CN108152505A - Method of immunity, for identifying the system of immunoassays and kit - Google Patents

Method of immunity, for identifying the system of immunoassays and kit Download PDF

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Publication number
CN108152505A
CN108152505A CN201611026623.1A CN201611026623A CN108152505A CN 108152505 A CN108152505 A CN 108152505A CN 201611026623 A CN201611026623 A CN 201611026623A CN 108152505 A CN108152505 A CN 108152505A
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Prior art keywords
antibody
antigen
reading
sample
amplification
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CN201611026623.1A
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CN108152505B (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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Boyang Biotechnology (Shanghai) Co Ltd
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Priority to CN201811400033.XA priority Critical patent/CN109470856B/en
Priority to CN201811401472.2A priority patent/CN109470867B/en
Priority to CN201811401320.2A priority patent/CN109470873B/en
Priority to CN201811400051.8A priority patent/CN109470863B/en
Priority to CN201611026623.1A priority patent/CN108152505B/en
Priority to CN201811401535.4A priority patent/CN109470869B/en
Priority to CN201811401317.0A priority patent/CN109470857B/en
Priority to CN201811401473.7A priority patent/CN109470868B/en
Priority to CN201811401545.8A priority patent/CN109470871A/en
Priority to CN201811400151.0A priority patent/CN109470864A/en
Priority to CN201811401321.7A priority patent/CN109470865B/en
Priority to CN201811401541.XA priority patent/CN109470870B/en
Application filed by Boyang Biotechnology (Shanghai) Co Ltd filed Critical Boyang Biotechnology (Shanghai) Co Ltd
Priority to EP17874359.7A priority patent/EP3546937A4/en
Priority to JP2019547751A priority patent/JP6980800B2/en
Priority to KR1020197014563A priority patent/KR102220361B1/en
Priority to PCT/CN2017/112145 priority patent/WO2018095314A1/en
Priority to US16/462,968 priority patent/US20190353664A1/en
Publication of CN108152505A publication Critical patent/CN108152505A/en
Priority to ZA2018/05983A priority patent/ZA201805983B/en
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    • 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
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • 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
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    • 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

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Abstract

The present invention provides a kind of method of immunity, which is characterized in that described method includes following steps：(1) chemiluminescence immunoassay reaction is carried out to the sample to be tested for containing object to be measured antigen (or antibody), excite and record chemiluminescent first time and second of reading, and the difference amplification between second and first time reading is denoted as A, (2) standard curve is done according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody), wherein the concentration of standard substance is less than the concentration for generating HOOK effects；(3) it if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the maximum value of the standard curve, is measured again after being diluted to sample.The invention further relates to a kind of for identifying the system of immunoassays and a kind of kit.The invention further relates to another method of immunity, another system for being used to identify immunoassays and another kits.

Description

Method of immunity, for identifying the system of immunoassays and kit
Technical field
The present invention relates to light-induced chemiluminescent technical fields, and in particular to a kind of method of immunity, one kind are used to identify The system of immunoassays and a kind of kit；And another method of immunity, another kind are for identify immunoassays System and another kit.
Background technology
Immunology detection is that the principle reacted based on antigen and antibody specific is carried out, due to its can utilize isotope, Measured object is shown for enzyme, chemiluminescent substance etc. or the amplification of signal, therefore it is micro- to be commonly used for detection protein, hormone etc. Measure bioactive substance.
Chemiluminescence immune assay is then to develop relatively rapid on-radiation immunoassay technology in recent years, and principle is profit The amplification of signal is carried out with chemiluminescent substance, and by its luminous intensity, immune cohesive process is directly measured, the method One of important directions of immunology detection are become.
Light-induced chemiluminescent method is one of common method of chemiluminescence analytical technique, intermolecular available for research biology Interaction is clinically mainly used for the detection of disease.The Technology Integration high molecular particle technology, organic synthesis, protein The research of the related fields such as chemistry and clinical detection.It is combined in a certain range by photosensitive particulate and luminous particle, is generated The transmission of ion oxygen energy, sends out optical signal, so as to be detected to sample to be tested.Wherein, filling thoughts inside photosensitive particulate Optical compounds, and luminophor and lanthanide series are filled with inside luminous particle.In swashing for red laser (600~700nm) It gives, photosensitive particulate releases the singlet oxygen ion (4 μ S) of upper state, and propagation distance is about 200nm.When photosensitive particulate and When the distance of luminous particle is close enough, the singlet oxygen ion of photosensitive particulate release can reach luminous particle, and pass through a system The chemical reaction of row, launches the light of 520~620nm high levels, and is detected by instrument.In this reaction system, particle Concentration is very low, and collision probability is smaller, and background signal is faint.Only photosensitive particulate and luminous particle by immune response combine with Afterwards, apparent light can just be launched, therefore the sensitivity of system is very high.In medical diagnosis on disease, common detection pattern includes three To four components：The luminous particle of envelope antigen or antibody, the antigen of biotin or digoxigenin labeled or antibody, Avidin or anti- The coated photosensitive particulate of digoxin neutralizes antigen or antibody etc..More than each component is resisted by two step more than incubation reactions with to be measured Former or antibody combines, and sample to be tested is qualitatively or quantitatively detected by the power of chemiluminescence amount.With it is traditional enzyme-linked Immunoassay method is compared, it has the characteristics that homogeneous, high sensitivity and easy to operate is easy to automation.Therefore, before applying Scape is very wide.
For in the detection pattern of double antibodies sandwich, when material concentration height to be detected is to a certain concentration, meeting is because be unable to shape Into dual anti-sandwich complex thus the phenomenon that signal value is relatively low, referred to as high dose-hook effect (HD-HOOK effects).Namely It says, high dose-hook effect refers in two-site sandwich immunization experiment, the high dose section of dose-effect curve, linearly Trend be not in platform-like it is unlimited after prolong, but be turned under it is curved, like a hook, the phenomenon that causing to generate false negative.
HD-HOOK effects often occur in immune detection, and incidence accounts for positive sample 30% or so.Due to HD- It is since its concentration is beyond the linear of detection kit that the presence of HOOK effects, which causes to be detected sample and cannot correctly be divided into, Range or concentration itself are exactly the value, so that misdiagnosis of the experiment, especially causes false negative rate to rise.
Specifically, on the one hand, in the sample for detecting high concentration, high dose-hook effect may cause to detect signal Relatively low, therefore sample is also read as relatively low concentration.The solution of the past is to increase the component of reagent, and sample to be tested is carried out Dilution carries out two-step method detection etc..
On the other hand, because of high dose-hook effect, when concentration of specimens is when being increased to certain value, signal can not be held Height of continuing rising, limits detection range.Previously mainly detection range is widened by optimizing antibody or improving antibody.
Conventional detection flow has following 5 steps：Determinand is added in reacting hole and reagent, the first step incubate, addition is led to With liquid, second step incubates and reading.
The detection method of the present invention is to be based on conventional detection flow, more in reaction process under the premise of reaction is not interrupted Secondary reading signal value, by the variation of observation signal come the actual concentration of judgement sample.
Invention content
The defects of in the presence of the prior art, the purpose of the present invention is to provide a kind of method of immunity, the party Method widens detection range by reading twice, in detection process, by by the amplification A of reading twice and a known system The maximum value of the standard curve of the amplification A of the reading twice of row standard substance compares, to judge whether sample to be tested needs to dilute It is measured again afterwards.
To achieve these goals and other related purposes, the present invention adopt the following technical scheme that：
The first aspect of the present invention provides a kind of method of immunity, which is characterized in that described method includes following steps： (1) chemiluminescence immunoassay reaction, excitation and record chemiluminescence are carried out to the sample to be tested for containing object to be measured antigen (or antibody) First time and second of reading, and the difference amplification between second and first time reading will be denoted as A, (2) are according to containing to be measured The amplification A of the reading twice of the known series of standards substance of target antigen (or antibody) does standard curve, wherein reference substance The concentration of matter is less than the concentration for generating HOOK effects；(3) if the sample to be tested of antigen containing object to be measured (or antibody) twice The amplification A of reading is more than the maximum value of the standard curve, then its concentration is more than upper limit of detection, and survey need to be diluted to sample It is fixed.
According to a preferred embodiment of the present invention, described method includes following steps：
(1) sample to be tested and the first antibody (or antigen) that will contain object to be measured antigen (or antibody) are coated luminous micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) first time reading：The photosensitive micro- of marker specific bond substance markers is added in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then into Row exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) Standard curve is done, wherein the concentration of standard substance is less than the concentration for generating HOOK effects；
(6) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the standard The maximum value of curve, then its concentration is more than upper limit of detection, and measure need to be diluted to sample
According to a preferred embodiment of the present invention, the known standard substance is positive control.
According to a preferred embodiment of the present invention, luminous particle refers to filled with luminophor and lanthanide series Close the high molecular particle of object；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, under red laser excitation, Singlet oxygen ion can be generated.
According to a preferred embodiment of the present invention, in step (2) and (3), with the red exciting light of 600~700nm Irradiation detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
According to a preferred embodiment of the present invention, the antigen refers to the substance with immunogenicity；The antibody Refer to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody, which refer to, specific to be tied Together in the antibody of the target antigen；First antigen and the second antigen, which refer to, can specifically bind to the anti-of the target antibody It is former.
The second aspect of the present invention is to provide a kind of system for identifying immunoassays, the system comprises：
Immune response device is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device, are used to excite and record chemiluminescent first time and second Secondary reading, and the difference amplification between second and first time reading is denoted as A,
Processor is used for the reading twice of the known series of standards substance according to antigen containing object to be measured (or antibody) Several amplification A does standard curve, and wherein the concentration of standard substance is less than the concentration for generating HOOK effects；If containing object to be measured The amplification A of the reading twice of the sample to be tested of antigen (or antibody) is more than the maximum value of the standard curve, then its concentration is more than Upper limit of detection need to be diluted sample measure
According to a preferred embodiment of the present invention, the application method of the system includes the following steps：
(1) sample to be tested and the first antibody (or antigen) that will contain object to be measured antigen (or antibody) are coated luminous micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) first time reading：The photosensitive micro- of marker specific bond substance markers is added in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then into Row exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) Standard curve is done, wherein the concentration of standard substance is less than the concentration for generating HOOK effects；
(6) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the standard The maximum value of curve, then its concentration is more than upper limit of detection, and measure need to be diluted to sample
The third aspect of the present invention is to provide a kind of kit, including the coated luminous particle of first antibody (or antigen), Mark the secondary antibody (or antigen) of substance markers, the photosensitive particulate of marker specific bond substance markers, which is characterized in that the examination The application method of agent box includes the following steps：(1) chemiluminescence is carried out to the sample to be tested for containing object to be measured antigen (or antibody) Immune response excites and records chemiluminescent first time and second of reading, and will be between second and first time reading Difference amplification is denoted as A, and (2) are according to the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) Amplification A does standard curve, and wherein the concentration of standard substance is less than the concentration for generating HOOK effects；(3) if resisted containing object to be measured The amplification A of the reading twice of the sample to be tested of former (or antibody) is more than the maximum value of the standard curve, then its concentration is more than inspection The upper limit is surveyed, measure need to be diluted to sample
According to a preferred embodiment of the present invention, the application method of the kit includes the following steps：
(1) sample to be tested and the first antibody (or antigen) that will contain object to be measured antigen (or antibody) are coated luminous micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) first time reading：The photosensitive micro- of marker specific bond substance markers is added in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then into Row exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) Standard curve is done, wherein the concentration of standard substance is less than the concentration for generating HOOK effects；
(6) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the standard The maximum value of curve, then its concentration is more than upper limit of detection, and measure need to be diluted to sample
The present invention also aims to provide a kind of method of immunity, detection range is widened by reading twice, In detection process, the amplification A of reading twice to be made comparisons with critical value, to judge whether sample to be tested is needed after diluting again It is measured.
To achieve these goals and other related purposes, the present invention adopt the following technical scheme that：
The fourth aspect of the present invention provides a kind of method of immunity, which is characterized in that described method includes following steps： (1) chemiluminescence immunoassay reaction, excitation and record chemiluminescence are carried out to the sample to be tested for containing object to be measured antigen (or antibody) First time and second of reading, and the difference amplification between second and first time reading will be denoted as A, (2) are according to containing to be measured The amplification of the reading twice A of one known standard substance of target antigen (or antibody) does critical value, and (3) will contain object to be measured antigen The amplification A of the reading twice of the sample to be tested of (or antibody) makes comparisons with critical value, if antigen containing object to be measured (or antibody) The amplification A of reading twice of sample to be tested be more than the critical value, then judge that the concentration of sample to be tested known is marked higher than described Quasi- material concentration；If the first time reading of the sample to be tested needs to carry out sample less than the known standard substance simultaneously It is measured again after dilution.
According to a preferred embodiment of the present invention, described method includes following steps：
(1) sample to be tested and the first antibody (or antigen) that will contain object to be measured antigen (or antibody) are coated luminous micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) first time reading：The photosensitive micro- of marker specific bond substance markers is added in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then into Row exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) it is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody) critical Value；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, If the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, judgement is treated The concentration of test sample sheet is higher than the known standard substance concentration；If signal value obtained by the first time reading of the sample to be tested simultaneously Less than the known standard substance, then need to be measured again after being diluted sample.
According to a preferred embodiment of the present invention, the known standard substance is positive control.
According to a preferred embodiment of the present invention, luminous particle refers to filled with luminophor and lanthanide series Close the high molecular particle of object；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, under red laser excitation, Singlet oxygen ion can be generated.
According to a preferred embodiment of the present invention, in step (2) and (3), with the red exciting light of 600~700nm Irradiation detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
According to a preferred embodiment of the present invention, the antigen refers to the substance with immunogenicity；The antibody Refer to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody, which refer to, specific to be tied Together in the antibody of the target antigen；First antigen and the second antigen, which refer to, can specifically bind to the anti-of the target antibody It is former.
The fifth aspect of the present invention provides a kind of system for identifying immunoassays, the system comprises：
Immune response device is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device, are used to excite and record chemiluminescent first time and second Secondary reading, and the difference amplification between second and first time reading is denoted as A,
Processor is used for the amplification A by the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) with facing Dividing value is made comparisons, if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than described critical Value then judges concentration of the concentration higher than the known standard substance of sample to be tested, if the first time of the sample to be tested simultaneously Reading then needs to be measured again after being diluted sample less than the known standard substance.
In a specific embodiment, the system that the present invention is used to identify immunoassays includes immune response device, Such as hold the container of solution；Chemiluminescence immunoassay reaction excitation and counting device, such as photon counting module and light-emitting diodes Pipe；And processor, such as computer, the reading is handled and mapped.This system for identifying immunoassays The application can be incorporated by reference for example with reference to the utility model patent CN201532646U of the applicant.
According to a preferred embodiment, the application method of the system includes the following steps：
(1) sample to be tested and the first antibody (or antigen) that will contain object to be measured antigen (or antibody) are coated luminous micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) first time reading：The photosensitive micro- of marker specific bond substance markers is added in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then into Row exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) it is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody) critical Value；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, If the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, judgement is treated The concentration of test sample sheet is higher than the concentration of the known standard substance；If signal obtained by the first time reading of the sample to be tested simultaneously Value then needs to be measured again after being diluted sample less than the known standard substance.
The sixth aspect of the present invention provides a kind of kit, including the coated luminous particle of first antibody (or antigen), mark Remember the secondary antibody (or antigen) of substance markers, the photosensitive particulate of marker specific bond substance markers, which is characterized in that the reagent The application method of box includes the following steps：(1) chemiluminescence is carried out to the sample to be tested for containing object to be measured antigen (or antibody) to exempt from Epidemic disease is reacted, and excites and record chemiluminescent first time and second of reading, and by the difference between second and first time reading Value amplification is denoted as A, and (2) are done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody) Critical value, (3) make comparisons the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) with critical value, If the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, judgement is treated The concentration of test sample sheet is higher than the concentration of the known standard substance；If the first time reading of the sample to be tested is less than described simultaneously Known standard substance then needs to be measured again after being diluted sample.
According to a preferred embodiment of the present invention, the application method of the kit includes the following steps：
(1) sample to be tested and the first antibody (or antigen) that will contain object to be measured antigen (or antibody) are coated luminous micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) first time reading：The photosensitive micro- of marker specific bond substance markers is added in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then into Row exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) it is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody) critical Value；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, If the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, judgement is treated The concentration of test sample sheet is higher than the concentration of the known standard substance；If signal obtained by the first time reading of the sample to be tested simultaneously Value then needs to be measured again after being diluted sample less than the known standard substance.
Here, it should be strongly noted that the above method is the method for non-disease diagnostic purpose, the method is used for double In antibody sandwich immunization or double antigens sandwich immunization detection process, detection range is widened by reading twice, with In detection process, the amplification A of reading twice is made comparisons with critical value, to judge sample to be tested carries out again after whether needing dilution It measures.
Preferably, the antigen refers to the substance with immunogenicity.Such as protein, polypeptide.Representative antigen packet Include (but not limited to)：Cell factor, tumor markers, Matrix metalloprotease-9, cardiovascular diabetes related protein etc..
The antibody refers to the immunoglobulin that can identify specific exotic that body generates.
In the embodiment of the present invention, the antigen or antibody are selected from HBsAb anti-HBs (HBsAb), people Chorionic gonadotrophin swashs and β subunits (HCG+ β), hepatitis B surface antigen (HBsAg), cancer antigen 125 (CA125), C peptides (CP), Ferritin (Ferr) and Anti-HCV.
It can be not particularly limited with the sample that the method for the present invention detects, can be any (or anti-containing object to be measured antigen Body) sample, representative example may include serum sample, urine specimen, saliva sample etc..Currently preferred sample is blood Final proof sheet.
Preferably, the first antibody and secondary antibody refer to the antibody that can specifically bind to the antigen.
For same antigen, corresponding first antibody and secondary antibody can be it is identical can also be it is different, It and can be in combination in the antigen.
First antigen and the second antigen refer to the antigen that can specifically bind to the target antibody.
For same antibody, corresponding first antigen and the second antigen can be it is identical can also be it is different, It and can be in combination in the antibody.
Preferably, it can be specifically bound between the marker and marker specific junction mixture.
It is furthermore preferred that the marker is biotin, the marker specific junction mixture is Streptavidin.
Preferably, the luminous particle refers to the high molecular particle filled with luminophor and lanthanide compound. Luminophor can be derivative of Dioxene (dioxine) or thioxene (thioxene) etc., and group of the lanthanides is first Plain compound can be Eu (TTA)3/ TOPO or Eu (TTA)3/ Phen etc., the particle can be by buying in the market.The table of luminous particle Face functional group can be the group of any energy connexin matter, and such as carboxyl, aldehyde radical, amido, epoxy ethyl or halogenated alkyl etc. are each The functional group of protein can be connected known to kind.
Preferably, the photosensitive particulate is filled with the high molecular particle of Photoactive compounds, can under red laser excitation To generate singlet oxygen ion.In the case that when it, distance is near enough with luminous particle, single line oxonium ion is transmitted to luminous particle, It is reacted with the luminophor in luminous particle, generates ultraviolet light, ultraviolet light further excites lanthanide compound, generates The photon of certain wavelength.Photoactive compounds can be phthalocyanine dye etc., which also can be by buying in the market.
Preferably, it in step (2) and (3), is irradiated with the red exciting light of 600~700nm, detects the transmitting of reaction solution Light quantity.The Detection wavelength for emitting light is 520~620nm.
Further, red laser (600~700nm) irradiation photosensitive particulate, the singlet oxygen ion of photosensitive particulate release, A part of singlet oxygen ion is received by luminous particle, so as to launch the light of 520~620nm high levels.
In detection range, the concentration of object to be measured antigen shows as the quantity of double-antibody sandwich compound, and and photon Number is directly proportional；But when object to be measured antigen concentration is excessively high, part determined antigen is combined respectively with single antibody, leads to dual anti-folder Heart compound is reduced, and optical signal is relatively low, it is impossible to reflect the actual concentration of object to be measured antigen.
Similarly, in detection range, the concentration of object to be measured antibody shows as the quantity of double antigens sandwich compound, and with Number of photons is directly proportional；But when object to be measured antibody concentration is excessively high, part test antibodies with single antigen binding, cause double respectively Antigen sandwich compound is reduced, and optical signal is relatively low, it is impossible to reflect the actual concentration of object to be measured antibody.
The method of the present invention by reading twice, compares the relationship between signal value amplification obtained by reading twice, so as to To play the role of widening detection range and distinguish HD-HOOK effect samples.The difference of reading is determined by following three aspects twice It is fixed：
In a first aspect, during first time reading, after photosensitive particulate is irradiated by red laser (600~700nm), single line is released State oxonium ion.After a part of singlet oxygen ion transport to luminous particle, by a series of chemical reaction, launch 520~ The light of 620nm high levels；And a part of singlet oxygen ion then with not by antibody (or antigen) combine object to be measured antigen (or Antibody) reaction so that the concentration of object to be measured antigen (or antibody) reduces.For the sample of low concentration, object to be measured antigen (or Antibody) after concentration declines, double antibodies sandwich compound is reduced, and second of read signal value can reduce；And for high concentration sample, it treats After surveying the reduction of target antigen (or antibody) concentration, double antibodies sandwich compound increases, and second of read signal value increases instead.
Second aspect, for low concentration sample, photosensitive particulate is in first time reading process by red laser (600 ~700nm) irradiation, after discharging singlet oxygen ion, energy is lost, and second of read signal can reduce.
The third aspect, for HD-HOOK effects, during first time reading, balance has not yet been reached in antigen-antibody reaction, The interval time of reading twice, reaction can still be carried out towards positive direction, and second of read signal can increase.
In conclusion the present invention carries out first time reading, the irradiation of photosensitive particulate stimulated luminescence when reaction not up to balances Discharge singlet oxygen, a part is transmitted to luminous particle, a part can with unbonded target antigen or antibody response to be detected, Consume part target antigen to be detected or antibody so that reaction balance is reverse mobile, and another aspect photosensitive particulate was exciting one It after secondary, is lost, when second of reading, the signal value of object to be measured antigen or the low sample of antibody concentration can reduce；And The combination of the double antibodies sandwich compound and photosensitive particulate of the high sample of concentration reaches far away balance in first time reading, second of reading Reaction can be moved towards positive reaction direction during number, so signal can increase, with the raising of object to be measured antigen (or antibody) concentration, The signal value of second of phot-luminescence and the amplitude that increases of first time signal value also increase.The amplification of signal and concentration of specimens positive It closes, the amplification A of signal twice is made comparisons with critical value, to judge sample to be tested is measured again after whether needing dilution.
Compared with prior art, beneficial effects of the present invention are：
(1) it the present invention is based on the disposable of light-induced chemiluminescent platform (luminescent oxygen channel) and the homogeneity of reaction, can realize The progress that multiple signal is measured without interrupting immune response is carried out to a reaction, detects the light letter in the differential responses time Number, the not examined scope limitation of the method effectively widens 100 times of detection range or more.
(2) method of the invention can differentiate whether the sample to be tested in double-antibody method detection needs to carry out again after diluting It measures, the method can significantly improve the accuracy of double antibody sandwich method immunoassays, and reduce double antibody sandwich method and be immunized The false negative rate of measure.
(3) method of the invention is easy to operate, can differentiate that non-disease diagnostic purpose double-antibody sandwich is exempted from simple and effectively The relatively low sample of reported concentrations caused by HD-HOOK effects during epidemic disease measures.
Description of the drawings
Fig. 1：HCG+ β are using signal value obtained by conventional method and concentration of specimens graph of relation；
Fig. 2：HCG+B using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation；
Fig. 3：Ferr is using signal value obtained by conventional method and concentration of specimens graph of relation；
Fig. 4：Ferr using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation；
Fig. 5：C peptides are using signal value obtained by conventional method and concentration of specimens graph of relation；
Fig. 6：C peptides using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 7：HBsAb is using signal value obtained by conventional method and concentration of specimens graph of relation；
Fig. 8：HBsAb using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Specific embodiment
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, according to record of the those skilled in the art to the grasp of the prior art and the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., 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；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODSINMOLECULAR BIOLOGY, Vo1.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present inventor has found after extensive and in-depth study, by setting up reading twice, and will reading twice Amplification A make comparisons with critical value, can determine whether sample to be tested whether need dilution after be measured again.
As described herein, term " first antibody " and " secondary antibody ", which refer to, can specifically bind to a certain antigen (as swollen Tumor markers) antibody.For same antigen (such as tumor markers), corresponding first antibody and secondary antibody can be Different can also be identical, and can be in combination in the antigen.Term " the first antigen " and " the second antigen " refer to It can specifically bind to the antigen of a certain antibody (such as hepatitis B surface antibody).For same antibody (such as hepatitis B surface antibody) It can also be identical, and can be in combination in described that speech, corresponding first antigen and the second antigen, which can be different, Antibody.
As described herein, term " antigen " refers to the substance with immunogenicity, such as protein, polypeptide.It is representative Antigen include but is not limited to：Cell factor, tumor markers, Matrix metalloprotease-9, cardiovascular diabetes related protein etc..
As described herein, term " tumor markers " refers in the generation and breeding of tumour, by tumour cell Either tumour cell is reacted by body caused by itself and is generated, reaction tumour exists and a kind of substance of growth. The representative tumor markers in this field include but is not limited to：Alpha-fetoprotein (AFP), cancer antigen 125 (CA125) etc..
The basic principle of double-antibody method：
The basic principle of double antibody sandwich method is well-known to those skilled in the art.Conventional way is by first antibody Solid phase carrier is fixed on, then by first antibody and antigen-reactive, then is reacted with the secondary antibody of label, finally carries out chemical hair Light or enzyme-linked chromogenic reaction detection signal.
The basic principle of light-induced chemiluminescent method：
The basic principle of light-induced chemiluminescent method is well-known to those skilled in the art.Conventional way is by photosensitive Particle and luminous particle combine in a certain range, generate the transmission of ion oxygen energy, send out optical signal, so as to sample to be tested It is detected.Wherein, filled with Photoactive compounds inside photosensitive particulate, and luminophor and lanthanum are filled with inside luminous particle Series elements.Under the excitation of red laser (600~700nm), photosensitive particulate releases singlet oxygen ion (4 μ of upper state S), propagation distance is about 200nm.When the distance of photosensitive particulate and luminous particle is close enough, the list of photosensitive particulate release Line state oxonium ion can reach luminous particle, and by a series of chemical reaction, launch the light of 520~620nm high levels, and It is detected by instrument.
In a preferred embodiment of the invention, the characteristics of first antibody is fixed in luminous particle is taken full advantage of, Simultaneously using biotin labeling secondary antibody, Streptavidin coating photosensitive particulate, by serum sample or antigen standard quality-control product Liquid is sequentially or simultaneously added in reaction vessel with the coated luminous particle of first antibody, biotin labeling secondary antibody, is added The photosensitive particulate of marked by streptavidin, so as to which following react occur：
(1) first antibody in luminous particle and corresponding antigen binding in serum sample or antigen standard quality-control product liquid, Form " antigen-first antibody-luminous particle " ternary complex；
(2) secondary antibody and corresponding antigen binding in serum sample or antigen standard quality-control product liquid, ultimately form " second Antibody-antigene-first antibody-luminous particle " double antibodies sandwich compound；
Biotin and Streptavidin specific binding so that double antibodies sandwich compound is combined with photosensitive particulate.
At this point, the distance between photosensitive particulate and luminous particle are less than 200nm, red laser (600~700nm) irradiation sense After light particles, the singlet oxygen of release can be received by luminous particle.By series of chemical, launch 520~620nm The light of high level, and sample to be tested is qualitatively or quantitatively detected by the power of chemiluminescence amount.
In another preferred embodiment of the invention, the spy that the first antigen is fixed in luminous particle is taken full advantage of Point, while the second antigen of biotin labeling is used, Streptavidin coating photosensitive particulate, by serum sample or antigen standard Quality Control The luminous particle of product liquid and the first antigen coat, the second antigen of biotin labeling are sequentially or simultaneously added in reaction vessel, then are added Enter the photosensitive particulate of marked by streptavidin, so as to which following react occur：
(1) the antibody product knot corresponding with serum sample or antigen standard quality-control product liquid of the first antigen in luminous particle It closes, forms " the-the first antigen of antibody-luminous particle " ternary complex；
(2) second antigens antibody corresponding with serum sample or antigen standard quality-control product liquid combines, and ultimately forms " second The-the first antigen of Ag-Ab-luminous particle " double antibodies sandwich compound；
Biotin and Streptavidin specific binding so that double antibodies sandwich compound is combined with photosensitive particulate.
At this point, the distance between photosensitive particulate and luminous particle are less than 200nm, red laser (600~700nm) irradiation sense After light particles, the singlet oxygen of release can be received by luminous particle.By series of chemical, launch 520~620nm The light of high level, and sample to be tested is qualitatively or quantitatively detected by the power of chemiluminescence amount.
Hereinafter, the relevant operation details further illustrated the present invention.
(1) first antibody (or antigen) coated luminous particle, is denoted as reagent 1, can purchase limited in rich positive biotechnology Company.
(2) secondary antibody (or antigen) can be with various markers known in the art and its specific junction mixture system into rower Note.Preferably by biotin-avidin system label secondary antibody (or antigen).Biotin labeling secondary antibody (or Antigen), reagent 2 is denoted as, can purchase in Bo Yang bio tech ltd.
(3) the coated photosensitive particulate of Streptavidin is denoted as general liquid, can purchase in Bo Yang bio tech ltd.
(4) standard items：
It is molten with the standard items for (being less than HD-HOOK effective concentrations) in the range of determined antigen (or antibody) configuration a certain concentration Liquid.By standard items, reagent 1,2 mixing of reagent, general liquid is added in after incubation reaction, after continuing incubation reaction for a period of time for the first time Reading (RLU1), then second of reading (RLU2) is carried out after incubating a period of time, calculate A=(RLU2/RLU1-1) x100%, root According to standard items RLU1 and the amplification A of reading does standard curve with calibration object concentration respectively twice；
(5) detection of sample：
It can be not particularly limited with the sample that the method for the present invention detects, can be any sample containing antigen (or antibody) Product, representative example may include serum sample, urine specimen, saliva sample etc..Preferred sample is blood serum sample.
(6) sample concentration calculates：
By sample to be tested, reading amplification A values are compared with the A of known standard items twice, if sample to be tested A is more than known standard The A of product, then this concentration of specimens is more than the concentration of known standard items；If this sample RLU1 is less than the RLU1 of this standard items simultaneously, Show the RLU1 of this sample it is low be due to caused by HD-HOOK effects, need dilution detect.
Embodiment 1 detects human chorionic gonadotrophin and verification present invention side of β subunits (HCG+ β) in human serum sample Method validity
Using the human chorionic gonadotrophin and β subunits (HCG+ of the production of Bo Yang biotechnologies (Shanghai) Co., Ltd. β) detection kit (light-induced chemiluminescent method) detects human chorionic gonadotrophin and β subunits (HCG+ in serum sample Content β).The kit includes calibration object 1- the calibration objects 6, (illuminating antibody, also that is, antibody is coated luminous micro- of reagent 1 Grain), reagent 2 (biotin labelled antibodies, that is, the antibody of biotin labeling).
To detected by Roche (be more than detection limit sample be diluted detect) 18 HCG+ β concentration patients serum's sample This, carries out conventional method and the method for the present invention detection respectively,
Common detection methods：By sample to be tested, the calibration object, (illuminating antibody, also that is, mouse monoclonal antibody is coated of reagent 1 Luminous particle) and reagent 2 (biotin labelled antibodies, that is, the mouse monoclonal antibody of biotin labeling) be separately added into reaction cup Afterwards, 37 DEG C of incubation 15min add in general liquid (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 10min, photon counting Device reading reads RLU, calculates to obtain concentration of specimens, the results are shown in table below.
Using method of reading twice of the invention：By sample to be tested, the calibration object, (illuminating antibody, also that is, murine monoclonal resists of reagent 1 The coated luminous particle of body) and reagent 2 (biotin labelled antibodies, that is, the mouse monoclonal antibody of biotin labeling), 37 DEG C of temperature 15min is educated, adds in general liquid (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min, reading RLU1,37 DEG C are continued temperature 7min, reading RLU2 are educated, and calculates amplification A=(RLU2/RLU1-1) X100% of second of signal value, as a result such as following table institute Show,
Table 1：
Note：The detection range of HCG+ β conventional detections is 0-10000mIU/ml, is beyond upper limit of detection sample display density ＞ 10000mIU/ml.
Roche testing results are as actual concentration, and by table 1 and Fig. 1 it is found that in conventional detection, concentration rises to 54531mIU/ml signal values are increased with concentration and are increased, and concentration continues to increase, and signal value is increased with HCG+ β concentration and reduced, i.e., Concentration is more than 54531mIU/ml then HD-HOOK, in conventional detection, detection range 0-10000mIU/ml, in detection It is ＞ 10000mIU/ml to limit sample display density.When HD-HOOK effects concentration of specimens persistently increases, signal continues to decline, will Occur ultrahigh concentration sample report into relatively low concentration levels, such as sample 18.So it cannot be differentiated in conventional detection to be measured The relatively low concentration that the testing result of sample is actual concentration or superelevation value sample is reported by HD-HOOK effects.
The relatively low sample of reported concentrations caused by the method for the present invention differentiates HOOK effects by reading twice.Each treat test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of index of judgement sample concentration.54531mIU/ml is increased to it is found that signal value continues with concentration by table 2 and Fig. 2, Zhi Houxin Number value starts to increase and decline with concentration, but amplification A is persistently to rise with concentration.Therefore the directly relatively A of sample to be tested Value and the A values of calibration object, you can judge the magnitude relationship of sample to be tested concentration and calibration object concentration.The amplification A of sample 10~18 The amplification (11.1%) of calibration object 6 is all higher than, and A values persistently increase, shows that the HCG+ β concentration of sample 10~18 is all higher than 10000mIU/ml, and concentration persistently increases, this is consistent with the concentration results of Roche, and 18 signal value of sample is less than calibration object 6, often Rule method detectable concentration is 8713.02mIU/ml, can differentiate that it, for HD-HOOK effect samples, needs to carry out by the method for the present invention Dilution detection.
Embodiment 2：Detect ferritin (Ferr) verification the method for the present invention validity in sample
Ferritin (Ferr) detection kit (chemiluminescence produced using Bo Yang biotechnologies (Shanghai) Co., Ltd. Method) detect ferritin in sample (purchased from Fitzgerald, Catalog No：Content 30-AF10).The kit includes Calibration object 1- calibration objects 6, reagent 1 (illuminating antibody, also that is, the coated luminous particle of antibody), reagent 2 (biotin labelled antibodies, That is, the antibody of biotin labeling).
Calibration object 1- calibration objects 6：Known concentration sample in conventional kit, concentration are much smaller than HOOK samples, make standard Curve calculates testing concentration.
The component that need to separately use：The general liquid of LiCA (photosensitive particulate of marked by streptavidin), the product are rich sun biology Scientific ＆ technical corporation produces the auxiliary reagent of light-induced chemiluminescent analysis system.It is tried with instrument and the detection of corresponding light-induced chemiluminescent method Agent box matches, for the detection of antigen, antibody.
The ferritin antigen of high concentration is subjected to gradient dilution, common detection methods and detection method are respectively adopted Measure the concentration value of the sample of the ferritin containing various concentration.
Common detection methods：By calibration object 1- calibration objects 6, sample to be tested the 1-15, (illuminating antibody, also that is, mouse is single of reagent 1 The coated luminous particle of clonal antibody) and reagent 2 (biotin labelled antibodies, that is, the mouse monoclonal antibody of biotin labeling) plus After entering reaction cup, 37 DEG C of incubation 15min add in the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubations 10min, photon counter reading read RLU, and the results are shown in table below.
Using method of reading twice of the invention：By calibration object 1- calibration objects 6, sample to be tested 1-15, and reagent 1 (illuminating antibody, also That is, the coated luminous particle of mouse monoclonal antibody) and reagent 2 (biotin labelled antibodies, that is, the murine monoclonal of biotin labeling Antibody), 37 DEG C of incubation 15min add in the general liquid of LiCA (photosensitive particulate of marked by streptavidin), and 37 DEG C of incubation 3min are read Number RLU1,37 DEG C are continued to incubate 7min, reading RLU2, and calculate the amplification A=(RLU2/RLU1-1) of second of signal value X100%, the results are shown in table below,
Table 2：
Note：The detection range of ferritin conventional detection is 0-2000ng/ml, is ＞ beyond upper limit of detection sample display density 2000ng/ml。
In conventional detection, by table 2 and Fig. 3 it is found that concentration rises to the increasing with concentration raising of 51000ng/ml signal values Height, concentration continue to increase, and signal value is increased with Ferr concentration and reduced, conventional detection ranging from 0-2000ng/ml, beyond detection Upper limit sample display density is ＞ 2000ng/ml.When HD-HOOK effects concentration of specimens persistently increases, when concentration is increased to 2550000ng/ml, signal drop to the signal of calibration object 6 hereinafter, just will appear ultrahigh concentration sample report into relatively low concentration Situation, such as sample 15.So the testing result that sample to be tested cannot be differentiated in conventional detection is actual concentration or superelevation The relatively low concentration that value sample is reported by HD-HOOK effects.
The relatively low sample of reported concentrations caused by the method for the present invention differentiates HOOK effects by reading twice.Each treat test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of index of judgement sample concentration.51000ng/ml is increased to it is found that signal value continues with concentration by table 2 and Fig. 4, later signal Value starts to increase and decline with concentration, but amplification A is persistently to rise with concentration.Therefore directly compare the A values of sample to be tested With the A values of calibration object, you can judge the magnitude relationship of sample to be tested concentration and calibration object concentration.The amplification A of sample 4~15 is big In the amplification (- 5.5%) of calibration object 6, show that the Ferr concentration of sample 4~15 is all higher than 2000ng/ml.This and actual concentrations phase Symbol, 15 signal value of sample are less than calibration object 6, and conventional method detectable concentration is 1860.97ng/ml, can be reflected by the method for the present invention It is not more than detection range sample for concentration, need to be diluted detection.
Embodiment 3：Detect C peptides (CP) verification the method for the present invention validity in sample
It is examined using C peptides (CP) detection kit (chemoluminescence method) of Bo Yang biotechnologies (Shanghai) Co., Ltd. production C peptides (are purchased from Fitzgerald, Catalog No in test sample sheet：Content 30-AC96).The kit includes calibration object 1- schools Quasi- product 6, reagent 1 (illuminating antibody, also that is, the coated luminous particle of antibody), reagent 2 (biotin labelled antibodies, that is, biotin The antibody of label).
Calibration object 1- calibration objects 6：Known concentration sample in conventional kit, concentration are much smaller than HOOK samples, make standard Curve calculates testing concentration.
The C peptides antigen of high concentration is subjected to gradient dilution, common detection methods are respectively adopted and detection method is surveyed The concentration value of the sample of the fixed peptides of C containing various concentration.
Common detection methods：By calibration object 1- calibration objects 6, sample to be tested the 1-15, (illuminating antibody, also that is, mouse is single of reagent 1 The coated luminous particle of clonal antibody) and reagent 2 (biotin labelled antibodies, that is, the mouse monoclonal antibody of biotin labeling) plus After entering reaction cup, 37 DEG C of incubation 15min add in the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubations 10min, photon counter reading read RLU, and the results are shown in table below.
Using method of reading twice of the invention：By calibration object 1- calibration objects 6, sample to be tested 1-15, and reagent 1 (illuminating antibody, also That is, the coated luminous particle of mouse monoclonal antibody) and reagent 2 (biotin labelled antibodies, that is, the murine monoclonal of biotin labeling Antibody), 37 DEG C of incubation 15min add in the general liquid of LiCA (photosensitive particulate of marked by streptavidin), and 37 DEG C of incubation 3min are read Number RLU1,37 DEG C are continued to incubate 7min, reading RLU2, and calculate the amplification A=(RLU2/RLU1-1) of second of signal value X100%, the results are shown in table below,
Table 3：
Note：The detection range of C peptide conventional detections is 0-30nng/ml, is ＞ beyond upper limit of detection sample display density 30ng/ml。
In conventional detection, by table 3 and Fig. 5 it is found that concentration rises to the increasing with concentration raising of 10000ng/ml signal values Height, concentration continue to increase, and signal value is increased with C peptide concentrations and reduced, conventional detection ranging from 0-30ng/ml, in detection It is ＞ 30ng/ml to limit sample display density.When concentration is increased to 33500000ng/ml, signal drop to the signal of calibration object 6 with Under, just it will appear ultrahigh concentration sample report into relatively low concentration levels, such as sample 16,17.So in conventional detection just not The testing result that sample to be tested can be differentiated is that actual concentration or superelevation value sample are reported relatively low by HD-HOOK effects Concentration.
The relatively low sample of reported concentrations caused by the method for the present invention differentiates HOOK effects by reading twice.Each treat test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of index of judgement sample concentration.10000ng/ml is increased to it is found that signal value continues with concentration by table 3 and Fig. 6, later signal Value starts to increase and decline with concentration, but amplification A is persistently to rise with concentration.Therefore directly compare the A values of sample to be tested With the A values of calibration object, you can judge the magnitude relationship of sample to be tested concentration and calibration object concentration.The amplification A of sample 5~17 is big In the amplification (- 4.9%) of calibration object 6, show that the C peptide concentrations of sample 5~17 are all higher than 30ng/ml, more than upper limit of detection.This with Actual concentrations are consistent, sample 16,17 signal values less than calibration object 6, conventional method detectable concentration be respectively 8.15ng/ml, 0.76ng/ml can differentiate that it is more than upper limit of detection sample by the method for the present invention, need to be diluted detection.
Embodiment 4：Detect hepatitis b virus s antigen (HBsAg) in human serum sample
Examination is detected using the hepatitis b virus s antigen (HBsAg) of Bo Yang biotechnologies (Shanghai) Co., Ltd. production HBsAg concentration in agent box (light-induced chemiluminescent method) detection sample, the kit include calibration object 1- calibration objects 6, reagent 1 (illuminating antibody, also that is, the coated luminous particle of antibody), reagent 2 (biotin labelled antibodies, that is, biotin labeling is anti- Body).
Calibration object 1- calibration objects 6：Known concentration sample in conventional kit, concentration are much smaller than HOOK samples, make standard Curve calculates testing concentration.
First with the method for the present invention testing calibration product 1- calibration objects 6, serum sample 1-15 to be measured：By determinand, reagent 1 After (the coated luminous particle of antibody) and reagent 2 (antibody of biotin labeling) add in reaction cup, 37 DEG C of incubation 15min are added in The general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min, reading RLU1,37 DEG C are continued to incubate 7min, Reading RLU2, and amplification A=(RLU2/RLU1-1) X100% of second of signal value is calculated, as a result such as following table.
Table 4：
The method of the present invention obtains the concentration of 15 serum samples as above：Shown in upper table, the amplification ratio with calibration object 6 is first passed through The sample more than upper limit of detection (336.56IU/mL) is relatively distinguished, i.e. A values are judged as HOOK effect samples more than 27%, push away It is detected after recommending dilution；And A is less than 27% for sample in detection range, directly can calculate concentration of specimens with calibration curve.
By detectable concentration variation after gradient dilution is carried out to sample to verify the reliability of conclusions, by sample 1- samples This 15 progress 2 is diluted diluting with 4 times, while is diluted sample with the undiluted former times sample of common detection methods detection, 2 With 4 times of diluted samples, judge whether the sample there are HOOK effects by the variation of concentration after observation dilution, sample after even diluting Concentration is increased instead as HOOK effect samples.Concentration can reduce non-HOOK effects sample after dilution.As a result it is as follows：
Table 5：
Detectable concentration increases after serum sample 1,2,3,5,6,9,12,13,14,15 dilutes, that is, proves HD-HOOK effects Sample, concentration are more than 336.56IU/mL, and concentration reduces after serum sample 4,7,8,10,11 dilutes, it was demonstrated that are not HD-HOOK effects Answer sample.It is identical with the result of the method for the present invention.
Embodiment 5：CA125 verifies the method for the present invention validity in detection human serum sample
(light swashs carbohydrate antigen 125 (CA125) detection kit produced using Bo Yang biotechnologies (Shanghai) Co., Ltd. Chemoluminescence method) detection sample in CA125 concentration, the kit include calibration object 1- calibration objects 6, reagent 1 (illuminating antibody, Also that is, the coated luminous particle of antibody), reagent 2 (biotin labelled antibodies, that is, the antibody of biotin labeling).
Calibration object 1- calibration objects 6：Known concentration sample in conventional kit, concentration are much smaller than HOOK samples, make standard Curve calculates testing concentration.
First with the method for the present invention testing calibration product 1- calibration objects 6, serum sample 1-18 to be measured：By determinand, reagent 1 After (the coated luminous particle of antibody) and reagent 2 (antibody of biotin labeling) add in reaction cup, 37 DEG C of incubation 15min are added in The general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min, reading RLU1,37 DEG C are continued to incubate 7min, Reading RLU2, and amplification A=(RLU2/RLU1-1) X100% of second of signal value is calculated, as a result such as following table.
Table 6：
The method of the present invention obtains the concentration of 18 serum samples as above：Shown in upper table, the amplification ratio with calibration object 6 is first passed through The sample more than upper limit of detection is relatively distinguished, i.e. A values are more than 7.4% sample being judged as more than upper limit of detection, recommend dilution After detect；And it is non-HOOK effects sample that A, which is less than 7.4%, directly can calculate concentration of specimens with calibration curve.
By detectable concentration variation after gradient dilution is carried out to sample to verify the reliability of conclusions, by sample 1- samples This 18 progress 2 is diluted diluting with 4 times, while is diluted sample with the undiluted former times sample of common detection methods detection, 2 With 4 times of diluted samples, judge whether the sample there are HOOK effects by the variation of concentration after observation dilution, sample after even diluting Concentration is increased instead as HOOK effect samples.Concentration can reduce non-HOOK effects sample after dilution.As a result it is as follows：
Table 7：
Detectable concentration increases after serum sample 16,17,18 dilutes, that is, proves HD-HOOK effect samples, serum sample 1- Concentration reduces after sample 15 dilutes, it was demonstrated that is not HD-HOOK effect samples.It is identical with the result of the method for the present invention.It is not dilute In the case of releasing sample, the former times sample of conventional method detection can then judge serum sample 16,17,18 by accident because of HD-HOOK effects For relatively low concentration samples.
Embodiment 6：Detect anti-HBs (HBsAb) verification the method for the present invention validity in sample
The anti-HBs detection kit produced using Bo Yang biotechnologies (Shanghai) Co., Ltd. HBsAbHBsAb (light-induced chemiluminescent method) (is purchased from BeiJing ZhongKe Jing Dasheng to detect anti-HBs in sample Object Technology Co., Ltd., Clone No：M2201 content).The kit includes calibration object 1- calibration objects 6, reagent 1 (the coated luminous particles of HBsAg) and reagent 2 (HBsAg of biotin labeling).
Calibration object 1- calibration objects 6：Known concentration sample in conventional kit, concentration are much smaller than HOOK samples, make standard Curve calculates testing concentration.
The HBsAb of high concentration is subjected to gradient dilution, common detection methods are respectively adopted and detection method measures The concentration value of the sample of the HBsAb containing various concentration.
Common detection methods：By calibration object 1- calibration objects 6, sample to be tested 1-14, (HBsAg is coated luminous micro- for reagent 1 Grain) and after reagent 2 (HBsAg of biotin labeling) adds in reaction cup, 37 DEG C of incubation 15min, adding in LiCA general liquid, (strepto- is close With the photosensitive particulate of element label), 37 DEG C of incubation 10min, photon counter reading reads RLU, and the results are shown in table below.
Using method of reading twice of the invention：By calibration object 1- calibration objects 6, sample to be tested 1-14, (HBsAg is coated for reagent 1 Luminous particle) and reagent 2 (HBsAg of biotin labeling), 37 DEG C of incubation 15min, addition LiCA general liquid (Streptavidin marks The photosensitive particulate of note), 37 DEG C of incubation 3min, reading RLU1,37 DEG C are continued to incubate 7min, reading RLU2, and calculate second and believe Number value amplification A=(RLU2/RLU1-1) X100%, the results are shown in table below,
Table 8：
Note：The detection range of HBsAb conventional detections is 0-1000mIU/ml, is ＞ beyond upper limit of detection sample display density 1000mIU/ml。
In conventional detection, by table 8 and Fig. 7 it is found that concentration rises to the increasing with concentration raising of 10000mIU/ml signal values Height, concentration continue to increase, and signal value is increased with HBsAb concentration and reduced, conventional detection ranging from 0-1000mIU/ml, beyond inspection It is ＞ 1000mIU/ml to survey upper limit sample display density.When concentration be increased to 335000mIU/ml and more than, signal drops to school The signal of quasi- product 6 is hereinafter, just will appear ultrahigh concentration sample report into relatively low concentration levels, such as sample 12,13,14.So The testing result that sample to be tested cannot be differentiated in conventional detection is actual concentration or superelevation value sample by HD-HOOK effects The relatively low concentration for influencing and reporting.
The relatively low sample of reported concentrations caused by the method for the present invention differentiates HOOK effects by reading twice.Each treat test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of index of judgement sample concentration.10000mIU/ml is increased to it is found that signal value continues with concentration by table 8 and Fig. 8, Zhi Houxin Number value starts to increase and decline with concentration, but amplification A is persistently to rise with concentration.Therefore the directly relatively A of sample to be tested Value and the A values of calibration object, you can judge the magnitude relationship of sample to be tested concentration and calibration object concentration.The amplification A of sample 8~14 is equal More than the amplification (35.9%) of calibration object 6, show that the HBsAb concentration of sample 8~14 is all higher than 1000mIU/ml, more than in detection Limit.This is consistent with actual concentrations, sample 12,13,14.Signal value is less than calibration object 6, and conventional method detectable concentration is respectively 802.57mIU/ml, 352.22mIU/ml, 147.9mIU/ml can differentiate that it is more than upper limit of detection sample by the method for the present invention This, need to be diluted detection.
Embodiment 7：The method of the present invention is applied in qualitative kit Anti-HCV
Using detecting reagent kit for antibody of hepatitis C virus (the chemistry hair of Bo Yang biotechnologies (Shanghai) Co., Ltd. production Light method) detect the content of Anti-HCV in sample.The kit includes reference material, negative control, positive control, reagent 1 (shine HCV antigens, also that is, the luminous particle of HCV antigen coats) and reagent 2 (biotin labeling HCV antigens, that is, biotin The HCV antigens of label).
Reference material, negative control, positive control：Reference material is used as referring to dense known to sample to be tested yin and yang attribute to judge Spend standard items；Negative control, positive control are used as judging the known concentration standard items of test validity.By high concentration Anti-HCV carries out gradient dilution, and common detection methods are respectively adopted and detection method measures Anti- containing various concentration The signal value of HCV samples.
Common detection methods：By a series of Anti-HCV samples of gradient dilution, reagent 1 (shine HCV antigens, also that is, The luminous particle of HCV antigen coats) and reagent 2 (biotin labeling HCV antigens, that is, the HCV antigens of biotin labeling) addition After reaction cup, 37 DEG C of incubation 15min add in the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubations 10min, photon counter reading read RLU, and the results are shown in Table 8.
Using method of reading twice of the invention：By a series of Anti-HCV samples of gradient dilution, (HCV that shines resists reagent 1 Original, also that is, the luminous particle of HCV antigen coats) and reagent 2 (biotin labeling HCV antigens, that is, the HCV of biotin labeling resists It is former), 37 DEG C of incubation 15min add in the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min, reading RLU1,37 DEG C are continued to incubate 7min, reading RLU2, and calculate the amplification A=(RLU2/RLU1-1) of second of signal value X100%, the results are shown in table below.
Table 9：
As shown above, after antigen diluent multiple is reduced to 10000 times, signal value is increased with concentration and is reduced, and is occurred HOOK effects, when concentration continues to rise to certain value (such as sample 12), RLU drops to reference value cut off hereinafter, conventional detection It will be judged by accident under method as a result negative, the method for the present invention then can first observe the amplification A=(RLU2/RLU1-1) of signal twice X100% compares the A values of sample to be tested and the A values (- 25%) of positive control, judges sample to be tested and positive with reference between Magnitude relationship, as shown above, the A values (47%) of sample 12 show that time sample is dense far above the A values (- 25%) of positive control Degree is positive sample, the not abundant of signal is because of HOOK effects, it should be diluted verification higher than positive control.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all can carry out modifications and changes under the spirit and scope without prejudice to the present invention to above-described embodiment.Cause This, those of ordinary skill in the art is complete without departing from disclosed spirit and institute under technological thought such as Into all equivalent modifications or change, should by the present invention claim be covered.

Claims (42)

1. a kind of method of immunity, which is characterized in that described method includes following steps：(1) to contain object to be measured antigen (or Antibody) sample to be tested carry out chemiluminescence immunoassay reaction, excite and record chemiluminescent first time and second of reading, and Difference amplification between second and first time reading is denoted as A, (2) are known according to antigen containing object to be measured (or antibody) The amplification A of the reading twice of series of standards substance does standard curve, and wherein the concentration of standard substance is less than generation HOOK effects Concentration；(3) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than standard song The maximum value of line, then be measured again after being diluted to sample.
2. method of immunity according to claim 1, which is characterized in that described method includes following steps：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) it is marked according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) The concentration of directrix curve, wherein standard substance is less than the concentration for generating HOOK effects；
(6) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the standard curve Maximum value, then be measured again after being diluted to sample.
3. method of immunity according to claim 1, which is characterized in that the known standard substance is positive control.
4. method of immunity according to claim 2, which is characterized in that luminous particle refers to filled with luminophor With the high molecular particle of lanthanide compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, red Under color laser excitation, singlet oxygen ion can be generated.
5. method of immunity according to claim 2, which is characterized in that in step (2) and (3), with 600~700nm The irradiation of red exciting light, detect the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
6. method of immunity according to claim 2, which is characterized in that the antigen refers to the object with immunogenicity Matter；The antibody refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody The antibody of the target antigen can be specifically bound to by referring to；First antigen and the second antigen refer to can specifically bind to it is described The antigen of target antibody.
7. a kind of system for identifying immunoassays, the system comprises：
Immune response device is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device are used to excite and record chemiluminescent first time and read for the second time Number, and the difference amplification between second and first time reading is denoted as A,
Processor is used for the reading twice of the known series of standards substance of basis antigen containing object to be measured (or antibody) Amplification A does standard curve, and wherein the concentration of standard substance is less than the concentration for generating HOOK effects；If antigen containing object to be measured The amplification A of the reading twice of the sample to be tested of (or antibody) is more than the maximum value of the standard curve, then sample is diluted It is measured again afterwards.
8. system according to claim 7, which is characterized in that the application method of the system includes the following steps：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) it is marked according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) The concentration of directrix curve, wherein standard substance is less than the concentration for generating HOOK effects；
(6) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the standard curve Maximum value, then be measured again after being diluted to sample.
9. system according to claim 7, which is characterized in that the known standard substance is positive control.
10. system according to claim 8, which is characterized in that luminous particle refers to filled with luminophor and group of the lanthanides The high molecular particle of element compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, in red laser Under excitation, singlet oxygen ion can be generated.
11. system according to claim 7, which is characterized in that in step (2) and (3), swashed with the red of 600~700nm Shine irradiation, detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
12. system according to claim 7, which is characterized in that the antigen refers to the substance with immunogenicity；It is described Antibody refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody refer to can be special Property is incorporated into the antibody of the target antigen；First antigen and the second antigen, which refer to, can specifically bind to the target antibody Antigen.
13. a kind of kit, including the coated luminous particle of first antibody (or antigen), mark substance markers secondary antibody (or Antigen), the photosensitive particulates of marker specific bond substance markers, which is characterized in that the application method of the kit includes as follows Step：(1) chemiluminescence immunoassay reaction, excitation and record chemistry are carried out to the sample to be tested for containing object to be measured antigen (or antibody) Luminous first time and second of reading, and the difference amplification between second and first time reading is denoted as A, (2) basis contains The amplification A of the reading twice of the known series of standards substance of object to be measured antigen (or antibody) does standard curve, acceptance of the bid The concentration of quasi- substance is less than the concentration for generating HOOK effects；(3) if the sample to be tested of antigen containing object to be measured (or antibody) The amplification A of reading is more than the maximum value of the standard curve twice, then is measured again after being diluted to sample.
14. kit according to claim 13, which is characterized in that the application method of the kit includes following step Suddenly：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) it is marked according to the amplification A of the reading twice of the known series of standards substance of antigen containing object to be measured (or antibody) The concentration of directrix curve, wherein standard substance is less than the concentration for generating HOOK effects；
(6) if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the standard curve Maximum value, then be measured again after being diluted to sample.
15. the kit according to claim 13 or 14, which is characterized in that the known standard substance is positive control.
16. system according to claim 14, which is characterized in that luminous particle refers to filled with luminophor and group of the lanthanides The high molecular particle of element compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, in red laser Under excitation, singlet oxygen ion can be generated.
17. system according to claim 14, which is characterized in that in step (2) and (3), with the red of 600~700nm Exciting light irradiates, and detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
18. system according to claim 14, which is characterized in that the antigen refers to the substance with immunogenicity；Institute It states antibody and refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody refer to can be special The opposite sex is incorporated into the antibody of the target antigen；First antigen and the second antigen, which refer to, can specifically bind to the target and resist The antigen of body.
19. a kind of method of immunity, which is characterized in that described method includes following steps：(1) to containing object to be measured antigen The sample to be tested of (or antibody) carries out chemiluminescence immunoassay reaction, excites and records chemiluminescent first time and second is read Number, and the difference amplification between second and first time reading is denoted as A, (2) are according to antigen containing object to be measured (or antibody) The amplification of the reading twice A of one known standard substance does critical value, and (3) will contain the sample to be tested of object to be measured antigen (or antibody) The amplification A of reading twice make comparisons with critical value, if the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) Several amplification A is more than the critical value, then the concentration of the sample to be tested is higher than the concentration of known standard substance.
20. a kind of method of immunity, which is characterized in that described method includes following steps：(1) to containing object to be measured antigen The sample to be tested of (or antibody) carries out chemiluminescence immunoassay reaction, excites and records chemiluminescent first time and second is read Number, and the difference amplification between second and first time reading is denoted as A, (2) are according to antigen containing object to be measured (or antibody) The amplification of the reading twice A of one known standard substance does critical value, and (3) will contain the sample to be tested of object to be measured antigen (or antibody) The amplification A of reading twice make comparisons with critical value, if the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) Several amplification A is more than the critical value, and the first time reading of the sample to be tested is less than the known standard substance simultaneously, then It is measured again after being diluted to sample.
21. method of immunity according to claim 19, which is characterized in that described method includes following steps：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) critical value is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody)；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, if The amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, then described to treat test sample This concentration is higher than the concentration of the known standard substance.
22. method of immunity according to claim 20, which is characterized in that described method includes following steps：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) critical value is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody)；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, if The amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, and described simultaneously treats Signal value obtained by the first time reading of test sample sheet is surveyed again less than the known standard substance after being then diluted to sample It is fixed.
23. the method for immunity according to claim 19 or 20, which is characterized in that the known standard substance is positive Control.
24. the method for immunity according to claim 21 or 22, which is characterized in that luminous particle refers to filled with luminous The high molecular particle of compound and lanthanide compound；The macromolecule that the photosensitive particulate is filled with Photoactive compounds is micro- Grain under red laser excitation, can generate singlet oxygen ion.
25. the method for immunity according to claim 21 or 22, which is characterized in that in step (2) and (3), with 600~ The red exciting light irradiation of 700nm detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
26. the method for immunity according to claim 21 or 22, which is characterized in that the antigen refers to immunogene The substance of property；The antibody refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and Two antibody refer to the antibody that can specifically bind to the target antigen；First antigen and the second antigen refer to and can specifically bind In the antigen of the target antibody.
27. a kind of system for identifying immunoassays, the system comprises：
Immune response device is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device are used to excite and record chemiluminescent first time and read for the second time Number, and the difference amplification between second and first time reading is denoted as A,
Processor is used for the amplification A and critical value of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) It makes comparisons, if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, The concentration of the sample to be tested is higher than the concentration of the known standard substance.
28. a kind of system for identifying immunoassays, the system comprises：
Immune response device is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device are used to excite and record chemiluminescent first time and read for the second time Number, and the difference amplification between second and first time reading is denoted as A,
Processor is used for the amplification A and critical value of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) It makes comparisons, if the amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, and Signal value obtained by the first time reading of the sample to be tested is less than the known standard substance simultaneously, then after being diluted to sample It is measured again.
29. system according to claim 27, which is characterized in that the application method of the system includes the following steps：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) critical value is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody)；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, if The amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, then described to treat test sample This concentration is higher than the concentration of the known standard substance.
30. system according to claim 28, which is characterized in that the application method of the system includes the following steps：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) critical value is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody)；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, if The amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, and described simultaneously treats Signal value obtained by the first time reading of test sample sheet is surveyed again less than the known standard substance after being then diluted to sample It is fixed.
31. system according to claim 27, which is characterized in that the known standard substance is positive control.
32. the system according to claim 29 or 30, which is characterized in that luminous particle refer to filled with luminophor and The high molecular particle of lanthanide compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, in red Under laser excitation, singlet oxygen ion can be generated.
33. the system according to claim 29 or 30, which is characterized in that in step (2) and (3), with 600~700nm's Red exciting light irradiation detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
34. system according to claim 27, which is characterized in that the antigen refers to the substance with immunogenicity；Institute It states antibody and refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody refer to can be special The opposite sex is incorporated into the antibody of the target antigen；First antigen and the second antigen, which refer to, can specifically bind to the target and resist The antigen of body.
35. a kind of kit, including the coated luminous particle of first antibody (or antigen), mark substance markers secondary antibody (or Antigen), the photosensitive particulates of marker specific bond substance markers, which is characterized in that the application method of the kit includes as follows Step：(1) chemiluminescence immunoassay reaction, excitation and record chemistry are carried out to the sample to be tested for containing object to be measured antigen (or antibody) Luminous first time and second of reading, and the difference amplification between second and first time reading is denoted as A, (2) basis contains The amplification of the reading twice A of one known standard substance of object to be measured antigen (or antibody) does critical value, and (3) will contain object to be measured The amplification A of the reading twice of the sample to be tested of antigen (or antibody) makes comparisons with critical value, if antigen containing object to be measured (or it is anti- Body) the amplification A of reading twice of sample to be tested be more than the critical value, then the concentration of the sample to be tested is known higher than described The concentration of standard substance.
36. a kind of kit, including the coated luminous particle of first antibody (or antigen), mark substance markers secondary antibody (or Antigen), the photosensitive particulates of marker specific bond substance markers, which is characterized in that the application method of the kit includes as follows Step：(1) chemiluminescence immunoassay reaction, excitation and record chemistry are carried out to the sample to be tested for containing object to be measured antigen (or antibody) Luminous first time and second of reading, and the difference amplification between second and first time reading is denoted as A, (2) basis contains The amplification of the reading twice A of one known standard substance of object to be measured antigen (or antibody) does critical value, and (3) will contain object to be measured The amplification A of the reading twice of the sample to be tested of antigen (or antibody) makes comparisons with critical value, if antigen containing object to be measured (or it is anti- Body) the amplification A of reading twice of sample to be tested be more than the critical value, and the first time reading of the sample to be tested simultaneously is low In the known standard substance, then it is measured again after being diluted to sample.
37. kit according to claim 35, which is characterized in that the application method of the kit includes following step Suddenly：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) critical value is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody)；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, if The amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, then described to treat test sample This concentration is higher than the concentration of the known standard substance.
38. kit according to claim 36, which is characterized in that the application method of the kit includes following step Suddenly：
(1) sample to be tested of object to be measured antigen (or antibody) and the coated luminous particle of first antibody (or antigen), mark will be contained Remember secondary antibody (or antigen) mixing of substance markers, incubate to obtain mixed liquor；
(2) first time reading：The photosensitive particulate of marker specific bond substance markers is added in the mixed liquor of step (1), temperature Exciting light irradiation is carried out after educating and detects transmitting light quantity, photon counter reading is calculated as RLU1；
(3) second of reading：After reaction solution after progress first time reading in step (2) is further incubated for, then swashed It shines and irradiates and detect transmitting light quantity, photon counter reading is calculated as RLU2；
(4) signal value obtained by second of reading of sample is calculated relative to the amplification A, A=of signal value obtained by first time reading (RLU2/RLU1-1) x100%；
(5) critical value is done according to the amplification of the reading twice A of a known standard substance of antigen containing object to be measured (or antibody)；
(6) the amplification A of the reading twice for the sample to be tested for containing object to be measured antigen (or antibody) is made comparisons with critical value, if The amplification A of the reading twice of the sample to be tested of antigen containing object to be measured (or antibody) is more than the critical value, and described simultaneously treats Signal value obtained by the first time reading of test sample sheet is surveyed again less than the known standard substance after being then diluted to sample It is fixed.
39. kit according to claim 35, which is characterized in that the known standard substance is positive control.
40. the kit according to claim 35 or 36, which is characterized in that luminous particle refers to filled with luminophor With the high molecular particle of lanthanide compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, red Under color laser excitation, singlet oxygen ion can be generated.
41. the kit according to claim 35 or 36, which is characterized in that in step (2) and (3), with 600~700nm The irradiation of red exciting light, detect the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
42. the kit according to claim 35 or 36, which is characterized in that the antigen refers to the object with immunogenicity Matter；The antibody refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody The antibody of the target antigen can be specifically bound to by referring to；First antigen and the second antigen refer to can specifically bind to it is described The antigen of target antibody.
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KR1020197014563A KR102220361B1 (en) 2016-11-22 2017-11-21 HD-HOOK effect sample identification and immunoassay methods, systems, kits and devices
EP17874359.7A EP3546937A4 (en) 2016-11-22 2017-11-21 Method, system, reagent kit and system for verifying hd-hook effect sample and for performing immunoassay
PCT/CN2017/112145 WO2018095314A1 (en) 2016-11-22 2017-11-21 Method, system, reagent kit and system for verifying hd-hook effect sample and for performing immunoassay
US16/462,968 US20190353664A1 (en) 2016-11-22 2017-11-21 Method, system, reagent kit, and device for determining hd-hook-effect sample and immunoassay
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019223691A1 (en) * 2018-05-21 2019-11-28 博阳生物科技（上海）有限公司 Chemiluminescence analytical method and system and kit using same
CN110823869A (en) * 2018-08-10 2020-02-21 上海索昕生物科技有限公司 Light-activated chemiluminescence detection device
CN113113079A (en) * 2021-02-25 2021-07-13 安徽桐康医疗科技股份有限公司 Method for identifying hook effect in quantitative immunochromatography test

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113125712B (en) * 2019-12-31 2023-06-16 科美博阳诊断技术(上海)有限公司 Homogeneous chemiluminescence detection kit for hepatitis C virus antibody and application thereof
CN115877012A (en) * 2021-09-27 2023-03-31 深圳市理邦精密仪器股份有限公司 Antigen concentration measuring method and measuring instrument
CN114594260A (en) * 2022-03-28 2022-06-07 河南中医药大学第一附属医院 Method for recognizing hook effect in HBsAg through light-activated chemiluminescence homogeneous immunoassay
CN116429754B (en) * 2023-02-13 2024-07-19 科美博阳诊断技术(上海)有限公司 HCV detection kit and using method thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2203836A (en) * 1987-04-24 1988-10-26 Dr Ch Ng Soo Ling Determination of analyte concentration
CN101055272A (en) * 2006-04-13 2007-10-17 奥林巴斯生命及材料科学欧洲有限公司 Immunoassay method
US20080213797A1 (en) * 2007-03-01 2008-09-04 Abbott Laboratories Immunoassays exhibiting a reduction in prozone phenomena
CN101326440A (en) * 2005-04-29 2008-12-17 金伯利-克拉克环球有限公司 Assay devices having detection capabilities within the hook effect region
CN101769929A (en) * 2008-12-30 2010-07-07 博阳生物科技（上海）有限公司 Surface antibody testing fine particles for hepatitis B virus, and preparation and application thereof
CN102341706A (en) * 2009-08-07 2012-02-01 爱科来株式会社 Method for Detecting Prozone Phenomenon, Analysis Method, Device for Detecting Prozone Phenomenon, and Analysis Device
CN102944672A (en) * 2012-11-16 2013-02-27 李方和 Method for qualitatively and quantitatively detecting target substance to be detected in blood serum by utilizing light initiated chemiluminescence immune assay
EP2790019A1 (en) * 2013-04-10 2014-10-15 Siemens Healthcare Diagnostics Products GmbH High dose hook detection
CN104991056A (en) * 2015-08-05 2015-10-21 武汉林勉生物技术有限公司 Method for serological test and quantitative analysis

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0968425B1 (en) * 1996-12-03 2003-06-25 SOINI, Erkki Biospecific, two photon excitation, fluorescence detection and device
JP3920448B2 (en) * 1998-03-18 2007-05-30 オリンパス株式会社 Prozone phenomenon judgment method
JP2000221195A (en) * 1999-02-03 2000-08-11 Toshiba Iyo System Engineering Kk Prozone judging method, storage medium storing the same and automatic analyzer using the same
DE10064827A1 (en) * 2000-12-22 2002-06-27 Dade Behring Marburg Gmbh Sandwich assay for detecting analyte, useful e.g. for hormones, with detection or correction of the hook effect by measuring detection signals twice
US20030027234A1 (en) * 2001-07-30 2003-02-06 Pandian Murugan R. Methods for detecting Down's syndrome
US20030109067A1 (en) * 2001-12-06 2003-06-12 Immunetech, Inc. Homogeneous immunoassays for multiple allergens
US7851209B2 (en) * 2003-04-03 2010-12-14 Kimberly-Clark Worldwide, Inc. Reduction of the hook effect in assay devices
US20050148096A1 (en) * 2004-01-07 2005-07-07 Cole Laurence A. Methods for detecting pregnancy
CN100489528C (en) * 2005-06-17 2009-05-20 上海透景生命科技有限公司 Method for indicating high dose hook effect
CN101251540B (en) * 2008-03-26 2012-12-05 博阳生物科技（上海）有限公司 Hepatitis B virus e antigen testing corpuscle, preparation and application thereof
WO2009126336A1 (en) * 2008-04-11 2009-10-15 Becton, Dickinson And Company Methods of controlling the sensitivity and dynamic range of a homogeneous assay
CN101281137A (en) * 2008-04-24 2008-10-08 博阳生物科技（上海）有限公司 Light activating chemical luminescence luminous immune detecting method
GB0809995D0 (en) * 2008-05-31 2008-07-09 Spd Swiss Prec Diagnostics Gmb Assay device
CN101620229B (en) * 2008-07-02 2013-04-17 博阳生物科技（上海）有限公司 Hepatits B virus e antibody assay kit and assay method thereof
CN101666801A (en) * 2008-09-02 2010-03-10 博阳生物科技（上海）有限公司 Hepatitis B virus surface antigen detection particles, preparation thereof and use thereof
CN101750487B (en) * 2008-12-02 2013-07-03 博阳生物科技（上海）有限公司 Dry method photic stimulation chemiluminescence immunoassay reagent kit and preparation and application thereof
CN101769927B (en) * 2008-12-30 2014-06-18 博阳生物科技（上海）有限公司 Detection particle of carcino-embryonic antigen as well as preparation and application thereof
CN101769931B (en) * 2008-12-30 2013-10-02 博阳生物科技（上海）有限公司 Fetus alpha globulin detection particles, preparation thereof and application thereof
US9347947B2 (en) * 2009-03-12 2016-05-24 Siemens Healthcare Diagnostics Inc. Immunoassays employing non-particulate chemiluminescent reagent
US20120220051A1 (en) * 2009-07-08 2012-08-30 Anp Technologies, Inc. Immunogenicity Assay
CN102323419B (en) * 2011-08-31 2013-10-09 内蒙古科慧生物科技有限责任公司 Kit and detection method for quantitative determination of digoxin
CN102338804A (en) * 2011-08-31 2012-02-01 内蒙古科慧生物科技有限责任公司 Quantitative determination kit for free human chorionic gonadotropin beta subunit (F-beta-HCG) and detection method thereof
CN102654501A (en) * 2012-05-21 2012-09-05 江苏省原子医学研究所 Method for detecting optically-excited chemiluminiscence of pepsinogen II and reagent kit
CN102749461B (en) * 2012-06-26 2015-09-02 博奥赛斯（天津）生物科技有限公司 Free β human chorionic gonadotrophin chemiluminescence immunoassay immue quantitative detection reagent box and preparation method thereof
CN103063845A (en) * 2012-12-18 2013-04-24 苏州浩欧博生物医药有限公司 Nanometer magnetic particle chemiluminiscence kit, preparation method and detection method of hepatitis B virus surface-antibody
CN103616510A (en) * 2013-12-06 2014-03-05 苏州长光华医生物医学工程有限公司 Hepatitis B surface antibody measurement kit and detection method thereof
CN103837675B (en) * 2014-03-07 2016-01-13 天津市南开医院 The homogeneous luminescent immune analysis method of polycomponent Simultaneous Quantitative Analysis and the kit used thereof
CN104730247A (en) * 2015-03-12 2015-06-24 广州市丰华生物工程有限公司 Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit
CN105044332B (en) * 2015-06-04 2017-10-10 天津起跑线生物信息技术有限公司 A kind of method for monitoring HOOK effects in immune colloid gold experiment
CN105785030A (en) * 2016-03-09 2016-07-20 博阳生物科技（上海）有限公司 Light-activating chemiluminescence immunoassay kit for serum specific IgE (immunoglobulin E)
CN105758835B (en) * 2016-05-04 2018-03-27 成都爱兴生物科技有限公司 A kind of homogeneous immunoassay POCT detection methods and the system using the detection method

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2203836A (en) * 1987-04-24 1988-10-26 Dr Ch Ng Soo Ling Determination of analyte concentration
CN101326440A (en) * 2005-04-29 2008-12-17 金伯利-克拉克环球有限公司 Assay devices having detection capabilities within the hook effect region
CN101055272A (en) * 2006-04-13 2007-10-17 奥林巴斯生命及材料科学欧洲有限公司 Immunoassay method
US20080213797A1 (en) * 2007-03-01 2008-09-04 Abbott Laboratories Immunoassays exhibiting a reduction in prozone phenomena
CN101769929A (en) * 2008-12-30 2010-07-07 博阳生物科技（上海）有限公司 Surface antibody testing fine particles for hepatitis B virus, and preparation and application thereof
CN102341706A (en) * 2009-08-07 2012-02-01 爱科来株式会社 Method for Detecting Prozone Phenomenon, Analysis Method, Device for Detecting Prozone Phenomenon, and Analysis Device
CN102944672A (en) * 2012-11-16 2013-02-27 李方和 Method for qualitatively and quantitatively detecting target substance to be detected in blood serum by utilizing light initiated chemiluminescence immune assay
EP2790019A1 (en) * 2013-04-10 2014-10-15 Siemens Healthcare Diagnostics Products GmbH High dose hook detection
CN104991056A (en) * 2015-08-05 2015-10-21 武汉林勉生物技术有限公司 Method for serological test and quantitative analysis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
S. AMARASIRI FERNANDO，ET AL: "Studies of the "hook"effect in the one-step sandwich immunoassay", 《JAURNAL OF IMMUNOLOGICAL METHODS》 *
马宏伟 等: "血清心肌肌钙蛋白Ⅰ光激化学发光免疫测定法的建立", 《检验医学》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019223691A1 (en) * 2018-05-21 2019-11-28 博阳生物科技（上海）有限公司 Chemiluminescence analytical method and system and kit using same
CN110823869A (en) * 2018-08-10 2020-02-21 上海索昕生物科技有限公司 Light-activated chemiluminescence detection device
CN113113079A (en) * 2021-02-25 2021-07-13 安徽桐康医疗科技股份有限公司 Method for identifying hook effect in quantitative immunochromatography test
CN113113079B (en) * 2021-02-25 2023-04-11 安徽桐康医疗科技股份有限公司 Method for identifying hook effect in quantitative immunochromatography test

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