CN109470862A - Method of immunity, the system for identifying immunoassays and kit - Google Patents
Method of immunity, the system for identifying immunoassays and kit Download PDFInfo
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- 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
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
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- 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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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/636—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited using an arrangement of pump beam and probe beam; using the measurement of optical non-linear properties
- G01N2021/637—Lasing effect used for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
Abstract
The present invention relates to a kind of method of immunity of fetus alpha globulin of light-induced chemiluminescent technical field, described method includes following steps: (1) carrying out chemiluminescence immunoassay reaction to the sample to be tested containing fetus alpha globulin, 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) the first standard curve and the second standard curve are done according to the first time of the known series of standards substance containing fetus alpha globulin reading and the amplification A ' read twice respectively；(3) the amplification A read by the first time reading of the sample to be tested containing fetus alpha globulin and twice is compared with the first standard curve and the second standard curve respectively, to determine the concentration of sample.The system and a kind of kit for detecting fetus alpha globulin that the invention further relates to a kind of for identifying fetus alpha globulin immunoassays.
Description
It is on November 22nd, 2016 that the application, which is the applying date, entitled " immune application No. is 201611034237.7
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 fetus alpha globulin immunoassays side
Method, a kind of system for identifying fetus alpha globulin immunoassays and a kind of kit for detecting fetus alpha globulin.
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 the methods of optimization antibody or raising antibody concentration.
Conventional detection process has following 5 steps: determinand being added in reacting hole and reagent, the first step are incubated, added
The general liquid of LiCA, second step incubate and reading.
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, this hairs
Bright method widens detection range by reading twice, and simplicity rapidly calculates testing concentration.
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 the method includes walking as follows
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 A for surveying the first time reading of the known series of standards substance of target antigen (or antibody) and reading twice is marked respectively
Directrix curve；(3) the amplification A and mark for the first time for containing the sample to be tested of object to be measured antigen (or antibody) being read and being read twice
Directrix curve is compared, to determine the concentration of sample.According to a preferred embodiment, the method for immunity 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
Do standard curve；
(6) determine that test substance concentration is the first transition in standard curve either in last transition by A value, then will
The RLU1 of sample to be tested substitutes into its corresponding standard curve and calculates concentration.
According to a preferred embodiment, the luminous particle refers to filled with luminophor and lanthanide compound
The high molecular particle of object；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, can under red laser excitation
To generate singlet oxygen ion.
According to a preferred embodiment, in the step (2) and (3), shone with the red exciting light of 600~700nm
It penetrates, detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
According to a preferred embodiment, the antigen refers to the substance with immunogenicity；The antibody refers to machine
The immunoglobulin that can identify specific exotic that body generates；The first antibody and secondary antibody, which refer to, can specifically bind to institute
State the antibody of target antigen；First antigen and the second antigen refer to the antigen that can specifically bind to the target antibody.
The second 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 for the first time of the known series of standards substance according to antigen containing object to be measured (or antibody)
Reading and the amplification A read twice do standard curve respectively, will contain the first of the sample to be tested of object to be measured antigen (or antibody)
Secondary reading and the amplification A read twice are compared with standard curve, to determine the concentration of sample.
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) according to the amplification A of the known series of standards substance of antigen containing object to be measured (or antibody) read twice
Do standard curve；
(6) determine that test substance concentration is the first transition in standard curve either in last transition by A value, then will
The RLU1 of sample to be tested substitutes into its corresponding standard curve and calculates concentration.
The third 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, (2) according to the first time reading of the known series of standards substance of antigen containing object to be measured (or antibody) with
The amplification A read twice does standard curve respectively；(3) first time that will contain the sample to be tested of object to be measured antigen (or antibody) reads
Number and the amplification A read twice are compared with standard curve, to determine the concentration of sample.
According to a preferred embodiment, 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
Do standard curve；
(6) determine that test substance concentration is the first transition in standard curve either in last transition by A value, then will
The RLU1 of sample to be tested substitutes into its corresponding standard curve and calculates concentration.
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, simplicity rapidly calculates its concentration.
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 insulin (INS), anti-HBs
(HBsAb), fetus alpha globulin (AFP) and thyroid-stimulating hormone (TSH).
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.The difference read twice is determined by following three aspects:
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 for comparing signal twice can widen detection range, in the detection process, it is dense that simplicity rapidly calculates its
Degree.
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 size of signal, which compares, twice can distinguish HD-HOOK effect sample, the not examined scope limitation of the method, effectively
Widen 100 times of detection range or more.
(2) method of the invention can 100% correctly identify double-antibody method detection in HD-HOOK effect sample, institute
The method of stating can significantly improve the accuracy of double antibody sandwich method immunoassays, and reduce the vacation of double antibody sandwich method immunoassays
Negative rate.
(3) method of the invention is easy to operate, detection range is widened by reading twice, and in the detection process, letter
Just testing concentration is rapidly calculated.
Detailed description of the invention
Fig. 1: AFP uses the graph of relation of signal value and concentration of specimens obtained by common detection methods.
Fig. 2: AFP using first time read signal obtained by the method for the present invention and amplification A and 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, 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 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 studies and read twice
Relationship between several amplification and concentration of specimens, widens detection range by reading twice, in the detection process, simplicity is fast
Its concentration is calculated fastly.
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: fetus alpha globulin (AFP), cancer antigen 125 (CA125)
Deng.
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
(or antigen) is fixed on solid phase carrier, then reacts first antibody (or antigen) with antigen (or antibody), then with label
Two antibody (or antigen) reaction, finally carries out chemiluminescence or enzyme-linked chromogenic reaction detects 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 the general liquid of LiCA, can purchase the limited public affairs of Yu Boyang biotechnology
Department.
(4) standard items:
With the series of standards product of determined antigen (or antibody) configuration concentration range very wide (crossing over HD-HOOK effective concentration)
Solution.Standard items, reagent 1, reagent 2 are mixed, the general liquid of LiCA is added after incubation reaction, after continuing incubation reaction for a period of time
Reading (RLU1) for the first time, then second of reading (RLU2) is carried out after incubating a period of time, it calculates A=(RLU2/RLU1-1)
X100%, the amplification A read according to the RLU1 of standard items and twice do standard curve with standard concentration respectively；Standard items
RLU1 and the standard curve of concentration were shown as in the non-HD-HOOK effective stage, and RLU1 is increased with the raising of concentration, were denoted as RLU1
First transition, after concentration is increased to the HD-HOOK effective stage, RLU1 is reduced with the raising of concentration, is denoted as the decline of RLU1
Section, the A of standard items and the standard curve of concentration show as A and increase with the raising of concentration, not by HD-HOOK effects.
(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 first read to amplification A value to substitute into the standard curve of standard items A and standard concentration twice, is judged
Sample to be tested concentration is first transition or last transition in RLU1, then the RLU1 of sample to be tested is substituted into place section
Sample to be tested concentration is calculated in the standard curve of standard items RLU1 and standard concentration.
Embodiment 1: conventional method and the method for the present invention detect fetus alpha globulin (AFP) sample respectively
Fetus alpha globulin detection kit (the light activation produced using Bo Yang biotechnology (Shanghai) Co., Ltd.
Luminescence method) detect the content of AFP in sample (purchased from Fitzgerald, Catalog No:30-1370).
The AFP antigen of high concentration is subjected to gradient dilution, common detection methods are respectively adopted and detection method is surveyed
The signal value of the sample of the fixed AFP containing various concentration.
Common detection methods: the AFP sample of gradient dilution, reagent 1 (the coated luminous particle of AFP first antibody) and reagent
After reaction cup is added in 2 (the AFP secondary antibodies of biotin labeling), 37 DEG C of incubation 15min, the general liquid of LiCA is added, and (strepto- is affine
The photosensitive particulate of element label), 37 DEG C of incubation 10min, photon counter reads RLU, and the results are shown in Table 1.
Using method of reading twice of the invention: will know the determinand sample of concentration, (AFP first antibody is coated to shine reagent 1
Particle) and reagent 2 (the AFP secondary antibody of biotin labeling), 37 DEG C of incubation 15min, the addition general liquid (Streptavidin of LiCA
The photosensitive particulate of label), 37 DEG C of incubation 3min read RLU1, and 37 DEG C are continued to incubate 7min, read RLU2, and calculate second
The amplification A=(RLU2/RLU1-1) × 100% of signal value, testing result is as follows:
Table 1:
By table 1 and Fig. 1 it is found that concentration from 5ng/ml to 10000ng/ml signal value with concentration increase and increase, concentration after
Height of continuing rising, signal value are increased with AFP concentration and are reduced, i.e., concentration is greater than 10,000ng/ml then HD-HOOK, in conventional detection,
The sample report concentration that antigen concentration is higher than this detection range will be relatively low (reported concentrations are respectively less than 10,000ng/ml).
The method of the present invention widens detection range by reading twice.Each sample to be tested successively detects signal value result
RLU1, RLU2, using amplification A=(RLU2/RLU1-1) X100% of second of reading as the index of judgement sample concentration ranges
One of.By table 1 and Fig. 2 it is found that signal value is increased to 10,000ng/ml with concentration is continuous, signal value starts to increase with concentration later
And decline, but amplification A is persistently to rise with concentration.
When AFP calibration object concentration is covered from 5ng/ml to 1,000,000ng/ml range is made with the method for the present invention
RLU1 and A standard curve (such as Fig. 2), increases with concentration, and A persistently rises, the RLU1 points of risings for 5ng/ml to 10,000ng/ml
The last transition in section and 10,000ng/ml to 1,000,000ng/ml.It is detected to obtain sample to be tested with the method for the present invention
RLU1, RLU2 and A.First pass through A value determine test substance concentration be 5ng/ml to 10,000ng/ml first transition either
10,000ng/ml to 1,000,000ng/ml last transition, then the RLU1 of test substance is substituted into its corresponding standard curve
Calculate exact concentration.
As shown in Table 1, when AFP concentration is 10,000ng/ml, there are signal peak, corresponding A 18%.If determinand A <
18%, then sample to be tested is not HD-HOOK sample, its RLU1 is substituted into standard curve of the concentration less than 10,000ng/ml and is calculated
Concentration；If A >=18%, sample to be tested is HD-HOOK sample, its RLU1 is substituted into the standard that concentration is greater than 10,000ng/ml
Curve calculates concentration, has widened 1,000,000ng/ml from 10,000ng/ml to will test the upper limit.
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 (15)
1. a kind of method of immunity of fetus alpha globulin, which is characterized in that described method includes following steps: (1) right
Sample to be tested containing fetus alpha globulin carries out chemiluminescence immunoassay reaction, excites and record chemiluminescent first time and the
Rereading, and the difference amplification between second and first time reading is denoted as A；(2) according to containing fetus alpha globulin
The first time reading of known series of standards substance and the amplification A ' read twice do standard curve respectively；(3) fetus will be contained
The amplification A that the first time of the sample to be tested of alpha globulin reads and reads twice is compared with standard curve, to determine sample
This concentration.
2. method of immunity according to claim 1, which is characterized in that described method includes following steps:
(a1) by second of sample to be tested and the coated luminous particle of first antibody, label substance markers containing fetus alpha globulin
Antibody mixing, 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) standard song is done according to the amplification A ' of the known series of standards substance containing fetus alpha globulin read twice
Line；
(a6) by A value determine fetus alpha globulin in sample to be tested concentration be standard curve first transition either
In last transition, then the RLU1 of sample to be tested is substituted into its corresponding standard curve and calculates concentration.
3. 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 produce.
4. method of immunity according to claim 2, which is characterized in that in step (a2) and (a3), with 600~
The red exciting light of 700nm irradiates, and detects the transmitting light quantity of reaction solution；The Detection wavelength for emitting light is 520~620nm.
5. method of immunity according to claim 2, which is characterized in that the antibody refers to capable of identifying for body generation
The immunoglobulin of specific exotic；The first antibody and secondary antibody, which refer to, can specifically bind to the fetus first bulb egg
White antibody.
6. a kind of system for identifying fetus alpha globulin 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 excite and record chemiluminescent first time and second is read
Number, and the difference amplification between second and first time reading is denoted as A,
Processor, is used for according to first time of the known series of standards substance containing fetus alpha globulin reading and twice
The amplification A of reading does standard curve respectively, and the first time of the sample to be tested containing fetus alpha globulin is read and is read twice
Amplification A ' be compared with standard curve, to determine the concentration of sample.
7. system according to claim 6, which is characterized in that the application method of the system includes the following steps:
(1) by second of sample to be tested and the coated luminous particle of first antibody, label substance markers containing fetus alpha globulin
Antibody mixing, incubates to obtain mixed liquor；
(2) it reads for the first time: adding the photosensitive particulate of marker specific bond substance markers in the mixed liquor of step (1), temperature
Exciting light irradiation is carried out after educating 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 being swashed
It shines and irradiates and detect transmitting light quantity, 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) × 100%；
(5) standard song is done according to the amplification A ' of the known series of standards substance containing fetus alpha globulin read twice
Line；
(6) by A value determine fetus alpha globulin in sample to be tested concentration be standard curve first transition either
In last transition, then the RLU1 of sample to be tested is substituted into its corresponding standard curve and calculates concentration.
8. system according to claim 7, which is characterized in that luminous particle refers to filled with luminophor and group of the lanthanides member
The high molecular particle of plain compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, swashs in red laser
It gives, can produce singlet oxygen ion.
9. 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.
10. system according to claim 7, which is characterized in that the antibody refers to that capable of identifying for body generation is specific outer
Carry out the immunoglobulin of object；The first antibody and secondary antibody, which refer to, can specifically bind to the anti-of the fetus alpha globulin
Body.
11. a kind of kit for detecting fetus alpha globulin including the coated luminous particle of first antibody, marks substance markers
The photosensitive particulate of secondary antibody antigen, marker specific bond substance markers, which is characterized in that the application method packet of the kit
It includes following steps: (1) chemiluminescence immunoassay reaction, excitation and record chemistry being carried out to the sample to be tested containing fetus alpha globulin
Luminous first time and reading for the second time, and the difference amplification between reading for the second time first time is denoted as A, (2) basis contains
The first time reading of the known series of standards substance of fetus alpha globulin and the amplification A ' read twice do standard respectively
Curve；(3) the amplification A ' and standard curve read by the first time reading of the sample to be tested containing fetus alpha globulin and twice
It is compared, to determine the concentration of sample.
12. kit according to claim 11, which is characterized in that the application method of the kit includes following step
It is rapid:
(b1) by second of sample to be tested and the coated luminous particle of first antibody, label substance markers containing fetus alpha globulin
Antibody mixing, incubates to obtain mixed liquor；
(b2) it reads for the first time: adding the photosensitive particulate of marker specific bond substance markers in the mixed liquor of step (b1),
Exciting light irradiation is carried out after incubation and detects transmitting light quantity, and photon counter reading is calculated as RLU1；
(b3) it reads for second: after the reaction solution after progress first time reading in step (b2) is further incubated for, then carrying out
Exciting light irradiates and detects transmitting light quantity, and photon counter reading is calculated as RLU2；
(b4) 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%；
(b5) standard song is done according to the amplification A ' of the known series of standards substance containing fetus alpha globulin read twice
Line；
(b6) by A value determine fetus alpha globulin in sample to be tested concentration be standard curve first transition either
In last transition, then the RLU1 of sample to be tested is substituted into its corresponding standard curve and calculates concentration.
13. kit according to claim 12, which is characterized in that luminous particle refers to filled with luminophor and lanthanum
The high molecular particle of series elements compound；The photosensitive particulate is filled with the high molecular particle of Photoactive compounds, swashs in red
Under light excitation, singlet oxygen ion can produce.
14. kit according to claim 12, which is characterized in that in step (b2) and (b3), 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.
15. kit according to claim 12, which is characterized in that the antibody refers to that capable of identifying for body generation is specific
The immunoglobulin of exotic；The first antibody and secondary antibody, which refer to, can specifically bind to the fetus alpha globulin
Antibody.
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| US20210208080A1 (en) * | 2018-05-21 | 2021-07-08 | Beyond Diagnostics (Shanghai) Co., Ltd | Chemiluminescence analytical method and system and kit using same |
| CN112114131A (en) * | 2019-06-21 | 2020-12-22 | 博阳生物科技（上海）有限公司 | Homogeneous phase chemiluminescence detection method and application thereof |
| CN113125699B (en) * | 2019-12-31 | 2024-03-26 | 科美博阳诊断技术(上海)有限公司 | beta-hCG homogeneous phase chemiluminescence detection kit and application thereof |
| CN116429752A (en) * | 2023-02-13 | 2023-07-14 | 科美博阳诊断技术(上海)有限公司 | AFP detection kit and application method thereof |
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| CN109470860B (en) | 2021-12-10 |
| CN109470874A (en) | 2019-03-15 |
| CN108132344A (en) | 2018-06-08 |
| CN109470861A (en) | 2019-03-15 |
| CN109470860A (en) | 2019-03-15 |
| CN109470861B (en) | 2021-12-07 |
| CN108132344B (en) | 2021-06-29 |
| CN109470866A (en) | 2019-03-15 |
| CN109470874B (en) | 2022-01-04 |
| CN109470862B (en) | 2021-12-03 |
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