CN107238710B - A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier - Google Patents

A method of based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier Download PDF

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CN107238710B
CN107238710B CN201710489337.7A CN201710489337A CN107238710B CN 107238710 B CN107238710 B CN 107238710B CN 201710489337 A CN201710489337 A CN 201710489337A CN 107238710 B CN107238710 B CN 107238710B
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张明翠
夏濛
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United Power Pharma Tech Co ltd
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Abstract

The present invention relates to a kind of method based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 targeted nano pharmaceutical carrier, in conjunction with antigen, specificity and the fluorescence of antibody response sensibility to SiO2- FO nanoparticle has carried out quantitative detecting analysis.Step includes: preparation SiO2- FO envelope antigen and immunogene will obtain antibody with high specificity, then fluorescence antibody are made in antibody on FITC label in immunogen injection to animal body, then establish direct competitive fluorescent immune method Accurate Determining SiO2The content of-FO.Detection method is easy to operate, high sensitivity, high specificity, high-throughput detection can be achieved.

Description

One kind being based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 The method of targeted nano pharmaceutical carrier
Technical field
The present invention relates to the quantitative detections of nano material, and in particular to one kind is examined based on direct competitive fluorescence immunoassay standard measure Survey folic acid functionalized SiO 2 (SiO2- FO) targeted nano pharmaceutical carrier method.
Background technique
The present invention relates to the quantitative detections of nano material, in particular to a kind of to be examined based on direct competitive fluorescence immunoassay standard measure Survey folic acid functionalized SiO 2 (SiO2- FO) targeted nano pharmaceutical carrier method.
Background technique
Cancer is to seriously endanger one of the disease of human health, and treatment of cancer is for improving the life quality of patient, mitigating Pain, the reduction death rate of patient has a very important significance.Chemotherapy is mainly used for the treatment of cancer in clinic at present Method, but current chemotherapeutics does not have targeting, normal cell can be also killed while killing cancer cell, so as to cause tight The toxic side effect of weight, hinders the development and application of chemotherapeutics, causes greatly to damage to patient's body and mind.Therefore, medicine is reduced Object toxic side effect, improvement drug distribution and metabolism, raising drug therapy efficiency etc. have become grinding for field of biomedicine Study carefully one of hot spot.
As the fast development and application, more and more researchers of nano material have turned one's attention to Nano medication load Body.Drug molecule can be wrapped up wherein or be adsorbed on its surface in 10~500nm, pass through targeting point by nano-carrier particle size Son is in conjunction with cell surface specific receptor or magnetic targeted is realized safely and effectively into the cell under cellular uptake effect Targeted drug conveying, therefore there is special value and significance in drug delivery.
Functionalized nano material is realizing targeting administration, slow releasing pharmaceutical, the in terms of performance for reducing drug Good application prospect out, it has also become the hot spot of newtype drug transportation system research in recent years.Silica nano material conduct One of the important member of inorganic nanoparticles material, since its preparation is easy, property is stable, good biocompatibility and surface are easily repaired Decorations, have played important function in field of biomedicine.One will be used as on targeting ligand modified with folic acid to silica nano material The novel targeted pharmaceutical carrier of kind is filled with new vitality for the treatment of cancer, provides to improve anti-tumor drug medicine-carried system New thinking and means has played important function in field of biomedicine.
Nanometer biotechnology is forward position and the hot issue of field of biotechnology, from the point of view of existing result of study, for receiving Rice material is typically limited to the distribution investigated it in vivo, degradation, drug release efficiency and toxicity applied to medical domain and grinds Study carefully etc..It is well known that nano material, because its special effect may have a certain impact to human body, different dosage may Different effect or side effect are generated to body.Therefore, whether the dose measurement of nano-medicament carrier may be used for investigating the material To be particularly important applied to clinic.And its structure, ingredient, grain are concentrated mainly on for the characterizing method of nano material at present The analysis such as degree, pattern and interface.It and is not also that very much, currently used method has inductance for the quantitative analysis of nano material Coupled plasma mass (ICP-MS), High performance liquid chromatography-inductively coupled plasma mass spectrometry method (HPLC-ICP-MS) with And gas-chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS), however these methods are not only cumbersome, instrument is high It is expensive, and stability and sensitivity is not high, and detection technique is still not mature enough.Therefore, for nano-carrier itself in organism Interior quantitative detection is still a very necessary and challenging job.
Summary of the invention
In order to solve the above technical problems, the present invention provides one kind based on direct competitive fluorescent immune method quantitative detection folic acid function Silica (SiO can be changed2- FO) targeted nano pharmaceutical carrier method.In conjunction with antigen, specificity and the fluorescence of antibody response Sensibility is to SiO2- FO nanoparticle has carried out quantitative detecting analysis.This method has easy to operate, high sensitivity, specificity By force, the features such as high-throughput detection can be achieved.
The technical scheme adopted by the invention is as follows:
One kind being based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano The method of pharmaceutical carrier, the described method comprises the following steps:
a、SiO2The preparation of-FO envelope antigen and immunogene；
B, anti-SiO2The preparation of-FO antibody；
C, the preparation of fluorescence antibody；
D, by SiO2- FO envelope antigen is coated in ELISA Plate after being coated liquid dilution, and various concentration is added in closing SiO2- FO standard items, the anti-SiO marked with FITC2- FO antibody is established direct competitive fluoroimmunoassay and is quantitatively examined as primary antibody Survey SiO2-FO；
E, with SiO2The logarithm of-FO standard concentration is abscissa, and fluorescence intensity level is that ordinate draws standard curve, from And quantitatively detect SiO2The concentration of-FO.
The linear equation of the standard curve is A=12434.4-1136.7logC, and wherein A is fluorescence intensity level, and C is SiO2- FO concentration.Its coefficient R=- 0.9992, the range of linearity 10-1-105Ng/mL, detection are limited to 0.035ng/mL.
The step a specifically includes the following steps:
A-1, by SiO2- FO is dissolved in PBS solution, obtains water-soluble SiO2- FO solution；
A-2, OVA is dissolved in PBS solution, the SiO that step a-1 is obtained then is added2- FO solution and glutaraldehyde solution, 2~5h of stirring is protected from light at 25~28 DEG C；It places reaction liquid into dialyse in bag filter and SiO can be obtained for 24 hours2- FO envelope antigen；
A-3, BSA is dissolved in PBS solution, the SiO that step a-1 is obtained then is added2- FO solution and glutaraldehyde solution, 2~5h of stirring is protected from light at 25~28 DEG C；It places reaction liquid into dialyse in bag filter and SiO can be obtained for 24 hours2- FO immunogene.
Further, the step a specifically includes the following steps:
A-1,10~20mg SiO is weighed2- FO is dissolved in 1~2mL PBS solution, and 1~2h of magnetic agitation obtains water-soluble The SiO of property2- FO solution；
A-2, it weighs 10~20mg OVA and is dissolved in 2~4mL PBS solution, and be added what step a-1 was obtained under stiring SiO2- FO solution is added dropwise 80~200 μ L, 25% glutaraldehyde solution again after mixing, is protected from light 2~5h of stirring at 25~28 DEG C； It places reaction liquid into bag filter, being collected afterwards for 24 hours with PBS dialysis can be obtained SiO2- FO envelope antigen, concentration be 2~ 4mg/mL；
A-3, it weighs 10~20mg BSA and is dissolved in 2~4mL PBS solution, and be added what step a-1 was obtained under stiring SiO2- FO solution is added dropwise 80~200 μ L, 25% glutaraldehyde solution again after mixing, is protected from light 2~5h of stirring at 25~28 DEG C； It places reaction liquid into bag filter, being collected afterwards for 24 hours with PBS dialysis can be obtained SiO2- FO immunogene, concentration are 2~4mg/ mL。
In the step a-2 and a-3, the molecular cut off of bag filter is 8000~14000Da.
The water-soluble SiO2- FO solution concentration is 5~15mg/mL.
The step b specifically includes the following steps:
B-1, first immunisation: by SiO2- FO immunogene and Freund's complete adjuvant in equal volume than mixing after, using dorsal sc The mode of multi-point injection is injected into White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；First immunisation is after three weeks Carry out booster immunization；
B-2, booster immunization: by SiO2- FO immunogene and incomplete Freund's adjuvant in equal volume than mixing after, take same Mode is injected into White Rabbit body, injects 8~10 points, and injection volume is 1~2mL/；Hereafter reinforced exempting from again every two weeks Serum titer is surveyed in epidemic disease, the blood sampling of intermediate Zhou Jinhang ear vein, until potency reaches 1:64000, then carries out last time booster immunization, And take a blood sample after a week from animal arteria carotis immune, it stands and antiserum is precipitated and is purified, obtain anti-SiO2- FO antibody, Concentration is 15~20mg/mL.
The step c specifically includes the following steps:
C-1, anti-SiO after purification is taken2- FO antibody, with carbonate buffer solution be diluted to every milliliter of inner protein 8~ Antibody-solutions are made in 15mg；
C-2, fluorescein isothiocynate (FITC) is dissolved in carbonate buffer solution, the FITC that concentration is 2mg/mL is made Solution；
C-3, FITC solution is slowly added in the antibody-solutions that step c-1 is obtained at 25~28 DEG C, magnetic agitation 4~ 6h；
C-4, the solution that step c-3 is obtained is filtered purifying with sephadex, obtains the anti-SiO of FITC label2- FO antibody.
In the step c-3, the ratio between volume of FITC solution and antibody-solutions is 1:10.
The step d specifically includes the following steps:
D-1, coating: use coating buffer by SiO2- FO envelope antigen dilutes 80 times, is coated with 96 hole elisa Plates, every hole 100 μ L, 4 DEG C of refrigerator overnights；
D-2, closing: drying, PBST solution wash 3 times, and 3~5min, washes away the SiO not being combined every time2- FO coating 1wt% casein is added in antigen, and every 200 μ L of hole is closed, and 37 DEG C of baking ovens incubate 1~2h；
D-3, sample-adding competition: drying, PBST solution wash 3 times, and 3~5min, washes away extra confining liquid every time, then will The SiO of 50 μ L various concentrations2The anti-SiO of-FO standard items and 50 μ L FITC label2- FO antibody divides gradient to be added in each hole, is allowed to Competitive reaction occurs, 37 DEG C of baking ovens incubate 2~4h；
D-4, detection: drying, PBST solution wash 3 times, and 3~5min, removes the SiO of free state every time2- FO standard items or Antibody conjugates, measuring each hole in excitation wavelength with multi-function microplate reader is 485nm, and launch wavelength is that the fluorescence at 528nm is strong Angle value.
The present invention provides one kind to be based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano pharmaceutical carrier method, realized in conjunction with antigen, the specificity of antibody response and the sensibility of fluorescence The immunization method is to SiO2The quantitative detection of-FO.
Compared with prior art, the present invention has the characteristics that following:
(1) the immune response effect for utilizing body, successfully prepares and has filtered out the anti-SiO of high-titer2- FO antibody is Establish the Immunoassay quantification detection SiO of high specific2- FO lays a good foundation；
(2) it is established using the sensibility of antigen, the specificity of antibody response and fluorescence a kind of based on direct competitive fluorescence Immunoassay quantitative detection SiO2The new method of-FO provides a kind of quick, accurate quantitative analysis detection for nano material from now on Method；
(3) this method is easy to operate, high sensitivity, high specificity, high-throughput detection can be achieved.
Detailed description of the invention
Fig. 1 is with SiO2The logarithm of-FO standard concentration is abscissa, and fluorescence intensity level is that the standard that ordinate is established is bent Line chart.
Specific embodiment
Freund's complete adjuvant, bovine serum albumin(BSA) (BSA) and chicken ovalbumin (OVA) buy spontaneous work bioengineering (Shanghai) limited liability company.
Other reagents can sale producer from the market be commercially available.
The present invention relates to each solution the preparation method comprises the following steps:
PBS solution (0.01mol/L pH=8.0): NaCl 8.0g, KCl 0.1g, NaH are weighed2PO4·2H200.106g、 Na2HPO4·12H2O 3.34g is dissolved in distilled water and is settled to 1000mL.
Carbonate buffer solution CB (0.5mol/L pH=9.6): Na is weighed2CO3 1.59g、NaHCO32.94g being dissolved in steaming In distilled water and it is settled to 100mL.
PBST solution (0.01mol/L pH=8.0): being added 500 μ L Tween-20 in 1000mL PBS, and mixing is equal It is even.
Coating buffer CB (0.05mol/L pH=9.6): Na is weighed2CO3 1.59g、NaHCO32.94g being dissolved in steaming In distilled water and it is settled to 1000mL.
1wt% casein solution: it is confining liquid, weighs 0.01g casein and is dissolved in 1mL PBS, is uniformly mixed.
MES buffer (0.1M, pH=5.5): 0.1921g MES is claimed to be dissolved in 10mL distilled water, with NaOH solution tune Save its pH to 5.5.
Embodiment 1
One kind being based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano The method of pharmaceutical carrier, the described method comprises the following steps:
a、SiO2The preparation of-FO envelope antigen and immunogene
A-1,10mg SiO is weighed2- FO is dissolved in 1mL PBS solution, and magnetic agitation 1h is to obtain concentration as 10mg/mL's SiO2- FO solution；
A-2, it weighs 10mg OVA and is dissolved in 2mL PBS, and the SiO that step a-1 is obtained is added in stirring2- FO solution mixes 90 μ L, 25% glutaraldehyde solution is added dropwise again after closing uniformly, stirring 4h is protected from light at 25 DEG C；Placing reaction liquid into molecular cut off is In the bag filter of 8000-12000Da, being collected afterwards for 24 hours with PBS dialysis can be obtained SiO2- FO envelope antigen, concentration are 3.33mg/mL, 4 DEG C of storages are stand-by；
A-3, it weighs 10mg BSA and is dissolved in 2mL PBS, and the SiO that step a-1 is obtained is added in stirring2- FO solution mixes 90 μ L, 25% glutaraldehyde solution is added dropwise again after closing uniformly, stirring 4h is protected from light at 25 DEG C；Placing reaction liquid into molecular cut off is In 8000-12000Da bag filter, being collected afterwards for 24 hours with PBS dialysis can be obtained SiO2- FO immunogene, concentration 3.33mg/ ML, 4 DEG C of storages are stand-by.
SiO in the step a-12- FO's (folic acid functionalized SiO 2) the preparation method comprises the following steps:
A-1-1, amino functional silica (SiO2–NH2) preparation
0.5h is mixed in 380 μ L ethyl orthosilicates and 12mL dehydrated alcohol；570 μ L, 26% ammonium hydroxide is added, at 25 DEG C Stirring is for 24 hours；400 μ L 3- aminopropyl triethoxysilanes are added, are persistently stirred for 24 hours at 25 DEG C；By reaction solution 10000rpm/ Min is centrifugated 20min, and precipitating is taken to be washed with dehydrated alcohol, and amino functional dioxy can be obtained in drying for 24 hours at 25 DEG C SiClx；
A-1-2, folic acid functionalized SiO 2 (SiO2- FO) preparation
It weighs 57.5mg folic acid and is dissolved in 2mL MES buffer, add 40.3mg 1- ethyl-(3- dimethylamino third Base) phosphinylidyne diimmonium salt hydrochlorate (EDC) and 17.25mg n-hydroxysuccinimide, stir 1h at room temperature；
Weigh 100mg SiO2–NH2It is added in above-mentioned mixed liquor after being dissolved in 5mL MES buffer, stirring is protected from light at 25 DEG C 24h；Reaction solution 10000rpm/min is centrifugated 10min, takes precipitating to be washed with distilled water and dehydrated alcohol, and at 25 DEG C Folic acid functionalized SiO 2 can be obtained in drying for 24 hours.
B, anti-SiO2The preparation of-FO antibody
Selecting the Male New Zealand White Rabbit that 4 weight are 2~2.5kg is immunization, first will purchase before experiment New zealand white rabbit raise 2 weeks or so, maintain its health status, wherein 3 are only used as immunization, the 4th is only used as blank pair According to blank control group is without any immune.
B-1, first immunisation: by SiO2- FO immunogene and Freund's complete adjuvant in equal volume than mixing after, using dorsal sc The mode of multi-point injection is injected into 3 experimental rabbit bodies, injects 8~10 points, and injection volume is 1mL/；The mode of immunity inoculation There are injecting immune, oral immunity, aerosol immunization etc., wherein injecting immune has subcutaneous injection, intracutaneous injection, intramuscular injection vein again The modes such as injection, dorsal sc injection is easy to operate, and drug diffusion is slower, is conducive to that body is stimulated to generate immune response, then Generate antibody；And male rabbit can have an impact experiment to avoid its physiological period as immunization.
B-2, booster immunization: first immunisation carries out booster immunization after three weeks.By SiO2- FO immunogene is not exclusively helped with Freund Agent in equal volume than mixing after, take same mode to be injected into 3 experimental rabbit bodies, inject 8~10 points, injection volume 1mL/ Only；Hereafter booster immunization again is carried out every two weeks, and serum titer is surveyed in intermediate Zhou Jinhang ear vein blood sampling, until potency reaches 1: 64000, then last time booster immunization is carried out, and take a blood sample after a week from animal arteria carotis immune, it stands and antiserum is precipitated simultaneously It is purified, obtains anti-SiO2- FO antibody, concentration 16.55mg/mL.
Incomplete Freund's adjuvant the preparation method comprises the following steps: weigh the wool grease of 50g, the atoleine for measuring 100mL is mixed It closes, Ultrasound Instrument repeated ultrasonic, is no more than 20min every time, prevents ultrasonic procedure medium temperature from spending height, cannot radiate in time, ultrasound makes To be uniformly mixed, until dripping to the mixing liquid in water and until indiffusion in half a minute, obtained oily liquids is Incomplete Freund's adjuvant, 4 DEG C of refrigerator storages are stand-by.
Antibody titer identification method particularly includes: envelope antigen is diluted 400 times with coating buffer CB, is coated on 96 holes On ELISA Plate, 4 DEG C of refrigerator overnights；Drying, is washed 3 times, each 3min with PBST solution, and every hole adds 200 μ L 1wt% caseins to seal Close liquid, 37 DEG C of closing 1h；Drying, PBST are washed 3 times, each 3min, and every hole is added 100 μ L and is diluted to the anti-of various concentration with PBS Serum, thinner ratio are 1/1000~1/128000,37 DEG C of incubation 2h；Drying, PBST are washed 3 times, each 3min, and every hole is added The goat anti-rabbit igg that 100 μ L are marked with the HRP that the diluted thinner ratio of PBS is 1/5000,37 DEG C of incubation 2h；Drying, PBST are washed 3 times, Each 3min, every hole are added 100 μ L o-phenylene diamine substrate liquid and carry out chromogenic reaction, 37 DEG C of incubation 0.5h；Every hole adds 50 μ L2mol/L H2SO4Terminate reaction；Finally absorbance value A of each hole at 490nm is measured with multi-function microplate reader.Comparative experiments The light absorption value A of group and blank control group under same antiserum extension rate, works as AExperimental group>=2 times of ABlank groupWhen corresponding maximum dilution Multiple, that is, sero-fast potency.
C, the preparation of fluorescence antibody
C-1, anti-SiO after purification is taken2- FO antibody 1mL is diluted to every milliliter of inner protein with carbonate buffer solution Antibody-solutions are made in 8.5mg；
C-2, it weighs 2mg fluorescein isothiocynate (FITC) and is dissolved in 1mL carbonate buffer solution, FITC solution is made；
C-3,0.1mLFITC solution is slowly added in 1mL antibody-solutions at 25 DEG C, magnetic agitation 5h；
C-4, the good solution of above-mentioned label is filtered purifying with sephadex, collects column lower end solution, obtains The anti-SiO of FITC label2- FO antibody, -20 DEG C of storages are stand-by.
D, the anti-SiO of the fluorescence marked is utilized2- FO antibody and SiO2- FO envelope antigen carries out direct competitive fluorescence immunoassay point Analysis experiment establishes standard curve in optimal conditions to reach quantitative detection SiO2The purpose of-FO
D-1, coating: use coating buffer by SiO2- FO envelope antigen dilutes 80 times, is coated with 96 hole elisa Plates.Every hole 100 μ L, 4 DEG C of refrigerator overnights；
D-2, closing: drying, PBST solution wash 3 times, and each 3min washes away the SiO not being combined2- FO coating is anti- It is former.1wt% casein is added, every 200 μ L of hole is closed and (reduced non-specific adsorption), and 37 DEG C of baking ovens incubate 1h；
D-3, sample-adding competition: drying, PBST solution wash 3 times, and each 3min washes away extra confining liquid.50 μ L are dense Degree is respectively 0.1ng/mL, 1ng/mL, 10ng/mL, 102ng/mL、103ng/mL、104ng/mL、105The SiO of ng/mL2- FO mark Quasi- product are added sequentially in each row of ELISA Plate, i.e., in triplicate, 50 μ L FITC are then added in each concentration gradient into each hole The anti-SiO of label2- FO antibody is allowed to that competitive reaction occurs, and 37 DEG C of baking ovens incubate 3h；
The SiO of various concentration2- FO standard items the preparation method comprises the following steps: with the PBS buffer solution of 0.01mol/L pH=8.0 By SiO2- FO is diluted to specified concentration.
D-4, detection: drying, PBST solution wash 3 times, and each 3min removes the SiO of free state2- FO standard items are anti- Body conjugate.Measuring each hole in excitation wavelength with multi-function microplate reader is 485nm, and launch wavelength is the fluorescence intensity at 528nm Value, same concentration gradient are averaged calculating fluorescence intensity level.
E, with SiO2The logarithm of-FO standard concentration is abscissa, and fluorescence intensity level is that ordinate draws standard curve.System The standard curve obtained is as shown in Figure 1, the linear equation of standard curve is A=12434.4-1136.7logC, and wherein A is that fluorescence is strong Angle value, C SiO2- FO concentration.Its coefficient R=- 0.9992, the range of linearity 10-1-105Ng/mL, detection are limited to 0.035ng/mL。
F, above each step is repeated, only by the SiO of the various concentration in step (3)2- FO standard items replace with unknown dense The SiO of degree2- FO prepare liquid, then measuring each hole in excitation wavelength with multi-function microplate reader is 485nm, launch wavelength 528nm The fluorescence intensity level at place seeks average fluorescent strength value.According to the linear equation A=12434.4- of above-mentioned standard curve 1136.7logC SiO can be calculated2The concentration of-FO prepare liquid.
Above method be many experiments verifying after optimal experimental method, the obtained standard curve of the method it is linear Relationship is best, and the range of linearity is most wide.
It is above-mentioned that direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 is based on to one kind referring to embodiment The detailed description that the method for targeted nano pharmaceutical carrier carries out, is illustrative without being restrictive, can be according to limited model It encloses and enumerates several embodiments, therefore the change and modification in the case where not departing from present general inventive concept, guarantor of the invention should be belonged to Within the scope of shield.

Claims (7)

1. one kind is based on direct competitive fluorescent immune method quantitative detection folic acid functionalized SiO 2 (SiO2- FO) targeted nano medicine The method of object carrier, which is characterized in that the described method comprises the following steps:
a、SiO2The preparation of-FO envelope antigen and immunogene；
B, anti-SiO2The preparation of-FO antibody；The anti-SiO2The potency of-FO antibody is 1:64000；
C, the preparation of fluorescence antibody；
D, by SiO2- FO envelope antigen is coated in ELISA Plate after being coated liquid dilution, closing, the SiO that various concentration is added2-FO Standard items, the anti-SiO marked with FITC2- FO antibody establishes direct competitive fluoroimmunoassay quantitative detection SiO as primary antibody2- FO；
E, with SiO2The logarithm of-FO standard concentration is abscissa, and fluorescence intensity level is that ordinate draws standard curve SiO2-FO The logarithm of standard concentration is abscissa, and fluorescence intensity level is that ordinate draws standard curve；
The linear equation of the standard curve is A=12434.4-1136.7logC, and wherein A is fluorescence intensity level, C SiO2-FO Concentration；
The step a specifically includes the following steps:
A-1,10 ~ 20mg SiO is weighed2- FO is dissolved in 1 ~ 2mL PBS solution, and 1 ~ 2h of magnetic agitation obtains water-soluble SiO2- FO solution；
A-2, it weighs 10 ~ 20mg OVA and is dissolved in 2 ~ 4mL PBS solution, and the SiO that step a-1 is obtained is added under stiring2-FO Solution is added dropwise 80 ~ 200 μ L, 25% glutaraldehyde solution again after mixing, is protected from light 2 ~ 5h of stirring at 25 ~ 28 DEG C；Reaction solution is set In bag filter, being collected afterwards for 24 hours with PBS dialysis can be obtained SiO2- FO envelope antigen, concentration are 2 ~ 4mg/mL；
A-3, it weighs 10 ~ 20mg BSA and is dissolved in 2 ~ 4mL PBS solution, and the SiO that step a-1 is obtained is added under stiring2-FO Solution is added dropwise 80 ~ 200 μ L, 25% glutaraldehyde solution again after mixing, is protected from light 2 ~ 5h of stirring at 25 ~ 28 DEG C；Reaction solution is set In bag filter, being collected afterwards for 24 hours with PBS dialysis can be obtained SiO2- FO immunogene, concentration are 2 ~ 4mg/mL.
2. the method according to claim 1, wherein the molecular cut off of bag filter is 8000 ~ 14000Da.
3. the method according to claim 1, wherein water-soluble SiO2- FO solution concentration is 5 ~ 15mg/mL.
4. the method according to claim 1, wherein the step b specifically includes the following steps:
B-1, first immunisation: by SiO2- FO immunogene and Freund's complete adjuvant in equal volume than mixing after, using dorsal sc multiple spot The mode of injection is injected into White Rabbit body, injects 8 ~ 10 points, and injection volume is 1 ~ 2mL/；First immunisation is added after three weeks It is strong immune；
B-2, booster immunization: by SiO2- FO immunogene and incomplete Freund's adjuvant in equal volume than mixing after, take same mode It is injected into White Rabbit body, injects 8 ~ 10 points, injection volume is 1 ~ 2mL/；Hereafter booster immunization again is carried out every two weeks, in Between the blood sampling of Zhou Jinhang ear vein survey serum titer, until potency reaches 1:64000, then carry out last time booster immunization, and It is immune to take a blood sample after a week from animal arteria carotis, it stands and antiserum is precipitated and is purified, obtain anti-SiO2- FO antibody, concentration For 15 ~ 20mg/mL.
5. the method according to claim 1, wherein the step c specifically includes the following steps:
C-1, anti-SiO after purification is taken2- FO antibody is diluted to every milliliter of 8 ~ 15mg of inner protein with carbonate buffer solution, is made Antibody-solutions；
C-2, fluorescein isothiocynate (FITC) is dissolved in carbonate buffer solution, it is molten that the FITC that concentration is 2mg/mL is made Liquid；
C-3, FITC solution is slowly added in the antibody-solutions that step c-1 is obtained at 25 ~ 28 DEG C, 4 ~ 6h of magnetic agitation；
C-4, the solution that step c-3 is obtained is filtered purifying with sephadex, obtains the anti-SiO of FITC label2-FO Antibody.
6. according to the method described in claim 5, it is characterized in that, in the step c-3, the body of FITC solution and antibody-solutions The ratio between product is 1:10.
7. the method according to claim 1, wherein the step d specifically includes the following steps:
D-1, coating: use coating buffer by SiO2- FO envelope antigen dilutes 80 times, is coated with 96 hole elisa Plates, 100 μ L of every hole, and 4 DEG C refrigerator overnight；
D-2, closing: drying, PBST solution wash 3 times, and 3 ~ 5min, washes away the SiO not being combined every time2- FO envelope antigen, 1wt% casein is added, every 200 μ L of hole is closed, and 37 DEG C of baking ovens incubate 1 ~ 2h；
D-3, sample-adding competition: drying, PBST solution wash 3 times, and 3 ~ 5min, washes away extra confining liquid every time, then by 50 μ L The SiO of various concentration2The anti-SiO of-FO standard items and 50 μ L FITC label2- FO antibody divides gradient to be added in each hole, is allowed to occur Competitive reaction, 37 DEG C of baking ovens incubate 2 ~ 4h；
D-4, detection: drying, PBST solution wash 3 times, and 3 ~ 5min, removes the SiO of free state every time2- FO standard items or antibody knot Object is closed, measuring each hole in excitation wavelength with multi-function microplate reader is 485nm, and launch wavelength is the fluorescence intensity level at 528nm.
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