CN110514648B - Chemiluminescent analysis POCT detection device and application thereof - Google Patents

Abstract

The invention relates to a chemiluminescent analysis POCT detection device and application thereof, belonging to the technical field of chemiluminescence. The chemiluminescent analysis POCT detection device is characterized in that the reagent card and the POCT detection device are separately designed and used in an integrated manner, and the sample to be detected is collected by using the reagent card, so that the chemiluminescent analysis POCT detection device is convenient to carry. Meanwhile, the detection method for performing chemiluminescent immunoassay POCT by using the device widens the detection range by selecting two readings after multiple times on the premise of not interrupting the reaction, and simply, conveniently and rapidly calculates the concentration of the to-be-detected object in the detection process. In addition, the method can accurately identify the HD-HOOK effect sample in the double-antibody sandwich method detection by 100%, can obviously improve the accuracy of the double-antibody sandwich method immunoassay, and reduce the false negative rate of the double-antibody sandwich method immunoassay.

Description

A chemiluminescence analysis POCT detection device and its application

Technical Field

The present invention belongs to the technical field of chemiluminescence, and in particular relates to a chemiluminescence analysis POCT detection device and an application thereof.

Background Art

Chemiluminescence analysis is a non-radioactive immunoassay technology that has developed rapidly in recent years. Its principle is to use chemiluminescent substances to amplify signals and directly measure the immune binding process with the help of its luminescence intensity. This method has become one of the important directions of immunological detection. Photoluminescence is one of the commonly used methods of chemiluminescence analysis technology. It can be used to study the interactions between biological molecules and is mainly used for disease detection in clinical practice. This technology integrates research in related fields such as polymer particle technology, organic synthesis, protein chemistry and clinical testing. Compared with traditional enzyme-linked immunosorbent assay methods, it has the characteristics of homogeneity, high sensitivity, simple operation and easy automation. Therefore, its application prospects are very broad.

In the double antibody sandwich detection mode of chemiluminescence, when the concentration of the substance to be detected reaches a certain concentration, the signal value will be low because the double antibody sandwich complex cannot be formed. This phenomenon is called the high-dose-hook effect (HD-HOOK effect). In other words, the high-dose-hook effect refers to the high-dose section of the dose-response curve in the double-site sandwich immunoassay. The linear trend is not a platform-like infinite extension, but a downward bend like a hook, resulting in a false negative phenomenon. The HD-HOOK effect often occurs in immunoassays, and its incidence accounts for about 30% of positive samples. Due to the existence of the HD-HOOK effect, the detected sample cannot be correctly distinguished as to whether its concentration exceeds the linear range of the detection kit or its concentration itself is this value, resulting in misdiagnosis of the experiment, especially leading to an increase in the false negative rate.

At the same time, the existing chemiluminescence method has the following disadvantages: A. The instrument system is bulky and occupies a large area. At the same time, due to the large test throughput, the reagent card uses 100 tests as a whole unit, which has requirements for the laboratory sample size; B. The instrument system and reagents are expensive and have high maintenance costs, which are not suitable for primary medical institutions; C. The instrument is large and cannot be carried into the diagnosis and treatment site; D. The chemiluminescence system mainly uses serum and plasma as samples, and generally cannot use whole blood, which limits its scope of use.

In recent years, a new point-of-care testing technology has emerged, which is a clinical test (bedside testing) performed next to the patient. It is referred to as POCT testing technology. The mainstream of POCT testing technology is fluorescence quantitative chromatography or colloidal gold, which is a rapid diagnosis technology that uses fluorescent microspheres or colloidal gold wrapped with fluorescent substances to perform immunological testing through membrane chromatography. However, since these two technologies are mainly release tests on NC membranes, and the CV of the membrane itself is more than 5%, the CV of POCT testing by solid phase membrane method is generally more than 10%, and the detection precision is very poor. For items with high sensitivity requirements such as cTnI, quantification becomes extremely difficult.

Therefore, there is an urgent need to provide a chemiluminescence analysis POCT detection device and its application that avoids the HD-HOOK effect over a wide range and has the characteristics of being rapid and portable.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a chemiluminescence analysis POCT detection device, which separately designs a reagent card and a POCT detection device, and uses them in an integrated manner. The reagent card is used to collect samples to be tested, which is easy to carry. At the same time, the detection method of chemiluminescence analysis POCT using the device, after multiple readings without interrupting the reaction, selects two readings to broaden the detection range, and in the detection process, simply and quickly calculates the concentration of the test object. In addition, the method of the present invention can 100% correctly identify HD-HOOK effect samples in double antibody sandwich detection, and the method can significantly improve the accuracy of double antibody sandwich immunoassay and reduce the false negative rate of double antibody sandwich immunoassay.

To this end, the first aspect of the present invention provides a chemiluminescence analysis POCT detection device, which comprises:

A reagent loading module, which is used to mix a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction to form a mixture to be detected;

A reaction module, which is used to provide a suitable temperature environment for the chemiluminescent reaction of the mixture to be tested obtained in the reagent loading module;

A detection module, which is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as readings RLUm and RLUk, respectively, and the difference increase between RLUm and RLUk is recorded as A, and the increase A=(RLUm/RLUk-1)×100%; and

A processor module, which is used to make a standard curve based on a series of known concentrations of standard substances containing the target molecule to be detected and the difference increase A' between the readings RLUm' and RLUk' of any two reactions; and compare the increase A with the standard curve to determine the concentration of the sample;

Wherein, n, m and k are all natural numbers greater than 0, and k＜m≤n, n≥2.

In some embodiments of the present invention, the device further comprises a light excitation module, which is used to excite the test mixture t times in succession to generate chemiluminescence, wherein t is a natural number greater than 0, and n≤t.

In other embodiments of the present invention, the device further comprises a reagent card for use with the reagent card, wherein the reagent card is provided with a sample hole for testing, a donor reagent hole and a receptor reagent hole; the sample hole for testing is used to hold the sample for testing, the donor reagent hole is used to hold the donor reagent, and the receptor reagent hole is used to hold the receptor reagent;

During detection, the reagent card is arranged in the POCT detection device. Under the control of the circuit control module, the reaction module is used to adjust the temperature of the reagent card and the substance in the reagent card, the reagent loading module is used to transfer the substance in the reagent card, the light excitation module is used to emit laser light t times in succession, and the detection module is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as the reading RLUm and the reading RLUk respectively, and the difference increase between RLUm and RLUk is recorded as A; the processor module makes a standard curve based on a series of standard substances containing the target molecule to be detected of known concentrations and the difference increase A' between the readings RLUm' and RLUk' of any two reactions; and the increase A is compared with the standard curve to determine the concentration of the sample;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

In some embodiments of the present invention, the reagent card is further provided with a diluent hole for containing diluent; the sample hole to be tested, the donor reagent hole, the receptor reagent hole and the diluent hole are all covered with a film to seal the hole openings.

In some embodiments of the present invention, the reagent card is further provided with a barcode area, and a barcode is provided in the barcode area.

In some embodiments of the present invention, the device further comprises a barcode scanning module, and the barcode scanning module is used to identify and read information in the barcode.

The second aspect of the present invention provides a method for performing chemiluminescence analysis POCT detection using the device as described in the first aspect of the present invention, comprising the following steps:

(1) mixing a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction to form a mixture to be detected;

(2) exciting the test mixture t times to generate chemiluminescence, and recording the chemiluminescence signal value n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn;

(3) selecting any two signal values from the n recorded chemiluminescent signal values, recording them as reading RLUm and reading RLUk, respectively, and recording the difference increment between RLUm and RLUk as A;

(4) preparing a standard curve based on a series of known concentrations of standard substances containing the target molecule to be detected and the difference between the readings RLUm' and RLUk' of any two reactions in step (2) and step (3);

(5) Determine the concentration of the sample by comparing the A value with a standard curve;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

In some embodiments of the present invention, the increase A=(RLUm/RLUk-1)×100%.

In some other embodiments of the present invention, n is greater than 2.

In some embodiments of the present invention, in step (1), the chemiluminescent reaction is a homogeneous chemiluminescent reaction.

In other embodiments of the present invention, in step (1), the reagents required for the chemiluminescent reaction include an acceptor reagent and a donor reagent; wherein:

The donor reagent comprises a donor, and the donor is capable of generating singlet oxygen in an excited state;

The receptor reagent comprises a receptor capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal.

In some embodiments of the present invention, the receptor is a polymer particle filled with a luminescent compound and a lanthanide compound.

In other embodiments of the present invention, the luminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxines, enol ethers, enamines, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether olefins, aryl imidazoles and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

In some embodiments of the present invention, the lanthanide compound is a europium complex.

In other embodiments of the present invention, the receptor comprises an olefin compound and a metal chelate, which is in a non-particulate form and is soluble in an aqueous medium.

In some embodiments of the present invention, the receptor binds directly or indirectly to a first specific binding partner of a target molecule to be detected.

In other embodiments of the present invention, the donor is a polymer particle filled with a photosensitive compound, which can generate singlet oxygen under red laser excitation.

In some embodiments of the present invention, the photosensitive compound is selected from one of methylene blue, rose bengal, porphyrin and phthalocyanine.

In other embodiments of the present invention, the donor is directly or indirectly bound to a label.

In some embodiments of the present invention, in step (1), the reagents required for the chemiluminescent reaction also include a second specific binding agent reagent for the target molecule to be detected; preferably, the second specific binding agent for the target molecule to be detected is directly or indirectly bound to the marker specific binding agent.

In other embodiments of the present invention, in step (1), the sample containing the target molecule to be detected is first mixed with the receptor reagent and the second specific binding agent reagent of the target molecule to be detected, and then mixed with the donor reagent.

In some embodiments of the present invention, in step (2), energy and/or active compounds are used to excite the test mixture to produce chemiluminescence; preferably, the test mixture is irradiated with red excitation light of 600 to 700 nm to excite it to produce chemiluminescence.

In some other embodiments of the present invention, in step (2), the detection wavelength for recording the chemiluminescent signal value is 520-620 nm.

In some embodiments of the present invention, the target molecule to be detected is an antigen or an antibody; wherein the antigen refers to a substance with immunogenicity, and the antibody refers to an immunoglobulin produced by the body that can recognize specific foreign substances.

In other embodiments of the present invention, the standard substance is a positive control.

In some preferred embodiments of the present invention, the method specifically comprises the following steps:

(a1) mixing a sample suspected of containing the antigen (or antibody) to be tested with a receptor reagent and then incubating for the first time; then mixing the mixture obtained in the first incubation with a donor reagent, and incubating for the second time to form a mixture to be tested;

(a2) exciting the test mixture with red excitation light of 600-700 nm t times to generate chemiluminescence, and recording the chemiluminescence signal value n times, with the detection wavelength being 520-620 nm; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn;

(a3) selecting any two signal values from the n recorded chemiluminescent signal values, recording them as readings RLUm and RLUk respectively, and recording the difference between RLUm and RLUk as A, where A=(RLUm/RLUk-1)×100%;

(a4) preparing a standard curve based on a series of positive control substances containing the target molecule to be detected with known concentrations and the difference between the readings RLUm' and RLUk' of any two reactions in step (a2) and step (a3);

(a5) determining whether the concentration of the target molecule to be measured is in the ascending interval or the descending interval of the standard curve according to the A value, and then substituting the RLU1 of the target molecule to be measured into the corresponding standard curve to calculate the concentration;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

The beneficial effects of the present invention are as follows: the chemiluminescence analysis POCT detection device of the present invention separately designs the reagent card and the POCT detection device, and uses them in an integrated manner. The reagent card is used to collect the sample to be tested, which is easy to carry. At the same time, the detection method of chemiluminescence analysis POCT using the device, after multiple readings without interrupting the reaction, selects two readings to broaden the detection range, and in the detection process, simply and quickly calculates the concentration of the test object. In addition, the method of the present invention can 100% correctly identify HD-HOOK effect samples in the double antibody sandwich method detection, and the method can significantly improve the accuracy of the double antibody sandwich method immunoassay and reduce the false negative rate of the double antibody sandwich method immunoassay.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below in conjunction with the accompanying drawings.

FIG1 is a graph showing the relationship between the signal value and A obtained by the method of the present invention and the sample concentration.

FIG2 is a graph showing the relationship between the signal value and A obtained by the method of the present invention for HCG+β and the sample concentration.

FIG3 is a graph showing the relationship between the signal value and A obtained by the method of the present invention and the sample concentration.

FIG4 is a graph showing the relationship between the signal value and A obtained by the method of the present invention for anti-HIV and the sample concentration.

FIG5 is a graph showing the relationship between the signal value and A obtained by the method of the present invention for MYO and the sample concentration.

FIG6 is a graph showing the relationship between the signal value and A of NT-proBNP obtained by the method of the present invention and the sample concentration.

FIG7 : A graph showing the relationship between the signal value and A obtained by the method of the present invention for PCT and the sample concentration.

FIG8 is a graph showing the relationship between the signal value and A of cTnI obtained by the method of the present invention and the sample concentration.

DETAILED DESCRIPTION

To make the present invention easy to understand, the present invention will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to be limiting.

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

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. 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, preferred methods and materials are now described.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (P. M. Wassarman and A. P. Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

I. Terminology

The term "chemiluminescence analysis and determination" used in the present invention means that a chemical reaction can produce a product in an electronically excited state. When the product molecule undergoes a radiation transition or transfers energy to other luminescent molecules so that the molecule undergoes a radiation transition again, a luminescence phenomenon occurs. This phenomenon of emitting light due to the absorption of chemical energy and the electronic excitation of molecules is called chemiluminescence. The method of using chemiluminescence to perform chemical analysis and determine the test object is called a chemiluminescence analysis and determination method.

It can be a liquid phase chemiluminescence analysis method, a gas phase chemiluminescence analysis method, or a solid phase chemiluminescence analysis method; preferably, a liquid phase chemiluminescence analysis method.

That is, it can be a common chemiluminescence analysis method (the energy supply reaction is a general chemical reaction), a biochemiluminescence analysis method (the energy supply reaction is a biochemical reaction; referred to as BCL), or an electrochemiluminescence analysis method (the energy supply reaction is an electrochemical reaction, referred to as ECL), etc.; preferably, it is a common chemiluminescence analysis method.

Not only a heterogeneous chemiluminescence analysis method but also a non-homogeneous chemiluminescence analysis method may be used, and a non-homogeneous chemiluminescence analysis method is preferred.

The term "target molecule to be detected" in the present invention can be an immune molecule, such as an antigen or an antibody; it can also be an inorganic compound, such as a metal ion, hydrogen peroxide, ^CN- or _NO2- ; it can also be an organic compound, such as oxalic acid, ascorbic acid, imine, acetylcholine, etc.; it can also be a sugar, such as glucose or lactose; it can also be an amino acid, a hormone, an enzyme, a fatty acid, a vitamin and a drug; preferably an immune molecule. The term "sample to be detected" in the present invention includes the target molecule to be detected. The term "mixed solution to be detected" in the present invention includes the sample to be detected.

The term "reagents required for chemiluminescent analysis and determination" mentioned in the present invention means that a chemical reaction must meet the following conditions to produce chemiluminescence: first, the reaction must provide sufficient excitation energy, and it must be provided by a certain step alone, because the energy released in the previous step of the reaction will disappear in the solution due to vibration relaxation and cannot emit light; second, there must be a favorable reaction process so that the energy of the chemical reaction can be accepted by at least one substance and generate an excited state; third, the excited state molecule must have a certain chemiluminescence quantum efficiency to release photons, or be able to transfer its energy to another molecule to make it enter an excited state and release photons.

The reagents required for chemiluminescent analysis include but are not limited to the following substances: (1) reactants in chemiluminescent reactions; (2) catalysts, sensitizers or inhibitors in chemiluminescent reactions; (3) reactants, catalysts, sensitizers, etc. in coupled reactions.

The term "successively" described in the present invention is a time feature, indicating that the number of multiple "stimulations" is distinguished according to time units.

The term "antibody" used in the present invention is used in the broadest sense, including antibodies of any isotype, antibody fragments that retain specific binding to antigens, 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 can be further conjugated with other moieties, such as biotin or streptavidin, etc.

The term "antigen" used in the present invention refers to substances with immunogenicity, such as proteins and polypeptides. Representative antigens include (but are not limited to): cytokines, tumor markers, metalloproteins, cardiovascular diabetes-related proteins, etc. The term "tumor marker" refers to a class of substances produced by tumor cells themselves or by the body's response to tumor cells during the occurrence and proliferation of tumors, which reflects the existence and growth of tumors. Representative tumor markers in this field include (but are not limited to): alpha-fetoprotein (AFP), cancer antigen 125 (CA125), etc.

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

The term "specific binding" used in the present invention refers to the mutual recognition and selective binding reaction between two substances, which refers to the conformational correspondence between the corresponding reactants from the perspective of three-dimensional structure.

The term "biotin" used in the present invention is widely present in animal and plant tissues. Its molecule has two ring structures, namely, the imidazolone ring and the thiophene ring, wherein the imidazolone ring is the main site for binding with streptavidin. Activated biotin can be coupled with almost all known biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, under the mediation of protein cross-linking agents; and "streptavidin" is a protein secreted by Streptomyces with a molecular weight of 65kD. The "streptavidin" molecule is composed of 4 identical peptide chains, each of which can bind to one biotin. Therefore, each antigen or antibody can be coupled with multiple biotin molecules at the same time, thereby producing a "tentacle effect" to improve the sensitivity of analysis.

In any desired case, any reagent used in the present invention, including antigens, antibodies, receptors or donors, can be conjugated with biotin or streptavidin according to actual needs.

The term "donor" as used herein refers to a sensitizer that can generate an active intermediate such as singlet oxygen that reacts with an acceptor after being activated by energy or an active compound. The donor can be photoactivated (such as dyes and aromatic compounds) or chemically activated (such as enzymes, metal salts, etc.).

In some specific embodiments of the present invention, the donor is a photosensitizer, which can be a photosensitizer known in the art, preferably a compound that is relatively stable to light and does not react effectively with singlet oxygen, and its non-limiting examples include compounds such as methylene blue, rose bengal, porphyrin, phthalocyanine and chlorophyll disclosed in U.S. Pat. No. 5,709,994 (the entire text of this patent document is cited as reference), and derivatives of these compounds with 1-50 atomic substituents, which are used to make these compounds more lipophilic or more hydrophilic, and/or as a linking group connected to a specific binding pair member. Examples of other photosensitizers known to those skilled in the art can also be used in the present invention, such as the contents described in U.S. Pat. No. 6,406,913, which is incorporated herein by reference.

In other specific embodiments of the present invention, the donor is other chemically activated sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide into singlet oxygen and water. Other examples of donors include: 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc., which release singlet oxygen when heated or directly absorb light.

The term "acceptor" used in the present invention refers to a compound that can react with singlet oxygen to produce a detectable signal. The donor is activated by energy or an active compound and releases high-energy singlet oxygen, which is captured by the receptor in a close distance, thereby transferring energy to activate the receptor.

In some specific embodiments of the present invention, the acceptor is a substance that undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose and simultaneously or subsequently emit light. Typical examples of these substances include, but are not limited to, enol ethers, enamines, 9-alkylidene xanthan gums, 9-alkylidene-N-alkyl acridans, aromatic vinyl ethers, diene oxides, dimethylthiophene, aromatic imidazoles, or lucigenin.

In other specific embodiments of the present invention, the acceptor is an olefin capable of reacting with singlet oxygen to form hydroperoxides or dioxetanes that can decompose into ketones or carboxylic acid derivatives; a stable dioxetanes that can be decomposed by the action of light; acetylenes that can react with singlet oxygen to form diketones; hydrazones or hydrazides that can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of acceptors that can be utilized according to the presently disclosed and claimed invention are described in U.S. Pat. No. US5340716 (which patent document is hereby incorporated by reference in its entirety).

In other specific embodiments of the present invention, the "donor" and/or "acceptor" can be coated on a substrate through functional groups to form "donor microspheres" and/or "acceptor microspheres". The "substrate" described in the present invention is a microsphere or particle known to those skilled in the art, which can be of any size, can be organic or inorganic, can be expandable or non-expandable, can be porous or non-porous, has any density, but preferably has a density close to that of water, preferably can float in water, and is composed of transparent, partially transparent or opaque materials. The substrate may or may not have an electric charge, and when it has an electric charge, it is preferably a negative charge. The substrate can be a solid (such as a polymer, metal, glass, organic and inorganic substances such as minerals, salts and diatoms), a small oil droplet (such as hydrocarbons, fluorocarbons, siliceous fluids), a vesicle (such as synthetic such as phospholipids, or natural such as cells, and cell organs). The matrix can be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silicon particles, metal sols, cells and microcrystalline dyes. The matrix is usually multifunctional or can be bound to the donor or receptor through specific or non-specific covalent or non-covalent interactions. There are many functional groups that are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxyl, sulfhydryl, etc. A non-limiting example of a matrix suitable for the present invention is a carboxyl-modified latex particle. The details of this matrix can be found in U.S. Patents US5709994 and US5780646 (these two patent documents are cited in their entirety as reference).

II. Specific implementation plan

The basic principle of the double antibody sandwich method:

The basic principle of the double antibody sandwich method is well known to those skilled in the art. The conventional method is to fix the first antibody to a solid phase carrier, then react the first antibody with the antigen, then react with the labeled second antibody, and finally perform chemiluminescence or enzyme-linked colorimetric reaction to detect the signal.

The present invention will be described in detail below.

To this end, the chemiluminescence analysis POCT detection device involved in the first aspect of the present invention comprises:

A reagent loading module, which is used to mix a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction to form a mixture to be detected;

A reaction module, which is used to provide a suitable temperature environment for the chemiluminescent reaction of the mixture to be tested obtained in the reagent loading module;

A detection module, which is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as readings RLUm and RLUk, respectively, and the difference increase between RLUm and RLUk is recorded as A, and the increase A=(RLUm/RLUk-1)×100%; and

A processor module, which is used to make a standard curve based on a series of known concentrations of standard substances containing the target molecule to be detected and the difference increase A' between the readings RLUm' and RLUk' of any two reactions; and compare the increase A with the standard curve to determine the concentration of the sample;

Wherein, n, m and k are all natural numbers greater than 0, and k＜m≤n, n≥2.

In some embodiments of the present invention, the device further comprises a light excitation module, which is used to excite the test mixture t times in succession to generate chemiluminescence, wherein t is a natural number greater than 0, and n≤t.

In other embodiments of the present invention, the device further comprises a reagent card for use with the reagent card, wherein the reagent card is provided with a sample hole for testing, a donor reagent hole and a receptor reagent hole; the sample hole for testing is used to hold the sample for testing, the donor reagent hole is used to hold the donor reagent, and the receptor reagent hole is used to hold the receptor reagent;

During detection, the reagent card is arranged in the POCT detection device. Under the control of the circuit control module, the reaction module is used to adjust the temperature of the reagent card and the substance in the reagent card, the reagent loading module is used to transfer the substance in the reagent card, the light excitation module is used to emit laser light t times in succession, and the detection module is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as the reading RLUm and the reading RLUk respectively, and the difference increase between RLUm and RLUk is recorded as A; the processor module makes a standard curve based on a series of standard substances containing the target molecule to be detected of known concentrations and the difference increase A' between the readings RLUm' and RLUk' of any two reactions; and the increase A is compared with the standard curve to determine the concentration of the sample;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

In some embodiments of the present invention, the reagent card is further provided with a diluent hole for containing diluent; the sample hole to be tested, the donor reagent hole, the receptor reagent hole and the diluent hole are all covered with a film to seal the hole openings.

In some embodiments of the present invention, the reagent card is further provided with a barcode area, and a barcode is provided in the barcode area.

In some embodiments of the present invention, the device further comprises a barcode scanning module, and the barcode scanning module is used to identify and read information in the barcode.

The second aspect of the present invention relates to a method for performing chemiluminescence analysis POCT detection using the device as described in the first aspect of the present invention, which comprises the following steps:

(1) mixing a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction to form a mixture to be detected;

(2) exciting the test mixture t times to generate chemiluminescence, and recording the chemiluminescence signal value n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn;

(3) selecting any two signal values from the n recorded chemiluminescent signal values, recording them as reading RLUm and reading RLUk, respectively, and recording the difference increment between RLUm and RLUk as A;

(4) preparing a standard curve based on a series of known concentrations of standard substances containing the target molecule to be detected and the difference between the readings RLUm' and RLUk' of any two reactions in step (2) and step (3);

(5) Determine the concentration of the sample by comparing the A value with a standard curve;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

In some embodiments of the present invention, the increase A=(RLUm/RLUk-1)×100%.

In other embodiments of the present invention, n is greater than 2. For example, n may be 3, 4 or 5. In the present invention, when n is greater than 2, the method has a higher detection sensitivity and a stronger ability to resist the HD-HOOK effect.

In some embodiments of the present invention, in step (1), the chemiluminescent reaction is a homogeneous chemiluminescent reaction.

In other embodiments of the present invention, in step (1), the reagents required for the chemiluminescent reaction include an acceptor reagent and a donor reagent; wherein:

The donor reagent comprises a donor, and the donor is capable of generating singlet oxygen in an excited state;

The receptor reagent comprises a receptor capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal.

In some embodiments of the present invention, the receptor is a polymer particle filled with a luminescent compound and a lanthanide compound.

In other embodiments of the present invention, the luminescent compound is selected from olefin compounds, preferably selected from dimethylthiophene, dibutanedione compounds, dioxines, enol ethers, enamines, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether olefins, aryl imidazoles and lucigenin and their derivatives, more preferably selected from dimethylthiophene and its derivatives.

In some embodiments of the present invention, the lanthanide compound is a europium complex.

In other embodiments of the present invention, the receptor comprises an olefin compound and a metal chelate, which is in a non-particulate form and is soluble in an aqueous medium.

In some embodiments of the present invention, the receptor binds directly or indirectly to a first specific binding partner of a target molecule to be detected.

In other embodiments of the present invention, the donor is a polymer particle filled with a photosensitive compound, which can generate singlet oxygen under red laser excitation.

In some embodiments of the present invention, the photosensitive compound is selected from one of methylene blue, rose bengal, porphyrin and phthalocyanine.

In other embodiments of the present invention, the donor is directly or indirectly bound to a label.

In some embodiments of the present invention, in step (1), the reagents required for the chemiluminescent reaction also include a second specific binding agent reagent for the target molecule to be detected; preferably, the second specific binding agent for the target molecule to be detected is directly or indirectly bound to the marker specific binding agent.

In other embodiments of the present invention, in step (1), the sample containing the target molecule to be detected is first mixed with the receptor reagent and the second specific binding agent reagent of the target molecule to be detected, and then mixed with the donor reagent.

In some embodiments of the present invention, in step (2), energy and/or active compounds are used to excite the test mixture to produce chemiluminescence; preferably, the test mixture is irradiated with red excitation light of 600 to 700 nm to excite it to produce chemiluminescence.

In some other embodiments of the present invention, in step (2), the detection wavelength for recording the chemiluminescent signal value is 520-620 nm.

In some embodiments of the present invention, the target molecule to be detected is an antigen or an antibody; wherein the antigen refers to a substance with immunogenicity, and the antibody refers to an immunoglobulin produced by the body that can recognize specific foreign substances.

In other embodiments of the present invention, the standard substance is a positive control.

In some preferred embodiments of the present invention, the method specifically comprises the following steps:

(a1) mixing a sample suspected of containing the antigen (or antibody) to be tested with a receptor reagent and then incubating for the first time; then mixing the mixture obtained in the first incubation with a donor reagent, and incubating for the second time to form a mixture to be tested;

(a2) exciting the test mixture with red excitation light of 600-700 nm t times to generate chemiluminescence, and recording the chemiluminescence signal value n times, with the detection wavelength being 520-620 nm; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn;

(a3) selecting any two signal values from the n recorded chemiluminescent signal values, recording them as readings RLUm and RLUk respectively, and recording the difference between RLUm and RLUk as A, where A=(RLUm/RLUk-1)×100%;

(a4) preparing a standard curve based on a series of positive control substances containing the target molecule to be detected with known concentrations and the difference between the readings RLUm' and RLUk' of any two reactions in step (a2) and step (a3);

(a5) determining whether the concentration of the target molecule to be measured is in the ascending interval or the descending interval of the standard curve according to the A value, and then substituting the RLU1 of the target molecule to be measured into the corresponding standard curve to calculate the concentration;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

It should be noted here that the above method is not for the purpose of disease diagnosis. The method is used to broaden the detection range by taking two readings during the double antibody sandwich immunoassay or double antigen sandwich immunoassay detection process, so that during the detection process, the increase A of the two readings is compared with the critical value to determine whether the sample to be tested needs to be diluted before measurement.

Preferably, the antigen refers to a substance with immunogenicity, such as a protein or a polypeptide. Representative antigens include (but are not limited to): cytokines, tumor markers, metalloproteins, cardiovascular diabetes-related proteins, etc.

The antibody refers to an immunoglobulin produced by the body that can recognize specific foreign substances.

In an embodiment of the present invention, the antigen or antibody is selected from alpha-fetoprotein (AFP), HBsAb hepatitis B virus surface antibody (HBsAb), human chorionic gonadotropin and β subunit (HCG+β), hepatitis B surface antigen (HBsAg), cancer antigen 125 (CA125), C-peptide (CP), ferritin (Ferr) and Anti-HCV, etc.

The sample that can be detected by the method of the present invention is not particularly limited, and can be any sample containing the target antigen (or antibody) to be detected, and representative examples can include serum samples, urine samples, saliva samples, etc. The preferred sample of the present invention is a serum sample.

Preferably, the marker and the marker-specific binding substance can specifically bind to each other.

More preferably, the label is biotin, and the label specific binding substance is streptavidin.

Preferably, the receptor refers to a polymer particle filled with a luminescent compound and a lanthanide compound. The luminescent compound may be a derivative of dioxene or thioxene, and the lanthanide compound may be Eu(TTA) ₃ /TOPO or Eu(TTA) ₃ /Phen, and the particles may be commercially available. The surface functional group of the receptor may be any group that can be linked to a protein, such as a carboxyl group, an aldehyde group, an amine group, an ethylene oxide group, a haloalkyl group, and other known functional groups that can be linked to a protein.

Preferably, the donor is a polymer particle filled with a photosensitive compound, which can generate singlet oxygen ions under red laser excitation. When the donor is close enough to the receptor, the singlet oxygen ions are transferred to the receptor and react with the luminescent compound in the receptor to generate ultraviolet light, which further excites the lanthanide compound to generate photons of a certain wavelength. The photosensitive compound can be a phthalocyanine dye, etc., and the particle can also be purchased on the market.

Within the detection range, the concentration of the target antigen to be detected is expressed as the number of double antibody sandwich complexes and is proportional to the number of photons; however, when the concentration of the target antigen to be detected is too high, some of the antigen to be detected will bind to a single antibody separately, resulting in a reduction in the double antibody sandwich complexes and a low light signal, which cannot reflect the actual concentration of the target antigen to be detected.

Similarly, within the detection range, the concentration of the target antibody to be tested is expressed as the number of double-antigen sandwich complexes and is proportional to the number of photons; but when the concentration of the target antibody to be tested is too high, some of the antibodies to be tested will bind to a single antigen separately, resulting in a reduction in the double-antigen sandwich complexes and a low light signal, which cannot reflect the actual concentration of the target antibody to be tested.

The method of the present invention, after multiple readings, selects two of the readings and compares the relationship between the signal value increases obtained from the two readings, thereby playing a role in broadening the detection range and distinguishing HD-HOOK effect samples. The difference between the two readings is determined by the following three aspects:

First, during the first reading, the donor is irradiated with a red laser (600-700nm) to release singlet oxygen ions. After a portion of the singlet oxygen ions are transferred to the receptor, they emit high-energy light of 520-620nm through a series of chemical reactions; while a portion of the singlet oxygen ions react with the target antigen (or antibody) to be tested that is not bound by the antibody (or antigen), thereby reducing the concentration of the target antigen (or antibody) to be tested. For low-concentration samples, after the concentration of the target antigen (or antibody) to be tested decreases, the double antibody sandwich complex decreases, and the signal value of the second reading will decrease; while for high-concentration samples, after the concentration of the target antigen (or antibody) to be tested decreases, the double antibody sandwich complex increases, and the signal value of the second reading increases instead.

Secondly, for low-concentration samples, the donor is irradiated by red laser (600-700nm) during the first reading process, and after releasing singlet oxygen ions, its energy is lost and the signal of the second reading will be reduced.

Thirdly, for the HD-HOOK effect, the antigen-antibody reaction has not yet reached equilibrium during the first reading. In the interval between the two readings, the reaction will still proceed in the positive direction, and the signal of the second reading will increase.

III. Examples

In order to make the present invention easier to understand, the present invention will be further described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The raw materials or components used in the present invention can be obtained by commercial routes or conventional methods unless otherwise specified.

Example 1: Chemiluminescence analysis POCT detection device

The chemiluminescent immunoassay POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd. and includes:

A reagent loading module, which is used to mix a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction to form a mixture to be detected;

A reaction module, which is used to provide a suitable temperature environment for the chemiluminescent reaction of the mixture to be tested obtained in the reagent loading module;

A detection module, which is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as readings RLUm and RLUk, respectively, and the difference increase between RLUm and RLUk is recorded as A, and the increase A=(RLUm/RLUk-1)×100%; and

A processor module, which is used to make a standard curve based on a series of known concentrations of standard substances containing the target molecule to be detected and the difference increase A' between the readings RLUm' and RLUk' of any two reactions; and compare the increase A with the standard curve to determine the concentration of the sample;

Wherein, n, m and k are all natural numbers greater than 0, and k＜m≤n, n≥2.

The device also includes a light excitation module, which is used to excite the mixture to be tested t times in succession to generate chemiluminescence, wherein t is a natural number greater than 0, and n≤t.

The device also includes a reagent card for use with the reagent card, which is provided with a sample hole to be tested, a donor reagent hole and a receptor reagent hole; the sample hole to be tested is used to hold the sample to be tested, the donor reagent hole is used to hold the donor reagent, and the receptor reagent hole is used to hold the receptor reagent;

During detection, the reagent card is arranged in the POCT detection device. Under the control of the circuit control module, the reaction module is used to adjust the temperature of the reagent card and the substance in the reagent card, the reagent loading module is used to transfer the substance in the reagent card, the light excitation module is used to emit laser light t times in succession, and the detection module is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as the reading RLUm and the reading RLUk respectively, and the difference increase between RLUm and RLUk is recorded as A; the processor module makes a standard curve based on a series of standard substances containing the target molecule to be detected of known concentrations and the difference increase A' between the readings RLUm' and RLUk' of any two reactions; and the increase A is compared with the standard curve to determine the concentration of the sample;

Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.

The reagent card is also provided with a diluent hole for containing the diluent; the sample hole to be tested, the donor reagent hole, the receptor reagent hole and the diluent hole are all covered with a film to seal the hole openings.

The reagent card is also provided with a barcode area, in which a barcode is provided, and the barcode is a one-dimensional code.

The device also includes a barcode scanning module, which is used to identify and read the information in the barcode. The barcode scanning module supports IC card scanning and printed barcode medium (paper or reagent card) scanning, and the information is read in by contact scanning or non-contact scanning, which can be infrared or radio frequency; the information includes but is not limited to the name of the detection and analysis project, standard curve, reagent components, batch number, expiration date, and manufacturer information.

The reagent card is in the shape of a circular single-person reagent card.

Example 2: Detection of alpha-fetoprotein (AFP) samples by conventional method and the method of the present invention

This example uses an alpha-fetoprotein (AFP) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. to detect the content of alpha-fetoprotein in the sample. The POCT detection device is developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of alpha-fetoprotein antigen is diluted in a gradient manner, and the signal values of samples containing different concentrations of alpha-fetoprotein are measured using a conventional detection method and the detection method of the present invention, respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 1.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 1 and Figure 1:

Table 1:

As can be seen from Table 1, the signal value increases with the increase of concentration from 50ng/ml to 51,200ng/ml. As the concentration continues to increase, the signal value decreases with the increase of alpha-fetoprotein concentration. That is, when the concentration is greater than 51,200ng/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A0), then HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 51,200ng/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 1 and Figure 1, the signal value continues to increase with the concentration to 51,200ng/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (e.g. 0-3,276,800 ng/ml) RLU2 and A standard curve (as shown in Figure 1), first determine whether the concentration of the analyte is in the ascending or descending range through the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 3: Detection of human chorionic gonadotropin and β subunit (HCG+β) samples by conventional method and the method of the present invention

The human chorionic gonadotropin and β subunit (HCG+β) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of human chorionic gonadotropin and β subunit in the samples. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

High concentrations of human chorionic gonadotropin and β subunit antigen are diluted in a gradient manner, and the signal values of samples containing different concentrations of human chorionic gonadotropin and β subunit are measured using conventional detection methods and the detection method of the present invention, respectively.

The conventional detection method includes the following steps

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 2.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 2 and Figure 2:

Table 2:

As can be seen from Table 2, the signal value increases with increasing concentration from 100mIU/ml to 102,400mIU/ml. If the concentration continues to increase, the signal value decreases with increasing concentrations of human chorionic gonadotropin and β subunit. That is, when the concentration is greater than 102,400mIU/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A ₀ ), then HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 102,400mIU/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 2 and Figure 2, the signal value continues to increase with the concentration to 51,200mIU/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (e.g. 0-6,553,600mIU/ml) RLU2 and A standard curve (as shown in Figure 2), first determine whether the concentration of the analyte is in the ascending or descending range by the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 4: Detection of Ferritin Samples by Conventional Method and the Method of the Present Invention

The ferritin content in the sample was detected using a ferritin detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of ferritin antigen is diluted in a gradient manner, and the signal values of samples containing ferritin of different concentrations are measured using the conventional detection method and the detection method of the present invention, respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 3.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 3 and Figure 3:

Table 3:

As can be seen from Table 3, the signal value increases with the increase of concentration from 50 ng/ml to 51,200 ng/ml. As the concentration continues to increase, the signal value decreases with the increase of ferritin concentration. That is, when the concentration is greater than 51,200 ng/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A ₀ ), HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 51,200 ng/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 3 and Figure 3, the signal value continues to increase with the concentration to 51,200ng/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (e.g. 0-3,276,800 ng/ml) RLU2 and A standard curve (as shown in Figure 3), first determine whether the concentration of the analyte is in the ascending or descending range through the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 5: Detection of human immunodeficiency virus antibody (anti-HIV) samples by conventional method and the method of the present invention

The human immunodeficiency virus antibody (anti-HIV) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of human immunodeficiency virus antibodies in the samples. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of human immunodeficiency virus antibody is diluted in a gradient manner, and the signal values of samples containing human immunodeficiency virus antibody of different concentrations are measured by conventional detection method and the detection method of the present invention respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the test substance of known concentration with reagent 1 (receptor solution bound to HIV antigen) and reagent 2 (HIV antigen solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 4.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to HIV antigen) and reagent 2 (HIV antigen solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 4 and Figure 4:

Table 4:

As can be seen from Table 4, the signal value increases with the increase of concentration from 25ng/ml to 25600ng/ml. When the concentration continues to increase, the signal value decreases with the increase of human immunodeficiency virus antibody concentration, that is, when the concentration is greater than 25600ng/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A ₀ ), HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 25600ng/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 4 and Figure 4, the signal value continues to increase with the concentration to 25600ng/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (such as 0-1638400ng/ml) RLU2 and A standard curve (as shown in Figure 4), first determine whether the concentration of the analyte is in the rising range or the falling range through the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 6: Detection of myoglobin (MYO) samples by conventional method and the method of the present invention

The myoglobin (MYO) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of myoglobin in the sample. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of myoglobin antigen is diluted in a gradient manner, and the signal values of samples containing different concentrations of myoglobin are measured using a conventional detection method and the detection method of the present invention, respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 5.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 5 and Figure 5:

Table 5:

As can be seen from Table 5, the signal value increases with the increase of concentration from 6ng/ml to 25600ng/ml. The concentration continues to increase, and the signal value decreases with the increase of human immunodeficiency virus antibody concentration, that is, when the concentration is greater than 25600ng/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A ₀ ), HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 25600ng/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 5 and Figure 5, the signal value continues to increase with the concentration to 25600ng/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (such as 0-409,600ng/ml) RLU2 and A standard curve (as shown in Figure 5), first determine whether the concentration of the analyte is in the rising or falling range through the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 7: Detection of N-terminal atrial natriuretic peptide (NT-proBNP) samples by conventional method and the method of the present invention

The N-terminal atrial natriuretic peptide (NT-proBNP) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of N-terminal atrial natriuretic peptide in the sample. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of N-terminal atrial natriuretic peptide antigen is diluted in a gradient manner, and the signal values of samples containing different concentrations of N-terminal atrial natriuretic peptide are measured using a conventional detection method and the detection method of the present invention, respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 6.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 6 and Figure 6:

Table 6:

As can be seen from Table 6, the signal value increases with the increase of concentration from 62.5pg/ml to 256000pg/ml. The concentration continues to increase, and the signal value decreases with the increase of N-terminal atrial natriuretic peptide concentration, that is, when the concentration is greater than 256000pg/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as _A0 ), then HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 256000pg/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 6 and Figure 6, the signal value continues to increase with the concentration to 256000pg/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (such as 0-4096000pg/ml) RLU2 and A standard curve (as shown in Figure 6), first determine whether the concentration of the analyte is in the rising range or the falling range through the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 8: Detection of procalcitonin (PCT) samples by conventional method and the method of the present invention

The procalcitonin (PCT) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the procalcitonin content in the sample. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of procalcitonin antigen is diluted in a gradient manner, and the signal values of samples containing procalcitonin of different concentrations are measured using the conventional detection method and the detection method of the present invention respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 7.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37°C for 7 minutes, and the photon counter reads the second reading, which is recorded as RLU2, and the increase of the second signal value is calculated as A = (RLU2/RLU1-1) × 100%. The detection results are shown in Table 7 and Figure 7:

Table 7:

As can be seen from Table 7, the signal value increases with the increase of concentration from 1 ng/ml to 12,500 ng/ml. If the concentration continues to increase, the signal value decreases with the increase of procalcitonin concentration. That is, when the concentration is greater than 12,500 ng/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A ₀ ), HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 12,500 ng/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 7 and Figure 7, the signal value continues to increase with the concentration to 12,500ng/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Make a full range (e.g. 0-312,500 ng/ml) RLU2 and A standard curve (as shown in Figure 7), first determine whether the concentration of the analyte is in the ascending or descending range through the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

Example 9: Detection of troponin I (cTnI) samples by conventional method and the method of the present invention

The troponin I (cTnI) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of troponin I in the samples. The POCT detection device was developed by Boyang Biotechnology (Shanghai) Co., Ltd.

The high concentration of troponin I antigen is diluted in a gradient manner, and the signal values of samples containing troponin I at different concentrations are measured using a conventional detection method and the detection method of the present invention, respectively.

The conventional detection method includes the following steps:

A. Mix the sample of the substance to be tested with known concentration with reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin), and incubate at 37°C for 10 min; then add reagent 3 (donor solution bound to streptavidin), and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the RLU. The results are shown in Table 8.

The steps of using the two-reading method of the present invention are as follows:

A. Incubate the sample of the test substance with known concentration, reagent 1 (receptor solution bound to mouse monoclonal antibody) and reagent 2 (mouse monoclonal antibody solution bound to biotin) at 37°C for 10 min, then add reagent 3 (donor solution bound to streptavidin) and incubate at 37°C for 2.5 min to obtain a reaction solution;

B. The reaction solution in step A is irradiated with laser, and the photon counter reads the first reading, which is recorded as RLU1; then the incubation is continued at 37° C. for 7 min, and the photon counter reads the second reading, which is recorded as RLU2. The increase of the second signal value is calculated as A=(RLU2/RLU1-1)×100%. The detection results are shown in Table 8 and FIG8 :

Table 8:

As can be seen from Table 8, the signal value increases with the increase of concentration from 0.2ng/ml to 500ng/ml. When the concentration continues to increase, the signal value decreases with the increase of troponin I concentration, that is, when the concentration is greater than 500ng/ml (this concentration is defined as the HD-HOOK inflection point, and its increase is defined as A ₀ ), HD-HOOK, in routine testing, the reported concentration of samples with antigen concentrations higher than this detection range will be lower (the reported concentrations are all less than 500ng/ml).

The method of the present invention uses two readings to broaden the detection range and indicate HD-HOOK samples or samples beyond the detection range. Each sample to be tested detects the signal value results RLU1 and RLU2 successively, and the RLU increase A of the second reading = (RLU2/RLU1-1) × 100% is used as one of the indicators for judging the sample concentration range. As shown in Table 8 and Figure 8, the signal value continues to increase with the concentration to 500ng/ml (defined as the rising interval), and then the signal value begins to decrease with the increase in concentration (defined as the falling interval), but the increase A continues to increase with the concentration. The RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.

Draw a full range (e.g. 0-62,500 ng/ml) RLU2 and A standard curve (as shown in Figure 8), first determine whether the concentration of the analyte is in the ascending or descending range by the A value of the analyte, and then substitute the RLU2 of the analyte into its corresponding standard curve to calculate the exact concentration.

It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims (28)

1. A chemiluminescence analysis POCT detection device, comprising: A reagent loading module, which is used to mix a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction to form a mixture to be detected; the reagent required for a chemiluminescent reaction includes an acceptor reagent and a donor reagent; A reaction module, which is used to provide a suitable temperature environment for the chemiluminescent reaction of the mixture to be tested obtained in the reagent loading module; A detection module, which is used to record the signal values of the chemiluminescence n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn; and any two signal values of the chemiluminescence signal values recorded for the n times are selected and recorded as readings RLUm and RLUk, respectively, and the difference increase between RLUm and RLUk is recorded as A, and the increase A=(RLUm/RLUk-1)×100%; and A processor module is used to make a standard curve based on a series of known concentrations of standard substances containing target molecules to be detected and the difference increase A' of the readings RLUm' and RLUk' of any two reactions; compare the increase A with the standard curve to determine the concentration of the sample; make a standard curve of the full-scale RLU2 and the increase A, first determine whether its concentration is in the rising range or the falling range through the increase A, and then substitute the RLU2 of the target molecule to be detected into its corresponding standard curve to calculate the exact concentration; Wherein, n, m and k are all natural numbers greater than 0, and k＜m≤n, n≥2; The device also includes a light excitation module, which is used to excite the mixture to be tested t times in succession to generate chemiluminescence, wherein t is a natural number greater than 0, and n≤t. 2. The device according to claim 1, characterized in that the device also includes a reagent card for use with the test, the reagent card is provided with a sample hole to be tested, a donor reagent hole and a receptor reagent hole; the sample hole to be tested is used to hold the sample to be tested, the donor reagent hole is used to hold the donor reagent, and the receptor reagent hole is used to hold the receptor reagent; During testing, the reagent card is set in the POCT testing device. Under the control of the circuit control module, the reaction module is used to adjust the temperature of the reagent card and the substance in the reagent card, and the reagent adding module is used to transfer the substance in the reagent card. 3. The device according to claim 2 is characterized in that the reagent card is also provided with a diluent hole, which is used to hold the diluent; the sample hole to be tested, the donor reagent hole, the receptor reagent hole and the diluent hole are all covered with a film to seal the hole openings. 4. The device according to claim 2 or 3, characterized in that the reagent card is also provided with a barcode area, and a barcode is provided in the barcode area. 5. The device according to claim 4 is characterized in that the device also includes a barcode scanning module, and the barcode scanning module is used to identify and read information in the barcode. 6. A method for performing chemiluminescence analysis POCT detection using the device according to any one of claims 1 to 5, comprising the following steps: (1) mixing a sample suspected of containing a target molecule to be detected with a reagent required for a chemiluminescent reaction and reacting the mixture to form a test mixture; the reagent required for a chemiluminescent reaction includes an acceptor reagent and a donor reagent; (2) exciting the test mixture t times to generate chemiluminescence, and recording the chemiluminescence signal value n times; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn; (3) Select any two signal values from the n recorded chemiluminescent signal values, record them as reading RLUm and reading RLUk respectively, and record the difference between RLUm and RLUk as A; the difference A=(RLUm/RLUk-1)×100%; (4) preparing a standard curve based on a series of known concentrations of standard substances containing the target molecule to be detected and the difference between the readings RLUm' and RLUk' of any two reactions in step (2) and step (3); (5) Determine the concentration of the sample by comparing the A value with the standard curve; make a standard curve of the full-scale RLU2 and the amplitude A, first determine whether its concentration is in the rising range or the falling range by the amplitude A, and then substitute the RLU2 of the target molecule to be measured into its corresponding standard curve to calculate the exact concentration; Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2. The method according to claim 6 , wherein n is greater than 2. 8. The method according to claim 6 or 7, characterized in that in step (1), the chemiluminescent reaction is a homogeneous chemiluminescent reaction. 9. The method according to claim 8, characterized in that in step (1), the donor reagent comprises a donor, and the donor can generate singlet oxygen in an excited state; The receptor reagent comprises a receptor capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal. 10. The method according to claim 9, wherein the receptor is a polymer particle filled with a luminescent compound and a lanthanide compound. The method according to claim 10 , wherein the luminescent compound is selected from olefin compounds. 12. The method according to claim 11, characterized in that the luminescent compound is selected from dimethylthiophene, dibutyl diacetyl compounds, dioxines, enol ethers, enamines, 9-alkylene xanthane, 9-alkylene-N-9,10 dihydroacridine, aryl ether olefins, aryl imidazoles and lucigenin and their derivatives. 13. The method according to claim 12, characterized in that the luminescent compound is selected from dimethylthiophene and its derivatives. 14. The method according to any one of claims 10 to 12, characterized in that the lanthanide compound is a europium complex. 15. The method of claim 9, wherein the receptor comprises an olefin compound and a metal chelate, which is in a non-particulate form and is soluble in an aqueous medium. 16. The method according to any one of claims 9, 10 and 15, characterized in that the receptor binds directly or indirectly to the first specific binding substance of the target molecule to be detected. 17. The method according to claim 9, characterized in that the donor is a polymer particle filled with a photosensitive compound, which can generate singlet oxygen under red laser excitation. 18. The method according to claim 17, wherein the photosensitive compound is selected from one of methylene blue, rose bengal, porphyrin and phthalocyanine. 19. The method according to claim 9 or 17, characterized in that the donor is directly or indirectly bound to a label. 20. The method according to claim 6, characterized in that in step (1), the reagents required for the chemiluminescent reaction also include a second specific binding agent reagent for the target molecule to be detected. 21. The method according to claim 20, characterized in that the second specific binding substance of the target molecule to be detected is directly or indirectly bound to the specific binding substance of the marker. 22. The method according to claim 21, characterized in that in step (1), the sample to be tested containing the target molecule to be tested is first mixed with a receptor reagent and a second specific binding agent reagent of the target molecule to be tested, and then mixed with a donor reagent. 23. The method according to claim 22, characterized in that in step (2), energy and/or active compounds are used to excite the test mixture to produce chemiluminescence. 24. The method according to claim 23, characterized in that the mixture to be tested is irradiated with red excitation light of 600-700 nm to excite chemiluminescence. 25. The method according to claim 23 or 24, characterized in that in step (2), the detection wavelength for recording the chemiluminescent signal value is 520-620 nm. 26. The method according to claim 25 is characterized in that the target molecule to be detected is an antigen or an antibody; wherein the antigen refers to a substance with immunogenicity, and the antibody refers to an immunoglobulin produced by the body that can recognize specific foreign substances. 27. The method according to claim 26, wherein the standard substance is a positive control. 28. The method according to claim 27, characterized in that the method specifically comprises the following steps: (a1) mixing a sample suspected of containing the antigen (or antibody) to be tested with a receptor reagent and then incubating for the first time; then mixing the mixture obtained in the first incubation with a donor reagent, and incubating for the second time to form a mixture to be tested; (a2) exciting the test mixture with red excitation light of 600-700 nm t times to generate chemiluminescence, and recording the chemiluminescence signal value n times, with the detection wavelength being 520-620 nm; wherein the chemiluminescence signal value recorded for the nth time is recorded as the reading RLUn; (a3) selecting any two signal values from the n recorded chemiluminescent signal values, recording them as readings RLUm and RLUk respectively, and recording the difference between RLUm and RLUk as A, where A=(RLUm/RLUk-1)×100%; (a4) preparing a standard curve based on a series of positive control substances containing the target molecule to be detected with known concentrations and the difference between the readings RLUm' and RLUk' of any two reactions in step (a2) and step (a3); (a5) determining whether the concentration of the target molecule to be measured is in the ascending interval or the descending interval of the standard curve according to the A value, and then substituting the RLU2 of the target molecule to be measured into its corresponding standard curve to calculate the concentration; Among them, t, n, m and k are all natural numbers greater than 0, and k＜m≤n≤t, n≥2.