CN105214097A - A kind of immunity enforcement of oral administration nanometer vaccine delivery body and detection technique - Google Patents
A kind of immunity enforcement of oral administration nanometer vaccine delivery body and detection technique Download PDFInfo
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Abstract
The present invention relates to PMMMA putamina layer that a kind of pH responds for the enforcement of oral vaccine delivery immunity and detection technique.Based on the micro-nano vaccine systems such as PLGA/ antigen, by surface free radical polymerization method, obtain poly-(methyl methacrylate-acrylic acid methyl ester .-methacrylic acid) (PMMMA) putamina layer that is stable, resistance to enzymolysis in acid condition and strengthen oral vaccine.Implemented the mode of immunity by this vaccine oral, experimenter (as tilapia) can obtain the good immunocompetence of high-level lasting effect in a short time.Invention introduces a set of through optimizing technical system for detecting immune effect, the non-reduced PAGE comprising fluorescently-labeled antigen-antibody complex detects, in Escherichia coli cell-the antibody coagulation detection of periplasmic space antigen presentation system, Small Volume Serum bacteriostatic experiment, immunoprotection and FITC labelled antigen bioluminescence imaging technology.
Description
Technical field
The present invention relates to and implement and detection technique field for oral vaccine immunity, specifically relate to application pH sensitivity, the macromolecule of hydrolysis strengthens vaccine protection in oral immunity process, assist antigen targeting to transport and the method for quick of absorption, immune response level and purposes.
Background technology
Oral vaccine is more convenient relative to injecting immune on the implementation, little to nervous system injury.Polypeptide as antigen safely, have no side effect, designability is strong.Although intestinal tract is distributed with abundant immunocyte and provides the foundation with the application being organized as oral vaccine, but because small intestinal is rich in multiple hydrolytic enzyme, become the crucial barrier that current oral vaccine is successfully developed, thus the report of current rare oral vaccine successful Application.
Immune oral vaccine is entered in order to obtain effectively to be absorbed by intestinal mucosa; invention a kind of with the complete coating antigen of PMMMA-PLGA, effectively " detouring " small intestinal, targeting pH value the higher and immune implementation method of the vaccine protective carrier of the large intestine that hydrolytic enzyme is few, this enforcement technology can at large intestine Co ntrolled release PLGA/ antigen nano-particle, promote that antigen is by Cell uptake.
Relative to traditional adjuvant; the outer strata (methyl methacrylate-acrylic acid methyl ester .-methacrylic acid) (PMMMA) of this carrier has pH responding ability; in dense hydrophobic state under acidity; completely isolated hydrolytic enzyme; protection vaccine; undergo phase transition under alkalescence, change hydrophily into and PLGA/ antigen is released.This carrier adopts surface free radical polymerization to react first on PLGA technique for packing, has filled up the defect that popular response can not wrap up inner core particles completely.
Immunity embodiment adopts mixed feedstuff; drain; the technology that chitosan (for stabilization of vaccines when neutral and alkalescence) soaks; animal subject tilapia is thrown something and fed; excite the specific antibody (adopting antigenic mark technology and non-reduced PAGE to verify) forming higher level in tilapia body in a short time; effective elimination pathogen (GBS bacterium), protection tilapia natural law reaches more than 210 days.In conjunction with living imaging analysis, the antigen (FITC) being subject to PMMMA-PLGA protection is mainly distributed in Ia large intestine, kidney and spleen, and the less liver abundant at digestive enzyme and small intestinal detect.
In testing process, have employed FITC labelled antigen first and apply non-reduced PAGE detectable antigens-antibody complex, fast and effeciently detect immune level.Based on it, detection technique also comprise based on Escherichia coli cell outer-pericentral siphon, the quick antibody coagulation detection of born of the same parents' endoantigen expression system, the bioluminescence imaging technology of micro-antiserum bacteriostatic experiment, immunoprotection and FITC labelled antigen.
The proposition of this patent implements-detection technique scheme based on the simple and easy preparation of above-mentioned new oral vaccine delivery body and corresponding complete immunity, improves the success rate of oral immunity, can be used for accurately identifying the immune level obtained by this vaccine oral rapidly.
Summary of the invention
The object of this invention is to provide a kind of responsive with pH, acid preparation method to the putamina layer of resistant to hydrolysis under weak base condition, this putamina layer can wrap up kernel, and then carries out oral immunity enforcement using it as vaccine delivery body and set up the Fast Detection Technique system of induction of immunity correlated performance.
The present invention is achieved through the following technical solutions:
The PMMMA putamina layer responded with a kind of pH strengthens oral vaccine, and corresponding immunity is implemented and immune level detection technique.Comprise: based on the granular system by PLGA/ antigen etc. with hydrophobic interfaces; by surface free radical polymerization method; acquisition is stablized in acid condition, poly-(methyl methacrylate-acrylic acid methyl ester .-methacrylic acid) (PMMMA) putamina layer of resistance to enzymolysis; strengthen the protection to medicines such as vaccines in order to packaging medicine kernel, and be intended to strengthen oral vaccine.This vaccine mode of throwing something and feeding mixes with feedstuff, and first-selection provides protection by chitosan in meta-alkali environment.Specific period adopts the optimum organization of detection technique to identify antibody horizontal; preferably, the non-reduced PAGE that detection technique comprises fluorescent labeled antigen-antibody complex detects, the antibody coagulation detection of bacillus coli antigen expression system, Small Volume Serum bacteriostatic experiment, immunoprotection and FITC labelled antigen bioluminescence imaging technology.
According to the present invention, described hypostracum has hydrophobic interfaces, and in an embodiment, poly lactic coglycolic acid (PLGA) is the slow release layer for antigen well known in the prior art.
According to the present invention, poly-(methyl methacrylate-acrylic acid methyl ester .-methacrylic acid) (PMMMA) putamina layer of described pH sensitivity, directly realize closely parcel in PLGA periphery by surface free radical polymerization reaction, this putamina layer is fine and close hydrophobic layer under acidic or neutral conditions, resistant function is had to enzymatic hydrolysis, and the degraded can effectively escaped in stomach, small intestinal and small intestinal intestinal absorption, in the large intestine that alkalescence improves, PMMMA presents hydrophily by phase in version, and discharging can by the PLGA/ antigen vesicle nano-particle of intestinal absorption.This release characteristics realizes vaccine in the release of large intestine environment and absorption.Preferably, described poly-(methyl methacrylate-acrylic acid methyl ester .-methacrylic acid) (PMMMA) putamina layer is by three kinds of precursors, i.e. the copolymer of methyl methacrylate, acrylic acid methyl ester. and methacrylic acid formation.The detection of immune level, based on the non-reduced PAGE detection method of antigen (FITC)-antibody complex, integrates the living imaging detection technique of the bacterial agglutination detection of bacillus coli antigen expression system, the antibacterial detection of micro-antiserum, immunoprotection and FITC labelled antigen.
In a preferred embodiment, described interior nucleoprotein is protein/polypeptide, is preferably the activated protein/polypeptide of tool.Preferred, described antigen protein is immunogenicity and antigenic albumen, such as, be the surface immunogen albumen of B race streptococcus (GBS) and the fusion rotein Trx-SIP of epoxythio albumen (Trx).
In a preferred embodiment, the responsive PMMMA control delivery of described pH is obtained by following preparation method: in the liquid phase containing PLGA/ antigen vaccine system, successively add methyl methacrylate, acrylic acid methyl ester., methacrylic acid in order, then add initiator and catalyst.More preferably, reaction temperature is controlled at 40-60 DEG C, pH regulator is to 6.0-7.0, under ultrasonication, the methyl methacrylate that hydrophobicity is stronger is first adsorbed to PLGA surface by hydrophobic interaction, and then add have hydrophobic acrylic acid methyl ester. absorption PLGA, finally in homodisperse emulsion system, add hydrophilic precursors methacrylic acid, and Ammonium persulfate. (APS) and N, N, N', N'-tetramethylethylenediamine (TEMED), at the Raolical polymerizable of PLGA surface catalysis three precursors, make the firm coated PLGA/ antigen of linear PMMMA macromolecule be obtained by reacting.
In a preferred embodiment, methyl methacrylate: acrylic acid methyl ester.: methacrylic acid volume ratio is 6-8:2-4:1-2, more preferably, volume ratio is 6.5:2.5:1-2; Also more preferably, volume ratio is 6.5:2.5:1 or 6.5:2.5:2.
Present invention also offers a kind of with PMMMA reinforcement kernel vaccine system, the implementation method realizing oral immunity and detection technique, in one embodiment, comprise the steps:
1) the pH sensitive membrane shell PMMMA of described PLGA/ antigen (Trx-SIP) the kernel vaccine system of preparation parcel, strengthens PLGA/ antigen vaccine system.
2) enhancement mode vaccine PMMMA-PLGA/Trx-SIP and food (containing feedstuff) mix, and soak, obtain more stable Edible vaccine system with chitosan solution.
3) above-mentioned Edible vaccine system peroral immunity in animal is adopted.
4) the immunoreactive non-reduced PAGE of anti serum and antigen detects, and solidifying, the micro-bacteriostasis of antiserum bacterium detects, and immunoprotection is tested, and bioluminescence imaging technology labelled antigen (FITC labelling) distribution in main organs.
According to the present invention, described step 1) in, described antigen is protein/polypeptide, by various method well known in the prior art, as PLGA is wrapped up polypeptide by the interface self-assembly method in emulsion, and preparation one-level vaccine system.The preparation method of pH sensitive layer PMMMA is preferably: in the liquid phase containing PLGA/ antigen, carry out Raolical polymerizable with methyl methacrylate (MMA), acrylic acid methyl ester. (MA) and methacrylic acid (MAA), prepare on PLGA surface the pH wrapping up PLGA/ antigen particles completely and respond to system.Preferably, Raolical polymerizable is caused with Ammonium persulfate. (APS).Preferred, N, N, N', N'-tetramethylethylenediamine (TEMED) is catalyst.
According to the present invention, described step 2) in, this food feedstuff mixes with PMMMA-PLGA/ antigen system and food/feedstuff to make.Be preferably: mix under neutrality or sour environment with PMMMA-PLGA/ antigen system and food, after vacuum is drained, after being soaked in chitosan solution (pH<7.0), after being soaked in rapidly the weak base buffer several seconds, appropriate drying is made, and vaccine is stablized in food.More preferably: the Edible vaccine obtained, through vacuum drying or lyophilization, fully removes moisture, protection antigen is not hydrolyzed rapidly in storage, thus extends the antigen active time.
According to the present invention, described step 3) in, animal is the vertebrates with gastrointestinal system.Be preferably: under the water temperature of 30 degrees Celsius, raise tilapia, adopt Trx-SIP as antigen, by step 1) and 2) prepare the feed vaccine of PMMMA-PLGA/Trx-SIP, vaccine divides bony fish tilapia of throwing something and feeding for three times, every tail fish is thrown something and fed with the intake of 20 μ gTrx-SIP/ gram body weight at every turn, every throw something and feed for twice between interval 1 week.
According to the present invention, described step 4) in, the present invention is directed to using of PMMMA-PLGA/ antigen oral vaccine and provide a kind of new system detecting method.Under albumen/peptide is in non-reducing state, detect antibody by non-reduced PAGE to be formed the specific recognition of antigen (isothiocyanate FITC labelling) and Antibody-antigen complex, relative to traditional nonreducing gel electrophoretic detection, the technology of labelling is directly perceived, highly sensitive, antibody is not subject to obviously affecting of FITC labelling to the specific binding of antigen.During bacterial agglutination detects, adopt serial dilution antibody respectively with in Escherichia coli cell, pericentral siphon-born of the same parents' external space antigen presentation system, and the bacteria suspension of escherichia coli extracellular expression reference protein system carries out mixed checking with coagulation, determines that antiserum is to the specific recognition of antigen presentation bacterium and combination.Thereafter adopt micro-bacteriostatic experiment, namely volume is that the antiserum of micro updating and pathogen suspension mix, and fully cultivates, spread plate on flat board, detects colony-forming units (CFU).Preferred: bacteria suspension adopts normal saline, and namely 0.9%NaCl suspends to antibacterial.Preferred: the mixed time is set as 48 hours, comprise 37 DEG C and mix 1 hour, 4 DEG C mix 47 hours, and the setting of 4 DEG C is to fully keep the activity of antibody and the performance of conjugated antigen under ex vivo environment; The dull and stereotyped broad spectrum of bacteria culture plate being preferably antibiotic-free, such as LB is dull and stereotyped; Dull and stereotyped training systern be 37 DEG C 3 days, 30 DEG C 4 days, to reach the object of fully cultivating cause of disease bacterium colony.Immunoprotection Setup Experiments is that throw something and feed group, PMMMA-PLGA/ antigen of antigen is thrown something and fed group, and does not take the blank group of immunity.Preferably, adopt Intraperitoneal injection method inoculation pathogen, such as, GBS bacteria suspension is carried out lumbar injection, inoculate the tilapia through three oral immunities, in 210 days, monitor the health status of fish continuously, statistics infection proportion.Bioluminescence imaging technology adopts method detectable antigens (FITC labelling) distribution in tilapia main organs of fluorescent tracing.Preferably, adopt isothiocyanate labelled antigen Trx-SIP, by step 1) and 2) prepare Edible vaccine, the tilapia of health is thrown something and fed, takes food after 36 hours and carry out living imaging analysis (accompanying drawing 1).This time vaccines through complete digestive system, and is in the cyclic metabolism stage, is beneficial to the accuracy ensureing result.
Accompanying drawing illustrates:
Fig. 1: bioluminescence imaging technology analyzes PMMMA-PLGA/Trx-SIP Targeting Performance.Vaccine of throwing something and feeding detects Trx-SIP (FITC labelling) distribution in vivo after 36 hours.Parameter: exciting light: 500nm; Utilizing emitted light: 530nm.
Detailed description of the invention
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples will contribute to the present invention is described, but not limit the present invention in any form.
Embodiment 1. prepares the pH sensitive membrane shell PMMMA of described PLGA/ antigen (Trx-SIP) the vaccine system of parcel.
Reference literature general " two emulsification mechanism " technology, preparation PLGA/Trx-SIP granule, analyze pattern with Flied emission transmission electron microscope (model: FEITecnaiG2F20S-TWIN), prepared granule has obvious core-shell structure copolymer cyst membrane structure.
According to the ratio 6.5:2.5:2 optimized, methyl methacrylate (MMA), acrylic acid methyl ester. (MA), methacrylic acid (MAA), Ammonium persulfate. (APS), N is added in deionized water successively under ultrasound condition, N, N', N'-tetramethylethylenediamine (TEMED), ultrasonic reaction 5 hours at 45 DEG C.With centrifugal the obtained core-shell structure copolymer nanosphere suspension of the rotating speed of >14000xg 30 minutes, be resuspended in ionized water, more than 3 times repeatedly, fully remove unreacted precursor.Be suspended from deionized water or corresponding buffer (pH<7.0), analyze pattern with Flied emission transmission electron microscope (model: FEITecnaiG2F20S-TWIN), prepared granule has obvious core-membrane shell structure.Adopt international mtt assay to analyze material toxicity, experimental result shows, each system obtained in experiment all has good bio-compatibility, and cell survival rate is at normal range (70%-120%).Illustrate that the PMMMA-PLGA/Trx-SIP obtained after reacting completely has good bio-compatible ability.
Prepared by embodiment 2. enhancement mode Edible vaccine
Food (adopting tilapia standard feed in this embodiment) and thickening agent, water are mixed with certain proportion, near pH regulator to 6.0, PMMMA-PLGA/Trx-SIP and feedstuff are mixed, make the coccoid granule that diameter is 3 millimeters, vacuum is drained, and soaks the several seconds with chitosan solution (pH<7.0), makes the surface of chitosan only wet particle, then put rapidly to pH7.0-7.2 aqueous environment, make chitosan film forming.The Edible vaccine particle surface stable through chitosan is smooth, can not scatter in the tilapia feeding environment of near neutral, can keep the concentration of nano-particle in feedstuff, and is easy to be ingested by fish.But under gastric acid effect, this chitosan layer can be dissolved rapidly, release PMMMA-PLGA/Trx-SIP.
Embodiment 3. is by above-mentioned Edible vaccine system oral immunity in tilapia.
Tilapia is raised the water temperatures of 30 degrees Celsius, pH controls at 6.5-7.0, adopt Trx-SIP as antigen, by embodiment 1) and 2) prepare the feed vaccine of PMMMA-PLGA/Trx-SIP, vaccine divides three tilapias of throwing something and feeding (body weight is about 12 grams for kind: Oreochromisniloticus × Oreochromisaureus, average 2 months age of a fish), every tail fish is thrown something and fed with the intake of 20 μ gTrx-SIP/ gram body weight at every turn, and the immunization interval time is 7 days.In order to keep stable condition of culture, changed once the water of fresh dechlorination every 2-3 days.Result of throwing something and feeding shows, the equal growing way of tested tilapia is normal, sign is healthy.The tilapia arranging the Trx-SIP that throws something and feeds is matched group, and feeding volume is 80 μ gTrx-SIP/ gram.
The tilapia immunoassay technology of embodiment 4. through throwing something and feeding
Culling heart blood method gathers the blood of tilapia, low speed (<3000xg) centrifugal 10-20min, obtains antiserum.Current techique labelling FITC on Trx-SIP albumen of reference literature, in PBS buffer, ultrafiltration and concentration after dialysis, mixes protein solution and antiserum with different ratios, and room temperature places 20min.Prepare non-reduced PAGE glue, resolving gel concentration 15%, concentrated gum concentration is 4%.2 × non-reduced sample-loading buffer and Trx-SIP/ anti-serum samples equal-volume is adopted to mix, electrophoresis under the condition of low-voltage, low temperature.Result shows, the tilapia of PMMMA-PLGA/Trx-SIP immunity forms the specific antibody of SIP (being mainly IgM), can form complex with Trx-SIP.
By antiserum serial dilutions, respectively with in Escherichia coli cell, the bacteria suspension (being suspended from 0.9%NaCl) of the outer Protein S LH expression system (contrast) of periplasmic space SIP antigen presentation system and Escherichia coli cell is mixed.Result shows that antibody capable effectively makes two kinds of SIP above express bacterium coagulation, but to the outer Protein S LH expression system of Escherichia coli cell without agglutination, illustrates that antibody has the character of sip protein specific bond.
Thereafter adopt micro-bacteriostatic experiment on flat board, detect the ability of antiserum elimination GBS bacterium.Antiserum (original content) 3 μ L and bacteria suspension (OD=0.7,0.9%NaCl normal saline suspends) equal-volume mix, 37 DEG C of 1 placement hour, place 47 hours for 4 DEG C.Coating antibiotic-free LB is dull and stereotyped, 37 DEG C cultivate 3 days, fully cultivate 4 days at 30 DEG C subsequently, detect colony-forming units (CFU).As shown in table 1, the tilapia antiserum through higher dosage PMMMA-PLGA/Trx-SIP immunity can eliminate GBS bacterium completely.The tilapia serum of Trx-SIP immunity does not show effective bacteriostasis substantially.
Table 1: the dull and stereotyped ability detecting antiserum elimination GBS bacterium
Note: feeding volume sets relative to tilapia body weight.Antiserum (original content) 3 μ L and bacteria suspension (OD=0.7,0.9%NaCl) equal-volume mix.Mixed standing time parameter: 37 DEG C 1 hour, 4 DEG C 47 hours.Slat chain conveyor time parameter: 37 DEG C of 3 days, 30 DEG C 4 days continuous culture.
Immunoprotection Setup Experiments is that PMMMA-PLGA/ antigen is thrown something and fed group, to throw something and feed without coating antigen group and not immune blank group, and tilapia average weight is 12 grams.Each group all adopts 20 tail tilapias to throw something and feed, one week interval time, totally three times.Inoculate the tilapia through three oral immunities, adopt Intraperitoneal injection mode to inoculate about 10
7individual GBS bacterial cell (bacteria suspension is prepared with 50 μ L sterilizing 0.9%NaCl).To be thrown something and fed mode by PMMMA-PLGA/Trx-SIP, on average throw something and feed 20 μ gTrx-SIP, the Trx-SIP without parcel of every gram of body weight tilapia is that every gram of body weight is thrown something and fed 80 μ g.The health status of fish is monitored continuously, statistics infection proportion in 210 days.As shown in table 2, PMMMA-PLGA putamina layer increases substantially the effect of Trx-SIP oral immunity, makes the time protected completely extend to more than 210 days from 1 month.
Bioluminescence imaging technology adopts the distribution of method detectable antigens in tilapia main organs of fluorescent tracing.Reference literature universal method adopts isothiocyanate FITC labelled antigen Trx-SIP, by step 1) and 2) prepare Edible vaccine, the tilapia of health is thrown something and fed, takes food and carry out living imaging analysis after 36 hours.Result shows; the antigen of PMMMA-PLGA parcel has the distribution of higher amount, the fish that PLGA/Trx-SIP is oral at Ia internal organs such as large intestine, kidney and spleens, then mainly detect at liver, small intestinal, large intestine; after not having the Trx-SIP protected to throw something and feed, mainly detect at liver.
Table 2: immune protection performance detects
Note: each group all adopts 20 tail tilapias to throw something and feed, and one week interval time, totally three times, records immune natural law from after first time immunity.Tilapia average weight: 12 grams, intraperitoneal inoculation GBS bacterium liquid measure: totally 10
7cFU (cleaning resuspended with sterilizing 0.9%NaCl), 50 μ L.By PMMMA-PLGA/Trx-SIP, on average every gram of body weight tilapia is thrown something and fed 20 μ gTrx-SIP, and the Trx-SIP without parcel is that every gram of body weight is thrown something and fed 80 μ g.
Note: all chemical reagent are all purchased from Aladdin Reagent Company.Allly relate to conversion, the step of antibacterial and cell culture is sterile working.
Claims (10)
1. a putamina layer carrier, is characterized in that, it consists of poly-(methyl methacrylate-acrylic acid methyl ester .-methacrylic acid).
2. the preparation method of putamina layer carrier described in claim 1, it is characterized in that, comprise the steps:, containing in the liquid phase of kernel, successively to add methyl methacrylate, acrylic acid methyl ester. and methacrylic acid in order, optionally add initiator again and catalyst reacts, parcel kernel.
3. preparation method according to claim 2, is characterized in that: react and carry out in ultrasonic, and temperature range is for 40-60 DEG C, pH are 6.0-7.0.
4. preparation method according to claim 2, is characterized in that: described initiator is Ammonium persulfate., and described catalyst is N, N, N', N'-tetramethylethylenediamine.
5. preparation method according to claim 2, is characterized in that: the volume ratio of described methyl methacrylate, acrylic acid methyl ester. and methacrylic acid is 6-8:2-4:1-2; More preferably, volume ratio is 6.5:2.5:1-2; Also more preferably, volume ratio is 6.5:2.5:1 or 6.5:2.5:2.
6. an oral vaccine system, comprises putamina layer carrier according to claim 1 and kernel vaccine system.
7. oral vaccine system according to claim 6, it is characterized in that: described kernel vaccine system has hydrophobic interface layer, this boundary layer inside is albumen or polypeptide antigen, do not get rid of all kinds of adjuvant, preferably, kernel vaccine system is the micro-nano granules that bio-compatibility is good, can be ingested by cell, is more preferably poly lactic coglycolic acid/antigen micro-nano granules; Described putamina layer carrier wraps up kernel vaccine system completely; Acidity this putamina layer to neutrallty condition is stablized; be applicable in stomach and small intestinal, protect kernel vaccine system; avoid antigen degradation and absorption, simultaneously at large intestine by pH response release kernel vaccine system, and then be applicable to the enhancing protection of oral vaccine, the transport of large intestine targeting and immunopotentiation.
8. the immunological embodiments of oral vaccine system described in claim 6, it is characterized in that: by oral enforcement immunity, preferably, employing makes Edible vaccine with food is mixed, preferred, adopt chitosan layer to form the rete in order to stabilization of vaccines outward at Edible vaccine, this rete is stable under alkalescence, avoids inner vaccination particles to be released.
9. oral immunity embodiment according to claim 8, is characterized in that: implemented once at interval of one week, at least immunity three times.
10. the effect detection method of the immunological embodiments described in claim 8 or 9, it is characterized in that: comprise in antiserum specific binding and antigen, causal organism ability eliminated by antiserum, immunoprotection, targeting ability measures assembled scheme, preferably, adopt non-reducing polyacrylamide gel electrophoresis detection antibody to be combined with fluorescently-labeled antigen and form complex, in Escherichia coli cell-bacterium of periplasmic space antigen presentation system is solidifying to be measured, the antibacterial detection of trace antiserum, the qualification of antagonism cause of disease immune protection, and use bioluminescence imaging technology to carry out fluorescently-labeled Antigen distribution detection, do not get rid of other relevant auxiliary detection means.
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Application publication date: 20160106 |