, preferably V2 is more than or equal to 10 mu L and less than or equal to 40 mu L, and more preferably V2 is more than or equal to 15 mu L and less than or equal to 25 mu L; v3 is more than or equal to 5 mu L and less than or equal to 50 mu L, preferably V3 is more than or equal to 10 mu L and less than or equal to 40 mu L, and more preferably V3 is more than or equal to 15 mu L and less than or equal to 25 mu L;

step M3, controlling the incubation module to incubate the first mixture, and reacting the first mixture at the temperature of K1 for T1 time to obtain a second mixture; wherein T1 is less than or equal to 15min; preferably, T1 is less than or equal to 13min; further preferably, T1 is less than or equal to 6min and less than or equal to 10min.

A fifth aspect of the invention relates to the use of a kit according to the second aspect of the invention, a method according to the third aspect of the invention or a device according to the fourth aspect of the invention in a photo-activated chemiluminescent detection technique.

A sixth aspect of the invention relates to the use of a method according to the first aspect of the invention for preparing a kit for detecting an N-terminal B-type brain natriuretic peptide in a test sample of a subject suspected to suffer from heart failure, thereby determining the level of an N-terminal B-type brain natriuretic peptide in the test sample; preferably, the sample to be tested is plasma or serum.

In order that the invention may be more readily understood, the invention will be further described in detail with reference to the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.

In the examples described below, the main instruments used in the experiments are shown in Table 1, and the reagents and materials (including biological materials, dilutions, sample information, etc.) used are shown in Table 2.

Table 1: instrument used in experiments

Sequence number	Name of the name	Device numbering	Manufacturer' s
				1	LiCAHT	BYG-222	Boyang
2	Oscillator (vortex)	BYD-045-04	VDRTEX

Table 2: reagents and raw materials employed in the experiments

Example 1: preparation of homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide

1. Preparation of acceptor and donor

The preparation method, the composition structure and the content of the components used as acceptors and donors in the present invention can be found in example 1 of chinese patent CN100429197C (which is incorporated herein by reference in its entirety).

2. Preparation of the first composition (R1)

(1) Pretreatment of

0.2mg of NBNP antibody (coating) to be treated is put into a dialysis bag (the molecular weight cut-off is 14 KD), the dialysis bag is put into a beaker, 100 times of 0.05M CB buffer solution with the volume of pH of 9.6 is added into the beaker, the beaker is placed on a magnetic stirrer for dialysis at the temperature of 2-8 ℃ and the dialysis solution is changed for at least 1 time and at least 4-5 hours each time, the NBNP antibody (coating) after dialysis is obtained, the NBNP antibody is sucked out and transferred into a clean centrifuge tube, and the protein concentration is sampled and measured.

(2) NBNP antibody (coating) and receptor binding Process

2mg of the receptor was added to a centrifuge tube, 0.05M CB buffer at pH9.6 was added, the mixture was centrifuged at 7500rpm for 15 minutes, the supernatant was discarded, 400ul of 0.05M CB buffer at pH9.6 was added to the centrifuge tube, and after ultrasonic washing, the mixture was centrifuged again.

200ul of 0.05M CB buffer with pH9.6 is added to re-suspend the receptor to make the concentration of the receptor be 10mg/ml, then 0.1mg of PCT Ab1 after dialysis is added, after uniform mixing, a centrifuge tube is placed on a vertical rotary mixer at 37 ℃ for uniform mixing at 25-40 rpm for overnight.

Cooling the centrifuge tube at 2-8deg.C for 10min to obtain 4ul of NaBH 8mg/ml ₄ The solution was immediately added to the centrifuge tube and mixed well, and reacted for 2 hours at room temperature at 25-40 rpm on a vertical rotating mixer.

32ul of 75mg/ml Gly solution was added to the centrifuge tube and mixed well, and reacted for 1 hour at 25-40 rpm on a vertical rotary mixer.

(3) Cleaning and compounding

After the centrifuge tube was weighed and leveled, it was centrifuged at 7500rpm for 5min, the supernatant was discarded, and the buffer of 0.1M PBST, pH7.4, was added, and the washing was performed by ultrasonic washing twice, and then the buffer for receptor preservation was used for washing once. Adding 0.08-0.12M Tris-HCl solution with pH value of 7.5-8.5, and mixing to obtain the final product. Wherein the concentration of component a in the first composition is 200 μg/mL.

3. Preparation of the second composition (R2)

(1) Pretreatment of

Loading 0.2mg of NBNP antibody to be treated into a dialysis bag (molecular weight cut-off of 3 KD), placing the dialysis bag into a beaker, and adding 100 times volume of 0.1M NaHCO with pH of 8.0 into the beaker ₃ And (3) placing the buffer solution on a magnetic stirrer for dialysis at the temperature of 2-8 ℃, changing the dialysis solution at least 1 time, dialyzing for at least 4-5 hours each time to obtain the NBNP antibody after dialysis, transferring the NBNP antibody into a clean centrifuge tube, and sampling and measuring the protein concentration.

(2) Binding of biotin to NBNP antibody

200ul of 0.1M NaHCO pH8.0 was taken ₃ Buffer solution is added into a centrifuge tube, 0.1mg of NBNP antibody after dialysis is added,mixing well. Then 8ul of prepared 5mg/ml biotin solution is added, the mixture is quickly and evenly mixed, and the mixture is reacted overnight at 25 to 40rpm on a vertical rotary mixer at the temperature of 2 to 8 ℃ to obtain the NBNP antibody combined with the biotin.

(3) Dialysis and formulation

Loading the NBNP antibody solution combined with biotin into a dialysis bag (the molecular weight cut-off is 3 KD), placing the dialysis bag into a beaker, adding 100 times of 0.1M PBS buffer solution with the volume of pH7.4 into the beaker, placing the beaker on a magnetic stirrer, dialyzing at 2-8 ℃, and changing the dialysis solution at least 1 time for at least 4-5 hours each time; and after the dialysis is finished, adding a second buffer solution to complete the preparation of the second composition for later use. Wherein the concentration of component b in the second composition is 8 μg/mL.

4. Preparation of the third composition (R3)

a. Photosensitive microsphere (donor) suspension treatment: sucking a certain amount of photosensitive microspheres, centrifuging in a high-speed refrigerated centrifuge, discarding the supernatant, adding a certain amount of MES buffer, performing ultrasonic treatment on an ultrasonic cell disruption instrument until the particles are resuspended, and adding the MES buffer to adjust the concentration of the photosensitive microspheres to 100mg/ml.

b. Preparing streptavidin solution: a quantity of streptavidin was weighed and dissolved to 8mg/ml in MES buffer.

c. Mixing: mixing the treated photosensitive microsphere (donor) suspension, 8mg/ml streptavidin solution and MES buffer solution in a volume ratio of 2:5:1, and rapidly and uniformly mixing to obtain a reaction solution.

d. The reaction: 25mg/ml NaBH is prepared by MES buffer solution ₃ CN solution was added in a volume ratio of 1:25 to the reaction mixture, and the mixture was swiftly mixed and reacted at 37℃for 48 hours.

e. Closing: preparing 75mg/ml Gly solution and 25mg/ml NaBH in MES buffer ₃ CN solution is added into the solution according to the volume ratio of 2:1:10 with the reaction solution, and the mixture is uniformly mixed and rotated at 37 ℃ for 2 hours. Then 200mg/ml BSA solution (MES buffer) was added thereto in a volume ratio of 5:8, and the mixture was swiftly mixed and reacted at 37℃for 16 hours.

f. Cleaning: and adding MES buffer solution into the reacted solution, centrifuging by a high-speed refrigerated centrifuge, discarding the supernatant, adding fresh MES buffer solution, suspending again by an ultrasonic method, centrifuging again, washing for 3 times, and finally suspending by a photosensitive reagent buffer solution to obtain a third composition. Wherein the concentration of component c in the third composition was 100. Mu.g/mL, and the composition was used as a general-purpose liquid.

5. And partitioning, assembling the first composition, the second composition and the third composition to obtain the homogeneous immunoassay kit for rapidly detecting the N-terminal B-type brain natriuretic peptide.

Implementation 2: preparation of homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide

Preparation of the first composition (R1)

The NBNP antibody (coating) was linked to the receptor as described in the examples of PCT/US2010/025433 to form a receptor that bound to the NBNP antibody (coating). Wherein the structure of the receptor is soluble, which is in a non-particulate form, and the concentration of the receptor (component a) that binds to the NBNP antibody (coating) in the first composition (R1) is 200 μg/mL.

The remaining components were prepared as in example 1.

Example 3: sensitivity detection of homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide

The experimental steps are as follows:

1. selecting a sample with moderate concentration, balancing to room temperature, uniformly mixing, and then carrying out gradient dilution with a negative sample until the concentration is lower than the detection lower limit;

2. the evenly mixed sample, R1 and R2 prepared in example 1 (added according to the experimental design sequence) are respectively added into an 8X 12 white board;

3. placing the white board with the sample into a LiCA HT instrument for reaction in the following reaction mode;

(1) Mix 40ul sample, 15ul R1 and 15ul R2;

(2) Incubation at 37℃for 10min;

(3) 175ul of universal solution (R3) was added;

(4) Incubation at 37℃for 2min;

(5) The readings were excited and the results are shown in table 3.

Table 3: kit sensitivity verification result

Example 4: effect of different incubation times on N-terminal B-brain natriuretic peptide detection

The experimental steps are as follows:

1. re-selecting 8 samples with uniform concentration distribution, balancing to room temperature, and uniformly mixing;

2. the concentrations of R1 and R2 prepared in the example 1 are respectively diluted to 100 mug/mL and 1 mug/mL, evenly mixed and marked;

3. adding the uniformly mixed sample, the prepared R1 and R2 into an 8X 12 white board according to a design table, and uniformly mixing;

4. the loaded white plate was placed in a LiCA HT instrument and reacted using two reaction modes:

the first reaction mode is:

(1) Mixing 25ul of sample, 25ul of R1 and 25ul of R2 uniformly;

(2) Incubation times at 37 ℃ are shown in table 4;

(3) 175ul of universal solution (R3) was added;

(4) Incubation at 37℃for 5min;

(5) The readings were excited and the results are shown in table 4.

The second reaction mode is:

(1) Mixing 25ul of sample, 25ul of R1 and 25ul of R2 uniformly;

(2) Incubation at 37℃for 10min;

(3) 175ul of the universal solution (R3;

(4) Incubation times at 37 ℃ are shown in table 5;

(5) The readings were excited and the results are shown in table 5.

Table 4: results of different first incubation times for N-terminal type B brain natriuretic peptide detection

Table 5: results of different second incubation times for N-terminal type B brain natriuretic peptide detection

As can be seen from Table 4, the first incubation time was significantly higher at 6min than at 3min, so the first incubation time should be longer than 6min.

As is clear from Table 5, since the second-stage incubation time has little influence on the signal, the reaction results are not greatly affected by the substantial shortening of the second-stage incubation time, but the second-stage incubation time is ensured to be 2 minutes or longer.

Example 5: influence of different sample addition amounts and concentrations of samples and reagents on N-terminal B-type brain natriuretic peptide detection

The experimental steps are as follows:

1. selecting samples with uniform concentration distribution, balancing to room temperature, and uniformly mixing;

2. the prepared R1 and R2 are shown in Table 6, and MAK 33 is added into the diluent adopted in the preparation;

3. adding uniformly mixed samples, and prepared R1 and R2 (added according to the experimental design sequence) into an 8X 12 white board respectively;

4. placing the white board with the sample into a LiCA HT instrument for reaction in the following reaction mode;

(1) See table 6;

(2) Incubation at 37℃for 10min;

(3) Adding a general liquid (R3);

(4) Incubation at 37℃for 2min;

(5) The readings were excited and the results are shown in Table 7.

Table 6: different sample addition amounts of sample and reagent

Table 7: results of different sample addition amounts and concentrations of samples and reagents on N-terminal B-type brain natriuretic peptide detection

(follow-up) Table 7: results of different sample addition amounts and concentrations of samples and reagents on N-terminal B-type brain natriuretic peptide detection

As can be seen from table 7, under the same sample loading conditions, decreasing the sample loading of R1 and R2 decreases the signal, and the signal-to-noise ratio decreases with decreasing sample loading of R1 and R2 because the high value is larger than the background sample.

Reducing the sample size increases the luminescence value, but since the high-end luminescence value increases without increasing the background amplitude, reducing the sample size reduces the sensitivity; however, increasing the sample size advances the HOOK effect, so that the sample loading amount needs to be reduced as much as possible on the premise of ensuring the sensitivity.

Example 6: the effect of indirect binding of NBNP antibody (coating) to the receptor via FITC-FITC antibody system on N-terminal type B brain natriuretic peptide detection.

The adopted kit is as follows:

kit 1: kit prepared in example 1

Kit 2: the procedure was the same as in kit 1 except that R1 was the NBNP antibody (coated) bound to the receptor by the FITC-FITC antibody system.

The experimental steps are as follows:

1. carrying out gradient dilution on the antigen, and uniformly mixing for later use;

2. respectively adding the uniformly mixed sample, R1 and R2 into a white board;

(1) Mixing 25ul of sample, 25ul of R1 and 25ul of R2 uniformly;

(2) Incubation at 37℃for 10min;

(3) 175ul of universal solution (R3) was added;

(4) Incubation at 37℃for 5min;

(5) Excitation readings, detection results are shown in table 8, and the relationship between signal and concentration is shown in fig. 1.

Table 8: detection result

NBNP sample pg/mL	Kit 1	Kit 2
			7.64	327	490
126.9	1364	2728
			482.2	6780	13559
1205	24331	48662
			6658	140168	280336
12500	165194	330387
			23147	426444	852888
32150	571234	1142468

As can be seen from table 8 and fig. 1, the use of the kit 2 significantly improves the signal value, and thus the detection sensitivity. It is shown that the indirect binding of NBNP antibody (coating) and receptor by FITC-FITC antibody system reduces the steric hindrance of NBNP antibody (coating) binding with antigen and improves the detection sensitivity.

Example 7: effect of different group modified receptors on detection of N-terminal B-type brain natriuretic peptide.

The adopted kit is as follows:

kit 1: kits were prepared as described in example 1 using aldehyde-modified acceptors.

Kit 2: a kit prepared as described in example 1 using a carboxy-modified acceptor.

The experimental steps are as follows:

1. balancing the sample to room temperature, and uniformly mixing;

(1) Mixing 25ul of sample, 25ul of R1 and 25ul of R2 uniformly;

(2) Incubation at 37℃for 10min;

(3) 175ul of universal solution (R3) was added;

(4) Incubation at 37℃for 5min;

(5) Excitation readings and detection results are shown in table 9.

Table 9: detection result

As can be seen from Table 9, the signal to noise ratio in the case of the kit prepared with the carboxyl-modified receptor was 43% higher than that in the case of the kit prepared with the aldehyde-modified receptor, so that the sensitivity of the detection can be improved by preparing the kit with the carboxyl-modified receptor.

Example 8: influence of surfactant addition in R3 on N-terminal B-type brain natriuretic peptide detection

The experimental steps are as follows:

1. adding surfactants with different concentrations into R3;

2. uniformly mixing 25ul, 125ul and 225ul of samples to be tested;

incubating for 10min at 3.37 ℃;

4. 175ul of universal solution (R3) was added;

incubating for 5min at 5.37 ℃;

6. the readings were excited and the results are shown in table 10.

Table 10: effect of adding surfactants with different concentrations to R3 on detection of N-terminal B-type brain natriuretic peptide

As can be seen from Table 10, R3 is added at various concentrationsAfter X-080, the signals are obviously improved, especiallyThe signal enhancement is most pronounced at concentrations of X-080 between 0.02 and 0.04 wt%. Therefore, after the surfactant is added into R3, the detection distinction degree can be improved, and the detection sensitivity is further improved.

Comparative example 1: effect of varying reagent concentration only on N-terminal B-brain natriuretic peptide detection

The experimental steps are as follows:

1. carrying out gradient dilution on a high-value sample to be detected, balancing the diluted sample and the high-value sample to room temperature, and uniformly mixing;

2. the concentrations of the prepared R1 and R2 are shown in Table 11, and MAK 33 is added into the diluent adopted in the preparation;

3. respectively adding a uniformly mixed sample to be tested and prepared R1 and R2 (added according to the experimental design sequence) into an 8X 12 white board;

(1) Mixing 25ul of sample to be tested, 25ul of R1 and 25ul of R2 uniformly;

(2) Incubation at 37℃for 15min;

(3) 175ul of universal solution (R3) was added;

(4) Incubation at 37℃for 10min;

(5) The readings were excited and the results are shown in table 12.

Table 11: concentration of formulated R1 and R2

Table 12: detection result

As can be seen from table 12, the signal to noise ratio of all experimental groups after changing the reagent concentration was significantly lower than that of the control group, indicating that the change of the reagent concentration alone did not improve the detection sensitivity. Specifically, under the same concentration of R1, the concentrations of the corresponding R2 and R3 are improved, the signal to noise ratio is reduced, and the detection sensitivity is further reduced; at the same time, increasing the concentration of R1 at the same concentration of R2 and R3 does not increase the signal to noise ratio, even when the concentration of R1 reaches 200 ug/ml.

Comparative example 2: comparison with Roche's brain natriuretic peptide precursor detection kit (electrochemiluminescence)

The experimental steps are as follows:

1. balancing a plurality of samples which are uniformly distributed and measured by Roche to room temperature, and uniformly mixing;

(1) Mixing 25ul of sample to be tested, 25ul of R1 and 25ul of R2 uniformly;

(2) Incubation at 37℃for 15min;

(3) 175ul of universal solution (R3) was added;

(4) Incubation at 37℃for 10min;

(5) The readings were excited and the results are shown in figure 2.

It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims

1. A light excitation chemiluminescence detection method for rapidly detecting N-terminal B-type brain natriuretic peptide comprises the following steps:

step R2, mixing the first mixture with V4 volume of the third composition, and then performing a second reaction to obtain a second mixture; the temperature of the second reaction is K2, and the time is T2;

wherein T1 is less than or equal to 15min, T2 is less than or equal to 5min, and T1 plus T2 is less than or equal to 15min;

the first composition is a solution of a receptor capable of reacting with reactive oxygen species to generate a detectable signal that binds to a first antibody or binding fragment thereof capable of specifically binding to a first epitope of an N-terminal B-type brain natriuretic peptide;

The second composition is a solution of a second antibody or binding fragment thereof that binds biotin; the second antibody or binding fragment thereof is capable of specifically binding to a second epitope of an N-terminal B-type brain natriuretic peptide, and the second epitope and the first epitope do not overlap;

the third composition is a solution of a donor capable of generating active oxygen in an excited state, bound to streptavidin.

2. The method of claim 1, wherein the third composition further comprises a surfactant;

preferably, the surfactant is a nonionic surfactant;

preferably, the surfactant is a linear nonionic surfactant;

preferably, the concentration of the surfactant is from 0.01 to 0.04wt%, more preferably from 0.02 to 0.04wt%.

3. The method according to claim 1 or 2, wherein the first antibody or binding fragment thereof binds indirectly to a receptor, preferably indirectly through a fluorescein isothiocyanate-fluorescein isothiocyanate antibody system; and/or the number of the groups of groups,

the acceptor is an aldehyde and/or carboxyl modified acceptor, preferably a carboxyl modified acceptor.

4. A method according to any one of claims 1 to 3, wherein the concentration of the receptor capable of reacting with active oxygen to generate a detectable signal bound to the primary antibody or binding fragment thereof in the first composition is 50 to 300 μg/mL, preferably 80 to 250 μg/mL, more preferably 100 to 200 μg/mL; and/or the number of the groups of groups,

The concentration of the second antibody or binding fragment thereof bound to biotin in the second composition is 1 to 15. Mu.g/mL, preferably 2 to 10. Mu.g/mL, more preferably 4 to 8. Mu.g/mL; and/or the number of the groups of groups,

in the third composition, the concentration of the donor capable of generating active oxygen in an excited state, which is bound to streptavidin, is 20 to 200. Mu.g/mL, preferably 30 to 150. Mu.g/mL, more preferably 50 to 100. Mu.g/mL.

5. The method according to any one of claims 1 to 4, wherein the mass ratio of the first antibody or binding fragment thereof to the receptor in the first composition is (0.1 to 0.9): 10, preferably (0.2 to 0.8): 10, more preferably (0.3 to 0.7): 10; and/or the number of the groups of groups,

in the second composition, the mass ratio of the biotin to the second antibody or the binding fragment thereof is 1 (1-10); preferably 1 (1) to 5).

6. The method according to any one of claims 1 to 5, wherein K1 and K2 are each independently selected from 35 to 45 ℃; preferably from 37 to 42 ℃.

7. The method according to any one of claims 1 to 6, wherein T1 is equal to or less than 13min, T2 is equal to or more than 2min, and T1+T2 is equal to or less than 13min;

preferably, T1 is more than or equal to 6min and less than or equal to 10min;

preferably, T1+T2 is less than or equal to 12min;

Preferably, T1 is 10min and T2 is 2min.

8. The method of any one of claims 1 to 7, wherein 5 μΙ_ V1 +.100 μΙ_; preferably, 10. Mu.L.ltoreq.V1.ltoreq.80. Mu.L; more preferably, 20. Mu.L.ltoreq.V1.ltoreq.60. Mu.L; and/or the number of the groups of groups,

v2 is less than or equal to 5 mu L and less than or equal to 50 mu L; preferably, 8. Mu.L.ltoreq.V2.ltoreq.40. Mu.L; more preferably, 10. Mu.L.ltoreq.V2.ltoreq.25. Mu.L; and/or the number of the groups of groups,

v3 is less than or equal to 5 mu L and less than or equal to 50 mu L; preferably, 8. Mu.L.ltoreq.V3.ltoreq.40. Mu.L; more preferably, 10. Mu.L.ltoreq.V3.ltoreq.25. Mu.L; and/or the number of the groups of groups,

v4 is more than or equal to 150 mu L and less than or equal to 250 mu L; preferably, 160. Mu.L.ltoreq.V4.ltoreq.220. Mu.L; more preferably 170. Mu.L.ltoreq.V4.ltoreq.200. Mu.L.

9. The method according to any one of claims 1 to 8, wherein in step R1, the sample to be tested is mixed with the first composition before the second composition is added; and/or the number of the groups of groups,

in the step R3, the second mixture is irradiated by excitation light with the wavelength of 600-700nm, the donor is excited to generate active oxygen, the acceptor reacts with the contacted active oxygen to generate 520-620nm emitted light, and the existence and/or the intensity of an emitted light signal are detected, so that whether N-terminal B-type brain natriuretic peptide exists in a sample to be detected is judged and/or the content of the N-terminal B-type brain natriuretic peptide is determined.

10. The method according to any one of claims 1 to 9, wherein the active oxygen is singlet oxygen; and/or the sensitivity of the method is 5pg/mL; and/or the detection limit is less than or equal to 10pg/mL.