CN110514646B

CN110514646B - Chemiluminescence analysis determination method, system and kit using same

Info

Publication number: CN110514646B
Application number: CN201810803878.7A
Authority: CN
Inventors: 杨阳; 赵卫国; 刘宇卉; 李临
Original assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd; Chemclin Diagnostics Corp
Current assignee: Kemei Boyang Diagnostic Technology Shanghai Co ltd; Chemclin Diagnostics Corp
Priority date: 2018-05-21
Filing date: 2018-07-20
Publication date: 2023-01-20
Anticipated expiration: 2038-07-20
Also published as: CN110514646A

Abstract

The invention relates to a chemiluminescence analysis determination method, a system using the chemiluminescence analysis determination method and a kit, belonging to the technical field of chemiluminescence. The method of the invention selects two readings to widen the detection range and indicate the HD-HOOK sample or the sample beyond the detection range after reading for multiple times, and simply, conveniently and rapidly calculates the concentration of the object to be detected in the detection process.

Description

Chemiluminescence analysis determination method, system and kit using same

Technical Field

The invention relates to the technical field of chemiluminescence, in particular to a chemiluminescence analysis determination method, a system using the chemiluminescence analysis determination method and a kit.

Background

Chemiluminescence analysis is a non-radioactive immunoassay technology which is developed rapidly in recent years, and the principle is that a chemiluminescence substance is used for amplifying a signal and an immunological binding process is directly measured by virtue of the luminous intensity, and the method is one of important directions of immunological detection. The light-activated chemiluminescence method is one of the common methods of chemiluminescence analysis technology, can be used for researching the interaction between biological molecules, and is mainly used for detecting diseases clinically. The technology integrates the researches in the related fields of polymer particle technology, organic synthesis, protein chemistry, clinical detection and the like. Compared with the traditional enzyme-linked immunoassay method, the method has the characteristics of homogeneous phase, high sensitivity, simple and convenient operation, easy automation and the like. Therefore, the application prospect is very wide.

In the detection mode of double antibody sandwich in chemiluminescence, when the concentration of a substance to be detected is high to a certain concentration, a phenomenon that a double antibody sandwich complex cannot be formed so that a signal value is low is called a high dose-HOOK effect (HD-HOOK effect). That is, the high dose-hook effect refers to the phenomenon that in the double-site sandwich immunization experiment, the linear trend of the high dose section of the dose response curve is not in a platform shape and extends backwards infinitely, but is in a downward curve shape like a hook, so that false negative is generated. The HD-HOOK effect frequently occurs in immunoassay, and the incidence rate of the HD-HOOK effect accounts for about 30 percent of that of positive samples. The existence of HD-HOOK effect can not correctly distinguish the detected sample as that the concentration exceeds the linear range of the detection kit or the concentration is the value, so that the experimental misdiagnosis especially leads to the increase of false negative rate.

Therefore, it is desirable to provide a chemiluminescence assay method which can broaden the detection range and avoid the HD-HOOK effect.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a chemiluminescence analysis and determination method.

To achieve the above and related objects, a first aspect of the present invention provides a chemiluminescent analytical assay method comprising the steps of:

(1) Mixing a sample to be detected suspected of containing target molecules to be detected with a reagent required by a chemiluminescent reaction, and reacting to form a mixture to be detected;

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

(3) Selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and amplifying the difference value of the RLUm and the RLUk as A; (ii) a

(4) Making a standard curve according to a series of standard substances with known concentration and containing target molecules to be detected and difference amplification A ' of the readings RLUm ' and RLUk ' of any two reactions in the steps (2) and (3);

(5) Determining the concentration of a target molecule to be detected by comparing the amplification A obtained in the step (3) with the standard curve obtained in the step (4);

wherein t, n, m and k are natural numbers larger than 0, k is more than m and less than or equal to n and less than or equal to t, and n is more than or equal to 2.

In some embodiments of the invention, the amplification a = (RLUm/RLUk-1) × 100%.

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

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

In some preferred embodiments of the present invention, in step (1), the reagents required for the chemiluminescent reaction to occur comprise an acceptor reagent and a donor reagent; wherein:

the donor reagent comprises a donor capable of generating singlet oxygen in an excited state;

the receptor agent comprises a receptor capable of reacting with singlet oxygen to produce a detectable chemiluminescent signal.

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

In some preferred embodiments of the present invention, the luminescent compound is selected from the group consisting of olefinic compounds, preferably selected from the group consisting of dimethylthiophene, dibutyldione compounds, dioxines, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10-dihydroacridines, aryletherenes, arylimidazoles, and lucigenins, and derivatives thereof, and more preferably selected from the group consisting of dimethylthiophene and derivatives thereof.

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

In some embodiments of the invention, the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and is soluble in an aqueous medium.

In some embodiments of the invention, the receptor is bound directly or indirectly to a first specific binding substance of the target molecule to be detected.

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

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

In some embodiments of the invention, the donor is bound to the label, either directly or indirectly.

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

In some embodiments of the present invention, in step (1), the test sample containing the target molecule to be tested is mixed with the acceptor reagent and the second specific binding agent for the target molecule to be tested, and then mixed with the donor reagent.

In some embodiments of the present invention, in step (2), the mixture to be tested is excited to generate chemiluminescence by using energy and/or an active compound; preferably, the mixture to be detected is irradiated by red excitation light with the wavelength of 600-700 nm to excite the mixture to generate chemiluminescence.

In other embodiments of the present invention, in step (2), the detection wavelength of the chemiluminescent signal value is recorded as 520 to 620nm.

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 immunoglobulin which is produced by the body and can recognize specific foreign matters.

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

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

(a1) Mixing a sample to be detected suspected to contain an antigen (or antibody) to be detected with a receptor reagent and then carrying out primary incubation; mixing the mixed solution obtained in the first incubation step with a donor reagent, and forming a mixture to be detected after the second incubation step;

(a2) Sequentially exciting the mixture to be detected t times by red exciting light with the wavelength of 600-700 nm to generate chemiluminescence, recording the signal value of the chemiluminescence n times, and detecting the wavelength of 520-620 nm; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn;

(a3) Selecting any two signal values in the chemiluminescent signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and recording the difference amplification of the RLUm and the RLUk as an amplification A, wherein the amplification A = (RLUm/RLUk-1). Times.100%;

(a4) Making a standard curve according to a series of positive control substances with known concentrations and the difference amplification A ' of the readings RLUm ' and RLUk ' of the positive control substances containing the target molecules to be detected and the random two reactions in the step (a 2) and the step (a 3);

(a5) Determining whether the concentration of the target molecules to be detected is in the rising interval or the falling interval of the standard curve according to the value A, and substituting RLum of the target molecules to be detected into the corresponding standard curve to calculate the concentration;

In a second aspect the present invention provides a system for using a chemiluminescent analytical assay method comprising:

a reaction device for carrying out a chemiluminescent reaction,

the excitation and reading device is used for exciting the mixture to be detected to generate chemiluminescence for t times in sequence, and recording the signal value of the chemiluminescence for n times; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn; selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as reading RLUm and RLUk, and amplifying the difference value of the RLUm and the RLUk as A;

the processor is used for making a standard curve according to a series of standard substances with known concentration and containing target molecules to be detected and the difference amplification A ' of the readings RLUm ' and RLUk ' of any two reactions; comparing the amplification A with the standard curve to determine the concentration of the target molecule to be detected;

In some embodiments of the invention, the method of using the system comprises the steps of:

(1) Mixing a sample to be detected suspected of containing target molecules to be detected with a reagent required for generating chemiluminescence immune reaction, and reacting to form a mixture to be detected;

(3) Selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and amplifying the difference value of the RLUm and the RLUk as A;

(5) Determining the concentration of the target molecule to be detected by comparing the amplification A obtained in step (3) with the standard curve obtained in step (4);

wherein t, n, m and k are natural numbers more than 0, k is more than m and less than or equal to n and less than or equal to t, and n is more than or equal to 2.

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

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

In some embodiments of the invention, the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium;

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

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

In some embodiments of the invention, the donor is bound to the label directly or indirectly.

In some embodiments of the invention, in step (1), the test sample containing the target molecule to be tested is mixed with the acceptor reagent and the second specific binder reagent for the target molecule to be tested, and then mixed with the donor reagent.

In some embodiments of the present invention, in step (2), the mixture to be tested is excited to generate chemiluminescence by using energy and/or an active compound; preferably, the mixture to be tested is irradiated by red excitation light with the wavelength of 600-700 nm to excite the mixture to generate chemiluminescence.

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

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

(a4) Making a standard curve according to a series of positive control substances with known concentration and containing target molecules to be detected and difference amplification A ' of the readings RLUm ' and RLUk ' of any two reactions in the step (a 2) and the step (a 3);

(a5) Determining whether the concentration of the target molecule to be detected is in an ascending interval or a descending interval of the standard curve according to the value A, and substituting RLum of the target molecule to be detected into the corresponding standard curve to calculate the concentration;

In a third aspect, the invention provides a kit comprising reagents required for a chemiluminescent assay, the method of use comprising the steps of:

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

(a1) Mixing a sample to be detected suspected to contain an antigen (or antibody) to be detected with a receptor reagent and then carrying out primary incubation; mixing the mixed solution obtained by the first incubation with a donor reagent, and forming a mixture to be detected after the second incubation;

(a3) Selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and amplifying the difference value of the RLUm and the RLUk as A, wherein the amplification A = (RLUm/RLUk-1). Times.100%;

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

In a fourth aspect the invention provides the use of a method according to the first aspect of the invention, a system according to the second aspect of the invention or a kit according to the third aspect of the invention for the detection of AFP.

The invention has the beneficial effects that:

(1) According to the method, after multiple readings are carried out, two readings are selected to widen the detection range and indicate the HD-HOOK sample or the sample beyond the detection range, and the concentration of the object to be detected is simply, conveniently and quickly calculated in the detection process.

(2) The method can accurately identify HD-HOOK effect samples in the double-antibody sandwich method detection by 100 percent, can obviously improve the accuracy of the double-antibody sandwich method immunoassay, and reduces the false negative rate of the double-antibody sandwich method immunoassay.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 is a standard curve of concentration and corresponding signal values in the examples.

FIG. 2 is a standard curve of the signal difference amplification A of concentration versus multiple readings in the examples.

FIG. 3: HCG + beta is obtained by adopting the method of the invention, and the relationship curve diagram of the signal value and A and the sample concentration is respectively obtained.

FIG. 4: and Ferr adopts a relation curve graph of the signal value and A obtained by the method of the invention and the sample concentration respectively.

FIG. 5: anti-HIV adopts the method of the invention to obtain the signal value and A and the relation curve chart of the sample concentration respectively.

FIG. 6: MYO adopts the relation curve graph of the signal value and A obtained by the method of the invention and the sample concentration respectively.

FIG. 7: NT-proBNP adopts the signal value and A that the method of the invention obtains to separately and the curve chart of the concentration relation of the sample.

FIG. 8: the PCT adopts a curve chart of the relationship between the signal value and A obtained by the method of the invention and the concentration of the sample respectively.

FIG. 9: cTnI is a curve diagram of the relationship between the signal value obtained by the method and the concentration of the sample, and the relationship between the signal value A and the concentration of the sample.

Detailed Description

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

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

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

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, sambrook et al: 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; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; (iii) 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.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.

Term of

The term "chemiluminescent assay" as used herein refers to a chemical reaction that produces an electronically excited product that emits light when the molecule undergoes radiative transitions or transfers energy to another molecule that emits light. This phenomenon, in which molecules emit light by electron excitation due to absorption of chemical energy, is called chemiluminescence. The method of performing chemical analysis and measuring an analyte by using chemiluminescence is called a chemiluminescence analysis measuring method.

The method can be a liquid phase chemiluminescence analysis measuring method, a gas phase chemiluminescence analysis measuring method or a solid phase chemiluminescence analysis measuring method; preferably a liquid phase chemiluminescent analytical assay

The method can be a common chemiluminescence analysis and determination method (energy supply reaction is general chemical reaction), a biological chemiluminescence analysis and determination method (energy supply reaction is biochemical reaction; BCL for short), an electrochemiluminescence analysis and determination method (energy supply reaction is electrochemical reaction; ECL for short) and the like; preferably a common chemiluminescence assay.

The measurement method may be not only a homogeneous chemiluminescence analysis measurement method but also a heterogeneous chemiluminescence analysis measurement method, and preferably a heterogeneous chemiluminescence analysis measurement method.

The term "target molecule to be detected" according to the present invention may be an immune molecule, such as an antigen or an antibody; or inorganic compounds, e.g. metal ions, hydrogen peroxide, CN ^- Or NO ₂ -; organic compounds are also possible, for example: oxalic acid, ascorbic acid, imine, acetylcholine, etc.; also sugars, such as: glucose or lactose; also amino acids, hormones, enzymes, fatty acids, vitamins and drugs; preferably an immune molecule. The term "sample to be tested" according to the present invention includes a target molecule to be tested. The term "mixed solution to be measured" in the present invention"which contains the sample to be tested.

The term "reagent required for chemiluminescence analysis determination" as used herein means that a chemical reaction for generating chemiluminescence must satisfy the following conditions: the first is that the reaction must provide sufficient excitation energy and be provided solely by a step, since the energy released by the previous reaction will disappear in solution due to vibrational relaxation and cannot emit light; secondly, there is a favorable reaction process, so that the energy of the chemical reaction can be accepted by at least one substance and an excited state is generated; thirdly, the excited molecule must have a certain chemiluminescent quantum efficiency to release a photon, or be able to transfer its energy to another molecule to bring it into an excited state and release a photon.

Reagents required for the chemiluminescent assay include, but are not limited to, the following: (1) reactants in a chemiluminescent reaction; (2) A catalyst, sensitizer or inhibitor in a chemiluminescent reaction; (3) Reactants in the coupling reaction, catalysts, sensitizers, etc.

The term "successively" as used herein is a time feature that indicates that the number of "activations" is differentiated by time unit.

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

The term "antigen" as used herein refers to a substance, such as a protein or polypeptide, that is immunogenic. Representative antigens include (but are not limited to): cell factors, tumor markers, metalloproteins, cardiovascular diabetes related proteins and the like. The term "tumor marker" refers to a class of substances produced by the tumor cells themselves or by the body's reaction to the tumor cells during the development and proliferation of tumors, which reflect the presence and growth of tumors. Representative tumor markers in the art include (but are not limited to): alpha-fetoprotein (AFP), cancer antigen 125 (CA 125), and the like.

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

The term "specific binding" as used herein refers to the mutual discrimination and selective binding reaction between two substances, and is the conformation correspondence between the corresponding reactants in terms of the three-dimensional structure.

The term "biotin" is widely present in animal and plant tissues, and has two cyclic structures on the molecule, namely, an imidazolone ring and a thiophene ring, wherein the imidazolone ring is the main part bound with streptavidin. Activated biotin can be conjugated to almost any biological macromolecule known in the art, including proteins, nucleic acids, polysaccharides, and lipids, and the like, mediated by a protein cross-linking agent; "streptavidin" is a protein secreted by Streptomyces and has a molecular weight of 65kD. The "streptavidin" molecule consists of 4 identical peptide chains, each of which is capable of binding one biotin. Therefore, each antigen or antibody can be simultaneously coupled with a plurality of biotin molecules, so that a 'tentacle effect' is generated to improve the analysis sensitivity.

Any reagent used in the present invention, including antigens, antibodies, acceptors or donors, can be conjugated with biotin or streptavidin, etc., as desired.

The term "donor" as used herein refers to a sensitizer capable of generating an active intermediate such as singlet oxygen that reacts with an acceptor upon activation by energy or an active compound. The donor may be photoactivated (e.g., dyes and aromatic compounds) or chemically activated (e.g., enzymes, metal salts, etc.).

In some embodiments of the invention, the donor is a photosensitizer which may be a photosensitizer known in the art, preferably a compound that is relatively light stable and does not react efficiently with singlet oxygen, non-limiting examples of which include compounds such as methylene blue, rose bengal, porphyrins, phthalocyanines, and chlorophylls disclosed in, for example, U.S. Pat. No. 5,5709994, which is incorporated herein by reference in its entirety, as well as derivatives of these compounds having 1 to 50 atom substituents that serve to render these compounds more lipophilic or more hydrophilic, and/or as a linker group to a member of a specific binding pair. Examples of other photosensitizers known to those skilled in the art may also be used in the present invention, for example as described in US patent No. US6406913, which is incorporated herein by reference.

In other embodiments of the invention, the donor is a chemically activated other sensitizer, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of donors include: 1, 4-dicarboxyethyl-1, 4-naphthalene endoperoxide, 9, 10-diphenylanthracene-9, 10-endoperoxide, etc., which release singlet oxygen upon heating or upon direct absorption of light by these compounds.

The term "acceptor" as used herein refers to a compound capable of reacting with singlet oxygen to produce a detectable signal. The donor is induced by energy or an active compound to activate and release singlet oxygen in a high energy state that is trapped by a close proximity acceptor, thereby transferring energy to activate the acceptor.

In some embodiments of the invention, the receptor is a substance that: it undergoes a chemical reaction with singlet oxygen to form an unstable metastable intermediate that can decompose with or following luminescence. Typical examples of such substances include, but are not limited to: enol ether, enamine, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acridin, aromatic vinyl ether, diepoxy ethylene, dimethyl thiophene, aromatic imidazole or lucigenin.

In other embodiments of the invention, the acceptor is an alkene capable of reacting with singlet oxygen to form a hydroperoxide or dioxetane that can be decomposed into ketones or carboxylic acid derivatives; a stable dioxetane which can be decomposed by the action of light; acetylenes which can react with singlet oxygen to form diketones; hydrazones or hydrazides which can form azo compounds or azocarbonyl compounds, such as luminol; and aromatic compounds that can form endoperoxides. Specific, non-limiting examples of receptors that can be utilized in accordance with the disclosed and claimed invention are described in U.S. patent No. US5340716, which is incorporated herein by reference in its entirety.

In other embodiments of the invention, the "donor" and/or "acceptor" may be coated onto the substrate via a functional group to form "donor microspheres" and/or "acceptor microspheres". The "matrix" according to the present invention is microspheres or microparticles known to the skilled person, of any size, which may be organic or inorganic, which may be expandable or non-expandable, which may be porous or non-porous, which have any density, but preferably have a density close to that of water, preferably are capable of floating in water, and which are made of a transparent, partially transparent or opaque material. The substrate may or may not have a charge, and when charged, is preferably negatively charged. The matrix may be a solid (e.g., polymers, metals, glass, organic and inorganic substances such as minerals, salts and diatoms), oil droplets (e.g., hydrocarbons, fluorocarbons, siliceous fluids), vesicles (e.g., synthetic such as phospholipids, or natural such as cells, and organelles). The matrix may be latex particles or other particles containing organic or inorganic polymers, lipid bilayers such as liposomes, phospholipid vesicles, oil droplets, silica particles, metal sols, cells and microcrystalline dyes. The matrix is generally multifunctional or capable of binding to a donor or recipient by specific or non-specific covalent or non-covalent interactions. Many functional groups are available or incorporated. Typical functional groups include carboxylic acid, acetaldehyde, amino, cyano, vinyl, hydroxy, mercapto, and the like. One non-limiting example of a matrix suitable for use in the present invention is a carboxyl modified latex particle. Details of such substrates can be found in U.S. patent nos. US5709994 and US5780646 (both of which are incorporated herein by reference in their entirety).

Detailed description of the preferred embodiments

Basic principle of double antibody sandwich method:

the basic principles of the double antibody sandwich method are well known to those skilled in the art. It is conventional practice to fix a primary antibody to a solid phase carrier, then to react the primary antibody with an antigen, then to react with a labeled secondary antibody, and finally to perform a chemiluminescent or enzyme-linked chromogenic reaction to detect a signal.

The present invention will be described in detail below.

The chemiluminescence assay measurement method according to the first aspect of the present invention comprises the steps of:

(3) Selecting any two signal values in the chemiluminescent signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and amplifying the difference value of the RLUm and the RLUk as A; (ii) a

(4) Making a standard curve according to a series of standard substances with known concentrations and containing target molecules to be detected and the difference amplification A ' of the readings RLUm ' and RLUk ' of the two reactions in the step (2) and the step (3);

In other embodiments of the present invention, n is greater than 2. For example, n can be 3, 4, or 5, and the like. In the invention, when n is more than 2, the method has higher detection sensitivity and stronger HD-HOOK effect resistance.

In some embodiments of the invention, the acceptor is a polymeric particle filled with a luminescent compound and a lanthanide compound.

In some preferred embodiments of the present invention, the luminescent compound is selected from the group consisting of olefin compounds, preferably from the group consisting of dimethylthiophene, dibutyldiketone compounds, dioxins, enol ethers, enamines, 9-alkylidenexanthanes, 9-alkylene-N-9, 10 dihydroacridines, arylethyletherenes, arylimidazoles, and lucigenins, and derivatives thereof, and more preferably from the group consisting of dimethylthiophene and derivatives thereof.

In some embodiments of the invention, the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium.

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

A system using a chemiluminescence assay measurement method according to a second aspect of the present invention includes:

a reaction device for performing a chemiluminescent reaction,

a processor, which is used for making a standard curve according to a series of standard substances with known concentration and containing target molecules to be detected and the difference amplification A ' of the readings RLUm ' and RLUk ' of any two reactions; comparing the amplification A with the standard curve to determine the concentration of the target molecule to be detected;

(2) Exciting the mixture to be detected to generate chemiluminescence for t times, and recording the signal value of the chemiluminescence for n times; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn;

In other embodiments of the present invention, n is greater than 2. For example, n can be 3, 4, or 5, etc. In the invention, when n is more than 2, the system has higher detection sensitivity and stronger HD-HOOK effect resistance.

the donor reagent comprises a donor which can generate singlet oxygen in an excited state;

in some embodiments of the invention, the receptor is bound directly or indirectly to a first specific binding member of the target molecule to be detected.

A kit according to a third aspect of the present invention comprises reagents required for a chemiluminescent assay, and the method of use comprises the steps of:

(3) Selecting any two signal values in the chemiluminescent signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and amplifying the difference value of the RLUm and the RLUk as A;

In other embodiments of the present invention, n is greater than 2. For example, n can be 3, 4, or 5, and the like. In the invention, when n is more than 2, the detection sensitivity of the kit is higher, and the HD-HOOK effect resistance is stronger.

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

In some embodiments of the invention, the method of using the kit specifically comprises the following steps:

A fourth aspect of the invention relates to the use of a method according to the first aspect of the invention, a system according to the second aspect of the invention or a kit according to the third aspect of the invention for AFP detection.

In this regard, it is specifically noted that the above method is a method for non-disease diagnosis purposes, and the method is used for widening the detection range, indicating the HD-HOOK sample or the beyond-detection-range sample by taking two readings after taking multiple readings in the detection process of the double antibody sandwich immunoassay or the double antigen sandwich immunoassay.

Preferably, the antigen refers to a substance having immunogenicity. Such as proteins, polypeptides. Representative antigens include (but are not limited to): cell factors, tumor markers, metalloproteins, cardiovascular diabetes related proteins and the like.

The antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters.

In the present invention, the antigen or antibody may be selected from alpha-fetoprotein (AFP), HBsAb hepatitis B virus surface antibody (HBsAb), human chorionic gonadotropin-beta subunit (HCG + beta), hepatitis B surface antigen (HBsAg), cancer antigen 125 (CA 125), C-peptide (CP), ferritin (Ferr), anti-HCV, and the like.

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

Preferably, the label is capable of specifically binding to a label-specific binding substance.

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

Preferably, the acceptor is a polymer particle filled with a luminescent compound and a lanthanide compound. The luminescent compound may be a derivative of Dioxane (dioxin) or thioxene (dimethylthiophene), etc., and the lanthanide compound may be Eu (TTA) ₃ TOPO or Eu (TTA) ₃ Phen, the particles are commercially available. The surface functional group of the receptor can be any group capable of linking to a protein, such as carboxyl, aldehyde, amine, epoxy ethyl, or haloalkyl groups.

Preferably, the donor is polymer particles filled with a photosensitive compound, and can generate singlet oxygen ions under excitation of red laser light. When the single oxygen ion is close enough to the receptor, the single oxygen ion is transferred to the receptor and reacts with the luminescent compound in the receptor to generate ultraviolet light, and the ultraviolet light further excites the lanthanide compound to generate photons with certain wavelength. The photosensitive compound may be a phthalocyanine dye or the like, and the fine particles may also be commercially available.

In the detection range, the concentration of the target antigen to be detected is expressed as the number of double-antibody sandwich complexes and is in direct proportion to the number of photons; however, when the concentration of the target antigen to be detected is too high, part of the antigen to be detected is combined with a single antibody respectively, so that the double-antibody sandwich complex is reduced, the optical signal is low, and the actual concentration of the target antigen to be detected cannot be reflected.

Similarly, in the detection range, the concentration of the target antibody to be detected is expressed as the number of the double-antigen sandwich compound and is in direct proportion to the number of photons; however, when the concentration of the target antibody to be detected is too high, part of the target antibody to be detected is combined with a single antigen respectively, so that the double-antigen sandwich compound is reduced, the optical signal is low, and the actual concentration of the target antibody to be detected cannot be reflected.

According to the method, after multiple readings are carried out, two readings are selected to widen the detection range and indicate the HD-HOOK sample or the over-detection-range sample. The difference between the two readings is determined by the following three aspects:

in the first reading, the donor is irradiated by red laser (600-700 nm) to release singlet oxygen ions. After a part of singlet oxygen ions are transferred to a receptor, high-energy-level light of 520-620 nm is emitted through a series of chemical reactions; and a part of the singlet oxygen ions react with the target antigen (or antibody) to be detected which is not bound by the antibody (or antigen), so that the concentration of the target antigen (or antibody) to be detected is reduced. For a sample with low concentration, after the concentration of the target antigen (or antibody) to be detected is reduced, the double-antibody sandwich compound is reduced, and the signal value of the second reading is reduced; for a high-concentration sample, after the concentration of the target antigen (or antibody) to be detected is reduced, the double-antibody sandwich complex is increased, and the signal value of the second reading is increased.

In the second aspect, for a low concentration sample, the donor is irradiated by red laser (600-700 nm) during the first reading, and after singlet oxygen ions are released, the energy of the donor is lost, and the second reading signal is reduced.

In a third aspect, for the HD-HOOK effect, the antigen-antibody reaction is not in equilibrium at the first reading, the reaction proceeds in the positive direction at the interval between the two readings, and the signal of the second reading increases.

In conclusion, when the reaction does not reach the equilibrium, the first reading is carried out, the donor is irradiated by the exciting light to release singlet oxygen, one part of the singlet oxygen is transferred to the receptor, one part of the singlet oxygen can react with the unbound target antigen or antibody to be detected, and part of the target antigen or antibody to be detected is consumed, so that the reaction equilibrium is reversely moved, on the other hand, the donor is consumed after being excited once, and when the second reading is carried out, the signal value of the sample with low concentration of the target antigen or antibody to be detected is reduced; the binding of the double antibody sandwich complex of the high concentration sample and the donor is far from equilibrium in the first reading, and the reaction moves towards the positive reaction in the second reading, so that the signal is increased.

Example III

In order that the present invention may be more readily understood, the following detailed description will proceed with reference being made to examples, which are intended to be illustrative only and are not intended to limit the scope of the invention. The starting materials or components used in the present invention may be commercially or conventionally prepared unless otherwise specified.

Example 1: detection of alpha-fetoprotein (AFP) samples Using conventional and inventive methods, respectively

In this example, the content of alpha-fetoprotein (AFP) in a sample was determined by using a kit (chemiluminescence method) for detecting alpha-fetoprotein (AFP) produced by boyang biotechnology (shanghai) ltd.

The high-concentration alpha-fetoprotein antigen is subjected to gradient dilution, and the signal values of samples containing different concentrations of alpha-fetoprotein are respectively measured by adopting a conventional detection method and the detection method.

The conventional detection method comprises the following steps:

mixing a sample (known standard substance) of an analyte with a known concentration with a reagent 1 (receptor solution bound to a murine monoclonal antibody) and a reagent 2 (murine monoclonal antibody solution bound to biotin), and incubating for 15min at 37 ℃; then adding a reagent 3 (donor solution combined with streptavidin), and incubating for 10min at 37 ℃ to obtain a reaction solution; the reaction solution was subjected to laser irradiation, and a photon counter read and RLU read, and the results are shown in Table 1.

The steps of the multiple reading method are as follows:

mixing a sample (known standard substance) of an analyte with a known concentration with a reagent 1 (receptor solution bound to a murine monoclonal antibody) and a reagent 2 (murine monoclonal antibody solution bound to biotin), and incubating at 37 ℃ for 15min; then reagent 3 (donor solution bound to streptavidin) was added, incubated at 37 ℃ for 1min, reading RLU1 (1 min), continued incubation at 37 ℃, re-reading at 5min for two incubation times, reading RLU2 (5 min), continued incubation at 37 ℃, re-reading at 10min for three incubation times, reading RLU3 (10 min), continued incubation at 37 ℃, re-reading at 20min for four incubation times, reading RLU4 (20 min), and the signal readings of two of them were selected to calculate the amplification A = (RLUm/RLUk-1). Times.100% of the difference in the secondary signals, k < m.ltoreq.n. The results are shown in table 1:

table 1:

as can be seen from Table 1, the signal value increases with increasing concentration from 50ng/ml to 51,200ng/ml, the signal value continues to increase, the signal value decreases with increasing alpha-fetoprotein concentration, that is, HD-HOOK is observed at a concentration greater than 51,200ng/ml, and in the conventional detection, the reported concentration of the sample with the antigen concentration higher than the detection range is lower (the reported concentrations are less than 51,200ng/ml).

According to the method, after multiple readings are carried out, two readings are selected to widen the detection range and indicate the HD-HOOK sample or the sample with the beyond detection range. After a series of samples with known concentrations are detected, signal value results are obtained, and the concentrations, the corresponding signal values and the amplification A are respectively plotted into standard curves (as shown in FIG. 1 and FIG. 2, respectively). And selecting RLU2 (5 min), obtaining RLU4 (20 min) and RLU1 (1 min) of the sample to be detected through secondary reading, and calculating the amplification A = (RLU 4/RLU 1-1). Times.100% to be used as one of indexes for judging the sample concentration interval. As can be seen from Table 1 and FIG. 1, the signal value increased to 51,200ng/ml with increasing concentration (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with increasing concentration (FIG. 2). RLU1 (1 min), RLU2 (5 min), RLU3 (10 min), RLU4 (20 min) and A of the sample to be detected are detected by the method.

Making RLU2 (5 min) and A standard curve of full scale (such as 0-3,276,800ng/ml), determining the concentration of the sample to be tested to be in an ascending interval or a descending interval according to the A value of the sample, and substituting the RLU2 (5 min) of the sample to the corresponding standard curve to calculate the exact concentration.

Example 2: detection of human chorionic gonadotropin and beta subunit (HCG + beta) samples according to conventional and inventive methods, respectively

The content of the human chorionic gonadotropin and the beta subunit in the sample is detected by adopting a detection kit (chemiluminescence method) for the human chorionic gonadotropin and the beta subunit (HCG + beta) produced by Boyang biotechnology (Shanghai) Limited.

The high-concentration human chorionic gonadotropin and beta subunit antigen are subjected to gradient dilution, and the signal values of samples containing different concentrations of human chorionic gonadotropin and beta subunit are respectively measured by adopting a conventional detection method and the detection method.

The conventional detection method comprises the following steps: mixing a sample of an analyte with a known concentration with a reagent 1 (receptor solution combined with the mouse monoclonal antibody) and a reagent 2 (mouse monoclonal antibody solution combined with biotin), and incubating for 15min at 37 ℃; reagent 3 (streptavidin-bound donor solution) was then added, incubated at 37 ℃ for 10min, the photon counter read, and the RLU read, with the results shown in table 2.

The invention adopts a double-reading method: samples of the analyte, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody), were incubated at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, further incubated at 37 ℃ for 7min, reading RLU2, and the second signal amplitude A = (RLU 2/RLU 1-1). Times.100% was calculated, as shown in Table 2 and FIG. 3:

table 2:

as can be seen from Table 2, the signal values at concentrations from 100mIU/ml to 102,400mIU/ml increase with increasing concentration, the signal values continue to increase with increasing concentrations of human chorionic gonadotropin and beta subunit, i.e., the signal values decrease with increasing concentrations of greater than 102,400mIU/ml (the concentration is defined as the HD-HOOK inflection point, and the amplification is defined as the A-HOOK inflection point) ₀ ) HD-HOOK, in the conventional detection, the sample with the antigen concentration higher than the detection range has lower report concentration (the report concentration is less than 102,400mIU/ml).

The method of the invention widens the detection range, indicates HD-HOOK samples or over-detection range samples through two times of reading. And (3) successively detecting signal value results RLU1 and RLU2 by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 2 and FIG. 3, the signal value increased with concentration to 51,200mIU/ml (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

Making full-scale RLU2 and A standard curve (such as FIG. 3) of 0-6,553,600mIU/ml, determining the concentration of the substance to be tested in the ascending interval or the descending interval according to the value A of the substance to be tested, and substituting the RLU2 of the substance to be tested into the corresponding standard curve to calculate the exact concentration.

Example 3: detection of ferritin (Ferr) samples in conventional and inventive methods, respectively

The ferritin (Ferr) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Inc. is used for detecting the ferritin content in the sample.

And (3) carrying out gradient dilution on the ferritin antigen with high concentration, and respectively determining the signal values of samples containing ferritin with different concentrations by adopting a conventional detection method and the detection method.

The conventional detection method comprises the following steps: after adding a sample of an analyte with known concentration, reagent 1 (a luminescent antibody, i.e., luminescent particles coated with a murine monoclonal antibody) and reagent 2 (a biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) to the reaction cup, incubating at 37 ℃ for 15min, adding LiCA Universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 10min, reading by a photon counter, and reading RLU, the results are shown in Table 3.

The invention adopts a double-reading method: samples of the analyte, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody), were incubated at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, further incubated at 37 ℃ for 7min, reading RLU2, and the second signal amplitude A = (RLU 2/RLU 1-1). Times.100% was calculated, as shown in Table 3 and FIG. 4:

table 3:

as can be seen from Table 3, the signal value at the concentration from 50ng/ml to 51,200ng/ml increases with increasing concentration, the concentration continues to increase, and the signal value decreases with increasing ferritin concentration, i.e., the concentration is greater than 51,200ng/ml (this concentration is defined as HD-HOOK inflection point, and the amplification is defined as A) ₀ ) Then HD-HOOK, in the conventional detection, the sample report concentration of the antigen concentration higher than the detection range will be lower (report concentration is less than 51,200ng/ml).

The method of the invention widens the detection range, indicates HD-HOOK samples or over-detection range samples through two times of reading. And (3) detecting signal value results RLU1 and RLU2 in sequence by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 3 and FIG. 4, the signal value increased with concentration to 51,200ng/ml (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

RLU2 and A standard curves (such as figure 4) of a full scale (such as 0-3,276,800ng/ml) are made, the concentration of the substance to be detected is determined to be in an ascending interval or a descending interval through the value of the A of the substance to be detected, and then the RLU2 of the substance to be detected is substituted into the corresponding standard curve to calculate the exact concentration.

Example 4: detection of human immunodeficiency virus antibody (anti-HIV) samples Using the conventional method and the method of the present invention

The content of the human immunodeficiency virus antibody (anti-HIV) in the sample is detected by adopting a detection kit (chemiluminescence method) for the human immunodeficiency virus antibody produced by Boyang biotechnology (Shanghai) Limited.

The high-concentration HIV antibody is subjected to gradient dilution, and the signal values of samples containing different concentrations of HIV antibody are respectively determined by adopting a conventional detection method and the detection method.

The conventional detection method comprises the following steps: after adding the analyte samples with known concentration, reagent 1 (luminescent reagent, i.e., luminescent particles coated with HIV antigen) and reagent 2 (biotin reagent, i.e., biotin-labeled HIV antigen) to the reaction cup, incubating at 37 ℃ for 15min, adding LiCA universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 10min, reading by a photon counter, and reading RLU, the results are shown in Table 4.

The invention adopts a double-reading method: samples of the analyte, reagent 1 (luminescent reagent, i.e., luminescent particles coated with HIV antigen) and reagent 2 (biotin reagent, i.e., biotin-labeled HIV antigen), were incubated at 37 ℃ for 15min, lico universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, incubation at 37 ℃ was continued for 7min, reading RLU2, and the second signal value amplification a = (RLU 2/RLU 1-1) × 100% was calculated, with the results shown in table 4 and fig. 5:

table 4:

as can be seen from Table 4, the signal value at the concentration of 25ng/ml to 25600ng/ml increases with increasing concentration, the concentration continues to increase, and the signal value decreases with increasing concentration of the human immunodeficiency virus antibody, i.e., the concentration is greater than 25600ng/ml (the concentration is defined as HD-HOOK inflection point, and the amplification is defined as A) ₀ ) HD-HOOK, in the conventional detection, the reported concentration of the sample with the antigen concentration higher than the detection range is lower (the reported concentration is less than 25600 ng/ml).

The method of the invention widens the detection range, indicates HD-HOOK samples or over-detection range samples through two times of reading. And (3) successively detecting signal value results RLU1 and RLU2 by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 4 and FIG. 5, the signal value increased to 25600ng/ml with increasing concentration (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with increasing concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

Making full-scale RLU2 and A standard curve (such as FIG. 5) of 0-1638400ng/ml, determining the concentration of the sample to be tested to be in ascending interval or descending interval according to the value of A, and substituting RLU2 of the sample to the corresponding standard curve to calculate the exact concentration.

Example 5: detection of Myoglobin (MYO) samples separately for conventional and inventive methods

The content of myoglobin in the sample was detected by myoglobin (myoglobin) detection kit (chemiluminescence method) produced by boyang biotechnology (shanghai) ltd.

And (3) carrying out gradient dilution on the high-concentration myoglobin antigen, and respectively determining the signal values of samples containing myoglobin with different concentrations by adopting a conventional detection method and the detection method.

The conventional detection method comprises the following steps: after adding a sample of an analyte of known concentration, reagent 1 (a luminescent antibody, i.e., luminescent particles coated with a murine monoclonal antibody) and reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled murine monoclonal antibody) to the cuvette, incubating at 37 ℃ for 15min, adding LiCA Universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 10min, reading with a photon counter, and reading RLU, the results are shown in Table 5.

The invention adopts a double-reading method: samples of analytes with known concentrations, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody), were incubated at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, further incubated at 37 ℃ for 7min, reading RLU2, and the second signal amplitude A = (RLU 2/RLU 1-1). Times.100% was calculated, as shown in Table 5 and FIG. 6:

table 5:

as can be seen from Table 5, the signal value at the concentration of 6ng/ml to 25600ng/ml increased with the increase in concentration, the signal value continued to increase with the increase in concentration of the HIV antibody, i.e., the signal value decreased at a concentration of 25600ng/ml or more (the concentration is defined as HD-HOOK inflection point, and the amplification is defined as A) ₀ ) Then HD-HOOK, in the conventional detection, the sample report concentration of the antigen concentration higher than the detection range will be lower (report concentration is less than 25600 ng/ml).

The method of the invention widens the detection range, indicates HD-HOOK samples or over-detection range samples through two times of reading. And (3) successively detecting signal value results RLU1 and RLU2 by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 5 and FIG. 6, the signal value increased to 25600ng/ml with increasing concentration (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with increasing concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

Making full-scale RLU2 and A standard curve (such as FIG. 6) of 0-409,600ng/ml, determining the concentration of the sample to be tested to be in the ascending interval or the descending interval according to the value of the sample A, and substituting the RLU2 of the sample to the corresponding standard curve to calculate the exact concentration.

Example 6: detection of samples of N-terminal atrial natriuretic peptide (NT-proBNP) by conventional and inventive methods, respectively

The content of N-terminal atrial natriuretic peptide (NT-proBNP) in a sample is detected by using an N-terminal atrial natriuretic peptide (NT-proBNP) detection kit (chemiluminescence method) produced by Boyang biotechnology (Shanghai).

And (3) carrying out gradient dilution on the high-concentration N-terminal atrial natriuretic peptide antigen, and determining the signal values of samples containing different concentrations of N-terminal atrial natriuretic peptide by respectively adopting a conventional detection method and the detection method.

The conventional detection method comprises the following steps: after adding a sample of an analyte of known concentration, reagent 1 (a luminescent antibody, i.e., luminescent particles coated with a murine monoclonal antibody) and reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled murine monoclonal antibody) to the cuvette, incubating at 37 ℃ for 15min, adding LiCA Universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 10min, reading with a photon counter, and reading RLU, the results are shown in Table 6.

The invention adopts a double-reading method: samples of analytes with known concentrations, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody), were incubated at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, further incubated at 37 ℃ for 7min, reading RLU2, and the second signal amplitude A = (RLU 2/RLU 1-1). Times.100% was calculated, as shown in Table 6 and FIG. 7:

table 6:

as can be seen from Table 6, the signal value at the concentration from 62.5pg/ml to 256000pg/ml increases with increasing concentration, the concentration continues to increase, and the signal value decreases with increasing concentration of the N-terminal atrial natriuretic peptide, i.e., the concentration is greater than 256000pg/ml (the concentration is defined as HD-HOOK inflection point, and the amplification is defined as A ₀ ) Then HD-HOOK, in the conventional detection, the sample with antigen concentration higher than the detection range will report lower concentration (report concentration is less than 256000 pg/ml).

The method of the invention widens the detection range, indicates HD-HOOK samples or over-detection range samples through two times of reading. And (3) detecting signal value results RLU1 and RLU2 in sequence by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 6 and FIG. 7, the signal value increased with concentration to 256000pg/ml (defined as the ascending region), and then the signal value began to decrease with increasing concentration (defined as the descending region), but the increase A continued to increase with concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

Making full-scale (such as 0-4096000 pg/ml) RLU2 and A standard curve (such as FIG. 7), determining the concentration of the substance to be tested in the rising interval or the falling interval according to the value of A, and substituting the RLU2 of the substance to be tested into the corresponding standard curve to calculate the exact concentration.

Example 7: separately detecting Procalcitonin (PCT) samples by the conventional method and the method of the present invention

The content of procalcitonin in the sample is detected by adopting a Procalcitonin (PCT) detection kit (chemiluminescence method) produced by Boyang biological technology (Shanghai) Limited company.

And (3) carrying out gradient dilution on the high-concentration procalcitonin antigen, and determining the signal values of samples containing different concentrations of procalcitonin by respectively adopting a conventional detection method and the detection method disclosed by the invention.

The conventional detection method comprises the following steps: after adding a sample of an analyte of known concentration, reagent 1 (a luminescent antibody, i.e., luminescent particles coated with a murine monoclonal antibody) and reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled murine monoclonal antibody) to the cuvette, incubation was carried out at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubation was carried out at 37 ℃ for 10min, a photon counter read, and RLU was read, and the results are shown in Table 7.

The invention adopts a double-reading method: samples of analytes with known concentrations, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody), were incubated at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, further incubated at 37 ℃ for 7min, reading RLU2, and the second signal amplitude A = (RLU 2/RLU 1-1). Times.100% was calculated, as shown in Table 7 and FIG. 8:

table 7:

as can be seen from Table 7, the signal value at a concentration from 1ng/ml to 12,500ng/ml increases with increasing concentration, the concentration continues to increase, and the signal value decreases with increasing procalcitonin concentration, i.e., the concentration is greater than 12,500ng/ml (the concentration is defined as HD-HOOK inflection point, and the amplification is defined as A) ₀ ) Then HD-HOOK, in the conventional detection, the sample report concentration of the antigen concentration higher than the detection range will be lower (report concentration is less than 12,500ng/ml).

The method widens the detection range and indicates the HD-HOOK sample or the over-detection range sample through two times of reading. And (3) successively detecting signal value results RLU1 and RLU2 by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 7 and FIG. 8, the signal value increased with concentration to 12,500ng/ml (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

Making full-scale (such as 0-312,500ng/ml) RLU2 and A standard curve (such as FIG. 8), determining the concentration in ascending or descending interval according to the value of A, and substituting RLU2 into corresponding standard curve to calculate the exact concentration.

Example 8: detection of troponin I (cTnI) samples according to the conventional and inventive methods, respectively

The content of troponin I in the sample was determined using a troponin I (cTnI) assay kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., ltd.

The troponin I antigen with high concentration is subjected to gradient dilution, and the signal values of samples containing troponin I with different concentrations are respectively measured by adopting a conventional detection method and the detection method of the invention.

The conventional detection method comprises the following steps: after adding a sample of an analyte of known concentration, reagent 1 (a luminescent antibody, i.e., luminescent particles coated with a murine monoclonal antibody) and reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled murine monoclonal antibody) to the cuvette, incubating at 37 ℃ for 15min, adding LiCA Universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 10min, reading with a photon counter, and reading RLU, the results are shown in Table 8.

The invention adopts a double-reading method: samples of analytes with known concentrations, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody), were incubated at 37 ℃ for 15min, liCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, further incubated at 37 ℃ for 7min, reading RLU2, and the second signal amplitude A = (RLU 2/RLU 1-1). Times.100% was calculated, as shown in Table 8 and FIG. 9:

table 8:

as can be seen from Table 8, the signal values at concentrations from 0.2ng/ml to 500ng/ml increased with increasing concentration, the concentration continued to increase, and the signal values decreased with increasing troponin I concentration, i.e., at concentrations greater than 500ng/ml (this concentration is defined as the HD-HOOK inflection point, and the amplification is defined as A) ₀ ) Then HD-HOOK, in the conventional detection, the sample report concentration of the antigen concentration higher than the detection range will be lower (report concentration is less than 500 ng/ml).

The method of the invention widens the detection range, indicates HD-HOOK samples or over-detection range samples through two times of reading. And (3) successively detecting signal value results RLU1 and RLU2 by each sample to be detected, and taking the RLU amplification A = (RLU 2/RLU 1-1). Times.100% of the second reading as one of indexes for judging the sample concentration interval. As can be seen from Table 8 and FIG. 9, the signal value increased with concentration to 500ng/ml (defined as the rising interval), and then the signal value began to decrease with increasing concentration (defined as the falling interval), but the increase A continued to increase with concentration. RLU1, RLU2 and A of the sample to be detected are detected by the method.

Making full-scale RLU2 and A standard curve (such as FIG. 9) of 0-62,500ng/ml, determining the concentration of the sample to be tested to be in an ascending interval or a descending interval according to the value of the sample A, and substituting the RLU2 of the sample to be tested into the corresponding standard curve to calculate the exact concentration.

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

Claims

1. A chemiluminescence analysis determination method capable of widening detection range and avoiding high-dose HOOK effect (HD-HOOK effect) comprises the following steps:

(2) Exciting the mixture to be detected to generate chemiluminescence for t times, and recording the signal value of the chemiluminescence reaction for n times; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn;

(3) Selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and recording the amplification of the RLUm and the RLUk as A;

(4) According to a series of standard substances with known concentrations and readings RLUm 'and amplification A' of RLUk 'of any two reactions obtained in the same steps in the step (2) and the step (3), making a first standard curve of the concentrations and the amplification A' of the series of standard substances with the target molecules to be detected, and making a second standard curve of the concentrations and the chemiluminescence signal values of the series of standard substances with the target molecules to be detected;

(5) Determining the concentration of the sample to be detected through the amplification A, judging whether the concentration of the sample to be detected is in an ascending interval or a descending interval according to the determined concentration of the sample to be detected, and substituting RLum of the target molecule to be detected into a standard curve corresponding to the RLum to calculate the concentration;

the method is a method for non-disease diagnostic purposes;

wherein t, n, m and k are natural numbers larger than 0, k is larger than m and is not less than n and is not more than t, and n is larger than 2.

2. The method according to claim 1, wherein the amplification a = (RLUm/RLUk-1) × 100%.

3. The method according to claim 1, wherein in step (1), the chemiluminescent reaction is a homogeneous chemiluminescent reaction.

4. The method according to any one of claims 1 to 3, wherein in step (1), the reagents required for the chemiluminescent reaction to occur comprise an acceptor reagent and a donor reagent; wherein:

5. The method of claim 4, wherein the receptor is a polymer particle filled with a luminescent compound and a lanthanide compound.

6. The method of claim 5, wherein the luminescent compound is selected from the group consisting of olefin compounds.

7. The method according to claim 6, wherein the luminescent compound is selected from the group consisting of dimethylthiophene, dibutyldiketone compounds, dioxins, enol ethers, enamines, 9-alkylene-N-9,10 dihydroacridines, arylethyletherenes, arylimidazoles, lucigenins and derivatives thereof.

8. The method of claim 7, wherein the luminescent compound is selected from the group consisting of dimethylthiophene and its derivatives.

9. The method of claim 5, wherein the lanthanide compound is a europium complex.

10. The method of claim 4, wherein the acceptor comprises an olefinic compound and a metal chelate, which is in non-particulate form and soluble in an aqueous medium.

11. The method of claim 4, wherein the receptor is bound directly or indirectly to a first specific binding substance of a target molecule to be detected.

12. The method of claim 4, wherein the donor is a polymer particle filled with a photosensitive compound that can generate singlet oxygen upon excitation by a red laser.

13. The method of claim 12, wherein the photoactive compound is selected from one of methylene blue, rose bengal, a porphyrin, and a phthalocyanine.

14. The method of claim 4, wherein the donor is bound to the label directly or indirectly.

15. The method of claim 4, wherein in step (1), the reagents required for the chemiluminescent reaction to occur further comprise a second specific binding reagent for the target molecule to be detected.

16. The method of claim 15, wherein the second specific binding substance of the target molecule to be detected is directly or indirectly bound to the specific binding substance of the label.

17. The method of claim 16, wherein in step (1), the test sample containing the target molecule to be tested is mixed with the acceptor reagent and the second specific binder reagent for the target molecule to be tested, and then mixed with the donor reagent.

18. The method according to any one of claims 1 to 3, wherein in step (2), the mixture to be tested is excited to chemiluminescence by energy and/or an active compound.

19. The method according to claim 18, wherein in the step (2), the mixture to be tested is irradiated with 600-700 nm red excitation light to excite chemiluminescence.

20. The method of claim 19, wherein in step (2), the chemiluminescent signal value is recorded at a detection wavelength of 520-620 nm.

21. The method of claim 1, wherein 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 immunoglobulin which is produced by the body and can recognize specific foreign matters.

22. The method of claim 1, wherein the standard substance is a positive control.

23. The method according to any of claims 19-20 or 22, characterized in that the method comprises in particular the steps of:

(a1) Mixing a sample to be detected suspected to contain an antigen or an antibody to be detected with a receptor reagent and then carrying out primary incubation; mixing the mixed solution obtained in the first incubation step with a donor reagent, and forming a mixture to be detected after the second incubation step;

(a2) Sequentially exciting the mixture to be detected to generate chemiluminescence for t times by using red exciting light with the wavelength of 600-700 nm, recording the signal value of the chemiluminescence reaction for n times, and detecting the wavelength of 520-620 nm; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn;

(a3) Selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as a reading RLUm and a reading RLUk, and recording the amplification of the RLUm and the RLUk as an A, wherein the amplification A = (RLUm/RLUk-1). Times.100%;

(a4) According to a series of standard substances with known concentration and readings RLUm 'and amplification A' of RLUk 'of any two times of reactions obtained in the same steps in the step (a 2) and the step (a 3), making a first standard curve of the concentration and the amplification A' of the series of standard substances with the target molecules to be detected, and making a second standard curve of the concentration and the chemiluminescence signal value of the series of standard substances with the target molecules to be detected;

(a5) Determining the concentration of the sample to be detected through the amplification A, judging whether the concentration of the sample to be detected is in an ascending interval or a descending interval according to the determined concentration of the sample to be detected, and substituting RLum of the target molecule to be detected into a standard curve corresponding to the RLum to calculate the concentration;

24. A system using the chemiluminescence assay measurement method according to any one of claims 1 to 23, which can broaden the detection range and avoid the high-dose HOOK effect (HD-HOOK effect), and which comprises:

a reaction device for performing a chemiluminescent reaction;

the excitation and reading device is used for exciting the mixture to be detected to generate chemiluminescence for t times in sequence, and recording the signal value of the chemiluminescence reaction for n times; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn; selecting any two signal values in the chemiluminescence signal values recorded for n times, respectively recording the signal values as reading RLUm and RLUk, and recording the amplification of the RLUm and the RLUk as A;

the processor is used for making a standard curve according to a series of standard substances with known concentration and containing target molecules to be detected, readings RLUm 'of any two reactions of the standard substances and amplification A' of RLUk ', making a first standard curve according to the concentration and the amplification A' of a series of standard substances with the target molecules to be detected, making a second standard curve according to the concentration and a chemiluminescence signal value of a series of standard substances with the target molecules to be detected, determining the concentration of a sample to be detected through the amplification A, judging whether the concentration of the sample to be detected is in an ascending interval or a descending interval according to the determined concentration of the sample to be detected, and substituting the RLUm of the target molecules to be detected into the corresponding standard curve to calculate the concentration;

wherein t, n, m and k are all natural numbers larger than 0, k is more than m and less than or equal to n and less than or equal to t, and n is more than 2.

25. The system of claim 24, wherein the method of using the system comprises the steps of:

26. The system of claim 25, wherein the amplification a = (RLUm/RLUk-1) × 100%.

27. The system of claim 25, wherein in step (1), the chemiluminescent reaction is a homogeneous chemiluminescent reaction.

28. The system of claim 25, wherein in step (1), the reagents required for the chemiluminescent reaction to occur comprise an acceptor reagent and a donor reagent; wherein:

29. The system of claim 28, wherein the receptor is a polymer particle filled with a luminescent compound and a lanthanide compound.

30. The system of claim 29, wherein the luminescent compound is selected from the group consisting of olefin compounds.

31. The system of claim 30, wherein the luminescent compound is selected from the group consisting of dimethylthiophene, dibutyldione compounds, dioxines, enol ethers, enamines, 9-alkylene-N-9,10 dihydroacridines, aryletherenes, arylimidazoles, and lucigenins and derivatives thereof.

32. The system of claim 31, wherein the luminescent compound is selected from the group consisting of dimethylthiophene and its derivatives.

33. The system of claim 29, wherein the lanthanide compound is a europium complex.

34. The system of claim 28, wherein the receptor comprises an olefinic compound and a metal chelate, in non-particulate form, and soluble in an aqueous medium.

35. The system of claim 28, wherein the receptor is directly or indirectly bound to a first specific binding member of the target molecule to be detected.

36. The system of claim 28, wherein the donor is a polymer particle filled with a photosensitive compound that generates singlet oxygen upon excitation by a red laser.

37. The system of claim 36, wherein the photoactive compound is selected from one of methylene blue, rose bengal, a porphyrin, and a phthalocyanine.

38. The system of claim 28, wherein the donor is bound to a label directly or indirectly.

39. The system of claim 28, wherein in step (1), the reagents required for the chemiluminescent reaction to occur further comprise a second specific binding agent for the target molecule to be detected.

40. The system of claim 39, wherein the second specific binding substance of the target molecule to be detected is directly or indirectly bound to the specific binding substance of the label.

41. The system of claim 39 or 40, wherein in step (1), the test sample containing the target molecule to be tested is mixed with the acceptor reagent and the second specific binding agent for the target molecule to be tested, and then mixed with the donor reagent.

42. The system of claim 25, wherein in step (2), the mixture to be tested is excited to chemiluminescence by energy and/or reactive compounds.

43. The system according to claim 42, wherein in the step (2), the mixture to be tested is irradiated by red excitation light with a wavelength of 600-700 nm to excite the mixture to generate chemiluminescence.

44. The system of claim 43 wherein in step (2) the detection wavelength at which said chemiluminescent signal value is recorded is 520-620 nm.

45. The system of claim 25, wherein 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 immunoglobulin which is produced by the body and can recognize specific foreign matters.

46. The system of claim 25, wherein the standard substance is a positive control.

47. The system according to claim 43 or 44, characterized in that the method comprises in particular the steps of:

(a2) Sequentially exciting the mixture to be detected t times by red exciting light with the wavelength of 600-700 nm to generate chemiluminescence, recording the signal value of the chemiluminescence reaction n times, and detecting the wavelength of 520-620 nm; wherein, the chemiluminescence signal value recorded for the nth time is recorded as a reading RLUn;

48. Use of a method according to any of claims 1-23, a system according to any of claims 24-47 for AFP detection.