CN107561268A - A kind of chemiluminescence immunoassay and its application based on the mark amplification of intelligent nano luminescent particulate - Google Patents
A kind of chemiluminescence immunoassay and its application based on the mark amplification of intelligent nano luminescent particulate Download PDFInfo
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Abstract
The invention discloses a kind of chemiluminescence immunoassay based on the mark amplification of intelligent nano luminescent particulate and its application.Including by the carboxyl of the antibody covalent coupling of one plant of determined antigen to intelligent nano luminescent microparticle surfaces, forming the intelligent nano luminescent particulate with magnifying tags effect；Another strain antibody of determined antigen is directly or indirectly coated on solid phase carrier, add intelligent nano luminescent particulate and the sample containing determined antigen, by the interaction of antibody-antigen-antibody, the intelligent nano luminescent particulate immune complex of solid phase carrier antibody determined antigen antibody is formed；After isolating immune complex, excimer is added, detects luminous intensity with chemiluminescence detector so as to which the concentration of determined antigen be calculated.The present invention can improve light emitting molecule and antibody labeling efficiency, strengthen luminous intensity.So that the present invention has a high sensitivity, the advantages such as detection range is wider.
Description
Technical field
The invention belongs to chemiluminescence immunoassay technology field, is related to a kind of based on intelligent nano luminescent particulate mark
The chemiluminescence immunoassay of amplification and its application.
Background technology
Chemiluminescence immune assay (chemiluminescence immunoassay, CLIA), is by with high sensitivity
Chemical luminescent detecting technology be combined with the immune response of high specific, for various antigens, haptens, antibody, hormone,
The detection and analysis technology of enzyme, aliphatic acid, vitamin and medicine etc., be after radioimmunology analysis, enzyme exempt from analysis, fluoroimmunoassay and when
Between a newest immunoassay growing up after resolved fluorometric immunoassay.Exempt from analytic approach compared with traditional enzyme,
CLIA has the characteristics of sensitivity is higher, detection time is shorter, labeling method is simpler and cost of material is lower.Although such as
This, with the gradual popularization of analytical control technology and clinically for detecting further carrying for testing molecule sensitivity requirement
Height, there is an urgent need to systematicness can further lift sensitivity on the basis of CLIA, reduce the new technology of detection time.
Existing document report, by light emitting molecule (acridine derivatives, N- (4- aminobutyls)-N- ethyl different luminols ABEI,
Enzyme etc.) and nanoparticle covalent coupling, then with the coated microballoon mark detection antibody of light emitting molecule, detection can be greatly improved
The mark ratio of light emitting molecule on antibody, while reduce in covalent coupling operation for antigen binding site on detection antibody
Destroy.This method can greatly improve CLIA detection sensitivity.But because light emitting molecule and albumen are marked micro- simultaneously
On ball, light emitting molecule can compete the coupling functional group on microballoon with antibody, cause labeling effciency relatively low, and shiner still can
Antibody sites are blocked, cause loss of activity.The coupling functional group Limited Number of other microparticle surfaces, causes it to mark amplification effect
It is limited.
Intelligent nanoparticle is a kind of high molecular nanometer particulate that response can be produced to environmental stimuli, and environmental stimuli can be with
It is temperature, pH value, solvent, salinity, light (ultraviolet light or visible ray), electric field and chemical substance etc..According to environmental stimuli
Response condition, intelligent nanoparticle can be divided into following several：Responsive to temperature type nanoparticle, pH responsive type nanometers are micro-
Grain, photaesthesia type nanoparticle, salt density value type nanoparticle, biomolecule responsive type nanoparticle and electric field-sensitive type nanometer are micro-
Grain etc..Wherein mostly important, most commonly used is responsive to temperature type (temperature sensitive type).Illustrated below by taking temperature sensitive type nanoparticle as an example
Invention.
Temperature sensitive type nanoparticle is different to the response forms of temperature change, and this one kind is more for most common and research below
's.When minor variations occur for temperature, the change of several times or even decades of times occurs because being swelled or shrinking for the volume of nanoparticle,
Near a certain critical-temperature, or even it can occur discontinuously to be mutated, i.e., so-called Volume-phase transition.Volume is undergone mutation critical
Temperature is referred to as lower critical solution temperature (Lower Critical solution Temperature, LCST).This nanometer is micro-
Grain gel often has a certain proportion of hydrophobic and hydrophilic radical, and the change of temperature can influence hydrophobic effect between these groups
With the power of hydrogen bond action, nanoparticle structure is set to change, so as to trigger Volume-phase transition.Such nanoparticle gel exists
When reaching lowest critical solution temperature, due to being hydrated the stretching, extension of macromolecular chain, the swellbility of gel can undergo mutation formula increase, with
Increase to space.The hydrogel that PAHG and methacrylic acid, acrylic acid are formed after covalent cross-linking polymerize
It is respectively provided with heat expansion temperature sensitivity.This gel has high temperature swelling, the temperature-responsive behavior of low-temperature shrink.When reason is low temperature
Hydrogen bond is formed in gel network, volume is shunk, and hydrogen bond is dissociated during high temperature, and gel is swelled.
The content of the invention
The purpose of the present invention is the above-mentioned deficiency for prior art, there is provided one kind is based on intelligent nano luminescent particulate mark
Remember the chemiluminescence immunoassay of amplification.
It is a further object of the present invention to provide the application of this method.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of chemiluminescence immunoassay based on the mark amplification of intelligent nano luminescent particulate, including it is to be measured by one plant
The antibody covalent coupling of antigen forms the intelligence with magnifying tags effect on the carboxyl of intelligent nano luminescent microparticle surfaces
Type nano luminescent particulate；Another strain antibody of determined antigen is directly or indirectly coated on solid phase carrier, added intelligent
Nano luminescent particulate and the sample containing determined antigen, by the interaction of antibody-antigene, form solid phase carrier-antibody-and treat
Survey Ag-Ab-intelligent nano luminescent particulate immune complex；It is cleaned isolate immune complex after, add and suitable swash
Stimulating food, the critical point that immune complex produces response to it to environmental stimuli is given before detection, in intelligent nano luminescent particulate
Hydrogen bond weaken, gel collapse, shiner exposure, produce strong luminous intensity, detected with chemiluminescence detector luminous strong
Spend so as to which the concentration of determined antigen be calculated；Described intelligent nano luminescent particulate contains carboxyl selected from microparticle surfaces, micro-
The responsive to temperature type nanoparticle of grain internal package luminescent substance, pH responsive types nanoparticle, photaesthesia type nanoparticle, salt are quick
Sense type nanoparticle, biomolecule responsive type nanoparticle or electric field-sensitive type nanoparticle.
As a kind of the excellent of chemiluminescence immunoassay based on the mark amplification of intelligent nano luminescent particulate of the present invention
Choosing：A kind of chemiluminescence immunoassay based on the mark amplification of temperature-sensitive nano luminous particle, including by one plant of determined antigen
Antibody covalent coupling lights on the carboxyl on temperature-sensitive nano luminous particle surface, forming the temperature-sensitive nano with magnifying tags effect
Particulate；Another strain antibody of determined antigen is directly or indirectly coated on solid phase carrier, adds temperature-sensitive nano luminous particle
And the sample containing determined antigen, by the interaction of antibody-antigene, formed solid phase carrier-antibody-determined antigen-antibody-
Temperature-sensitive nano luminous particle immune complex；It is cleaned isolate immune complex after, addition be preheated to minimum Critical Solution
The excimer of temperature, heating immune complex temperature is to lowest critical solution temperature before detecting, in temperature-sensitive nano luminous particle
Hydrogen bond weakens, gel collapse, and shiner exposure produces strong luminous intensity, and luminous intensity is detected with chemiluminescence detector
So as to which the concentration of determined antigen be calculated；Wherein, described temperature-sensitive nano luminous particle is surface simultaneous with carboxyl and two
The Isopropyl amide base of parent's property, and particle diameter for the internal package luminescent substance of 50-200 nanometers by N-isopropylacrylamide and
The hydrogel fines that methacrylic acid or acrylic acid are formed after covalent cross-linking polymerize.
Described temperature-sensitive nano luminous particle is preferably prepared by the following method to obtain：Prepared using the method for emulsion polymerization
The P of N-isopropylacrylamide and methacrylic acid copolymer (NIPAm-co-MAA) nano-particle；Acted on simultaneously in ultrasound and stirring
Under conditions of luminescent substance be added dropwise to be dispersed with the system of P (NIPAm-co-MAA) nano-particle, make luminescent substance
Fully swelling enters in poly- P (NIPAm-co-MAA) nano-particle, obtains P (NIPAm-co-MAA)/acridinium ester composite nano-granule
Son is temperature-sensitive nano luminous particle.
Described P (NIPAm-co-MAA) nano-particle is preferably prepared by the following method：Prepare monomer isopropyl propylene
Acid amides, methacrylic acid and crosslinking agent methylene-bisacrylamide (MBA), wherein methacrylic acid and monomer isopropyl propylene
The mol ratio of acid amides dosage is 1：19 to 1：22, crosslinking agent methylene-bisacrylamide and monomer N-isopropylacrylamide dosage
Mol ratio is 1：61 to 1：65.Be pre-mixed uniformly after emulsify, emulsify uniformly after agitating and heating reaction 7-8 hours, product pass through from
P (NIPAm-co-MAA) nano-particle is obtained after the heart, washing.
Described luminescent substance preferably can produce chemiluminescent material with exciting liquid or substrate-function；It is further excellent
Select appointing in N- (4- ammonia butyl) different Rumi of-N- ethyls, acridinium ester, acridine sulfonamide, horseradish peroxidase, alkaline phosphatase
Meaning is a kind of.
When described luminescent substance is selected from acridinium ester, described lower critical solution temperature is 40 DEG C, the choosing of described excimer
Salpeter solution from the 0.2M containing 0.5wt% hydrogenperoxide steam generators and the 0.5M sodium hydroxides containing 1wt%Tween-20 are molten
Liquid.
Described solid phase carrier, which preferably is selected from magnetic particle, ELISA Plate etc., can be used as immunoassay solid support.
Antibody on covalent coupling to temperature-sensitive nano luminous particle carboxyl and directly or indirectly it is coated with solid phase carrier
Pairing of the antibody preferred pin to same antigen to be checked monoclonal antibody.
Chemiluminescence immunoassay of the present invention based on the mark amplification of temperature-sensitive nano luminous particle is in preparationization
Learn the application in electrochemiluminescent immunoassay detection kit.
A kind of chemiluminescence immune detection reagent kit, based on the change based on the mark amplification of temperature-sensitive nano luminous particle
Luminescent immunoassay is learned to prepare；Described detection kit also includes chemiluminescence exciting liquid system.
Described kit is preferably the chemiluminescence immune detection reagent kit for detecting Troponin I.
The chemiluminescence immune detection reagent kit of described detection Troponin I preferably comprises surface covalent coupling flesh calcium
The temperature-sensitive nano luminous particle of protein I monoclonal antibody 1, streptavidin-biotin system are coated with another plant of Troponin I indirectly
The sensitization magnetic particle of monoclonal antibody 2, exciting liquid 1：The salpeter solution of 0.2M containing 0.5wt% hydrogenperoxide steam generators, swash
Lotion 2：0.5M sodium hydroxide solutions containing 1wt%Tween-20；Described Troponin I monoclonal antibody 1 and flesh calcium egg
White I monoclonal antibodies 2 are pairing antibody.
Beneficial effect
The carrier that the present invention is marked based on intelligent nanoparticle as light emitting molecule, now just with most popular temperature
Illustrate exemplified by quick nanoparticle.First, obtained by NIPAm (N-isopropylacrylamide) and MAA (methacrylic acid) polymerizations
Polymer, while light emitting molecule is added to temperature-sensitive nano interparticle, surface has been obtained simultaneous with carboxyl and amphipathic
The temperature-sensitive nano luminous particle of Isopropyl amide base.On the one hand because it adds shiner, Isopropyl amide base in swelling process
When temperature is relatively low, mutual hydrogen bond action is strong, can pin shiner for gel state, can greatly promote the hair of particulate
Light thing load capacity, than traditional load capacity for improving the 1-2 order of magnitude in the method for microparticle surfaces by covalent coupling；Therefore
Intelligent nanoparticle can be as the amplification system of mark.On the other hand, the carboxyl of microparticle surfaces can be used for coupled antibody.
Due to not having the competition of shiner on microparticle surfaces, the labeling effciency of antibody can be greatly promoted, and reduces shiner to antibody
The interference of activity so that immune response progress is more abundant, and immune detection scope is expanded.
Chemiluminescence immunoassay of the present invention based on the mark amplification of intelligent nano luminescent particulate, in traditional magnetization
Learn and organically incorporate intelligent nano material strain relief shiner technology and nanoparticle in luminescence immunoassay technical foundation
The mark amplifying technique of son, it is possible to increase light emitting molecule and antibody labeling efficiency, strengthen luminous intensity so that the present invention has spirit
Sensitivity is high, the advantages such as detection range is wider.
Brief description of the drawings
The technology path schematic diagram of Fig. 1 present invention：
Wherein：1st, the anti-human CTNI monoclonal antibodies covalent coupling of mouse is on the carboxyl of temperature-sensitive nano luminous particle, and 2, another
Strain antibody is directly or indirectly coated on solid phase carrier, adds temperature-sensitive nano luminous particle and sample to be tested, forms solid phase
Carrier-antibody-determined antigen-antibody-temperature-sensitive nano luminous particle immune complex.3rd, add and be pre-heated to 40 DEG C and excite
Thing.Hydrogen bond in microballoon weakens, and gel collapse, shiner starts to expose.After shiner is exposed to nano-particle surface, it can produce
Stronger luminous intensity.
Fig. 2 is without amplification system, only polystyrene nanospheres amplification system, and the CTNI of temperature sensitive particulate mark amplification system
Examination criteria curve
The correlation of CTNI concentration and the measured value of Siemens in the serum specimen of the system measurements determination of Fig. 3 present invention
Embodiment
Below in conjunction with drawings and examples, the present invention is further illustrated, though following examples based on temperature sensitive type to be received
Illustrate the method for the present invention exemplified by the chemiluminescence immunoassay detection CTNI of rice luminous particle mark amplification, but should not
Therefore the scope of the present invention is limited.The inventive point of the present invention is luminescent substance parcel wrapping carboxylic intelligence with surface
Type nano-particle surface forms intelligent nano luminescent particulate and is used for coupled antibody, and disclosed all types of intelligence in the prior art
The preparation of energy type nanoparticle, therefore, all intelligent nanoparticles each fall within protection scope of the present invention.
The laboratory apparatus and reagent used in following examples：
A. laboratory apparatus
Heating magnetic stirring apparatus (German IKA), transmission electron microscope (Japanese JEOL), thermostat water bath (HH-60, often
Zhou Guohua Electrical Appliances Co., Ltd), constant-temperature table (Taicang science and education equipment factory), micropipettor (Thermo companies).Centrifuge
(German Eppendorf companies), Magneto separate frame (think happy chromatographic technique development centre in Tianjin) again, bag filter (Shanghai Taibo).
B. experiment reagent
CTNI standard items (Biospacific companies), (Hytest, the name of an article cTnI Antibody of CTNI monoclonal antibodies 1
[3A10A12] article No. 32-141), CTNI monoclonal antibodies 2 (BDbioscience, name of an article G265-8, article No. 554717), PBS-
T buffer solutions (KH2PO4 3.35g/L,Na2HPO4-12H2O 17.9g/L,KCl 0.2g/L,NaCl 8.77g/L,Tween-
200.5g/L, pH6.96), calf serum (Gibco companies), CTNI standard dilutions are the PBS-T containing 20% calf serum
Buffer solution, if other reagents and consumptive material are purchased from Sigma-Aldrich companies without specified otherwise.
Embodiment 1
1. the preparation of temperature-sensitive nano luminous particle：
The preparation of A.P (NIPAm-co-MAA) nano-particle
Compolymer/nano particle (the P of N-isopropylacrylamide and methacrylic acid is prepared using the method for emulsion polymerization
(NIPAm-co-MAA))：50mg lauryl sodium sulfate (SDS) and 100mg potassium peroxydisulfate are added in three-necked flask, is added
100mL deionized water dissolvings.Take monomer N-isopropylacrylamide 2mL, methacrylic acid 0.1mL and crosslinking agent di-2-ethylhexylphosphine oxide third
Acrylamide (MBA) 0.05mL, be pre-mixed uniformly after, be added in three-necked flask, emulsification uniformly after at 70 DEG C agitating and heating
Reaction 8 hours.Centrifugation (rotating speed 10000rpm, centrifuging 30min) removes liquid after collection of products, then is resuspended with deionized water, such as
This is repeated 3 times, and obtains P (NIPAm-co-MAA) nano-particle.
The preparation of B.P (NIPAm-co-MAA) nano-particle/acridinium ester light-emitting composite
100mg P (NIPAm-co-MAA) nano-particle is taken, adds 50mL deionized waters to disperse, and adds 10mg SDS,
Make system fully emulsified.Acridinium ester 1mg is dissolved in 1mL dichloromethane, under conditions of ultrasound and stirring act on simultaneously,
It is added dropwise to and is dispersed with the system of P (NIPAm-co-MAA) nano-particle, continues stirring after being added dropwise to complete and ultrasound 1 is small
When, acridinium ester is fully swelled into nano-particle.Centrifugation (rotating speed 10000rpm, centrifuging 30min) removes after collection of products
Liquid, then be resuspended with deionized water, be so repeated 3 times, obtain P (NIPAm-co-MAA)/acridinium ester composite nanoparticle, i.e., it is warm
Quick nano luminescent particulate.
CTNI monoclonal antibodies are coupled in temperature-sensitive nano luminous particle and closed with BSA by 2
A. free acridinium ester, specific steps are gone into temperature-sensitive nano luminous particle obtained above cleaning：It is above-mentioned temperature sensitive to receive
Rice luminous particle solution removes supernatant in 10 minutes in 14000 revs/min of lower high speed centrifugations, is delayed with 50mM pH=5.5 MES
Fliud flushing cleaning disperses again, is repeated twice above step.
B. the MES buffer solution EDAC and Sulfo-NHS that 50mM pH=5.5 are added in solution lights above-mentioned temperature-sensitive nano
The activated carboxylic of microparticle surfaces be NHS esters after eccentric cleaning twice.
C. the temperature-sensitive nano luminous particle after activation is weighed, is 1 according to the monoclonal antibody of coupling and the mass ratio of nanoparticle：20
Calculate the amount of CTNI monoclonal antibodies 1.CTNI monoclonal antibodies are diluted to required concentration with 50mM pH=8.0 borate buffer, slowly to
The temperature-sensitive nano luminous particle being added dropwise in the solution of CTNI monoclonal antibodies 1 after activation.After being sufficiently stirred two hours of reaction at room temperature, to
Avtive spot vacant in BSA solution closing temperature-sensitive nano luminous particle is added in system so that BSA is final dense in system
Spend for 0.25%, after being sufficiently stirred closing 20 minutes high speed centrifugation washing remove free BSA and CTNI monoclonal antibodies.
3 antibody indirects are coated on Myoglobin carrier
3.1 streptavidins are coated with the synthesis of magnetic bead
A. by buffer solution needed for the cleaning replacement of carboxyl magnetic bead.Specific steps：1ml carboxyls magnetic bead is taken to place micro- magnetic in centrifuge tube
Property separator frame on Magnetic Isolation, after about 25S, draw supernatant liquid, add 200-1000ul 50mM PH=5.5MES bufferings
Liquid is blown and beaten scattered again, is repeated twice above step.
B. the EDAC and NHS of MES preparations are added in suspension MES magnetic microsphere, is cleaned three times after centrifugation
C. weigh magnetic microsphere, be 1 according to the monoclonal antibody of coupling and the mass ratio of magnetic microsphere：20 calculate CTNI monoclonal antibodies
Amount.CTNI monoclonal antibodies are diluted to required concentration with 50mM PH=7.6 phosphate buffer, the CTNI that will slowly prepare
Monoclonal antibody is added drop-wise in scattered magnetic microsphere.Room temperature is sufficiently stirred reaction 12 hours, and BSA and glycine are added into system
The concentration of vacant site on solution closing magnetic microsphere, BSA and glycine is 0.2%, clear after being sufficiently stirred closing 1 hour
Wash, store.
3.2CTNI labeling of monoclonal antibody biotins：
It is 10mg/ml that biotin is dissolved into concentration with DMSO, is 10 according to biotin-CTNI monoclonal antibodies 2：1 molar ratio computing
The amount of monoclonal antibody 2 and biotin is calculated, after the concentration needed for 50mM PH=7.6 phosphate buffer is diluted to of CTNI monoclonal antibodies 2,
Certain density biotin is added dropwise into the CTNI monoclonal antibodies diluted.After reacting half an hour at 37 DEG C, dialysis removes uncombined
Biotin.
4. the preparation of exciting liquid system：
A. exciting liquid 1：The salpeter solution of 0.2M containing 0.5% hydrogenperoxide steam generator.
B. exciting liquid 2：0.5M sodium hydroxide solutions containing 1%Tween-20.
Embodiment 2
To confirm the significant advantage that amplification system is marked based on temperature-sensitive nano luminous particle of the present invention, we design following
Parallel contrast test.The CTNI standard concentrations gradient of comparable sodium with traditional double antibodies sandwich system, only pass through polystyrene
The mark amplification system of nanosphere, and the mark amplification system of the present invention detect respectively, and record luminous value fit standard song
Line.The concentration of CTNI used detection antibody is equal in three above system.
A. traditional CLIA double-antibody methods:
Compound concentration gradient 0,0.003,0.008,0.023,0.069,0.206,0.617 is diluted with PBS,
1.852,5.556,16.667,50.000ng/mL CTNI standard items.In reaction cup, the coated magnetic of streptavidin is added
Microballoon 10 μ L, the biotinylated μ L of mono- plant of monoclonal antibody of CTNI, 50 μ L, CTNI standard items 100, and directly marked acridine
The CTNI another kind monoclonal antibodies of derivative.After 37 DEG C of warm bath 20min, cleaned three times with PBS-T cleaning fluid Magnetic Isolations.First
Exciting liquid 1 and 2 is added afterwards, the luminous value caused by chemiluminescence detector measure various concentrations CTNI standard items.With reference to the U.S.
The Clinical Laboratory Standard committee (CLSI) issue《It is determined that the scheme of detection lower bound and quantitative test limit》(EP17-A) it is literary
Part, the quantitative test limit LOQ under this reaction system is measured.LOQ：This laboratory is according to clinical requirement setting detection CTNI
Overall error target be 20%, estimate (TE)=bias+2 × coefficient of variation (CV) of overall error.
B. polystyrene nanospheres mark amplification system：
Surface carboxyl groups polystyrene nanospheres cleaned repeatedly with 50mM pH5.5 MES buffer solutions after add EDC and
The activated carboxylic of nanometer ball surface is that eccentric cleaning is twice again after NHS esters by Sulfo-NHS.
The temperature-sensitive nano luminous particle after activation is weighed, is 1 according to the monoclonal antibody of coupling and the mass ratio of nanoparticle：20 meters
The amount of CTNI monoclonal antibodies 1 is calculated, CTNI monoclonal antibodies are diluted to required concentration with 50mM pH8.0 borate buffer, it is slowly mono- to CTNI
The nanosphere being added dropwise in anti-solution after activation.After being sufficiently stirred two hours of reaction at room temperature, BSA solution is added into system
Close avtive spot vacant on nanosphere so that ultimate densities of the BSA in system is 0.25%, is sufficiently stirred 20 points of closing
Clock can obtain the coated polystyrene nanospheres of CTNI monoclonal antibodies.
According to acridine derivatives：CTNI monoclonal antibodies are 1：20 mol ratio mark is above-mentioned to be coupled the poly- of CTNI monoclonal antibodies
Styrene nanosphere.It is slowly added dropwise after acridine derivatives are fully dissolved with DMSO and is sent out into the temperature-sensitive nano for being coupled CTNI monoclonal antibodies
In light particles solution, overnight, dialysis removes the acridine derivatives being not associated with solution for lucifuge reaction at 4 DEG C.It can obtain a word used for translation
The polystyrene nanospheres for being coated with CTNI monoclonal antibodies of pyridine ester mark, abbreviation sensitization polystyrene nanospheres.
It is 0,0.003,0.008,0.023,0.069,0.206,0.617 with PBS dilution compound concentration gradient,
1.852,5.556,16.667,50.000ng/mL CTNI standard items.In reaction cup, the coated magnetic of streptavidin is added
The μ L of microballoon 10, the biotinylated quick pipe/polyhenylethylene nano of μ L, 50ul of mono- plant of monoclonal antibody of CTNI, 50 μ L, CTNI standard items 100
Ball.After 37 DEG C of warm bath 20min, cleaned three times with PBS-T cleaning fluid Magnetic Isolations.Exciting liquid 1 and 2 is successively added, is sent out with chemistry
Luminous value caused by optical detector measure various concentrations CTNI standard items, with calculating the rate of recovery after five parameter curves.With reference to
National Committee of Clinical Laboratory Standards (CLSI) issue《It is determined that the scheme of detection lower bound and quantitative test limit》(EP17-
A) file, the quantitative test limit LOQ under this reaction system is measured.LOQ：This laboratory is according to clinical requirement setting detection
CTNI overall error target is 20%, estimate (TE)=bias+2 × coefficient of variation (CV) of overall error.
C. based on the temperature-sensitive nano luminous particle of embodiment 2 mark amplification system：
Compound concentration gradient 0,0.003,0.008,0.023,0.069,0.206,0.617 is diluted with PBS,
1.852,5.556,16.667,50.000ng/mL CTNI standard items.In reaction cup, the coated magnetic of streptavidin is added
Microballoon 10 μ L, the biotinylated μ L of mono- plant of monoclonal antibody of CTNI, 50 μ L, CTNI standard items 100, and 50ul sensitization is temperature sensitive receives
Rice luminous particle.After 37 DEG C of warm bath 20min, cleaned three times with PBS-T cleaning fluid Magnetic Isolations.Exciting liquid 1 and 2 is successively added,
The luminous value caused by chemiluminescence detector measure various concentrations CTNI standard items.Entrusted with reference to U.S. clinical Laboratory Standard
Member's meeting (CLSI) issue《It is determined that the scheme of detection lower bound and quantitative test limit》(EP17-A) file, under this reaction system
Quantitative test limit LOQ is measured.LOQ：This laboratory sets the overall error target for detecting CTNI as 20% according to clinical requirement,
The estimate (TE) of overall error=bias+2 × coefficient of variation (CV).
As a result such as table 1, shown in table 2, in the range of the 80%-120% rate of recovery, the LOQ of traditional double-antibody method is only
For 0.069ng/mL, the LOQ of the mark amplification system of polystyrene nanospheres is 0.023ng/mL, about 3 times raising.It is poly-
The high-specific surface area of styrene nanosphere allows more antibody to be coupled on nanosphere, therefore can be formed and more exempted from
Epidemic disease complex.In addition, antibody and the coated nanosphere of closed protein can provide more acridine derivatives marker sites, increase
The labelled amount of luminescent substance is added, so as to improve luminous value and sensitivity.After system of the present invention, LOQ is
0.008ng/mL, compared traditional pattern, there is more than 8.6 times of raising.Compared with the amplification body of polystyrene nanospheres
System, also there is 3 times of raising.This result confirms the mark amplification property of system of the present invention, not only realizes nanosphere
Mark amplification enhancing performance, while the characteristic of the heated swelling exposure shiner using temperature-sensitive nano luminous particle are further big
Width improves sensitivity and luminous value.
Table 1 is without amplification system, only polystyrene nanospheres amplification system, and the CTNI of temperature sensitive particulate mark amplification system
Detect the influence of the rate of recovery
Table 2 is without amplification system, only polystyrene nanospheres amplification system, and the CTNI of temperature sensitive particulate mark amplification system
The determination of the quantitative test limit of detection
CTNI concentration is related to the measured value of Siemens in the serum specimen of the system measurements determination of the present invention of embodiment 3
Property contrast experiment
To verify the accuracy of system reagent box of the present invention, more parts of serum specimens are tried with Siemens's CTNI chemiluminescence detections
After agent test CTNI contents, suitable 60 parts of wherein gradient is picked out.CTNI contents are determined respectively with the system of the present invention, are mapped
Compare correlation.As seen from Figure 3, two kinds of systems show correlation well, R2Value up to 0.9896.Confirm body of the present invention
System and according to the system kit except with hypersensitivity, outside broader detection range, also splendid accuracy.
Claims (11)
1. a kind of chemiluminescence immunoassay based on the mark amplification of intelligent nano luminescent particulate, it is characterised in that including inciting somebody to action
The antibody covalent coupling of one plant of determined antigen has magnifying tags effect on the carboxyl of intelligent nano luminescent microparticle surfaces, being formed
The intelligent nano luminescent particulate of fruit；Another strain antibody of determined antigen is directly or indirectly coated on solid phase carrier, added
Enter intelligent nano luminescent particulate and the sample containing determined antigen, by the interaction of antibody-antigene, form solid phase and carry
Body-antibody-determined antigen-antibody-intelligent nano luminescent particulate immune complex；It is cleaned isolate immune complex after,
Suitable excimer is added, the critical point that immune complex produces response to it to environmental stimuli, intelligent nanometer are given before detection
Hydrogen bond in luminous particle weakens, and gel collapse, shiner exposure, produces strong luminous intensity, uses chemiluminescence detector
Luminous intensity is detected so as to which the concentration of determined antigen be calculated；Described intelligent nano luminescent particulate contains selected from microparticle surfaces
There is carboxyl, interparticle wraps up responsive to temperature type nanoparticle, pH responsive types nanoparticle, the photaesthesia type nanometer of luminescent substance
Particulate, salt density value type nanoparticle, biomolecule responsive type nanoparticle or electric field-sensitive type nanoparticle.
2. the chemiluminescence immunoassay according to claim 1 based on the mark amplification of intelligent nano luminescent particulate,
It is characterized in that including by the carboxyl of the antibody covalent coupling of one plant of determined antigen to responsive to temperature type nano luminescent microparticle surfaces
On, form the responsive to temperature type nano luminescent particulate with magnifying tags effect；By another strain antibody of determined antigen directly or
Person is coated on solid phase carrier indirectly, adds responsive to temperature type nano luminescent particulate and the sample containing determined antigen, by anti-
The interaction of body-Ag-Ab, form solid phase carrier-antibody-determined antigen-antibody-responsive to temperature type nano luminescent particulate
Immune complex；It is cleaned isolate immune complex after, add and be preheated to the excimer of lowest critical solution temperature, detection
To lowest critical solution temperature, the hydrogen bond in responsive to temperature type nano luminescent particulate weakens preceding heating immune complex temperature, coagulates
Glue collapses, shiner exposure, produces strong luminous intensity, detects luminous intensity with chemiluminescence detector so as to be calculated
The concentration of determined antigen；Wherein, described responsive to temperature type nano luminescent particulate is surface simultaneous with carboxyl and amphipathic
Isopropyl amide base, and particle diameter for 50-200 nanometers internal package luminescent substance by N-isopropylacrylamide and methyl-prop
The hydrogel fines that olefin(e) acid or acrylic acid are formed after covalent cross-linking polymerize.
3. chemiluminescence immunoassay according to claim 2, it is characterised in that described responsive to temperature type nanometer hair
Light particles are prepared by the following method to obtain：N-isopropylacrylamide and methacrylic acid are prepared using the method for emulsion polymerization
Compolymer/nano particle P (NIPAm-co-MAA)；Luminescent substance is added dropwise under conditions of ultrasound and stirring act on simultaneously
It is dispersed with compolymer/nano particle P (NIPAm-co-MAA) system, luminescent substance is fully swelled into compolymer/nano particle P
(NIPAm-co-MAA) in, it is responsive to temperature type nano luminescent to obtain P (NIPAm-co-MAA)/acridinium ester composite nanoparticle
Particulate.
4. chemiluminescence immunoassay according to claim 2, it is characterised in that described luminescent substance is can be with
Exciting liquid or substrate-function produce chemiluminescent material；It is preferred that N- (4- ammonia butyl) different Rumi of-N- ethyls, acridinium ester, a word used for translation
Any one in pyridine sulfonamide, horseradish peroxidase, alkaline phosphatase.
5. chemiluminescence immunoassay according to claim 4, it is characterised in that described luminescent substance is selected from acridine
During ester, described lowest critical solution temperature is 40 DEG C, and described excimer, which is selected from, contains 0.5wt% hydrogenperoxide steam generators
0.2M salpeter solution and the 0.5M sodium hydroxide solutions containing 1wt%Tween-20.
6. chemiluminescence immunoassay according to claim 2, it is characterised in that described solid phase carrier is selected from magnetic
Particulate, ELISA Plate.
7. chemiluminescence immunoassay according to claim 1 or 2, it is characterised in that covalent coupling is to can only responsive type
Antibody on nano luminescent particulate carboxyl and the antibody being directly or indirectly coated with solid phase carrier are for same to be checked anti-
The monoclonal antibody of former pairing.
8. the chemiluminescence immunoassay point based on the mark amplification of intelligent nano luminescent particulate any one of claim 1-7
Application of the analysis method in chemiluminescence immune detection reagent kit is prepared.
A kind of 9. chemiluminescence immune detection reagent kit, it is characterised in that based on described in claim any one of 1-7 based on intelligence
It is prepared by the chemiluminescence immunoassay of energy type nano luminescent particulate mark amplification；Described detection kit is also comprising chemistry hair
Light exciting liquid system.
10. kit according to claim 9, it is characterised in that described kit is the chemistry of detection Troponin I
Electrochemiluminescent immunoassay detection kit.
11. kit according to claim 9, it is characterised in that the chemiluminescence immunoassay of described detection Troponin I
Detection kit includes the surface covalent coupling responsive to temperature type nano luminescent particulate of Troponin I monoclonal antibody 1, chain parent
It is coated with the sensitization magnetic particle of another plant of Troponin I monoclonal antibody 2, exciting liquid 1 indirectly with element-biotin system：Contain
The 0.2M of 0.5wt% hydrogenperoxide steam generators salpeter solution, exciting liquid 2：0.5M sodium hydroxides containing 1wt%Tween-20 are molten
Liquid；Described Troponin I monoclonal antibody 1 and Troponin I monoclonal antibody 2 are pairing antibody.
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| CN109470690A (en) * | 2018-10-19 | 2019-03-15 | 浙江大学 | The antigen detection method of electrochemical luminescence is differentiated based on current potential |
| CN110567928A (en) * | 2019-09-30 | 2019-12-13 | 上海交通大学 | multi-element detection method based on quantum dot fluorescent nanospheres |
| CN113125422A (en) * | 2021-04-16 | 2021-07-16 | 合肥工业大学 | Preparation method of chemiluminescent hydrogel microspheres, prepared hydrogel microspheres and application thereof |
| CN114504727A (en) * | 2022-04-02 | 2022-05-17 | 广州纳丽生物科技有限公司 | Polydopamine photothermal conversion effect microneedle and preparation method thereof |
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| WO2017081152A1 (en) * | 2015-11-10 | 2017-05-18 | Administracion General De La Comunidad Autonoma De Euskadi | Diagnostic methods and devices |
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| CN105556289A (en) * | 2013-10-02 | 2016-05-04 | 古河电气工业株式会社 | Method for detecting target substance |
| WO2017081152A1 (en) * | 2015-11-10 | 2017-05-18 | Administracion General De La Comunidad Autonoma De Euskadi | Diagnostic methods and devices |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109470690A (en) * | 2018-10-19 | 2019-03-15 | 浙江大学 | The antigen detection method of electrochemical luminescence is differentiated based on current potential |
| CN110567928A (en) * | 2019-09-30 | 2019-12-13 | 上海交通大学 | multi-element detection method based on quantum dot fluorescent nanospheres |
| CN110567928B (en) * | 2019-09-30 | 2021-09-28 | 上海交通大学 | Multi-element detection method based on quantum dot fluorescent nanospheres |
| CN113125422A (en) * | 2021-04-16 | 2021-07-16 | 合肥工业大学 | Preparation method of chemiluminescent hydrogel microspheres, prepared hydrogel microspheres and application thereof |
| CN114504727A (en) * | 2022-04-02 | 2022-05-17 | 广州纳丽生物科技有限公司 | Polydopamine photothermal conversion effect microneedle and preparation method thereof |
| CN114504727B (en) * | 2022-04-02 | 2022-08-23 | 广州纳丽生物科技有限公司 | Polydopamine photothermal conversion effect microneedle and preparation method thereof |
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