CN108204959B

CN108204959B - Method for identifying HD-HOOK Effect sample and system for identifying HD-HOOK Effect in immunoassay

Info

Publication number: CN108204959B
Application number: CN201611034252.1A
Authority: CN
Inventors: 杨阳; 赵卫国; 张向辉
Original assignee: Beyond Diagnostics Shanghai Co ltd
Current assignee: Kemei Boyang diagnostic technology (Shanghai) Co., Ltd
Priority date: 2016-11-22
Filing date: 2016-11-22
Publication date: 2020-10-30
Anticipated expiration: 2036-11-22
Also published as: CN108204959A

Abstract

The invention provides a method for identifying HD-HOOK effect samples, which is characterized by comprising the following steps: performing chemiluminescence immune reaction on a calibrator, a peak calibrator and a sample to be detected containing a target antigen (or antibody) to be detected, exciting and recording a first reading and a second reading of chemiluminescence, recording the amplification A of the difference between the second reading and the first reading of the peak calibrator as R0, comparing whether the amplification A between the second reading and the first reading of the sample to be detected is greater than R0, if the amplification A is greater than R0, the sample has HD-HOOK effect, and if the amplification A is less than R0, the sample does not have HD-HOOK effect. The invention also relates to a system and a kit for identifying an immunoassay.

Description

Method for identifying HD-HOOK Effect sample and system for identifying HD-HOOK Effect in immunoassay

Technical Field

The invention relates to the technical field of light-activated chemiluminescence, in particular to a method for identifying a HD-HOOK effect sample, a system for identifying HD-HOOK effect in immunoassay and a kit.

Background

Immunological detection is based on the principle of antigen-antibody specific reaction, and is often used for detecting a trace amount of bioactive substances such as proteins and hormones because it allows display of a sample or amplification of a signal using an isotope, enzyme, chemiluminescent substance, or the like.

Chemiluminescence immunoassay is a non-radioactive immunoassay which is developed rapidly in recent years, and the principle is that a chemiluminescence substance is used for amplifying signals 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. The photosensitive particles and the luminescent particles are combined in a certain range to generate ion oxygen energy transfer and send out optical signals, so that a sample to be detected is detected. Wherein, the photosensitive particle is filled with photosensitive compound, and the luminescent particle is filled with luminescent compound and lanthanide. Under the excitation of red laser (600-700nm), the photosensitive particles release singlet oxygen ions (4 muS) in a high energy state, and the propagation distance is about 200 nm. When the distance between the photosensitive particles and the luminescent particles is close enough, singlet oxygen ions released by the photosensitive particles can reach the luminescent particles, and through a series of chemical reactions, high-level light of 520-620nm is emitted and detected by an instrument. In the reaction system, the concentration of particles is very low, the collision probability is small, and the background signal is weak. Only after the photosensitive particles and the luminescent particles are combined through immune reaction can obvious light be emitted, so that the sensitivity of the system is high. In disease diagnosis, the detection modes commonly used comprise three to four components: luminescent particles coated with antigen or antibody, biotin or digoxigenin-labeled antigen or antibody, avidin or digoxigenin-coated photosensitive particles, neutralizing antigen or antibody, and the like. The components are combined with the antigen or the antibody to be detected through more than two incubation reactions, and qualitative or quantitative detection is carried out on the sample to be detected through the intensity of chemiluminescence. 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 double-antibody sandwich detection mode, 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.

In particular, on the one hand, in the detection of samples with high concentrations, high dose-hook-like effects may lead to a lower detection signal, and the samples are therefore interpreted as lower concentrations. The prior solution is to add the components of the reagent and dilute the sample to be detected or carry out two-step detection and the like.

On the other hand, because of the high dose-hook effect, when the concentration of the sample rises to a certain value, the signal does not rise continuously, limiting the detection range. The detection range is widened mainly by optimizing the antibody or increasing the antibody concentration.

The conventional detection process has the following 5 steps: the reaction wells are filled with the analyte and reagent, the first incubation step, the LiCA universal solution addition, the second incubation step and the reading step.

The detection method is based on a conventional detection process, and on the premise of not interrupting the reaction, the signal value is read for many times in the reaction process, and the real epidemic of the sample is judged by observing the change of the signal.

Disclosure of Invention

In view of the defects in the prior art, the present invention aims to provide a method for effectively identifying a sample with HD-HOOK effect (high dose-HOOK effect), so as to easily and rapidly identify the sample with HD-HOOK effect and calculate the concentration of the sample, thereby preventing the sample with high concentration of antigen (or antibody) from being mistakenly identified as a sample with low concentration or even false negative.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the present invention, there is provided a method for identifying HD-HOOK effect samples, the method comprising the steps of: performing chemiluminescence immune reaction on a calibrator, a peak calibrator and a sample to be detected containing a target antigen (or antibody) to be detected, exciting and recording a first reading and a second reading of chemiluminescence, recording the amplification A of the difference between the second reading and the first reading of the peak calibrator as R0, comparing whether the amplification A between the second reading and the first reading of the sample to be detected is greater than R0, if the amplification A is greater than R0, the sample has HD-HOOK effect, and if the amplification A is less than R0, the sample does not have HD-HOOK effect.

According to a preferred embodiment of the invention, the method comprises the steps of:

(1) mixing a calibrator, a peak calibrator, a sample to be detected containing a target antigen (or antibody) to be detected, luminescent particles coated by a first antibody (or antigen) and a second antibody (or antigen) marked by a marker, and incubating to obtain a mixed solution;

(2) the first reading: adding photosensitive particles marked by the marker specific binding substances into the mixed solution obtained in the step (1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;

(3) and (4) reading for the second time: further incubating the reaction solution subjected to the first reading in the step (2), irradiating excitation light and detecting the amount of emitted light, wherein the reading of a photon counter is counted as RLU 2;

(4) calculating the amplification A of the signal value obtained by the second reading of the sample relative to the signal value obtained by the first reading, wherein A is (RLU2/RLU1-1) x 100%;

(5) recording the increase a in the difference between the second and first readings of the peak calibrator as R0;

(6) and comparing the two-time reading amplification A value of the sample to be detected with R0, and if A is greater than or equal to R0, identifying the sample as an HD-HOOK effect sample.

In the context of the present invention, the term "peak calibrator" refers to a sample containing a specific concentration of an analyte, wherein the concentration at which the linear trend begins to curve downward in the high dose segment of the analyte dose response curve of a double antibody sandwich immunoassay is the concentration of the analyte in the peak calibrator.

According to a preferred embodiment of the invention, the two-time reading amplification A value of the sample to be detected is compared with R0, if A is more than or equal to R0, the sample to be detected is an HD-HOOK effect sample and needs to be diluted; if A is less than R0, the sample concentration is calculated directly from the calibration curve.

According to a preferred embodiment of the present invention, the light-emitting particles refer to polymer particles filled with a light-emitting compound and a lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and can generate singlet oxygen ions under the excitation of red laser.

According to a preferred embodiment of the present invention, in the steps (2) and (3), the amount of light emitted from the reaction solution is detected by irradiating the reaction solution with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.

According to a preferred embodiment of the invention, the antigen refers to a substance having immunogenicity; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters; the first and second antibodies refer to antibodies that can specifically bind to the target antigen; the first antigen and the second antigen refer to antigens that can specifically bind to the target antibody.

A second aspect of the invention provides a system for identifying an HD-HOOK effect in an immunoassay, the system comprising:

an immunoreaction device for performing a chemiluminescent immunoreaction,

a chemiluminescent immune response excitation and counting device for exciting and recording a first and second reading of chemiluminescence,

a processor for determining the presence of an HD-HOOK effect sample based on the difference amplification A between the second and first readings of the sample under test.

According to a preferred embodiment of the invention, the system comprises:

an immunoreaction device for performing a chemiluminescent immunoreaction,

a processor for comparing whether the increase A in the difference between the second and first readings of the sample under test is greater than the increase R0 in the difference between the second and first readings of the peak calibrator, the sample having an HD-HOOK effect if greater than R0 and no HD-HOOK effect if less than R0,

wherein a second reading of the chemiluminescence is obtained by re-exciting and reading the same immune response after a time interval.

In a specific embodiment, the system for identifying an immunoassay of the present invention comprises an immunoreaction device, such as a container for holding a solution; chemiluminescent immune response excitation and counting devices, such as photon counting modules and light emitting diodes; and a processor, such as a computer, for processing and mapping the readings. Such a system for identifying immunoassays can be referred to, for example, in the applicant's utility model patent CN201532646U, which is incorporated by reference into the present application.

According to a preferred embodiment of the invention, the method of using the system comprises the following steps:

A third aspect of the present invention provides a kit comprising a calibrator, a peak calibrator, luminescent microparticles coated with a first antibody (or antigen), a second antibody (or antigen) labeled with a labeling substance, and photosensitive microparticles labeled with a labeling substance-specific binding substance, wherein the method for using the kit comprises the steps of: performing chemiluminescence immune reaction on the calibrator, the peak calibrator and a sample to be detected containing a target antigen (or antibody) to be detected, exciting and recording a first reading and a second reading of chemiluminescence, and determining the existence of the HD-HOOK effect sample according to the difference amplification A between the second reading and the first reading of the sample to be detected.

According to a preferred embodiment of the present invention, the method of using the kit comprises the steps of: performing chemiluminescence immune reaction on the calibrator, the peak calibrator and a sample to be detected containing a target antigen (or antibody) to be detected, exciting and recording a first reading and a second reading of chemiluminescence, and comparing whether the difference amplification A between the second reading and the first reading of the sample to be detected is larger than the amplification R0 of the difference between the second reading and the first reading of the peak calibrator, wherein if the difference amplification A is larger than R0, the sample has HD-HOOK effect, and if the difference amplification A is smaller than R0, the sample does not have HD-HOOK effect.

According to a preferred embodiment of the present invention, the method of using the kit comprises the steps of:

Here, it should be particularly noted that the above-described method is a method for non-disease diagnosis purposes, which is used to easily and rapidly select the HD-HOOK effect sample in the double antibody sandwich immunoassay or double antigen sandwich immunoassay detection process, so as to prevent the high concentration antigen (or antibody) sample from being mistakenly identified as the low concentration antigen (or antibody) sample.

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 an embodiment of the invention, the antigen or antibody is selected from hepatitis b surface antigen (HBsAg), hepatitis b surface antibody (HBsAb), cancer antigen 125(CA125), ferritin (Ferr) and C-peptide (CP).

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 thereof may include a serum sample, a urine sample, a saliva sample, and the like. Preferred samples of the invention are serum samples.

Preferably, the first antibody and the second antibody refer to antibodies that can specifically bind to the antigen.

The respective first and second antibodies may be the same or different for the same antigen, and may bind to the antigen simultaneously.

The first antigen and the second antigen refer to antigens that can specifically bind to the target antibody.

The respective first and second antigens may be the same or different for the same antibody and may bind to said antibody simultaneously.

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 light-emitting fine particles are polymer fine particles filled with a light-emitting compound and a lanthanide compound. The luminescent compound may be a derivative of Dioxane or Thioxene, and the lanthanide compound may be Eu (TTA)₃TOPO or Eu (TTA)₃Phen et al, the particles are commercially available. The surface functional group of the luminescent particle can be any group capable of linking with protein, such as carboxyl, aldehyde, amine, epoxy ethyl or halogenated alkyl, etc. various known functional groups capable of linking with protein.

Preferably, the photosensitive particles are polymer particles filled with a photosensitive compound, and can generate singlet oxygen ions under excitation of red laser. When the single oxygen ion is close enough to the luminous particles, the single oxygen ion is transferred to the luminous particles to react with the luminous compound in the luminous particles 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 microparticles are also commercially available.

Preferably, in the steps (2) and (3), the amount of light emitted from the reaction solution is detected by irradiating the reaction solution with 600 to 700nm of red excitation light. The detection wavelength of the emitted light is 520-620 nm.

Furthermore, the photosensitive particles are irradiated by red laser (600-700nm), singlet oxygen ions released by the photosensitive particles are received by the luminescent particles, and therefore 520-620nm high-energy-level light is emitted.

In the detection range, the concentration of the target antigen to be detected is expressed as the number of the double-antibody sandwich compound 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 the single antibody respectively, so that the double-antibody sandwich compound 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, the relationship between the signal value amplification obtained by two readings is compared through two readings, so that the effects of widening the detection range and distinguishing the HD-HOOK effect samples can be achieved. The difference between the two readings is determined by three aspects:

in the first aspect, during the first reading, the photosensitive particles are irradiated by red laser (600-700nm) to release singlet oxygen ions. After a part of singlet oxygen ions are transferred to the luminescent particles, high-level light with the wavelength of 520-620nm 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 HD-HOOK effect sample, after the concentration of the target antigen (or antibody) to be detected is reduced, the double-antibody sandwich compound is increased, and the signal value of the second reading is increased.

In the second aspect, for a low-concentration sample, after the photosensitive particles are irradiated by red laser (600-700nm) in the first reading process and singlet oxygen ions are released, the energy of the photosensitive particles 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 summary, when the reaction does not reach the equilibrium, the first reading is performed, the photosensitive particles are irradiated by the excitation light to release singlet oxygen, a part of the singlet oxygen is transmitted to the luminescent particles, and a part of the singlet oxygen can react with the unbound target antigen or antibody to be detected, so that part of the target antigen or antibody to be detected is consumed, the reaction equilibrium moves reversely, on the other hand, the photosensitive particles are consumed after being excited once, and when the second reading is performed, the signal value of the sample with low concentration of the target antigen or antibody to be detected is reduced; the combination of the double-antibody sandwich compound of the sample with high concentration and the photosensitive particles is far from reaching the balance during the first reading, and the reaction moves towards the positive reaction direction during the second reading, so that the signal is increased, and the increasing amplitude of the signal value of the second photo-excitation light and the first signal value is increased along with the increase of the concentration of the target antigen (or antibody) to be detected. The amplification of the signal is positively correlated with the concentration of the sample, and comparison of the amplification of the two signals can indicate that one sample with low signal value and high amplification is the HD-HOOK effect.

Compared with the prior art, the invention has the beneficial effects that:

(1) based on the non-washing of a light-activated chemiluminescence platform (luminous oxygen channel) and the uniformity of the reaction, the invention can realize multiple signal measurement on one reaction without interrupting the immune reaction, detect the optical signals at different reaction times, and distinguish HD-HOOK effect samples by comparing the sizes of the two signals, and the method is not limited by the detection range and effectively widens the detection range by more than 100 times.

(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.

(3) The method of the invention is simple to operate, and can simply, conveniently and effectively eliminate false negative samples caused by HD-HOOK effect in double-antibody sandwich immunoassay for non-disease diagnosis.

Drawings

FIG. 1: and (3) carrying out a curve graph of the relation between the signal value and the sample concentration obtained by the Ferr by adopting a conventional detection method.

FIG. 2: ferr adopts the first reading signal obtained by the method of the invention and the relationship curve of the amplification A and the sample concentration.

FIG. 3: and C, a curve chart of the relation between the signal value obtained by the conventional detection method and the sample concentration.

FIG. 4: peptide C the first reading obtained by the method of the invention and the relationship curve between the amplification A and the concentration of the sample.

FIG. 5: HBsAb is a graph of signal value versus sample concentration using conventional detection methods.

FIG. 6: HBsAb first readings and amplification A obtained by the method of the invention are plotted against sample concentration.

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

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 Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, 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.

The inventor of the invention finds that the false negative caused by HD-HOOK effect in double-antibody sandwich immunoassay can be simply, conveniently and effectively eliminated and the accuracy of the double-antibody sandwich immunoassay can be improved by setting up two readings and researching the relation between the amplification of the two readings and whether the sample is the HD-HOOK effect sample.

As used herein, the terms "first antibody" and "second antibody" refer to antibodies that specifically bind to an antigen (e.g., a tumor marker). The respective first and second antibodies may be different or the same for the same antigen (e.g., a tumor marker), and may bind to the antigen simultaneously. The terms "first antigen" and "second antigen" refer to antigens that specifically bind to an antibody, such as hepatitis b surface antibody. For the same antibody (e.g., hepatitis b surface antibody), the corresponding first and second antigens may be different or the same, and may bind to the antibody simultaneously.

As used herein, the term "antigen" refers to a substance that is immunogenic, e.g., a protein, polypeptide. Representative antigens include (but are not limited to): cell factors, tumor markers, metalloproteins, cardiovascular diabetes related proteins and the like.

As used herein, the term "tumor marker" refers to a substance produced by the tumor cells themselves or by the body's reaction to the tumor cells during the development and proliferation of tumors, which reflects the presence and growth of tumors. Representative tumor markers in the art include (but are not limited to): alpha-fetoprotein (AFP), cancer antigen 125(CA125), and the like.

The basic principle of the double antibody sandwich method:

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

The basic principle of the light-activated chemiluminescence method:

the basic principles of light-activated chemiluminescence are well known to those skilled in the art. Conventionally, photosensitive particles and luminescent particles are combined in a certain range to generate ion oxygen energy transfer and emit light signals, so that a sample to be detected is detected. Wherein, the photosensitive particle is filled with photosensitive compound, and the luminescent particle is filled with luminescent compound and lanthanide. Under the excitation of red laser (600-700nm), the photosensitive particles release singlet oxygen ions (4 muS) in a high energy state, and the propagation distance is about 200 nm. When the distance between the photosensitive particles and the luminescent particles is close enough, singlet oxygen ions released by the photosensitive particles can reach the luminescent particles, and through a series of chemical reactions, high-level light of 520-620nm is emitted and detected by an instrument.

In a preferred embodiment of the present invention, the feature that the first antibody is fixed on the luminescent particles is fully utilized, meanwhile, the second antibody is labeled by biotin, the photosensitive particles are coated by streptavidin, the serum sample or the antigen standard quality control liquid, the luminescent particles coated by the first antibody and the biotin-labeled second antibody are sequentially or simultaneously added into a reaction container, and then the photosensitive particles are labeled by streptavidin, so that the following reactions occur:

(1) the first antibody on the luminescent particles is combined with corresponding antigen in a serum sample or an antigen standard quality control liquid to form an 'antigen-first antibody-luminescent particles' ternary complex;

(2) the second antibody is combined with corresponding antigen in a serum sample or an antigen standard quality control liquid to finally form a double-antibody sandwich compound of 'second antibody-antigen-first antibody-luminescent particles';

biotin and streptavidin specifically bind, allowing the double-antibody sandwich complex to bind with the photosensitive microparticles.

At this time, the distance between the photosensitive particles and the luminescent particles is less than 200nm, and after the photosensitive particles are irradiated by red laser (600-700nm), the released singlet oxygen can be received by the luminescent particles. Through a series of chemical reactions, the high-energy-level light of 520-620nm is emitted, and qualitative or quantitative detection is carried out on the sample to be detected through the intensity of the chemiluminescence.

In another preferred embodiment of the present invention, the feature that the first antigen is fixed on the luminescent particles is fully utilized, meanwhile, the second antigen is labeled by biotin, the streptavidin-coated photosensitive particles are added into the reaction vessel in sequence or simultaneously with the luminescent particles coated by the first antigen and the biotin-labeled second antigen, and then the streptavidin-labeled photosensitive particles are added, so as to generate the following reactions:

(1) combining the first antigen on the luminescent particles with corresponding antibody volume in a serum sample or an antigen standard quality control liquid to form an antibody-first antigen-luminescent particle ternary complex;

(2) the second antigen is combined with a corresponding antibody in a serum sample or an antigen standard quality control product liquid to finally form a double-antibody sandwich compound of 'second antigen-antibody-first antigen-luminescent particles';

At this time, the distance between the photosensitive particles and the luminescent particles is less than 200nm, and after the photosensitive particles are irradiated by red laser (600-700nm), the released singlet oxygen can be received by the luminescent particles. Through a series of chemical reactions, 520-620nm high-energy-level light is emitted, and qualitative or quantitative detection is carried out on a sample to be detected through the intensity of chemiluminescence.

The principle of the HD-HOOK effect-like method is indicated:

in the method of the present invention, a sample or a standard substance to be tested (including a calibrator and a peak calibrator), a first antibody (or antigen) coated on a luminescent particle, and a second antibody (or antigen) labeled with biotin are added into a reaction well, and streptavidin-coated photosensitive particles are added after incubation for a period of time (normally detecting the concentration of the antigen or antibody, the method is the same as the common method without the indication function of the HOOK effect). The following reactions then take place simultaneously:

(1) the first antibody (or antigen) on the luminescent particles is combined with the corresponding antigen (or antibody) in the sample to be detected or the antigen (or antibody) standard solution to form an 'antigen-first antibody-luminescent particles' ternary complex (or 'antibody-first antigen-luminescent particles' ternary complex); the second antibody (or antigen) is combined with the corresponding antigen (or antibody) in the serum sample or the antigen (or antibody) standard solution, and finally a 'second antibody-antigen-first antibody-luminescent particle' double-antibody sandwich complex (or 'second antigen-antibody-first antigen-luminescent particle' double-antibody sandwich complex) is formed;

(2) biotin and streptavidin are specifically combined, so that the double-antibody sandwich compound and the photosensitive particles are combined together,

at this time, the distance between the photosensitive particles and the luminescent particles is less than 200nm, and after the photosensitive particles are irradiated by red laser (600-700nm), the released singlet oxygen can be received by the luminescent particles. Emitting 520-620nm high-energy-level light through a series of chemical reactions, and carrying out qualitative or quantitative detection on a sample to be detected through the intensity of chemiluminescence to obtain a first reading RLU 1;

(3) after further incubation of the reaction solution after the first reading, a second reading RLU2 was taken;

(5) selecting a sample with a linear trend starting to bend downwards as a peak value calibrator in a high dose section of a dose response curve of an object to be detected, and detecting the peak value calibrator according to the steps (1) to (4), wherein the A value of the peak value calibrator is used as an HD-HOOK effect boundary value R0;

(6) and comparing the value A of the two-time reading amplification of the sample to be detected with the HD-HOOK effect boundary value R0, and if A is more than or equal to R0, identifying the sample as the HD-HOOK effect sample.

The details of the operation of the present invention will be further described below.

(1) First antibody (or antigen) -coated luminescent particles, labeled reagent 1, are available from Boyang Biotechnology Inc.

(2) The second antibody (or antigen) may be labeled with various art-known labels and their specific binder systems. It is preferred to label the secondary antibody (or antigen) by the biotin-avidin system. Biotin-labeled secondary antibodies (or antigens), designated reagent 2, are commercially available from Boyang Biotechnology Ltd.

(3) Streptavidin-coated photosensitive particles, designated LiCA Universal liquid, are available commercially from Boyang Biotech Ltd.

(4) And (3) standard substance:

preparing a series of standard solutions with a certain concentration range (the concentration of the calibrator 1-6 is lower than the concentration of HD-HOOK effect, and the concentration of the peak calibrator is equal to the concentration of HD-HOOK effect) by using an antigen (or an antibody) to be detected. Uniformly mixing the standard substance, the reagent 1 and the reagent 2, adding LiCA universal solution after incubation reaction, continuously performing the first reading (RLU1) after the incubation reaction is performed for a period of time, performing the second reading (RLU2) after the incubation reaction is performed for a period of time, calculating the A ═ 100% (RLU2/RLU1-1) x, and respectively making a standard curve according to the RLU1 of the standard substance and the amplification A of the two readings and the concentration of the standard substance; the A value of the peak calibrator was taken as the HD-HOOK effect cut-off R0.

(5) And (3) detection of the sample:

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), and representative examples may include a serum sample, a urine sample, a saliva sample, and the like. Preferred samples are serum samples.

(6) And (3) calculating the concentration of the sample:

firstly, comparing the two-time reading amplification A value of a sample to be detected with an HD-HOOK effect boundary value R0, if A is smaller than R0, the sample is not an HD-HOOK effect sample, and substituting RLU1 of the sample to be detected into a standard curve of standard RLU1 and standard concentration to calculate the concentration of the sample to be detected; if A is greater than or equal to R0, the sample is identified as HD-HOOK effect sample, and dilution detection is needed.

Example 1: detection of hepatitis B Virus surface antigen (HBsAg) in human serum samples

The concentration of HBsAg in a sample is detected using a kit according to the present invention relating to a method for identifying an HD-HOOK sample, which comprises a calibrator 1-calibrator 6, a peak calibrator, a reagent 1 (a luminescent antibody, i.e. antibody-coated luminescent particles), and a reagent 2 (a biotin-labeled antibody, i.e. a biotin-labeled antibody).

Calibrator 1-calibrator 6: the concentration of the known concentration sample in the conventional kit is far less than that of the HOOK sample, and a standard curve is made to calculate the concentration of the substance to be detected.

Selecting a peak value calibration product: the known HOOK sample is diluted in gradient, the signal value is detected conventionally, and the sample with the highest signal value is selected as the peak value calibrator, i.e. the sample with the signal value smaller than the concentration has no HOOK effect, while the sample with the signal value higher than the concentration has the HOOK effect. The A value is marked as R0 and is used as the critical value for judging whether the object to be detected is HOOK or not.

The other components used are as follows: LiCA universal liquid (streptavidin-labeled photosensitive particles), which is an auxiliary reagent of a light-activated chemiluminescence analysis system produced by Boyang Biotech company. The kit is matched with an instrument and a corresponding light-activated chemiluminescence detection kit for use, and is used for detecting antigens and antibodies.

Firstly, detecting a calibrator 1-6, a peak calibrator and a to-be-detected serum sample 1-15 by using the method of the invention: after adding the analyte, reagent 1 (antibody-coated luminescent particles) and reagent 2 (biotin-labeled antibodies) to the reaction cup, incubating at 37 ℃ for 15min, adding LiCA universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 3min, reading RLU1, continuing incubating at 37 ℃ for 7min, reading RLU2, and calculating the increase A of the second signal value to be (RLU2/RLU1-1) X100%, wherein the detection results are as follows:

table 1:

the concentration of 15 serum samples obtained by the method of the invention is as follows: as shown in table 1, the sample with the HOOK effect is distinguished by comparing with the amplification R0 of the peak value calibrator, i.e. if the a value is greater than 31%, the sample is determined to be the HOOK effect sample, and detection after dilution is recommended; and if A is less than 31%, the sample is not the HOOK effect sample, and the sample concentration can be directly calculated by using the calibration curve.

The reliability of the conclusion is verified by detecting the concentration change after the sample is subjected to gradient dilution, 2-time dilution and 4-time dilution are carried out on the samples 1-15, meanwhile, the undiluted original-time sample, the 2-time diluted sample and the 4-time diluted sample are detected by a conventional detection method, whether the sample has a HOOK effect or not is judged by observing the change of the diluted concentration, and namely, if the concentration of the diluted sample is increased, the HOOK effect sample is obtained. The concentration of the non-HOOK effect samples decreased after dilution. The results are as follows:

table 2:

the serum samples 1, 2, 3, 5, 6, 9, 12, 13, 14 and 15 are diluted to detect the increase of concentration, namely, the serum samples are proved to be HD-HOOK effect samples, and the serum samples 4, 7, 8, 10 and 11 are diluted to detect the decrease of concentration, thereby being proved not to be HD-HOOK effect samples. The results are exactly the same as for the process of the invention.

Example 2: verification of effectiveness of the method of the invention by detecting CA125 in human serum samples

The concentration of CA125 in a sample is detected using a kit according to the present invention, which comprises calibrators 1 to 6, a peak calibrator, a reagent 1 (a luminescent antibody, i.e., an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled antibody).

Firstly, detecting a calibrator 1-6, a peak calibrator and a to-be-detected serum sample 1-18 by using the method of the invention: after adding the analyte, reagent 1 (antibody-coated luminescent particles) and reagent 2 (biotin-labeled antibodies) to the reaction cup, incubating at 37 ℃ for 15min, adding LiCA universal solution (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 3min, reading RLU1, continuing incubating at 37 ℃ for 7min, reading RLU2, and calculating the increase A of the second signal value to be (RLU2/RLU1-1) X100%, wherein the detection results are as follows:

table 3:

the concentration of 18 serum samples obtained by the method of the invention is as follows: as shown in table 3, the sample with the HOOK effect is distinguished by comparing with the amplification R0 of the peak value calibrator, i.e. if the a value is greater than 18.7%, the sample is determined to be the HOOK effect sample, and the detection is recommended after dilution; and when A is less than 18.7%, the sample is not the HOOK effect sample, and the sample concentration can be directly calculated by using the calibration curve.

The reliability of the conclusion is verified by detecting the concentration change after the sample is subjected to gradient dilution, 2-time dilution and 4-time dilution are carried out on the samples 1-18, meanwhile, the undiluted original-time sample, the 2-time diluted sample and the 4-time diluted sample are detected by a conventional detection method, whether the sample has a HOOK effect or not is judged by observing the change of the diluted concentration, and namely, if the concentration of the diluted sample is increased, the HOOK effect sample is obtained. The concentration of the non-HOOK effect samples decreased after dilution. The results are as follows:

table 4:

the serum samples 16, 17 and 18 are diluted to detect the increase of concentration, namely, the serum samples are proved to be HD-HOOK effect samples, and the serum samples 1 to 15 are diluted to detect the decrease of concentration, so that the serum samples are not the HD-HOOK effect samples. The results are exactly the same as for the process of the invention.

Example 3: the detection of ferritin (Ferr) in a sample verifies the effectiveness of the method

The kit involved in the method for identifying the HD-HOOK sample according to the invention is used for detecting the content of ferritin (purchased from Fitzgerald, Catalog No: 30-AF10) in the sample. The kit includes calibrators 1-6, a peak calibrator, reagent 1 (a luminescent antibody, i.e., an antibody-coated luminescent particle), reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled antibody).

Selecting a peak value calibration product: the known HOOK sample is diluted in gradient, the signal value is detected conventionally, the sample with the highest signal value is selected as the peak value calibrator, i.e. the sample 9 in the experiment, the HOOK effect does not occur when the sample is less than the concentration, and the HOOK effect occurs when the sample is more than the concentration. The A value is marked as R0 and is used as the critical value for judging whether the object to be detected is HOOK or not.

And (3) carrying out gradient dilution on the ferritin antigen with high concentration, and respectively determining the concentration 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 calibrators 1-6, peak calibrators and samples to be tested 1-15, reagent 1 (luminoantibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) to the reaction cup, 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, the results are shown in the following table.

The invention adopts a double-reading method: calibrators 1-6, peak calibrator and test samples 1-15, reagent 1 (luminogenic antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotinylated antibody, i.e., biotinylated murine monoclonal antibody) were incubated at 37 ℃ for 15min, LiCA Universal solution (streptavidin-labeled light-sensitive particles) was added, incubated at 37 ℃ for 3min, RLU1 read, and incubated at 37 ℃ for 7min, RLU2 read, and the second signal value amplitude A ═ X100% (RLU2/RLU1-1) was calculated, with the following measurements:

table 5:

note: the detection range of the routine detection of ferritin is 0-2000ng/ml, and the sample concentration is more than 2000ng/ml when the sample exceeds the upper limit of the detection.

As can be seen from Table 5 and FIG. 1, when the samples diluted by high concentration antigen gradient were detected conventionally, the signal value increased with increasing concentration when the concentration increased to 51000ng/ml, the concentration continued to increase, and the signal value decreased with increasing Ferr concentration, i.e. the concentration was greater than 51000ng/ml, HD-HOOK, and the sample 9 at 51000ng/ml was the peak value calibrator, and R0 was 13.9%.

In a conventional assay, the detection range is 0-2000ng/ml, samples above the upper limit of detection show concentrations > 2000 ng/ml. As the HD-HOOK effect sample concentration continues to rise, the signal continues to fall, and a condition of reporting an ultra-high concentration sample as a lower concentration, such as sample 15, occurs. Therefore, in the conventional detection, whether the detection result of the sample to be detected is the real concentration or the reported lower concentration of the ultrahigh-value sample influenced by the HD-HOOK effect cannot be distinguished.

The method of the invention identifies the HOOK sample by two readings. And (3) detecting signal value results RLU1 and RLU2 in sequence for each sample to be detected, and taking the amplification A of the second reading (RLU2/RLU1-1) X100% as one of indexes for judging the concentration of the sample. As can be seen from Table 5 and FIG. 2, the signal value increased with concentration to 51000ng/ml, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration. Therefore, the A value of the sample to be detected and the A value of the calibrator are directly compared, and the relation between the concentration of the sample to be detected and the concentration of the calibrator can be judged. The amplification A of the samples 10-15 is larger than the amplification R0 (13.9%) of the peak value calibrator, and the Ferr concentrations of the samples 10-15 are larger than 51000ng/ml, and the samples are HD-HOOK samples. This corresponds to the actual concentration, the signal value of sample 15 is lower than that of calibrator 6, the conventional method has a detection concentration of 1860.97ng/ml, and the HD-HOOK effect sample can be identified by the method of the present invention, and needs to be diluted for detection.

Example 4: detection of C Peptide (CP) in sample to verify effectiveness of the method

The kit involved in a method for identifying HD-HOOK samples according to the invention was used to detect the content of C-peptide (purchased from Fitzgerald, Catalog No: 30-AC96) in the samples. The kit includes calibrators 1-6, a peak calibrator, reagent 1 (a luminescent antibody, i.e., an antibody-coated luminescent particle), reagent 2 (a biotin-labeled antibody, i.e., a biotin-labeled antibody).

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

table 6:

note: the detection range of the conventional detection of C peptide is 0-30ng/ml, and the sample shows a concentration of more than 30ng/ml beyond the upper limit of the detection.

As can be seen from Table 6 and FIG. 3, when the samples diluted by high concentration antigen gradient were detected conventionally, the signal value increased with the increase of concentration when the concentration increased to 10000ng/ml, the concentration continued to increase, and the signal value decreased with the increase of C peptide concentration, i.e., when the concentration was greater than 10000ng/ml, HD-HOOK, sample 9 of 10000ng/ml was the peak value calibrator, and R0 was 23.3%.

In a conventional assay, the detection range is 0-30ng/ml, samples above the upper limit of detection show concentrations > 30 ng/ml. When the HD-HOOK effect sample concentration continues to rise and the signal continues to fall, a situation in which the ultra-high concentration sample is reported as a lower concentration, such as samples 16 and 17, occurs. Therefore, in the conventional detection, whether the detection result of the sample to be detected is the real concentration or the reported lower concentration of the ultrahigh-value sample influenced by the HD-HOOK effect cannot be distinguished.

Example 5: detection of hepatitis B Virus surface antibody (HBsAb) in a sample to verify the effectiveness of the method of the invention

The kit related to the method for identifying the HI) -HOOK sample is adopted to detect the hepatitis B virus surface antibody (purchased from Beijing Zhongke Kyoda Biotech Co., Ltd., Clone No: concentration of M2201). The kit includes calibrators 1-6, a peak calibrator, reagent 1 (luminescent antigen, i.e., antigen-coated luminescent particles), reagent 2 (biotin-labeled antigen, i.e., biotin-labeled antigen).

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

The conventional detection method comprises the following steps: the calibrator 1 to calibrator 6, the peak calibrator, and the samples to be tested 1 to 14, the reagent 1 (HBsAg-coated luminescent particles) and the reagent 2 (biotin-labeled HBsAg) were added to the reaction cuvette, incubated at 37 ℃ for 15min, the LiCA universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 10min, the photon counter read, and RLU was read, with the results shown in the following table.

The invention adopts a double-reading method: the calibrator 1-calibrator 6, the peak calibrator and the samples to be tested 1-14, the reagent 1 (HBsAg-coated luminescent particles) and the reagent 2 (biotin-labeled HBsAg) were incubated at 37 ℃ for 15min, LiCA universal solution (streptavidin-labeled photosensitive particles) was added, the mixture was incubated at 37 ℃ for 3min, the reading RLU1 was taken, the incubation was continued at 37 ℃ for 7min, the reading RLU2 was taken, and the second signal value amplification A ═ RLU2/RLU1-1) X100% was calculated, and the following detection results were obtained:

table 7:

note: the detection range of the HBsAb routine detection is 0-1000mIU/ml, and the sample with the concentration of more than 1000mIU/ml is shown above the upper detection limit.

As can be seen from table 7 and fig. 5, when the sample is subjected to routine detection and high concentration HBsAb gradient dilution, the signal value increases with the increase of the concentration when the concentration increases to 10000mIU/ml, the concentration continues to increase, the signal value decreases with the increase of the concentration of HBsAb, that is, when the concentration is greater than 10000mIU/ml, HD-HOOK is detected, the sample 9 of 10000mIU/ml is a peak value calibrator, and R0 is 37.5%.

In the conventional detection, the detection range is 0-1000mIU/ml, and the sample with the concentration higher than the upper detection limit is more than 1000 mIU/ml. When the HD-HOOK effect sample concentration continues to rise and the signal continues to fall, reporting of the ultra-high concentration sample as a lower concentration condition, such as samples 12, 13, 14, occurs. Therefore, in the conventional detection, whether the detection result of the sample to be detected is the real concentration or the reported lower concentration of the ultrahigh-value sample influenced by the HD-HOOK effect cannot be distinguished.

The method of the invention identifies the HOOK sample by two readings. And (3) detecting signal value results RLU1 and RLU2 in sequence for each sample to be detected, and taking the amplification A of the second reading (RLU2/RLU1-1) X100% as one of indexes for judging the concentration of the sample. As can be seen from Table 7 and FIG. 6, the signal value increased to 10000mIU/ml with increasing concentration, and then the signal value began to decrease with increasing concentration, but the increase A continued to increase with increasing concentration. Therefore, the A value of the sample to be detected and the A value of the calibrator are directly compared, and the relation between the concentration of the sample to be detected and the concentration of the calibrator can be judged. The amplification A of the samples 10-14 is larger than the amplification R0 (37.5%) of the peak value calibrator, which indicates that the HBsAb concentrations of the samples 10-14 are larger than 10000mIU/ml, and the samples are HD-HOOK samples. This corresponds to the actual concentration, the signal values of the samples 12, 13 and 14 are lower than that of the calibrator 6, and the detection concentrations are 802.57mIU/ml, 352.22mIU/ml and 147.9mIU/ml respectively by the conventional method, so that the HD-HOOK effect sample can be identified by the method of the invention and needs to be subjected to dilution detection.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims

1. A method of identifying HD-HOOK effect samples, the method comprising the steps of: performing chemiluminescence immune reaction on a calibrator, a peak calibrator and a sample to be detected containing a target antigen or antibody to be detected, exciting and recording a first reading and a second reading of chemiluminescence, recording the amplification A of the difference between the second reading and the first reading of the peak calibrator as R0, comparing whether the amplification A between the second reading and the first reading of the sample to be detected is greater than R0, if so, the sample has HD-HOOK effect, and if not, the sample does not have HD-HOOK effect;

the a ═ 100 (RLU2/RLU1-1) x, where RLU1 and RLU2 are the first and second readings of chemiluminescence, respectively.

2. Method according to claim 1, characterized in that it comprises the following steps:

(1) mixing a calibrator, a peak calibrator, a to-be-detected sample containing a to-be-detected target antigen, a first antibody-coated luminescent particle and a second antibody labeled by a label, and incubating to obtain a mixed solution; or mixing the calibrator, the peak calibrator, the sample to be detected containing the target antibody to be detected, the luminescent particles coated by the first antigen and the second or antigen marked by the marker, and incubating to obtain a mixed solution;

3. The method according to claim 1 or 2, characterized in that the two read amplification A values of the sample to be tested are compared with R0, if A is more than or equal to R0, the sample to be tested is HD-HOOK effect sample and needs to be diluted; if A is less than R0, the sample concentration is calculated directly from the calibration curve.

4. The method according to claim 2, wherein the luminescent particles are polymer particles filled with a luminescent compound and a lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and can generate singlet oxygen ions under the excitation of red laser.

5. The method according to claim 2, wherein in the steps (2) and (3), the amount of the emitted light of the reaction solution is detected by irradiating the reaction solution with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.

6. The method according to claim 2, wherein the antigen is an immunogenic substance; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters; the first and second antibodies refer to antibodies that can specifically bind to the target antigen; the first antigen and the second antigen refer to antigens that can specifically bind to the target antibody.

7. A system for identifying an HD-HOOK effect in an immunoassay, the system comprising:

an immunoreaction device for performing a chemiluminescent immunoreaction,

a processor for determining the presence of an HD-HOOK effect sample based on the difference amplification A between the second and first readings of the sample under test;

8. The system of claim 7, the system comprising:

9. The system of claim 7, wherein the method of using the system comprises the steps of:

(1) mixing a calibrator, a peak calibrator, a to-be-detected sample containing a to-be-detected target antigen, a first antibody-coated luminescent particle and a second antibody labeled by a label, and incubating to obtain a mixed solution; or mixing the calibrator, the peak calibrator, the sample to be detected containing the target antibody to be detected, the first antigen-coated luminescent particles and the second antigen marked by the marker, and incubating to obtain a mixed solution;

10. The system of claim 9, wherein the two read amplification a values of the sample to be tested are compared with R0, and if a is greater than or equal to R0, the sample to be tested is an HD-HOOK effect sample and needs to be diluted; if A is less than R0, the sample concentration is calculated directly from the calibration curve.

11. The system of claim 9, wherein the luminescent particles are polymer particles filled with luminescent compound and lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and can generate singlet oxygen ions under the excitation of red laser.

12. The system according to claim 9, wherein in the steps (2) and (3), the amount of the emitted light of the reaction solution is detected by irradiating the reaction solution with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.

13. The system of claim 9, wherein the antigen is an immunogenic substance; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters; the first and second antibodies refer to antibodies that can specifically bind to the target antigen; the first antigen and the second antigen refer to antigens that can specifically bind to the target antibody.

14. A kit comprising a calibrator, a peak calibrator, luminescent particles coated with a first antibody or antigen, a second antibody or antigen labeled with a label, and photosensitive particles labeled with a label-specific binding substance, wherein the method of using the kit comprises the steps of: performing chemiluminescence immunoreaction on a calibrator, a peak calibrator and a sample to be detected containing a target antigen or antibody to be detected, exciting and recording first and second readings of chemiluminescence, and determining the existence of an HD-HOOK effect sample according to the difference amplification A between the second and first readings of the sample to be detected;

15. The kit according to claim 14, wherein the method of using the kit comprises the steps of: performing chemiluminescence immune reaction on the calibrator, the peak calibrator and a sample to be detected containing a target antigen or antibody to be detected, exciting and recording a first reading and a second reading of chemiluminescence, and comparing whether the difference amplification A between the second reading and the first reading of the sample to be detected is larger than the amplification R0 of the difference between the second reading and the first reading of the peak calibrator, wherein if the difference amplification A is larger than R0, the sample has HD-HOOK effect, and if the difference amplification A is smaller than R0, the sample does not have HD-HOOK effect.

16. The kit according to claim 14, wherein the method of using the kit comprises the steps of:

17. The kit of claim 14, wherein the two-time reading amplification A value of the sample to be tested is compared with R0, if A is greater than or equal to R0, the sample to be tested is HD-HOOK effect sample and needs to be diluted; if A is less than R0, the sample concentration is calculated directly from the calibration curve.

18. The kit according to claim 16, wherein the luminescent particles are polymer particles filled with a luminescent compound and a lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and can generate singlet oxygen ions under the excitation of red laser.

19. The kit according to claim 16, wherein in the steps (2) and (3), the amount of the emitted light of the reaction solution is detected by irradiating the reaction solution with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.

20. The kit according to claim 16, wherein the antigen is an immunogenic substance; the antibody refers to immunoglobulin which is produced by an organism and can recognize specific foreign matters; the first and second antibodies refer to antibodies that can specifically bind to the target antigen; the first antigen and the second antigen refer to antigens that can specifically bind to the target antibody.