CN105181958A - Method for quantitatively assaying 80nmAuNPs on the basis of indirect competitive enzyme-linked immunosorbent assay - Google Patents

Method for quantitatively assaying 80nmAuNPs on the basis of indirect competitive enzyme-linked immunosorbent assay Download PDF

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CN105181958A
CN105181958A CN201510567972.3A CN201510567972A CN105181958A CN 105181958 A CN105181958 A CN 105181958A CN 201510567972 A CN201510567972 A CN 201510567972A CN 105181958 A CN105181958 A CN 105181958A
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80nmaunps
antibody
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张明翠
张月
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Chongqing Moda Biotechnology Co ltd
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Anhui Normal University
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Abstract

The invention provides a method for quantitatively assaying 80nmAuNPs on the basis of indirect competitive enzyme-linked immunosorbent assay, which adopts indirect competitive enzyme-linked immunosorbent assay in the technology of enzyme-linked immunosorbent assay and utilizes the specific binding of antigens and antibodies to assay 80nmAu particles. Compared with the prior art, the method successfully synthesizes haptens into holoantigens by the action of modifier, making great contribution to the production of antibodies. By utilizing the immune response effect of the body, the method successfully prepares an anti-80nmAuNPs polyclonal antibody with high specificity, the shelf life of the antibody is long, and therefore feasibility is high; by utilizing indirect competitive enzyme-linked immunosorbent assay in the technology of enzyme-linked immunosorbent assay to carry out a type of quantitative assay, the invention of the method provides a highly feasible method for the quantitative assay of future nanomaterials. The method is easy to operate, highly feasible, highly sensitive and low in assay limit, and can realize high-throughput assay.

Description

A kind of method quantitatively detecting 80nmAuNPs based on Indirect cELISA
Technical field
The present invention relates to the quantitative detection of nano material, particularly a kind of method quantitatively detecting 80nmAuNPs based on Indirect cELISA.
Background technology
The inevitable progress along with science and technology of development of society, along with 20 the end of the century nanometer technology appearance, nano material is the emerging and material science developed rapidly.Nano material refers to the super-fine material of particle size at nanometer scale (1nm ~ 100nm), has small-size effect, large specific surface area, quantum size effect and macro quanta tunnel effect.Just because of the character that these are special, nano material obtains and applies more and more widely in life is produced, and such as, nano material has very important application in biology sensor, bioprobe, pharmaceutical carrier, medical imaging, suncream etc.But science and technology is all have two faced, nano material is while producing to the life of people to bring convenience, and it is also the problem worried most that its safety issue also becomes that global people pay close attention to most.
Front nano biological effect brings new opportunity and new method by giving disease early diagnosis and efficient treatment; Negative nano biological effect is called that its research nano-substance of nanotoxicology is to the potential negative effect of health living environment and social safety etc.2003, Nature, Science, in 1 year, successively delivered editor's article 4 times, and American Chemical Society and European many scholarly journals are also published an article one after another afterwards, inquire into nano biological effect with the scientists of every field.
Have investigation display, nm of gold is to microorganism, and cancer cell etc. have certain toxicological effect, and in daily life, the approach of usual contact nanometer particle is divided into: respiratory system, skin and intestines and stomach.Due to the specific surface area that nano particle is larger, the toxicity of the nano material of same material different size is also not quite similar, and therefore, seems particularly important to the quantitative detection of nano material.According to the document reported, can x-ray photoelectron spectroscopy be passed through, x-ray diffraction, scanning electron microscope, projection electron microscope, atomic force microscope etc. to the qualitative analysis of nano particle.
But the quantitative detection for nano particle is still an international headache at present, the existing research of the quantitative detecting method to nano particle mainly contains: ultraviolet spectrophotometry surveys the content of nanoparticle in nano pulp, nanometer plating solution; Inductively coupled plasma-mass spectrometry and thin-layer chromatography and Inductively coupled plasma-mass spectrometry coupling is utilized quantitatively to detect nm of gold.Two class methods all have certain limitation, first kind method is only confined to the measurement to metal nanoparticle, quantitative detection for non pinetallic nano particle has significant limitation, and first to be mixed with solution when metal nanoparticle is measured, find the specific solute that can dissolve nano material to be measured, secondly also to carry out to this solute the relation that ultraviolet spectrophotometry sets up its wavelength and absorbance, the last mensuration wavelength being determined nano material to be measured again by pairing comparision, method is comparatively complicated, operates also relatively loaded down with trivial details; The pre-service of Equations of The Second Kind method sample is comparatively loaded down with trivial details, and complicated operation is time-consuming, and experimental apparatus used is also relatively costly.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of method quantitatively detecting 80nmAuNPs based on Indirect cELISA, make use of two and anti-be exaggerated detection signal, improve the sensitivity of experimental analysis, namely optical number by measuring the anti-compound of antigen---primary antibodie---two reaches the object quantitatively detected, method is quick, simple, can carry out high throughput assay.
A kind of method quantitatively detecting 80nmAuNPs based on Indirect cELISA provided by the invention, comprises the following steps:
The preparation of a, 80nmAuNPs;
B, 80nmAuNPs envelope antigen and immunogenic preparation;
Prepared by c, 80nmAuNPs antibody:
By the 80nmAuNPs immunogen injection that obtains in animal body, obtain 80nmAuNPs antibody;
D, utilize the 80nmAu antibody of the high specific that purifying is good and 80nmAu envelope antigen to carry out indirect competitive enzyme-linked immunosorbent analytic approach, reached the object of the 80nmAu quantitatively detecting unknown concentration by Criterion curve.
In step a, the preparation method of 80nmAuNPs is: by HAuCl 4after solution mixes with distilled water, ebuillition of heated, then add trisodium citrate, continue reaction, be cooled to room temperature, obtain 80nmAuNPs.
Further, the preparation method that step a prepares 80nmAuNPs is specially: get 500 μ LHAuCl 4solution and 99mL distilled water are in there-necked flask, boiling is heated to heat collecting type constant-temperature heating magnetic stirring apparatus, process need 15min ~ 30min, adds trisodium citrate fast, the rapid blackening of reactant liquor in the solution of boiling, then earth look red is become, after continuing reaction 35min, remove heating arrangement, stir and be cooled to room temperature, collecting 4 DEG C of lucifuges stores stand-by, and this earth red solution is 80nmAuNPs.
Further, step a adds 0.7mL1wt% citric acid three sodium solution;
HAuCl in step a 4the preparation method of solution is: get 1gHAuCl 44H 2o powder dissolution, in 50mL ultrapure water, obtains the HAuCl of 16.5mg/mL 4solution;
Because the impact of amount on nm of gold size of reductive agent trisodium citrate is most important, the amount of size and trisodium citrate is inversely proportional to, and is 0.7mL1wt% trisodium citrate through exploring the amount drawing best trisodium citrate.
Because of HAuCl in step a 4easy deliquescence, so need disposable by pulverous HAuCl 4be mixed with solution sealing to preserve, again due to HAuCl 4very easily corroding metal, so metal key should do not used in obtain solution process to weigh gold chloride, the compound method in this experiment is for getting 1gHAuCl 4powder dissolution, in 50mL ultrapure water, seals 4 DEG C of Refrigerator stores.
Because 80nmAuNPs is small-molecule substance, belong to haptens, and haptens only has immunoreactivity and do not possess immunogenicity.In order to allow 80nmAuNPs have immunogenicity, its surface need be carried out modifying and then connect upper macromolecular substances becomes existing immunogenicity and has again immunoreactive holoantigen.
80nmAuNPs envelope antigen and to be immunogenicly prepared as in step b:
Get 80nmAuNPs solution prepared by step a, concentration is 0.048mg/mL, after adding thioglycolic acid TGA stirring 10min ~ 25min under the state stirred, preferred stirring 15min, continue to add N-Hydroxysuccinimide NHS and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine EDAC lucifuge stirring 2h ~ 5h, preferred stirring 3h, regulate pH=9 ~ 10, add 1wt% bovine serum albumin(BSA) BSA or 1wt% chicken ovalbumin OVA ice bag lucifuge stirring 3h ~ 8h (preferred 5h), obtain corresponding immunogene and envelope antigen, gained solution is placed in bag filter, distill water dialysis 8h ~ 15h (preferred 12h), collecting 4 DEG C of lucifuges stores stand-by,
Further, in step b, the volumetric usage of thioglycolic acid TGA and N-Hydroxysuccinimide NHS, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine EDAC than the volumetric usage for 2:1:2,80nmAuNPs, thioglycolic acid TGA, bovine serum albumin(BSA) BSA or chicken ovalbumin OVA than being 400:1:10.Because large-sized nm of gold is easily reunited, therefore, the amount ratio of mentioned reagent is that inventor probes into and obtains, unnecessary or be less than above proportioning, all can not realize the present invention.
Described thioglycolic acid TGA is for regulating activator, and described N-Hydroxysuccinimide NHS is coupling agent;
The alkali regulating pH used is NaOH;
Concentration Wei≤9.8% of described middle thioglycolic acid TGA, because it is aobvious acid, need in experimentation to adjust pH to 7.0 with NaOH, the concentration of N-Hydroxysuccinimide NHS is 0.09mol/L, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine EDAC concentration is 0.05mol/L, bovine serum albumin(BSA) BSA concentration is 10mg/mL, and chicken ovalbumin OVA concentration is 10mg/mL.Because large-sized nm of gold is easily reunited
In step c, 80nmAuNPs preparation method for antibody is:
By immunogene and freund adjuvant miscible with volume ratio 1:1, repeatedly immunity is carried out to animal, obtained 80nmAuNPs antibody.
Further, in step c, 80nmAuNPs preparation method for antibody is specially: by immunogene with Freund's complete adjuvant equal-volume than hybrid injection in animal, adopt the mode of dorsal sc multi-point injection, inject 8 ~ 10 points, inoculation amount is each 1mL/.First immunisation is after three weeks, carry out first time booster immunization, immunogene being mixed with freund 's incomplete adjuvant equal-volume is expelled in animal again, injection volume is each 1mL/, after this booster immunization is carried out again week about, immune time is 7 ~ 8 times altogether, and middle week blood sampling survey is tired, until antibody titer meets experiment demand.
Adjuvant: Cucumber is injected in vivo in advance or with antigen simultaneously, can strengthen body to the immune response of this antigen or change immune response, such assistant is called adjuvant.
Adjuvant is divided into Freund's complete adjuvant and freund 's incomplete adjuvant two kinds, and the preparation method of freund 's incomplete adjuvant is: wool grease and whiteruss ultrasonic by the amount of 1:2, until mix, in ultrasonic procedure, note in time heat radiation.In freund 's incomplete adjuvant, add the Bacille Calmette-Guerin of killing namely become Freund's complete adjuvant, because non-medical organization acquisition Bacille Calmette-Guerin is comparatively difficult, therefore, the Freund's complete adjuvant in this experiment is directly bought and Sigma company.
Select Male New Zealand White Rabbit as immunization in the present invention, body weight is about 2kg, rabbit is the time that first the male rabbit of health of 2 ~ 5 months is raised about two weeks before the experiments were performed age, keeps the animation of its health.
Have selected some healthy Male New Zealand White Rabbits as immunization in experimentation.Body weight is about 2kg, and rabbit is the time that first the male rabbit of health of 2 ~ 5 months is raised about two weeks before the experiments were performed age, keeps the animation of its health.The position of the inoculation adopted and mode have multiple, are mainly divided into: eye droppings Nasal immunization, aerosol immunization, drinking-water immunity, under the wing thorn kind and intracutaneous, subcutaneous or intramuscular injection, concrete injecting method is depending on immune animal species.This experiment have employed the mode of dorsal sc multi-point injection, and at dorsal sc injection 8 ~ 10 points of every rabbit, total injection volume is only about 1mL/.
Steps d indirect competitive enzyme-linked immunosorbent analytic approach is:
(1) bag quilt: by dilution ratio be the 80nmAuNPs-OVA envelope antigen bag of 1/50-1/500 by 96 hole ELISA Plate, every hole 100 μ L, 4 DEG C of refrigerator overnight;
(2) close: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away not combined on 80nmAuNPs envelope antigen, add closed chicken ovalbumin 1wt%OVA to close, every hole 200 μ L, closes the site that not coated antigen combines, 37 DEG C of baking oven incubation 1 ~ 2h;
(3) application of sample competition: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away unnecessary confining liquid, every hole adds the standard items of 50 μ L80nmAuNPs or 80nmAuNPs sample solution to be measured and 50 μ L80nmAuNPs antibody and to be at war with reaction, baking oven incubation 1 ~ 2h, makes the AuNPs in determinand or standard items and envelope antigen compete anti-80nmAuNPs antibody simultaneously;
(4) enzyme-added: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away liquid to be measured or the antibody of not combined free state, add the goat anti-rabbit igg (two resist) of horseradish peroxidase-labeled, every hole 100 μ L (combining for the anti-80nmAuNPs antibody (primary antibodie) that AuNPs is combined in envelope antigen in previous step) baking oven incubation 2 ~ 4h;
(5) develop the color: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away not combined two and resist, add o-phenylenediamine, every hole 100 μ L, baking oven incubation colour developing 0.5 ~ 1h;
(6) stop: every hole adds 50 μ L, the H of 2mol/L 2sO 4cessation reaction, microplate reader measures the light absorption value of each hole at 490nm place;
(7) drawing standard curve, by typical curve calculate survey the concentration of unknown concentration 80nmAuNPs.
The invention provides a kind of method quantitatively detecting 80nmAuNPs based on Indirect cELISA, utilize the Indirect cELISA in enzyme-linked immuno assay technology, by the preparation of 80nmAu polyclonal antibody, utilize the specific binding of antigen-antibody quantitative have detected 80nmAu particle.Compared with prior art, the present invention has following feature: haptens, by the effect of dressing agent, is successfully synthesized holoantigen by (1), has made major contribution for antibody is able to generation.(2) utilize the immune response effect of body, successfully prepare the 80nmAuNPs polyclonal antibody that specificity is high, the shelf-life of antibody is longer, and therefore, feasibility is high.(3) utilize the Indirect cELISA in enzyme-linked immuno assay technology to carry out a kind of quantitative detection to 80nmAuNPs, the invention of the method is that the quantitative detection of nano material from now on provides a kind of higher method of feasibility.(4) the method is simple to operate, and feasibility is high, highly sensitive, and detection limit is low.
Accompanying drawing explanation
Fig. 1 is the typical curve that embodiment 1 obtains;
Fig. 2 is the typical curve that embodiment 2 obtains.
Embodiment
1, experiment reagent and instrument:
(1) reagent
Gold chloride (HAuCl 44H 2o), sodium citrate, citric acid, thioglycolic acid (TGA), N-Hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine (EDAC), chicken egg white (OVA), bovine serum albumin(BSA) (BSA), the goat anti-rabbit igg of HRP mark, sodium chloride, potassium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate dihydrate, sodium carbonate, sodium bicarbonate, Tween-20, wool grease, whiteruss, ether, Freund's complete adjuvant, o-phenylenediamine, hydrogen peroxide, NaOH, sulfuric acid, ultrapure water.
(2) instrument
Electrothermostat incubator 303-1, DF-101S type collecting and distributing type constant-temperature heating magnetic stirring apparatus, PHS-3C type accurate pH meter, KQ3200E type ultrasonic cleaner, TGL-16G type high speed tabletop centrifuge, UV-4100 type ultraviolet-visible spectrophotometer, multi-functional microplate reader, magnetic stirring apparatus, 96 hole ELISA Plate, disposable syringe (1mL, 5mL).
(3) solution
1) 2wt% chlorauric acid solution: 1g gold chloride powder is dissolved in 50mL distilled water;
2) 1wt% citric acid three sodium solution: take 0.1g trisodium citrate and be dissolved in 9.9mL distilled water, mix;
3) dilution PBS (0.01mol/LpH=7.4): take NaCl8.0g, KCl0.1g, NaH 2pO 42H 200.29g, Na 2hPO 412H 2o2.96g to be dissolved in distilled water and to be settled to 1000mL;
4) washing lotion PBST: add 500 μ LTween-20 in 1000mLPBS, mix;
5) bag is buffered liquid CB (0.05mol/LpH=9.6): take Na 2cO 31.59g, NaHCO 32.94g to be dissolved in distilled water and to be settled to 1000mL;
6) substrate solution: take 1.85gNa 2hPO 412H 2o, 0.51gC 6h 8o 7to be dissolved in distilled water and to be settled to 50mL, taking 4mg o-phenylenediamine and be dissolved in the above-mentioned solution of 10mL, add 15 μ L30%H before use 2o 2;
7) confining liquid: take 1mgOVA and be dissolved in 1mLPBS, mix;
8) stop buffer: 2mol/LH 2sO 4;
Embodiment 1
Quantitatively detect a method of 80nmAuNPs based on Indirect cELISA, comprise the following steps:
(1), the synthesis of 80nmAuNPs holoantigen
1) preparation of 80nmAuNPs
Get 500 μ L2%wtHAuCl 4with 99mL distilled water in there-necked flask, boiling is heated to heat collecting type constant-temperature heating magnetic stirring apparatus, be heated to the time-consuming 15min ~ 30min of boiling process, 0.7mL1% trisodium citrate is added fast again in the solution of boiling, after continuing boiling 35min, remove heating arrangement, stir and be cooled to room temperature, collecting 4 DEG C of lucifuges stores stand-by, and this earth red solution is 80nmAuNPs.
2) 80nmAuNPs envelope antigen and immunogenic preparation
Get the above-mentioned 80nmAuNPs solution of 8mL, add after 20 μ L9.8% thioglycolic acids (TGA) stir 15min under the state stirred, continue to add 10 μ L0.09mol/L N-Hydroxysuccinimide (NHS) and 20 μ L0.05mol/L1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimine (EDAC) lucifuge stirring 3h, NaOH adjusts pH9 ~ 10, add 200 μ L10mg/mLBSA or 10mg/mLOVA ice bag stir 5h and correspondingly obtain immunogene and envelope antigen, gained solution is placed in bag filter, distill water dialysis 12h, collecting 4 DEG C of lucifuges stores stand-by.
(2) preparation of 80nmAuNPs polyclonal antibody
1) preparation of freund adjuvant
Take the wool grease of 50g, measure the whiteruss mixing of 100mL, Ultrasound Instrument repeated ultrasonic, be no more than 20min at every turn, prevent temperature in ultrasonic procedure too high, can not dispel the heat in time, ultrasonic making it mixes, until to be dripped to by this mixing material in water and indiffusion in half a minute, the oily liquids obtained is freund 's incomplete adjuvant, and 4 DEG C of refrigerator storage are stand-by.Due to non-medical organization, the more difficult acquisition of Bacille Calmette-Guerin, so Freund's complete adjuvant can only be bought from Reagent Company, this is tested Freund's complete adjuvant used and buys from sigma company.
2) 80nmAuNPs preparation method for antibody
In order to strengthen the former immune response of body fight, during first immunisation, immunogene need be mixed with Freund's complete adjuvant equal-volume ratio and being subcutaneously injected in male rabbit body.Mix by the immunogene of equal-volume ratio and freund 's incomplete adjuvant during booster immunization and carry out hypodermic injection again, carry out booster immunization week about, antibody titer is surveyed in middle week blood sampling, booster immunization six times altogether, antibody titer reaches experiment demand and desirable blood obtains antiserum, and purifying obtains 80nmAuNPs antibody.Concrete immunologic process is as follows:
Get 4 body weight be the healthy Male New Zealand White Rabbit of about 2kg as animal used as test, wherein 3 as immunization, and the 4th as blank.During first immunisation, it is even to get isopyknic 80nmAuNPs immunogene and Freund's complete adjuvant emulsification, is subcutaneously injected into the back of 3 male rabbits, often dorsal injection 8 ~ 10 points of only male rabbit, injection volume be 1mL/ only.First time booster immunization is carried out after 3 weeks, with the immunogene of equal-volume ratio and the potpourri of freund 's incomplete adjuvant during booster immunization, injecting method is with first time immunity, carry out a booster immunization week about, injection volume is 1mL/ later, and middle all venous blood collections survey serum titer, until serum titer meets experiment demand, carry out last booster immunization, altogether booster immunization six times, get blood purifying and obtain antibody.
3) qualification of antibody titer
The tiring of antibody is used to weigh immune effect, and in order to detect the quality of obtained 80nmAuNPs antibody mass, this experiment takes tiring of indirect competitive enzyme-linked immunosorbent assay antibody.Concrete grammar: with CB, 80nmAuNPs-OVA is diluted to a series of concentration and is coated in 96 hole ELISA Plate, 4 DEG C of refrigerator overnight, morning next day, 3 times are washed with washing lotion PBST, each 3min, removing is not wrapped by upper envelope antigen, every hole 200 μ L1%OVA closes, 37 DEG C of closed 1h, PBST washes 3 times, each 3min, add the antiserum (dilution ratio is 1/1000 ~ 1/64000) with PBS dilution, every hole 100 μ L, 37 DEG C of incubation 2h, PBST washes 3 times, each 3min, add the goat anti-rabbit igg that dilution ratio is the HRP mark of 1/1000, every hole 100 μ L, 37 DEG C of incubation 2.5h, PBST washes 3 times, each 3min, add substrate solution colour developing, every hole 100 μ L, 37 DEG C of incubation colour developing 0.5h, add 2mol/LH 2sO 4cessation reaction.490nm place light absorption value is surveyed by multi-functional microplate reader.
(3) indirect competitive enzyme-linked immunosorbent analytic approach quantitatively detects 80nmAuNPs
Because the indirect competitive enzyme-linked immunosorbent analytic approach in luminosity enzyme-linked immunosorbent assay is that the goat anti-rabbit igg (namely two resist) utilizing HRP to mark detects the anti-80nmAu antibody (i.e. primary antibodie) be combined with envelope antigen, light signal finally by the anti-compound of detectable antigens primary antibodie two reaches the object of quantitative detectable antigens, the method utilizes the optical signal detecting antigen of the compound of antigen primary antibodie and Direct cELISA to have lower detection limit than only, higher sensitivity, therefore this experiment adopts Indirect cELISA quantitatively to detect 80nmAuNPs.Specific experiment process is as follows:
1) bag quilt: wrap by 96 hole ELISA Plate with the 80nmAu-OVA that concentration is 21.7 μ g/mL, every hole 100 μ L, 4 DEG C, refrigerator spends the night.
2) close: morning next day, take out ELISA Plate, PBST washs 3 times, each 3min, adds 1%OVA and closes, every hole 200 μ L, 37 DEG C of closed 1h.
3) application of sample competition: PBST washs 3 times, each 3min, every hole adds 50 μ L homemade 80nmAuNPs titers or liquid to be measured and 50 μ L80nmAuNPs antibody, 37 DEG C of competition 2h.
4) enzyme-added: PBST washs 3 times, each 3min, every hole adds the goat anti-rabbit igg that 100 μ L dilution ratios are the HRP mark of 1/1000,37 DEG C of incubation 2.5h.
5) develop the color: PBST washs 3 times, each 3min, every hole adds 100 μ L substrate solution 37 DEG C colour developing 0.5h.
6) stop: every hole adds 50 μ L2mol/LH 2sO 4cessation reaction, surveys 490nm place light absorption value in multi-functional microplate reader.
(4) drawing standard curve
As shown in Figure 1, the linear equation of typical curve is obtained typical curve: A=0.862-0.076logC, coefficient R 2=0.96, the range of linearity is 10 -3-10 3, detect be limited to 0.0063ng/mL. by typical curve calculate survey unknown concentration 80nmAuNPs concentration be 0.96ng/mL, the recovery is 82%-101%.
Embodiment 2
Quantitatively detect a method of 80nmAuNPs based on Indirect cELISA, comprise the following steps:
(1), the synthesis of 80nmAuNPs holoantigen
1) preparation of 80nmAuNPs
Get 500 μ L2wt%HAuCl 4with 99mL distilled water in there-necked flask, boiling is heated to heat collecting type constant-temperature heating magnetic stirring apparatus, be heated to the time-consuming 15min ~ 30min of boiling process, 0.7mL1wt% trisodium citrate is added fast again in the solution of boiling, after continuing boiling 35min, remove heating arrangement, stir and be cooled to room temperature, collecting 4 DEG C of lucifuges stores stand-by, and this earth red solution is 80nmAuNPs.
2) 80nmAuNPs envelope antigen and immunogenic preparation
Get the above-mentioned 80nmAuNPs solution of 8mL, add after 20 μ L9.8% thioglycolic acids (TGA) stir 13min under the state stirred, continue to add 10 μ L0.09mol/L N-Hydroxysuccinimide (NHS) and 20 μ L0.05mol/L1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimine (EDAC) lucifuge stirring 4h, add 180 μ L10mg/mLBSA or 10mg/mLOVA ice bag stir 5h and correspondingly obtain immunogene and envelope antigen, gained solution is placed in bag filter, distill water dialysis 12h, collects 4 DEG C of lucifuges and stores stand-by.
(2) preparation of 80nmAuNPs polyclonal antibody
1) preparation of freund adjuvant
Take the wool grease of 50g, measure the whiteruss mixing of 100mL, Ultrasound Instrument repeated ultrasonic, be no more than 20min at every turn, prevent temperature in ultrasonic procedure too high, can not dispel the heat in time, ultrasonic making it mixes, until to be dripped to by this mixing material in water and indiffusion in half a minute, the oily liquids obtained is freund 's incomplete adjuvant, and 4 DEG C of refrigerator storage are stand-by.Due to non-medical organization, the more difficult acquisition of Bacille Calmette-Guerin, so Freund's complete adjuvant can only be bought from Reagent Company, this is tested Freund's complete adjuvant used and buys from sigma company.
2) 80nmAuNPs preparation method for antibody
In order to strengthen the former immune response of body fight, during first immunisation, immunogene need be mixed with Freund's complete adjuvant equal-volume ratio and being subcutaneously injected in male rabbit body.Mix by the immunogene of equal-volume ratio and freund 's incomplete adjuvant during booster immunization and carry out hypodermic injection again, carry out booster immunization week about, antibody titer is surveyed in middle week blood sampling, booster immunization six times altogether, antibody titer reaches experiment demand and desirable blood obtains antiserum, and purifying obtains 80nmAuNPs antibody.Concrete immunologic process is as follows:
Get 4 body weight be the healthy Male New Zealand White Rabbit of about 2kg as animal used as test, wherein 3 as immunization, and the 4th as blank.During first immunisation, it is even to get isopyknic 80nmAuNPs immunogene and Freund's complete adjuvant emulsification, is subcutaneously injected into the back of 3 male rabbits, often dorsal injection 8 ~ 10 points of only male rabbit, and injection volume is only about 1mL/.First time booster immunization is carried out after 3 weeks, with the immunogene of equal-volume ratio and the potpourri of freund 's incomplete adjuvant during booster immunization, injecting method is with first time immunity, carry out a booster immunization week about, injection volume is 1mL/ later, and middle all venous blood collections survey serum titer, until serum titer meets experiment demand, carry out last booster immunization, altogether booster immunization six times, get blood purifying and obtain antibody.
3) qualification of antibody titer
The tiring of antibody is used to weigh immune effect, and in order to detect the quality of obtained 80nmAuNPs antibody mass, this experiment takes tiring of indirect competitive enzyme-linked immunosorbent assay antibody.Concrete grammar: with CB, 80nmAu is diluted to a series of concentration and is coated in 96 hole ELISA Plate, 4 DEG C of refrigerator overnight, morning next day, 3 times are washed with washing lotion PBST, each 3min, removing is not wrapped by upper envelope antigen, every hole 200 μ L1%OVA closes, 37 DEG C of closed 1h, PBST washes 3 times, each 3min, add the antiserum (dilution ratio is 1/1000 ~ 1/64000) with PBS dilution, every hole 100 μ L, 37 DEG C of incubation 2h, PBST washes 3 times, each 3min, add the goat anti-rabbit igg that dilution ratio is the HRP mark of 1/1000, every hole 100 μ L, 37 DEG C of incubation 2.5h, PBST washes 3 times, each 3min, add substrate solution colour developing, every hole 100 μ L, 37 DEG C of incubation colour developing 0.5h, add 2mol/LH 2sO 4cessation reaction.490nm place light absorption value is surveyed by multi-functional microplate reader.
(3) indirect competitive enzyme-linked immunosorbent analytic approach quantitatively detects 80nmAuNPs
Because the indirect competitive enzyme-linked immunosorbent analytic approach in luminosity enzyme-linked immunosorbent assay is that the goat anti-rabbit igg (namely two resist) utilizing HRP to mark detects 80nmAuNPs antibody (i.e. primary antibodie), light signal finally by the anti-compound of detectable antigens primary antibodie two reaches the object of quantitative detectable antigens, the method utilizes the optical signal detecting antigen of the compound of antigen primary antibodie and Direct cELISA to have lower detection limit than only, higher sensitivity, therefore this experiment adopts Indirect cELISA quantitatively to detect 80nmAuNPs.Specific experiment process is as follows:
1) bag quilt: wrap by 96 hole ELISA Plate with the 80nmAu-OVA that concentration is 21.7 μ g/mL, every hole 100 μ L, 4 DEG C, refrigerator spends the night.
2) close: morning next day, take out ELISA Plate, PBST washs 3 times, each 3min, adds 1%OVA and closes, every hole 200 μ L, 37 DEG C of closed 1h.
3) application of sample competition: PBST washs 3 times, each 3min, every hole adds 50 μ L homemade 80nmAuNPs titers or liquid to be measured and 50 μ L80nmAuNPs antibody, 37 DEG C of competition 2h.
4) enzyme-added: PBST washs 3 times, each 3min, every hole adds the goat anti-rabbit igg that 100 μ L dilution ratios are the HRP mark of 1/1000,37 DEG C of incubation 2.5h.
5) develop the color: PBST washs 3 times, each 3min, every hole adds 100 μ L substrate solution 37 DEG C colour developing 0.5h.
6) stop: every hole adds 50 μ L2mol/LH 2sO 4cessation reaction, surveys 490nm place light absorption value in multi-functional microplate reader.
(4) drawing standard curve
As shown in Figure 2, the linear equation of typical curve is obtained typical curve: A=1.18-0.145logC, coefficient R 2=0.994, the range of linearity is 10 -3-10 3, detect be limited to 0.0057ng/mL. by typical curve calculate survey unknown concentration 80nmAuNPs concentration be 9.48ng/mL, the recovery is 103.9%-117%.

Claims (10)

1. quantitatively detect a method of 80nmAuNPs based on Indirect cELISA, it is characterized in that, said method comprising the steps of:
The preparation of a, 80nmAuNPs;
B, 80nmAuNPs envelope antigen and immunogenic preparation;
Prepared by c, 80nmAuNPs antibody:
By the 80nmAuNPs immunogen injection that obtains in animal body, obtain 80nmAuNPs antibody;
D, utilize the 80nmAu antibody of the high specific that purifying is good and 80nmAu envelope antigen to carry out indirect competitive enzyme-linked immunosorbent analytic approach, reached the object of the 80nmAu quantitatively detecting unknown concentration by Criterion curve.
2. method according to claim 1, is characterized in that, in step a, the preparation method of 80nmAuNPs is: by HAuCl 4after solution mixes with distilled water, ebuillition of heated, then add trisodium citrate, continue reaction, be cooled to room temperature, obtain 80nmAuNPs.
3. method according to claim 2, is characterized in that, the preparation method that step a prepares 80nmAuNPs is specially: get 500 μ LHAuCl 4solution and 99mL distilled water are in there-necked flask, boiling is heated to heat collecting type constant-temperature heating magnetic stirring apparatus, process need 15min ~ 30min, adds trisodium citrate fast, the rapid blackening of reactant liquor in the solution of boiling, then earth look red is become, after continuing reaction 35min, remove heating arrangement, stir and be cooled to room temperature, collecting 4 DEG C of lucifuges stores stand-by, and this earth red solution is 80nmAuNPs.
4. method according to claim 3, is characterized in that, step a adds 0.7mL1wt% citric acid three sodium solution.
5. method according to claim 1, it is characterized in that, 80nmAuNPs envelope antigen and to be immunogenicly prepared as in step b: get 80nmAuNPs solution prepared by step a, after adding thioglycolic acid TGA stirring 10-25min under the state stirred, continue to add N-Hydroxysuccinimide NHS and 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine EDAC lucifuge stirring 2-5h, regulate pH=9 ~ 10, add 1wt% bovine serum albumin(BSA) BSA or 1wt% chicken ovalbumin OVA ice bag lucifuge stirring 5h, obtain and correspondingly obtain immunogene and envelope antigen, gained solution is placed in bag filter, distill water dialysis 8-15h, collecting 4 DEG C of lucifuges stores stand-by.
6. method according to claim 5, it is characterized in that, 80nmAuNPs envelope antigen and to be immunogenicly prepared as in step b: in step b, the volumetric usage of thioglycolic acid TGA and N-Hydroxysuccinimide NHS, 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine EDAC is than being 2:1:2.
7. method according to claim 5, is characterized in that, in step b, the volumetric usage of 80nmAuNPs, thioglycolic acid TGA, bovine serum albumin(BSA) BSA or chicken ovalbumin OVA is than being 400:1:10.
8. method according to claim 1, is characterized in that, in step c, 80nmAuNPs preparation method for antibody is:
By immunogene and freund adjuvant miscible with volume ratio 1:1, repeatedly immunity is carried out to animal, obtained 80nmAuNPs antibody.
9. the method according to claim 1 or 8, it is characterized in that, in step c, 80nmAuNPs preparation method for antibody is specially: by immunogene with Freund's complete adjuvant equal-volume than hybrid injection in animal, adopt the mode of dorsal sc multi-point injection, inject 8 ~ 10 points, inoculation amount is each 1mL/.First immunisation is after three weeks, carry out first time booster immunization, immunogene being mixed with freund 's incomplete adjuvant equal-volume is expelled in animal again, injection volume is each 1mL/, after this booster immunization is carried out again week about, immune time is 7 ~ 8 times altogether, and middle week blood sampling survey is tired, until antibody titer meets experiment demand.
10. method according to claim 1, is characterized in that, steps d indirect competitive enzyme-linked immunosorbent analytic approach is:
(1) bag quilt: by dilution ratio be the 80nmAuNPs-OVA envelope antigen bag of 1/50-1/500 by 96 hole ELISA Plate, every hole 100 μ L, 4 DEG C of refrigerator overnight;
(2) close: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away not combined on 80nmAuNPs envelope antigen, add closed chicken ovalbumin 1wt%OVA to close, every hole 200 μ L, closes the site that not coated antigen combines, 37 DEG C of baking oven incubation 1 ~ 2h;
(3) application of sample competition: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away unnecessary confining liquid, every hole adds the standard items of 50 μ L80nmAuNPs or 80nmAuNPs sample solution to be measured and 50 μ L80nmAuNPs antibody and to be at war with reaction, baking oven incubation 1 ~ 2h, makes the AuNPs in determinand or standard items and envelope antigen compete anti-80nmAuNPs antibody simultaneously;
(4) enzyme-added: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away liquid to be measured or the antibody of not combined free state, add the goat anti-rabbit igg (two resist) of horseradish peroxidase-labeled, every hole 100 μ L (combining for the anti-80nmAuNPs antibody (primary antibodie) that AuNPs is combined in envelope antigen in previous step) baking oven incubation 2 ~ 4h;
(5) develop the color: PBST washs 96 hole ELISA Plate 3 times, each 3 ~ 5min, wash away not combined two and resist, add o-phenylenediamine, every hole 100 μ L, baking oven incubation colour developing 0.5 ~ 1h;
(6) stop: every hole adds 50 μ L, the H of 2mol/L 2sO 4cessation reaction, microplate reader measures the light absorption value of each hole at 490nm place;
(7) drawing standard curve, by typical curve calculate survey the concentration of unknown concentration 80nmAuNPs.
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