CN104316679A - Application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay - Google Patents
Application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay Download PDFInfo
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Abstract
The invention discloses application of hyperbranched polyglycerol modified magnetic nanoparticle microspheres in chemiluminescence immune assay. By utilizing the hyperbranched polyglycerol modified magnetic nanoparticle microspheres, a nanometer microsphere material of which the surface is hydrophilic and which is enriched in functional groups is prepared. When a chemiluminescence diagnostic agent based on the hyperbranched polyglycerol modified magnetic nanoparticle microspheres is actually used, the reagent can well inhibit nonspecific adsorption of proteins, and the antibody coupling amount is 2-3 times higher than that of commercial magnetic microspheres. In the project-based performance analysis, the diagnostic agent based on the magnetic microspheres can greatly enhance the reaction between antigens and antibodies. When the reagent is compared with a commercial magnetic microsphere reagent, the sensitivity is improved by 4-5 times, and the upper detection limit is improved by 1.3 times, so that the detection range is improved by nearly 6.5 times.
Description
Technical field
The invention belongs to biological detection reagent field, relate to the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay.
Background technology
Chemiluminescence immunoassay is by the immune response analytical technology of high specific, and chemiluminescence reaction system and super-sensitive luminescence assays technology combine, for the detection analytical technology of various antigen, haptens, antibody, enzyme, hormone and medicine etc.It is the up-to-date immunoassay grown up after radiating immuning analysis technology, enzyme-linked immuno assay technology, fluoroimmunoassay technology and Time-resolved fluoroimmunoassay.
Chemiluminescence immune assay comprises two parts, i.e. immune response system and chemiluminescence system.Immune response system utilizes immunoreactive high specific principle, is out also combined with specific label by thing to be detected separating trap from the material of sample complexity to be detected is formed; Chemiluminescence system utilizes specific process that label is participated in redox reaction and goes, the oxidation of oxidized dose of label, forms the intermediate of an excited state, and the intermediate of excited state is also unstable, launches photon soon and gets back to ground state.The photon launched changes electric signal into by photoelectric effect and carries out following digital process after being received by light signal measuring apparatus.Immune response system constructs the relation of test substance and label, and chemiluminescence system constructs the relation of label content and electric signal, and the amount of test substance can be converted into electric signal thus by computing by both connected applications.
Chemiluminescence immune analysis method has the advantages such as highly sensitive, high specificity, the range of linearity are wide due to it, is day by day subject to the favor of people, is used widely in the field such as life science, clinical medicine and environment, food, medicine.Detect with regard to reagent performance own with regard to chemiluminescence immune assay, sensitivity and linear measurement range is wherein most important two performance parameters.This is because more and more need in actual applications to detect low enrichment analysis matter sample, developing high-sensitive chemiluminescence immune analysis method, is the important directions of immune analysis method development in recent years.The testing result of the simultaneously wider range of linearity will provide to clinical detection and help: the 1. accurate location of the different phase (early stage, mid-term, late period) of disease, thus can formulate more specificity, more targetedly therapeutic scheme by assist physician; 2. can carry out immediate analysis to the result for the treatment of, make medical personnel to pinpoint the problems in early days and to adjust therapeutic scheme in time, avoid bringing larger Operative risk and economic pressures to patient.
In the kit developing process of reality, the optimization of lowest detectable limit and sensing range widen two paradox often.The method of conventional optimization lowest detectable limit is general all to sacrifice upper limit of detection for cost, such as: the lowest detectable limit 1. by regulating Nano microsphere particle diameter to optimize kit, less particle diameter will provide larger antibody bonded area under equal quality concentration, thus contributes to the range of linearity widening immunoassay.Larger particle diameter is by the performance of raising reaction system under the tested effluent condition of low concentration, lowest detectable limit can be optimized, but the increase of particle diameter can bring the decline of solid phase carrier total surface area, cause the antibody amount be combined on carrier to decline, thus affect upper limit of detection; 2. optimize lowest detectable limit by relatively reducing the package amount of antibody on solid phase carrier, but and upper one similar, the minimizing of antibody amount can sacrifice upper limit of detection equally; 3. carry out the relative content of analysans in increase system by increasing sample application amount thus optimize lowest detectable limit, but after the lifting of determinand content, upper limit of detection must show decline in the analysis of high concentration sample.In a word, if will by only optimizing a condition, the performance of Optimization analyses reagent in lowest detectable limit and upper limit of detection two indices simultaneously, conventional method be almost helpless.In actual development process, simultaneously traditional scheme acts on contrary variable from two optimizing, and gets compromise effect.Therefore these Optimization Works will inevitably be unusual elapsed times and do not have optimum efficiency.
Magnetic microsphere is as component---solid phase carrier crucial in chemiluminescence immunoassay system, and itself character can play very important impact for the result of immunoassay.At present conventional commercialization magnetic microsphere is made up of polystyrene shell and superparamagnetism magnetic fluid kernel, and functional group (such as: amino, carboxyl etc.) is by the method introducing of copolymerization, grafting or parcel.The magnetic microsphere of this mode has following two remarkable defects: first, polystyrene is a kind of very hydrophobic material, direct application inevitably can produce the consequence of non-specific adsorption, this can cause the impurity in sample to be also adsorbed on microsphere surface, the interference of impurity not only can increase the background values of reflection, causes the fluctuation of reacting baseline, even can cause false positive or false-negative result time serious, directly have influence on the confidence level of detection, form mistaken diagnosis or fail to pinpoint a disease in diagnosis; The second, no matter be the functional group which kind of mode above-mentioned is introduced, its density all can not break through the maximum theory density of monolayer surface, thus limits antibody coupling amount thereon.
Strengthening signal and reducing noise is two important steps optimizing chemiluminescence immunoassay reagent, reduce noise and mainly on solid phase carrier, cover layer of substance, after this layer of material occupies most site on microballoon, can non-specific adsorption be effectively reduced, but its generation can not be stopped completely.Normally used material is BSA albumen, casein, gelatin, but research finds that the effect of this kind of material is unsatisfactory, and simultaneously in order to seek best experiment condition, experiment gropes also to need to expend huge manpower and time.Strengthen signal and mainly concentrate on signal probe amplification aspect, this just requires that nano particle can load marker as much as possible, even can use such as biotin---the process of this magnifying tags of Streptavidin.
Polyglycol (PEG) is the hydroaropic substance used in biological medicine direction widely, the feature of its biocompatibility and nonimmunogenic to make at it in a lot of field as material modified use, the such as modification of antibody, albumen, nucleotide, liposome, the surface modification etc. of polymkeric substance and polymer microballoon.In immunoassay, the PEG of nanoparticle surface can effectively reduce non-specific adsorption, reduces background values, improves the sensitivity of reaction, reduces false positive and false negative result.But linear PEG polymkeric substance is owing to only having functional group in polymer end base section in its structure, therefore one or two avtive spots are only had to be available for coupling in whole polymer architecture, binding ability is more weak, and coupling efficiency is not high, and this point limits it and applies widely.
Different from linear polymer, dissaving polymer (degree of branching is at least more than or equal to 2) comprises more functional group in structure bifurcation.Have good dissolubility as hyperbranched poly unification, after dissolving, viscosity is lower also better with mobility, uses simple and convenient.Moreover dissaving polymer synthetic method is also fairly simple, be mainly divided into three kinds: the first, utilize multi-functional monomer polycondensation to prepare; The second, in the polymer precursor prepared, carry out grafting and modifying, the 3rd, utilize the ring-opening reaction of epoxy to prepare hyperbranched structure.
Hyperbranched polyglycidyl ether is a kind of special dissaving polymer, there is the backbone structure of similar polyglycol, branched structure is rich in hydroxyl, and this part hydroxyl, on the one hand for chemical coupling provides a large amount of binding site, brings good hydrophilicity to polymkeric substance again on the other hand.These design features make hyperbranched polyglycidyl ether have a large amount of functional end-groups, have good biocompatibility, highly dissoluble and high rheological variation.Therefore from strengthening signal and reducing the performance that these two aspects of noise all improve reagent, remarkable using value is had from making it at biological reagent.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay is provided.
Another object of the present invention is to provide a kind of kit containing hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere.
Object of the present invention realizes by following technical scheme:
The application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay.
The application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, comprise following methods: be coated on nano-magnetic microsphere surface by amino effect after hyperbranched polyglycidyl ether is modified and form hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere, the carboxyl of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere is activated to the molecular detection of rear coupling energy and testing molecule specific binding, define hyperbranched polyglycidyl ether nanometer Ci Wei Qiu ?molecular detection compound solid phase carrier, by hyperbranched polyglycidyl ether nanometer Ci Wei Qiu ?molecular detection compound solid phase carrier and sample to be tested and the upper luminescent substance of mark can with the capture molecules hybrid reaction of testing molecule specific binding after, define hyperbranched polyglycidyl ether nanometer Ci Wei Qiu ?molecular detection compound Gu phase Zai Ti ?wait survey point Zi ?catch point Zi ?the compound of luminescent substance, compound is isolated through cleaning, add excimer wherein, luminescent substance in compound is subject to exciting generation chemiluminescence, finally detect with chemiluminescence detector the concentration that luminous intensity calculates the target analytes in sample to be tested.
The described application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, described nano-magnetic microsphere size is 1 μm ~ 10 μm.
The described application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, described hyperbranched polyglycidyl ether is polymerized by trimethylolpropane and diglycidyl; The described succinimidyl carbonate that is modified to is modified.
The described application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, described molecular detection and the coupling mode of nano-magnetic microsphere comprise the modes such as carboxyl coupling, amino coupled, hydroxyl coupling, aldehyde radical coupling, chloromethyl coupling, sulfydryl coupling.
The described application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, the test substance of detection can be antigen, antibody, incomplete antibody, haptens, small molecule chemicals, metallic element etc.; Molecular detection be can with the antibody of test substance specific binding, antigen, haptens, incomplete antibody, Small molecular etc.; Capture molecules be can with the material of testing molecule or molecular detection or both compound specific bindings.
The described application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, described luminescent substance be selected from comprise N ?(4 ?ammonia butyl) ?N ?the different luminol of ethyl, acridinium ester, acridine sulfonamide, horseradish peroxidase, alkaline phosphatase etc.
The described application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay, described excimer be selected from containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L salpeter solution, containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L sulfuric acid solution, containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L hydrochloric acid solution, containing 0.5% ?5%Tween ?20 0.1 ?1mol/L sodium hydroxide solution, containing 0.5% ?5%Tween ?80 0.1 ?1mol/L sodium hydroxide solution, or containing 0.5% ?5%TritonX ?100 0.1 ?1mol/L sodium hydroxide solution.
A kind of chemiluminescence immune detection reagent kit, comprise hyperbranched polyglycidyl ether decorated nanometer Ci Wei Qiu of the present invention ?molecular detection compound, chemiluminescence exciting liquid system, connect the capture molecules of luminescent substance.
Wherein, described chemiluminescence exciting liquid system comprises exciting liquid 1 and exciting liquid 2, exciting liquid 1 be selected from containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L salpeter solution, containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L sulfuric acid solution or containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L hydrochloric acid solution.Exciting liquid 2 be selected from containing 0.5% ?5%Tween ?20 0.1 ?1mol/L sodium hydroxide solution, containing 0.5% ?5%Tween ?80 0.1 ?1mol/L sodium hydroxide solution, containing 0.5% ?5%TritonX ?100 0.1 ?1mol/L sodium hydroxide solution; Described chemiluminescence exciting liquid system loading methods, for first to add exciting liquid 1, after the 2s of interval, then adds exciting liquid 2.
Prepare a method for described kit, comprise the following steps:
1) preparation of hyperbranched polyglycidyl ether: the initiating agent using trimethylolpropane as reaction, adopts CH
3oK carries out part deprotonation to the hydroxyl of TMP, and deprotonation part accounts for 10% of total part, is then slowly dripped by diglycidyl and enters system initiated polymerization, temperature of reaction 95 ~ 100 DEG C, react 10 ~ 12 hours; Reaction terminates afterproduct and is dissolved in methyl alcohol, by strongly acidic cation-exchange (AMBERJET
tM1500H Resin) neutralization; Polymkeric substance by separating out in acetone, dissolves in methyl alcohol, repeats to purify for twice, and finally drying 13 ~ 16 hours in the vacuum of 75 ~ 85 DEG C, obtains hyperbranched polyglycidyl ether;
2) modification of hyperbranched polyglycidyl ether: hyperbranched polyglycidyl ether is carried out modifying obtaining the hyperbranched polyglycidyl ether modified of DSC with succinimidyl carbonate (DSC) in DMF solution;
3) hyperbranched polyglycidyl ether is to the modification of magnetic microsphere: the hyperbranched polyglycidyl ether solution dripping DSC modification prepared by previous step in amino-magnetic microspheres solution, hyperbranched polyglycidyl ether entered DSC modification rear surface a large amount of succinimide esters (NHS ester), this part active ester can be reacted with the amino on amino-magnetic microballoon, react 0.5 ~ 1h in shaking table after, add the PB damping fluid of 50mM again, now unnecessary NHS ester is hydrolyzed, finally use 50mM PBS solution (pH=7.5) cleaning, magnetic separation rack is used to be separated magnetic microsphere in cleaning process, after cleaning terminates, use 50mM PBS solution (pH=7.5) resuspended, therefore a large amount of carboxyl is left in advance on magnetic microsphere surface,
4) hyperbranched polyglycidyl ether modified magnetic microballoon coupling molecular detection: activated carboxylic is carried out to the magnetic microsphere of hyperbranched polyglycidyl ether modification prepared by previous step, usual method for add 1 ?(3 ?dimethylamino-propyl) ?3 ?ethyl-carbodiimide hydrochloride (EDC), its molar content be carboxyl molar content 1 ?10 times, mix rear concussion reaction 30 minutes, activation terminates rear centrifugal use 50mM PBS solution (pH=7.5) cleaning, after molecular detection 100mM sodium bicarbonate buffer liquid (pH=8.0) is diluted to, join in magnetic microsphere solution, be placed in 200 ~ 220rpm 35 ~ 37 DEG C and shake bed reaction 2.5 ~ 3.5h, reaction terminates rear use, 50mM PBS solution (pH=7.5) cleans suspended magnetic microballoon, obtained hyperbranched polyglycidyl ether nanometer Ci Wei Qiu ?molecular detection compound solid phase carrier,
5) mark of luminescent substance: capture molecules joins in 50mM PBS damping fluid (pH=8.0), add the luminescent substance of 0.3 ~ 1mmol/L, mixing, room temperature lucifuge reaction 20 ~ 30min, use the unconjugated luminescent substance of 10mM PBS damping fluid (pH=6.5) dialysis removing, finally add 50% and heavily steam glycerine, Zhi Yu ?20 DEG C of preservations;
6) exciting liquid 1 and exciting liquid 2 is prepared: exciting liquid 1 is the salpeter solution of the 0.2M containing 0.5% superoxol; Exciting liquid 2 be containing 1%Tween ?20 0.5M sodium hydroxide solution.
Prepare a method for mentioned reagent box, preferably include following steps:
The preparation of hyperbranched polyglycidyl ether: in the there-necked flask being furnished with mechanical raking and micro-constant flow pump, adds 0.24g trimethylolpropane (TMP), as the initiating agent of reaction, adopts 3.7M CH
3oK (being dissolved in methyl alcohol) carries out part deprotonation to the hydroxyl of TMP, and deprotonation part accounts for 10% of total part, and unnecessary methyl alcohol is removed by heating evaporation.Then the diglycidyl of 50mL slowly drips and enters system initiated polymerization, notes the rate of addition of diglycidyl, drips too fastly may cause implode.Temperature of reaction 95 DEG C, reacts 12 hours;
Reaction terminates afterproduct and is dissolved in methyl alcohol, by strongly acidic cation-exchange (AMBERJET
tM1500H Resin) neutralization.Polymkeric substance, by separating out in acetone, dissolves in methyl alcohol, repeats to purify for twice.Last in the vacuum of 80 DEG C dry 15 hours.Obtain hyperbranched polyglycidyl ether;
The modification of hyperbranched polyglycidyl ether: hyperbranched polyglycidyl ether is dissolved in dimethyl formamide (DMF), be mixed with the solution of 10mg/mL, get 1mL, add 20mg succinimidyl carbonate (DSC) to react under normal temperature, obtain the hyperbranched polyglycidyl ether (DSC ?HPG) that DSC modifies.
Hyperbranched polyglycidyl ether is to the modification of magnetic microsphere: get 5mg amino-magnetic microballoon (particle size is 1 μm ~ 10 μm), convert according to solid content 5mg/ml, removes supernatant and retains solid matter.Drip in magnetropism microspheres solution afterwards 500 μ L DSC ?HPG solution, in shaking table, react 30min.After reaction terminates, in reaction system, add 1mL 50mMPBS solution continue reaction 30min, after unnecessary NHS Ester hydrolysis, 50mM PBS solution (pH=7.5) is used to clean three times, magnetic separation rack is used to be separated magnetic microsphere in cleaning process, after cleaning terminates, use 1mL 50mM PBS solution (pH=7.5) resuspended;
Hyperbranched polyglycidyl ether modified magnetic microballoon coupling molecular detection: activate the carboxyl of microballoon, activation terminates rear use 500 μ L 50mM PBS solution (pH=7.5) and cleans magnetic microsphere.After molecular detection 100mM sodium bicarbonate buffer liquid (pH=8.0) is diluted to 1mg/mL, join in magnetic microsphere solution, be placed in 220rpm 37 DEG C and shake bed reaction 3h.Reaction terminates rear use 400 μ L 50mM PBS solution (pH=7.5) and cleans suspended magnetic microballoon, cleans three times, obtained hyperbranched polyglycidyl ether magnetic microsphere molecular detection compound;
The mark of luminescent substance: capture molecules joins in 50mM PBS damping fluid (pH=8.0), adds the luminescent substance of 0.5mmol/L, mixing, room temperature lucifuge reaction 20min.Use 10mM PBS damping fluid (pH=6.5) dialysis removing unconjugated luminescent substance, finally add 50% and heavily steam glycerine, Zhi Yu ?20 DEG C of preservations;
Preparation exciting liquid 1 and exciting liquid 2: exciting liquid 1 is the salpeter solution of the 0.2M containing 0.5% superoxol; Exciting liquid 2 be containing 1%Tween ?20 0.5M sodium hydroxide solution.
Beneficial effect:
Patent of the present invention introduces hyperbranched polyglycidyl ether magnetic microsphere in chemiluminescence immunoassay, utilize the feature of the highly hydrophilic of hyperbranched polyglycidyl ether and branching high functional group densities, the performance of chemiluminescence immune assay reagent in lowest detectable limit and upper limit of detection can be improved simultaneously.Specifically, hyperbranched polyglycidyl ether water wettability is strong, the non-specific adsorption of the non-targeted albumen caused because of hydrophobic effect in testing process can be reduced in widely, can background be reduced largely and reduce noise, thus the lowest detectable limit of reagent can be optimized; The magnetic microsphere that the feature of branching high functional group densities makes to have modified this molecule when coated antibody as solid phase carrier, can improve package amount, thus widen sensing range.The water wettability of hyperbranched polyglycidyl ether makes the superincumbent antibody of coupling can keep its conformation better in addition, thus improves the ability of conjugated antigen.
Patent utilization hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere of the present invention, prepares surface hydrophilic and is rich in the nanospheres of functional group.When reality uses this chemiluminescence diagnostic reagent based on this hyperbranched polyglycidyl ether magnetic microsphere, find that reagent can be good at the non-specific adsorption of Profilin, antibody coupling amount also more commercial magnetic microsphere exceed 2 ?3 times.In the performance evaluation of project, find that the diagnostic reagent based on this kind of magnetic microsphere can reaction greatly between enhancement antigen antibody, find when contrasting with commercialization magnetic microsphere reagent, sensitivity improve 4 ?5 times, upper limit of detection improves 1.3 times, thus sensing range improves 6.5 times nearly.During other performance such as linear dependence, repeatability, the recovery, stability for reagent, analyze the requirement finding all to reach GB or rower, reagent superior performance.
The invention of this reagent can solve the problem of ubiquitous non-specific adsorption in current diagnosis reagent very well, very wide for some linear requirements, the project that sensitivity requirement is very high, use this reagent can also obtain good effect, therefore this diagnostic reagent has higher clinical examination using value.
Accompanying drawing explanation
Fig. 1 the technology of the present invention route schematic diagram
Linear correlation figure between Fig. 2 hGH detection system and contrast detection system
Fig. 3 detects reagent typical curve based on the hGH of hyperbranched polyglycidyl ether modified magnetic microballoon
Fig. 4 detects reagent typical curve based on the hGH of common carboxyl group magnetic microsphere
Fig. 5 hGH detects the Virus monitory value under reagent difference inspection number of days
Linear correlation figure between Fig. 6 TPO-Ab detection system and comparison system
Fig. 7 detects reagent typical curve based on the TPO-Ab of hyperbranched polyglycidyl ether modified magnetic microballoon
Fig. 8 detects reagent typical curve based on the TPO-Ab of commercialization carboxyl magnetic microsphere
Fig. 9 TPO-Ab detects the Virus monitory value under reagent difference inspection number of days
Embodiment
Embodiment 1 is based on the preparation method of hyperbranched polyglycidyl ether modified magnetic microballoon diagnostic reagent
The preparation of hyperbranched polyglycidyl ether
In the there-necked flask being furnished with mechanical raking and micro-constant flow pump, add 0.24g trimethylolpropane (TMP), as the initiating agent of reaction, adopt 3.7M CH
3oK (being dissolved in methyl alcohol) carries out part deprotonation to the hydroxyl of TMP, and deprotonation part accounts for 10% of total part, and unnecessary methyl alcohol is removed by heating evaporation.Then the diglycidyl of 50mL slowly drips and enters system initiated polymerization, notes the rate of addition of diglycidyl, drips too fastly may cause implode.Temperature of reaction 95 DEG C, reacts 12 hours.
Reaction terminates afterproduct and is dissolved in methyl alcohol, by strongly acidic cation-exchange (AMBERJET
tM1500H Resin) neutralization.Polymkeric substance, by separating out in acetone, dissolves in methyl alcohol, repeats to purify for twice.Last in the vacuum of 80 DEG C dry 15 hours.Hyperbranched polyglycidyl ether is colourless viscous liquid.
The modification of hyperbranched polyglycidyl ether
Hyperbranched polyglycidyl ether is dissolved in dimethyl formamide (DMF), be mixed with the solution of 10mg/mL, get 1mL, add 20mg succinimidyl carbonate (DSC) to react under normal temperature, obtain the hyperbranched polyglycidyl ether (DSC ?HPG) that DSC modifies.
Hyperbranched polyglycidyl ether is to the modification of magnetic microsphere
Get 5mg amino-magnetic microballoon (particle size is 1 μm ~ 10 μm), convert according to solid content 5mg/ml, remove supernatant and retain solid matter.Drip in magnetropism microspheres solution afterwards 500 μ L DSC ?HPG solution, in shaking table, react 30min.After reaction terminates, in reaction system, add 1mL 50mM PBS solution continue reaction 30min, after unnecessary NHS Ester hydrolysis, 50mM PBS solution (pH=7.5) is used to clean three times, magnetic separation rack is used to be separated magnetic microsphere in cleaning process, after cleaning terminates, use 1mL 50mM PBS solution (pH=7.5) resuspended.
Hyperbranched polyglycidyl ether modified magnetic microballoon coupling hGH antibody
The carboxyl of the magnetic microsphere that hyperbranched polyglycidyl ether is modified is activated, usual method is for adding EDC, its molar content is 10 times of carboxyl molar content, mix rear concussion reaction 30 minutes, activation terminates rear centrifugal use 500 μ L 50mMPBS solution (pH=7.5) and cleans magnetic microsphere.After hGH ?5802 antibody (manufacturer Medixm AB article No. 100042) is diluted to 1mg/mL with 100mM sodium bicarbonate buffer liquid (pH=8.0), joins in magnetic microsphere solution, be placed in 220rpm37 DEG C and shake bed reaction 3h.Reaction terminates rear use 400 μ L 50mM PBS solution (pH=7.5) and cleans suspended magnetic microballoon, clean three times, obtain hyperbranched polyglycidyl ether Ci Wei Qiu ?5802 compounds.
Commercialization carboxyl magnetic microsphere (the manufacturer JSR diagnostic reagent of reagent in contrast in embodiment 2, article No. MS160/Carboxyl) the same with the coupling method of hGH antibody, only commodity in use carboxyl magnetic microsphere substitutes the magnetic microsphere that hyperbranched polyglycidyl ether is modified.
The mark of acridinium ester
Get hGH ?5801 antibody (manufacturer Medixm AB article No. 100041) join in 50mM PBS damping fluid (pH=8.0), add the acridinium ester of 0.5mmol/L, mixing, room temperature lucifuge reaction 20min.Use 10mM PBS damping fluid (pH=6.5) dialysis removing unconjugated acridinium ester, finally add 50% and heavily steam glycerine, Zhi Yu ?20 DEG C of preservations.
Chemiluminescence exciting liquid system
Preparation exciting liquid 1 and exciting liquid 2: exciting liquid 1 is the salpeter solution of the 0.2M containing 0.5% superoxol; Exciting liquid 2 be containing 1%Tween ?20 0.5M sodium hydroxide solution.
The diagnostic reagent of embodiment 2 hyperbranched polyglycidyl ether modified magnetic microballoon exploitation utilizes the checking of double-antibody method in hGH project in chemiluminescence immune assay
By hyperbranched polyglycidyl ether modified magnetic microballoon-hGH ?5802 antibody complex 30 μ l, hGH ?5801 antibody-acridinium ester compound 120 μ l, join in 100 μ l sample to be analyzed, react 15 minutes.Due to specific immune response formed hyperbranched polyglycidyl ether modified magnetic microballoon-hGH ?the immune complex of 5802 antibody-hGH-5801 antibody-acridinium esters, use magnetic separation frame to carry out Separation of Solid and Liquid and remove supernatant, leave and take sediment.Cleaning fluid is used to clean compound, add excimer 1, interval 2s adds excimer 2,9 carbon atoms of hydrogen peroxide addition acridinium ester acridine ring in excimer 1, generated negative oxygen ion in the basic conditions, attack carbonyl in molecule, leaving group, generates unstable intermediate 1,2-dioxetane ketone, the open loop of 1,2-dioxetane ketone generates acridone, discharges photon simultaneously.Interval 0.5s starts to measure the chemiluminescence luminous intensity produced, and the photon technique time is 0.5-3s.
Samples detection relevance evaluation
The human growth hormone (HGH) using Roche company to produce respectively measure kit (Electrochemiluminescince) and the present invention build hGH detection system 40 parts of concentration measured at the hGH of 0.01 ~ 32.49ng/mL scope sample to be analyzed, then calculate the present invention institute build hGH detection system and contrast detection system between linear dependence, and calculate 5,10,15 and 20ng/mL calibrating anticipated deviation (Bx) and relative deviation (Bx/x).
As shown in Figure 2, the present invention build system and comparison system equation of linear regression be y=0.9083x+0.4067, linearly dependent coefficient is r
2=0.9913 (r=0.9956); As shown in table 1, the present invention build system 5,10,15, the anticipated deviation of a 20ng/mL4 calibrating point is up to 1.43ng/mL, expection relative deviation is up to 7.15%, show the present invention build systems axiol-ogy sample to be analyzed and can obtain the result basically identical with Roche detection system, accuracy in detection is high, can meet clinical practice demand well.
Table 1 anticipated deviation and relative deviation calculate
The assessment of sensing range and contrast
Lowest detectable limit
Lowest detectable limit, upper limit of detection detect an important parameter of reagent, and the detection reagent that lowest detectable limit is low, upper limit of detection is high represents that this Product checking performance is high.This paper is except the lowest detectable limit and upper limit of detection that built hGH are detected to reagent are assessed, and the hGH also prepared based on commercialization carboxyl microballoon detects reagent, and to the two lowest detectable limit with upper limit of detection is assessed and comparison.
Adopt hGH that the present invention builds to detect reagent and contrast agents and prepare hGH detection reagent respectively to people's negative serum sample replication 20 times based on commercialization carboxyl microballoon, obtain the luminous intensity of sample, calculate the mean value of the luminous intensity recorded respectively
with standard deviation (SD), calculate detection average and add twice standard deviation.According to the fit equation measuring concentration and luminous intensity, calculate concentration value, namely this concentration be detection system minimum detectability.Result is as shown in table 2, and the hGH detection system being solid phase carrier with hyperbranched polyglycidyl ether magnetic microsphere, can reduce the luminous intensity of dummy, reduces noise, improves the lowest detectable limit of detection system.
Table 2 lowest detectable limit is assessed
Upper limit of detection is assessed:
Get hGH recombinant protein, with normal saline dilution to 0.01,0.02,0.05,0.5,5,15,25,35,45, a 65ng/mL10 concentration level, use hGH that the present invention builds to detect reagent and contrast agents and prepare hCG detection reagent to each concentration level replication 4 times based on commercialization carboxyl magnetic microsphere, take theoretical concentration as x-axis, detection luminous value is y-axis, draws scatter diagram.As shown in Figure 3, the dull gradient distribution within the scope of 0 ~ 65ng/mL of result display embodiment 1 reagent, when concentration of specimens is more than 45ng/mL, reaction system is tending towards saturated, detects and enters plateau.And contrast agents dull gradient distribution (as shown in Figure 4) within the scope of 0 ~ 35ng/mL, after concentration of specimens exceeds above-mentioned scope, because antigen amount is excessive, the detection antibody that excessive antigen consumption is too much and occur hook effect.From data, the upper limit of detection of this paper institute method for building up is higher than control group 1.3 times.In literary composition, the bent approximating method of reagent mark being solid phase carrier based on hyperbranched polyglycidyl ether modified magnetic microballoon used is five parameter fittings, and formula is y=Amin+ (Amax-Amin)/(1+ (x0/x) ^h) ^s
Wherein each parameter is
Amin＝210.46098
Amax＝1.62E+06
x0＝45.02666
h＝7.65128
s＝0.1452
The recovery is tested
Select hGH concentration be 15ng/mL sample based on sample, be divided into 4 parts, every part of volume is 1mL.HGH antigen is not added in analyzing samples 1; 0.125mL 60ng/mL hGH antigen adjustment concentration is added to 20ng/mL in analyzing samples 2; 0.286mL 60ng/mL hGH antigen adjustment concentration is added to 25ng/mL in analyzing samples 3; 0.5mL 60ng/mL hGH antigen adjustment concentration is added to 30ng/mL in analyzing samples 4.Use the present invention to build hGH detection system and measure hGH concentration in above-mentioned analyzing samples, each sample replication 3 times, the recovery of computational analysis sample and ratio system error.Measurement result is as shown in table 3, showing that system that the present invention builds reclaims CONCENTRATION DISTRIBUTION when adding concentration 5,10 and 15ng/mL is 4.97,10.15 and 14.92ng/mL, the recovery is respectively 99.40%, 101.50% and 99.47%, average recovery rate is 100.02%, and ratio system error is 0.02%.Show that the present invention builds hGH detection system, recovering effect is good, and accuracy in detection is high, can meet actual operation requirements.
Table 3 recovery
Reperformance test
Adopt the present invention to build hGH detection system and respectively repeatability mensuration carried out to quality controlled serum 1 (5ng/mL) and quality controlled serum 2 (10ng/mL), each quality controlled serum replication 20 times, investigation the present invention build the repeatability of system.Result display (see table 4), the coefficient of variation that detection system is measured at 5ng/mL Quality Control point and 10ng/mL Quality Control point is respectively 5.58% and 2.50%.Show that the present invention builds systems axiol-ogy precision high, reproducible, clinical request for utilization can be met.
Table 4 repeatability
Stability assessment
HGH is placed in 37 DEG C of constant incubators after measuring reagent, investigates the reference serum that concentration is respectively low high three concentration in 0.50,5.00 and 20.00ng/ml, each sample replication 3 times, amounts to 7 days, calculate the average of testing result every day every 1d.As shown in Figure 5, hGH that the present invention builds detects reagent in 37 DEG C of preservations after 7 days to result, and testing result is in a slight decrease with first day testing result, but maximum relative deviation is not less than 15%, shows that reagent stability is good, can preserve for a long time.
Embodiment 3
The diagnostic reagent of hyperbranched polyglycidyl ether modified magnetic microballoon exploitation utilizes the checking of indirect method in TPO-Ab project in chemiluminescence immune assay
Nano-magnetic microsphere-TPO antigen (the manufacturer Hytest article No. 8RTPO) compound of hyperbranched polyglycidyl ether modification is prepared, for the chemiluminescence immune assay of following TPO-Ab according to the method for embodiment 1.Commercialization carboxyl magnetic microsphere (the manufacturer JSR diagnostic reagent of reagent in contrast in the present embodiment, article No. MS160/Carboxyl) with the coupling method of TPO antigen with embodiment 1, only commodity in use carboxyl magnetic microsphere substitutes the magnetic microsphere that hyperbranched polyglycidyl ether is modified.
Nano-magnetic microsphere-TPO the antigenic compound that hyperbranched polyglycidyl ether is modified is joined in 20 μ L sample to be analyzed, mixing, 37 DEG C of temperature bath 10min, nano-magnetic microsphere-TPO the antigenic compound that hyperbranched polyglycidyl ether is modified catches TPO-Ab in sample to be detected to solid phase carrier, after cleaning three times, add anti-(Beijing Bo Sheng longitude and latitude Science and Technology Ltd. of manufacturer of acridinium ester label TPO-Ab bis-again, article No. 131108), irrelevant antibody in sample to be analyzed is removed in cleaning, acridinium ester label TPO-Ab bis-anti-binding is at the TPO-Ab on magnetic microsphere, reaction terminates rear use 400 μ L 50mM PBS solution (pH=7.5) and cleans immune complex, repeated washing 3 times, remove reactant liquor and retain the immune complex formed, add excimer 1, excimer 2 is added after the 2s of interval, 9 carbon atoms of hydrogen peroxide addition acridinium ester acridine ring in excimer, generated negative oxygen ion in the basic conditions, attack carbonyl in molecule, leaving group is left away, and generates unstable intermediate 1,2-dioxetane ketone, the open loop of 1,2-dioxetane ketone generates acridone, discharges photon simultaneously.Interval 0.5s-3s measures the chemiluminescence intensity produced.
Samples detection relevance evaluation
Respectively use Roche company produce thyroid peroxidase antibody measure kit (Electrochemiluminescince) and the present invention build TPO-Ab detection reagent the to be analyzed sample of 40 parts of TPO-Ab concentration at 15.36 ~ 461.90IU/mL is measured, both calculating linear dependence to evaluate the linear comparability of the method for the invention and conventional method, and calculates the method for the invention examines and determine point anticipated deviation and relative deviation in 50,100,200 and 400IU/mL.
As shown in Figure 6, the present invention build reagent and comparison system equation of linear regression be y=0.9774x+3.5116, linearly dependent coefficient r
2=0.9824 (r=0.9912); As shown in table 5, reagent that the present invention builds is up to 5.54IU/mL at the anticipated deviation of 50,100,200,400IU/mL 4 calibrating points, and relative deviation is up to 4.76%, can meet application demand well.Show the present invention build reagent and detect sample to be analyzed and can obtain the result basically identical with Roche detection system, accuracy in detection is high, can meet clinical practice demand well.
Table 5 anticipated deviation and relative deviation
The assessment of sensing range and contrast
The assessment of lowest detectable limit
Adopt TPO-Ab that the present invention builds to detect reagent and contrast agents and prepare TPO-Ab detection reagent respectively to people's negative serum sample replication 20 times based on commercialization carboxyl microballoon, obtain the luminous intensity of sample, calculate the mean value of the luminous intensity recorded respectively
with standard deviation (SD), calculate detection average and add twice standard deviation.According to the fit equation measuring concentration and luminous intensity, calculate concentration value, namely this concentration be detection system minimum detectability.Result is as shown in table 6, and the TPO-Ab detection system being solid phase carrier with polyglycidyl ether modified magnetic microballoon, can reduce the luminous intensity of dummy, reduces noise, the lowest detectable limit of optimum detection system.
Table 6 lowest detectable limit is assessed
The Pre-Evaluation of upper limit of detection
Get TPO-Ab calibration object, with calf serum be diluted to 4,8,16,30,60,120,240,480,600, a 1200IU/mL10 concentration level, use TPO-Ab that the present invention builds to detect reagent and contrast agents and prepare TPO-Ab detection reagent to each concentration level replication 4 times based on commercialization carboxyl microballoon, take theoretical concentration as x-axis, detection luminous value is y-axis, draws scatter diagram.As shown in Figure 7, result shows reagent that the present invention builds dull gradient distribution within the scope of 0 ~ 1200IU/mL, and when concentration of specimens is more than 600IU/mL, reaction system is tending towards saturated, detects and enters plateau.Contrast agents (as shown in Figure 8) is in dull gradient distribution within the scope of 0 ~ 480IU/mL in addition, after concentration of specimens exceeds above-mentioned scope, consumes too much detection antibody and occurs hook effect.From data, the upper limit of detection of this paper institute method for building up is higher than control experiment group 2.5 times.In literary composition, the bent approximating method of reagent mark being solid phase carrier based on hyperbranched polyglycidyl ether magnetic microsphere used is five parameter fittings, and formula is y=Amin+ (Amax-Amin)/(1+ (x0/x) ^h) ^s
Wherein each parameter is
Amin＝1764.48268
Amax＝582910.2428
x0＝604.20119
h＝52.55252
s＝0.01954
The recovery is tested
Select TPO-Ab concentration be 200IU/mL sample based on sample, be divided into 4 parts, every part of volume is 1mL.TPO-Ab is not added in analyzing samples 1; Add TPO-Ab in analyzing samples 2 and adjust concentration to 250IU/mL; Add TPO-Ab in analyzing samples 3 and adjust concentration to 300IU/mL; Add TPO-Ab in analyzing samples 4 and adjust concentration to 400IU/mL.The method of the invention is used to carry out TPO-Ab concentration determination to above-mentioned analyzing samples, each sample replication 3 times, the recovery of computational analysis sample and ratio system error.Result display (see table 7), the method of the invention add concentration be 50,100 and 200IU/mL time recovery concentration be respectively 48.98IU/mL, 96.79IU/mL and 205.35IU/mL, the recovery is respectively 97.96%, 96.79% and 102.68%, average recovery rate is 99.14%, and ratio system error is 0.86%.Recovering effect is good, and accuracy in detection is high, can meet Clinical practice requirement.
Table 7 recovery and ratio system error
Reperformance test
TPO-Ab detects reagent to carry out repeatability to quality controlled serum 1 (100IU/mL) and quality controlled serum 2 (300IU/mL) respectively and measures, each quality controlled serum replication 20 times, investigation civilian build the repeatability of reagent.Experimental result is as follows: result display (see table 8), and the measurement coefficient of variation that detection reagent is determined at 100IU/mL Quality Control point and 300IU/mL Quality Control point is respectively 6.75% and 2.60%.Show that reagent that the present invention builds detects precision high, reproducible, clinical request for utilization can be met.
Table 8 repeatability
Stability assessment
TPO-Ab is placed in 37 DEG C of constant incubators after measuring reagent, investigates the reference serum that concentration is respectively low high three concentration in 50,200 and 400IU/mL, each sample replication 3 times, amounts to 7 days, calculate the average of testing result every day every 1d.As shown in Figure 9, TPO-Ab that the present invention builds detects reagent in 37 DEG C of preservations after 7 days to result, and testing result is in a slight decrease with first day testing result, but maximum relative deviation is not less than 15%, shows that reagent stability is good, can preserve for a long time.
Claims (10)
1. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere in chemiluminescence immune assay.
2. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere according to claim 1 in chemiluminescence immune assay, it is characterized in that comprising following methods: be coated on nano-magnetic microsphere surface by amino effect after hyperbranched polyglycidyl ether is modified and form hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere, the carboxyl of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere is activated to the molecular detection of rear coupling energy and testing molecule specific binding, define hyperbranched polyglycidyl ether nano-magnetic microsphere-molecular detection compound solid phase carrier, by hyperbranched polyglycidyl ether nano-magnetic microsphere-molecular detection compound solid phase carrier and sample to be tested and the upper luminescent substance of mark can with the capture molecules hybrid reaction of testing molecule specific binding after, define the compound of hyperbranched polyglycidyl ether nano-magnetic microsphere-molecular detection compound solid phase carrier-testing molecule-capture molecules-luminescent substance, compound is isolated through cleaning, add excimer wherein, luminescent substance in compound is subject to exciting generation chemiluminescence, finally detect with chemiluminescence detector the concentration that luminous intensity calculates the target analytes in sample to be tested.
3. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere according to claim 1 in chemiluminescence immune assay, is characterized in that described nano-magnetic microsphere size is 1 μm ~ 10 μm.
4. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere according to claim 1 in chemiluminescence immune assay, is characterized in that described hyperbranched polyglycidyl ether is polymerized by trimethylolpropane and diglycidyl; The described succinimidyl carbonate that is modified to is modified.
5. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere according to claim 1 in chemiluminescence immune assay, is characterized in that the test substance detected is antigen, antibody, incomplete antibody, haptens, small molecule chemicals or metallic element; Molecular detection be can with the antibody of test substance specific binding, antigen, haptens, incomplete antibody or Small molecular; Capture molecules be can with the material of testing molecule or molecular detection or both compound specific bindings.
6. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere according to claim 1 in chemiluminescence immune assay, it is characterized in that described luminescent substance be selected from comprise N ?(4 ?ammonia butyl) ?N ?the different luminol of ethyl, acridinium ester, acridine sulfonamide, horseradish peroxidase or alkaline phosphatase.
7. the application of hyperbranched polyglycidyl ether decorated nanometer magnetic microsphere according to claim 1 in chemiluminescence immune assay, it is characterized in that described excimer be selected from containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L salpeter solution, containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L sulfuric acid solution, containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L hydrochloric acid solution, containing 0.5% ?5%Tween ?20 0.1 ?1mol/L sodium hydroxide solution, containing 0.5% ?5%Tween ?80 0.1 ?1mol/L sodium hydroxide solution, or containing 0.5% ?5%TritonX ?100 0.1 ?1mol/L sodium hydroxide solution.
8. a chemiluminescence immune detection reagent kit, is characterized in that comprising hyperbranched polyglycidyl ether decorated nanometer magnetic according to claim 2 micro-ball ?molecular detection compound, chemiluminescence exciting liquid system, connects the capture molecules of luminescent substance.
9. chemiluminescence immune detection reagent kit according to claim 8, it is characterized in that described chemiluminescence exciting liquid system comprises exciting liquid 1 and exciting liquid 2, exciting liquid 1 be selected from containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L salpeter solution, containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L sulfuric acid solution or containing 0.1% ?1% hydrogen peroxide 0.1mol/L ?1mol/L hydrochloric acid solution; Exciting liquid 2 be selected from containing 0.5% ?5%Tween ?20 0.1 ?1mol/L sodium hydroxide solution, containing 0.5% ?5%Tween ?80 0.1 ?1mol/L sodium hydroxide solution or containing 0.5% ?5%TritonX ?100 0.1 ?1mol/L sodium hydroxide solution; Described chemiluminescence exciting liquid system loading methods, for first to add exciting liquid 1, after the 2s of interval, then adds exciting liquid 2.
10. prepare a method for kit described in claim 8 or 9, comprise the following steps:
1) preparation of hyperbranched polyglycidyl ether: the initiating agent using trimethylolpropane as reaction, adopts CH
3oK carries out part deprotonation to the hydroxyl of TMP, and deprotonation part accounts for 10% of total part, is then slowly dripped by diglycidyl and enters system initiated polymerization, temperature of reaction 95 ~ 100 DEG C, react 10 ~ 12 hours; Reaction terminates afterproduct and is dissolved in methyl alcohol, is neutralized by strongly acidic cation-exchange; Polymkeric substance by separating out in acetone, dissolves in methyl alcohol, repeats to purify for twice, and finally drying 13 ~ 16 hours in the vacuum of 75 ~ 85 DEG C, obtains hyperbranched polyglycidyl ether;
2) modification of hyperbranched polyglycidyl ether: carry out hyperbranched polyglycidyl ether succinimidyl carbonate modifying obtaining the hyperbranched polyglycidyl ether modified of DSC;
3) hyperbranched polyglycidyl ether is to the modification of magnetic microsphere: the hyperbranched polyglycidyl ether solution dripping DSC modification prepared by previous step in amino-magnetic microspheres solution, 0.5 ~ 1h is reacted in shaking table, after reaction terminates, in reaction system, add 50mM PBS solution continue reaction 0.5 ~ 1h, after unnecessary NHS Ester hydrolysis, use the 50mM PBS solution cleaning of pH=7.5, magnetic separation rack is used to be separated magnetic microsphere in cleaning process, after cleaning terminates, use the 50mM PBS solution of pH=7.5 resuspended;
4) hyperbranched polyglycidyl ether modified magnetic microballoon coupling molecular detection: the carboxyl of the magnetic microsphere of hyperbranched polyglycidyl ether modification prepared by previous step is activated, activation terminates the 50mM PBS solution cleaning of rear use pH=7.5, after the 100mM sodium bicarbonate buffer liquid of molecular detection pH=8.0 is diluted to, join in magnetic microsphere solution, be placed in 200 ~ 220rpm 35 ~ 37 DEG C and shake bed reaction 2.5 ~ 3.5h, reaction terminates rear use, the 50mM PBS solution cleaning suspended magnetic microballoon of pH=7.5, obtained hyperbranched polyglycidyl ether nano-magnetic microsphere-molecular detection compound solid phase carrier,
5) mark of luminescent substance: capture molecules joins in the 50mM PBS damping fluid of pH=8.0, add the luminescent substance of 0.3 ~ 1mmol/L, mixing, room temperature lucifuge reaction 20 ~ 30min, use the unconjugated luminescent substance of 10mM PBS damping fluid dialysis removing of pH=6.5, finally add 50% and heavily steam glycerine, be placed in-20 DEG C of preservations;
6) exciting liquid 1 and exciting liquid 2 is prepared: exciting liquid 1 is the salpeter solution of the 0.2M containing 0.5% superoxol; Exciting liquid 2 be containing 1%Tween ?20 0.5M sodium hydroxide solution.
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