CN109470869B - Immunoassay method, system and kit for identifying immunoassay - Google Patents

Immunoassay method, system and kit for identifying immunoassay Download PDF

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CN109470869B
CN109470869B CN201811401535.4A CN201811401535A CN109470869B CN 109470869 B CN109470869 B CN 109470869B CN 201811401535 A CN201811401535 A CN 201811401535A CN 109470869 B CN109470869 B CN 109470869B
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detected
sample
antibody
reading
concentration
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CN109470869A (en
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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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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G01N33/5764—Hepatitis B surface antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Abstract

The invention relates to a system for identifying an immunoassay, belonging to the technical field of light-activated chemiluminescence, which comprises the following components: an immunoreaction device for performing a chemiluminescent immunoreaction; a chemiluminescent immunoreaction excitation and counting device for exciting and recording a first and second reading of chemiluminescence and multiplying the difference between the second and first reading by A; 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, 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, the concentration of the sample to be detected is higher than that of the known standard substance; and simultaneously, if the signal value obtained by the first reading of the sample to be detected is lower than that of the known standard substance, diluting the sample and then determining.

Description

Immunoassay method, system and kit for identifying immunoassay
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-activated chemiluminescence, in particular to an immunoassay method, a system for identifying immunoassay and a kit; as well as another immunoassay method, another system for identifying an immunoassay, and another 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 (600-700 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;
FIG. 3: a relation curve graph of a signal value and a sample concentration obtained by the Ferr by adopting a conventional method;
FIG. 4: a graph of the relationship between the signal value and A obtained by Ferr by adopting the method of the invention and the sample concentration respectively;
FIG. 5: c peptide adopts a relation curve graph of a signal value obtained by a conventional method and sample concentration;
FIG. 6: c peptide adopts the method of the invention to obtain the signal value and A which are respectively related to the concentration curve of the sample.
FIG. 7: a curve graph of the relation between the signal value and the sample concentration of the HBsAb obtained by adopting a conventional method;
FIG. 8: HBsAb is obtained by the method of the invention, and the signal value and A are respectively plotted with the concentration of the sample.
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 IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
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 BDA0001876424360000151
Figure BDA0001876424360000161
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.
Example 2: the detection of ferritin (Ferr) in a sample verifies the effectiveness of the method
The ferritin (Ferr) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Inc. was used to detect the ferritin content (purchased from Fitzgerald, Catalog No: 30-AF10) in the samples. 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).
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.
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.
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 calibrator 1-calibrator 6, sample to be tested 1-15, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) 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, samples to be tested 1-15, reagent 1 (luminogenic antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) were incubated at 37 ℃ for 15min, LiCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, and further incubated at 37 ℃ for 7min, reading RLU2, and the second signal value amplification A ═ X100% (RLU2/RLU1-1) was calculated, with the results shown in the following table,
table 2:
Figure BDA0001876424360000181
Figure BDA0001876424360000191
note: the detection range of the routine detection of ferritin is 0-2000ng/ml, and the sample shows a concentration of >2000ng/ml beyond the upper limit of detection.
In the conventional detection, as can be seen from Table 2 and FIG. 3, the signal value increases with increasing concentration when the concentration rises to 51000ng/ml, the signal value continues to increase with increasing Ferr concentration, the conventional detection range is 0-2000ng/ml, and the sample shows a concentration of >2000ng/ml beyond the upper detection limit. Reporting of the ultra-high concentration sample as a lower concentration, such as sample 15, occurs when the HD-HOOK effect sample concentration continues to rise and when the concentration rises to 2550000ng/ml, the signal falls below the signal of the calibrator 6. 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 2 and FIG. 4, 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 4-15 is larger than that (-5.5%) of the calibrator 6, which indicates that the Ferr concentrations of the samples 4-15 are larger than 2000 ng/ml. This corresponds to the actual concentration, the signal value of the sample 15 is lower than that of the calibrator 6, the detection concentration by the conventional method is 1860.97ng/ml, and the sample with the concentration exceeding the detection range can be identified by the method of the present invention, and the dilution detection is needed.
Example 3: detection of C Peptide (CP) in sample to verify effectiveness of the method
The content of C peptide (purchased from Fitzgerald, Catalog No: 30-AC96) in the sample was determined using C Peptide (CP) assay kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Ltd. 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).
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.
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.
The conventional detection method comprises the following steps: after adding calibrator 1-calibrator 6, sample to be tested 1-15, reagent 1 (luminescent antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) 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, samples to be tested 1-15, reagent 1 (luminogenic antibody, i.e., luminescent particles coated with murine monoclonal antibody) and reagent 2 (biotin-labeled antibody, i.e., biotin-labeled murine monoclonal antibody) were incubated at 37 ℃ for 15min, LiCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, and further incubated at 37 ℃ for 7min, reading RLU2, and the second signal value amplification A ═ X100% (RLU2/RLU1-1) was calculated, with the results shown in the following table,
table 3:
Figure BDA0001876424360000201
Figure BDA0001876424360000211
note: the detection range for routine detection of C-peptide was 0-30ng/ml, with samples above the upper detection limit showing concentrations >30 ng/ml.
In the conventional assay, as can be seen from Table 3 and FIG. 5, the signal value increased with increasing concentration as the concentration increased to 10000ng/ml, the signal value continued to increase, and the signal value decreased with increasing C peptide concentration, the conventional assay range was 0-30ng/ml, and the sample above the upper limit of assay showed a concentration of >30 ng/ml. When the concentration rises to 33500000ng/ml and the signal falls below that of the calibrator 6, reporting of the ultra-high concentration sample as a lower concentration condition, such as samples 16, 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.
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 3 and FIG. 6, the signal value increased to 10000ng/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 5-17 is larger than that of the calibrator 6 (-4.9%), which indicates that the C peptide concentrations of the samples 5-17 are larger than 30ng/ml and exceed the upper detection limit. The actual concentration is consistent, the signal values of the samples 16 and 17 are lower than that of the calibrator 6, the detection concentrations of the conventional method are respectively 8.15ng/ml and 0.76ng/ml, and the samples which exceed the upper detection limit can be identified by the method of the invention and need to be subjected to dilution detection.
Example 4: detection of hepatitis B Virus surface antigen (HBsAg) in human serum samples
The concentration of HBsAg in a sample is detected by using a hepatitis B virus surface antigen (HBsAg) detection kit (light-activated chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Inc., wherein the kit comprises a calibrator 1-6, 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.
Firstly, detecting a calibrator 1-6 and a serum sample to be detected 1-15 by using the method of the invention: after adding the analyte, reagent 1 (antibody-coated luminescent particles) and reagent 2 (biotin-labeled antibody) to the cuvette, incubation was carried out at 37 ℃ for 15min, LiCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubation was carried out at 37 ℃ for 3min, reading RLU1, incubation was continued at 37 ℃ for 7min, reading RLU2, and the second signal value was calculated as the increase A ═ X100% (RLU2/RLU1-1), and the results are shown in the following table.
Table 4:
Figure BDA0001876424360000221
Figure BDA0001876424360000231
the concentration of 15 serum samples obtained by the method of the invention is as follows: as shown in the table above, samples exceeding the upper limit of detection (336.56IU/mL) are distinguished by comparing with the amplification of the calibrator 6, i.e. if the a value is greater than 27%, the samples are judged to be HOOK effect samples, and the dilution is recommended for detection; and when A is less than 27%, the sample is in the detection range, and the concentration of the sample 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 5:
Figure BDA0001876424360000241
the serum samples 1, 2, 3, 5, 6, 9, 12, 13, 14 and 15 are diluted to be detected to be increased in concentration, namely, the serum samples are proved to be HD-HOOK effect samples, the concentration is higher than 336.56IU/mL, and the serum samples 4, 7, 8, 10 and 11 are diluted to be reduced in 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 5: 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 carbohydrate antigen 125(CA125) detection kit (photo-activated chemiluminescence method) manufactured by bosyang biotechnology (shanghai) ltd, which includes a calibrator 1-6, 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).
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.
Firstly, detecting a calibrator 1-6 and a serum sample to be detected 1-18 by using the method of the invention: after adding the analyte, reagent 1 (antibody-coated luminescent particles) and reagent 2 (biotin-labeled antibody) to the cuvette, incubation was carried out at 37 ℃ for 15min, LiCA Universal solution (streptavidin-labeled photosensitive particles) was added, incubation was carried out at 37 ℃ for 3min, reading RLU1, incubation was continued at 37 ℃ for 7min, reading RLU2, and the second signal value was calculated as the increase A ═ X100% (RLU2/RLU1-1), and the results are shown in the following table.
Table 6:
Figure BDA0001876424360000251
Figure BDA0001876424360000261
the concentration of 18 serum samples obtained by the method of the invention is as follows: as shown in the table above, samples exceeding the upper limit of detection are distinguished by comparing with the amplification of the calibrator 6, i.e. if the value a is greater than 7.4%, the samples exceeding the upper limit of detection are judged, and detection after dilution is recommended; and when A is less than 7.4%, 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 7:
Figure BDA0001876424360000262
Figure BDA0001876424360000271
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. In the case of undiluted samples, the original-fold samples are detected by the conventional method, and the serum samples 16, 17 and 18 are erroneously determined as low-concentration samples due to the HD-HOOK effect.
Example 6: detection of hepatitis B Virus surface antibody (HBsAb) in a sample to verify the effectiveness of the method of the invention
The content of hepatitis B virus surface antibody (obtained from Beijing Zhongke Kyoda Biotechnology Co., Ltd., Clone No: M2201) in the sample was determined by using HBsAbHBsAb (light-activated chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. The kit comprises a calibrator 1-calibrator 6, a reagent 1(HBsAg coated luminescent particles) and a reagent 2 (biotin-labeled HBsAg).
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.
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 test samples 1 to 14, the reagent 1 (HBsAg-coated luminescent particles) and the reagent 2 (biotin-labeled HBsAg) were added to the reaction cuvette, and then incubated at 37 ℃ for 15min, LiCA Universal solution (streptavidin-labeled photosensitive particles) was added, and then incubated at 37 ℃ for 10min, and then the result was read by a photon counter, and RLU was read, as shown in the following table.
The invention adopts a double-reading method: the calibrator 1 to calibrator 6, the test samples 1 to 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, incubation was carried out at 37 ℃ for 3min, reading RLU1 and further incubation was carried out at 37 ℃ for 7min, reading RLU2, and the second signal value amplification A ═ RLU2/RLU1-1) X100% was calculated, with the results shown in the following table,
table 8:
Figure BDA0001876424360000281
Figure BDA0001876424360000291
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.
In the conventional detection, as can be seen from Table 8 and FIG. 7, the signal value increased with the increase in concentration as the concentration increased to 10000mIU/ml, the concentration continued to increase, and the signal value decreased with the increase in HBsAb concentration, the conventional detection range was 0-1000mIU/ml, and the sample above the upper limit of detection showed a concentration of >1000 mIU/ml. When the concentration rises to 335000mIU/ml and above, the signal drops below that of the calibrator 6, and reporting of the ultra-high concentration samples as lower concentrations, 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 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 8 and FIG. 8, 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 8-14 is larger than that of the calibrator 6 (35.9%), which indicates that the HBsAb concentrations of the samples 8-14 are larger than 1000mIU/ml and exceed the upper limit of detection. This corresponds to the actual concentration, samples 12, 13, 14. The signal value is lower than that of a calibrator 6, the detection concentrations are 802.57mIU/ml, 352.22mIU/ml and 147.9mIU/ml respectively by the conventional method, and the signal can be identified as a sample exceeding the upper limit of detection by the method of the invention and needs to be subjected to dilution detection.
Example 7: the method of the invention is applied to the Anti-HCV qualitative kit
The Anti-HCV content in the sample is detected by using a hepatitis C virus antibody detection kit (chemiluminescence method) produced by Boyang biotechnology (Shanghai) Co. The kit comprises a reference, a negative control, a positive control, a reagent 1 (a luminescent HCV antigen, i.e., a luminescent microparticle coated with an HCV antigen) and a reagent 2 (a biotin-labeled HCV antigen, i.e., a biotin-labeled HCV antigen).
Reference, negative control, positive control: the reference substance is a standard substance with known concentration which is used as a reference to judge whether the sample to be detected is positive or negative; the negative control and the positive control are standard substances with known concentrations for judging the effectiveness of the test. And (3) carrying out gradient dilution on the Anti-HCV with high concentration, and respectively determining the signal values of samples containing Anti-HCV with different concentrations by adopting a conventional detection method and the detection method disclosed by the invention.
The conventional detection method comprises the following steps: after a series of Anti-HCV samples diluted in gradient, reagent 1 (luminescent HCV antigen, i.e., HCV antigen-coated luminescent particles) and reagent 2 (biotin-labeled HCV antigen, i.e., biotin-labeled HCV antigen) were added to the reaction cuvette, incubation was performed at 37 ℃ for 15min, LiCA universal solution (streptavidin-labeled photosensitive particles) was added, incubation was performed at 37 ℃ for 10min, a photon counter was read, RLU was read, and the results are shown in table 8.
The invention adopts a double-reading method: a series of Anti-HCV samples diluted in gradient, reagent 1 (luminescent HCV antigen, i.e. HCV antigen-coated luminescent particles) and reagent 2 (biotin-labeled HCV antigen, i.e. biotin-labeled HCV antigen), were incubated at 37 ℃ for 15min, lico universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ℃ for 3min, reading RLU1, incubation at 37 ℃ was continued for 7min, reading RLU2, and the second signal value increase a ═ X100% (RLU2/RLU1-1) was calculated, with the results shown in the table below.
Table 9:
Figure BDA0001876424360000301
Figure BDA0001876424360000311
as shown in the above table, after the dilution multiple of the antigen is reduced to 10000 times, the signal value is reduced along with the increase of the concentration, the HOOK effect occurs, when the concentration continues to increase to a certain value (as in the sample 12), the RLU decreases below the reference value cut off, the result is judged to be negative by mistake under the conventional detection method, the method of the invention can firstly observe the increase a of the signal twice (RLU2/RLU1-1) X100%, compare the a value of the sample to be detected with the a value of the positive control (-25%), judge the magnitude relation between the sample to be detected and the positive reference, as shown in the above table, the a value (47%) of the sample 12 is far higher than the a value of the positive control (-25%), which indicates that the secondary sample has a higher concentration than the positive control, and is the positive sample, and the insuffirrity of the signal is due to the HOOK effect, and the dilution verification should be performed.
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 (8)

1. A system for identifying an immunoassay, the system comprising:
an immunoreaction device for performing a light-activated chemiluminescent reaction,
the light-activated chemiluminescence reaction excitation and counting device is used for exciting and recording first and second readings of chemiluminescence, the difference between the second and first readings is increased and recorded as A, and the light-activated chemiluminescence reaction excitation and counting device comprises a photon counting module and a light-emitting diode;
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, 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, the concentration of the sample to be detected is higher than the concentration of the standard substance used in the critical value test.
2. A system for identifying an immunoassay, the system comprising:
an immunoreaction device for performing a light-activated chemiluminescent reaction,
the light-activated chemiluminescence reaction excitation and counting device is used for exciting and recording first and second readings of chemiluminescence, the difference between the second and first readings is increased and recorded as A, and the light-activated chemiluminescence reaction excitation and counting device comprises a photon counting module and a light-emitting diode;
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, 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 larger than the critical value and the signal value obtained by the first reading of the sample to be detected is lower than a standard substance used in critical value testing, diluting the sample and then determining.
3. The system of claim 1, wherein the method of using the system comprises the steps of:
(1) mixing a sample to be detected containing a target antigen to be detected with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution; or mixing a sample to be detected containing a target antibody to be detected with the first antigen-coated luminescent particles and a second 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) multiplied by 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, the concentration of the sample to be detected is higher than that of the known standard substance.
4. The system of claim 2, wherein the method of using the system comprises the steps of:
(1) mixing a sample to be detected containing a target antigen to be detected with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution; or mixing a sample to be detected containing a target antibody to be detected with the first antigen-coated luminescent particles and a second 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) and 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 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 determining.
5. The system of claim 3 or 4, wherein the known standard is a positive control.
6. The system according to claim 3 or 4, 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.
7. The system according to claim 3 or 4, 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.
8. The system of claim 3 or 4, 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.
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