CN106501525A - A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle - Google Patents
A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle Download PDFInfo
- Publication number
- CN106501525A CN106501525A CN201610870668.0A CN201610870668A CN106501525A CN 106501525 A CN106501525 A CN 106501525A CN 201610870668 A CN201610870668 A CN 201610870668A CN 106501525 A CN106501525 A CN 106501525A
- Authority
- CN
- China
- Prior art keywords
- lipid vesicle
- albumen
- envelop rate
- polypeptide
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/683—Total protein determination, e.g. albumin in urine involving metal ions
- G01N33/6833—Copper, e.g. Folin-, Lowry-, biuret methods
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle, belongs to pharmaceutical analysiss technical field.The lipid vesicle solution for being enclosed with albumen or polypeptide for preparing is diluted by the present invention, taking the lipid vesicle solution after dilution adds nonionic surfactant to carry out breakdown of emulsion, take the lipid vesicle solution before and after breakdown of emulsion again, be separately added into BCA reactant liquors, develop the color under uniform temperature and time；And absorbance is read at 562nm, protein concentration and envelop rate are calculated according to standard curve.Difference of the method using parcel albumen and developer reaction rate in the outer floating preteinses of lipid vesicle and lipid vesicle, measurement lipid vesicle protein drug envelop rate, without the need for separating to floating preteinses, easy to operate, reproducible, loaded down with trivial details and offset issue of the conventional method when lipid vesicle protein drug envelop rate is measured is solved, the associated production application and research field for needing lipid vesicle protein drug provides an effective method foundation.
Description
Technical field
The present invention relates to a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle, belongs to pharmaceutical analysiss
Technical field.
Background technology
Polypeptide, the research of protein medicaments lipid vesicle are when previous very active field.Lipid vesicle is by fat
Matter composition, the internal closure vesicle for water phase, can wrap up hydrophilic and lyophobic dust in water phase and film in which respectively.According to composition
Difference, lipid vesicle has the species such as liposome, carrier, class plastid, ethosome, but its structure is ball-shaped closure vesicle.
After albumen is oral can be subject to gastrointestinal tract acid, the destruction of alkali condition, body fluid and enzyme and degrade and inactivate.And with lipid vesicle be
Due to the protective effect of double-layer of lipoid after carrier is oral, it is possible to decrease palliating degradation degree of the various factors to protein medicaments, medicine is improved
The stability of thing；Oral lipid vesicle also has the characteristics of wide variety, selectivity and adjuvanticity are strong, and untoward reaction is low simultaneously,
Gain great popularity especially as mucosa-immune antigen vectors.At present envelop rate be important indicator in lipid vesicle quality evaluation it
One.The assay method of report has following several at present：(1) dialysis：Using semipermeable membrane molecular size difference, by replacing in time
Foreign minister reaches detached purpose.The method is simple, accurate, reproducible, has the disadvantage time-consuming longer, and consumed medium is more, Yi Zao
Into drug leakage.(2) supercentrifugation：Separated with the gravity difference of pastille lipid vesicle using free drug.But the party
Method is relatively costly, and each batch of sample room poor reproducibility, leads as centrifugal force loses drug leakage in lipid vesicle compared with conference
Cause the envelop rate of medicine low.(3) gel column chromatography：Conventional chromatographic column is polydextran gel (Sephadex) post and fine jade
Sepharose (Sepharose) post, is separated using the difference of lipid vesicle and free drug relative molecular mass, but is deposited
Elution time length, elution volume be big, the low problem of drug level.(4) ultrafiltration membrance filter method：Filter can be passed through using free drug
Film, and pastille lipid vesicle is trapped within filter membrane to separate free drug and lipid vesicle.The shortcoming of the method is film medicine-feeding
Thing small volume, film are stronger to the absorption of medicine, cause envelop rate higher.Above-mentioned various lipid vesicle entrapment efficiency algoscopys are deposited
There is certain error in a common problem, i.e. gained envelop rate.Such as dialysis, chromatography and filter membrane method, all it is profit
Separated with molecular size difference, thus more difficult by lipid vesicle and medicine crystal and/or lipid vesicle chip separation, affect bag
The result that envelope rate is determined；And three kinds of methods all take longer, there are much relations with drug level, in the choosing of chromatographic column or filter membrane
Select and there is also difficulty；Though centrifuging relative measurement speed, easily causes the seepage of medicine in lipid vesicle, instrument is deposited
Substantially relying on, use cost is higher.Therefore, it is highly desirable to find other measuring methods, both fast and convenient, and can be accurately
The encapsulating situation of ground reflection medicine.
At present laboratory research determine the common method of protein content mainly have Folin- phenol reagent process (Lowry methods),
Coomassie brilliant blue (Coomassie Brilliant Blue G-250, CBB) staining, bicinchoninic acid
(Bicinchoninic acid, BCA) method etc., wherein Lowry methods are owned by France in chemical method with BCA.The measuring principle of Lowry methods is
Protein is acted on forming copper-peptide bond complex with basic cupric sulfate, and the latter makes phenol reagent with tryptophan and L-Tyrosine collective effect
In phosphomolybdic acid and phosphotungstic acid reduction generate blue compound；The measuring principle of BCA methods is reduced into Asia for albumen quality and price copper ion
Copper ion, the latter are combined generation aubergine complex in alkaline solution with BCA.Bradford is owned by France in dye binding method,
I.e. Coomassie brilliant G-250 is changed into blueness, shade and egg with protein in an acidic solution in rufous after being combined
The proportional relation of white concentration.
The scope that various methods determine protein concentration is as follows：It is 10～100 μ that Lowry method standard proteins concentration arranges scope
g/mL；BCA method standard protein concentration is arranged on 20～1000 μ g/mL；Bradford methods are 31.25～2000 μ g/mL.Per
The method of kind has its advantage and limitation, in protein example often can disturb protein concentration containing some chemical reagent
Determine.The principal element for affecting Lowry methods to determine albumen has some aminoacid, nonionic surfactant, sucrose, Containing Sulfur
Compound；The principal element for affecting BCA methods to survey albumen has sucrose, carbamide, EDTA, affects the principal element of Bradford methods to have sweet
Oil, acetic acid, detergent and some alkaline buffer systems.For lipid vesicle protein drug, select BCA methods reduce non-
Interference when ionic surface active agent is detected to protein concentration.According to the literature, BCA methods determine protein concentration not by the overwhelming majority
The impact of the chemical substance in sample, can be with compatible sample up to 5% SDS, Triton X-100, Tween-20, Tween-
60th, the surfactant such as Tween-80.
Content of the invention
Present invention aim to address in existing lipid vesicle albumen or polypeptide drug entrapment efficiency determination method deficiency,
A kind of fast and convenient envelop rate computational methods are provided.
According to the envelop rate computational methods of the present invention, it is characterised in that using albumen inside and outside lipid vesicle or polypeptide with aobvious
The reaction rate difference of color reagent realizes the calculating of envelop rate, the method comprising the steps of：
（1）Conventional preparation is enclosed with the lipid vesicle solution of albumen or polypeptide；
（2）By step（1）In lipid vesicle solution in total protein concentration be diluted in OK range；
（3）Take a certain amount of step（2）In lipid vesicle solution, add demulsifier, make lipid vesicle dissolve；
（4）By step（2）And step（3）Lipid vesicle solution, add to 96 orifice plates by 20 μ L of every hole respectively, then in every hole
Add 200 μ L carry out the reagent of chromogenic reaction with albumen or polypeptide；
（5）In step（4）The bovine serum albumin standard substance of 0.5 mg/mL are separately added in the standard sample wells of 96 orifice plates, by 0,
1st, 2,4,8,12,16,20 μ L sample-addings, adding 200 μ L can carry out the reagent of chromogenic reaction with albumen or polypeptide；
（6）By step（4）With（5）96 orifice plates react a period of time at a certain temperature, until there is color change；
（7）By step（6）96 orifice plates be positioned over microplate reader, select 562 nm places reading absorbance.According to standard curve meter
Calculate step（4）Each porin concentration.
（8）According to formula lipid vesicle albumen envelop rate=(C Always-C Trip)/C Always×100%
Computational envelope rate, in formula,C AlwaysThe total protein concentration of breakdown of emulsion lipid vesicle is represented,C TripMeasured by untreated lipid vesicle
Protein concentration.
Wherein, step（1）The material wrapped up in the lipid vesicle can be replaced other can be occurred mutually with detectable
The material of effect；The lipid vesicle, is the lipid molecular that can form imitated vesicle structure, and its composition includes phospholipid, cholesterol, table
Face activating agent and other lipoid molecules etc..
Step（2）Total protein concentration scope after the lipid vesicle dilution should be at 20～1000 μ g/mL.
Step（3）The demulsifier be include and be not limited to Triton X-100, Tween 20 or Tween 80 etc. non-from
Sub- surfactant and other kinds of demulsifier；The demulsifier final concentration of 0.1%～0.5%.
Step（4）Or（5）Described in can with albumen or polypeptide carry out chromogenic reaction reagent be BCA reaction reagents；
The BCA reaction reagents are by being purchased from the BCA determination of protein concentration test kits of the green skies Bioisystech Co., Ltd in Shanghai
Add 1 volume BCA reagent B (50 by 50 volume BCA reagent As:1) it is formulated.
Step（6）Described in reaction temperature be 37 C～60 C；The response time is 20～40 minutes；
96 orifice plate therein can also change common quartz colorimetric utensil into and be detected.
Compared with prior art, the present invention has the advantages that：
The inventive method can avoid the separation of conventional floating preteinses/peptide molecule or removal step, one step of energy from quickly directly surveying
The content of floating preteinses and total protein in lipid vesicle is measured, so as to calculate the albumen envelop rate in lipid vesicle, is suitable for big
When scale lipid vesicle wraps up the optimization of protein/polypeptide, the quick measure of envelop rate and compare.Its principle is BCA
Contain copper ion in reaction reagent, it is known that bivalent cupric ion is reduced into univalent copper ion by protein under conditions of alkalescence, one
Valency copper ion and bicinchoninic acid（BCA）Interact and produce sensitive color reaction.The BCA of two molecules chelate a copper from
Son, the empurpled reaction complex of shape.The water miscible complex shows strong light absorptive, absorbance and egg at 562 nm
White concentration has good linear relationship in broad range, therefore can extrapolate protein concentration by light absorption value.According to above-mentioned anti-
Principle is answered, on the one hand using the catalytic action of the outer floating preteinses of lipid vesicle, makes BCA, with copper ion, chromogenic reaction occur, according to
Absorbance can calculate the concentration of floating preteinsesC Trip；On the other hand, due to the sealing of lipid vesicle structure, copper ion without
Method is smoothly had an effect with the parcel albumen in lipid vesicle, only broken when dissolving is carried out to lipid vesicle by surfactant
Bad, after discharging the albumen wrapped up in lipid vesicle, chemical colour reaction could occur with copper ion and BCA together with floating preteinses
Reaction, so that obtain the concentration of total protein further according to absorbanceC Always , quick calculating lipid can reach according to below equation therefore
The purpose of vesicle protein entrapment efficiency.
Lipid vesicle albumen envelop rate=(C Always-C Trip)/C Always×100%
In addition, the method can be additionally used in the separating effect for evaluating floating preteinses.For example, with dialysis or centrifugal ultrafiltration pipe by floating preteinses
After removing, can to process after lipid vesicle sampling be directly added into developer, and separately after sampling plus surfactant breakdown of emulsion again plus
Developer, the numerical value difference as measured by both substantially, show that separation method used is preferable to the elimination effect of floating preteinses, i.e.,
Separate effectively, on the contrary then anti-；The residual volume of separate after floating preteinses can be calculated simultaneously accordingly, as shown in Figure 4.
In sum, according to the invention provides a kind of joint BCA detection albumen principles and surfactant are to lipid capsule
The method of bubble protein drug envelop rate.Conventional method is this method solved when lipid vesicle protein drug envelop rate is measured, is needed
The problem of floating preteinses medicine is previously isolated from, time saving and energy saving.Using microplate reader and the method, extensive lipid vesicle can be carried out
The measure of protein drug envelop rate and calculating, easy to operate, reproducible, it is the associated production for needing lipid vesicle protein drug
Application and research field provide an effective method foundation.
Description of the drawings
Fig. 1 is lipid vesicle albumen entrapment efficiency determination principle schematic of the present invention；
Fig. 2 is lipid vesicle protein concentration measurement standard curve in the embodiment of the present invention 1；
Fig. 3 is lipid vesicle total protein concentration in the embodiment of the present invention 2（+Triton X-100）With floating preteinses concentration（-
Triton X-100）Measured value changes over result；
Fig. 4 is that the embodiment of the present invention 3 uses centrifugal ultrafiltration method（UF）After separating floating preteinses, initial total protein concentration（Initial
+Triton）With total protein concentration after ultra-filtration and separation（UF+Triton）Measured value changes over result.
Specific embodiment
BCA reaction reagents described in the embodiment of the present invention is by the BCA eggs for being purchased from the green skies Bioisystech Co., Ltd in Shanghai
Add 1 volume BCA reagent B (50 by 50 volume BCA reagent As in white concentration measuring kit:1) it is formulated.
Embodiment 1：
Routinely reverse phase evaporation prepares the liposome of parcel bovine serum albumin (BSA), specific as follows：Weigh 20 mg Semen sojae atricolor
Lecithin and 5 mg cholesterol, with round-bottomed flask is added to after 9 mL ether dissolutions, add the bovine serum albumin of 3 mL, 1 mg/mL
In vain（BSA）Solution, water bath sonicator form stable white emulsion suspension liquid in 2～5 minutes, and Rotary Evaporators are evaporated removing organic solvent,
Remaining liq is taken out, the process of ice-water bath Probe Ultrasonic Searching becomes clarification to solution and obtains final product liposome solutions.Liposome solutions are diluted 10
After times, concentration is 0.1 mg/mL.Take 96 orifice plates, by dilution after liposome solutions by per 20 μ L of hole be loaded；After separately taking dilution
Liposome solutions, add Triton X-100 to final concentration of 0.5%, same by the sample-adding per 20 μ L of hole；Per hole in standard sample wells
The BSA standard substance of 0.5 mg/mL are added, is loaded by 0,1,2,4,8,12,16,20 μ L, is added standard dilutions less than 20 μ L
Supply；After all holes add 200 μ L BCA reaction reagents, 40 min are placed in 40 C and color change occur, be immediately placed in enzyme mark
Instrument, selects to read absorbance at 562 nm.Protein concentration and corresponding light absorption value according to gauge orifice draws standard curve, such as attached
Shown in Fig. 2, after linear fitting, gained regression equation is y=0.015+1.483 x（Y is absorbance, and X is protein concentration, single
Position is mg/mL）, further according to this Equation for Calculating sample concentration.Dense as total protein with the lipid body opening gained for adding Triton X-100
DegreeC Always =0.455, with the lipid body opening gained without Triton X-100 as floating preteinses concentrationC Trip =0.176, according to formula meter
It is 61.2% to calculate envelop rate.
Lipid vesicle albumen envelop rate=(C Always-C Trip)/C Always×100%
Embodiment 2：
Routinely film dispersion method prepares the carrier of parcel bovine serum albumin (BSA), specific as follows：Weigh 30 mg Semen sojae atricolor
Lecithin, 6 mg cholesterol, 2 mg NaTDCs, with round-bottomed flask is added to after the dissolving of appropriate chloroform, Rotary Evaporators are evaporated
Organic solvent is removed, makes drag form a uniform thin film；Flask is placed in vacuum desiccator evacuation at least 3 hours,
To completely remove the organic solvent of remnants, the BSA solution of 5 mL, 1 mg/mL is added, vibration makes thin film aquation, at water bath sonicator
Reason 30 minutes, becomes clarification to solution and obtains final product transmission liquid solution, takes 10 times of appropriate transmission liquid solution dilution standby.96 orifice plates are taken, will
Transmission liquid solution after dilution is loaded by per 20 μ L of hole；The transmission liquid solution after dilution is separately taken, and Triton X-100 is added to end
Concentration is 0.25%, same by the sample-adding per 20 μ L of hole；Add the BSA standard substance of 0.5 mg/mL in standard sample wells per hole, by 0,1,
2nd, 4,8,12,16,20 μ L sample-addings, add standard dilutions to supply less than 20 μ L；All holes add 200 μ L BCA reaction reagents
Afterwards, 20 min are placed in 50 C, is immediately placed in microplate reader, select at 562 nm, to read absorbance.Plate one is swept every 10 min
Secondary, a hour is recorded continuously, afterwards at room temperature, after colour developing, the 3rd, 7,24 little recurrence of disease at the same time next year are swept once, under record different time
Absorbance change.Each porin concentration is calculated according to measurement gained standard curve every time, is floating preteinses concentration as shown in Figure 3
（-Triton X-100）With total protein concentration（+Triton X-100）Time changing curve.Between the figure can be seen that when measuring
After more than 1h, there is substantially change in the starting of floating preteinses and total protein concentration, it is therefore desirable to be chosen at institute's measured value in 1h, according to
Formula computational envelope rate.In this example, according to the calculating that measurement 30 minute when institute's value carries out envelop rate, to add Triton X-
100 transmission body opening gained is total concentrationC Always =0.434, with the transmission body opening gained without Triton X-100 as CfC Trip
=0.298, it is 31.2% according to formula computational envelope rate.
Lipid vesicle albumen envelop rate=(C Always-C Trip)/C Always×100%
Embodiment 3：
Liposome solutions are prepared according to embodiment 1, take 1 mL liposome solutions adopt molecular cut off for 100 kD ultrafiltration from
Free albumen is removed by centrifugation by heart pipe, then with final concentration of 0.1% Triton X-100 by separation before and after liposome
Breakdown of emulsion, after respectively taking 20 μ L, plus 200 μ L BCA reaction reagents, places 20 min in 50 C, is immediately placed in microplate reader, select 562
Absorbance is read at nm.Plate is swept once every 10 min equally, continuously one hour of record, afterwards at room temperature, after colour developing
3rd, 7,24 little recurrence of disease at the same time next year are swept once, record the absorbance change under different time.Calculated according to measurement gained standard curve every time
Each porin concentration, is initial total protein concentration as shown in Figure 4（Initial+Triton）Dense with total protein after ultra-filtration and separation
Degree（UF+Triton）Time changing curve.The figure can be seen that total protein concentration substantially reduces after ultrafiltration centrifugation, and number
Value does not change with the change of time, and the separating effect of floating preteinses is described very well, and the present embodiment is surveyed when being chosen at 30 min
Value, initial total protein concentration are 0.375, and after separating, total concentration value is parcel protein concentrationC Bag =0.148, bag is calculated according to formula
Envelope rate is 39.5%.
Lipid vesicle albumen envelop rate=C Bag/C Always×100%
Embodiment 4：
Use bovine serum albumin instead ovalbumin, transmission liquid solution is prepared by embodiment 2.To prepared liposome solutions,
First adopt molecular cut off free ovalbumin to be removed for the bag filter dialysis of 100 kD, then take the biography before and after a certain amount of separation
Pass 0.5 % Tween of liquid solution final concentration, 80 breakdowns of emulsion, as described in Example 3 plus BCA reaction reagents colour developing, measure parcel egg
White concentrationC Bag , it is 38.2% according to formula computational envelope rate.
Lipid vesicle albumen envelop rate=C Bag/C Always×100%
Embodiment 5：
Use bovine serum albumin instead human albumin, liposome solutions are prepared by embodiment 1.Molten to prepared liposome
Liquid, is separated off free ovalbumin using gel chromatographic columnses, then takes a certain amount of liposome solutions before and after separation, use final concentration
0.5 % Tween, 20 breakdowns of emulsion, such as embodiment 4 are separately added into BCA reaction reagents, colour developing, measure parcel protein concentrationC Bag , according to
Formula computational envelope rate is 66.7%.
Lipid vesicle albumen envelop rate=C Bag/C Always×100%
Embodiment 6：
Use bovine serum albumin instead human serum albumin, liposome solutions, computational envelope rate is prepared by embodiment 2.Meanwhile, right
Liposome is separated with floating preteinses by prepared liposome solutions using conventional supercentrifugation, then takes a certain amount of supernatant, plus
BCA reaction reagents, colour developing, measure floating preteinses concentrationC Trip , it is 35.4% according to formula computational envelope rate.
Lipid vesicle albumen envelop rate=(C Always-C Trip)/C Always×100%
Claims (9)
1. a kind of determine lipid vesicle in albumen or polypeptide drug envelop rate method, it is characterised in that using lipid vesicle
The reaction rate difference of inside and outside albumen or polypeptide and colour reagent realizes the calculating of envelop rate, the method comprising the steps of：
（1）Conventional preparation is enclosed with the lipid vesicle of albumen or polypeptide；
（2）By step（1）In lipid vesicle in total protein concentration be diluted in OK range；
（3）Take a certain amount of step（2）In lipid vesicle solution, add demulsifier, make lipid vesicle dissolve；
（4）By step（2）And step（3）In lipid vesicle solution, add to 96 orifice plates respectively, then add in every hole can and egg
White or polypeptide carries out the reagent of chromogenic reaction；
（5）In step（4）Described in 96 orifice plates standard sample wells in be separately added into bovine serum albumin standard substance, add energy
The reagent of chromogenic reaction is carried out with albumen or polypeptide；
（6）By step（5）96 orifice plates react a period of time at a certain temperature, until there is color change；
（7）By step（6）96 orifice plates be positioned over microplate reader, read absorbance；Calculate each porin concentration；
（8）Envelop rate is calculated according to each porin concentration.
2. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be,
Step（2）In lipid vesicle after the dilution, total protein concentration is 20～1000 μ g/mL.
3. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be,
Step（3）The demulsifier is to include and be not limited to the nonionic tables such as Triton X-100, Tween 20 or Tween 80
Face activating agent and other kinds of demulsifier；The demulsifier final concentration of 0.1%～0.5%.
4. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be, step（4）Or（5）Described in can with albumen or polypeptide carry out chromogenic reaction reagent be BCA reaction reagents.
5. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be,
Step（5）The addition bovine serum albumin standard concentration is 0.5 mg/mL；The addition BCA reaction reagents 200
μL.
6. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be,
Step（6）Described in reaction temperature be 37 C～60 C；The response time is 20～40 minutes.
7. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be,
Step（7）The absorbance is read at 562 nm.
8. a kind of method for determining albumen or polypeptide drug envelop rate in lipid vesicle according to claim 1, which is special
Levy and be, step（8）Described according to each porin concentration calculate envelop rate be according to formula
Lipid vesicle albumen envelop rate=(C Always-C Trip)/C Always×100%
Computational envelope rate, in formula,C AlwaysThe total protein concentration of breakdown of emulsion lipid vesicle is represented,C TripMeasured by untreated lipid vesicle
Protein concentration.
9. albumen or polypeptide drug envelop rate in a kind of measure lipid vesicle according to claim 1-8 any one
Method, it is characterised in that 96 described orifice plates replace with common quartz colorimetric utensil and detected.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610870668.0A CN106501525A (en) | 2016-09-30 | 2016-09-30 | A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610870668.0A CN106501525A (en) | 2016-09-30 | 2016-09-30 | A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106501525A true CN106501525A (en) | 2017-03-15 |
Family
ID=58290849
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610870668.0A Pending CN106501525A (en) | 2016-09-30 | 2016-09-30 | A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106501525A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109619368A (en) * | 2018-11-29 | 2019-04-16 | 广东海洋大学 | A kind of bacteriocin CAMT2 soybean lecithin nano liposomes and its preparation method and application |
| CN109662238A (en) * | 2018-11-29 | 2019-04-23 | 广东海洋大学 | A kind of bacteriocin CAMT2 soybean lecithin nano liposomes and application |
| CN110658272A (en) * | 2019-09-23 | 2020-01-07 | 佛山科学技术学院 | Method for detecting encapsulation rate of composite polysaccharide liposome |
| CN111307977A (en) * | 2020-03-12 | 2020-06-19 | 湖北盛齐安生物科技股份有限公司 | Drug-loading detection method of drug-loading vesicle |
| CN111982999A (en) * | 2020-08-20 | 2020-11-24 | 点斗基因科技(南京)有限公司 | Detection method of loaded small interfering RNA liposome in external preparation |
| CN115508567A (en) * | 2022-11-23 | 2022-12-23 | 迈杰转化医学研究(苏州)有限公司 | Quality control material for detecting PD-L1 and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102144974A (en) * | 2011-04-19 | 2011-08-10 | 四川大学 | Liposome preparation for anticoagulant thrombolytic difunctional fusion protein and preparation method thereof |
| CN102172418A (en) * | 2011-02-18 | 2011-09-07 | 上海交通大学医学院附属新华医院 | Acellular matrix material capable of sustainedly releasing growth factors |
| CN106177934A (en) * | 2016-08-08 | 2016-12-07 | 华南农业大学 | A kind of haemophilus parasuis subunit vaccine and preparation method thereof |
-
2016
- 2016-09-30 CN CN201610870668.0A patent/CN106501525A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102172418A (en) * | 2011-02-18 | 2011-09-07 | 上海交通大学医学院附属新华医院 | Acellular matrix material capable of sustainedly releasing growth factors |
| CN102144974A (en) * | 2011-04-19 | 2011-08-10 | 四川大学 | Liposome preparation for anticoagulant thrombolytic difunctional fusion protein and preparation method thereof |
| CN106177934A (en) * | 2016-08-08 | 2016-12-07 | 华南农业大学 | A kind of haemophilus parasuis subunit vaccine and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 邓璇 等: "HV12p-rPA血栓靶向脂质体的制备及体内溶栓实验", 《药物生物技术》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109619368A (en) * | 2018-11-29 | 2019-04-16 | 广东海洋大学 | A kind of bacteriocin CAMT2 soybean lecithin nano liposomes and its preparation method and application |
| CN109662238A (en) * | 2018-11-29 | 2019-04-23 | 广东海洋大学 | A kind of bacteriocin CAMT2 soybean lecithin nano liposomes and application |
| CN109662238B (en) * | 2018-11-29 | 2022-02-01 | 广东海洋大学 | Bacteriocin CAMT2 soybean lecithin nano-liposome and application thereof |
| CN109619368B (en) * | 2018-11-29 | 2022-02-01 | 广东海洋大学 | Bacteriocin CAMT2 soybean lecithin nano-liposome and preparation method and application thereof |
| CN110658272A (en) * | 2019-09-23 | 2020-01-07 | 佛山科学技术学院 | Method for detecting encapsulation rate of composite polysaccharide liposome |
| CN111307977A (en) * | 2020-03-12 | 2020-06-19 | 湖北盛齐安生物科技股份有限公司 | Drug-loading detection method of drug-loading vesicle |
| CN111307977B (en) * | 2020-03-12 | 2022-12-16 | 湖北盛齐安生物科技股份有限公司 | Drug-loading detection method of drug-loading vesicle |
| CN111982999A (en) * | 2020-08-20 | 2020-11-24 | 点斗基因科技(南京)有限公司 | Detection method of loaded small interfering RNA liposome in external preparation |
| CN115508567A (en) * | 2022-11-23 | 2022-12-23 | 迈杰转化医学研究(苏州)有限公司 | Quality control material for detecting PD-L1 and preparation method and application thereof |
| CN115508567B (en) * | 2022-11-23 | 2023-03-10 | 迈杰转化医学研究(苏州)有限公司 | Quality control material for detecting PD-L1 and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106501525A (en) | A kind of method of albumen or polypeptide drug envelop rate in measure lipid vesicle | |
| CN108279309B (en) | Detection test strip and detection method for PLA2R antibody | |
| CN101852800B (en) | Colloidal gold test strip for semi-quantitatively detecting concentration of tacrolimus drug in human whole blood and detection method | |
| JP2000507696A (en) | Cation selectivity sensor | |
| CN103698535B (en) | Platelet-activating factor acetylhydro-lase immue quantitative detection reagent box and preparation, method of operating | |
| CN105652008A (en) | Human Lp-PLA2 biotin-streptavidin fluorescence immunochromatographic assay card and preparation method thereof | |
| CN107831163A (en) | A kind of chemiluminescence detection kit of thyroglobulin and preparation method thereof | |
| JP7352831B2 (en) | Immunochromatography test piece, measurement kit, and measurement method | |
| CN107533061A (en) | For capturing the composition and method of allochthon | |
| CN104215777B (en) | A kind ofly detect the method for haemoglobin in conjunction with phosphorylation alpha-synapse nucleoprotein | |
| CN102680689A (en) | Kit for detecting creatine kinase isoenzyme and preparation and use methods thereof | |
| CN104360073A (en) | Troponin diagnosis test paper strip for coupled immunomagnetic beads | |
| CN105793707A (en) | Immunochromatography-assisted detection method | |
| KR20180032512A (en) | Reagent Composition for Detecting Glycated hemoglobin and Detection Method of Glycated hemoglobin Using Thereof | |
| CN104215779B (en) | A kind ofly detect the method for haemoglobin in conjunction with alpha-synapse nucleoprotein | |
| CN106093423A (en) | A kind of test kit measuring retinol binding protein and preparation method thereof | |
| CN109897080A (en) | High selection hypersensitive liver cancer-specific peroxynitrite probe and its application | |
| WO2020153461A1 (en) | Antigen measuring method and measuring apparatus | |
| US20150276731A1 (en) | Filtration Detection Device and Use Thereof | |
| KR102029798B1 (en) | Reagent Composition for Detecting Glycated albumin and Detection Method of Glycated albumin Using Thereof | |
| Huang et al. | Receptor antibody time‐resolved A2 phospholipase bead immunochromatography and its application in idiopathic membranous nephropathy | |
| US20070248950A1 (en) | Supported polydiacetylene 3-D arrays for flourescent or phosphorescent detection | |
| CN104764878A (en) | An immunocolloidal gold test strip for detecting penicilloic acid and a preparing method thereof | |
| WO2018056762A1 (en) | Reagent composition for measuring glycated albumin and method for measuring glycated albumin using same | |
| WO2009007858A2 (en) | Method for detecting and quantifying analytes on a solid support with liposome-encapsulated fluorescent molecules |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170315 |
|
| RJ01 | Rejection of invention patent application after publication |