CN109470873B - Immunoassay method and system for human chorionic gonadotropin and beta subunit - Google Patents

Immunoassay method and system for human chorionic gonadotropin and beta subunit Download PDF

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CN109470873B
CN109470873B CN201811401320.2A CN201811401320A CN109470873B CN 109470873 B CN109470873 B CN 109470873B CN 201811401320 A CN201811401320 A CN 201811401320A CN 109470873 B CN109470873 B CN 109470873B
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chorionic gonadotropin
human chorionic
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杨阳
赵卫国
张向辉
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Kemei Boyang Diagnostic Technology Shanghai Co ltd
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Abstract

The invention relates to an immunoassay method of human chorionic gonadotropin and beta subunit, a system for identifying immunoassay of the human chorionic gonadotropin and the beta subunit and a kit for detecting the human chorionic gonadotropin and the beta subunit, belonging to the technical field of light-activated chemiluminescence; and another method for immunoassay of human chorionic gonadotropin and beta subunit, another system for immunoassay for identifying human chorionic gonadotropin and beta subunit, and another kit for detecting human chorionic gonadotropin and beta subunit.

Description

Immunoassay method and system for human chorionic gonadotropin and beta subunit
The present application is a divisional application of the chinese patent application entitled "immunoassay method, system and kit for identification of immunoassay" filed on 2016, 22/11/2016, and filed on application No. 201611026623.1.
Technical Field
The invention relates to the technical field of light-induced chemiluminescence, in particular to an immunoassay method of human chorionic gonadotropin and beta subunit, an immunoassay system for identifying the human chorionic gonadotropin and the beta subunit, and a kit for detecting the human chorionic gonadotropin and the beta subunit; and another method for immunoassay of human chorionic gonadotropin and beta subunit, another system for immunoassay for identifying human chorionic gonadotropin and beta subunit, and another kit for detecting human chorionic gonadotropin and beta subunit.
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 (600-700 nm), 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 520-620 nm 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 the substance to be detected is high to a certain concentration, the phenomenon that the signal value is low because a double antibody sandwich complex cannot be formed is called 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, the high dose-hook effect may lead to a lower detection signal, and the sample is therefore interpreted as a lower concentration. 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 or improving the antibody.
The conventional detection process has the following 5 steps: adding the substance to be detected and the reagent into the reaction hole, carrying out the first step of incubation, adding the universal solution, carrying out the second step of incubation and reading.
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 concentration of the sample is judged by observing the change of the signal.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an immunoassay method, which widens the detection range through two readings, so that in the detection process, whether a sample to be detected needs to be diluted or not is judged by comparing the amplification A of the two readings with the maximum value of a standard curve of the amplification A of the two readings of a series of known standard substances, and then the detection is carried out.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
in a first aspect, the present invention provides an immunoassay method, comprising the steps of: (1) carrying out chemiluminescence immune reaction on 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 marking the difference amplification between the second reading and the first reading as A, (2) making a standard curve according to the amplification A of two readings of a series of known standard substances containing the target antigen (or antibody) to be detected, wherein the concentration of the standard substances is lower than that generating HOOK effect; (3) if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the maximum value of the standard curve, the concentration exceeds the upper detection limit, and the sample needs to be diluted and measured.
According to a preferred embodiment of the invention, the method comprises the steps of:
(1) mixing a sample to be detected containing a target antigen (or antibody) to be detected with 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) making a standard curve according to the amplification A of two readings of a series of known standard substances containing target antigens (or antibodies) to be detected, wherein the concentration of the standard substances is lower than that generating the HOOK effect;
(6) if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the maximum value of the standard curve, the concentration exceeds the upper detection limit, and the sample needs to be diluted for measurement
According to a preferred embodiment of the invention, the known standard substance is a positive control.
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 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 a second reading of chemiluminescence and multiplying the difference between the second and the first reading by A,
a processor for making a standard curve based on the amplification A of two readings of a known series of standard substances containing the target antigen (or antibody) to be measured, wherein the concentration of the standard substances is lower than the concentration producing the HOOK effect; if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the maximum value of the standard curve, the concentration exceeds the upper detection limit, and the sample needs to be diluted for measurement
According to a preferred embodiment of the invention, the method of using the system comprises the following steps:
(1) mixing a sample to be detected containing a target antigen (or antibody) to be detected with 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) making a standard curve according to the amplification A of two readings of a series of known standard substances containing target antigens (or antibodies) to be detected, wherein the concentration of the standard substances is lower than that generating the HOOK effect;
(6) if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the maximum value of the standard curve, the concentration exceeds the upper detection limit, and the sample needs to be diluted for measurement
A third aspect of the present invention provides a kit comprising 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: (1) carrying out chemiluminescence immune reaction on 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 marking the difference amplification between the second reading and the first reading as A, (2) making a standard curve according to the amplification A of two readings of a series of known standard substances containing the target antigen (or antibody) to be detected, wherein the concentration of the standard substances is lower than that generating HOOK effect; (3) if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the maximum value of the standard curve, the concentration exceeds the upper detection limit, and the sample needs to be diluted for measurement
According to a preferred embodiment of the present invention, the method of using the kit comprises the steps of:
(1) mixing a sample to be detected containing a target antigen (or antibody) to be detected with 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) making a standard curve according to the amplification A of two readings of a series of known standard substances containing target antigens (or antibodies) to be detected, wherein the concentration of the standard substances is lower than that generating the HOOK effect;
(6) if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the maximum value of the standard curve, the concentration exceeds the upper detection limit, and the sample needs to be diluted for measurement
The present invention also provides an immunoassay method, which widens the detection range by reading twice, and compares the amplification a of the reading twice with a critical value during the detection process to determine whether the sample to be detected needs to be diluted before the detection.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
a fourth aspect of the present invention provides an immunoassay method characterized by comprising the steps of: (1) performing chemiluminescence immune reaction on 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 marking the difference amplification between the second reading and the first reading as A, (2) taking the amplification A of the two readings of a known standard substance containing the target antigen (or antibody) to be detected as a critical value, (3) comparing the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected with the critical value, and if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is greater than the critical value, judging that the concentration of the sample to be detected is greater than the concentration of the known standard substance; and if the first reading of the sample to be detected is lower than the known standard substance at the same time, the sample needs to be diluted and then measured.
According to a preferred embodiment of the invention, the method comprises the steps of:
(1) mixing a sample to be detected containing a target antigen (or antibody) to be detected with 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) taking the two-time reading amplification A of a known standard substance containing a target antigen (or antibody) to be detected as a critical value;
(6) comparing the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected with a critical value, and if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is greater than the critical value, judging that the concentration of the sample to be detected is higher than the concentration of the known standard substance; and if the signal value obtained by the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then measuring.
According to a preferred embodiment of the invention, the known standard substance is a positive control.
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 fifth aspect of the invention provides a system for identifying 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 a second reading of chemiluminescence and multiplying the difference between the second and the first reading by A,
and the processor is used for comparing the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected with a critical value, judging that the concentration of the sample to be detected is higher than that of the known standard substance if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is larger than the critical value, and diluting the sample and then determining if the first reading of the sample to be detected is lower than that of the known standard substance.
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, the method of use of the system comprises the steps of:
(1) mixing a sample to be detected containing a target antigen (or antibody) to be detected with 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) taking the two-time reading amplification A of a known standard substance containing a target antigen (or antibody) to be detected as a critical value;
(6) comparing the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected with a critical value, and if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is greater than the critical value, judging that the concentration of the sample to be detected is greater than that of the known standard substance; and if the signal value obtained by the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then measuring.
A sixth aspect of the present invention provides a kit comprising 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: (1) performing chemiluminescence immune reaction on 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 marking the difference amplification between the second reading and the first reading as A, (2) taking the amplification A of the two readings of a known standard substance containing the target antigen (or antibody) to be detected as a critical value, (3) comparing the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected with the critical value, and if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is greater than the critical value, judging that the concentration of the sample to be detected is greater than that of the known standard substance; and if the first reading of the sample to be detected is lower than the known standard substance at the same time, the sample needs to be diluted and then measured.
According to a preferred embodiment of the present invention, the method of using the kit comprises the steps of:
(1) mixing a sample to be detected containing a target antigen (or antibody) to be detected with 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) taking the two-time reading amplification A of a known standard substance containing a target antigen (or antibody) to be detected as a critical value;
(6) comparing the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected with a critical value, and if the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to be detected is greater than the critical value, judging that the concentration of the sample to be detected is greater than that of the known standard substance; and if the signal value obtained by the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then measuring.
It should be particularly noted that the above method is a method for non-disease diagnosis, and is used for widening the detection range by two readings in the detection process of the double antibody sandwich immunoassay or the double antigen sandwich immunoassay, so as to compare the amplification a of the two readings with a critical value in the detection process to determine whether the sample to be detected needs to be diluted and then perform the determination.
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 the group consisting of HBsAb hepatitis B virus surface antibody (HBsAb), human chorionic gonadotropin and beta subunit (HCG + beta), hepatitis B surface antigen (HBsAg), cancer antigen 125(CA125), C-peptide (CP), ferritin (Ferr) and Anti-HCV.
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 (600-700 nm), singlet oxygen ions released by the photosensitive particles are received by the luminescent particles, and therefore 520-620 nm 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 (600-700 nm) 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 520-620 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 high concentration samples, 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 (600-700 nm) 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 the amplification A of the two signals is compared with a critical value to judge whether the sample to be detected needs to be diluted and then is measured.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention can realize signal measurement for a reaction for many times without interrupting the immune reaction based on the non-washing of a light-activated chemical luminescence platform (luminescence oxygen channel) and the uniformity of the reaction, and detect the optical signals in different reaction times, 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 be used for identifying whether the sample to be detected in the double-antibody sandwich method detection needs to be diluted and then is detected, the method can be used for remarkably improving the accuracy of the double-antibody sandwich method immunoassay and reducing 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 identify the sample with low report concentration caused by HD-HOOK effect in the double-antibody sandwich immunoassay for non-disease diagnosis.
Drawings
FIG. 1: a relation curve graph of a signal value and a sample concentration obtained by HCG + beta by adopting a conventional method;
FIG. 2: 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.
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, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and 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.
The inventor of the invention finds that whether the sample to be measured needs to be diluted or not can be judged by setting two readings and comparing the amplification A of the two readings with the critical value through extensive and intensive research.
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 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 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 (600-700 nm), 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 520-620 nm 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 (600-700 nm), the released singlet oxygen can be received by the luminescent particles. Through a series of chemical reactions, 520-620 nm 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.
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';
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 (600-700 nm), the released singlet oxygen can be received by the luminescent particles. Through a series of chemical reactions, 520-620 nm 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 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 as universal liquid, are commercially available from Boyang Biotechnology Ltd.
(4) And (3) standard substance:
preparing a standard solution within a certain concentration range (lower than the HD-HOOK effect concentration) by using the antigen (or antibody) to be detected. Mixing the standard substance, the reagent 1 and the reagent 2 uniformly, adding the universal solution after incubation reaction, continuously performing incubation reaction for a period of time, then performing first reading (RLU1), performing second reading (RLU2) after another period of time, calculating A (RLU2/RLU1-1) x 100%, 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;
(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:
comparing the two-time reading amplification value A of the sample to be detected with the value A of the known standard substance, and if the sample A to be detected is greater than the value A of the known standard substance, the concentration of the sample is greater than that of the known standard substance; if the sample RLU1 is smaller than the standard RLU1, it indicates that the sample RLU1 is low due to HD-HOOK effect, requiring dilution detection.
Example 1: the effectiveness of the method is verified by detecting human chorionic gonadotropin and beta subunit (HCG + beta) in a human serum sample
The content of the human chorionic gonadotropin and the beta subunit (HCG + beta) in the serum sample is detected by adopting a human chorionic gonadotropin and beta subunit (HCG + beta) detection kit (a light-activated chemiluminescence method) produced by Boyang biotechnology (Shanghai) Limited. The kit comprises a calibrator 1-calibrator 6, 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).
The serum samples of 18 patients with HCG + beta concentration obtained by Roche detection (dilution detection of samples exceeding the detection limit) are respectively detected by the conventional method and the method of the invention,
the conventional detection method comprises the following steps: after a sample to be tested, a calibrator, a reagent 1 (a luminescent antibody, namely, luminescent particles coated by a mouse monoclonal antibody) and a reagent 2 (a biotin-labeled antibody, namely, a biotin-labeled mouse monoclonal antibody) are respectively added into a reaction cup, incubation is carried out at 37 ℃ for 15min, a universal solution (streptavidin-labeled photosensitive particles) is added, incubation is carried out at 37 ℃ for 10min, a photon counter reads, RLU is read, the concentration of the sample is calculated, and the results are shown in the following table.
The invention adopts a double-reading method: the test sample, calibrator, reagent 1 (luminogenic antibody, i.e., luminogenic microparticles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) were incubated at 37 ℃ for 15min, a universal solution (streptavidin-labeled photosensitive microparticles) was added, the incubation was continued at 37 ℃ for 3min, reading RLU1, incubation was continued at 37 ℃ for 7min, reading RLU2, and the second signal value amplification A ═ X100% (RLU2/RLU1-1) was calculated, and the results are shown in the following table,
table 1:
Figure GDA0003264025580000151
Figure GDA0003264025580000161
note: the detection range of HCG + beta routine detection is 0-10000mIU/ml, and the sample concentration is more than 10000mIU/ml when the detection is beyond the upper limit.
The Roche detection result is used as the real concentration, as can be seen from table 1 and fig. 1, in the conventional detection, the signal value increases with the increase of the concentration when the concentration rises to 54531mIU/ml, the concentration continues to rise, the signal value decreases with the increase of the HCG + β concentration, that is, the concentration is greater than 54531mIU/ml, then HD-HOOK, in the conventional detection, the detection range is 0-10000mIU/ml, the sample with the concentration higher than the upper limit of the detection is greater than 10000mIU/ml, when the concentration of the HD-HOOK effect sample continues to rise, the signal continues to fall, and the ultrahigh concentration sample is reported to be a lower concentration, for example, 18. 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 sample with lower reported concentration caused by HOOK effect through 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 1 and FIG. 2, the signal value increased with concentration to 54531mIU/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 10-18 is larger than that of the calibrator 6 (11.1%), and the A values are continuously increased, which indicates that the HCG + beta concentrations of the samples 10-18 are larger than 10000mIU/ml, and the concentration is continuously increased, which is consistent with the concentration result of Roche, the signal value of the sample 18 is lower than that of the calibrator 6, the detection concentration of the sample 18 is 8713.02mIU/ml by a conventional method, and the samples can be identified as HD-HOOK effect samples by the method of the invention and need to be diluted for 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 (42)

1. A method for immunoassay of human chorionic gonadotropin and beta subunit, said method comprising the steps of: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing the human chorionic gonadotropin and the beta subunit, exciting and recording a first reading and a second reading of chemiluminescence, and marking the difference amplitude between the second reading and the first reading as A, (2) making a standard curve according to the amplitude A' of the two readings of a series of known standard substances containing the human chorionic gonadotropin and the beta subunit, wherein the concentration of the standard substances is lower than that generating HOOK effect; (3) and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is larger than the maximum value of the standard curve, diluting the sample and then testing.
2. The immunoassay method according to claim 1, comprising the steps of:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) making a standard curve based on the amplification A' of two readings of a known series of standards containing human chorionic gonadotropin and beta subunit, wherein the concentration of the standard is lower than the concentration producing the HOOK effect;
(a6) and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is larger than the maximum value of the standard curve, diluting the sample and then testing.
3. The immunoassay method according to claim 1, wherein the standard substance is a positive control.
4. The immunoassay method according to claim 2, wherein the light-emitting particles are 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.
5. The immunoassay method according to claim 2, wherein in the steps (a2) and (a3), the amount of light emitted from the reaction solution is detected by irradiating with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.
6. The immunoassay method according to claim 2, wherein the antibody is an immunoglobulin produced by an organism and capable of recognizing a specific foreign substance; the first and second antibodies refer to antibodies that can specifically bind to the human chorionic gonadotropin and beta subunit.
7. A system for an immunoassay for identifying human chorionic gonadotropin and beta subunit, said system comprising:
an immunoreaction device for performing a light-activated chemiluminescent reaction,
a light activated chemiluminescent reaction excitation and counting device for exciting and recording a first and a second reading of chemiluminescence and multiplying the difference between the second and the first reading by A,
a processor for developing a calibration curve based on the amplification a' of two readings of a known series of standards comprising human chorionic gonadotropin and beta subunits, wherein the concentrations of the standards are lower than the concentrations that produce the HOOK effect; and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is larger than the maximum value of the standard curve, diluting the sample and then testing.
8. The system of claim 7, wherein the method of using the system comprises the steps of:
(1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody 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) multiplied by 100%;
(5) making a standard curve based on the amplification A' of two readings of a known series of standards containing human chorionic gonadotropin and beta subunit, wherein the concentration of the standard is lower than the concentration producing the HOOK effect;
(6) and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is larger than the maximum value of the standard curve, diluting the sample and then testing.
9. The system of claim 7, wherein the standard substance is a positive control.
10. The system of claim 8, 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.
11. The system according to claim 8, wherein in the steps (2) and (3), the reaction solution is irradiated with 600-700 nm red excitation light, and the amount of the emitted light is detected; the detection wavelength of the emitted light is 520-620 nm.
12. The system of claim 8, wherein the antibody is an immunoglobulin produced by the body that recognizes a specific foreign object; the first and second antibodies refer to antibodies that can specifically bind to the human chorionic gonadotropin and beta subunit.
13. The use method of a kit for detecting human chorionic gonadotropin and beta subunit, wherein the kit comprises a first antibody coated luminescent particle, a second antibody marked by a marker and a photosensitive particle marked by a marker specific binding substance, and the use method of the kit comprises the following steps: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing the human chorionic gonadotropin and the beta subunit, exciting and recording a first reading and a second reading of chemiluminescence, and marking the difference amplitude between the second reading and the first reading as A, (2) making a standard curve according to the amplitude A' of the two readings of a series of known standard substances containing the human chorionic gonadotropin and the beta subunit, wherein the concentration of the standard substances is lower than that generating HOOK effect; (3) and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is larger than the maximum value of the standard curve, diluting the sample and then testing.
14. The method for using the kit according to claim 13, wherein the method for using the kit comprises the following steps:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) making a standard curve based on the amplification A' of two readings of a known series of standards containing human chorionic gonadotropin and beta subunit, wherein the concentration of the standard is lower than the concentration producing the HOOK effect;
(a6) and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is larger than the maximum value of the standard curve, diluting the sample and then testing.
15. The method of using the kit according to claim 13 or 14, wherein the standard substance is a positive control.
16. The method of using the kit according to claim 14, 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.
17. The method of using the kit according to claim 14, wherein in the steps (a2) and (a3), the amount of the emitted light of 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.
18. The method of using the kit according to claim 14, wherein the antibody is immunoglobulin produced by the body and capable of recognizing a specific foreign body; the first and second antibodies refer to antibodies that can specifically bind to the human chorionic gonadotropin and beta subunit.
19. A method for immunoassay of human chorionic gonadotropin and beta subunit, said method comprising the steps of: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing the human chorionic gonadotropin and the beta subunit, exciting and recording a first reading and a second reading of chemiluminescence, and marking the difference amplitude between the second reading and the first reading as A, (2) taking the two-reading amplification A' of a known standard substance containing the human chorionic gonadotropin and the beta subunit as a critical value, (3) comparing the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with the critical value, and if the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is greater than the critical value, the concentration of the sample to be detected is higher than that of the known standard substance.
20. A method for immunoassay of human chorionic gonadotropin and beta subunit, said method comprising the steps of: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing human chorionic gonadotropin and beta subunit, exciting and recording a first reading and a second reading of chemiluminescence, and marking the difference amplitude between the second reading and the first reading as A, (2) taking the two-reading amplification A' of a known standard substance containing the human chorionic gonadotropin and the beta subunit as a critical value, (3) comparing the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with the critical value, and if the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is larger than the critical value and the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then carrying out detection.
21. The immunoassay method according to claim 19, comprising the steps of:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) taking the two read amplification A' of a known standard substance containing human chorionic gonadotropin and beta subunit as a critical value;
(a6) comparing the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is greater than the critical value, the concentration of the sample to be tested is higher than that of the known standard substance.
22. The immunoassay method according to claim 20, comprising the steps of:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) taking the two read amplification A' of a known standard substance containing human chorionic gonadotropin and beta subunit as a critical value;
(a6) comparing the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is larger than the critical value and the signal value obtained by the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then detecting.
23. The immunoassay method of claim 19 or 20, wherein the known standard substance is a positive control.
24. The immunoassay method according to claim 21 or 22, wherein the light-emitting particles are 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.
25. The immunoassay method according to claim 21 or 22, wherein in the steps (a2) and (a3), the amount of light emitted from the reaction solution is detected by irradiating with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.
26. The immunoassay method according to claim 21 or 22, wherein the antibody is an immunoglobulin produced by an organism and capable of recognizing a specific foreign substance; the first and second antibodies refer to antibodies that can specifically bind to the human chorionic gonadotropin and beta subunit.
27. A system for an immunoassay for identifying human chorionic gonadotropin and beta subunit, said system comprising:
an immunoreaction device for performing a light-activated chemiluminescent reaction,
a light activated chemiluminescent reaction excitation and counting device for exciting and recording a first and a second reading of chemiluminescence and multiplying the difference between the second and the first reading by A,
and the processor is used for comparing the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is greater than the critical value, the concentration of the sample to be detected is higher than the concentration of the standard substance used in the critical value determination.
28. A system for an immunoassay for identifying human chorionic gonadotropin and beta subunit, said system comprising:
an immunoreaction device for performing a light-activated chemiluminescent reaction,
a light activated chemiluminescent reaction excitation and counting device for exciting and recording a first and a second reading of chemiluminescence and multiplying the difference between the second and the first reading by A,
and the processor is used for comparing the amplification A of the two readings of the sample to be measured containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be measured containing the human chorionic gonadotropin and the beta subunit is larger than the critical value and the signal value obtained by the first reading of the sample to be measured is lower than the standard substance used in the critical value measurement, diluting the sample and then measuring.
29. The system of claim 27, wherein the method of using the system comprises the steps of:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) taking the two read amplification A' of a known standard substance containing human chorionic gonadotropin and beta subunit as a critical value;
(a6) comparing the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is greater than the critical value, the concentration of the sample to be tested is higher than that of the known standard substance.
30. The system of claim 28, wherein the method of using the system comprises the steps of:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) taking the two read amplification A' of a known standard substance containing human chorionic gonadotropin and beta subunit as a critical value;
(a6) comparing the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is larger than the critical value and the signal value obtained by the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then detecting.
31. The system of claim 27, wherein the standard substance is a positive control.
32. The system of claim 29 or 30, 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.
33. The system according to claim 29 or 30, wherein in the steps (a2) and (a3), the amount of the emitted light of the reaction solution is detected by irradiating with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.
34. The system of claim 29 or 30, wherein the antibody is an immunoglobulin produced by the body that recognizes a specific foreign body; the first and second antibodies refer to antibodies that can specifically bind to the human chorionic gonadotropin and beta subunit.
35. The use method of a kit for detecting human chorionic gonadotropin and beta subunit, wherein the kit comprises a first antibody coated luminescent particle, a second antibody marked by a marker and a photosensitive particle marked by a marker specific binding substance, and the use method of the kit comprises the following steps: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing the human chorionic gonadotropin and the beta subunit, exciting and recording a first reading and a second reading of chemiluminescence, and marking the difference amplitude between the second reading and the first reading as A, (2) taking the two-reading amplification A' of a known standard substance containing the human chorionic gonadotropin and the beta subunit as a critical value, (3) comparing the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with the critical value, and if the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is greater than the critical value, the concentration of the sample to be detected is higher than that of the known standard substance.
36. The use method of a kit for detecting human chorionic gonadotropin and beta subunit, wherein the kit comprises a first antibody coated luminescent particle, a second antibody marked by a marker and a photosensitive particle marked by a marker specific binding substance, and the use method of the kit comprises the following steps: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing human chorionic gonadotropin and beta subunit, exciting and recording a first reading and a second reading of chemiluminescence, and marking the difference amplitude between the second reading and the first reading as A, (2) taking the two-reading amplification A' of a known standard substance containing the human chorionic gonadotropin and the beta subunit as a critical value, (3) comparing the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with the critical value, and if the two-reading amplification A of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is larger than the critical value and the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then carrying out detection.
37. The method for using the kit according to claim 35, wherein the method for using the kit comprises the following steps:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) taking the two read amplification A' of a known standard substance containing human chorionic gonadotropin and beta subunit as a critical value;
(a6) comparing the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be tested containing the human chorionic gonadotropin and the beta subunit is greater than the critical value, the concentration of the sample to be tested is higher than that of the known standard substance.
38. The method for using the kit according to claim 36, wherein the method for using the kit comprises the following steps:
(a1) mixing a sample to be detected containing human chorionic gonadotropin and beta subunit with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) 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) multiplied by 100%;
(a5) taking the two read amplification A' of a known standard substance containing human chorionic gonadotropin and beta subunit as a critical value;
(a6) comparing the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit with a critical value, and if the amplification A of the two readings of the sample to be detected containing the human chorionic gonadotropin and the beta subunit is larger than the critical value and the signal value obtained by the first reading of the sample to be detected is lower than the known standard substance, diluting the sample and then detecting.
39. The method of using the kit of claim 35, wherein the known standard is a positive control.
40. The method for using the kit according to claim 35 or 36, 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.
41. The method of using the kit according to claim 37 or 38, wherein in the steps (a2) and (a3), the amount of the emitted light of 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.
42. The method of using the kit of claim 35 or 36, wherein the antibody is an immunoglobulin produced by the body and capable of recognizing a specific foreign body; the first and second antibodies refer to antibodies that can specifically bind to the human chorionic gonadotropin and beta subunit.
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