CN101290319B - H5N1 type highly pathogenic avian influenza nanometer quantum point detection method - Google Patents

Abstract

The invention discloses a method for detecting a nano quantum dot of the highly pathogenic bird flu of H5N1, belonging to the disease inspection field. The method comprises the following steps that: (1) a monoclonal antibody or a polyclonal antibody of the bird flu of H5N1 is prepared and purified; (2) a water-solubility carboxylation nano quantum dot is prepared; (3) the nano quantum dot is used for marking the monoclonal antibody or the polyclonal antibody of the bird flu of H5N1; and (4) the sample is detected. The method has the advantages that the nano quantum dot is applied to the diagnosis of the highly pathogenic bird flu of H5N1, the method has high speed, stability, convenient detection of high flux, low condition requirement, simple and convenient operation and easy popularization; the method has the characteristics of simple operation, high speed and high sensitivity, etc. The method provides an effective way to detect and diagnose the highly pathogenic bird flu of H5N1 in China and has good popularization and application potential in the aspect of quarantine inspection in the process of import and export.

Description

The nanometer quantum point detection method of H5N1 type highly pathogenic bird flu

Technical field

The present invention relates to the nanometer quantum point detection method of H5N1 type highly pathogenic bird flu, is more specifically a kind of quantum dot-labeled avian influenza virus monoclonal antibody or many anti-methods that detects that adopts ultrasonic emulsification modification preparation, belongs to inspection and quarantine field.

Background technology

Bird Flu (is called for short bird flu, avian influenza, A I) be the bird deadly infectious disease that is caused by A type influenza virus, be decided to be the category-A infectious disease by International Office of Epizootics, wherein the H5N1 subtype avian influenza is the most frequent a kind of highly pathogenic bird flu that breaks out in recent years, bring huge economic loss to domestic fowl farming, also threatened the mankind's life and health.Infect the case of bird flu along with China has also found the people, the prevention and control of highly pathogenic bird flu are particularly important.The control key of bird flu is to set up the method for Rapid diagnosis of Avian Influenza virus, and existing diagnosis of avian influenza can not satisfy the needs of the quick diagnosis of China's highly pathogenic bird flu, therefore, set up the task of top priority that the Rapid diagnosis of Avian Influenza technology has become China's prevention and controlled highly pathogenic bird flu.

Fast detecting to bird flu is to control the basic prerequisite of this disease.Because clinical symptoms and the pathological change of bird flu are different because of the difference of kind, the length of time, course of disease length, accompanying infection situation and the infection strain power etc. of infection fowl, therefore, the symptom of bird flu depends on the separation of cause of disease always and identifies.The diagnosis of at present bird flu be mainly to separate and serological test detects virus or the antibody of detection infected chicken is identified by virus.Wherein serodiagnosis comprises six kinds of detection methods such as agar gel diffusion test (AGP), blood clotting (HA) and hemagglutination-inhibition test (HI), neuraminidase inhibition test (NI), neutralization test (NT), immunofluorescence technique (IFT), enzyme linked immunosorbent assay (ELISA).In recent years, along with the development of Protocols in Molecular Biology, the RT-PCR method also is applied to the detection of bird flu gradually.

The deficiencies in the prior art part is:

1. the separation of virus identifies it is a kind of reliable method of diagnosis AI, but length consuming time is cultivated in the virus separation, needs at least 7d, is unfavorable for the rational of quick diagnosis and immune programme for children, is difficult to competent needs to highly pathogenic AI quick diagnosis.

2. the virus antigenicity specific test is carried out in agar diffusion (AGP) test, although this method is simple, susceptibility is relatively poor, is prone to false positive.Carrying out HA when experiment, should first go out non-specific non-aggegation inhibiting factor.It is good that blood coagulation tests (HA) and blood clotting suppress experiment (HI) specificity, but its process is loaded down with trivial details time-consuming, but its operation is relatively loaded down with trivial details, must possess the standard virus of certain hemagglutinative titer and the red blood cell of fresh preparation simultaneously.Serological testing generally can only just can have antibody positive to occur to making a definite diagnosis at last retrospective diagnostic value in 23 weeks of morbidity even for more time.Neuraminic acid enzyme test (NI) test is much more complicated than the HI test, and general diagnostic test chamber can not be completed, and should complete by specializing in the Reference Lab that bird flu detects.Neutralization test (NT) is a kind of more responsive and special serological method, but its complex operation, the material that takes consuming time is clinically used hardly.

3.ELISA be that development in recent years gets a kind of comparatively ripe method, susceptibility is higher, shortcoming is that the susceptibility of test is subjected to the impact of sample quality very large.Negative findings does not show and does not infect avian influenza virus, can only be interpreted as not detecting.Immunofluorescence technique (IF) is fluorescence antibody (fluoresent antibody, FA) technology, being identify and locate specific antigen in the influenza infection cell, is mainly nucleoprotein (NP) or the stromatin (MP antigen) of influenza virus.What the detection of avian influenza virus was adopted usually is direct fluorescence method, this method is used for diagnosis and has the characteristics such as quick, easy and responsive, but false positive (non-specific fluorescence) appears sometimes, IiT also can be used for detecting the reaction of NP and MP antigen and antibody, and its susceptibility is very high.But preparation is had relatively high expectations to antigen, needs with non-ionic detergent, the virion of purifying to be carried out cracking.

4.RT-PCR method is a kind of special method of sensitivity, sensitivity will be higher than ELISA, and because this technical conditions requirement is high, and the field sample is complicated and changeable, disturbs greatly, and sample process is difficult, therefore detects on a large scale more difficult.

Nano-quantum point (quantum dot, QD) can be described as again semiconductor nanocrystals body (semiconductornanocrystal), is a kind of stable, water-soluble, the nanocrystal of size between 2～20nm that is comprised of II-VI family or III-V family element.That research is more at present is CdS, CdSe, CdTe, ZnS etc.In recent years, semiconductor-quantum-point is because its unique character more and more is subject to people's attention, and it is multidisciplinary that its research contents relates to physics, chemistry, material, biology etc., become an emerging cross discipline.

Quantum dot is because particle diameter is very little, and electronics and hole can be with to become the discrete energy levels structure with molecular structure by quantum confinement continuously.Therefore the optics behavior is very similar to some large molecules, can emitting fluorescence.Compare with traditional fluorescent dye, quantum dot has many good qualities: inorganic crystallites can be born exciting with light and launch repeatedly, and organic molecule can decompose. lasting stability can allow the researchist observe for more time cell and tissue, and carries out without difficulty the modifying interface connection.Measuring in the benefit of a maximum is that abundant color is arranged.The complicacy of living things system often need to be observed several components simultaneously, if dye with dye molecule, need different wave length to excite, measure in point and do not have this problem, use the nanocrystal of different sizes (and then different color) to come the different biomolecule of mark.Use single light source just can make the different particles can be by immediately monitoring.And from the ultraviolet ray to ruddiness, quantum dot has excitation spectrum scope widely, can with the size of quantum dot with become to assign to regulate excitation spectrum.The fluorescence emission spectrum peak of quantum dot with narrow symmetry simultaneously so just can use the quantum dot of different spectral characteristics simultaneously, and that emission spectrum does not occur is overlapping.Therefore, nano-quantum point is as a kind of novel fluorescence labeling material, and the application in biological field has more and more received closing general concern, has particularly obtained considerable progress at methods for clinical diagnosis.

Summary of the invention

First technical matters that the present invention will solve is to provide a kind of nanometer quantum point detection method of H5N1 type highly pathogenic bird flu.

Second technical matters that the present invention will solve is to provide a kind of carboxylated nano-quantum point of new type water-solubility that labelled antibody carries out biological detection that can be used for.

The 3rd technical matters that the present invention will solve is to provide the method that the ultrasonic emulsification modification prepares quantum dot.

For achieving the above object, the present invention is by the following technical solutions:

The nanometer quantum point detection method of H5N1 type highly pathogenic bird flu comprises the steps:

(1) preparation and purifying H5N1 type bird flu monoclonal antibody or polyclonal antibody;

(2) the water-soluble carboxyl nano-quantum point of preparation;

(3) nano-quantum point mark H5N1 type bird flu monoclonal antibody or polyclonal antibody;

(4) sample determination.

Described (1) preparation and the method for purifying H5N1 type bird flu monoclonal antibody are: with the concentrated H5N1 type avian influenza virus of purifying as immunizing antigen immunity 8 Balb/c mouse in all ages; Adaptive immune splenocyte and SP2/0 Fusion of Cells obtain hybridoma; The clone of screening positive hybridoma cell cultivates the hybridoma cell strain that obtains stably excreting antibody; Hybridoma cell strain is injected into the Balb/c mouse peritoneal induces with external the ascites that the ascites method obtains to contain monoclonal antibody; Adopt sad-saturated sulfuric acid amine method to concentrate and purifying ascites acquisition monoclonal antibody.

Described (1) preparation and the method for purifying H5N1 type bird flu polyclonal antibody are: the H5N1 type avian influenza virus that concentrates with purifying is as immunizing antigen, immune new zealand white rabbit; Heart blood sampling after 10 days, separation of serum; How anti-saturated ammonium sulfate is slightly carried; DEAE-cellulose chromatography crosses that the IgG of purifying puts in bag filter after post, concentrates with the solid polyethylene glycol embedding and obtains polyclonal antibody.

The method of described (3) nano-quantum point mark H5N1 type bird flu monoclonal antibody or polyclonal antibody is: preparation aldehyde radical slide; The method of utilizing crosslinking chemical EDC to be connected covalent bond to connect with sulfo-NHS is carried out the quantum dot-labeled of antibody.

Slide is difficult for permeating and spreading on its surface due to sample and its hydrophobicity, particularly fluorescence background are hanged down characteristics such as being convenient to Fluirescence observation, thereby by our carrier as experiment reaction in this subject study.It is in the most frequently used approach of surface of glass slide ankyrin that surface of glass slide is carried out aldehyde radical.Adopt amino silane and glutaraldehyde successively to carry out amination and aldehyde radical reaction, form the aldehyde radical of activation in surface of glass slide, the amino condensation reaction of the aldehyde radical of activation and protein, thus the method for formation covalent bond is at the surface of glass slide fixing protein.

Described preparation aldehyde radical slide refers to: with slide washing lotion (the dense H2SO4350ml+H2O40ml of K2CrO7 20g+) soaked overnight, then use a large amount of deionized water rinsings, after 60 ℃ of oven dry 2h, slide is immersed in the ethanolic solution of 5%APTES, effect 60min carries out the amination of surface of glass slide, take out washing, after drying, slide is immersed the PBS (0.2mol/L that contains 5% glutaraldehyde, pH8.0) carry out the aldehyde radical of slide in solution, the cleaning of PBS solution is dried rear standby.

The carboxyl reaction on EDC and quantum dot surface generates the acyl group isourea, but the easily hydrolysis in aqueous solution of acyl group isourea generates again the carboxyl product.If add sulfo-NHS in solution; the acyl group isourea just very easily reacts with sulfo-NHS; generation has the sulfo-NHS ester of amine reactivity, and the amino reaction on sulfo-NHS ester and antibody surface generates stable acid amides, thereby carries out the quantum dot-labeled of antibody.According to condition test, the experiment condition of the quantum dot-labeled antibody that we determine at last is that the best working concentration of coupling agent EDC and sulfo-NHS is 2.0mM and 5.0mM, the concentration of quantum dot is 3.0mM, the ratio of antibody and quantum dot the best is 3:1, the optimal pH of antibody labeling is 7.5, and the best reaction time is 2h.

The described method of utilizing crosslinking chemical EDC to be connected covalent bond to connect with sulfo-NHS is carried out the quantum dot-labeled of antibody and is referred to: the activation damping fluid (0.1MMES that adds 1ml in the eppendorf of 1.5ml, 0.5M NaCl, PH6.0), then add appropriate EDC and sulfo-NHS solution; Add the 20ul quantum dot solution in solution, ceaselessly mixed solution, react 20min under room temperature again; Add 1.4ul (20mM) 2 mercapto ethanol at room temperature to react 10min, the quantum dot solution that obtains activating; In the quantum dot solution of activation, add 1ml to contain the PBS solution (0.01M, pH7.5) of appropriate H5N1 type avian influenza antibody, and regulate PH to 7.5 with NaOH solution, under room temperature, stirring reaction is 2 hours; Little molecule and unreacted antibody is completely removed in 4000rpm centrifuging 1 minute; With 5ml deionized water dissolving precipitation, repeat quantum dot-labeled bird flu monoclonal antibody solution is carried out dissolution/precipitation 2 times, at last with 2ml deionized water dissolving precipitation, 4 ℃ of preservations.

The method of described (4) sample determination is: the surface of glass slide at aldehyde radical drips sample allantoic fluid to be checked, hatches 2h for 37 ℃; With washing slide 3 times, each 5min; Then drip in surface of glass slide the PBS-Tween solution that contains 5%BSA, hatch 45min for 37 ℃; Use 0.01M, the PBS washing slide of pH7.2 3 times after each 5min, adds quantum dot-labeled avian influenza antibody, hatches 45min for 37 ℃, uses 0.01M, the PBS washing slide of pH7.2 3 times, each 5min; Dry under room temperature after at the fluorescence microscopy Microscopic observation, the yin and yang attribute of judgement sample.

A kind ofly can be used for the carboxylated nano-quantum point of new type water-solubility that labelled antibody carries out biological detection, adopt following method to prepare.

Prepare the method for water-soluble carboxyl nano-quantum point, comprise the steps:

(1) preparation CdSe core-shell quanta dots;

(2) preparation CdSe/CdS core-shell quanta dots;

(3) mercaptoacetic acid is modified the CdSe/CdS quantum dot;

(4) the hydrophobic self-assembled modified quantum dot of amphiphilic macromolecular;

(5) ultrasonic emulsification modified quantum dot.

Present normally used quantum dot surface all is hydrophobicity by a large amount of organic solvent molecules coatings, and biomacromolecule or medicine are generally dissolvings or are dispersed in aqueous solution, so quantum dot and they can not fully approach more directly effectively combination, therefore to obtain stable fluorescence probe, mostly first the quantum dot surface will be modified.

Utilize between IIB family's atom such as Zn, Cd and sulphur atom effectively bonding action, with the bifunctional molecule with sulfydryl, the organic molecule that replaces the QDs surface as mercaptan carboxylic acid's class, dithiothreitol (DTT) (DTT), thiocholine etc., so that it changes water wettability into from hydrophobicity, then the functional group by the other end can be connected with biomacromolecule, and this method is simple, good reproducibility.But due to use be little molecule as connector, after modifying, original organic molecule can not get effectively substituting to protection and the stabilization of quantum dot, so stability is bad.

Although the quantum point grain diameter with the hydrophobic self-assembled modified quantum dot gained of amphiphilic macromolecular is less, this kind method purification ratio is more difficult, and environment is unfriendly.

Emulsification is conducive to form the formation of microcapsules, yet if be simple mechanical raking, formed microcapsules system is stable not.Ultrasonication helps emulsification and can form air pocket, keeps emulsion-stabilizing.So it is water-soluble modified that we adopt the method for ultrasonic emulsification to realize.

The quantum dot of the ultrasonic emulsification modification preparation of adopting has method innovation, thicker and the tight densification of macromolecule layer can better be protected quantum dot, avoids surrounding medium to infiltrate and causes destruction such as the light degradation of the luminescent properties of quantum dot, photobleaching, and oozing out of Cd ion and produce toxicity; The method purifying is simple, only needs centrifugally, and generally water-soluble modified (self-assembled modified by hydrophobic effect) needed the steps such as film-concentrated-gel filtration-again is concentrated; Adopt the O/W method, consumption of organic solvent is few, time-saving energy-saving, environmental friendliness; The self-assembled modified nanoparticle surface of hydrophobic effect is rough, and the nanoparticle surface of ultrasonic emulsification preparation is bright and clean, is not easy to cause non-specific adsorption, can better be applied in the biological detection aspect.

This paper has developed the CdSe/CdS core-shell quanta dots, and it is water-soluble modified to adopt the method for ultrasonic emulsification that quantum dot has been carried out, and has set up the detection method that nano-quantum point is used for H5N1 type avian influenza virus.This detection method detection speed is fast, only needs 4 hours from the processing of sample to going out result.Testing result is stable, and fluorescence is difficult for occuring cancellation, the sample that detected observable result still behind tens of skies.And the method for quantum dot-labeled avian influenza antibody is very simple, only need simple centrifugally just can carry out purifying to marked product.And the fluorescein-labelled method of traditional antibody need to react and spend the night, and marked product also needs dialysis or chromatography to carry out purifying, and whole process is consuming time, and complex operation.It is 10 that the method detects high dilution to chick embryo allantoic liquid ^-6, substantially suitable with the sensitivity that chicken embryo virus is separated, higher than the detection sensitivity of RT-PCR.

Advantage of the present invention is: nano-quantum point is applied to the diagnostic method that H5N1 type highly pathogenic bird flu detects, the method fast, stablize, be convenient to that high flux detects, conditional request is low, easy and simple to handle, be easy to promote, have simple to operate, fast and the characteristics such as sensitivity is high.The foundation of the method will provide for the detection and diagnosis of China's highly pathogenic bird flu a kind of effective means, have good promotion and application prospect in the import and export quarantine.

The present invention will be further described below in conjunction with the drawings and specific embodiments, is not limitation of the present invention.Every any this area of doing according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.

Description of drawings

Fig. 1 is absorption spectrum (a) and the fluorescence spectrum (b) of the CdSe/CdS QDs of three kinds of different-grain diameter sizes.

Fig. 2 is the ultra-violet absorption spectrum that mercaptoacetic acid is modified CdSe/CdS oil-soluble quantum dot.

Fig. 3 be the self-assembled modified quantum dot of amphiphilic macromolecular material TEM photo 190,000 *.

Fig. 4 (a) is the ultra-violet absorption spectrum of the hydrophobic self-assembled modified quantum dot of amphiphilic macromolecular.Fig. 4 (b) is the fluorescence spectrum after modification.

Fig. 5 is the TEM photo after the ultrasonic emulsification modified quantum dot.

Fig. 6 is the ultra-violet absorption spectrum of ultrasonic emulsification modified quantum dot.

Fig. 7 be aldehyde radical slide result verification as a result figure (a) add goat anti-rabbit igg; (b) add goat-anti chicken IgG.

Fig. 8 is the reaction principle figure of quantum dot-labeled antibody.

Fig. 9 is the centrifugal quantum dot-labeled many anti-rear peak shape figure that cross post of precipitation.

Figure 10 is the centrifugal quantum dot-labeled product reaction of precipitation fluorescence picture.

Figure 11 was the how anti-product figure of the quantum dot-labeled bird flu of post purifying.

Figure 12 was each component of post purifying and antigen-reactive fluorescence picture.

Figure 13 was the quantum dot-labeled bird flu monoclonal antibody figure of post purifying.

Figure 14 is the peak shape figure that crosses post after the quantum dot-labeled monoclonal antibody product of precipitation centrifugal purification.

Figure 15 a, b, c and d represent that respectively quantum dot-labeled H5N1 type bird flu monoclonal antibody detects ewcastle disease F48E9, IBDV, duck plague virus and DHV figure as a result.

Figure 16 is the figure as a result that the inventive method and indirect immuno fluorescent method detect the allantoic fluid that does not infect avian influenza virus.

Embodiment

Main material and instrument

1.HT nutrient culture media (Sigma company), HATA selects nutrient culture media (Sigma company), DMEM high glucose medium (CIBCOBRL company), Fusion of Cells agent (Sigma company), Benzylpenicillin sodium salt (North China pharmacy), streptomycin sulphate (North China pharmacy), sodium dodecylsulphonate (SDS) (Sigma company), caprylic acid (Beijing chemical reagents corporation), the sheep anti-mouse igg of HRP mark (glad through biological products company limited of section), polyglycol (PEG, molecular weight 6000) (glad through biological products company limited of section), gel filtration filler SephadexG-200 (Amersham bioscience), bird flu H 5 N 1, newcastle disease virus (NDV), egg drop syndrome virus (EDS76V) is preserved for this laboratory, SPF chicken embryo is available from Cimmeria animal health-care product company limited, the BALB mouse is available from institute of lab animals, Beijing, the SP2/0 cell is preserved for this laboratory, new zealand white rabbit is available from Beijing Experimental Animal Center.

automatic water purification machine (U.S. PAL1.CO), CO2gas incubator (SANYO GS), inverted microscope (Japanese Olympus), supercentrifuge (U.S. Optime), enzyme connection detector (U.S. Thermo Labsystems), desk-top electronic balance (German SATORIOVS), constant water bath box (Finland HETO), micropore Tissue Culture Plate (), electrophoresis set of equipments (American I nvitrogen), inverted fluorescence microscope (Japanese Olympus), centrifuge5810R tabletop refrigerated centrifuge (German eppendorf company), AKTA Basic protein purification instrument (AmershamBiosciences).

2. the preparation of quantum dot

three n-octyl phosphorous oxide (TOPO, 90%, Aldrich), three normal-butyl phosphorus (TBP, 90%, TCI, Japan), octadecylamine (ODA, 90%, ACROS), octadecylene (ODE, 90%, ACROS), elemental sulfur (S, 99.9%Aldrich), oleic acid (OA, 90%, Aldrich), cadmium oxide (CdO, 99.5%, Aldrich) and Se powder (100mesh, 99.99%, Aldrich), toluene, ethanol, methyl alcohol, normal hexane, chloroform, it is pure that methylene chloride and acetone are analysis, available from prestige reagent company of section of University Of Tianjin.

UV-2450 ultraviolet-visible pectrophotometer (Japanese Shimadzu company), P JEM-100CX II type transmission electron microscope (erkin Elmer Inc.), F-4500 fluorospectrophotometer (Hitachi), G2F20 transmission electron microscope (Tecnai), SHJM-1 type digital display constant temperature stirs electric jacket (Juancheng, Shandong Province welfare scientific research apparatus factory), Hettich Mikro22R type refrigerated centrifuge (JEOL company), electronic balance ALC-100.4 (Beijing Sai Duolisi balance company limited)

3. antibody is quantum dot-labeled

EDC (1-ethyl-3-(3-dimethylamino) carbodiimide hydrochloride, Pierce), sulfo-NHS (sulfo-N-N-Hydroxysuccinimide, Acrose), (3-aminomethyl) triethoxysilane (APTES, ((3-Aminopropyl), triethoxysilane, Alfa Acesar), gel filtration filler Superdex200 (Amersham bioscience), gel filtration filler sepharose6Fast Flow (Amersham bioscience).

UV-2450 ultraviolet-visible pectrophotometer (Japanese Shimadzu company), centrifuge5810R tabletop refrigerated centrifuge (German eppendorf company), BX51 is just putting fluorescent microscope (Japanese Olympus), AKTA Basic protein purification instrument (Amersham Biosciences), wd-9403f ultraviolet transilluminator (Liuyi Instruments Plant, Beijing).

4. the detection of sample

Bovine serum albumin (BSA, Beijing chemical reagents corporation), Tween-20 (Beijing chemical reagents corporation), ewcastle disease F48E9, IBDV, duck plague virus and DHV are retained (enforcement of the present invention also can be chosen other similar virus, not impact effect) by this laboratory.

BE500 constant incubator (German MEMMERT), BX51 are just being put fluorescent microscope (Japanese Olympus).

Embodiment 1: the breeding of virus, purifying and concentrated

1. the breeding of virus

Get the AIV H5N1 virus, do 1 * 10 ⁴Doubly dilution, allantoic cavity inoculation instar chicken embryo on the 10th, 0.2ml/ piece, 37 ℃ of cultivations, per sunshine, egg was 3 times, eliminated dead germ dead in 24 hours, collected the 24-96 hour chicken embryo of death not yet.Put 4 ℃ cooling, aseptic collection allantoic fluid.

Virus deactivation and purifying

(1) deactivation: add formaldehyde to make its final concentration in the allantoic fluid of collecting and be 0.1%, be placed in 37 ℃ of biochemical cultivation case deactivations.

(2) differential centrifugation 4000rpm, 4 ℃ centrifugal 45 minutes, discard precipitation.Supernatant is in 100,000 * g, 4 ℃ of ultracentrifugations 2 hours, and collecting precipitation is with the resuspended virus of PBS (pH7.4) of former allantoic fluid 1/20 volume ,-80 ℃ of preservations.

(3) gel filtration 0.01M, the PBS of pH7.4 is as equilibrium liquid and eluent.Get 1g SephadexG-200, boil 30min with distilled water, fully fill post after swelling, with the abundant balance columns bed of equilibrium liquid.To slightly put forward virus and pack in post, and after virus liquid all enters chromatographic column, close end opening and complete loading, and then add eluent to carry out wash-out, coutroi velocity is 0.3ml/min, and the 2ml/ pipe is according to the OD of each pipe ₂₈₀Value and HA valency are collected satisfactory pipe eluent.

(4) eluent concentrated will be collected respectively manages the eluent bag filter of packing into, and use solid polyethylene glycol (PEG, molecular weight 6000) embedding 10h concentrates.Take out viral concentrate, measure its OD with ultraviolet spectrophotometer ₂₆₀And OD ₂₈₀, according to formula: protein concentration (mg/ml)=1.45 * OD ₂₈₀-0.74 * OD ₂₆₀, calculate the concentration of virus protein, and measure the HA valency of protein concentrate.

3. result

Get the 200ml allantoic fluid and carry out purifying, differential centrifugation, with the outstanding precipitation of 1/20 PBS worm of first volume, ultraviolet spectrophotometer is measured its OD ₂₈₀And OD ₂₆₀, and according to formula: virus protein concentration (mg/ml)=(1.45 * OD ₂₈₀-0.74 * OD ₂₆₀) * extension rate, the content that calculates virus protein is 1.3564mg/ml.

After gel filtration, collect altogether eluent 20 pipes (1.0ml/ pipe), according to the OD that respectively collects component of ATAK BASIC protein purification instrument demonstration ₂₈₀, merge OD ₂₈₀Be in each pipe of first seam, obtain altogether 10ml, with fixedly ethylene glycol (PEG, molecular weight 6000) embedding, after 10h, volume is 1.5ml.Recording the concentrating virus protein concentration with ultraviolet spectrophotometer is 2.6859mg/ml.

The hemagglutination test result shows, the HA valency of the viral concentrate that obtains is 1:2 ¹¹

Embodiment 2: preparation and purifying H5N1 type bird flu monoclonal antibody

1. mouse immune program

With the concentrated virus of embodiment 1 purifying as immunizing antigen immunity Balb/c mouse in 8 age in week.Altogether immunity is four times, and immunity for the first time is with the complete emulsification of equivalent Fu Shi adjuvant, for the second time and immunity for the third time add the Fu Shi Freunds incomplete adjuvant of equivalent, do not add adjuvant the 4th time.2 weeks of each immune interval.The viral dosage of each immunity is every mouse 150 μ g, and immunization route is nape subordinate multi-point injection.After 2 weeks of the 4th immunity, tail is fed the HA valency that serum is measured in blood sampling, if serum titer is reasonable, merges first three sky and carries out booster immunization one time, with not adding the viral antigen of adjuvant through lumbar injection.

2. the foundation of hybridoma

(1) preparation of SP2/0 cell: before merging, 24-48h enlarges cultivation with SP2/0 cell sub-bottle, and 6h before merging discards nutrient solution in bottle, changes new liquid.During fusion, select the SP2/0 cell that form is good, be logarithmic growth, it is blown down from the bottle wall gently, be collected in the 50ml centrifuge tube, the centrifugal 3min of 1500rpm suspends with the 5mlDMEM basic culture solution and precipitates, once centrifugal again, then standby with counting after the DEME Eddy diffusion.

(2) preparation of feeder cells: will be without the ICR eyeball of mouse sacrificed by exsanguination of immunity; The mouse of putting to death is put in 75% alcohol soak 5min, change super-clean bench over to, be placed in aseptic waist dish; Peel off mouse skin, aseptic taking-up spleen is put into the plate that fills the 10mlDMEM basic culture solution, picks out connective tissue, and rinsing gently; Another fills in the plane ware of 10ml DMEM basic culture solution with the dirty immigration of Pi, overstocks Pi with the blunt nosed gripping repeatedly of sterilization dirty, fully discharges splenocyte wherein; The DMEM that will contain splenocyte moves in centrifuge tube, and the centrifugal 3min of 1500rpm abandons supernatant; With the resuspended precipitation of 10mlDMEM basic culture solution, inhale and blow evenly, 1500rpm, centrifugal 3min abandons supernatant.This step repeats 2-3 time; It is standby that precipitation selects nutrient culture media to suspend with 10ml HAT.

(3) preparation of immune spleen cell: after reinforced immunological 72h, immune mouse is extractd the eyeball bloodletting and separation of serum contrasts as antibody positive; The preparation of immune spleen cell is with the preparation process of above-mentioned feeder cells; At last, precipitation is standby with DMEM basic culture solution suspension counting.

(4) Fusion of Cells: get respectively 2.4 * 10 ⁸Individual splenocyte and contain 6.0 * 10 ⁷The suspension of individual SP2/0 cell joins in a 50ml centrifuge tube, gently mixing; The centrifugal 3min of 1500rpm discards supernatant as far as possible; Gently at the bottom of shock tube, make the loose even one-tenth pasty state of sedimentation cell with palm; With the agent of pipette, extract 1ml Fusion of Cells, make transfer pipet tip exposing cell, slowly add fusion agent, cell is fully contacted with fusion agent, be controlled at 60s and add; Add 25mlDMEM liquid to stop the effect of Fusion of Cells agent, method is as follows: slowly added 1mlDMEM liquid, added 4mlDMEM liquid, and then added 20mlDMEM liquid in 3 minutes in the 2nd minute in the 1st minute; Centrifuge tube is placed in 37 ℃ of CO ₂The standing 5min of incubator; The centrifugal 3min of 1500rpm/min abandons supernatant, selects the nutrient culture media fused cell with 105mlHAT, and the 10ml feeder cells suspension for preparing is all added, and is gently that mixing with cells is even; The suspension cell drop is entered in 6 96 orifice plates, and every hole 200 μ l are placed in 37 ℃ of CO ₂Cultivate in incubator; Merge second day, select nutrient culture media half amount to change liquid with HAT, the last merging the 4th day, then select nutrient culture media half amount to change liquid once with HAT, used HT instead and select nutrient culture media half amount to change liquid in the 8th day.Record the Growth of Cells situation every day.

(5) obtain positive hybridoma cell: (HI) detects with hemagglutination test.First detect with the 4 AIV H5 of unit hypotype antigen hemagglutinating antigens, for positive hole, then detect once with 8 units antigen hemagglutinating antigens, screen the strong positive hole.Blood cell used is the chicken red blood cell of 0.5% (V/V) concentration, directly supernatant stoste is carried out HI and detects; Screening: the cell gram gallery after merging grows at 1/10 o'clock that covers the cell hole bottom area, with establishing the HI method of getting well, its culture supernatant is detected.For the cell hole with 8 unit antigen hemagglutinating antigen tests positive, in time clone; 4 unit antigen hemagglutinating antigen tests positive, and 8 unit antigens detect the cell that is negative, and change cells frozen storing liquid, put into-80 ℃ of Refrigerator stores.

(6) clone of positive hybridoma cell (employing limiting dilution assay): prepared feeder cells clone's the same day, preparation process joins the feed cell suspension for preparing in 96 orifice plates as previously mentioned, 100 μ l/ holes; With detecting to inhale gently with suction pipe for the cell hole of strong positive, cell is uniformly dispersed, is drawn in the DMEM liquid that 1ml contains 20% hyclone, the suspension that takes a morsel carries out cytometer and sets; Select nutrient solution dilution positive hybridoma to 20/ml, 10/ml, 5/ml with HT; The cell suspension that dilution is good is added in 96 orifice plates of feeder cells, 100 μ l/ holes, and each dilutability is added to 32 holes; Every day observation of cell growing state.Selected cell culture fluid half amount to change liquid with HT on the 4th day; Treat that in the hole, the cell liquid flavescence detects; Selecting only has the positive hole of growing on a clone again to carry out cloning, until positive colony reaches 100%; After obtaining the hybridoma cell strain of stably excreting antibody, in time enlarge cultivation and frozen.

3. the frozen and recovery of cell

(1) cell is frozen: select to be in the cell of exponential phase, gently cell is blown down from the bottle wall; The centrifugal 3min of 1500rpm abandons supernatant; With cryopreserving liquid, cell precipitation is suspended, be sub-packed in cell cryopreservation tube, the 1.5ml/ pipe is added a cover and is obturaged, and indicates the cell code name; Cryopreservation tube is placed in the small beaker of absolute ethyl alcohol, changes-80 ℃ of refrigerator overnight over to after 4 ℃ of standing 2h of refrigerator, then it is moved into long preservation in liquid nitrogen container.

(2) recovery of cell: take out cryopreservation tube from liquid nitrogen pipe dress, put into rapidly 37 ℃ of water-baths, rock gently it is melted as early as possible; The centrifugal 3min of 1500rpm abandons supernatant; To precipitate suspension with the DMEM liquid that contains 20% serum, move into the cell bottle, and put in the CO2 incubator and cultivate.

4. the preparation of monoclonal antibody (adopting the external ascites method that induces)

Select multiparity Balb/c mouse, lumbar injection sterilization paraffin oil 0.5ml/ only; After 10-14 days, hybridoma is blown and beaten, the centrifugal 3min of 1500rpm abandons supernatant, uses the 5mlDMEM Eddy diffusion, counts standby; Every mouse peritoneal inoculation hybridoma 10 ⁶Individual; After 7-10 days, visible mouse web portion obviously increases, and gathers ascites; With the centrifugal 10min of ascites 3000rpm, collect supernatant ,-20 ℃ save backup.

5. monoclonal antibody concentrates and purifying (adopting sad-saturated sulfuric acid amine method)

Get ascites 10ml, add 0.06M Acetate-acetate buffer solution (pH4.0) 20ml (stirring of limit edged); After with 1M NaOH, ascites pH being transferred to 4.8, add while stirring caprylic acid 0.33ml, stirring at room 30min; Ascites is abandoned precipitation through the centrifugal 30min of 6000rpm, and supernatant transfers to 7.2 with 1M NaOH with pH, the limit edged stirs, and slowly adds isopyknic saturated ammonium sulfate, after stirring 30min, after the centrifugal 30min of 6000rpm/min, supernatant discarded, precipitation is dissolved in 6mlPBS; Slowly add 4ml saturated ammonium sulfate amine, and constantly stir, after 30min, the centrifugal 30min of 6000rpm/min is dissolved in precipitation in appropriate PBS, moves in bag filter and dialyses.

6. the evaluation of monoclonal antibody

(1) the HI valency is measured: with the ascites 3000rpm that collects, the 10min centrifugal treating discards impurity, measures the HI valency.

(2) the ELISA mensuration of tiring: coated: the H5 virus of purifying with differential centrifugation is as envelope antigen, with the coating buffer dilution, and 100 μ l/ holes, 4 ℃ are spent the night; Washing: dry coating buffer, with cleansing solution washing 3 times, 3min/ time; Add confining liquid, 100 μ l/ holes, 37 ℃ of 2h. dry, the same washing; Add two anti-: with PBS, HRP mark sheep anti-mouse igg press the working concentration dilution, 100 μ l/ holes, 37 ℃ of 60min. dryings, the same washing; Add substrate: every hole adds freshly prepared substrate solution, 100 μ l/ holes, 37 ℃, 15min.

G: stop buffer: every hole adds 100 μ l 1M H ₂SO ₄Cessation reaction; Reading: enzyme connection detector detects the OD in each hole automatically ₂₈₀

The mensuration of antigen coated concentration and antibody dilution: envelope antigen is done 1:20,1:40,1:80,1:160,1:320,1:640,1:1280,1:2560 dilution, the mouse positive and negative serum are done 1:2500,1:5000,1:10000,1:20000,1:40000,1:80000 dilution, indirect elisa method is measured each hole 0D655, square formation method defined antigen optimum dilution degree.

(3) monoclonal antibody ELISA titration: ascites is made doubling dilution from 1:10, measured it by the indirect ELISA method of determining and tire.

(4) cross matching: with the antigen hemagglutinating antigen of H7, H9, NDV, EDS76V, ascites is done the HI cross reaction respectively.The cross reaction that has been judged to of HI reaction is arranged, without the no cross reaction that is judged to of HI reaction.

(5) mensuration of monoclonal antibody heavy chain, light chain molecular weight: adopt SDS-PAGE to detect the monoclonal antibody molecular weight, ascites sad-the saturated ammonium sulfate purified product makes the electrophoresis sample.Concentrated glue and resolving gel concentration are respectively 5% and 12%.

7. the qualification result of monoclonal antibody

After measured, the HI that does the bird flu H5 hypotype monoclonal antibody of preparation tires and is that it is 106 that 1:211, ELISA tire.Blood clotting for H7 and H9 suppresses active, only H5 is had blood and presses down activity, illustrates that prepared antibody is the specific monoclonal antibody of anti-H5 subtype avian influenza hemagglutinin.With sad-saturated sulfuric acid amine method purified monoclonal antibody, then carry out the SDS-PAGE electrophoresis, at about 50KD and 27KD place, an obvious band is arranged respectively, heavy chain is about 50KD, and light chain is about 27KD.

Embodiment 3: preparation and purifying H5N1 type bird flu polyclonal antibody

With the concentrated H5N1 type avian influenza virus of purifying as immunizing antigen, the body weight of 3 health of immunity is at 2kg left and right bull new zealand white rabbit, immune programme for children such as table 1.Immune every 2 weeks of minor tick of first three time, the rear 10d ear edge vein exploitating blood of immunity for the third time, separation of serum, detect tiring of rabbit anteserum with agar immunodiffusion (AGID), more than if the fine jade expansion is tired and reached 1:16, slowly inject booster immunization 1 time with the concentrated recombinant protein that does not add adjuvant from ear vein, heart blood sampling after 10d, separation of serum is used for the extraction of IgG of the recombinant protein of the anti-AIV H5 of rabbit hypotype.

Table 1 rabbit immune programme for children

2. the purification of polyclonal antibody

(1) saturated ammonium sulfate is slightly carried: get 20ml serum, add physiological saline 20ml, then add saturated ammonium sulfate solution 10ml, make into 20% ammonium sulfate, the limit edged stirs, after abundant mixing, and standing 30min.The centrifugal 20min of 3000rpm abandons precipitation.Add to add again saturated ammonium sulfate 30ml in supernatant, make into 50% ammonium sulfate, abundant mixing, standing 30min.The centrifugal 20min of 3000rpm, the tears supernatant.Add 20ml physiological saline in precipitation, make it dissolving, then add saturated ammonium sulfate 10ml, after abundant mixing, standing 30in.The centrifugal 20min of 3000rpm abandons supernatant, and redissolve/precipitation 2-3 time is with 10ml physiological saline solution precipitation.4 ℃ of dialysed overnight in physiological saline, centre are changed liquid for several times (with the SO in the BaCl inspection dislysate of l% ₄ ²', until without SO ₄ ^2-Till).The centrifugal precipitation of going, supernatant is the IgG that slightly carries, and the IgG that slightly carries is dissolved in PBS (0.01M, pH7.2) be used for post.

(2) DEAE-cellulose chromatography is crossed post: use 0.0175M, the PBS of pH6.7 is as equilibrium liquid and eluent.Get appropriate DEAE-cellulose in the sodium hydroxide solution of 0.5M, soak 1h, frequently stir, use the Buchner funnel suction filtration, wash with distilled water, then suction filtration, until filtrate near neutrality till.Cellulose is soaked in the HCl of 0.5M, same suction filtration is extremely medium-sized, then cellulose is soaked in the NaOH of 0.5M again, and same the processing is washed till medium-sized.Then carry out carrying out wash-out after balance, dress post, loading.Connect the wash-out bottle, open the post end opening and begin wash-out, the sulfo group sodium sulphate with 20% checks, collects eluent when beginning to produce white precipitate, until in collection liquid without protein, be the IgG of purifying.

The IgG that purifies is put in bag filter, concentrate with solid polyethylene glycol (PEG, molecular weight 6000) embedding 10h; Measure the concentration of albumen.

3. Anti-TNF-α body measurement

Measuring its concentration through ultraviolet spectrophotometer is 2.8345mg/ml.

Embodiment 4: the preparation of water-soluble carboxyl quantum dot

1.CdSe the preparation of core-shell quanta dots

Get the 0.1429g selenium powder, be dissolved in 1ml octadecylene and 1.2mlTBP (trioctylphosphine phosphorus), sealing saves backup.Take the 0.3mmol cadmium oxide in there-necked flask, 0.4ml oleic acid and 4ml octadecylene are heated to cadmium oxide after maintenance 30min and dissolve fully in argon atmosphere.Take 0.5gTOPO (trioctylphosphine oxide), add in above-mentioned mixed solution under room temperature.After logical argon gas 45min, mixed solution is heated to 260 ℃, adds fast the selenium solution that has prepared again, after reaction 2min, continues stirring and is cooled to room temperature.After twice of CdSe quantum dot use acetone precipitation, the washing for preparing, be dissolved in methenyl choloride standby.

2.CdSe/CdS the preparation of core-shell quanta dots

Take the 51.9mg cadmium oxide, be dissolved in 0.4ml oleic acid and 10ml octadecylene.After logical argon gas 30min, mixed solution is heated to 270 ℃, and this moment, solution was yellowish transparence, and cool to room temperature is standby.Take sulphur powder 12.8mg, be dissolved in the 10ml octadecylene, after logical argon gas 30min, mixed solution is heated to 170 ℃, and solution is the yellow transparent shape, and cool to room temperature is standby.

Get 2mlCdSe core quantum dot, add the 5ml octadecylene, octadecylamine, each 0.5g of TOPO after logical argon gas 40min, are heated to 100 ℃ in flask, keep 10min removing unnecessary methenyl choloride, then are heated to 220 ℃, and this moment, keeping system was stable.According to data in literature, calculate every layer of required Cd of quantum dot of preparation ²⁺, S ^2-Amount.First dropwise add the required cadmium solution of ground floor, after 8min, add the required sulphur solution of ground floor, after 25min, then add the required cadmium solution of the second layer, after 8min, add the required sulphur solution of the second layer equally.After twice of CdSe/CdS quantum dot use acetone precipitation, the washing for preparing, the lucifuge sealing is kept in refrigerator stand-by.

3. the finishing of nano-quantum point

(1) mercaptoacetic acid is modified the CdSe/CdS quantum dot: first with acetone washing three times, precipitation is got in centrifuging to CdSe/CdS oil-soluble quantum dot, then disperses CdSe/CdS oil-soluble quantum dot with the chloroform dissolving.Getting excessive mercaptoacetic acid joins in CdSe/CdS oil-soluble quantum dot chloroformic solution, lucifuge stirred 6 hours, the TOPO/TOP organic molecule on oil-soluble quantum dot surface is mostly just substituted by water miscible mercaptoacetic acid molecule, thereby realizes water-solubleization of oil-soluble quantum dot.React after 6 hours, centrifuging and taking precipitation, then disperse with the PBS dissolving, more centrifugal, three times to remove excessive mercaptoacetic acid and other organic molecules repeatedly.

(2) the hydrophobic self-assembled modified quantum dot of amphiphilic macromolecular: get appropriate oil-soluble quantum dot powder and amphiphilic three block macromoleculars are dissolved in the chloroform/ethanol solution of 3:1.Stirred for several is hour to remove the chloroform organic solvent.After chloroform is removed (when can't smell the chloroform smell), add appropriate PBS buffer solution, more ultrasonic dispersion 3 minutes.Resulting solution removing a large amount of unconjugated macromolecules, is crossed after film the solution optical clear that becomes after the 0.22um film.The resulting coating solution of crossing is concentrated to after finite concentration after gel chromatographic columns with super filter tube again, further removes unconjugated macromolecule.Collect component and be concentrated to debita spissitudo with standby with super filter tube again.

(3) ultrasonic emulsification modification: get the oil-soluble quantum dot of appropriate molar ratios/three block macromoleculars, be dissolved in methylene chloride.After dissolve complete, pipette quantum dot/Polymer Solution with the 1ml syringe of clean dried.Take the F-68 emulsifiers dissolve of 6.0mg in deionized water, vigorous stirring obtains putting under ultrasonic probe after uniform emulsion, and ultrasonic probe stretches into below liquid level 0.5cm, and syringe needle and ultrasonic probe bottom are parallel.Start ultrasonic, 50 watts of power, the working time is set to 5 seconds, the quiescent interval is 3 seconds.When ultrasonic carrying out, slowly inject quantum dot/Polymer Solution, solution became milky and send bright fluorescence under portable ultra violet lamp this moment.Inject completely, rapidly emulsion is placed on magnetic and stirs on instrument and stir to keep the stable emulsion state, and further remove the methylene chloride organic solvent.After half an hour, precipitation is got in centrifuging, and 3 times to remove macromolecule and the F-68 emulsifying agent molecule that there is no combination repeatedly.

4. measure

The uv absorption of the CdSe quantum dot that makes and emission spectrum as shown in Figure 1, the wide and continuous distribution of its absorption spectrum has 4 exciton absorption peaks on absorption spectrum, show the strong quantum effect of products therefrom.The first exciton absorption peak is sharp-pointed, and along with particle diameter becomes large and red shift.Emission spectrum is narrow and symmetrical, and there is no the long wave conditions of streaking, and half-peak breadth only has 23nm.Along with the particle size of product is different, its emission spectrum is also different, and it is large that particle diameter becomes, and its emission spectrum is red shift thereupon also.The particle diameter distribution very narrow (can find out its monodispersity matter from the HRTEM photo) of so narrow spectrum peak explanation product.

Fig. 2 is the ultra-violet absorption spectrum that mercaptoacetic acid is modified CdSe/CdS oil-soluble quantum dot.Can find out on scheming, after modifying, the first exciton absorption peak becomes smooth, the first almost not skew of exciton absorption peak, modification be described after spectral characteristic change very few.

Follow the tracks of mercaptoacetic acid and modify the stability of afterproduct, a little sediment appears in the quantum dot after modification after a week, and it is relevant to the concentration of quantum dot solution the degree of deposited phenomenon to occur, and concentration is larger, deposited phenomenon occurs more obvious.The organic matter layer on oil-soluble quantum dot surface is replaced by hydrophilic mercaptoacetic acid, and the firm degree that the Cd-S bond is closed has determined the aggregation extent of quantum dot.After mercaptoacetic acid is modified, owing to having destroyed original quantum dot surface chemical structure, and in the air water solution medium, the little molecule of mercaptoacetic acid also can be due to the quantum dot surface photo oxidation, light degradation and break away from quantum dot, quantum dot concentration is larger, and its chance of assembling each other is larger, thereby strengthens the formation of precipitation.

Amphiphilic macromolecular self assembly core is by the hydrophobic effect combination.The TOPO/TOP on oil-soluble quantum dot surface is all the alkyl hydrophobic chains that contain 8 carbon atoms, and three block macromoleculars that we adopt contain abundant water wettability carboxylic group, through the modification of part carboxyalkyl, makes it to connect the octylame with 8 carbon atoms.Be combined with the 8 carbon atom hydrophobic effects on quantum dot surface by 8 carbon atoms on macromolecule like this, carry out self assembly on the quantum dot surface, and the carboxyl on macromolecule provides water wettability, make water-solubility of oil soluble quantum dots.

Fig. 3 be the self-assembled modified quantum dot of amphiphilic macromolecular material TEM photo 190,000 *.

Fig. 4 a is the ultra-violet absorption spectrum of the hydrophobic self-assembled modified quantum dot of amphiphilic macromolecular.Macromolecular material absorbs and refraction due to light is also had, and after water-soluble modification, the first exciton absorption peak becomes smooth, and the maximum absorption band red shift a little, show that there is the macromolecule parcel on the quantum dot surface.Fig. 4 b is the fluorescence spectrum after modification.Its half-peak breadth does not have to change substantially, but maximum emission wavelength has red shift (585-596nm), and further illustrating the quantum dot surface has by macromolecule and wrap up.

Fig. 5 is the TEM photo after the ultrasonic emulsification modified quantum dot.Can find out that particle diameter is even on scheming, nano grain surface is smooth, and is spherical in shape.And realized the single parcel of quantum dot.Pore inside each particle is oil-soluble CdSe/CdS QDs.The ultrasonic emulsification modification, can form dense tight macromolecule layer on quantum dot surface, can better protect quantum dot, avoid surrounding medium to infiltrate and the destruction of causing the luminescent properties of quantum dot (as light degradation, photobleaching, and oozing out of Cd ion and produce toxicity); Purifying is simple, only needs centrifugally, and generally water-soluble modified (self-assembled modified by hydrophobic effect) needed the steps such as film-concentrated-gel filtration-again is concentrated.Adopt 0/W, consumption of organic solvent is few, time-saving energy-saving, environmental friendliness; The self-assembled modified nanoparticle surface of hydrophobic effect is rough, and the nanoparticle surface of ultrasonic emulsification preparation is bright and clean, is not easy to cause non-specific adsorption, can better be applied in the biological detection aspect.

Fig. 6 is the ultra-violet absorption spectrum of ultrasonic emulsification modified quantum dot.Macromolecular material absorbs and refraction due to light is also had, and after water-soluble modification, the first exciton absorption peak becomes smooth, shows that the quantum dot surface has macromolecule parcel and maximum absorption band almost not to change, and illustrates that the original spectral characteristic of quantum dot is kept.Fluorescence spectrum after modification sees that maximum emission wavelength has red shift (612-619nm), and further illustrating the quantum dot surface has by macromolecule and wrap up.

Embodiment 5: antibody quantum dot-labeled

1. the preparation of aldehyde radical slide

With slide washing lotion (the dense H2SO4350ml+H2040ml of K2CrO720g+) soaked overnight, then use a large amount of deionized water rinsings, after 60 ℃ of oven dry 2h, slide is immersed in the ethanolic solution of 5%APTES, effect 60min carries out the amination of surface of glass slide, take out washing, after drying, slide is immersed the PBS (0.2mol/L that contains 5% glutaraldehyde, pH8.0) carry out the aldehyde radical of slide in solution, the cleaning of PBS solution is dried rear standby.

In order to verify the effect of aldehyde radical slide, draw a circle on slide with PAPpen, mark the position of solution reaction.Add rabbit igg in circle, 37 ℃ of temperature are incubated 2h, PBS-T (0.01mol/L, pH7.2,0.05%Tween20) washing 4 times, 10s/ time; Under equal conditions, then to contain the 0.02mol/L PBS solution sealing 1h of 1%BSA.Distilled water cleans, and after drying, adds the IgG (as shown in Figure 7) of quantum dot-labeled goat anti-rabbit igg and quantum dot-labeled goat-anti chicken, hatches 30min, PBS-T washing 4 times, and each 10min rinses the fluorescence microscopy Microscopic observation at last with distilled water.As shown in Figure 7, add goat anti-rabbit igg can see orange-yellow fluorescence, and add the IgG of goat-anti chicken there is no fluorescence, thereby prove the aldehyde radical surface of glass slide energy ankyrin that we make, and can keep the activity of institute's ankyrin.

2. with crosslinking chemical EDC and sulfo-NHS, the method that adopts covalent bond to connect is carried out the quantum dot-labeled of antibody

Shown in Figure 8, the carboxyl reaction on EDC and quantum dot surface generates the acyl group isourea, but the easily hydrolysis in aqueous solution of acyl group isourea generates again the carboxyl product.If add sulfo-NHS in solution; the acyl group isourea just very easily reacts with sulfo-NHS; generation has the sulfo-NHS ester of amine reactivity, and the amino reaction on sulfo-NHS ester and antibody surface generates stable acid amides, thereby carries out the quantum dot-labeled of antibody.

Concrete reaction conditions is as follows:

(1) add 0.4mg (2.0mM) EDC and 1.1mg (5.0mM) sulfo-NHS in the activation damping fluid (0.1M MES, 0.5M NaCl, PH6.0) of 1ml.

(2) add 20ul quantum dot solution (3.0mM) in solution, ceaselessly mixed solution, at room temperature react 20min.

(3) add 1.4ul (20mM) 2 mercapto ethanol at room temperature to react 10min, the quantum dot solution that obtains activating.

(4) connect at 1ml (antibody and quantum dot molecular number ratio are 3:1) that adds the required mark of bird flu in damping fluid (PBS, PH7.5).And regulate PH to 7.5 with NaOH solution, at room temperature stirring reaction is 2 hours.

(5) the 4000rpm centrifuging is 1 minute, removes little molecule and unreacted antibody completely.

(6) suck and throw aside supernatant.

(7) with 2ml deionized water dissolving precipitation, use the centrifugal or quantum dot-labeled product of gel filler Sepharose6FF filter method purifying repeatedly.

3. the purifying of quantum dot-labeled product

(1) precipitation centrifugal purification: the centrifugal 30min of reacted solution 14000rpm.Get its precipitation, after the deionized water washing, obtain quantum dot-labeled antibody complex with appropriate PBS (PH7.4,0.01M) dissolution precipitation.

(2) gel filtration purifying and concentrated (carrying out on AKTA Basic protein purification instrument): get appropriate Sepharose6FF filler, carry out suction filtration with nutsch filter, and wash 2-3 time with distilled water.Volume ratio by 1:1 in the filler that filters adds distilled water, mixing; Filler is fallen continuously as in post, open pumping unit, and wash with distilled water, until till the liquid level position of filler no longer descended, mark is the liquid level position of filler at this moment; Unclamp column cap, the position of adjusting column cap is the following 2-3mm of filler liquid level; Use 0.01M, the PBS effect equilibrium liquid of pH7.4, flow velocity is 2ml/min, the real-time OD of Observe and measure ₂₈₀The value curve is to OD ₂₈₀The value curve stops balance when stabilizing to straight line; Add quantum dot-labeled antibody-solutions with autopipette, use 0.01M, the PBS of pH7.4 is as eluent, and flow velocity is 2ml/min; Collect eluent, every pipe 1ml is until the OD that shows ₂₈₀The value curve stops when being a straight straight line collecting; According to OD ₂₈₀The OD at each peak that the value curve shows ₂₈₀Be worth, merge the eluent at each peak, wherein the eluent at peak 1 is quantum dot-labeled antibody-solutions; The quantum dot-labeled antibody-solutions of purifying is packed in bag filter.Embedding 10h concentrates with solid polyethylene glycol (PEG, molecular weight 6000).

4. result is measured

(1) how anti-quantum dot-labeled bird flu is

We cross post to the solution that last PBS dissolution precipitation obtains with gel filtration filler sepharose6FF.As equilibrium liquid and eluent, obtain a spike with PBS (0.1M, pH7.4), peak shape as shown in Figure 9.Find out thus, precipitating the quantum dot-labeled many anti-products that obtain after centrifugal is quantum dot-labeled many anti-compounds, and by centrifugal free antibody, unreacted little molecule coupling agent and other the intermediate product etc. that do not connect of having removed.

The collection component that merges this peak, be fixed with on the surface seal and wash on the slide of bird flu antigen after, drip the collection component at this peak, after washing, at the fluorescence microscopy Microscopic observation, can observe orange-yellow fluorescence (as Figure 10).

Find out thus, precipitating the quantum dot-labeled many anti-products that obtain after centrifugal is quantum dot-labeled many anti-compounds, and by centrifugal free antibody, unreacted little molecule coupling agent and other the intermediate product etc. that do not connect of having removed.Merge the supernatant in the washing of precipitate process, measure D280, D260, calculate free antibody concentration in supernatant, according to the amount of the antibody that adds before reaction, our the quantum dot-labeled efficient that calculates antibody is 41.83%. thus

Get the antibody-solutions after quantum dot-labeled, without centrifugal, directly carry out purifying with gel filtration filler sepharose6FF, with PBS (0.1M, pH7.4) as equilibrium liquid and eluent, the spectrogram that obtains such as Figure 11.

Merge the collection component at peak 1, peak 2 and peak 3, observe under ultraviolet transilluminator, peak 1 component of collection is sent orange-yellow fluorescence, and the component at peak 2 and peak 3 is not sent fluorescence.Be fixed with on the surface seal and wash on the slide of bird flu H5NI antigen after, drip the collection component at each peak, after washing, at the fluorescence microscopy Microscopic observation, can observe and add the slide of peak 1 component to send orange-yellow fluorescence, can prove peak 1 resists (as Figure 12 a) for quantum dot-labeled bird flu more, and the slide that adds the component at peak 2 and peak 3 is not observed fluorescence (as Figure 12 b, 12c) under fluorescent microscope, thereby proves that peak 1 component is the how anti-compound of quantum dot-labeled bird flu for our target product.

Measure respectively the absorbance of peak 1, peak 2, peak 3 components, the volume of collecting according to each peak and concentration can be calculated bird flu how anti-quantum dot-labeled efficient is 42.64%.

Thus, we can find out, can carry out purifying to the reaction product that quantum dot-labeled bird flu resists more with precipitating centrifugal method with crossing post, and two kinds of methods obtain the quantum dot-labeled bird flu efficient that resist more and are respectively 41.83% and 42.64%.

(2) quantum dot-labeled bird flu monoclonal antibody

We have adopted the same method quantum dot-labeled bird flu monoclonal antibody of preparation, and have carried out purifying with precipitating the product of centrifugal method with crossing post to mark respectively:

After the component of getting peak 1 is added to and seals and wash on the slide that the surface is fixed with bird flu antigen, drip this peak component, after washing, at the fluorescence microscopy Microscopic observation, can observe the slide that drips peak 1 component and send orange-yellow fluorescence, can prove that peak 1 is quantum dot-labeled bird flu monoclonal antibody.

The quantum dot-labeled thing that obtains by precipitating centrifugal method, cross post and obtain later on a spike (as Figure 14): by calculating, precipitate centrifugal and method gel filtration quantum dot-labeled how anti-product is carried out purifying, obtain at last the productive rate 45.38% and 46.18 of the quantum dot-labeled how anti-compound of bird flu.

Can find out from above-mentioned experiment, can put to scalar monoclonal antibody and many anti-purifying that carries out of mark bird flu by the method that precipitates centrifugal and gel filtration, precipitate the productive rate of the centrifugal target product that obtains a little less than gel filtration, but during its operating cost of gel filtration, also need after gel filtration to concentrate, still adopt the precipitation centrifugal method quantum dot-labeled product is carried out purifying.

Embodiment 6: the detection of sample

1. detection method: the surface of glass slide at aldehyde radical drips sample allantoic fluid to be checked, hatches 2h. for 37 ℃.Wash slide 3 times with PBS (0.01M, pH7.2), each 5min.Then drip in surface of glass slide the PBS-Tween solution that contains 5%BSA, hatch 45min for 37 ℃.With PBS (0.01M, pH7.2) the washing slide is 3 times, after each 5min, add quantum dot-labeled avian influenza antibody, hatch 45min for 37 ℃, with PBS (0.01M, pH7.2) the washing slide is 3 times, each 5min, after drying under room temperature at the fluorescence microscopy Microscopic observation, the yin and yang attribute of judgement sample.

2. the non-specific research of reduction method

After hatching negative sample on slide, seal and wash, then add the how monoclonal antibody of anti-and bird flu mark of quantum dot-labeled bird flu, carry out the non-specific research of following method:

(1) adding respectively concentration is that 0.5%, 0.8%, 1.0% and 1.5% BSA seals, the effect of research variable concentrations BSA sealing.

(2) experimental concentration is in 0.1,0.05,0.02,0.01,0.005 buffer system, and it is non-specific that sample detection occurs, and determines the ionic strength of buffer solution.

(3) in, Tween-20 concentration fixes on really that to add respectively concentration in the PBS damping fluid be 0.01%, 0.05%, 0.1%, 0.2% and 0.5% Tween-20, the concentration of Tween-20 in the test cleansing solution.

Result: by add the sealing effect research of variable concentrations BSA on negative sample, our selected concentration is that 1%BSA is as confining liquid.Due to quantum dot finishing carboxyl, electronegative, so ionic strength is too high, can cause Electrostatic Absorption, we have suitably reduced the ionic strength of buffer solution, to reduce non-specific adsorption.Through experiment, the PBS that we have adopted 0.01M, pH7.4 is buffer solution.In order to reduce non-specific adsorption, we add the Tween of variable concentrations in cleansing solution PBS.Be determined by experiment, we select 0.1% PBS-T as lavation buffer solution.How anti-checking by experiment in this method, is better than quantum dot-labeled bird flu with its specificity outline of quantum dot-labeled monoclonal antibody.

3. the specificity of method

(1) the bird flu monoclonal antibody of Marker selection or how anti-, detect respectively common bird virus with quantum dot-labeled antibody: ewcastle disease F48E9, IBDV, duck plague virus and DHV detect, observe and false positive results, the specificity of verification method whether occur.

(2) get a chick embryo allantoic liquid that does not infect avian influenza virus, method and the indirect immunofluorescence set up by this research institute respectively detect, and at the fluorescence microscopy Microscopic observation, relatively two kinds of methods is non-specific.

Result: (1) Figure 15 a, b, c and d represent that respectively quantum dot-labeled H5N1 type bird flu monoclonal antibody detects ewcastle disease F48E9, IBDV, duck plague virus and DHV, and false positive results does not appear in the results show.The specificity of method of proof is better.(2) method and the indirect immuno fluorescent method set up by this research respectively detect the allantoic fluid that does not infect avian influenza virus, two kinds of result such as Figure 16 that method detects, and a is the testing result of this research method for building up, b is the result that immunofluorescence detects.As can be seen from Figure 16, the detection method that this research institute sets up, it is non-specific better, does not observe nonspecific fluorescence, and a small amount of non-specific fluorescence occurred with immunofluorescence method.The specificity of proof the method is better than the method for immunofluorescence.

4. the sensitivity study of method

Get the allantoic fluid of infection and make 10 times of gradient dilutions, the method for then setting up with this research institute detects, and determines the sensitivity of method.Result: checking by experiment, we are when detecting allantoic fluid, and the method that this research institute sets up can detect the highly diluted multiple that amasss embryo allantoic liquid and be ^10-6

5. the stability study of testing result

The method of setting up with this research institute detects positive sample, then at room temperature places after 1 day, 3 days, 5 days, 10 days, 15 and 30 days and observes testing result again.Result: prove by experiment, the sample that detected was at room temperature placed after 30 days still can observe bright fluorescence.

Claims (8)

1. for detection of the preparation method of the nano-quantum point labelled antibody of H5N1 type HPAIV, it is characterized in that comprising the steps:

(1) preparation and purifying H5N1 type bird flu monoclonal antibody or polyclonal antibody;

(2) the water-soluble carboxyl nano-quantum point of preparation;

(3) nano-quantum point mark H5N1 type bird flu monoclonal antibody or polyclonal antibody; Gained monoclonal antibody or polyclonal antibody namely can be applicable to the mensuration of H5N1 type HPAIV in sample;

The water-soluble carboxyl nano-quantum point of described (2) preparation is to adopt following method preparation:

A. prepare the CdSe core-shell quanta dots;

B. prepare the CdSe/CdS core-shell quanta dots;

C. mercaptoacetic acid is modified the CdSe/CdS quantum dot;

D. the hydrophobic self-assembled modified quantum dot of amphiphilic macromolecular;

E. ultrasonic emulsification modified quantum dot;

The method of the hydrophobic self-assembled modified quantum dot of described d. amphiphilic macromolecular is: get in the chloroform/ethanol solution that quantum dot powder and amphiphilic three block macromoleculars are dissolved in 3: 1, stirring is to remove the chloroform organic solvent, after being removed, chloroform adds PBS buffer solution, ultrasonic dispersion is 3 minutes again, resulting solution after the 0.22um film to remove unconjugated macromolecule, after crossing film, solution is concentrated rear after gel chromatographic columns with super filter tube again, further removes unconjugated macromolecule;

The method of described (3) nano-quantum point mark H5N1 type bird flu monoclonal antibody or polyclonal antibody is: preparation aldehyde radical slide; The method of utilizing crosslinking chemical EDC to be connected covalent bond to connect with sulfo-NHS is carried out the quantum dot-labeled of antibody.

2. the preparation method of the nano-quantum point labelled antibody for detection of H5N1 type HPAIV according to claim 1, it is characterized in that: the method for described (1) preparation and purifying H5N1 type bird flu monoclonal antibody is: with the concentrated H5N1 type avian influenza virus of purifying as immunizing antigen immunity 8 Balb/c mouse in all ages; Adaptive immune splenocyte and SP2/0 Fusion of Cells obtain hybridoma; The clone of screening positive hybridoma cell cultivates the hybridoma cell strain that obtains stably excreting antibody; Hybridoma cell strain is injected into the Balb/c mouse peritoneal induces with external the ascites that the ascites method obtains to contain monoclonal antibody; Adopt sad-saturated sulfuric acid amine method to concentrate and purifying ascites acquisition monoclonal antibody.

3. the preparation method of the nano-quantum point labelled antibody for detection of H5N1 type HPAIV according to claim 1, it is characterized in that: described (1) preparation and the method for purifying H5N1 type bird flu polyclonal antibody are: the H5N1 type avian influenza virus that concentrates with purifying is as immunizing antigen, immune new zealand white rabbit; Heart blood sampling after 10 days, separation of serum; How anti-saturated ammonium sulfate is slightly carried; DEAE-cellulose chromatography crosses that the IgG of purifying puts in bag filter after post, concentrates with the solid polyethylene glycol embedding and obtains polyclonal antibody.

4. the preparation method of the nano-quantum point labelled antibody for detection of H5N1 type HPAIV according to claim 1, it is characterized in that: the method for described (3) nano-quantum point mark H5N1 type bird flu monoclonal antibody or polyclonal antibody is: preparation aldehyde radical slide; The method of utilizing crosslinking chemical EDC to be connected covalent bond to connect with sulfo-NHS is carried out the quantum dot-labeled of antibody.

5. the preparation method of the nano-quantum point labelled antibody for detection of H5N1 type HPAIV according to claim 4, it is characterized in that: described preparation aldehyde radical slide refers to: with slide washing lotion soaked overnight, then use a large amount of deionized water rinsings, after 60 ℃ of oven dry 2h, slide is immersed in the ethanolic solution of 5%APTES, effect 60min carries out the amination of surface of glass slide, take out washing, after drying, slide is immersed the aldehyde radical that carries out slide in the PBS solution that contains 5% glutaraldehyde, the cleaning of PBS solution is dried rear standby;

Described slide washing lotion is K ₂CrO ₇The dense H of 20g+ ₂SO ₄350ml+H ₂O 40ml; Described PBS solution is 0.2mol/L, pH8.0.

6. the preparation method of the nano-quantum point labelled antibody for detection of H5N1 type HPAIV according to claim 4, it is characterized in that: the described method of utilizing crosslinking chemical EDC to be connected covalent bond to connect with sulfo-NHS is carried out the quantum dot-labeled of antibody and is referred to: add the activation damping fluid of 1ml in the eppendorf of 1.5ml, then add appropriate EDC and sulfo-NHS solution; Add the 20ul quantum dot solution in solution, ceaselessly mixed solution, react 20min under room temperature again; Add the 2 mercapto ethanol of 1.4ul 20mM at room temperature to react 10min, the quantum dot solution that obtains activating; In the quantum dot solution of activation, add 1ml to contain the PBS solution of appropriate H5N1 type avian influenza antibody, and regulate PH to 7.5 with NaOH solution, under room temperature, stirring reaction is 2 hours; Little molecule and unreacted antibody is completely removed in 4000rpm centrifuging 1 minute; With 5ml deionized water dissolving precipitation, repeat quantum dot-labeled bird flu monoclonal antibody solution is carried out dissolution/precipitation 2 times, at last with 2ml deionized water dissolving precipitation, 4 ℃ of preservations;

Described activation damping fluid is 0.1M MES, 0.5M NaCl, PH6.0; Described PBS solution is 0.01M, pH7.5.

7. method for preparing water-soluble carboxyl nano-quantum point is characterized in that adopting following method to prepare:

(1) preparation CdSe core-shell quanta dots;

(2) preparation CdSe/CdS core-shell quanta dots;

(3) mercaptoacetic acid is modified the CdSe/CdS quantum dot;

(4) the hydrophobic self-assembled modified quantum dot of amphiphilic macromolecular;

(5) ultrasonic emulsification modified quantum dot;

The method of the hydrophobic self-assembled modified quantum dot of described (4) amphiphilic macromolecular is: get in the chloroform/ethanol solution that quantum dot powder and amphiphilic three block macromoleculars are dissolved in 3: 1, stirring is to remove the chloroform organic solvent, after being removed, chloroform adds PBS buffer solution, ultrasonic dispersion is 3 minutes again, resulting solution after the 0.22um film to remove unconjugated macromolecule, after crossing film, solution is concentrated rear after gel chromatographic columns with super filter tube again, further removes unconjugated macromolecule.

8. water-soluble carboxyl nano-quantum point by the preparation of the described method of claim 7.

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