CN101975856B - Preparation method of fluorescence antibody for detecting Newcastle disease virus and solid-phase immunofluorescence detection assay kit - Google Patents
Preparation method of fluorescence antibody for detecting Newcastle disease virus and solid-phase immunofluorescence detection assay kit Download PDFInfo
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Abstract
The invention relates to a preparation method of fluorescence antibody for detecting a Newcastle disease virus and a solid-phase immunofluorescence detection assay kit. The preparation method comprises the following steps of: respectively deriving R-phycoerythrin (RPE) and an antibody resisting the Newcastle disease virus (NDV) by using a cross-linking agent SPDP (N-succinimidyl-3-(2-pyridyldithiol) propionate), cross-linking the derivatives in a proper molar ratio, and purifying through HPLC (High Performance Liquid Chromatography) to prepare an RPE marked NDV fluorescence antibody. The solid-phase immunofluorescence detection assay kit is formed from the fluorescence antibody, an NDV-resisting antibody, an agarose microsphere, diluted hydrochloric acid and a washing liquid. The assay kit comprises the following detection flows of: coating an activated microsphere with the antibody, washing, combining with a sample to be measured, washing, combining with the fluorescence antibody, fully washing, exciting by blue and green light in a fluorescence microscope, observing and judging the result. The prepared fluorescence antibody has the advantages of high yield, high purity, bright orange fluorescence and good stability; and the assay kit has signal enrichment action by using a spherical carrier and can increase detection sensitivity. The invention is applicable to the rapid detection of the Newcastle disease virus.
Description
Technical field
The present invention relates to a kind of preparation method and solid-phase fluorescence immunoassay kit that detects the fluorescence antibody of Avian pneumo-encephalitis virus, belong to the immunofluorescence detection field.
Technical background
Ewcastle disease is the deadly infectious disease of serious harm aquaculture, by World Organization for Animal Health, is classified as category-A zoonosis, simultaneously it or Amphixenosis, the threat mankind's public health security.It is the essential items for inspection of bird and animality goods inspection and quarantining for import/export thereof that Avian pneumo-encephalitis virus detects, once World Organization for Animal Health requires to detect Avian pneumo-encephalitis virus in bird and animal product thereof, must destroy on the spot.Fast, sensitive detection method is most important to effective prevention and control of the making a definite diagnosis in time of epidemic disease, epidemic situation and prediction, early warning.The detection method of ewcastle disease mainly contains at present: (1) Virus Isolation; (2) serology detects, as HA-HI etc.; (3) molecular Biological Detection, as RT-PCR etc.But, above-mentioned detection method respectively has relative merits, as virus is separated and serological Identification length consuming time, serology detects needs their early stage and convalescence paired sera, the PCR detection sensitivity is high, experiment condition and operator's technical merit are had relatively high expectations, and HA-HI method commonly used is simple and easy to do, but sensitivity is low.
Immunofluorescence assay is a kind of time-honored labeled analysis method, has the specificity of antigen-antibody reaction and the sensitivity of fluoroscopic examination concurrently, and sensitivity is up to 10
-6mg/mL, be widely used in Diagnosis of Infectious Diseases.The specificity of immunofluorescence assay and susceptibility depend on the characteristic of specificity, affinity, titre and the fluorescent dye of antibody.Fluoroscopic examination often is subject to the impact of background fluorescence in serum and other biological sample.Take serum as example, and the fluorescence emission spectrum overlaid of the background fluorescence of 400-600nm wave band and FITC commonly used, disturb excessively, affects testing result; On the other hand, traditional fluorescent dye (as FITC etc.) thering is toxicity, easily cancellation, above-mentioned factor causes immunofluorescence analysis method for a long time relatively to lag behind the labelling methods such as ELISA, chemoluminescence method.Improving immunofluorescence assay sensitivity and specific approach has: (1) finds outstanding fluorescent dye, as selected, molar extinction coefficient is large, fluorescence quantum yield is high, stoke shift is large, the dyestuff of emission red fluorescence; (2) be combined with the immunological techniques such as monoclonal antibody, antibody sandwich method, antibody coating technique, particle enrichment fluoroimmunoassay; (3) adopt the solid phase detection method, as to take inert material microballoon, 96 orifice plates etc. be solid phase carrier.The solid-phase fluorescence immunoassay method can be used for detecting soluble antigen or antibody, and conventional solid phase carrier, as nylon membrane, nitrocellulose filter, cellulose acetate membrane, 96 orifice plates etc. possess lower background fluorescence usually, can disturb or reduce the sensitivity of fluoroscopic examination.The solid phase carrier that the present invention adopts is a kind of microballoon, by agarose, through cyanogen bromide-activated, is prepared, and without background fluorescence, because the surface area of ball type carrier is large, has the signal inrichment, can significantly improve the sensitivity of fluoroscopic examination.
Phycoerythrin is the main water miscible photosynthetic pigments albumen of the class in the red algae cell, can send bright orange colour fluorescence, with traditional fluorescent dye, compare, that phycoerythrin has is water-soluble, avirulent, molar extinction coefficient is large, fluorescence quantum yield is high, stoke shift is large, emission orange colour fluorescence, bias light disturb little, be difficult for the characteristics such as cancellation, be the desirable fluorescent dye of a class.Marine Red Alga Polysiphonia Urceolata is China coast wild marine red alga distributed more widely, contains a large amount of typical three peak type R-PEs (R-phycoerythrin, RPE), and the stable state of RPE is (α β)
6γ, the composition of RPE and crystal structure characteristics have determined its high stability.The molecular weight of Marine Red Alga Polysiphonia Urceolata RPE is about 240kDa, and maximum absorption wavelength lays respectively at 498,540,565nm, and the maximum fluorescence emission peak position is in 575nm.1 Marine Red Alga Polysiphonia Urceolata RPE molecule contains 34 color bases, and molar extinction coefficient is 24.1 * 10
5cm
-1m
-1fluorescence quantum yield is 82%, the fluorescence intensity of the phycoerythrin of 1 molecule at least is equivalent to 30 FITC or 100 rhodamine molecules in comparable wavelength, the stoke shift of phycoerythrin is up to 75nm, and emitting fluorescence is positioned at nearly red light district, thereby the bias light interference is little, contribute to improve the sensitivity of fluoroscopic examination, the sensitivity of phycoerythrin fluoroscopic examination is 5-10 times of FITC.And the stoke shift of traditional fluorescent dye (as FITC etc.) is little, emitting fluorescence and excitation spectrum partly overlap, living beings background fluorescence serious interference.The discovery of phycoerythrin and application significantly improve the sensitivity of immunofluorescence assay.Phycoerythrin is better than traditional synthetic fluorescent dye, can replace FITC and detects for immunofluorescence or coordinate the FITC use to realize that the Two Colour Fluorescence discriminating under single excitation source detects.
The fluorescent dyes such as RPE need be cross-linked into the fluorescence antibody probe with the biomacromolecule such as antibody when the fluoroscopic examination, during protein cross, ubiquity is difficult to the shortcoming of quantitatively controlling, often cause the yield of fluorescent marker low, mix in label a large amount of unconjugated fluorescent dyes is arranged, cause the production cost of label high, the fluoroscopic examination sensitivity.And the many height of crosslinker concentration also can cause fluorescent dye structural change, protein active to lose even fluorescence forfeiture, the too high immunocompetence that also can affect antibody of while crosslinker concentration, therefore select the concentration, cross-linking method etc. of suitable crosslinking chemical kind, crosslinking chemical directly to affect the quality of fluorescence probe, thereby affect the sensitivity of fluoroscopic examination.Chemical cross-linking agent SPDP is special-shaped difunctional chemical cross-linking agent, the reaction conditions gentleness, and the chemical group of introducing has light absorptive, is beneficial to the control of cross-linking process.By controlling suitable mol ratio, to carry out the liquid phase of RPE and antibody crosslinked, can make the crosslinked yield of fluorescent marker of development be greater than 90%, and efficiency, the quality of probe significantly improve, and cost significantly reduces, and promotes its immunofluorescence for disease to detect.
Summary of the invention
The present invention relates to a kind of preparation method and solid-phase fluorescence immunoassay kit that detects the fluorescence antibody of Avian pneumo-encephalitis virus.
The Marine Red Alga Polysiphonia Urceolata R-PE (RPE) that the fluorescence antibody of the detection Avian pneumo-encephalitis virus of the present invention's design is used is three peak type phycoerythrin, at the 498nm place, strong light absorptive is arranged, the maximum fluorescence emission peak position is in 575nm, under blue green light excites, the orange colour fluorescence of RPE energy emitting bright, therefore Marine Red Alga Polysiphonia Urceolata RPE is better than three Xing Heer peak, the peak type phycoerythrin in other source, it have nontoxicity, water-soluble large, fluorescence bright and be difficult for cancellation, stability advantages of higher, detection sensitivity is significantly higher than traditional fluorescent dye, is outstanding fluorescent dye.With the difunctional chemical cross-linking agent SPDP of abnormal shape, that RPE, antibody is crosslinked with suitable mol ratio liquid phase after deriving, the fluorescence antibody cross-linking efficiency of the RPE mark of preparation is high, purity is high, fluorescence is bright, antibody activity is good, good stability.
The solid phase carrier that the solid-phase fluorescence immunoassay kit the present invention relates to is used is a kind of microballoon, by agarose, through cyanogen bromide-activated, is prepared, and freeze-drying is preserved, and microsphere diameter is 60-200um, without background fluorescence, purchased from Amersia company.This is microsphere supported needs before use with 1mM dilute hydrochloric acid solution swelling 30min, with the hydrochloric acid of same concentration, rinses repeatedly, finally uses 50mM pH 8PBS (containing 100mM NaCl) to get express developed and obtains activating gel.The microballoon of activation should be combined with antibody or antigen immediately.
Solution of the present invention is as follows:
RPE preparation: adopt anion exchange chromatography separation and purification from marine red alga-Marine Red Alga Polysiphonia Urceolata.Get the Marine Red Alga Polysiphonia Urceolata of freezing preservation, the 20mM acetate buffer solution (pH5.8) that adds 6 times of volumes, 4 ℃ of lower swelling 24h, the multilayer filtered through gauze, extruding obtains the brown leaching liquor, adding solid ammonium sulfate to concentration in leaching liquor is 60% (w/v), place 24h for 4 ℃, centrifugal collecting precipitation, precipitation is dissolved in the acetate buffer solution (pH5.8) of 20mM, then use acetate buffer solution (pH5.8) dialysis of 20mM, dislysate anion exchange purifying, DEAE Sepharose Fast Flow is 20mM acetate buffer solution (pH5.8) (containing 50mM NaCl) pre-equilibration for post, rinse out sample excessive on anion-exchange column and impurity with 20mM acetate buffer solution (pH5.8), can obtain a large amount of pure RPE with 20mM acetate buffer solution (pH4.0) (containing 50mMNaCl) one-step elution, it is pure that the Marine Red Alga Polysiphonia Urceolata RPE purity of purifying reaches electrophoresis, the purity Index A
620/ A
280>5.2, structural formula is (α β)
6γ, molecular weight is about 240kDa, and maximum absorption band lays respectively at 498,540,565nm, the room temperature fluorescence emission peak is positioned at 575nm, 4 ℃ of the RPE ammonium sulfate precipitations of purifying keep in Dark Place, and before using, with the 50mM pH7.5PBS desalination of fully dialysing, adjustment concentration is 10mg/mL.
Avian pneumo-encephalitis virus polyclonal antibody preparation: Avian pneumo-encephalitis virus La Sota strain 10
4doubly dilution, allantoic cavity inoculation 9-10 age in days SPF chicken embryo, every embryo 0.2mL, hatch for 37-38 ℃, collect the dead germ of inoculation 24-120h, the allantoic fluid of survival embryo, hemagglutination test (HA test) detection titre>1: 256, steriling test is qualified, with 37 ℃ of deactivation 24h of 0.3% formaldehyde, add No. 10 white oils of sterilizing and Tween-80 in 1: 3 ratio, be emulsified into the water-in-oil type oil emulsion inactivated vaccine with colloid mill.The oil-adjuvant inactivated against Newcastle disease immunity SPF chicken in 4 week age of preparation, neck hypodermic injection 1.0mL/ only, head exempts from after 10 days the same dose booster immunization once, two exempt from blood sampling after 1 week, measure Avian pneumo-encephalitis virus HI antibody in serum, when HI tire>gather serum 1: 256 the time, the anion-exchange chromatography purifying obtains the anti-new castle disease virus polyclonal antibody ,-20 ℃ of freezing preservations.
The preparation of RPE mark fluorescent antibody: utilize Heterobifunctional Reagent SPDP derivative pyridine disulfide group on RPE, antibody respectively, then with DTT also the derivant of original antibody introduce free-SH, the RPE that will carry the pyridine disulfide group with carry the antibody of sulfydryl by the crosslinked fluorescence antibody for preparing of certain mol proportion.By SPDP by 50-100: 1 mol ratio mixes with RPE, antibody respectively, aluminium foil seal after in room temperature revolving reaction 2h, ultrafiltration is centrifugal removes unnecessary SPDP.Get DTT by 50-100: 1 mol ratio and antibody derivatives fully mix, the standing reaction of room temperature 1h, and ultrafiltration is centrifugal removes unnecessary DTT.Antibody-HS and RPE derivant mix with mol ratio at 1: 1, aluminium foil seal after in 20-25 ℃ of oscillating reactions 20h.Add NEM and seal unnecessary sulfydryl, room temperature revolving reaction 60min.
The purifying of the fluorescence antibody of RPE mark: adopt the HPLC method, carry out on the liquid chromatography workstation.Adopt molecular sieve high pressure liquid chromatography post purifying, liquid phase post model TSK G3000sw (specification 7.5mm * 60cm), mobile phase is 50mM PBS pH 7.5, flow velocity 0.5mL/min detects wavelength 190-800nm.The fluorescence labeling antiantibody is eluted at first.Collect first eluting peak, carry out spectroscopy, antibody activity, electrophoresis detection.The ratio that the eluting peak area of target product accounts for all eluting peak areas is cross-linking efficiency.
The spectral detection of RPE mark fluorescent antibody: absorption spectrum is measured on ultraviolet-visible spectrophotometer, the interval 250-700nm of scanning wavelength.Absorption spectrum detects can judge whether the crosslinked cross-linking reaction that whether successfully reaches causes the RPE sex change.The room temperature fluorescence emission spectrum is measured on fluorospectrophotometer, excitation wavelength 498nm, and the fluorescent scanning scope is 520-700nm.In ultraviolet region, the absorbance of RPE mark fluorescent antiantibody is apparently higher than contrast RPE, and the absorption peak position is identical, this be because RPE surface-crosslinked antibody molecule, and antiantibody has stronger light absorption at 280nm; In the 400-700nm scope, the absorption spectrum of cross-linking agent is similar with the absorption peak that contrasts RPE and absorbance, be due to antibody in this scope without light absorption.RPE mark photoactivated antibody with contrast RPE under the exciting of 498nm exciting light, the room temperature fluorescence emission wavelength all is positioned at 575nm, and the change of stoke shift does not occur, and still have the characteristic fluorescence emission spectrum of RPE, and fluorescent brightness is without obvious reduction.
The bioactivity of RPE mark fluorescent antibody: antibody titer adopts Microhemagglutination inhibition test method (HI) to measure.Prepare 4 unit detectable antigens with the corresponding ewcastle disease allantotoxicon of deactivation, reaction is carried out on V-type 96 orifice plates.What the high dilution that the red cell agglutination of take suppresses was fluorescence antibody tires.The HI of fluorescent-labeled antibody tires and answers >=1: 16, and concentration can concentrate by centrifugal ultrafiltration when low.
The electrophoresis detection of RPE mark fluorescent antibody: SDS-PAGE carries out on vertical panel discontinuous electro-phoresis device, and resolving gel concentration is 12.5%, and concentrated gum concentration is 5%, 218V constant voltage electrophoresis, coomassie brilliant blue R_250 dyeing, observations after decolouring.Electrophoresis finds that RPE mark fluorescent antibody had both had α, β, the γ subunit band of RPE, has again H, the L chain band of antibody, shows RPE and antibody linked success.
The Detection of Stability of RPE mark fluorescent antibody: in the RPE mark fluorescent antibody of purifying, add 0.02% sodium azide, 4 ℃ keep in Dark Place, every 30 days, measure-inferior fluorescence spectrum and antibody titer, the room temperature fluorescence emission spectrum is measured on fluorospectrophotometer, antibody titer adopts the hemagglutination-inhibition test method to measure, and observes the variation of fluorescent brightness and antibody activity.The fluorescence antibody stability of RPE mark is better, in the phosphate buffer of 0.05mol/L, preserves 120 days for 4 ℃, and reducing does not appear in the fluorescence intensity of fluorescence antibody, and the antibody titer of fluorescence antibody maintains 1: 32.
Solid-phase fluorescence immunoassay kit assembling and using: the solid-phase fluorescence immunoassay kit is comprised of the anti-new castle disease virus fluorescence antibody of the agarose microbeads carrier of cyanogen bromide-activated, RPE mark, anti-new castle disease virus polyclonal antibody, cleansing solution, watery hydrochloric acid etc.The using method of solid-phase fluorescence immunoassay kit is: during microsphere supported use, need use the watery hydrochloric acid swelling, get the Ago-Gel freeze-dried powder of 0.3g cyanogen bromide-activated, be placed in 1mM hydrochloric acid swelling 30min, hydrochloric acid with same concentration rinses 5 times, get express developed and obtain 1mL activation gel with 50mM pH 8PBS (containing 100mMNaCl), with 2mL concentration, being 5mg/ml immediately, the anti-new castle disease virus polyclonal antibody mixes, 20 ℃ of slow oscillation action 12h, the monoethanolamine sealing that adds 2mL 0.1M, 20 ℃ of slow oscillation action 4h, the centrifugal supernatant that goes, repeatedly rinse gel with 50mM pH 8PBS, centrifugal acquisition coupling has the Ago-Gel of antibody, be made into the suspending liquid of 10% (v/v) with the PBS of 100mM pH 7.5.Get the Ago-Gel 50uL that coupling has anti-new castle disease virus antibody, be placed in clean test tube, add virus liquid 100uL to be checked, 37 ℃ of incubation 2h, rinse 10 times with the PBS of 100mM pH7.5.The fluorescence antibody 100uL that adds 5ug/ml concentration, 37 ℃ of incubation 2h, rinse 5 times with the PBS of 100mM pH 7.5.Draw a small amount of microballoon, drip on the slide of low fluorescence background, cover plate, glycerine damping fluid mounting, excite with blue green light under fluorescent microscope, observation, result of determination.Positive microballoon excites the orange colour fluorescence of lower emitting bright at ruddiness, negative microballoon is without fluorescence.By fluorescence intensity and background staining power with+mean, be divided into ++ ++ (strong positive), +++(strong positive), ++ (than strong positive) ,+(the weak positive) ,-(feminine gender) Pyatyi.Fluorescence intensity reaches ++ more than be judged to be the positive.SPF chicken serum negative control and PBS blank are established in test.
Susceptibility, specificity, repeatability detect: (1) by the Avian pneumo-encephalitis virus serial dilution, utilizes the solid-phase fluorescence immunoassay kit to carry out one by one fluoroscopic examination to each dilutability with PBS, usings the highest titre of the detected virus of energy as the sensitivity detected.(2) replace Avian pneumo-encephalitis virus with avian influenza virus, IBV, infectious bursal disease virus, ILTV, bird pox virus etc. respectively, carry out fluoroscopic examination by above-mentioned solid phase immunofluorescence detection agent box, excite the lower fluorescence that all do not detect at blue green light, prove that the specificity of kit is good.(3) utilize kit to same dilution Avian pneumo-encephalitis virus sample duplicate detection 3 times, result is consistent, shows that the repeatability of kit is better.
With FITC mark fluorescent antibody test Avian pneumo-encephalitis virus, compare: the carbonic acid buffer dialysed overnight of 50mM pH 9 for the newcastle epidemic disease antibody of purifying, adjustment concentration is 10mg/ml.FITC with above-mentioned carbonic acid buffer preparation 0.1mg/ml.Antibody is placed in to 4 ℃ of slow dialysis 24h that stir of FITC solution of 10 times of antibody volumes, the phosphate buffer dialysis that then rapidly labelled antibody is placed in to 50mM pH 7.2, frequent exchange buffering liquid.The antibody probe that replaces the RPE mark with the antibody probe of FITC mark, detect Avian pneumo-encephalitis virus by the solid-phase fluorescence immunoassay method of setting up.
In a word, the fluorescence antibody fluorescence of RPE mark is bright, and long preservation, without obvious decay, is compared with the fluorescence antibody of FITC mark, and fluorescence is brighter, and sensitivity is higher, disturbed by the living beings background fluorescence little.The present invention adopts microsphere supported self without background fluorescence, can the interfere with fluorescence detection result, and because the surface area of ball type carrier is large, can be coated with more antibody or antigen, thereby in conjunction with more fluorescence probe, be beneficial to the signal enrichment, can improve the sensitivity that immunofluorescence detects.With existing solid phase carriers such as nitrocellulose membrane, 96 orifice plates, compare, background fluorescence disturbs less, and fluorescence is brighter, and detection sensitivity is higher.
The accompanying drawing explanation
The HPLC purifying figure that accompanying drawing 1 is RPE mark anti-new castle disease virus fluorescence antibody.Liquid phase post model is TSK G3000sw, the PBS that mobile phase is 50mM pH7.5, and flow velocity 0.5mL/min, the elution time of RPE mark anti-new castle disease virus fluorescence antibody, anti-new castle disease virus antibody, RPE is for being respectively 19.57min, 27.32,31.26min.
The fluorescence spectrum that accompanying drawing 2 is RPE mark fluorescent antibody (dotted line) and RPE (solid line).RPE mark fluorescent antibody with contrast RPE under the exciting of 498nm exciting light, the room temperature fluorescence emission wavelength all is positioned at 575nm, stoke shift does not occur and change, and still has the characteristic fluorescence emission spectrum of RPE.
The SDS-PAGE electrophoresis result that accompanying drawing 3 is RPE mark anti-new castle disease virus fluorescence antibody.RPE mark fluorescent antibody had both had α, β, the γ subunit band of RPE, had again H, the L chain band of antibody.
Accompanying drawing 4 is RPE mark fluorescent antibody solid-phase fluorescence immunoassay result.RPE mark fluorescent antibody positive microballoon emitting bright orange colour fluorescence, and negative microballoon can't detect fluorescence.
Embodiment:
The RPE preparation: the Marine Red Alga Polysiphonia Urceolata of freezing preservation adds the 20mM acetate buffer solution (pH5.8) of 6 times of volumes, 4 ℃ of lower swelling 24h, the multilayer filtered through gauze, extruding obtains the brown leaching liquor, it is 60% (w/v) that leaching liquor adds solid ammonium sulfate to concentration, place 24h for 4 ℃, centrifugal collecting precipitation, precipitation is dissolved in the acetate buffer solution (pH5.8) of 20mM, then use 4 ℃ of dialysed overnight of acetate buffer solution (pH5.8) of 20mM, dislysate is added to the DEAE Sepharose Fast Flow anion-exchange column by 20mM acetate buffer solution (pH5.8) (containing 50mM NaCl) pre-balance, rinse out sample excessive on anion-exchange column and impurity with 20mM acetate buffer solution (pH5.8), just can obtain a large amount of pure RPE with 20mM acetate buffer solution (pH4.0) (containing 50mM NaCl) one-step elution, it is pure that the Marine Red Alga Polysiphonia Urceolata RPE purity of purifying reaches electrophoresis, the purity Index A
620/ A
280>5.2, structural formula is (α β)
6γ, molecular weight is about 240kDa, and maximum absorption band lays respectively at 498,540,565nm, the room temperature fluorescence emission peak is positioned at 575nm, 4 ℃ of the RPE ammonium sulfate precipitations of purifying keep in Dark Place, and before using, with the 50mM pH7.5PBS desalination of fully dialysing, adjustment concentration is 10mg/mL.
Avian pneumo-encephalitis virus polyclonal antibody preparation: Avian pneumo-encephalitis virus La Sota strain 10
4doubly dilution, allantoic cavity inoculation 9-10 age in days SPF chicken embryo, every embryo 0.2mL, hatch for 37-38 ℃, collect the dead germ of inoculation 24-120h, the allantoic fluid of survival embryo, hemagglutination test (HA test) detection titre>1: 256, steriling test is qualified, with 37 ℃ of deactivation 24h of 0.3% formaldehyde, add No. 10 white oils of sterilizing and Tween-80 in 1: 3 ratio, be emulsified into the water-in-oil type oil emulsion inactivated vaccine with colloid mill.The oil-adjuvant inactivated against Newcastle disease immunity SPF chicken in 4 week age of preparation, neck hypodermic injection 1.0mL/ only, head exempts from after 10 days the same dose booster immunization once, two exempt from blood sampling after 1 week, measure Avian pneumo-encephalitis virus HI antibody in serum, when HI tire>gather 1: 256 time serum, DEAE Sepharose Fast Flow anion-exchange chromatography purifying, 50mM pH7PBS+0-1M NaCL gradient elution obtains pure anti-new castle disease virus polyclonal antibody ,-20 ℃ of freezing preservations.
The preparation of RPE mark Avian pneumo-encephalitis virus antibody fluorescence probe: SPDP mixes with RPE, Avian pneumo-encephalitis virus antibody respectively by the mol ratio of 100: 1, aluminium foil seal after in room temperature revolving reaction 2h, ultrafiltration is centrifugal removes unnecessary SPDP.Get DTT and fully mix by mol ratio and the Avian pneumo-encephalitis virus antibody derivatives of 100: 1, the standing reaction of room temperature 1h, ultrafiltration is centrifugal removes unnecessary DTT.Avian pneumo-encephalitis virus antibody-HS and RPE derivant mix with mol ratio at 1: 1, aluminium foil seal after in 20-25 ℃ of oscillating reactions 20h.Seal unnecessary sulfydryl with the NEM solution 20ul of 10mg/ml, room temperature revolving reaction 60min.NEM in reaction need not remove.
The purifying of the fluorescence antibody of RPE mark and cross-linking efficiency analysis: on the liquid chromatography workstation, adopt the HPLC method to carry out cross-linking efficiency analysis and the purifying of fluorescence antibody, molecular sieve high pressure liquid chromatography post model TSK G3000sw (specification 7.5mm * 60cm), mobile phase is 50mM PBS pH7.5, flow velocity 0.5mL/min, detect wavelength 190-800nm.The fluorescence labeling antiantibody is eluted at first in 19.57min.Collect first eluting peak.The ratio that the eluting peak area of target product accounts for all eluting peak areas is cross-linking efficiency, and the crosslinked yield that the analysis showed that fluorescence probe is 95%.
The spectral detection of RPE mark fluorescent antibody: absorption spectrum is measured on ultraviolet-visible spectrophotometer, the interval 250-700nm of scanning wavelength.Absorption spectrum detects can judge whether the crosslinked cross-linking reaction that whether successfully reaches causes the RPE sex change.The room temperature fluorescence emission spectrum is measured on fluorospectrophotometer, excitation wavelength 498nm, and the fluorescent scanning scope is 520-700nm.In ultraviolet region, the absorbance of RPE mark fluorescent antibody is higher than contrast RPE, and the absorption peak position is identical, this be because RPE surface-crosslinked antibody molecule, and antibody has stronger light absorption at 280nm; In the 400-700nm scope, the absorption spectrum of cross-linking agent is similar with the absorption peak that contrasts RPE and absorbance, be due to antibody in this scope without light absorption.RPE mark photoactivated antibody with contrast RPE under the exciting of 498nm exciting light, the room temperature fluorescence emission wavelength all is positioned at 575nm, and the change of stoke shift does not occur, and still have the characteristic fluorescence emission spectrum of RPE, and fluorescent brightness is without obvious reduction.
The bioactivity of RPE mark fluorescent antibody: antibody titer adopts Microhemagglutination inhibition test method (HI) to measure.Prepare 4 unit detectable antigens with the corresponding ewcastle disease allantotoxicon of deactivation, reaction is carried out on V-type 96 orifice plates.What the high dilution that the red cell agglutination of take suppresses was fluorescence antibody tires.It is 1: 32 that the HI of fluorescent-labeled antibody tires.
The electrophoresis detection of RPE mark fluorescent antiantibody: SDS-PAGE carries out on vertical panel discontinuous electro-phoresis device, and resolving gel concentration is 12.5%, and concentrated gum concentration is 5%.The constant voltage electrophoresis, voltage is 218V.By the dyeing of 0.25% (w/v) coomassie brilliant blue R_250, observations after decolouring.The SDS-PAGE electrophoresis finds that RPE mark fluorescent antibody had both had α, β, the γ subunit band of RPE, has again H, the L chain band of antibody, and electrophoresis result confirms RPE and antibody linked success.
The Detection of Stability of RPE mark fluorescent antibody: in the RPE mark fluorescent antibody of purifying, add 0.02% sodium azide, 4 ℃ keep in Dark Place, measured first order fluorescence spectrum and antibody titer every 30 days, the room temperature fluorescence emission spectrum is measured on fluorospectrophotometer, antibody titer adopts the hemagglutination-inhibition test method to measure, and observes the variation of fluorescent brightness and antibody activity.Preserve the fluorescence antibody good stability that test confirms the RPE mark of development.RPE mark fluorescent antibody in the phosphate buffer of 0.05mol/L 4 ℃ preserve 120 days, the fluorescence intensity of fluorescence antibody remains unchanged, the antibody titer of fluorescence antibody maintains 1: 32, occurs reducing.
Solid-phase fluorescence immunoassay kit assembling and using: the solid-phase fluorescence immunoassay kit is comprised of the anti-new castle disease virus fluorescence antibody of the agarose microbeads carrier of cyanogen bromide-activated, RPE mark, anti-new castle disease virus polyclonal antibody, cleansing solution, watery hydrochloric acid etc.The using method of solid-phase fluorescence immunoassay kit is: during microsphere supported use, need use the watery hydrochloric acid swelling, get the Ago-Gel freeze-dried powder of 0.3g cyanogen bromide-activated, be placed in 1mM hydrochloric acid swelling 30min, hydrochloric acid with same concentration rinses 5 times, get express developed and obtain 1mL activation gel with 50mM pH 8PBS (containing 100mM NaCl), with 2mL concentration, being 5mg/ml immediately, the anti-new castle disease virus polyclonal antibody mixes, 20 ℃ of slow oscillation action 12h, the monoethanolamine sealing that adds 2mL 0.1M, 20 ℃ of slow oscillation action 4h, the centrifugal supernatant that goes, repeatedly rinse gel with 50mM pH 8PBS, centrifugal acquisition coupling has the Ago-Gel of antibody, be made into the suspending liquid of 10% (v/v) with the PBS of 100mM pH 7.5.Get the Ago-Gel 50uL that coupling has antibody, be placed in clean test tube, add virus liquid 100uL to be checked, 37 ℃ of incubation 2h, rinse 10 times with the PBS of 100mM pH 7.5.The fluorescent-labeled antibody 100uL that adds 5ug/ml concentration, 37 ℃ of incubation 2h, rinse 5 times with the PBS of 100mM pH 7.5.Draw a small amount of microballoon, drip on the slide of low fluorescence background, cover plate, glycerine damping fluid mounting, excite with blue green light under fluorescent microscope, observation, result of determination.Positive microballoon excites the orange colour fluorescence of lower emitting bright at ruddiness, negative microballoon is without fluorescence.By fluorescence intensity and background staining power with+mean, be divided into ++ ++ (strong positive), +++(strong positive), ++ (than strong positive) ,+(the weak positive) ,-(feminine gender) Pyatyi.Fluorescence intensity reaches ++ more than be judged to be the positive.SPF chicken serum negative control and PBS blank are established in test.At blue green light, excite lower positive microballoon to be bright orange colour fluorescence, negative findings is without fluorescence.
Preparation Avian pneumo-encephalitis virus allantotoxicon, measuring viral red cell agglutination valency is 2
9, EID
50be 10
-7.2, the protein concentration of virus is 40.3mg/mL.With PBS, by the allantotoxicon serial dilution, utilize the solid-phase immunity fluorescence method of setting up to carry out one by one fluoroscopic examination to each dilutability.The least concentration that kit can detect Avian pneumo-encephalitis virus is 4.03 * 10
-7mg/ml.
Replace Avian pneumo-encephalitis virus with avian influenza virus, IBV, infectious bursal disease virus, ILTV, bird pox virus etc. respectively, carry out fluoroscopic examination by above-mentioned solid phase immunofluorescence detection agent box, excite the lower fluorescence that all do not detect at blue green light, prove that the specificity of kit is good.
Utilize the solid-phase fluorescence immunoassay kit to same dilution Avian pneumo-encephalitis virus sample duplicate detection 3 times, result is consistent, shows that the repeatability of detection kit is better.
FITC labelled antibody fluorescence probe detects Avian pneumo-encephalitis virus: the carbonic acid buffer dialysed overnight of 50mM pH 9 for the Avian pneumo-encephalitis virus antibody of purifying, adjustment concentration is 10mg/ml.FITC with above-mentioned carbonic acid buffer preparation 0.1mg/ml.Antibody is placed in to 4 ℃ of slow dialysis 24h that stir of FITC solution of 10 times of antibody volumes, the phosphate buffer dialysis that then rapidly labelled antibody is placed in to 50mM pH 7.2, frequent exchange buffering liquid.The antibody probe that replaces the RPE mark with the antibody probe of FITC mark, detect Avian pneumo-encephalitis virus with the solid-phase fluorescence immunoassay kit.The FITC label probe detects positive microballoon at blue-light excited lower transmitting green fluorescence, and the least concentration that can detect ewcastle disease is 4.03 * 10
-6mg/ml, low 10 times of remolding sensitivity RPE label probe.
Table 1RPE labelled antibody fluorescence probe detects Avian pneumo-encephalitis virus
Claims (1)
1. a preparation method who detects the fluorescence antibody of Avian pneumo-encephalitis virus, it is characterized in that: adopt the direct wash-out anion exchange chromatography of a step by Marine Red Alga Polysiphonia Urceolata separation and purification R-PE RPE, preparation process is: the Marine Red Alga Polysiphonia Urceolata of getting freezing preservation, add 20mM pH5.8 acetate buffer solution, 4 ℃ of swelling 24h, multilayer gauze parcel, extruding, filter and obtain the brown leaching liquor, it is 60%w/v that leaching liquor adds solid ammonium sulfate to concentration, place 24h for 4 ℃, centrifugal collecting precipitation, precipitation is dissolved in the acetate buffer solution of 20mM pH5.8, then the acetate buffer solution of 20mM pH5.8 is dialysed, dislysate is added to the anion-exchange column containing 50mM NaCl acetate buffer solution pre-equilibration with 20mM pH5.8, rinse out sample excessive on anion-exchange column and impurity with 20mM pH5.8 acetate buffer solution, just can obtain a large amount of pure R-PE RPE with 20mM pH4.0 containing 50mM NaCl acetate buffer solution one-step elution, it is pure that the Marine Red Alga Polysiphonia Urceolata R-PE RPE purity of purifying reaches electrophoresis, the purity Index A
565/ A
280>5.2, structural formula is (α β)
6γ, molecular weight is about 240kDa, maximum absorption band lays respectively at 498, 540, 565nm, the room temperature fluorescence emission peak is positioned at 575nm, 4 ℃ of the R-PE RPE ammonium sulfate precipitations of purifying keep in Dark Place, before using with the 50mM pH7.5 phosphate buffer PBS desalination of fully dialysing, adjustment concentration is 5mg/mL, use mol ratio 50-100: 1 chemical cross-linking agent N-succinimide 3-(2-pyridine radicals two sulphur) propionic ester SPDP is respectively by R-PE RPE, anti-new castle disease virus antibody mixes, aluminium foil seal after in room temperature revolving reaction 2h, centrifugal unnecessary N-succinimide 3-(2-pyridine radicals two sulphur) the propionic ester SPDP that removes of ultrafiltration, dithiothreitol (DTT) DTT is by 50-100: 1 mol ratio and antibody derivatives fully mix, the standing reaction of room temperature 1h, ultrafiltration is centrifugal removes unnecessary dithiothreitol (DTT) DTT, antibody one HS and R-PE RPE derivant mix with mol ratio at 1: 1, aluminium foil seal after in 20-25 ℃ of oscillating reactions 20h, add N mono-ethyl Malaysia phthalimide NEM and seal unnecessary sulfydryl, room temperature revolving reaction 60min, the high-efficient liquid phase chromatogram HPLC purifying, collect the cross-linking agent eluting peak, detect the room temperature fluorescence emission spectrum of cross-linking agent on fluorospectrophotometer, Microhemagglutination inhibition method is measured the antibody titer of cross-linking agent, detect the qualified 4 ℃ of preservations of 0.02% sodium azide that add, the anti-new castle disease virus fluorescence antibody purity of the R-PE RPE mark that adopts described method to prepare is high, orange colour fluorescence is bright, stable in properties, can be used for the quick of Avian pneumo-encephalitis virus, differentiate and detect.
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| CN110531081B (en) * | 2018-05-24 | 2022-09-06 | 中国科学院苏州纳米技术与纳米仿生研究所 | Newcastle disease virus antibody detection kit |
| CN111308063B (en) * | 2020-02-12 | 2023-07-07 | 湖州正熙医学检验实验室有限公司 | Phycoerythrin immunofluorescence probe labeling method |
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