CN104634961A - Preparation method and detection method of rabies virus homogeneous fluorescent composite microsphere - Google Patents
Preparation method and detection method of rabies virus homogeneous fluorescent composite microsphere Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Abstract
The invention discloses a preparation method and detection method of a rabies virus homogenous fluorescent composite microsphere. Polystyrene nano microsphere is taken as the functionalized internal core to mark the fluorescent substance Eu3+, and at the same time a complexing agent BHHCT is used to mark recombinant rabies virus antigens so as to produce the rabies virus homogeneous fluorescent composite microsphere. The prepared rabies virus homogenous fluorescent composite microsphere is used to detect rabies virus antibody without dissociation or enhancement, moreover, the surface of the homogenous fluorescent composite microsphere is covered by a protective layer made of amino silane, and thus the external affection on the fluorescent signals is reduced, the fluorescence has a long life, so that the requirements of time-resolved fluoro-immune assay can be fulfilled. The provided detection method by using the rabies virus homogenous composite microsphere has the advantages of high sensitivity, low detection cost, and reduced operation steps and reaction time, the automatic operation can be achieved more easily, and the linear range of instrument reading is wider.
Description
Technical field
The present invention relates to preparation method and the detection method thereof of rabies viruses homogeneous fluorescence complex microsphere.
Background technology
The glycoprotein (being called for short RVG) of rabies viruses (Rabies virus, RV) is the virus protein that body uniquely can be stimulated to produce neutralizing antibody, is also good detectable antigens simultaneously.Therefore, rabies virus glycoprotein is carried out high expression in Escherichia coli, the recombinant protein after the method validation purifying such as Western blot can produce specific reaction with RV positive serum, and this is that the development of RV antibody assay kit is laid a good foundation.
Time-resolved fluoroimmunoassay (Time-resolved Fluoroimmunoassy, TrFIA) lanthanide chelate labelled antigen, antibody, polypeptide, hormone etc. are utilized, occur as after the reaction such as antigen/antibody immune response, biotin/avidin reaction, add and strengthen lyolysis from lanthanide series, its fluorescence signal is strengthened, excite through exogenous pulsed light again, and adopt the photoelectric technologies such as time delay reading to detect the specific fluorescence of lanthanide series, thus reach the object that sample analysis is detected.Due to luminescent substance europium ion and sequestrant combine after and the antigen of bag quilt or antibody carry out immune response, the fluorescence signal that the Ag-Ab-Europium label compound formed occurs through excitation in alkalescent damping fluid is very weak, must add and strengthen liquid (acetic acid of β-one body, TOPO, Triton X-100, pH2-3 and the acid solution of adjacent benzene potassium hydrogen phthalate), make rare earth ion from complex compound from getting off, be combined with new complex compound and form macromolecular micro-capsule, this micro-capsule by energy transferring to Eu
3+, block the quenching effect that water produces, make original fluorescence signal strengthen nearly 1,000,000 times, be beneficial to fluorescence measurement.Visible, it is indispensable for strengthening liquid in TrIFA.
Summary of the invention
The object of the present invention is to provide preparation method and the detection method thereof of rabies viruses homogeneous fluorescence complex microsphere.
The technical solution used in the present invention is:
The preparation method of rabies viruses homogeneous fluorescence complex microsphere, comprises the following steps:
1) Properties of Polystyrene Nano Particles is prepared;
2) Eu
3+mark: Properties of Polystyrene Nano Particles is suspended in absolute ethyl alcohol, adds complexing agent BHHCT, lucifuge reaction 15 ~ 25min, with absolute ethanol washing, after Tris-HCl damping fluid is resuspended, add EuCl
3, lucifuge reaction 16 ~ 25min; Centrifugal, then use the resuspended precipitation of absolute ethyl alcohol, add amino silane, lucifuge reaction 1.5 ~ 2.5 hours, washing, obtains homogeneous fluorescence complex microsphere;
3) mark of antigen:
A, get homogeneous fluorescence complex microsphere, be resuspended in absolute ethyl alcohol, add carbodiimides, lucifuge reaction 23 ~ 35min;
B, centrifugal, phosphate buffer is resuspended, adds rabies virus antigen, lucifuge reaction 12 ~ 20h;
C, add 20%BSA solution, reaction 22 ~ 35min;
D, centrifugal, washing, obtains the homogeneous fluorescence complex microsphere of the mark rabies virus antigen for detecting rabies viruses.
Preferably, in step 1), polyvinylpyrrolidone, potassium persulfate, styrene are dissolved in ethanol water with mass ratio 25:0.6:100, under nitrogen protection, carry out polyreaction; Centrifugal, by the resuspended precipitation of absolute ethyl alcohol, add amino silane, lucifuge reaction 1.6 ~ 3 hours, washing, obtained Properties of Polystyrene Nano Particles.
Preferably, under ultrasound condition, 52 ~ 65 DEG C, carry out polyreaction.
Preferably, step 2) in, the mass ratio of BHHCT and Properties of Polystyrene Nano Particles is 1:50.
Preferably, in step 3), the mass ratio of homogeneous fluorescence complex microsphere and carbodiimides is 1:1.
The detection method of rabies viruses, comprises the following steps:
1), after recombination staphylococcus aureus albumin A being diluted with the phosphate buffer of pH7.4,0.02mol/L, join in ELISA Plate hole, every hole 100 μ L, place 13 ~ 18 hours for 2 ~ 8 DEG C;
2) every hole adds 200 μ L confining liquids, places 13 ~ 18 hours for 2 ~ 8 DEG C;
3) discard liquid in plate, wash plate, dry;
4) join in ELISA Plate hole by standard items or measuring samples, every hole 100 μ L, hatches for 37 DEG C;
5) discard liquid in plate, wash plate, dry;
6), after rabies viruses homogeneous fluorescence complex microsphere dilution method described in Claims 1 to 5 any one prepared, join in ELISA Plate hole, every hole 100 μ L, hatches for 37 DEG C;
7) discard liquid in plate, wash plate, dry;
8) with the time-resolved fluorescence mode detection fluorescence signal of multi-functional microplate reader, excitation wavelength is 330nm, and wavelength of transmitted light is 610nm;
9) with the logarithm value of standard concentration for horizontal ordinate, the logarithm value recording Relative fluorescence units is ordinate, obtain typical curve, contrast measuring samples detected value, result of determination.
Preferably, in step 1), the dilute concentration of recombination staphylococcus aureus albumin A is 2 μ g/mL.
Preferably, in step 6), the extension rate of rabies viruses homogeneous fluorescence complex microsphere is 5000 times.
Preferably, step 4) hatches 60min, and step 6) is hatched.
Preferably, confining liquid contains the phosphate buffer of pH7.4,0.05mol/L, 4%BSA, the NaN of 0.01%
3.
The invention has the beneficial effects as follows:
The present invention on traditional TrFIA basis, with Nano microsphere as fluorescent material Eu
3+carrier, under the polymerization of functionalization kernel, namely form homogeneous fluorescence complex microsphere, be then cross-linked RVG by chelation.The bi-functional complexing agent BHHCT that the present invention uses is owing to only having a chlorine sulfonephthalein base, and one end is connected with rare earth element, and one end is connected with antigen or antibody, avoids the cross-linking reaction between protein molecule, and due to fluorescent material Eu
3+directly do not contact with biomacromolecule, can not biologically active be affected, not need to carry out dissociating and strengthening.Further, homogeneous fluorescence complex microsphere surface is covered with the protective seam that amino silane is formed, and fluorescence signal is affected by the external environment reduction, and the stability constant of rabies viruses homogeneous fluorescence complex microsphere is up to 10
10, fluorescence lifetime reaches hundreds of microsecond, well can meet the needs of time resolved fluoro-immunoassay.
The preparation method of rabies viruses homogeneous fluorescence complex microsphere of the present invention is simple, and testing cost is low, decreases operation steps and reaction time, more easily realizes automation mechanized operation; Instrument readings is higher, and the range of linearity is wider, and detection sensitivity is apparently higher than ELISA.
Accompanying drawing explanation
Fig. 1 is the stereoscan photograph of Properties of Polystyrene Nano Particles.
Fig. 2 is the Eu of homogeneous fluorescence complex microsphere
3+fluorescence spectrum.
Fig. 3 is the typical curve that embodiment 2 homogeneous fluorescence complex microsphere detection method is set up.
Embodiment
The key improving TrFIA sensitivity is rare earth element complexing agent.The bi-functional complexing agent 4 that the present invention uses; 4 '-two (l ", 1 ", 1 "; 2 "; 2 ", 3 ", 3 "-seven fluoro-4 "; 6 "-acetyl butyryl-6 "-Ji)-chlorosulfonyl-adjacent diphenyl benzene (BHHCT) is a kind of stable Eu that can send intense fluorescence that (Yuan; J, et al., 1998) such as Yuan research and develop
3+complexing agent, the quadridentate group contained by it is greatly improved in sensitivity than beta-diketon class complexing agent in the past.BHHCT is owing to only having a chlorine sulfonephthalein base, and one end is connected with rare earth element, and one end is connected with antigen or antibody, avoids the cross-linking reaction between protein molecule.
In following examples, the recombinant protein A (recombinant protein SPA) of aureus cell wall used is purchased from Sino Biological Inc.; Fluorescent chelate BHHCT, recombinant rabies poison antigen RVG is presented by Beijing Thymopetidum Injection thing technology company; Anti-canine distemper virus antibody, anti-dog parvovirus antibody are purchased from Hytest; ELISA kit is purchased from Beijing North Rui Da Pharmaceutical Technology Co., Ltd; Standard items quality controlled serum is preserved by this laboratory.
embodiment 1
The preparation of rabies viruses homogeneous fluorescence complex microsphere:
(1) preparation of functionalization kernel Properties of Polystyrene Nano Particles
2.5g polyvinylpyrrolidone (PVP), 0.06g potassium persulfate (KPS), 10g styrene (St) are joined in 250ml absolute ethyl alcohol/aqueous medium to dissolve and is placed in ultrasound reactor, logical nitrogen, open ultrasonic generator, carry out polyreaction at 60 DEG C, obtain Properties of Polystyrene Nano Particles dispersion liquid.Centrifugal collecting precipitation, suspends with absolute ethyl alcohol, adds amino silane, and stirring at room temperature is reacted, and collects Properties of Polystyrene Nano Particles after absolute ethanol washing, and absolute ethyl alcohol suspends and saves backup.
(2) Eu
3+marking nano microballoon
Nano microsphere suspending liquid is concentrated into 20mg/mL, adds the ethanolic solution of the 4mg/mL BHHCT of 0.5mL to 5mL suspending liquid, room temperature lucifuge stirring reaction 20min; With absolute ethanol washing, Tris-HCl damping fluid is resuspended, adds the 0.01mol/L EuCl of 0.5mL
3solution, room temperature lucifuge stirring reaction 20min; Suspend with absolute ethyl alcohol after collected by centrifugation again, add amino silane, room temperature lucifuge stirring reaction 2 hours, with collecting precipitation after 1mL absolute ethanol washing, obtained homogeneous fluorescence complex microsphere, absolute ethyl alcohol suspends and saves backup.
(3) mark of rabies virus antigen RVG
A, get the 20mg/mL homogeneous fluorescence complex microsphere suspending liquid of 100 μ L, 0.05mol/L PB 6.0 washs 3 times, ultrasound suspending, and add 20 μ L carbodiimides (EDC, 100mg/mL, Sigma) solution, room temperature lucifuge reacts 30 minutes;
B, centrifugal collecting precipitation, the 0.05mol/L PB 6.0 of 100 μ L suspends, and adds 2mg RVG, and room temperature lucifuge reacts 16 hours;
C, add the 20%BSA solution of 100 μ L, room temperature reaction 30 minutes;
D, 0.05mol/L Tris 8.0 washs 3 times, and be suspended in 100 μ L conserving liquid (0.05mol/L Tris 8.0,1%BSA, 0.1% PVP40), 2 ~ 8 DEG C keep in Dark Place for subsequent use.
Shown in Fig. 1 is the stereoscan photograph that particle diameter is about the Properties of Polystyrene Nano Particles of 60nm.Fig. 1 (A) is obtained Properties of Polystyrene Nano Particles dispersion liquid, and as can be seen from the figure its microballoon size is comparatively consistent, does not bond and distribution uniform.Fig. 1 (B) is the stereoscan photograph of prepared homogeneous fluorescence complex microsphere, visible, wraps and is not changed in shape by the microballoon of europium, does not bond and distribution uniform, namely morphologically as broad as long with kernel.
Fig. 2 is the Eu of homogeneous fluorescence complex microsphere
3+fluorescence spectrum, Eu
3+exciting light at 330nm, wavelength of transmitted light concentrates on 610-620nm, and fluorescence signal is strong, and Stokes displacement has the gap of 290-300nm, can find out that microsphere fluorescence signal is launched stable.
embodiment 2
One, the detection method of rabies viruses:
(1) pre-coated: SPA bag to be buffered liquid (pH7.4,0.02mol/L phosphate buffer) and to join 96 microwell plates, every hole 100 μ L, place 16 hours for 2 ~ 8 DEG C;
(2) close: every hole adds the 200 μ L confining liquid (NaN of 0.05mol/L PB 7.4,4%BSA, 0.01%
3), 2 ~ 8 DEG C 16 hours;
(3) wash: discard liquid in plate, wash plate 3 times with cleansing solution (0.01mol/L PB 7.4,0.9%NaCl, 0.1%Tween-20), 37 DEG C dry up rear sealing and save backup;
(4) with standard serum and sample: join in the ELISA Plate hole of pre-coated SPA by standard serum or the measuring samples handled well, every hole 100 μ L, hatches for 37 DEG C;
(5) wash: discard liquid in plate, cleansing solution washes plate 5 times, pats dry;
(6) homogeneous fluorescence complex microsphere is added: with dilution (0.05mol/L Tris-HCl 8.0,0.5%Casein-Na, 5%NBS) dilute embodiment 1 prepare rabies viruses homogeneous fluorescence complex microsphere, join in the ELISA Plate hole after washing, every hole 100 μ L, hatches for 37 DEG C;
(7) wash: discard liquid in plate, cleansing solution washes plate 5 times, pats dry;
(8) with the time-resolved fluorescence mode detection fluorescence signal of multi-functional microplate reader, value read in record.The excitation wavelength of microplate reader is 330nm, and wavelength of transmitted light is 610nm.
two,the optimization of Rabies Virus Detection method:
1, SPA bag is by the optimization of concentration
Be buffered liquid (pH7.4,0.02mol/L phosphate buffer) with bag and SPA is carried out serial dilution (being respectively 0.5 μ g/mL, 1 μ g/mL, 2 μ g/mL, 5 μ g/mL, 10 μ g/mL), carry out pre-coated.Operate by the method for step one.Testing result is in table 1, and when bag is 2 μ g/mL by concentration, signal to noise ratio (S/N ratio) S/N reaches the highest, and namely the interference of background fluorescence is minimum, therefore 2 μ g/mL are that the suitableeest bag of SPA is by working concentration.
The different SPA bag of table 1 is by the testing result of concentration
| Bag is by concentration | 0.5μg/mL | 1μg/mL | 2μg/mL | 5μg/mL | 10μg/mL |
| 0.5 IU/mL | 22301 | 36723 | 45775 | 57176 | 82237 |
| 0 IU/mL | 2198 | 2219 | 2318 | 3572 | 6400 |
| S/N | 10.15 | 16.55 | 19.75 | 16.01 | 12.85 |
2, the optimization of homogeneous fluorescence complex microsphere extension rate
With the SPA coated elisa plate of 2 μ g/mL optium concentrations, with dilution, homogeneous fluorescence complex microsphere is diluted by 1000,2000,5000,10000 times of gradients, operate by the method for step one respectively.Testing result is in table 2, and when homogeneous fluorescence complex microsphere extension rate is 5000, the ratio of signal to noise ratio (S/N ratio) S/N reaches the highest, and background interference is low, and fluorescent value is high, therefore 5000 is the suitableeest extension rates of homogeneous fluorescence complex microsphere.
The testing result of the different extension rates of table 2 homogeneous fluorescence antibody complex microsphere
| Homogeneous fluorescence complex microsphere extension rate | 1000 | 2000 | 5000 | 10000 |
| 0.5 IU/mL | 75472 | 52347 | 46553 | 32201 |
| 0 IU/mL | 5071 | 3459 | 2303 | 2056 |
| S/N | 14.88 | 15.13 | 20.21 | 15.66 |
3, the optimization in reaction time
According to method described in step one, SPA concentration is 2 μ g/mL, and homogeneous fluorescence complex microsphere extension rate is 5000, adopts different time to carry out incubation reaction: single stage method and step (6) 40min incubation; Two-step approach and step (4) 40min+step (6) 40min incubation; Two-step approach and step (4) 60min+ step (6) 60min incubation.
Testing result is in table 3, and when using two-step approach (60min+60min) to carry out incubation, the ratio of signal to noise ratio (S/N ratio) S/N reaches the highest, therefore optimum reacting time is two-step approach (60min+60min).
The testing result of table 3 differential responses time
| Reaction time | Single stage method (60min) | Two-step approach (40min+40min) | Two-step approach (60min+60min) |
| 0.5 IU/mL | 34785 | 40152 | 50783 |
| 0 IU/mL | 2337 | 2214 | 2392 |
| S/N | 14.88 | 18.14 | 21.23 |
4, the detection method after optimizing is as follows:
(1) pre-coated: the SPA of 2 μ g/mL is joined 96 microwell plates, every hole 100 μ L, places 16 hours for 2 ~ 8 DEG C;
(2) close: every hole adds 200 μ L confining liquids, 2 ~ 8 DEG C 16 hours;
(3) wash: discard liquid in plate, wash plate 3 times with cleansing solution, 37 DEG C dry up rear sealing and save backup;
(4) with standard serum and sample: join in pre-coated ELISA Plate hole by standard serum or the measuring samples handled well, every hole 100 μ L, hatches 60min for 37 DEG C;
(5) wash: discard liquid in plate, cleansing solution washes plate 5 times, pats dry;
(6) homogeneous fluorescence complex microsphere is added: by rabies viruses homogeneous fluorescence complex microsphere with dilution (0.05mol/L Tris-HCl 8.0,0.5%Casein-Na, 5%NBS) dilute 5000 times, join in the ELISA Plate hole after washing, every hole 100 μ L, hatches 60min for 37 DEG C;
(7) wash: discard liquid in plate, cleansing solution washes plate 5 times, pats dry;
(8) with the time-resolved fluorescence mode detection fluorescence signal of multi-functional microplate reader, value read in record.The excitation wavelength of microplate reader is 330nm, and wavelength of transmitted light is 610nm.
5, the foundation of typical curve
The preparation of standard items: with antibody diluent (10% calf serum, the NaN of 0.01%
3) dilution obtain 6 standard serum concentration (antibody concentration is respectively 0 IU/mL, 0.05IU/mL, 0.5 IU/mL, 5 IU/mL, 20 IU/mL, 40 IU/mL).
The detection method of above-mentioned optimization is adopted to measure the Relative fluorescence units (RLU) of 6 standard items, each standard items are surveyed and are asked its mean value for twice, according to double-log function, (horizontal ordinate is the logarithm value of standard concentration again, ordinate is the logarithm value of RLU mean value) process obtain typical curve, analyze.The results are shown in Table 4, Fig. 3, the typical curve related coefficient of known homogeneous fluorescence complex microsphere detection technique is 0.994, indicates linear dependence good.
The testing result of table 4 standard items
| Concentration is IU/ml (x) | RLU-1 | RLU-2 | AVG（y） | logx | logy |
| 0 | 2259 | 1989 | 2124 | —— | 3.33 |
| 0.05 | 19187 | 19463 | 19325 | -1.30 | 4.29 |
| 0.5 | 171871 | 179463 | 175667 | -0.30 | 5.24 |
| 5 | 1440490 | 1411587 | 1426039 | 0.70 | 6.15 |
| 20 | 3101883 | 3117215 | 3109549 | 1.30 | 6.49 |
| 40 | 6382510 | 6612785 | 6497648 | 1.60 | 6.81 |
embodiment 3
1, the sensitivity experiment of the inventive method
With antibody concentration be respectively 0 IU/mL, 0.01 IU/mL, 0.02 IU/mL, 0.05 IU/mL, 0.1 IU/mL, 0.2 IU/mL, 0.5 IU/mL, 1 IU/mL, 2 IU/mL, 4 IU/mL, 6 IU/mL, 8 IU/mL, 12 IU/mL standard serum make reference, the detection method optimized according to embodiment 2 carries out sensitivity test, and contrast with ELISA method, coefficient of variation analysis compares the difference of sensitivity by the equation of linear regression of calculated curve.
Homogeneous fluorescence complex microsphere detection method of the present invention detects the standard items of 0.01 IU/mL, and namely its value of reading 5508 has the difference of 2.5 times with the result 2075 of 0 IU/mL reference material, and therefore the minimum detectability of the inventive method is not less than 0.01 IU/mL.And use ELISA method, when standard concentration is 0.2 IU/mL, its value of reading is 0.046, can not obviously distinguish with the result 0.044 of 0IU/mL standard items, and namely its minimum detectability is not less than 0.2 IU/mL.Can find out that the minimum detectability of the inventive method will exceed about 20 times than ELISA.The results are shown in Table 5.
The sensitivity technique of table 5 homogeneous fluorescence complex microsphere
| Reference material (IU/mL) | ELISA（OD450） | Microballoon detects (RLU) |
| 0 | 0.044 | 2075 |
| 0.01 | 0.04 | 5508 |
| 0.02 | 0.043 | 8376 |
| 0.05 | 0.044 | 14878 |
| 0.1 | 0.047 | 53627 |
| 0.2 | 0.046 | 71776 |
| 0.5 | 0.081 | 157404 |
| 1 | 0.122 | 285101 |
| 2 | 0.203 | 516395 |
| 4 | 0.357 | 935331 |
| 6 | 0.427 | 1223962 |
| 8 | 0.566 | 1494135 |
| 12 | 0.801 | 2298050 |
2, the specificity experiments of the inventive method
The detection method optimized by embodiment 2 detects canine distemper virus positive serum, canine parvovirus positive serum and each 5 parts of negative serum, and two kinds of positive serum detected values and negative serum detected value is analyzed.The results are shown in Table 6, the measured value of canine distemper virus positive serum and canine parvovirus positive serum and negative serum does not have difference substantially, illustrates for this two-strain positive serum, and the inventive method there will not be false positive when detecting Rabies virus antibody sample.
Table 6 specificity experiments result
| Numbering | Canine distemper virus positive serum | Canine parvovirus positive serum | Negative serum |
| 1 | 2475 | 2218 | 2154 |
| 2 | 3211 | 1732 | 2388 |
| 3 | 2901 | 2195 | 2461 |
| 4 | 2175 | 2247 | 2032 |
| 5 | 277 | 2173 | 2141 |
3, the detection of clinical sample
At Futian Area of Shenzhen City, Guangdong Province (F), the serum totally 30 parts of dog is collected by Luohu District (L) and Nanshan District (N) each pet clinic, another collection 8 parts of blood serum samples be detected with FAVN goldstandard, carry out the detection of rabies antibody respectively to it by the detection method of optimization of the present invention and ELISA method.
Contrast 30 parts of Virus monitory found that, two kinds of method coincidence rates are 72%.8 parts are detected by these two kinds of methods with the blood serum sample that FAVN goldstandard is detected: FAVN detects complete in positive; When the inventive method detects, a sample is in critical value, and ELISA method detects this sample and is equally also in critical value, and ELISA method detects and also has another increment product to be in critical value, and namely ELISA method detects has two increment product to be in critical value.Visible, the sensitivity and linear measurement range of the homogeneous fluorescence complex microsphere method that the present invention sets up all has exceeded ELISA method, and the coincidence rate of the measurement result of the inventive method and FAVN method reaches 98%, and ELISA is only 91%.Because the test sample of FAVN is for up to 96h, and the present invention only needs 2h, greatly can shorten detection time.Result shows that the homogeneous fluorescence complex microsphere method that the present invention sets up can carry out sample mensuration fast and accurately.
Claims (10)
1. the preparation method of rabies viruses homogeneous fluorescence complex microsphere, comprises the following steps:
1) Properties of Polystyrene Nano Particles is prepared;
2) Eu
3+mark: Properties of Polystyrene Nano Particles is suspended in absolute ethyl alcohol, adds complexing agent BHHCT, lucifuge reaction 15 ~ 25min, with absolute ethanol washing, after Tris-HCl damping fluid is resuspended, add EuCl
3, lucifuge reaction 16 ~ 25min; Centrifugal, then use the resuspended precipitation of absolute ethyl alcohol, add amino silane, lucifuge reaction 1.5 ~ 2.5 hours, washing, obtains homogeneous fluorescence complex microsphere;
3) mark of antigen:
A, get homogeneous fluorescence complex microsphere, be resuspended in absolute ethyl alcohol, add carbodiimides, lucifuge reaction 23 ~ 35min;
B, centrifugal, phosphate buffer is resuspended, adds rabies virus antigen, lucifuge reaction 12 ~ 20h;
C, add 20%BSA solution, reaction 22 ~ 35min;
D, centrifugal, washing, obtains the homogeneous fluorescence complex microsphere of the mark rabies virus antigen for detecting rabies viruses.
2. preparation method according to claim 1, is characterized in that: in step 1), polyvinylpyrrolidone, potassium persulfate, styrene is dissolved in ethanol water with mass ratio 25:0.6:100, carries out polyreaction under nitrogen protection; Centrifugal, by the resuspended precipitation of absolute ethyl alcohol, add amino silane, lucifuge reaction 1.6 ~ 3 hours, washing, obtained Properties of Polystyrene Nano Particles.
3. preparation method according to claim 2, is characterized in that: under ultrasound condition, 52 ~ 65 DEG C, carries out polyreaction.
4. preparation method according to claim 1, is characterized in that: step 2) in, the mass ratio of BHHCT and Properties of Polystyrene Nano Particles is 1:50.
5. preparation method according to claim 1, is characterized in that: in step 3), and the mass ratio of homogeneous fluorescence complex microsphere and carbodiimides is 1:1.
6. the detection method of rabies viruses, comprises the following steps:
1), after recombination staphylococcus aureus albumin A being diluted with the phosphate buffer of pH7.4,0.02mol/L, join in ELISA Plate hole, every hole 100 μ L, place 13 ~ 18 hours for 2 ~ 8 DEG C;
2) every hole adds 200 μ L confining liquids, places 13 ~ 18 hours for 2 ~ 8 DEG C;
3) discard liquid in plate, wash plate, dry;
4) join in ELISA Plate hole by standard items or measuring samples, every hole 100 μ L, hatches for 37 DEG C;
5) discard liquid in plate, wash plate, dry;
6), after rabies viruses homogeneous fluorescence complex microsphere dilution method described in Claims 1 to 5 any one prepared, join in ELISA Plate hole, every hole 100 μ L, hatches for 37 DEG C;
7) discard liquid in plate, wash plate, dry;
8) with the time-resolved fluorescence mode detection fluorescence signal of multi-functional microplate reader, excitation wavelength is 330nm, and wavelength of transmitted light is 610nm;
9) with the logarithm value of standard concentration for horizontal ordinate, the logarithm value recording Relative fluorescence units is ordinate, obtain typical curve, contrast measuring samples detected value, result of determination.
7. detection method according to claim 6, is characterized in that: in step 1), and the dilute concentration of recombination staphylococcus aureus albumin A is 2 μ g/mL.
8. detection method according to claim 6, is characterized in that: in step 6), and the extension rate of rabies viruses homogeneous fluorescence complex microsphere is 5000 times.
9. the detection method according to claim 6 ~ 8 any one, is characterized in that: step 4) hatches 60min, and step 6) is hatched.
10. detection method according to claim 6, is characterized in that: confining liquid contains the phosphate buffer of pH7.4,0.05mol/L, 4%BSA, the NaN of 0.01%
3.
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| CN105623651A (en) * | 2016-03-07 | 2016-06-01 | 江苏医诺万细胞诊疗有限公司 | Composite microsphere marker for conducting fluorescent marking on rear earth and preparation method of composite microsphere marker |
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| CN107290523A (en) * | 2017-05-23 | 2017-10-24 | 西北农林科技大学 | The method that antibody capture albumen and reporter gene fusion recombinant protein detect antibody |
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| CN105203747A (en) * | 2015-09-22 | 2015-12-30 | 宁波瑞源生物科技有限公司 | Method for closing labeled antibody fluorescent particles through arginine |
| CN105623651A (en) * | 2016-03-07 | 2016-06-01 | 江苏医诺万细胞诊疗有限公司 | Composite microsphere marker for conducting fluorescent marking on rear earth and preparation method of composite microsphere marker |
| CN107064488A (en) * | 2017-05-02 | 2017-08-18 | 中国农业科学院兰州兽医研究所 | A kind of total antibody solid phase of CSFV serum blocks the preparation method of antigen used in ELISA kit |
| CN107064502A (en) * | 2017-05-02 | 2017-08-18 | 中国农业科学院兰州兽医研究所 | A kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody and its detection method |
| CN107064502B (en) * | 2017-05-02 | 2019-07-12 | 中国农业科学院兰州兽医研究所 | A kind of multiple ELISA detection kit of pig virus infectious disease serum IgG antibody and its detection method |
| CN107290523A (en) * | 2017-05-23 | 2017-10-24 | 西北农林科技大学 | The method that antibody capture albumen and reporter gene fusion recombinant protein detect antibody |
| CN109632920A (en) * | 2018-11-20 | 2019-04-16 | 武汉市农业科学院 | A kind of preparation method of electrochemical signals marker material |
| CN109632920B (en) * | 2018-11-20 | 2021-05-14 | 武汉市农业科学院 | Preparation method of electrochemical signal marking material |
| CN114345250A (en) * | 2021-12-28 | 2022-04-15 | 深圳技术大学 | Preparation method of biosensor based on polystyrene microspheres |
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