CN109470864A - Method of immunity, the system for identifying immunoassays and kit - Google Patents

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

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CN109470864A
CN109470864A CN201811400151.0A CN201811400151A CN109470864A CN 109470864 A CN109470864 A CN 109470864A CN 201811400151 A CN201811400151 A CN 201811400151A CN 109470864 A CN109470864 A CN 109470864A
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sample
antibody
antigen
amplification
concentration
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杨阳
赵卫国
张向辉
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Boyang Biotechnology (Shanghai) Co Ltd
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G01N33/5764—Hepatitis B surface antigen
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—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
    • 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

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Abstract

The present invention relates to a kind of kits of light-induced chemiluminescent technical field, it includes first antibody (or antigen) coated luminous particle, mark the secondary antibody (or antigen) of substance markers, the photosensitive particulate of marker specific bond substance markers, the application method of the kit includes: that (1) carries out chemiluminescence immunoassay reaction to the sample to be tested of antigen containing object to be measured (or antibody), it excites and records chemiluminescent first time and second reads, and the difference amplification between second and first time reading is denoted as A, (2) 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), (3) the amplification A of the sample to be tested for containing object to be measured antigen (or antibody) read twice is made comparisons with critical value, if antigen containing object to be measured (or antibody) to test sample This amplification A read twice is greater than the critical value, then the concentration of the sample to be tested is higher than the concentration of the known standard substance.

Description

Method of immunity, the system for identifying immunoassays and kit
It is on November 22nd, 2016 that the application, which is the applying date, entitled " immune application No. is 201611026623.1 The divisional application of the Chinese patent application of measuring method, the system for identifying 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, a kind of for identifying 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 technique
Immunology detection be based on antigen and antibody specific reaction principle carry out, due to its can use isotope, Enzyme, chemiluminescent substance etc. carry out display or the amplification of signal to measured object, therefore are commonly used for detecting protein, hormone etc. micro- Measure bioactive substance.
Chemiluminescence immune assay is then to develop relatively rapid on-radiation immunoassay technology in recent years, and principle is benefit The amplification of signal is carried out with chemiluminescent substance, and by its luminous intensity, immune cohesive process is directly measured, the method Have become one of the important directions of immunology detection.
Light-induced chemiluminescent method is one of common method of chemiluminescence analytical technique, and it is intermolecular to can be used for studying biology Interaction, is clinically mainly used for the detection of disease.The technology incorporates high molecular particle technology, organic synthesis, protein The research of the related fieldss such as chemistry and clinical detection.It is combined in a certain range by photosensitive particulate and luminous particle, is generated The transmitting of ion oxygen energy issues optical signal, to detect to sample to be tested.Wherein, thoughts are filled 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 column, launches the light of 520~620nm high level, 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 resist The coated photosensitive particulate of digoxin neutralizes antigen or antibody etc..The above each component is resisted by the above incubation reaction of two steps with to be measured Former or antibody combines, and is qualitatively or quantitatively detected by the power of chemiluminescence amount to sample to be tested.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 automate.Therefore, before applying Scape is very wide.
It, can be because being unable to shape when material concentration height to be detected is to a certain concentration in the detection pattern of double antibodies sandwich At dual anti-sandwich complex to the relatively low phenomenon of signal value, referred to as high dose-hook effect (HD-HOOK effect).Namely It says, high dose-hook effect refers in two-site sandwich immunization experiment, the high dose section of dose-effect curve, linearly Trend instead of in platform-like it is unlimited after prolong, be turned under curved, like a hook, lead to the phenomenon that generating false negative.
In immune detection frequent occurrence, incidence accounts for positive sample 30% or so to HD-HOOK effect.Due to HD- It is since its concentration is beyond the linear of detection kit that the presence of HOOK effect, 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, when detecting the sample of high concentration, high dose-hook effect may cause detection signal Relatively low, therefore sample is also read as relatively low concentration.The solution of the past is to increase the component of reagent, is carried out to sample to be tested Dilution or progress 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.Detection range is mainly previously widened by optimization antibody or raising antibody.
Conventional detection process has following 5 steps: determinand being added in reacting hole and reagent, the first step incubate, addition is led to It is incubated and is read with liquid, second step.
Detection method of the invention is to be based on conventional detection process, more in reaction process under the premise of not interrupting reaction Secondary reading signal value, by the variation of observation signal come the actual concentration of judgement sample.
Summary of the invention
For the defect 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 the detection process, to pass through the amplification A that will be read twice and a known system The maximum value of the standard curve for the amplification A of column standard substance read twice compares, to judge whether sample to be tested needs to dilute It is measured again afterwards.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
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 of antigen containing object to be measured (or antibody) First time and second read, and the difference amplification between second and first time reading is denoted as A, (2) are according to containing to be measured The amplification A of the known series of standards substance of target antigen (or antibody) read twice does standard curve, wherein reference substance The concentration of matter is lower than the concentration for generating HOOK effect；(3) if the sample to be tested of antigen containing object to be measured (or antibody) twice The amplification A of reading is greater 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) by the sample to be tested for containing object to be measured antigen (or antibody) and first antibody (or antigen) it is coated shine it is micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive micro- of marker specific bond substance markers in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(3) it reads for second: after the 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) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (RLU2/RLU1-1) x100%；
(5) according to the amplification A of the known series of standards substance of antigen containing object to be measured (or antibody) read twice Standard curve is done, wherein the concentration of standard substance is lower than the concentration for generating HOOK effect；
(6) if the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice is greater than the standard The maximum value of curve, then its concentration is more than upper limit of detection, and measurement 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, It can produce singlet oxygen ion.
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:
It is immunoreacted device, is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device, are used to exciting and recording 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 lower than the concentration for generating HOOK effect；If containing object to be measured The amplification A of the sample to be tested of antigen (or antibody) read twice is greater than the maximum value of the standard curve, then its concentration is more than Upper limit of detection need to be diluted measurement to sample
According to a preferred embodiment of the present invention, the application method of the system includes the following steps:
(1) by the sample to be tested for containing object to be measured antigen (or antibody) and first antibody (or antigen) it is coated shine it is micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive micro- of marker specific bond substance markers in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(3) it reads for second: after the 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) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (RLU2/RLU1-1) x100%；
(5) according to the amplification A of the known series of standards substance of antigen containing object to be measured (or antibody) read twice Standard curve is done, wherein the concentration of standard substance is lower than the concentration for generating HOOK effect；
(6) if the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice is greater than the standard The maximum value of curve, then its concentration is more than upper limit of detection, and measurement 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: that (1) carries out chemiluminescence to the sample to be tested of antigen containing object to be measured (or antibody) It is immunoreacted, excites and record chemiluminescent first time and second reads, and will be between second and first time reading Difference amplification is denoted as A, and (2) are read twice according to 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 lower than the concentration for generating HOOK effect；(3) if it is anti-containing object to be measured The amplification A of the sample to be tested of former (or antibody) read twice is greater than the maximum value of the standard curve, then its concentration is more than inspection The upper limit is surveyed, measurement 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) by the sample to be tested for containing object to be measured antigen (or antibody) and first antibody (or antigen) it is coated shine it is micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive micro- of marker specific bond substance markers in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(3) it reads for second: after the 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) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (RLU2/RLU1-1) x100%；
(5) according to the amplification A of the known series of standards substance of antigen containing object to be measured (or antibody) read twice Standard curve is done, wherein the concentration of standard substance is lower than the concentration for generating HOOK effect；
(6) if the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice is greater than the standard The maximum value of curve, then its concentration is more than upper limit of detection, and measurement need to be diluted to sample
The object of the invention is also to provide a kind of method of immunity, widen detection range by reading twice, In the detection process, the amplification A read twice to be made comparisons with critical value, to judge after whether sample to be tested needs to dilute again It is measured.
To achieve the goals above and other related purposes, the present invention adopts the following technical scheme:
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 of antigen containing object to be measured (or antibody) First time and second read, and the difference amplification between second and first time reading is 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 sample to be tested of (or antibody) read twice makes comparisons with critical value, if antigen containing object to be measured (or antibody) The amplification A read twice of sample to be tested be greater than the critical value, then judge that the concentration of sample to be tested is higher than the known mark Quasi- material concentration；If the first time reading of the sample to be tested needs to carry out sample lower 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) by the sample to be tested for containing object to be measured antigen (or antibody) and first antibody (or antigen) it is coated shine it is micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive micro- of marker specific bond substance markers in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(3) it reads for second: after the 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) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (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 sample to be tested for containing object to be measured antigen (or antibody) read twice is made comparisons with critical value, If the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice be greater than the critical value, judge to The concentration of test sample sheet is higher than the known standard substance concentration；If the first time reading gained signal value of the sample to be tested simultaneously Lower 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, It can produce singlet oxygen ion.
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:
It is immunoreacted device, is used to implement chemiluminescence immunoassay reaction,
Chemiluminescence immunoassay reaction excitation and counting device, are used to exciting and recording chemiluminescent first time and second Secondary reading, and the difference amplification between second and first time reading is denoted as A,
Processor is used to contain the amplification A read twice of the sample to be tested of object to be measured antigen (or antibody) and face Dividing value is made comparisons, if the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice is greater than described critical Value then judges that the concentration of sample to be tested is higher than the concentration of the known standard substance, if the first time of the sample to be tested simultaneously Reading then needs to be measured again after being diluted sample lower 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) by the sample to be tested for containing object to be measured antigen (or antibody) and first antibody (or antigen) it is coated shine it is micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive micro- of marker specific bond substance markers in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(3) it reads for second: after the 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) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (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 sample to be tested for containing object to be measured antigen (or antibody) read twice is made comparisons with critical value, If the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice be greater than the critical value, judge to The concentration of test sample sheet is higher than the concentration of the known standard substance；If the first time reading gained signal of the sample to be tested simultaneously Value then needs to be measured again after being diluted sample lower 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: that (1) carries out chemiluminescence to the sample to be tested of antigen containing object to be measured (or antibody) and exempts from Epidemic disease reaction, exciting and record chemiluminescent first time and second reads, 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 sample to be tested for containing object to be measured antigen (or antibody) read twice with critical value, If the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice be greater than the critical value, judge to 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 lower 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) by the sample to be tested for containing object to be measured antigen (or antibody) and first antibody (or antigen) it is coated shine it is micro- Secondary antibody (or antigen) mixing of grain, label substance markers, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive micro- of marker specific bond substance markers in the mixed liquor of step (1) , exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(3) it reads for second: after the 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) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (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 sample to be tested for containing object to be measured antigen (or antibody) read twice is made comparisons with critical value, If the amplification A of the sample to be tested of antigen containing object to be measured (or antibody) read twice be greater than the critical value, judge to The concentration of test sample sheet is higher than the concentration of the known standard substance；If the first time reading gained signal of the sample to be tested simultaneously Value then needs to be measured again after being diluted sample lower than the known standard substance.
Here, the method is used for double it should be strongly noted that the above method is the method for non-disease diagnostic purpose In antibody sandwich immunization or double antigens sandwich immunization detection process, widen detection range by reading twice, with In detection process, the amplification A read twice is made comparisons with critical value, to judge whether sample to be tested needs to carry out again after diluting Measurement.
Preferably, the antigen refers to the substance with immunogenicity.Such as protein, polypeptide.Representative antigen packet It includes (but being 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 swash and β subunit (HCG+ β), hepatitis B surface antigen (HBsAg), cancer antigen 125 (CA125), C peptide (CP), Ferritin (Ferr) and Anti-HCV.
It can be not particularly limited, be can be any (or anti-containing object to be measured antigen with the sample that the method for the present invention detects 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 be also possible to it is different, And it 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 be also possible to it is different, And it 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 the derivative etc. of Dioxene (dioxine) or thioxene (thioxene), group of the lanthanides member 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 close 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 can also 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, to launch the light of 520~620nm high level.
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 in conjunction with single antibody, leads to dual anti-folder respectively Heart compound is reduced, and optical signal is relatively low, cannot 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, cannot reflect the actual concentration of object to be measured antibody.
Method of the invention reads the relationship between gained signal value amplification by reading twice more twice, so as to To play the role of widening detection range and distinguish HD-HOOK effect sample.The difference read twice is determined by following three aspects It is fixed:
In a first aspect, after photosensitive particulate is irradiated by red laser (600~700nm), releasing single line when reading for the first time 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 level；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 decline, double antibodies sandwich compound is reduced, and second of read signal value can reduce；And for high concentration sample, to 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 effect, when reading for the first time, balance is had not yet been reached in antigen-antibody reaction, The interval time read 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, Part target antigen to be detected or antibody are consumed, so that reaction balance is reverse mobile, another aspect photosensitive particulate was exciting one It after secondary, is lost, when second reads, the signal value of object to be measured antigen or the low sample of antibody concentration can be reduced；And The combination of the double antibodies sandwich compound and photosensitive particulate of the high sample of concentration reaches far away balance when reading first time, second of reading Reaction can be mobile towards positive reaction direction when 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 and concentration of specimens positive of signal It closes, the amplification A of signal twice is made comparisons with critical value, to judge whether sample to be tested needs to be measured again after diluting.
Compared with prior art, the invention has the benefit that
(1) it the present invention is based on the homogeneity of the disposable of light-induced chemiluminescent platform (luminescent oxygen channel) and reaction, is able to achieve Progress of the multiple signal measurement without interrupting immune response is carried out to a reaction, detects to believe in the light of differential responses time Number, the not examined scope limitation of the method effectively widens 100 times of detection range or more.
(2) whether the sample to be tested that method of the invention can identify in double-antibody method detection needs to carry out again after diluting Measurement, the method can significantly improve the accuracy of double antibody sandwich method immunoassays, and it is immune to reduce double antibody sandwich method The false negative rate of measurement.
(3) method of the invention is easy to operate, can identify non-disease diagnostic purpose double-antibody sandwich simple and effectively and exempt from The relatively low sample of reported concentrations caused by HD-HOOK effect in epidemic disease measurement.
Detailed description of the invention
Fig. 1: HCG+ β is using signal value obtained by conventional method and concentration of specimens graph of relation；
Fig. 2: HCG+ β using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation；
Fig. 3: Ferr 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 peptide is using signal value obtained by conventional method and concentration of specimens graph of relation；
Fig. 6: C peptide using signal value obtained by the method for the present invention and A respectively with concentration of specimens graph of relation.
Fig. 7: HBsAb 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 term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present 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, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc. MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present inventor has found after extensive and in-depth study, is read twice by setting up, and will read twice Amplification A make comparisons with critical value, can determine whether sample to be tested whether need dilute 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 be can be Different being also possible to is 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) Speech, corresponding first antigen and the second antigen can be different be also possible to it is identical, and can be in combination in described 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 reaction is generated by body caused by itself, reaction tumour exists and a 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 It is fixed on solid phase carrier, then by first antibody and antigen-reactive, then is reacted with the secondary antibody of label, chemical hair is finally carried out Light or enzyme-linked chromogenic reaction detect 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 transmitting of ion oxygen energy, optical signal are issued, thus to sample to be tested It is detected.Wherein, it is filled with Photoactive compounds inside photosensitive particulate, and is filled with luminophor and lanthanum 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 level, and It is detected by instrument.
In a preferred embodiment of the invention, the feature that first antibody is fixed in luminous particle is taken full advantage of, Biotin labeling secondary antibody is used simultaneously, and Streptavidin is coated with photosensitive particulate, by serum sample or antigen standard quality-control product Liquid and the coated luminous particle of first antibody, biotin labeling secondary antibody are sequentially or simultaneously added in reaction vessel, add The photosensitive particulate of marked by streptavidin, so that following reaction 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 is coated with 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 plus Enter the photosensitive particulate of marked by streptavidin, so that following reaction occur:
(1) the first antigen in luminous particle is tied with antibody corresponding in serum sample or antigen standard quality-control product liquid product It closes, forms " the-the first antigen of antibody-luminous particle " ternary complex；
(2) second antigens ultimately form " second in conjunction with corresponding antibody in serum sample or antigen standard quality-control product liquid 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, further illustrating relevant operation details of the invention.
(1) first antibody (or antigen) coated luminous particle, is denoted as reagent 1, and it is limited to can purchase Yu Boyang biotechnology Company.
(2) secondary antibody (or antigen) can be marked with various markers known in the art and its specific junction mixture system Note.Secondary antibody (or antigen) is marked preferably by biotin-avidin system.Biotin labeling secondary antibody (or Antigen), it is denoted as reagent 2, can purchase Yu Boyang Biotechnology Co., Ltd.
(3) the coated photosensitive particulate of Streptavidin is denoted as general liquid, can purchase Yu Boyang Biotechnology Co., Ltd.
(4) standard items:
It is molten with the standard items for (being lower than HD-HOOK effective concentration) within the scope of determined antigen (or antibody) configuration a certain concentration Liquid.Standard items, reagent 1, reagent 2 are mixed, general liquid is added after incubation reaction, after continuing incubation reaction for a period of time for the first time It reads (RLU1), then carries out second of reading (RLU2) after incubating a period of time, calculate A=(RLU2/RLU1-1) x100%, root According to standard items RLU1 and twice the amplification A that reads does standard curve with standard concentration respectively；
(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:
Sample to be tested is read to amplification A value twice compared with the A of known standard items, if sample to be tested A is greater than known standard The A of product, then this concentration of specimens is greater 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 to need to dilute detection as caused by HD-HOOK effect.
Embodiment 1: human chorionic gonadotrophin and β subunit (HCG+ β) the verifying present invention in detection human serum sample Method validity
The human chorionic gonadotrophin and β subunit (HCG+ produced using Bo Yang biotechnology (Shanghai) Co., Ltd. β) detection kit (light-induced chemiluminescent method) detects human chorionic gonadotrophin and β subunit (HCG+ in serum sample Content β).The kit include calibration object 1- calibration object 6, reagent 1 (illuminating antibody, also that is, antibody it is coated shine it is micro- Grain), reagent 2 (biotin labelled antibodies, that is, the antibody of biotin labeling).
To detected by Roche (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 are added 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, as a result as shown in the table.
Using method of reading twice of the invention: by sample to be tested, the calibration object, (illuminating antibody, also that is, murine monoclonal is anti-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, is added general liquid (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min read RLU1,37 DEG C of continuation temperature 7min is educated, RLU2 is read, and calculate 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 detection is 0-10000mIU/ml, be beyond upper limit of detection sample display density > 10000mIU/ml。
Roche testing result is as actual concentration, and by table 1 and Fig. 1 it is found that in conventional detection, concentration is risen to 54531mIU/ml signal value increases with concentration and is increased, and concentration continues to increase, and signal value is increased with HCG+ β concentration and reduced, i.e., Concentration is greater than 54531mIU/ml then HD-HOOK, in conventional detection, detection range 0-10000mIU/ml, beyond in detection Limiting sample display density is > 10000mIU/ml, and when HD-HOOK effect concentration of specimens persistently increases, signal is continued to decline, will Occur ultrahigh concentration sample report at relatively low concentration levels, such as sample 18.So cannot be differentiated in conventional detection to be measured The testing result of sample is actual concentration or the relatively low concentration that superelevation value sample is reported by HD-HOOK effects.
The method of the present invention identifies the relatively low sample of reported concentrations caused by HOOK effect by reading twice.Each to test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of the index of judgement sample concentration.By table 1 and Fig. 2 it is found that signal value is increased to 54531mIU/ml, Zhi Houxin with concentration is continuous 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 The A value of value and calibration object, that is, can determine whether the size relation of sample to be tested concentration Yu calibration object concentration.The amplification A of sample 10~18 It is all larger than the amplification (11.1%) of calibration object 6, and A value persistently increases, shows that the HCG+ β concentration of sample 10~18 is all larger than 10000mIU/ml, and concentration persistently increases, this is consistent with the concentration results of Roche, and 18 signal value of sample is lower than calibration object 6, often Rule method detectable concentration is 8713.02mIU/ml, can identify it by the method for the invention as HD-HOOK effect sample, need to carry out Dilution detection.
Embodiment 2: ferritin (Ferr) verifies the method for the present invention validity in detection sample
Ferritin (Ferr) detection kit (chemiluminescence produced using Bo Yang biotechnology (Shanghai) Co., Ltd. Method) detect the content of ferritin in sample (purchased from Fitzgerald, Catalog No:30-AF10).The kit includes Calibration object 1- calibration object 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 object 6: the known concentration sample in conventional kit, concentration are much smaller than HOOK sample, 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 The auxiliary reagent of scientific & technical corporation's production light-induced chemiluminescent analysis system.It is tried with instrument and the detection of corresponding light-induced chemiluminescent method Agent box matches, the detection for 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 object 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 are added the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubations 10min, photon counter reading, reads RLU, as a result as shown in the table.
Using present invention method of reading twice: by calibration object 1- calibration object 6, sample to be tested 1-15, 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 are added the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min, read Number RLU1,37 DEG C are continued to incubate 7min, read RLU2, and calculate the amplification A=(RLU2/RLU1-1) of second of signal value X100%, as a result as shown in the table,
Table 2:
Note: the detection range of ferritin conventional detection is 0-2000ng/ml, be 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 value Height, concentration continue to increase, and signal value is increased with Ferr concentration and reduced, and conventional detection range is 0-2000ng/ml, beyond detection Upper limit sample display density is > 2000ng/ml.When HD-HOOK effect 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 cannot differentiate sample to be tested in conventional detection is actual concentration or superelevation The relatively low concentration that value sample is reported by HD-HOOK effects.
The method of the present invention identifies the relatively low sample of reported concentrations caused by HOOK effect by reading twice.Each to test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of the index of judgement sample concentration.By table 2 and Fig. 4 it is found that signal value is increased to 51000ng/ml with concentration is continuous, signal later Value starts to increase and decline with concentration, but amplification A is persistently to rise with concentration.Therefore directly compare the A value of sample to be tested With the A value of calibration object, that is, it can determine whether the size relation of sample to be tested concentration Yu 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 larger than 2000ng/ml.This and actual concentrations phase Symbol, 15 signal value of sample are lower than calibration object 6, and conventional method detectable concentration is 1860.97ng/ml, can reflect by the method for the invention It is not more than detection range sample for concentration, need to be diluted detection.
Embodiment 3: C peptide (CP) verifies the method for the present invention validity in detection sample
It is examined using C peptide (CP) detection kit (chemoluminescence method) of Bo Yang biotechnology (Shanghai) Co., Ltd. production The content of C peptide (being purchased from Fitzgerald, Catalog No:30-AC96) in test sample sheet.The kit includes the school calibration object 1- 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 object 6: the known concentration sample in conventional kit, concentration are much smaller than HOOK sample, make standard Curve calculates testing concentration.
The C peptide 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 peptide of C containing various concentration.
Common detection methods: by calibration object 1- calibration object 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 are added the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubations 10min, photon counter reading, reads RLU, as a result as shown in the table.
Using present invention method of reading twice: by calibration object 1- calibration object 6, sample to be tested 1-15, 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 are added the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min, read Number RLU1,37 DEG C are continued to incubate 7min, read RLU2, and calculate the amplification A=(RLU2/RLU1-1) of second of signal value X100%, as a result as shown in the table,
Table 3:
Note: the detection range of C peptide conventional detection is 0-30ng/ml, is > 30ng/ beyond upper limit of detection sample display density 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 value Height, concentration continue to increase, and signal value is increased with C peptide concentration and reduced, and conventional detection range is 0-30ng/ml, beyond in detection Limiting sample display density is > 30ng/ml.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 at 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 method of the present invention identifies the relatively low sample of reported concentrations caused by HOOK effect by reading twice.Each to test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of the index of judgement sample concentration.By table 3 and Fig. 6 it is found that signal value is increased to 10000ng/ml with concentration is continuous, signal later Value starts to increase and decline with concentration, but amplification A is persistently to rise with concentration.Therefore directly compare the A value of sample to be tested With the A value of calibration object, that is, it can determine whether the size relation of sample to be tested concentration Yu 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 concentration of sample 5~17 is all larger than 30ng/ml, is more than upper limit of detection.This with Actual concentrations are consistent, sample 16,17 signal values be lower than calibration object 6, conventional method detectable concentration be respectively 8.15ng/ml, 0.76ng/ml, can identify it by the method for the invention is that need to be diluted detection more than upper limit of detection sample.
Embodiment 4: hepatitis b virus s antigen (HBsAg) in detection human serum sample
Examination is detected using the hepatitis b virus s antigen (HBsAg) of Bo Yang biotechnology (Shanghai) Co., Ltd. production Agent box (light-induced chemiluminescent method) detects HBsAg concentration in sample, and the kit includes calibration object 1- calibration object 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 object 6: the known concentration sample in conventional kit, concentration are much smaller than HOOK sample, make standard Curve calculates testing concentration.
First use the method for the present invention testing calibration product 1- calibration object 6, serum sample 1-15 to be measured: by determinand, reagent 1 After reaction cup is added in (the coated luminous particle of antibody) and reagent 2 (antibody of biotin labeling), 37 DEG C of incubation 15min are added The general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min read RLU1, and 37 DEG C are continued to incubate 7min, RLU2 is read, and calculates amplification A=(RLU2/RLU1-1) X100% of second of signal value, as a result such as following table.
Table 4:
The concentration that the method for the present invention obtains 15 serum samples is as above: shown in upper table, first passing through the amplification ratio with calibration object 6 The sample more than upper limit of detection (336.56IU/mL) is relatively distinguished, i.e. A value is judged as HOOK effect sample greater than 27%, pushes away It is detected after recommending dilution；And A is sample in detection range less than 27%, directly can calculate concentration of specimens with calibration curve.
Changed by detectable concentration after carrying out gradient dilution to sample to verify the reliability of conclusions, by sample 1- sample This 15 carries out 2 and is diluted to dilute with 4 times, while being diluted sample with the undiluted former times sample of common detection methods detection, 2 With 4 times of diluted samples, judge whether the sample has HOOK effect, sample after even diluting by the variation of concentration after observation dilution Concentration increases as HOOK effect sample instead.Concentration can reduce non-HOOK effect sample after dilution.As a result 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 effect Sample, concentration are greater 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 biotechnology (Shanghai) Co., Ltd. Chemoluminescence method) detection sample in CA125 concentration, the kit include calibration object 1- calibration object 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 object 6: the known concentration sample in conventional kit, concentration are much smaller than HOOK sample, make standard Curve calculates testing concentration.
First use the method for the present invention testing calibration product 1- calibration object 6, serum sample 1-18 to be measured: by determinand, reagent 1 After reaction cup is added in (the coated luminous particle of antibody) and reagent 2 (antibody of biotin labeling), 37 DEG C of incubation 15min are added The general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubation 3min read RLU1, and 37 DEG C are continued to incubate 7min, RLU2 is read, and calculates amplification A=(RLU2/RLU1-1) X100% of second of signal value, as a result such as following table.
Table 6:
The concentration that the method for the present invention obtains 18 serum samples is as above: shown in upper table, first passing through the amplification ratio with calibration object 6 The sample more than upper limit of detection is relatively distinguished, i.e. A value is greater than 7.4% sample being judged as more than upper limit of detection, recommends dilution After detect；And A is non-HOOK effect sample less than 7.4%, directly can calculate concentration of specimens with calibration curve.
Changed by detectable concentration after carrying out gradient dilution to sample to verify the reliability of conclusions, by sample 1- sample This 18 carries out 2 and is diluted to dilute with 4 times, while being diluted sample with the undiluted former times sample of common detection methods detection, 2 With 4 times of diluted samples, judge whether the sample has HOOK effect, sample after even diluting by the variation of concentration after observation dilution Concentration increases as HOOK effect sample instead.Concentration can reduce non-HOOK effect sample after dilution.As a result as follows:
Table 7:
Detectable concentration increases after serum sample 16,17,18 dilutes, that is, proves HD-HOOK effect sample, serum sample 1- Concentration reduces after sample 15 dilutes, it was demonstrated that is not HD-HOOK effect sample.It is identical with the result of the method for the present invention.It is not dilute In the case where releasing sample, conventional method detects former times sample, then can judge serum sample 16,17,18 by accident because of HD-HOOK effect For relatively low concentration samples.
Embodiment 6: anti-HBs (HBsAb) verify the method for the present invention validity in detection sample
The anti-HBs detection kit produced using Bo Yang biotechnology (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 object 6, reagent 1 (the coated luminous particle of HBsAg) and reagent 2 (HBsAg of biotin labeling).
Calibration object 1- calibration object 6: the known concentration sample in conventional kit, concentration are much smaller than HOOK sample, make standard Curve calculates testing concentration.
The HBsAb of high concentration is subjected to gradient dilution, common detection methods and detection method measurement are respectively adopted The concentration value of the sample of the HBsAb containing various concentration.
Common detection methods: by calibration object 1- calibration object 6, sample to be tested 1-14, reagent 1 (HBsAg is coated shine it is micro- Grain) and after reaction cup is added in reagent 2 (HBsAg of biotin labeling), 37 DEG C of incubations 15min, (strepto- is close for the addition general liquid of LiCA With the photosensitive particulate of element label), 37 DEG C of incubation 10min, photon counter reading reads RLU, as a result as shown in the table.
Using method of reading twice of the invention: by calibration object 1- calibration object 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, the addition general liquid of LiCA (Streptavidin mark The photosensitive particulate of note), 37 DEG C of incubation 3min read RLU1, and 37 DEG C are continued to incubate 7min, read RLU2, and calculate second and believe Number value amplification A=(RLU2/RLU1-1) X100%, as a result as shown in the table,
Table 8:
Note: the detection range of HBsAb conventional detection is 0-1000mIU/ml, be 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 value Height, concentration continue to increase, and signal value is increased with HBsAb concentration and reduced, and conventional detection range is 0-1000mIU/ml, beyond inspection Surveying upper limit sample display density is > 1000mIU/ml.When concentration is increased to 335000mIU/ml or more, signal drops to calibration The signal of product 6 is hereinafter, just will appear ultrahigh concentration sample report at 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 effect shadow The relatively low concentration rung and reported.
The method of the present invention identifies the relatively low sample of reported concentrations caused by HOOK effect by reading twice.Each to test sample This successively detects signal value result RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as One of the index of judgement sample concentration.By table 8 and Fig. 8 it is found that signal value is increased to 10000mIU/ml, Zhi Houxin with concentration is continuous 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 The A value of value and calibration object, that is, can determine whether the size relation of sample to be tested concentration Yu calibration object concentration.The amplification A of sample 8~14 is equal Greater than the amplification (35.9%) of calibration object 6, show that the HBsAb concentration of sample 8~14 is all larger than 1000mIU/ml, is more than in detection Limit.This is consistent with actual concentrations, sample 12,13,14.Signal value is lower than calibration object 6, and conventional method detectable concentration is respectively 802.57mIU/ml, 352.22mIU/ml, 147.9mIU/ml, can identify it by the method for the invention is more than upper limit of detection sample This, need to be diluted detection.
Embodiment 7: the method for 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 biotechnology (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 antigen, also that is, the luminous particle of HCV antigen coat) and reagent 2 (biotin labeling HCV antigen, that is, biotin The HCV antigen of label).
Reference material, negative control, positive control: reference material is used as with reference 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 and detection method measurement Anti- containing various concentration is respectively adopted The signal value of HCV sample.
Common detection methods: by a series of Anti-HCV samples of gradient dilution, reagent 1 (shine HCV antigen, also that is, The luminous particle of HCV antigen coat) and reagent 2 (biotin labeling HCV antigen, that is, the HCV antigen of biotin labeling) addition After reaction cup, 37 DEG C of incubation 15min are added the general liquid of LiCA (photosensitive particulate of marked by streptavidin), 37 DEG C of incubations 10min, photon counter reading, reads RLU, 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 is anti-for reagent 1 Original, also that is, the luminous particle of HCV antigen coat) and reagent 2 (biotin labeling HCV antigen, that is, the HCV of biotin labeling is anti- It is former), 37 DEG C of incubation 15min are added 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, read RLU2, and calculate the amplification A=(RLU2/RLU1-1) of second of signal value X100%, as a result as shown in the table.
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 effect, 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 value of sample to be tested and the A value (- 25%) of positive control, judges sample to be tested and positive referring between Size relation, as shown above, the A value (47%) of sample 12 are much higher than the A value (- 25%) of positive control, show that time sample is dense Degree is higher than positive control, is positive sample, the not abundant of signal is because of HOOK effect, it should be diluted verifying.
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 without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.

Claims (8)

1. a kind of kit, including the coated luminous particle of first antibody (or antigen), mark the secondary antibody of substance markers (or anti- It is former), the photosensitive particulates of marker specific bond substance markers, which is characterized in that the application method of the kit includes following step It is rapid: (1) chemiluminescence immunoassay reaction, excitation and record chemistry hair to be carried out to the sample to be tested of antigen containing object to be measured (or antibody) The first time of light and second reads, and the difference amplification between second and first time reading is denoted as A, (2) according to containing to The amplification of the reading twice A ' for surveying a known standard substance of target antigen (or antibody) does critical value, and (3) will contain object to be measured The amplification A of the sample to be tested of antigen (or antibody) read twice makes comparisons with critical value, if antigen containing object to be measured (or it is anti- Body) the amplification A read twice of sample to be tested be greater than the critical value, then the concentration of the sample to be tested is higher than described known The concentration of standard substance.
2. a kind of kit, including the coated luminous particle of first antibody (or antigen), mark the secondary antibody of substance markers (or anti- It is former), the photosensitive particulates of marker specific bond substance markers, which is characterized in that the application method of the kit includes following step It is rapid: (1) chemiluminescence immunoassay reaction, excitation and record chemistry hair to be carried out to the sample to be tested of antigen containing object to be measured (or antibody) The first time of light and second reads, and the difference amplification between second and first time reading is denoted as A, (2) according to containing to The amplification of the reading twice A ' for surveying a known standard substance of target antigen (or antibody) does critical value, and (3) will contain object to be measured The amplification A of the sample to be tested of antigen (or antibody) read twice makes comparisons with critical value, if antigen containing object to be measured (or it is anti- Body) sample to be tested the amplification A read twice be greater than the critical value, and simultaneously the sample to be tested first time reading it is low In the known standard substance, then it is measured again after being diluted to sample.
3. kit according to claim 1, which is characterized in that the application method of the kit includes the following steps:
(a1) sample to be tested and first antibody (or antigen) coated luminous particle, mark of object to be measured antigen (or antibody) will be contained Secondary antibody (or antigen) mixing for remembering substance markers, incubates to obtain mixed liquor；
(a2) it reads for the first time: adding the photosensitive particulate of marker specific bond substance markers in the mixed liquor of step (a1), Exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(a3) it reads for second: after the reaction solution after progress first time reading in step (a2) is further incubated for, then carrying out Exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(a4) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (RLU2/RLU1-1) × 100%；
(a5) 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)；
(a6) the amplification A of the sample to be tested for containing object to be measured antigen (or antibody) read twice is made comparisons with critical value, such as The amplification A of the sample to be tested of fruit antigen containing object to be measured (or antibody) read twice is greater than the critical value, then described to be measured The concentration of sample is higher than the concentration of the known standard substance.
4. kit according to claim 2, which is characterized in that the application method of the kit includes the following steps:
(a1) sample to be tested and first antibody (or antigen) coated luminous particle, mark of object to be measured antigen (or antibody) will be contained Secondary antibody (or antigen) mixing for remembering substance markers, incubates to obtain mixed liquor；
(a2) it reads for the first time: adding the photosensitive particulate of marker specific bond substance markers in the mixed liquor of step (a1), Exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(a3) it reads for second: after the reaction solution after progress first time reading in step (a2) is further incubated for, then carrying out Exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(a4) it calculates second of sample and reads amplification A, A=that gained signal value reads gained signal value relative to first time (RLU2/RLU1-1) × 100%；
(a5) 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)；
(a6) the amplification A of the sample to be tested for containing object to be measured antigen (or antibody) read twice is made comparisons with critical value, such as The amplification A of the sample to be tested of fruit antigen containing object to be measured (or antibody) read twice is greater than the critical value, and described simultaneously The first time reading gained signal value of sample to be tested is surveyed after being then diluted to sample again lower than the known standard substance It is fixed.
5. kit according to claim 1, which is characterized in that the known standard substance is positive control.
6. kit according to claim 1 or 2, 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 produce.
7. kit according to claim 3 or 4, which is characterized in that in step (a2) and (a3), 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.
8. kit according to claim 1 or 2, which is characterized in that the antigen refers to the substance with immunogenicity； The antibody refers to the immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody refer to can Specifically bind to the antibody of the target antigen；First antigen and the second antigen, which refer to, can specifically bind to the target The antigen of antibody.
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