CN109470862B - Immunoassay method, system and kit for identifying immunoassay - Google Patents
Immunoassay method, system and kit for identifying immunoassay Download PDFInfo
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Abstract
The invention relates to an immunoassay method of fetal alpha globulin, belonging to the technical field of light-activated chemiluminescence, which comprises the following steps: (1) carrying out chemiluminescence immune reaction on a sample to be detected containing fetal alpha globulin, 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) respectively making a first standard curve and a second standard curve according to the amplification A' of the first reading and the two readings of a series of known standard substances containing the fetal alpha globulin; (3) and comparing the first reading and the amplification A of the two readings of the sample to be detected containing the fetal alpha globulin with the first standard curve and the second standard curve respectively to determine the concentration of the sample. The invention also relates to a system for immunoassay for identification of fetal alpha globulin and a kit for detection of fetal alpha globulin.
Description
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. 201611034237.7.
Technical Field
The invention relates to the technical field of light-activated chemiluminescence, in particular to a fetal alpha globulin immunoassay method, a system for identifying the fetal alpha globulin immunoassay and a kit for detecting the fetal alpha globulin.
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 methods such as optimizing antibodies or increasing antibody concentration.
The conventional detection process has the following 5 steps: the reaction wells are filled with the analyte and reagent, the first incubation step, the LiCA universal solution addition, the second incubation step and the reading step.
The detection method is based on a conventional detection process, and on the premise of not interrupting the reaction, the signal value is read for many times in the reaction process, and the real 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 by reading twice and calculates the concentration of an object to be detected simply, conveniently and quickly.
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) respectively making a standard curve according to the first reading and the amplification A of the two readings of a series of known standard substances containing the target antigen (or antibody) to be detected; (3) the concentration of the sample is determined by comparing the first reading and the increase a of the two readings of the test sample containing the target antigen (or antibody) to the standard curve. According to a preferred embodiment, the immunoassay 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;
(6) and determining whether the concentration of the substance to be detected is in a rising interval or a falling interval of the standard curve according to the value A, and substituting the RLU1 of the sample to be detected into the corresponding standard curve to calculate the concentration.
According to a preferred embodiment, the luminescent particles refer to polymer particles filled with a luminescent compound and a lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and can generate singlet oxygen ions under the excitation of red laser.
According to a preferred embodiment, in the steps (2) and (3), the emitted light quantity of the reaction solution is detected by irradiating the reaction solution with 600-700 nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.
According to a preferred embodiment, 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,
and the processor is used for making a standard curve according to the first reading and the amplification A of the two readings of a known series of standard substances containing the target antigen (or antibody) to be detected, and comparing the first reading and the amplification A of the two readings of the sample to be detected containing the target antigen (or antibody) to the standard curve to determine the concentration of the sample.
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) 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;
(6) and determining whether the concentration of the substance to be detected is in a rising interval or a falling interval of the standard curve according to the value A, and substituting the RLU1 of the sample to be detected into the corresponding standard curve to calculate the concentration.
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) respectively making a standard curve according to the first reading and the amplification A of the two readings of a series of known standard substances containing the target antigen (or antibody) to be detected; (3) the concentration of the sample is determined by comparing the first reading and the increase a of the two readings of the test sample containing the target antigen (or antibody) to the standard curve.
According to a preferred embodiment, the method of use of 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;
(6) and determining whether the concentration of the substance to be detected is in a rising interval or a falling interval of the standard curve according to the value A, and substituting the RLU1 of the sample to be detected into the corresponding standard curve to calculate the concentration.
Here, it should be particularly noted that the above-mentioned method is a method for non-disease diagnosis purposes, and the method 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 calculate the concentration of the antibody in the detection process simply, conveniently and rapidly.
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 Insulin (INS), hepatitis b virus surface antibody (HBsAb), fetal alpha globulin (AFP) and Thyrotropin (TSH).
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 being 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 detection range can be widened. 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 detection range can be widened by comparing the amplification of the two signals, so that the concentration of the sample can be simply, conveniently and quickly calculated in the detection process.
Compared with the prior art, the invention has the beneficial effects that:
(1) based on the non-washing of a light-activated chemiluminescence platform (luminous oxygen channel) and the uniformity of the reaction, the invention can realize multiple signal measurement on one reaction without interrupting the immune reaction, detect the optical signals at different reaction times, and distinguish HD-HOOK effect samples by comparing the sizes of the two signals, and the method is not limited by the detection range and effectively widens the detection range by more than 100 times.
(2) The method can accurately identify HD-HOOK effect samples in the double-antibody sandwich method detection by 100 percent, can obviously improve the accuracy of the double-antibody sandwich method immunoassay, and reduces the false negative rate of the double-antibody sandwich method immunoassay.
(3) The method is simple to operate, the detection range is widened by reading twice, and the concentration of the substance to be detected is simply, conveniently and quickly calculated in the detection process.
Drawings
FIG. 1: AFP adopts the relation curve graph of signal value and sample concentration obtained by the conventional detection method.
FIG. 2: AFP adopts the first reading signal obtained by the method of the invention and the curve graph of the relationship between the amplification A and the sample concentration.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.
The inventor of the invention finds that the concentration can be simply and rapidly calculated in the detection process by setting up two readings, researching the relationship between the amplification of the two readings and the concentration of the sample and widening the detection range through the two readings.
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): fetal alpha globulin (AFP), cancer antigen 125(CA125), and the like.
The basic principle of the double antibody sandwich method:
the basic principles of the double antibody sandwich method are well known to those skilled in the art. It is common practice to fix a first antibody (or antigen) to a solid phase carrier, then react the first antibody (or antigen) with the antigen (or antibody), then react with a labeled second antibody (or antigen), and finally perform a chemiluminescent or enzyme-linked chromogenic reaction to detect a signal.
The basic principle of the light-activated chemiluminescence method:
the basic principles of light-activated chemiluminescence are well known to those skilled in the art. Conventionally, photosensitive particles and luminescent particles are combined in a certain range to generate ion oxygen energy transfer and emit light signals, so that a sample to be detected is detected. Wherein, the photosensitive particle is filled with photosensitive compound, and the luminescent particle is filled with luminescent compound and lanthanide. Under the excitation of red laser (600-700 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 LiCA Universal liquid, are available commercially from Boyang Biotech Ltd.
(4) And (3) standard substance:
a series of standard solutions with wide concentration range (spanning HD-HOOK effect concentration) are prepared by using the antigen (or antibody) to be detected. Uniformly mixing the standard substance, the reagent 1 and the reagent 2, adding LiCA universal solution after incubation reaction, continuously performing the first reading (RLU1) after the incubation reaction is performed for a period of time, performing the second reading (RLU2) after the incubation reaction is performed for a period of time, calculating the A ═ 100% (RLU2/RLU1-1) x, and 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 respectively; the standard curve of RLU1 and concentration of the standard substance shows that in a non-HD-HOOK effect stage, RLU1 increases with the increase of concentration and is marked as an increasing interval of RLU1, RLU1 decreases with the increase of concentration after the concentration increases to an HD-HOOK effect stage and is marked as a decreasing interval of RLU1, and the standard curve of A and concentration of the standard substance shows that A increases with the increase of concentration and is not influenced by HD-HOOK effect.
(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:
the two-time reading amplification A value of the sample to be detected is substituted into a standard curve of the standard substance A and the standard substance concentration, whether the concentration of the sample to be detected is in an ascending interval or a descending interval of the RLU1 is judged, and then the RLU1 of the sample to be detected is substituted into the standard curve of the standard substance RLU1 and the standard substance concentration in the interval to be detected to calculate the concentration of the sample to be detected.
Example 1: conventional and inventive methods separately detect a fetal alpha-globulin (AFP) sample
A fetal alpha globulin detection kit (light-activated chemiluminescence method) produced by Boyang biotechnology (Shanghai) Limited is adopted to detect the content of AFP (purchased from Fitzgerald, Catalog No: 30-1370) in a sample.
The high-concentration AFP antigen is subjected to gradient dilution, and the signal values of samples containing different concentrations of AFP are respectively determined by adopting a conventional detection method and the detection method provided by the invention.
The conventional detection method comprises the following steps: after adding the AFP sample, reagent 1(AFP primary antibody coated luminescent particles) and reagent 2 (biotin-labeled AFP secondary antibody) in a reaction cup, after gradient dilution, incubating at 37 ℃ for 15min, adding LiCA Universal liquid (streptavidin-labeled photosensitive particles), incubating at 37 ℃ for 10min, reading RLU with a photon counter, and the results are shown in Table 1.
The invention adopts a double-reading method: incubating a sample of an object to be detected, a reagent 1 (luminescent particles coated by an AFP primary antibody) and a reagent 2 (a biotin-labeled AFP secondary antibody) at a known concentration for 15min at 37 ℃, adding LiCA universal solution (streptavidin-labeled photosensitive particles), incubating for 3min at 37 ℃, reading RLU1, continuing incubating for 7min at 37 ℃, reading RLU2, and calculating the increase A of a second signal value to be (RLU2/RLU 1-1). times.100%, wherein the detection results are as follows:
table 1:
as can be seen from Table 1 and FIG. 1, the signal values from 5ng/ml to 10000ng/ml increase with increasing concentration, the concentration continues to increase, the signal values decrease with increasing AFP concentration, i.e., HD-HOOK is observed at concentrations greater than 10,000ng/ml, and in the conventional assay, the reported concentration of the sample with antigen concentration higher than this assay range will be lower (the reported concentration is less than 10,000 ng/ml).
The method of the invention widens the detection range by reading twice. And (3) detecting signal value results RLU1 and RLU2 in sequence by 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 sample concentration interval. As can be seen from Table 1 and FIG. 2, the signal value increased to 10,000ng/ml with increasing concentration, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with increasing concentration.
When the AFP calibrant concentration covered the range from 5ng/ml to 1,000,000ng/ml, RLU1 and A standard curves were generated using the method of the present invention (see FIG. 2), where A continued to rise as the concentration increased, and RLU1 was divided into a rising interval of 5ng/ml to 10,000ng/ml and a falling interval of 10,000ng/ml to 1,000,000 ng/ml. RLU1, RLU2 and A of the sample to be detected are detected by the method. Firstly, the A value is used for determining that the concentration of the substance to be detected is in a rising interval of 5ng/ml to 10,000ng/ml or a falling interval of 10,000ng/ml to 1,000,000ng/ml, and then the RLU1 of the substance to be detected is substituted into the corresponding standard curve to calculate the exact concentration.
As can be seen from Table 1, at an AFP concentration of 10,000ng/ml, there is a signal peak, corresponding to 18% A. If the A of the object to be detected is less than 18%, the sample to be detected is not an HD-HOOK sample, and the RLU1 of the sample to be detected is substituted into a standard curve with the concentration of less than 10,000ng/ml to calculate the concentration; if A is more than or equal to 18 percent, the sample to be detected is an HD-HOOK sample, and the RLU1 of the sample is substituted into a standard curve with the concentration of more than 10,000ng/ml to calculate the concentration, so that the upper limit of detection is widened from 10,000ng/ml to 1,000,000 ng/ml.
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 (12)
1. A method for immunoassay of fetal alpha globulin, comprising the steps of: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing fetal alpha globulin, 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) respectively making a standard curve according to the first reading and the amplification A' of the two readings of a series of known standard substances containing the fetal alpha globulin; (3) comparing the first reading of the sample to be detected containing the fetal alpha globulin and the amplification A of the two readings with a standard curve, determining whether the concentration of the fetal alpha globulin in the sample to be detected is in an ascending interval or a descending interval of the standard curve according to the value A, and substituting the first reading of the sample to be detected into the corresponding standard curve to determine the concentration of the sample;
the a = (RLU2/RLU1-1) x100%, where RLU1 and RLU2 are the first and second readings of chemiluminescence, respectively;
the method is a method for non-diagnostic purposes.
2. The immunoassay method according to claim 1, comprising the steps of:
(a1) mixing a sample to be detected containing fetal alpha globulin with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution; the first antibody and the second antibody refer to antibodies that can specifically bind to the fetal alpha globulin;
(a2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (a 1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(a3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (a 2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(a4) calculating the amplification A of the signal value obtained by the second reading of the sample relative to the signal value obtained by the first reading, wherein A = (RLU2/RLU1-1) × 100%;
(a5) making a standard curve according to the amplification A' of two readings of a series of known standard substances containing the fetal alpha globulin;
(a6) and determining whether the concentration of the fetal alpha globulin in the sample to be detected is in a rising interval or a falling interval of the standard curve according to the value A, and substituting the RLU1 of the sample to be detected into the corresponding standard curve to calculate the concentration.
3. The immunoassay method according to claim 2, wherein the light-emitting particles are polymer particles filled with a light-emitting compound and a lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and generate singlet oxygen ions under the excitation of red laser.
4. The immunoassay method according to claim 2, wherein in the steps (a 2) and (a 3), the amount of light emitted from the reaction solution is detected by irradiating with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.
5. A system for identifying a fetal alpha globulin immunoassay, the system comprising:
an immunoreaction device for performing a light-activated chemiluminescent reaction,
a light activated chemiluminescent reaction excitation and counting device for exciting and recording a first and a second reading of chemiluminescence and multiplying the difference between the second and the first reading by A,
the processor is used for respectively making a standard curve according to the first reading and the amplification A' of the two readings of a series of known standard substances containing the fetal alpha globulin, comparing the first reading and the amplification A of the two readings of a sample to be detected containing the fetal alpha globulin with the standard curve, determining whether the concentration of the fetal alpha globulin in the sample to be detected is in an ascending interval or a descending interval of the standard curve according to the value A, and substituting the first reading of the sample to be detected into the corresponding standard curve to determine the concentration of the sample;
the a = (RLU2/RLU1-1) x100%, where RLU1 and RLU2 are the first and second readings of chemiluminescence, respectively.
6. The system of claim 5, wherein the method of using the system comprises the steps of:
(1) mixing a sample to be detected containing fetal alpha globulin with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution; the first antibody and the second antibody refer to antibodies that can specifically bind to the fetal alpha globulin;
(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 = (RLU2/RLU1-1) × 100%;
(5) making a standard curve according to the amplification A' of two readings of a series of known standard substances containing the fetal alpha globulin;
(6) and determining whether the concentration of the fetal alpha globulin in the sample to be detected is in a rising interval or a falling interval of the standard curve according to the value A, and substituting the RLU1 of the sample to be detected into the corresponding standard curve to calculate the concentration.
7. The system of claim 6, 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 generate singlet oxygen ions under the excitation of red laser.
8. The system according to claim 6, 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.
9. A method of using a kit for detecting fetal alpha globulin, the kit comprising a first antibody-coated luminescent particle, a second antibody labeled with a label, and a photosensitive particle labeled with a label-specific binding substance, the first antibody and the second antibody being antibodies that specifically bind to the fetal alpha globulin; the method for using the kit is characterized by comprising the following steps: (1) carrying out light-activated chemiluminescence reaction on a sample to be detected containing fetal alpha globulin, 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) respectively making a standard curve according to the first reading of a series of known standard substances containing the fetal alpha globulin and the amplification A' of the two readings; (3) comparing the first reading of the sample to be detected containing the fetal alpha globulin and the amplification A of the two readings with a standard curve, determining whether the concentration of the fetal alpha globulin in the sample to be detected is in an ascending interval or a descending interval of the standard curve according to the value A, and substituting the first reading of the sample to be detected into the corresponding standard curve to determine the concentration of the sample;
the a = (RLU2/RLU1-1) x100%, where RLU1 and RLU2 are the first and second readings of chemiluminescence, respectively.
10. The method for using the kit according to claim 9, wherein the method for using the kit comprises the following steps:
(b1) mixing a sample to be detected containing fetal alpha globulin with luminescent particles coated by a first antibody and a second antibody marked by a marker, and incubating to obtain a mixed solution; the first antibody and the second antibody refer to antibodies that can specifically bind to the fetal alpha globulin;
(b2) the first reading: adding photosensitive particles labeled with the label-specific binding substances into the mixed solution obtained in the step (b 1), irradiating excitation light after incubation, detecting the quantity of emitted light, reading by a photon counter, and counting as RLU 1;
(b3) and (4) reading for the second time: further incubating the reaction solution after the first reading in step (b 2), irradiating excitation light and detecting the amount of emitted light, reading by a photon counter, and counting as RLU 2;
(b4) 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 = (RLU2/RLU1-1) × 100%;
(b5) making a standard curve according to the amplification A' of two readings of a series of known standard substances containing the fetal alpha globulin;
(b6) and determining whether the concentration of the fetal alpha globulin in the sample to be detected is in a rising interval or a falling interval of the standard curve according to the value A, and substituting the RLU1 of the sample to be detected into the corresponding standard curve to calculate the concentration.
11. The method of using the kit according to claim 10, wherein the light-emitting particles are polymer particles filled with a light-emitting compound and a lanthanide compound; the photosensitive particles are polymer particles filled with photosensitive compounds, and generate singlet oxygen ions under the excitation of red laser.
12. The method of using the kit according to claim 10, wherein in the steps (b 2) and (b 3), the amount of the emitted light of the reaction solution is detected by irradiating the reaction solution with 600 to 700nm red excitation light; the detection wavelength of the emitted light is 520-620 nm.
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| US20210208080A1 (en) * | 2018-05-21 | 2021-07-08 | Beyond Diagnostics (Shanghai) Co., Ltd | Chemiluminescence analytical method and system and kit using same |
| CN112114131A (en) * | 2019-06-21 | 2020-12-22 | 博阳生物科技（上海）有限公司 | Homogeneous phase chemiluminescence detection method and application thereof |
| CN113125699B (en) * | 2019-12-31 | 2024-03-26 | 科美博阳诊断技术(上海)有限公司 | beta-hCG homogeneous phase chemiluminescence detection kit and application thereof |
| CN116429752B (en) * | 2023-02-13 | 2024-07-19 | 科美博阳诊断技术(上海)有限公司 | AFP detection kit and application method thereof |
| CN119147750A (en) * | 2024-11-15 | 2024-12-17 | 科美诊断技术股份有限公司 | Photo-excitation chemiluminescence detection reagent for APOE4, kit and application |
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Also Published As
| Publication number | Publication date |
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| CN108132344B (en) | 2021-06-29 |
| CN109470874A (en) | 2019-03-15 |
| CN109470860B (en) | 2021-12-10 |
| CN109470861B (en) | 2021-12-07 |
| CN109470860A (en) | 2019-03-15 |
| CN108132344A (en) | 2018-06-08 |
| CN109470861A (en) | 2019-03-15 |
| CN109470862A (en) | 2019-03-15 |
| CN109470874B (en) | 2022-01-04 |
| CN109470866A (en) | 2019-03-15 |
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