CN110146692A - One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit - Google Patents

One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system and detection kit Download PDF

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CN110146692A
CN110146692A CN201910449405.6A CN201910449405A CN110146692A CN 110146692 A CN110146692 A CN 110146692A CN 201910449405 A CN201910449405 A CN 201910449405A CN 110146692 A CN110146692 A CN 110146692A
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buffer
antibody
streptavidin
magnetic bead
acridinium ester
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孟令敏
阚洪晶
孙成艳
高威
何浩会
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Dirui Medical Technology Co Ltd
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    • 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
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    • 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
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • 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
    • G01N33/54326—Magnetic particles

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Abstract

The present invention provides a kind of based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system detection kit, while additionally providing the preparation process and detection method of the kit, belongs to vitro diagnostic techniques field.The reaction system of the kit includes: magnetic bead of antibody or antigen, the antibody of biotin labeling or antigen, the Streptavidin coating with carboxyl or Tosyl functional group of acridinium ester label, and the magnetic bead particle size range is 0.1-5 μm.The present invention, which is blended or adds the method for Streptavidin by using the magnetic bead of different-grain diameter and biotinylated derivative and Streptavidin MagneSphere are premixed three kinds of schemes, solves the problems, such as the interference of immunoreagent biotin.The kit detection range of linearity of the invention is wide, and the right > 0.99 of Linear Quasi, specificity is good, and high sensitivity, detectable concentration is low, and antibiotin interference performance is up to 100ng/mL or more.

Description

One kind being based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine System and detection kit
Technical field
The invention belongs to vitro diagnostic techniques fields, and in particular to one kind is based on acridinium ester chemiluminescent, Streptavidin Magnetic bead-biotin iodine system detection kit, while additionally providing the preparation process and detection method of the kit.
Background technique
Immuno analytical method experienced enzyme and exempt from analytical technology (EIA), radiating immuning analysis technology (RIA), fluorescence immunoassay point Analysis technology (FIA), Time-resolved fluoroimmunoassay (TRFIA) and chemiluminescence immunoassay technology (CLIA) several ranks Section.Wherein, CLIA is as state-of-the-art immuno analytical method, and not only high degree of automation, detection speed are fast, but also have compared with High sensitivity and analysis specificity.CLIA according to the difference of light emitting molecule, can be divided into adamantane system, Luminol, Different luminol system, ruthenium bipyridilium systems and acridinium ester system etc..Luminol, adamantane system are due to there is the participation of enzyme, easily It is influenced by factors such as environmental pH, temperature；Ruthenium bipyridyl electrochemiluminescsystem system is higher to the performance requirement of instrument and equipment, to clear It washes condition and requires harshness；Different luminol is low compared with luminol luminescence system luminous efficiency；Acridinium ester chemiluminescent system and otherization It learns luminescence system to compare, with background, low, marking process is simply, quantum yield is not reduced, fluorescent lifetime is short after label, stablizes The advantages that property is good, is now widely used in vitro diagnostic techniques.
Application of the acridinium ester in immunochemiluminometry starts from 1979, is currently applied to chemiluminescence immune assay Acridinium ester be divided into using Abbott Laboratories' chemiluminescence immunoassay reagent as the amides acridinium ester of representative and with Siemens's chemiluminescence immunoassay Reagent is two kinds of DMAE class of representative, and the two luminescent properties are close.Acridinium ester chemiluminescent is flash type, in chemiluminescence immunoassay Analysis field has advantage compared to other techniques, and acridinium ester chemiluminescent emitted luminescence intensity after starting agent 0.4s is added reaches Maximum, shine in half-life period 0.9s, 2s terminates substantially, may be implemented quickly to detect.Acridinium ester label simple process, label are anti- A step is answered to complete, luminescence process only needs under alkaline condition, can directly shine, not need other through hydrogen peroxide oxidation Shine catalyst.
Streptavidin is a kind of protein secreted by streptomycete, it can specifically bind with biotin, they Between binding force be the strongest non-covalent bond binding force being currently known.Because of this characteristic, Streptavidin-biotin is anti- System is answered to be widely used in purifying and detection field.Streptavidin exists in the form of homotetramer, often rubs Your tetrameric molecule is in combination with four moles of biotin molecule, so having signal amplification in field of immunodetection.
Summary of the invention
It is anti-based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin amplification that the object of the present invention is to provide one kind Answer system and detection kit.Detection kit sensitivity and specificity with higher.
Present invention firstly provides one kind to be based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine body System, comprising:
The antibody or antigen of acridinium ester label, the antibody of biotin labeling or antigen, Streptavidin coating have carboxyl Or the magnetic bead of Tosyl functional group；
0.1-5 μm of the magnetic bead partial size.
Preferably, the preparation method of the antibody of the acridinium ester label or antigen, comprising:
(1) it is equipped with acridinium ester mother liquor
Take antibody or antigen to be marked, before marking using phosphate buffer (PB) the replacement antibody of pH7.4 25mM or The included buffer of person's antigen, first it is dissolved into 0.1 before acridinium ester label in anhydrous n,N-Dimethylformamide (DMF)~ The acridinium ester mother liquor of 100mg/mL；
(2) acridinium ester label
Under the conditions of being protected from light, take the antibody for replacing buffer or antigen and acridinium ester mother liquor according to molar concentration 1: The ratio of (1-200) is marked, the PB that label buffer is pH7.4 25mM, and it is small that mixed label 1-12 is hanged under the conditions of being protected from light When, it purifies after reaction, obtains the antibody or antigen of acridinium ester label；
The acridinium ester is N- hydroxysuccinimide (NHS)-acridinium ester, and structural formula is as shown in Equation 1:
In formula 1, R1:R2And R3Independently selected from: H orOne of, n =1-12；
R4:M=1-12；R5:
Preferably, the preparation method of the antibody of the biotin labeling or antigen, comprising:
(1) biotin mother liquor is prepared
Antibody or antigen to be marked are taken, replaces antibody using the phosphate buffer (PB) of pH7.4 25mM before marking Or the buffer that antigen is included, first it is dissolved into 0.1 before biotin labeling in anhydrous n,N-Dimethylformamide (DMF)~ The biotin mother liquor of 100mg/mL；
(2) biotin labeling
Under the conditions of being protected from light, take the antibody for replacing buffer or antigen and biotin mother liquor according to molar concentration 1: The ratio of (1-200) is marked, and label buffer is pH7.4 25mM PB, mixed label is hanged under the conditions of being protected from light 1-12 hours, It purifies after reaction, obtains the antibody or antigen of biotin labeling；
The structural formula of the biotin is as shown in formula 2 or formula 3:
Wherein, n=1-13,
Preferably, the preparation method of magnetic bead of the Streptavidin coating with carboxyl or Tosyl functional group, Include:
When magnetic bead be carboxyl magnetic bead when, should the preparation method comprises the following steps:
Streptavidin is dissolved in 2- (N- morpholine) ethanesulfonic acid (MES) buffer, is made into concentration 1-10mg/mL's Buffer, MES pH of cushioning fluid range are 5.5~6.7；
Carboxyl magnetic bead is taken, using MES buffer solution for cleaning, the activation of EDC HCl activation buffer is then added, EDC HCl is used Amount is the 1%~100% of magnetic bead quality, and the priming reaction time is 5-60min., and supernatant is removed after the completion of activation, strepto- is added Avidin solution, Streptavidin dosage are the 0.1%~10% of magnetic bead dosage, and concentration of the magnetic bead in coating reaction solution is 1- 50mg/mL, coating the reaction time be 1~for 24 hours, coating reaction carries out at room temperature；
When magnetic bead is Tosyl functional group magnetic bead, preparation method includes:
Streptavidin is dissolved in phosphate (PB) buffer, the buffer of concentration 1-10mg/mL, PB buffering are made into Liquid pH value range is 7.0~8.0；
Then the magnetic bead for taking Tosyl functional group is added PB buffer and is resuspended, added strepto- using PB buffer solution for cleaning Avidin solution and ammonium sulfate, shake up coating 4-48h using vortex mixer under the conditions of 37 DEG C, and magnetic bead is dense in coating reaction solution Degree range is 1-50mg/mL, and Streptavidin dosage is the 0.1%~10% of magnetic bead quality, the reaction density range of ammonium sulfate For 0.1~2.5mol/L；
Streptavidin MagneSphere in the antibody or antigen of the biotin labeling and the mixture of Streptavidin MagneSphere Partial size be 1~5 μm；
The present invention provides a kind of detection kit based on above-mentioned reaction system, is named as kit A, kit A packet It includes:
Magnetic particle reaction liquid Ra1, acridinium ester label reaction solution Ra2 and biotinylated derivative reaction solution Ra3；
The magnetic particle reaction liquid Ra1 includes Streptavidin MagneSphere 1, Streptavidin MagneSphere 2, first surface activity Agent, sodium chloride, the first buffer, the first preservative and the first protein stabilizer；
Or include: Streptavidin MagneSphere 1, first surface activating agent, sodium chloride, Streptavidin, the first buffer, First preservative and the first protein stabilizer；
The partial size of the Streptavidin MagneSphere 1 is 2.5~5 μm, and the partial size of Streptavidin MagneSphere 2 is 0.1~2 μ m；
The acridinium ester label reaction solution Ra2 antibody comprising acridinium ester label or antigen, sodium chloride, the second buffering Liquid, the second preservative, second surface activating agent and the second protein stabiliser；
The biotinylated derivative reaction solution Ra3 antibody comprising biotin labeling or antigen, sodium chloride, third buffering Liquid, third preservative, third surfactant and third protein stabiliser.
The present invention provides a kind of detection kit based on above-mentioned reaction system, is named as kit B, kit B packet It includes:
Magnetic particle reaction liquid Rb1, acridinium ester label reaction solution Rb2 and project dilution Rb3；
The magnetic particle reaction liquid RbThe mixing of 1 antibody or antigen and Streptavidin MagneSphere comprising biotin labeling Object, sodium chloride, first surface activating agent, the first buffer, the first preservative and the first protein stabilizer；
Streptavidin MagneSphere in the antibody or antigen of the biotin labeling and the mixture of Streptavidin MagneSphere Partial size be 1~5 μm；
The acridinium ester label reaction solution Rb2 antibody comprising acridinium ester label or antigen, sodium chloride, the second buffering Liquid, the second preservative, second surface activating agent and the second protein stabiliser；
The project dilution Rb3 comprising sodium chloride, third buffer, third preservative, third surfactant and Third protein stabiliser.
Preferably, the first buffer, the second buffer described in kit A, B and third buffer are independent selected from 2- (N- morpholine) ethanesulfonic acid (MES), piperazine-NN- bis- (2-ethanesulfonic acids) (PIPES), 3- (N- code coffee base) -2- hydroxypropionate sodium (MOPSO), Tris- hydrochloric acid or two (2- ethoxy) imido grpup three (methylol) methane (Bistris), buffering range pH5.0- 8.5, concentration 10-600mmoL/L.
Preferably, the first preservative, the second preservative described in kit A, B and third preservative is independent is selected from NaN3, Proclin 300 or Proclin 950, mass concentration 0.01%-1%.
Preferably, first surface activating agent, second surface activating agent described in kit A, B and third surfactant It is independent selected from tween, polyethylene glycol, poloxamer, Qula be logical and choline chloride, mass concentration 0.01%-5%.
Preferably, the first protein stabilizer, the second protein stabiliser described in kit A, B and third protein stabiliser Independent one in bovine serum albumin(BSA) (BSA), casein, cow's serum Gamma globulin (BGG), egg protein, Fish protein Kind or several, mass concentration 0.1%-10%.
Preferably, the preparation of the mixture of the antibody of the biotin labeling or antigen and Streptavidin MagneSphere 3 Method, comprising:
Take Streptavidin MagneSphere 3, using the buffer solution for cleaning of PB pH7.4, be then added biotin labeling antibody or Antigen premixes 5min.-18h under the conditions of 37 DEG C, and magnetic bead reaction density range is 1-50mg/mL, premixes reaction solution as magnetic bead guarantor Liquid storage.
The principle of the present invention
Acridinium ester chemiluminescent dynamics and chemiluminescence principle are detailed in Fig. 1, H2O2Electrophilic add occurs at acridinium ester C-9 Peroxide is generated at reaction, peroxide is through the ketonic decomposition of transition state dichloroethane at the N- methylpyridone and CO of excitation state2, The former issues the photon of about λ=430nm back to ground state.Using immune response specific between antibody and antigen, change is used It learns luminescence analyzer and analyzes acridinium ester luminous signal, the quantitative or qualitative analysis of measured object may be implemented.
Biotin interference is always immunity detection reagent especially Streptavidin-biotin system immunologic function test reagent Box wants to solve the problems, such as, the people that out-patient has more than 7% takes biotin, about 7.4% blood samples of patients sample biotin Content is more than the lowest threshold 10ng/mL for the biotin interference immune detection that immunologic function test reagent platform is claimed.The present invention passes through It is blended using the magnetic bead of different-grain diameter or the method for addition Streptavidin and by biotinylated derivative and Streptavidin The three kinds of schemes of magnetic bead premixing reduce because of the biotin in sample in conjunction with magnetic bead surfaces Streptavidin occupy-place due to reagent are caused to be examined The problem of surveying the appearance deviation of result.The magnetic bead surfaces product of different-grain diameter is different with non-specific adsorption ability.The magnetic of small particle Bead surface product is bigger than large-sized magnetic bead, and Streptavidin protein load is higher, it is possible in conjunction with more biotins, thus It can reduce because biotin occupy-place bring is interfered in sample.But the magnetic bead of small particle not inhale by easy cleaning, surface non-specificity It is attached higher, influence the sensitivity of reagent.The magnetic bead blending of two kinds of partial sizes can reach optimal reagent sensitivity and and antibiotin Interference performance.The project not high to sensitivity requirement is properly added Streptavidin and is designed by specific reaction process, can also Reduce the biotin interference in sample.Biotinylated derivative and Streptavidin MagneSphere premix system because of biotin reaction object Magnetic bead surfaces are integrated to, so not will receive the biotin interference in sample.
Beneficial effects of the present invention
The present invention provides a kind of based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system Detection kit, while additionally providing the preparation process and detection method of the kit.It is total by using the magnetic bead of different-grain diameter It mixes or adds the method for Streptavidin and biotinylated derivative and Streptavidin MagneSphere are premixed into three kinds of schemes and solve The problem of immunoreagent biotin interference, the sample type that the present invention is applicable in has: disease marker, drugs, drug, cause of disease are micro- Biology, environmental contaminants.The kit detection range of linearity of the invention is wide, the right > 0.99 of Linear Quasi, specific good, sensitivity Height, detectable concentration is low, and antibiotin interference performance is up to 100ng/mL or more.The test philosophy of kit can be double antibody folder Heart method, dual-antigen sandwich method, indirect method, prize law, competition law, neutralisation, the combination being also possible between these methodologies.Root According to the standard curve of acridinium ester luminous intensity (RLU) and measured object concentration, it can realize measured object quantitative or qualitative point Analysis.
Detailed description of the invention
Fig. 1 is acridinium ester chemiluminescent dynamics of the present invention and chemiluminescence principle schematic diagram.
Specific embodiment
Present invention firstly provides one kind to be based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine body System, comprising:
The antibody or antigen of acridinium ester label, the antibody of biotin labeling or antigen, Streptavidin coating have carboxyl Or the magnetic bead of Tosyl functional group；
0.1-5 μm of the magnetic bead partial size.
According to the present invention, the antibody of the acridinium ester label or the preparation method of antigen, comprising: (1) be equipped with acridinium ester Mother liquor
Take antibody or antigen to be marked, before marking using phosphate buffer (PB) the replacement antibody of pH7.4 25mM or The included buffer of person's antigen, it is dissolved into 0.1 before acridinium ester label in anhydrous n,N-Dimethylformamide (DMF) in advance~ The acridinium ester mother liquor of 100mg/mL；
(2) acridinium ester label technique
Under the conditions of being protected from light, take the antibody for replacing buffer or antigen and acridinium ester mother liquor according to molar concentration 1: The ratio of (1-200) is marked, the PB that label buffer is pH7.4 25mM, and it is small that mixed label 1-12 is hanged under the conditions of being protected from light When, it is purified after reaction using AKTA purifying instrument, obtains the antibody or antigen of acridinium ester label；
The acridinium ester be DMAE type acridinium ester, structural formula such as formula 1:
In formula 1, R1:R2And R3Independently selected from: H andOne of, n= 1-12；
R4:M=1-12；R5:
According to the present invention, the antibody of the biotin labeling or the preparation method of antigen, comprising:
(1) biotin mother liquor is prepared
Antibody or antigen to be marked are taken, replaces antibody using the phosphate buffer (PB) of pH7.4 25mM before marking Or the buffer that antigen is included, it is dissolved into 0.1 before biotin labeling in anhydrous n,N-Dimethylformamide (DMF) in advance~ The biotin mother liquor of 100mg/mL；
(2) biotin labeling technique
Under the conditions of being protected from light, take the antibody for replacing buffer or antigen and biotin mother liquor according to molar concentration 1: The ratio of (1-200) is marked, and label buffer is pH7.4 25mM PB, mixed label is hanged under the conditions of being protected from light 1-12 hours, It is purified after reaction using AKTA purifying instrument, obtains the antibody or antigen of biotin labeling；
The structural formula of the biotin is as shown in formula 2 or formula 3:
Wherein, n=1-13；
It is prepared by the Streptavidin MagneSphere
According to the present invention, the preparation side of the magnetic bead of the Streptavidin coating with carboxyl or Tosyl functional group Method, comprising:
(1) Streptavidin is coated with carboxyl functional group magnetic bead
Streptavidin is dissolved in 10mM 2- (N- morpholine) ethanesulfonic acid (MES) buffer, concentration 1- is made into The buffer of 10mg/mL, MES pH of cushioning fluid range are 5.5~6.7；
Carboxyl magnetic bead is taken, three times using 10mM MES buffer solution for cleaning, the activation of EDC HCl activation buffer is then added, EDC HCl is being made into the concentrated solution that concentration range is 0.01-50mg/mL, EDC using preceding in 10mM MES buffer in advance HCl dosage is the 1%~100% of magnetic bead quality, and the priming reaction time is 5-60min., removes supernatant after the completion of activation, fast Solution of streptavidin is added in speed, and Streptavidin dosage is the 0.1%~10% of magnetic bead dosage, and magnetic bead is in coating reaction solution Concentration be 1-50mg/mL, coating the reaction time be 1~for 24 hours, coating reaction carries out at room temperature, reaction process use Vortex mixer mixes.After reaction three times using 10mM MES buffer solution for cleaning, it is then resuspended to Streptavidin MagneSphere and saves It is saved in liquid.
It is 25mM Tris, 0.1% tween, 0.01%BSA, 0.2% NaN that the magnetic bead, which saves liquid,3。
(2) Streptavidin is coated with Tosyl functional group magnetic bead
Streptavidin is dissolved in 10mM phosphate (PB) buffer, the buffer of concentration 1-10mg/mL, PB are made into PH of cushioning fluid range is 7.0~8.0.
Then three times using 10mM PB buffer solution for cleaning appropriate PB buffer weight is added in the magnetic bead for taking Tosyl functional group It is outstanding, appropriate solution of streptavidin and ammonium sulfate are added, shakes up coating 4-48h, sulfuric acid using vortex mixer under the conditions of 37 DEG C Ammonium is dissolved in the concentrated solution for being made into that concentration range is 2-10mol/L in 10mM PB buffer in advance, and magnetic bead is in coating reaction solution Concentration range be 1-50mg/mL, Streptavidin dosage be magnetic bead quality 0.1%~10%, the reaction density of ammonium sulfate Range is 0.1~2.5mol/L.After reaction three times using 10mM PB buffer solution for cleaning, it is then resuspended to Streptavidin Magnetic bead saves to be saved in liquid.
It is 25mM Tris, 0.1% tween, 0.01%BSA, 0.2% NaN that the magnetic bead, which saves liquid,3。
The present invention provides a kind of detection kit based on above-mentioned reaction system, is named as kit A, kit A packet It includes:
Magnetic particle reaction liquid Ra1, acridinium ester label reaction solution Ra2 and biotinylated derivative reaction solution Ra3；
The magnetic particle reaction liquid Ra1 includes Streptavidin MagneSphere 1, Streptavidin MagneSphere 2, first surface activity Agent, sodium chloride, the first buffer, the first preservative and the first protein stabilizer；It is also preferable to include blocking agents；
Or include: Streptavidin MagneSphere 1, first surface activating agent, sodium chloride, Streptavidin, the first buffer, First preservative and the first protein stabilizer；It is also preferable to include blocking agents；
The partial size of the Streptavidin MagneSphere 1 is 2.5~5 μm, and the partial size of Streptavidin MagneSphere 2 is 0.1~2 μ m；The mass concentration of the Streptavidin MagneSphere 1 and Streptavidin MagneSphere 2 is preferably 0.03%-0.1%.
The acridinium ester label reaction solution Ra2 antibody comprising acridinium ester label or antigen, sodium chloride, the second buffering Liquid, the second preservative, second surface activating agent and the second protein stabiliser；It is also preferable to include serum and blocking agent；
The biotinylated derivative reaction solution Ra3 antibody comprising biotin labeling or antigen, sodium chloride, third buffering Liquid, third preservative and third surfactant and third protein stabiliser, it is also preferable to include serum and blocking agent.
According to the present invention, the antibody or antigen of the acridinium ester label are preferably people's epididymal proteins 4 of acridinium ester label (HE4) cytokeratin 19 fragment (CYFRA21-1) antibody of antibody or acridinium ester label, the antibody of the acridinium ester label Concentration is preferably 0.1-3 μ g/mL.
According to the present invention, the antibody or antigen of the biotin labeling are preferably people's epididymal proteins 4 of biotin labeling (HE4) cytokeratin 19 fragment (CYFRA21-1) antibody of antibody or biotin labeling, the antibody of the biotin labeling Concentration is preferably 0.5-8 μ g/mL.
According to the present invention, the kit A magnetic particle reaction liquid RaStreptavidin mass concentration in 1 is preferably 0.03%-0.1%.
According to the present invention, NaCl content described in kit A is preferably 10-600mmol/L.
According to the present invention, the first buffer, the second buffer described in kit A and the preferably independent choosing of third buffer From 2- (N- morpholine) ethanesulfonic acid (MES), piperazine-NN- bis- (2-ethanesulfonic acids) (PIPES), 3- (N- code coffee base) third sulphur of -2- hydroxyl Sour sodium (MOPSO), Tris- hydrochloric acid or two (2- ethoxy) imido grpup three (methylol) methane (Bistris), buffering range are PH5.0-8.5, concentration 10-600mmoL/L, more preferably 25-400mmoL/L.
According to the present invention, the first preservative, the second preservative described in kit A and the preferred NaN of third preservative3、 Proclin300 or Proclin 950, mass concentration range are 0.01%-1%.
According to the present invention, first surface activating agent, second surface activating agent and third surfactant described in kit A It is preferred that tween, polyethylene glycol, poloxamer, Qula are led to and choline chloride, mass concentration 0.01%-5%.
According to the present invention, the first protein stabilizer, the second protein stabiliser described in kit A and third protein stabiliser are excellent It is selected as one of bovine serum albumin(BSA) (BSA), casein, cow's serum Gamma globulin (BGG), egg protein, Fish protein or several Kind, the mass concentration range of the protein stabiliser is 0.1%-10%.
According to the present invention, serum described in kit A is preferably cow's serum, human serum, sheep blood serum, and the Serology Quality is dense Degree range is 1%-50%.
According to the present invention, blocking agent described in kit A can be used mouse IGG or Roche, Scantibody, The blocking agent of Meridian commercialization, blocking agent is preferably 10-500 μ g/mL, more preferably 50-200 μ g/ using concentration range mL。
According to the present invention, the preparation method of the kit A, comprising:
Step 1: RaThe preparation of 1 reaction solution
First buffer, first surface activating agent, the first preservative and the first protein stabilizer are mixed, R is configured toa1 Basis buffer；
Streptavidin MagneSphere 1 and Streptavidin MagneSphere 2 are taken, is cleaned with the PBS buffer solution of pH7.2, then by magnetic bead It is resuspended to RaIn 1 basis buffer, it is made into the R of mass concentration 0.03%-0.1%a1 reaction solution, the PBS buffer solution include 20mmol/L PB and 150mmol/L sodium chloride；
Alternatively, by the first buffer, first surface activating agent, the first protein stabilizer, Streptavidin, NaCl and first Preservative mixing, is configured to Ra1 basis buffer；
Streptavidin MagneSphere magnetic bead 1 is taken, is cleaned with the PBS buffer solution of pH7.2, then magnetic bead is resuspended to Ra1 basis In buffer, it is made into the R of mass concentration 0.03%-0.1%a1 reaction solution.The PBS buffer solution include 20mmol/L PB with 150mmol/L sodium chloride；
Step 2 RaThe preparation of 2 reaction solutions
Sodium chloride, the second protein stabiliser, the second buffer, the second preservative and second surface activating agent are mixed, matched R is madea2 basis buffers；
The antibody of acridinium ester label is taken to be placed in RaIn 2 basis buffers, it is made into the R that concentration is 0.1-3 μ g/mLa2 reaction solutions；
Step 3 RaThe preparation of 3 reaction solutions
Sodium chloride, third protein stabiliser, third buffer, third preservative and third surfactant are mixed, matched R is madea3 basis buffers.
The antibody of biotin labeling is taken to be placed in RaIn 3 basis buffers, it is made into the R of concentration 0.5-8 μ g/mLa3 reaction solutions.
The preparation of step 4 calibration object
The preparation of calibration object is prepared according to the difference of antibody or antigen according to those skilled in the art's conventional method.
According to the present invention, the preparation method of the Streptavidin MagneSphere 1 and Streptavidin MagneSphere 2, including
Streptavidin is dissolved in 10mM MES buffer, the buffer of concentration 1-10mg/mL, MES buffering are made into Liquid pH value is 6.0.
Taking appropriate carboxyl magnetic bead, (particle size range for the magnetic bead 1 or magnetic bead 2 that the partial size of carboxyl magnetic bead is prepared as needed is selected Select, be selected from 2.5~5 μm or 0.1~2 μm), three times using 10mM MES buffer solution for cleaning, EDC HCl activation buffering is then added Liquid activation, EDC HCl are being the dense molten of 0.01-50mg/mL using the preceding concentration range that is made into 10mM MES buffer in advance Liquid, EDC HCl dosage are the 1%~100% of magnetic bead quality, and the priming reaction time is 5-60min..It is removed after the completion of activation Clear liquid is rapidly added
Solution of streptavidin, Streptavidin dosage are the 0.1%~10% of magnetic bead dosage, and magnetic bead is in coating reaction solution In concentration be 1-50mg/mL, coating the reaction time be 1~for 24 hours, coating reaction carries out at room temperature, reaction process makes It is mixed with vortex mixer.After reaction three times using 10mM MES buffer solution for cleaning, it is then resuspended to Streptavidin MagneSphere and protects It is saved in liquid storage.
It is 25mM Tris, 0.1% tween, 0.01%BSA, 0.2% NaN that the magnetic bead, which saves liquid,3。
The present invention provides a kind of detection kit based on above-mentioned reaction system, is named as kit B, kit B packet It includes:
Magnetic particle reaction liquid Rb1, acridinium ester label reaction solution Rb2 and project dilution Rb3；
The magnetic particle reaction liquid RbMixture, the chlorine of 1 antigen comprising biotin labeling and Streptavidin MagneSphere 3 Change sodium, first surface activating agent, the first buffer, the first preservative and the first protein stabilizer；It is also preferable to include serum and resistance Disconnected agent；
The grain of Streptavidin MagneSphere 3 in the antigen of the biotin labeling and the mixture of Streptavidin MagneSphere 3 Diameter is 1~5 μm；
The acridinium ester label reaction solution Rb2 include the antibody of acridinium ester label, sodium chloride, the second buffer, the Two preservatives, second surface activating agent and the second protein stabiliser；It is also preferable to include serum and blocking agent；
The project dilution Rb3 comprising sodium chloride, third buffer, third preservative, third surfactant and Third protein stabiliser, it is also preferable to include serum and blocking agent.
According to the present invention, the mixture of the antigen and Streptavidin MagneSphere 3 of biotin labeling described in kit B is preferred For thyroxine (T4) antigen and the mixture of Streptavidin MagneSphere 3 or the progesterone of biotin labeling of biotin labeling (P) mixture of antigen and Streptavidin MagneSphere 3, the antigen of the biotin labeling and mixing for Streptavidin MagneSphere 3 Closing amount of substance concentration is preferably 0.03-0.1%.
According to the present invention, the antibody of acridinium ester label described in kit B is preferably the thyroxine of acridinium ester label (T4) progesterone (P) antibody of antibody or acridinium ester label, the antibody concentration of the acridinium ester label is preferably 1-800ng/mL.
According to the present invention, the first buffer, the second buffer described in kit B and the preferably independent choosing of third buffer From 2- (N- morpholine) ethanesulfonic acid (MES), piperazine-NN- bis- (2-ethanesulfonic acids) (PIPES), 3- (N- code coffee base) third sulphur of -2- hydroxyl Sour sodium (MOPSO), Tris- hydrochloric acid or two (2- ethoxy) imido grpup three (methylol) methane (Bistris), buffering range are PH5.0-8.5, more preferable pH6.0-7.4, concentration 10-600mmoL/L, more preferably 25-400mmoL/L.
According to the present invention, the first preservative, the second preservative described in kit B and the preferred NaN of third preservative3、 Proclin 300 or Proclin 950, mass concentration range are 0.01%-1%.
According to the present invention, first surface activating agent, second surface activating agent and third surfactant described in kit B It is preferred that tween, polyethylene glycol, poloxamer, Qula are led to and choline chloride, mass concentration 0.01%-5%.
According to the present invention, the first protein stabilizer, the second protein stabiliser described in kit B and third protein stabiliser are excellent It is selected as one of bovine serum albumin(BSA) (BSA), casein, cow's serum Gamma globulin (BGG), egg protein, Fish protein or several Kind, the mass concentration range of the protein stabiliser is 0.1%-10%.
According to the present invention, serum described in kit B is preferably cow's serum, human serum, sheep blood serum, and the Serology Quality is dense Degree range is 1%-50%.
According to the present invention, blocking agent described in kit B can be used mouse IGG or Roche, Scantibody, The blocking agent of Meridian commercialization, blocking agent is preferably 10-500 μ g/mL, more preferably 50-200 μ g/ using concentration range mL。
According to the present invention, the sodium chloride concentration in Rb1, Rb2 and Rb3 reaction solution is preferably 10-600mmoL/L.
According to the present invention, the system of the mixture of the antibody or antigen and Streptavidin MagneSphere 3 of the biotin labeling Preparation Method, comprising:
Step 1: the preparation of Streptavidin MagneSphere 3
Streptavidin is dissolved in PB buffer, the Streptavidin buffer of concentration 1-10mg/mL is made into, PB is slow Fliud flushing pH value range is 7.0~8.0.
The magnetic bead of Tosyl functional group is taken, the partial size of magnetic bead is 1~5 μm, using PB buffer solution for cleaning, is then added appropriate PB buffer is resuspended, and adds solution of streptavidin and ammonium sulfate, shakes up coating 4- using vortex mixer under the conditions of 37 DEG C 48h.Ammonium sulfate is dissolved in the concentrated solution for being made into that concentration range is 2-10mol/L in 10mM PB buffer in advance, and magnetic bead is being coated with Concentration range in reaction solution is 1-50mg/mL, and Streptavidin dosage is the 0.1%~10% of magnetic bead quality, ammonium sulfate Reaction density range is 0.1~2.5mol/L.PB buffer solution for cleaning is used after reaction, is then resuspended to Streptavidin magnetic Pearl saves to be saved in liquid.It is 25mM Tris, 0.1% tween, 0.2% NaN that the magnetic bead, which saves liquid,3。
Step 2: the antibody or antigen and Streptavidin MagneSphere 3 of biotin labeling premix
Streptavidin MagneSphere 3 is taken, using the buffer solution for cleaning of PB pH7.4, the antigen of biotin labeling is then added, The antigen dosage of every milligram of corresponding biotin labeling of Streptavidin MagneSphere 3 is 20ng, and reaction is premixed under the conditions of 37 DEG C 5min.-18h, magnetic bead reaction density range are 1-50mg/mL, and premix reaction solution is that magnetic bead saves liquid.After premix, use Then the buffer solution for cleaning of PB pH7.4 is saved in liquid in magnetic bead and is saved.It is preferably 25mM Tris that the magnetic bead, which saves liquid, 0.1% tween, 0.2% NaN3。
According to the present invention, the kit B's the preparation method is as follows:
Step 1 RbThe preparation of 1 reaction solution
First buffer, sodium chloride, the first protein stabilizer, the first preservative and first surface activating agent are mixed, matched R is madeb1 basis buffer；
The mixture of the antibody of biotin labeling or antigen and Streptavidin MagneSphere 3 is placed in RbIn 1 basis buffer, It is made into the R that magnetic bead concentration is 0.03%-0.1%b1 reaction solution；
Step 2 RbThe preparation of 2 reaction solutions
Sodium chloride, the second buffer, the second preservative, the second protein stabiliser and second surface activating agent are mixed, matched R is madeb2 basis buffers；
The antibody or antigen for taking acridinium ester label are placed in RbIn 2 basis buffers, it is made into the R that concentration is 1-800ng/mLb2 Reaction solution；
Step 3 RbThe preparation of 3 reaction solutions
Sodium chloride, third buffer, third protein stabiliser, third preservative and third surfactant are mixed, matched R is madeb3 reaction solutions；
Step 4: calibration object
The preparation of calibration object is prepared according to the difference of antibody or antigen according to those skilled in the art's conventional method.
The present invention also provides the detection methods of above two kit, specifically include:
Method 1
The first step, by sample, Ra1(Rb1)、Ra3(Rb3), reaction solution sequence is added in reaction cup, is incubated for 0-25 minutes；The Cleaning solution cleaning is added in two steps；R is added in third stepa2(Rb2) it is incubated for 5-25 minutes；Cleaning solution cleaning is added in 4th step；5th step Preexciting liquid and exciting liquid are added in reaction mixture, luminescence-producing reaction is excited.It is measured and is generated using chemiluminescent analyzer Light quantity subnumber, the content of measured object is directly proportional to light quantity subnumber in sample or inverse ratio.
Method 2
The first step, by sample, Ra3(Rb3)、Ra2(Rb2) reaction solution sequence is added in reaction cup, is incubated for 0-25 minutes；The R is added in two stepsa1(Rb1) it is incubated for 5-25 minutes；Cleaning solution cleaning is added in third step；Preexciting liquid and exciting liquid are added 4th step Into reaction mixture, luminescence-producing reaction is excited.The light quantity subnumber generated, measured object in sample are measured using chemiluminescent analyzer Content is directly proportional to light quantity subnumber or inverse ratio.
Method 3
The first step, by sample, Ra1(Rb1)、Ra3(Rb3) reaction solution sequence is added in reaction cup, is incubated for 0-25 minutes；The R is added in two stepsa2(Rb2) it is incubated for 5-25 minutes；Cleaning solution cleaning is added in third step；Preexciting liquid and exciting liquid are added 4th step Into reaction mixture, luminescence-producing reaction is excited.The light quantity subnumber generated, measured object in sample are measured using chemiluminescent analyzer Content is directly proportional to light quantity subnumber or inverse ratio.
Method 4
The first step, by sample, Ra3(Rb3)、Ra1(Rb1)、Ra2(Rb2) reaction solution sequence is added in reaction cup, is incubated for 10- 30 minutes；Cleaning solution cleaning is added in second step；Preexciting liquid and exciting liquid are added in reaction mixture by third step, excitation hair Light reaction.The light quantity subnumber generated is measured using chemiluminescent analyzer, the content of measured object and light quantity subnumber are at just in sample Than or inverse ratio.
Further detailed description is done to the present invention combined with specific embodiments below.
1 kit A of embodiment
1.1 kits constitute as follows:
The preparation method of 1.2 kits
(1)RaThe preparation of 1 reaction solution
The preparation of Streptavidin MagneSphere 1 and Streptavidin MagneSphere 2 specifically:
Taking partial size is respectively the magnetic bead of 3 μm and 1 μm Tosyl functional groups, three times using 10mM PB buffer solution for cleaning, then PB buffer is added to be resuspended, adds solution of streptavidin and ammonium sulfate, shakes up coating using vortex mixer under the conditions of 37 DEG C For 24 hours, ammonium sulfate is dissolved in the concentrated solution for being made into that concentration range is 4mol/L in 10mM PB buffer in advance, and magnetic bead is anti-in coating Answering the concentration range in liquid is 20mg/mL, and Streptavidin dosage is the 2% of magnetic bead quality, the reaction density range of ammonium sulfate For 1.6mol/L.After reaction three times using 10mM PB buffer solution for cleaning, it is then resuspended to Streptavidin MagneSphere and saves liquid In save backup, obtain Streptavidin MagneSphere 1 and Streptavidin MagneSphere 2；
Streptavidin MagneSphere 1 and Streptavidin MagneSphere 2 are taken, partial size is respectively 3 μm and 1 μm, is delayed with the PBS of pH7.2 Fliud flushing is cleaned three times, and then magnetic bead is resuspended to suitable RaIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into The R of magnetic bead mass concentration 0.1%a1 reaction solution.
(2)RaThe preparation of 2 reaction solutions
A. the HE4 antibody of acridinium ester label is prepared:
Appropriate HE4 antibody is taken, is carried out with the acridinium ester (3mg/mL) being dissolved in DMF according to the ratio of molar concentration 1:3 Label, label buffer are pH7.4PB (20mmol/L), mark time 6h, and reaction process needs are protected from light.Label reaction terminates Afterwards, it is purified using AKTA purifying instrument (G25 gel column), flow velocity 3mL/min, the stoste in collecting pipe after purification uses ultraviolet point Light photometer carries out antibody and quantifies, and quantitative concentrations are 256.1 μ g/mL.
B. R is prepareda2 reaction solutions:
Take the HE4 antibody stoste of appropriate acridinium ester label in RaIn 2 basis buffers, being made into HE4 antibody concentration is 0.2 μ The R of g/mLa2 reaction solutions.
(3)RaThe preparation of 3 reaction solutions
A. the HE4 antibody of biotin labeling is prepared:
Appropriate HE4 antibody is taken, is carried out with the biotin (3mg/mL) being dissolved in DMF according to the ratio of molar concentration 1:10 Label, label buffer are pH7.4PB (20mmol/L), mark time 3h, and reaction process needs are protected from light.Label reaction terminates Afterwards, it is purified using AKTA purifying instrument (G25 gel column), flow velocity 3mL/min, the stoste in collecting pipe after purification uses ultraviolet point Light photometer carries out antibody and quantifies, and quantitative concentrations are 213.6 μ g/mL.
B. R is prepareda3 reaction solutions:
Take the HE4 antibody stoste of appropriate biotin labeling in RaIn 3 basis buffers (other ingredients in addition to antibody), match The R for being 1.4 μ g/mL at concentrationa3 reaction solutions.
The test method of 1.3 kits
The first step, by 30 μ L samples, 60 μ L Ra2、60μL Ra3 reaction solutions sequence is added in reaction cup, is incubated for 10 minutes； Second step continuously adds 50 μ L R in reaction cupa1 reaction solution is incubated for 10 minutes；Cleaning solution, cleaning five is added in third step It is secondary；Preexciting liquid and exciting liquid are added in reaction mixture by the 4th step, are excited luminescence-producing reaction, are measured the light quantum of generation Number.
The minimum detection limit reference table 1 of HE4 kit prepared by embodiment 1, linear reference table 2, repeated reference table 3, With Roche reagent clinical correlation and the anti-interference data reference table 4 of biotin.
Table 1
Table 2
Table 3
Table 4
1 kit A of comparative example formula is as follows:
The preparation method of 1.2 kits
(1)RaThe preparation of 1 reaction solution
Take Streptavidin MagneSphere 1,3 μm of partial size, cleaned three times with the PBS buffer solution of pH7.2, then by magnetic bead be resuspended to Suitable RaIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into the R of magnetic bead mass concentration 0.1%a1 reaction solution.
(2)RaThe preparation of 2 reaction solutions
With embodiment 1.
(3)RaThe preparation of 3 reaction solutions
With embodiment 1.
The test method of 1.3 kits
With embodiment 1
The minimum detection limit reference table 5 of HE4 kit prepared by comparative example 1, the anti-interference data reference table 6 of biotin.
Table 5
2 kit A of comparative example formula is as follows:
The preparation method of 1.2 kits
(1)RaThe preparation of 1 reaction solution
Take Streptavidin MagneSphere 2,1 μm of partial size, cleaned three times with the PBS buffer solution of pH7.2, then by magnetic bead be resuspended to Suitable RaIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into the R of magnetic bead mass concentration 0.1%a1 reaction solution.
(2)RaThe preparation of 2 reaction solutions
With embodiment 1.
(3)RaThe preparation of 3 reaction solutions
With embodiment 1.
The test method of 1.3 kits
With embodiment 1
The minimum detection limit reference table 6 of HE4 kit prepared by comparative example 2, the anti-interference data reference table 7 of biotin.
Table 6
Table 7
Embodiment Sample a measured value (pmol/L) Sample b measured value (pmol/L) Deviation
Embodiment 1 256.12 247.67 - 3.3%
Comparative example 1 246.10 201.53 - 18.1%
Comparative example 2 251.05 265.28 5.7%
Sample a: biotin interferes seronegativity；
Sample b: biotin interferes seropositivity, is prepared into sample b, biological cellulose content after adding biotin in sample a 100ng/mL。
From the experimental data of the embodiment of the present invention 1 and comparative example 1 and 2 it can be seen that embodiment 1 is in sensitivity and antibiont It is better than comparative example 1 and 2 in terms of element interference comprehensive performance.Can illustrate: the magnetic bead blending of two kinds of partial sizes can promote kit Detection limit and antibiotin interference performance.
2 kit A of embodiment
1.1 kits constitute as follows:
The preparation method of 1.2 kits
(1)RaThe preparation of 1 reaction solution
Streptavidin MagneSphere 1 is taken, 2.8 μm of partial size, is cleaned three times with the PBS buffer solution of pH7.2, then magnetic bead is resuspended To suitable RaIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into the R of mass concentration 0.03%a1 reaction solution.
(2)RaThe preparation of 2 reaction solutions
A. the CYFRA21-1 antibody of acridinium ester label is prepared:
Appropriate CYFRA21-1 antibody is taken, with the acridinium ester (3mg/mL) that is dissolved in DMF according to the ratio of molar concentration 1:50 Example is marked, and label buffer is pH7.4 PB (20mmol/L), marks time 12h, and reaction process needs are protected from light.Label is anti- It after answering, is purified using AKTA purifying instrument (G25 gel column), flow velocity 3mL/min, the stoste in collecting pipe after purification uses Ultraviolet specrophotometer carries out antibody and quantifies, and quantitative concentrations are 248.1 μ g/mL.
B. R is prepareda2 reaction solutions:
Take the CYFRA21-1 antibody stoste of appropriate acridinium ester label in RaIn 2 basis buffers, it is anti-to be made into CYFRA21-1 Bulk concentration is the R of 1 μ g/mLa2 reaction solutions.
(3)RaThe preparation of 3 reaction solutions
A. the CYFRA21-1 antibody of biotin labeling is prepared:
Appropriate CYFRA21-1 antibody is taken, with the biotin (3mg/mL) that is dissolved in DMF according to the ratio of molar concentration 1:40 Example is marked, and label buffer is pH7.4PB (20mmol/L), marks time 8h, and reaction process needs are protected from light.Label reaction After, it is purified using AKTA purifying instrument (G25 gel column), flow velocity 3mL/min, the stoste in collecting pipe after purification uses purple Outer spectrophotometer carries out antibody and quantifies, and quantitative concentrations are 278.6 μ g/mL.
B. R is prepareda3 reaction solutions:
Take the CYFRA21-1 antibody stoste of appropriate biotin labeling in RaIn 3 basis buffers (in addition to antibody it is other at Point), it is made into the R that concentration is 4 μ g/mLa3 reaction solutions.
The test method of 1.3 kits
The first step, by 50 μ L samples, 90 μ L Ra1、90μL Ra3 reaction solutions sequence is added in reaction cup, is incubated for 20 minutes； Second step continuously adds 70 μ L R in reaction cupa2 reaction solutions are incubated for 5 minutes；Cleaning solution is added in third step, cleans five times； Preexciting liquid and exciting liquid are added in reaction mixture by the 4th step, excite luminescence-producing reaction.The light quantity subnumber generated is measured, The content of measured object is directly proportional to light quantity subnumber in sample.
The minimum detection limit reference table 8 of CYFRA21-1 kit prepared by embodiment 2, linear reference table 9, repeatability ginseng Table 10 is examined, with Roche reagent clinical correlation and biotin anti-interference ability reference table 11.
Table 8
Table 9
Table 10
Table 11
3 kit A of comparative example
1.1 kits are constituted
The preparation method of 1.2 kits
(1)RaThe preparation of 1 reaction solution
Streptavidin MagneSphere 1 is taken, 2.8 μm of partial size, is cleaned three times with the PBS buffer solution of pH7.2, then magnetic bead is resuspended To suitable RaIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into the R of mass concentration 0.03%a1 reaction solution.
(2)RaThe preparation of 2 reaction solutions
With embodiment 2
(3)RaThe preparation of 3 reaction solutions
With embodiment 2
The test method of 1.3 kits
With embodiment 2
The object element anti-interference ability reference table 12 of CYFRA21-1 kit prepared by comparative example 3.
Table 12
Embodiment Sample c measured value (ng/mL) Sample d measured value (ng/mL) Deviation
Embodiment 2 190.01 185.02 - 2.63%
Comparative example 3 181.23 125.38 - 30.8%
Sample c: biotin interferes seronegativity；
Sample d: biotin interferes seropositivity, and biotin is added in sample c and is prepared into sample d, biological cellulose content 100ng/mL。
From the experimental data of the embodiment of the present invention 2 and comparative example 3 it can be seen that the antibiotin interference performance of embodiment 2 Better than comparative example 3.Illustrate to be properly added Streptavidin in formula, antibiotin interference performance can be promoted.
3 kit B of embodiment
1.1 kits are constituted
The preparation method of 1.2 kits
(1)RbThe preparation of 1 reaction solution
3 preparation method of Streptavidin MagneSphere
Streptavidin is dissolved in 10mM phosphate (PB) buffer, the Streptavidin for being made into concentration 2mg/mL is slow Fliud flushing, PB pH of cushioning fluid are 7.4.
The magnetic bead 3 of appropriate Tosyl functional group is taken, 1 μm of partial size, three times using 10mM PB buffer solution for cleaning, is then added suitable It measures PB buffer to be resuspended, adds Streptavidin buffer and ammonium sulfate, shake up coating using vortex mixer under the conditions of 37 DEG C 4h.Ammonium sulfate is dissolved in the concentrated solution for being made into that concentration range is 4mol/L in 10mM PB buffer in advance, and magnetic bead is reacted in coating Concentration range in liquid is 20mg/mL, and Streptavidin dosage is the 2% of magnetic bead quality, and the reaction density range of ammonium sulfate is 1.6mol/L.After reaction three times using 10mM PB buffer solution for cleaning, it is then resuspended to Streptavidin MagneSphere and saves in liquid It saves.
It is 25mM Tris, 0.1% tween, 0.2% NaN that the magnetic bead, which saves liquid,3。
It is prepared by the mixture of Streptavidin MagneSphere 3 and biotin labeling T4 antibody
Take Streptavidin MagneSphere 3, using 10mM PB pH7.4 buffer solution for cleaning three times, then be added biotin mark Remember T4 antibody, 20min. is premixed under the conditions of 37 DEG C, magnetic bead reaction density is 20mg/mL, and every milligram of Streptavidin MagneSphere is used Biotin dosage is 20ng.After premix, using 10mM PB pH7.4 buffer solution for cleaning three times, then magnetic bead save It is saved in liquid.
It is 25mM Tris, 0.1% tween, 0.2% NaN that the magnetic bead, which saves liquid,3。
In use, taking the T4 antibody of biotin labeling and the mixture of Streptavidin MagneSphere 3, buffered with the PBS of pH7.2 Liquid cleans three times, and then magnetic bead is resuspended to suitable RbIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into matter Measure the R of concentration 0.1%b1 reaction solution.
(2)RbThe preparation of 2 reaction solutions
A. the T4 antibody of acridinium ester label is prepared:
Appropriate T4 antibody is taken, is carried out with the acridinium ester (3mg/mL) being dissolved in DMF according to the ratio of molar concentration 1:100 Label, label buffer are pH7.4PB (20mmol/L), mark time 2h, and reaction process needs are protected from light.Label reaction terminates Afterwards, it is purified using AKTA purifying instrument (G25 gel column), flow velocity 3mL/min, the stoste in collecting pipe after purification uses ultraviolet point Light photometer carries out antibody and quantifies, and quantitative concentrations are 281.5 μ g/mL.
B. R is preparedb2 reaction solutions:
Take the T4 antibody stoste of appropriate acridinium ester label in RbIn 2 basis buffers, being made into T4 antibody concentration is 50ng/mL Rb2 reaction solutions.
(3)RbThe preparation of 3 reaction solutions
According to Rb3 reaction formula of liquid are prepared.
The test method of 1.3 kits
The first step, by 100 μ L samples, 70 μ L Rb3、50μL Rb1、70μL Rb2 reaction solutions sequence is added in reaction cup, incubates It educates 30 minutes；Cleaning solution is added in second step, cleans five times；Preexciting liquid and exciting liquid are added to reaction mixture by third step In, excite luminescence-producing reaction.The light quantity subnumber generated is measured using chemiluminescent analyzer, the content and light quantity of measured object in sample Subnumber is inversely proportional.
The minimum detection limit reference table 13 of FT4 kit prepared by embodiment 3, linear reference table 14, repeated reference table 15, with Siemens reagent clinical correlation reference table 16.
Table 13
Table 14
Table 15
Table 16
4 kit B of comparative example
1.1 kits are constituted
The preparation method of 1.2 kits
(1)RbThe preparation of 1 reaction solution
Streptavidin MagneSphere 3 is taken, is cleaned three times with the PBS buffer solution of pH7.2, then magnetic bead is resuspended to suitable RbIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into the R of mass concentration 0.1%b1 reaction solution.
(2)RbThe preparation of 2 reaction solutions
With embodiment 3.
(3)RbThe preparation of 3 reaction solutions
According to Rb3 reaction formula of liquid are prepared.
The test method of 1.3 kits
With embodiment 3.
The biotin anti-interference ability and embodiment 3 of FT4 kit prepared by comparative example 4 compare reference table 17.
Table 17
Embodiment Sample e measured value (ng/dL) Sample f measured value (ng/dL) Deviation
Embodiment 3 4.87 4.79 - 1.7%
Comparative example 4 4.95 5.87 18.6%
Sample e: biotin interferes seronegativity；
Sample f: biotin interferes seropositivity, and biotin is added in sample e and prepares sample f, biological cellulose content 100ng/mL。
From the experimental data of the embodiment of the present invention 3 and comparative example 4 it can be seen that 3 biotinylated derivative of embodiment and strepto- Avidin magnetic bead premixes system biotin anti-interference ability and is better than non-premix system comparative example 4.
4 kit B of embodiment
1.1 kits are constituted
The preparation method of 1.2 kits
(1)RbThe preparation of 1 reaction solution
3 preparation method of Streptavidin MagneSphere
Streptavidin is dissolved in 10mM phosphate (PB) buffer, the Streptavidin for being made into concentration 2mg/mL is slow Fliud flushing, PB pH of cushioning fluid are 7.4.
The magnetic bead of appropriate Tosyl functional group is taken, 3 μm of partial size, three times using 10mM PB buffer solution for cleaning, is then added suitable It measures PB buffer to be resuspended, adds appropriate Streptavidin buffer and ammonium sulfate, shaken up under the conditions of 37 DEG C using vortex mixer It is coated with 18h.Ammonium sulfate is dissolved in the concentrated solution for being made into that concentration range is 6mol/L in 10mM PB buffer in advance, and magnetic bead is wrapping It is 40mg/mL by the concentration range in reaction solution, Streptavidin dosage is the 4% of magnetic bead quality, the reaction density of ammonium sulfate Range is 1.8mol/L.After reaction three times using 10mM PB buffer solution for cleaning, it is then resuspended to Streptavidin MagneSphere and protects It is saved in liquid storage.
It is 25mM Tris, 0.1% tween, 0.2% NaN that the magnetic bead, which saves liquid,3。
It is prepared by the P antibody mixture of Streptavidin MagneSphere 3 and biotin labeling
Take appropriate Streptavidin MagneSphere 3, using 10mM PB pH7.4 buffer solution for cleaning three times, then be added biology The P antibody of element label, premixes 20min. under the conditions of 37 DEG C, and magnetic bead reaction density is 20mg/mL, every milligram of Streptavidin magnetic 3 biotin dosage of pearl is 100ng.After premix, using 10mM PB pH7.4 buffer solution for cleaning three times, then in magnetic bead It saves and is saved in liquid.
It is 25mM Tris, 0.1% tween, 0.1%BSA, 0.2% NaN that the magnetic bead, which saves liquid,3。
In use, the P antibody of appropriate biotin labeling and the mixture of Streptavidin MagneSphere 3 are taken, with the PBS of pH7.2 Three times, then magnetic bead is resuspended to suitable R for buffer solution for cleaningbIn 1 basis buffer (other ingredients in addition to magnetic bead), match At the R of mass concentration 0.05%b1 reaction solution.
(2)RbThe preparation of 2 reaction solutions
A. the P antibody of acridinium ester label is prepared
Appropriate P antibody is taken, is marked with the acridinium ester (3mg/mL) being dissolved in DMF according to the ratio of molar concentration 1:1 Note, label buffer are pH7.4 PB (20mmol/L), mark time 12h, and reaction process needs are protected from light.Label reaction terminates Afterwards, it is purified using AKTA purifying instrument (G25 gel column), flow velocity 3mL/min, the stoste in collecting pipe after purification uses ultraviolet point Light photometer carries out antibody and quantifies, and quantitative concentrations are 268.5 μ g/mL.
B. R is preparedb2 reaction solutions
Take the P antibody stoste of appropriate acridinium ester label in RbIn 2 basis buffers, being made into P antibody concentration is 300ng/mL Rb2 reaction solutions.
(3)RbThe preparation of 3 reaction solutions
According to Rb3 reaction formula of liquid are prepared.
The test method of 1.3 kits
The first step, by 50 μ L samples, 50 μ L Rb2 reaction solutions sequence is added in reaction cup, is incubated for 10 minutes, second step exists 50 μ L R are added in reaction cupb3、50μL Rb1 reaction solution is incubated for 10 minutes；Cleaning solution is added in third step, cleans five times；4th Step, preexciting liquid and exciting liquid are added in reaction mixture, excite luminescence-producing reaction.It is measured and is produced using chemiluminescent analyzer Raw light quantity subnumber, the content of measured object is inversely proportional with light quantity subnumber in sample.
The minimum detection limit reference table 18 of P kit prepared by embodiment 4, linear reference table 19, repeated reference table 20, With Roche reagent clinical correlation reference table 21.
Table 18
Table 19
Table 20
Table 21
5 kit B of comparative example
1.1 kits are constituted
The preparation method of 1.2 kits
(1)RbThe preparation of 1 reaction solution
Streptavidin MagneSphere 3 is taken, is cleaned three times with the PBS buffer solution of pH7.2, then magnetic bead is resuspended to suitable RbIn 1 basis buffer (other ingredients in addition to magnetic bead), it is made into the R of mass concentration 0.05%b1 reaction solution.
(2)RbThe preparation of 2 reaction solutions
With embodiment 4
(3)RbThe preparation of 3 reaction solutions
According to Rb3 reaction formula of liquid are prepared.
The test method of 1.3 kits
With embodiment 4
The anti-interference data of biotin and embodiment 4 of P kit prepared by comparative example 5 compare reference table 22.
Table 22
Sample g: biotin interferes seronegativity；
Sample h: biotin interferes seropositivity, and biotin is added in sample g and prepares sample h, biological cellulose content 100ng/mL。
From the experimental data of the embodiment of the present invention 4 and comparative example 5 it can be seen that example IV biotinylated derivative and strepto- Avidin magnetic bead premixes system biotin anti-interference ability and is better than non-premix system comparative example 5.

Claims (11)

1. one kind is based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine system, which is characterized in that packet It includes:
The antibody or antigen of acridinium ester label, the antibody of biotin labeling or antigen, Streptavidin coating with carboxyl or The magnetic bead of Tosyl functional group；
The magnetic bead partial size is 0.1-5 μm.
2. according to claim 1 a kind of based on acridinium ester chemiluminescent, Streptavidin-biotin iodine body System, which is characterized in that the antibody of the acridinium ester label or the preparation method of antigen, comprising:
(1) it is equipped with acridinium ester mother liquor
Taking antibody to be marked, perhaps the preceding phosphate buffer using pH7.4 25mM of antigen label replaces antibody or antigen certainly The buffer of band is first dissolved into the acridinium ester of 0.1~100mg/mL before acridinium ester label in anhydrous n,N-Dimethylformamide Mother liquor；
(2) acridinium ester label
Under the conditions of being protected from light, take the antibody for replacing buffer or antigen and acridinium ester mother liquor according to molar concentration 1:(1- 200) ratio is marked, the PB that label buffer is pH7.4 25mM, mixed label is hanged under the conditions of being protected from light 1-12 hours, instead It is purified after answering, obtains the antibody or antigen of acridinium ester label；
The acridinium ester is N- hydroxysuccinimide-acridinium ester, and structural formula is as shown in Equation 1:
In formula 1, R1:R2And R3Independently selected from: H orOne of, n=1-12；
R4:M=1-12；R5:
3. according to claim 1 a kind of based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine System, which is characterized in that the antibody of the biotin labeling or the preparation method of antigen, comprising:
(1) biotin mother liquor is prepared
Taking antibody to be marked, perhaps the preceding phosphate buffer for using pH7.4 25mM of antigen label replaces antibody or antigen Included buffer is first dissolved into the biology of 0.1~100mg/mL before biotin labeling in anhydrous n,N-Dimethylformamide Plain mother liquor；
(2) biotin labeling
Under the conditions of being protected from light, take the antibody for replacing buffer or antigen and biotin mother liquor according to molar concentration 1:(1- 200) ratio is marked, and label buffer is pH7.4 25mM PB, mixed label is hanged under the conditions of being protected from light 1-12 hours, instead It is purified after answering, obtains the antibody or antigen of biotin labeling；
The structural formula of the biotin is as shown in formula 2 or formula 3:
Wherein, n=1-13；
4. according to claim 1 a kind of based on acridinium ester chemiluminescent, Streptavidin MagneSphere-biotin iodine System, which is characterized in that the preparation method of the magnetic bead of the Streptavidin coating with carboxyl or Tosyl functional group, Include:
When magnetic bead be carboxyl magnetic bead when, should the preparation method comprises the following steps:
Streptavidin is dissolved in 2- (N- morpholine) ethanesulfonic acid buffer, the buffer of concentration 1-10mg/mL, 2- are made into (N- morpholine) ethanesulfonic acid buffer pH value range is 5.5~6.7；
Carboxyl magnetic bead is taken, is cleaned using 2- (N- morpholine) ethanesulfonic acid buffer, 1-3- (dimethylamino-propyl)-then is added 3- ethyl carbon dimethylamino hydrochloride (EDC HCl) activation buffer activation, EDC HCl dosage be magnetic bead quality 1%~ 100%, the priming reaction time is 5-60min, and supernatant is removed after the completion of activation, solution of streptavidin is added, strepto- is affine Plain dosage is the 0.1%~10% of magnetic bead dosage, and concentration of the magnetic bead in coating reaction solution is 1-50mg/mL, when coating is reacted Between for 1~for 24 hours, coating reaction carries out at room temperature；
When magnetic bead is Tosyl functional group magnetic bead, preparation method includes:
Streptavidin is dissolved in phosphate buffer, the buffer of concentration 1-10mg/mL, phosphate buffer pH are made into Being worth range is 7.0~8.0；
The magnetic bead for taking Tosyl functional group, is cleaned using phosphate buffer, and phosphate buffer is then added and is resuspended, adds Solution of streptavidin and ammonium sulfate shake up coating 4-48h using vortex mixer under the conditions of 37 DEG C, and magnetic bead is in coating reaction solution Concentration range be 1-50mg/mL, Streptavidin dosage be magnetic bead quality 0.1%~10%, the reaction density of ammonium sulfate Range is 0.1~2.5mol/L.
5. the detection kit based on reaction system described in claim 1, which is characterized in that be named as kit A, the reagent Box A includes:
Magnetic particle reaction liquid Ra1, acridinium ester label reaction solution Ra2 and biotinylated derivative reaction solution Ra3；
The magnetic particle reaction liquid Ra1 includes Streptavidin MagneSphere 1, Streptavidin MagneSphere 2, first surface activating agent, chlorine Change sodium, the first buffer, the first preservative and the first protein stabilizer；
It or include: Streptavidin MagneSphere 1, first surface activating agent, sodium chloride, Streptavidin, the first buffer, first Preservative and the first protein stabilizer；
The partial size of the Streptavidin MagneSphere 1 is 2.5~5 μm, and the partial size of Streptavidin MagneSphere 2 is 0.1~2 μm；
The acridinium ester label reaction solution Ra2 antibody comprising acridinium ester label or antigen, sodium chloride, the second buffer, the Two preservatives, second surface activating agent and the second protein stabiliser；
The biotinylated derivative reaction solution Ra3 antibody comprising biotin labeling or antigen, sodium chloride, third buffer, the Three preservatives, third surfactant and third protein stabiliser.
6. the detection kit based on reaction system described in claim 1, which is characterized in that be named as kit B, the reagent Box B includes:
Magnetic particle reaction liquid Rb1, acridinium ester label reaction solution Rb2 and project dilution Rb3；
The magnetic particle reaction liquid RbMixture, the chlorine of 1 antibody comprising biotin labeling or antigen and Streptavidin MagneSphere Change sodium, first surface activating agent, the first buffer, the first preservative and the first protein stabilizer；
The grain of Streptavidin MagneSphere in the antibody or antigen of the biotin labeling and the mixture of Streptavidin MagneSphere Diameter is 1~5 μm；
The acridinium ester label reaction solution Rb2 antibody comprising acridinium ester label or antigen, sodium chloride, the second buffer, the Two preservatives, second surface activating agent and the second protein stabiliser；
The project dilution Rb3 include sodium chloride, third buffer, third preservative, third surfactant and third egg White stabilizer.
7. detection kit according to claim 5 or 6, which is characterized in that the first buffer described in kit A, B, Second buffer and third buffer are independent selected from 2- (N- morpholine) ethanesulfonic acid, piperazine-NN- bis- (2-ethanesulfonic acids), 3- (N- Code coffee base) -2- hydroxypropionate sodium, Tris- hydrochloric acid or two (2- ethoxy) imido grpup three (methylol) methane, buffering range be PH5.0-8.5, concentration 10-600mmoL/L.
8. detection kit according to claim 5 or 6, which is characterized in that the first preservative described in kit A, B, Second preservative and third preservative are independent selected from NaN3, Proclin 300 or Proclin 950, mass concentration be 0.01%-1%.
9. detection kit according to claim 5 or 6, which is characterized in that the activity of first surface described in kit A, B Agent, second surface activating agent and third surfactant it is independent selected from tween, polyethylene glycol, poloxamer, Qula be logical and chlorine Change choline, mass concentration 0.01%-5%.
10. detection kit according to claim 5 or 6, which is characterized in that the first protein described in kit A, B is steady It is independent selected from bovine serum albumin(BSA), casein, cow's serum Gamma to determine agent, the second protein stabiliser and third protein stabiliser One or more of globulin, egg protein, Fish protein, mass concentration 0.1%-10%.
11. detection kit according to claim 6, which is characterized in that the antibody or antigen of the biotin labeling With the preparation method of the mixture of Streptavidin MagneSphere, comprising:
Streptavidin MagneSphere is taken, using the buffer solution for cleaning of PB pH7.4, the antibody or antigen of biotin labeling is then added, 5min.-18h is premixed under the conditions of 37 DEG C, magnetic bead reaction density range is 1-50mg/mL, and premix reaction solution is that magnetic bead saves liquid.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1143368A (en) * 1994-03-05 1997-02-19 曼海姆泊灵格股份公司 Agent for suppressing interference in immuno assaying
WO2006083305A2 (en) * 2004-06-23 2006-08-10 University Of Texas System Methods and compositions for the detection of biological molecules using a two particle complex
CN104316679A (en) * 2014-10-29 2015-01-28 南京基蛋生物科技有限公司 Application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay
CN108344869A (en) * 2018-02-05 2018-07-31 苏州长光华医生物医学工程有限公司 A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application
CN109060783A (en) * 2018-10-25 2018-12-21 成都博奥新景医学科技有限公司 A kind of single part chemical luminous immune detection method and system based on bar magnet method
CN109187973A (en) * 2018-08-17 2019-01-11 迪瑞医疗科技股份有限公司 For detecting the chemiluminescence immunoassay kit of neuronspecific enolase

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1143368A (en) * 1994-03-05 1997-02-19 曼海姆泊灵格股份公司 Agent for suppressing interference in immuno assaying
WO2006083305A2 (en) * 2004-06-23 2006-08-10 University Of Texas System Methods and compositions for the detection of biological molecules using a two particle complex
CN104316679A (en) * 2014-10-29 2015-01-28 南京基蛋生物科技有限公司 Application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay
CN108344869A (en) * 2018-02-05 2018-07-31 苏州长光华医生物医学工程有限公司 A kind of hepatitis B surface antigen chemiluminescence immune detection reagent kit and its application
CN109187973A (en) * 2018-08-17 2019-01-11 迪瑞医疗科技股份有限公司 For detecting the chemiluminescence immunoassay kit of neuronspecific enolase
CN109060783A (en) * 2018-10-25 2018-12-21 成都博奥新景医学科技有限公司 A kind of single part chemical luminous immune detection method and system based on bar magnet method

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