CN116465700A - Method for detecting biological activity of active protein and polypeptide in medicine gel - Google Patents
Method for detecting biological activity of active protein and polypeptide in medicine gel Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/50—Fibroblast growth factors [FGF]
- G01N2333/501—Fibroblast growth factors [FGF] acidic FGF [aFGF]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/475—Assays involving growth factors
- G01N2333/50—Fibroblast growth factors [FGF]
- G01N2333/503—Fibroblast growth factors [FGF] basic FGF [bFGF]
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention discloses a method for detecting biological activity of active protein and polypeptide drugs in carbomer type gel, which comprises the steps of diluting the gel by using DMEM test culture solution containing fetal calf serum and heparin sodium and using calcium ions to release the active protein from the gel and detecting the biological activity of the active protein. The fetal bovine serum and heparin sodium can protect active proteins in the diluted gel, can protect active proteins released from a carbomer skeleton after calcium ions are added for a short period of time, avoid protein inactivation caused by overlong waiting time from dilution and protein release to detection, and improve detection accuracy.
Description
[ field of technology ]
The invention belongs to the technical field of active protein detection, and particularly relates to a method for detecting the biological activity of active protein and polypeptide drugs in carbomer type gel, which is particularly suitable for detecting the biological activity of active aFGF in acidic fibroblast growth factor (aFGF) gel.
[ background Art ]
Carbomers (carbomers) are acrylic acid crosslinked resins obtained by crosslinking acrylic acid with pentaerythritol or the like, and are classified into carbomers 934, carbomers 940, carbomers 941, and the like according to the viscosity. Carbomer is white loose powder, and has strong hygroscopicity. Can be quickly swelled in water, but is not dissolved, and the aqueous dispersion is acidic and has low viscosity. When the aqueous alkali is used for neutralization, the aqueous alkali is gradually dissolved in water, the viscosity is rapidly increased, and semitransparent gel with certain strength and elasticity is formed when the concentration is high. A maximum viscosity or consistency is reached between pH6 and 11. Carbomer gels have remarkable plastic rheological properties. The gel agent using carbomer as matrix has the advantages of quick drug release, no greasiness, easy spreading, lubrication and comfort, no irritation to skin and mucous membrane, etc., and is especially suitable for treating seborrheic dermatosis. Salt electrolyte, strong acid can reduce viscosity of carbomer gel, alkaline earth metal ion and cationic polymer can be combined with the salt electrolyte to form insoluble salt. The neutralized carbomer is a gel matrix, has important applications such as thickening and suspending, has simple process and good stability, and is widely applied to emulsion, cream and gel.
The acidic fibroblast growth factor (aFGF) is one of members of Fibroblast Growth Factor (FGF) family of cells derived from mesoderm and neuroectoderm, is a multifunctional cell growth factor, has a trace active substance with wide mitogenic action, is mainly distributed in organs or tissues such as brain, pituitary, nervous tissue, retina, adrenal gland, heart and bone, has a small content of other tissues, and exists in serum and body fluid at a very low concentration. It is an endogenous active factor closely related to the healing process of tissue wound, and has obvious promoting effects on tissue wound healing, nerve injury repair and blood vessel regeneration. The composition is used for the following wound surfaces: deep II degree burn wound, chronic ulcer wound (including residual wound after trauma, diabetic ulcer, vascular ulcer and bedsore) to promote wound healing.
The rh-aFGF gel adopts carbomer as a gel matrix, and the carbomer hydrogel is of a reticular structure, so that the rh-aFGF is more stable, the drug is slowly released, and meanwhile, the wound surface seepage can be absorbed, so that a highly hydrophilic gel is formed, the secretion of the wound surface and the bacterial infection are controlled, the dissolution of the drug is accelerated, the slow release effect is realized to a certain extent, the effect is more durable, the wound surface tissue healing is promoted, the healing speed is accelerated, and the effect of the rh-aFGF is more direct and effective.
Carbomer hydrogel is of a network structure and is important for stabilizing the activity of rh-aFGF, but because a trace amount of drug protein in the carbomer hydrogel bound in a three-dimensional network structure is difficult to release completely, the content and the biological activity of the drug protein in a drug cannot be accurately measured when the gel drug is detected. The Chinese patent application CN2012104293 61.9.9 discloses a method for releasing protein drugs from the three-dimensional network structure of carbomer hydrogel, and further detecting the protein content of the drugs in the drug gel, which adopts the technical means that gel is diluted or undiluted, and a solution containing calcium ions is added as a releasing agent. Although the method can release rh-bFGF, the addition of electrolyte ions can destroy the biological activity of part of rh-bFGF protein, in the actual detection process, long intervals are often needed among dilution, configuration and sample injection detection of samples, and after the rh-bFGF is released from the hydrogel with a net structure, the interval is long, the inactivation of the protein is often caused, so that the method disclosed by CN201210429361.9 causes the 'distortion' of the detection of the active protein.
[ invention ]
In order to overcome the defects in the prior art, the invention provides a method for detecting the biological activity of active proteins and polypeptides in a medicine gel, and in order to achieve the technical effects, the invention adopts the following technical scheme:
the invention provides a method for detecting the biological activity of protein in a medicine gel, which is characterized by comprising the following steps:
(1) Diluting the medicine gel with DMEM test culture solution; (2) Adding a protein release agent to completely release the protein from the gel; (3) detecting the biological activity of the protein in the drug.
Further, the invention provides a detection method for detecting the biological activity of the protein in the medicine gel, and the DMEM test culture solution comprises fetal bovine serum and heparin sodium.
Further, the invention provides a detection method for detecting the biological activity of protein in the medicine gel, wherein the DMEM test culture solution contains 1-2% of fetal bovine serum and 10-30ug/ml of heparin sodium, preferably 1.5% of fetal bovine serum and 20ug/ml of heparin sodium.
Furthermore, the invention provides a detection method for detecting the biological activity of the protein in the medicine gel, wherein 1ml to 8ml of DMEM test culture solution, preferably 2ml to 6ml of DMEM test culture solution, more preferably 3ml of DMEM test culture solution is added into each gram of gel to be detected.
Furthermore, the invention provides a detection method for detecting the biological activity of the protein in the medicine gel, and the release agent is prepared by dissolving calcium ion-containing aqueous solution, preferably calcium salt, in deionized water or purified water.
Furthermore, the invention provides a detection method for detecting the biological activity of the protein in the medicine gel, wherein 0.2 ml-1.0 ml of release agent is added to each gram of gel to be detected, and preferably 0.5 ml-1.0 ml of release agent is added to each gram of gel to be detected.
Further the present invention provides a method for detecting the biological activity of a protein in a pharmaceutical gel, said calcium salt being selected from the group consisting of water-soluble calcium salts, preferably one or more of calcium chloride, calcium sulphate, calcium nitrate, calcium bicarbonate.
The invention further provides a detection method for detecting the biological activity of protein in a medicine gel, wherein the concentration range of calcium ions in the aqueous solution containing the calcium ions is as follows: 0.015mol/L to 0.15mol/L, preferably 0.1 to 0.15mol/L.
Further, the invention provides a detection method for detecting the biological activity of the protein in the medicine gel, which is suitable for detecting the biological activity of the protein in the gel medicine with carbomer as a gel matrix.
Further provided is a method for detecting the biological activity of a protein in a pharmaceutical gel, which is suitable for detecting the biological activity of a protein in a protein gel, preferably the protein is selected from acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF).
The beneficial effects are that:
according to the invention, the DMEM test culture solution containing fetal calf serum and heparin sodium is used for diluting the medicine gel, so that the active protein in the diluted gel can be protected, and the problem of partial inactivation of the protein after calcium ions are added is solved.
The DMEM test culture solution containing the fetal calf serum and the heparin sodium can protect the active protein released from the carbomer skeleton after calcium ions are added for a short period of time, avoid protein inactivation caused by overlong detection waiting time after dilution, and improve detection accuracy.
[ detailed description ] of the invention
Example 1: taking the example of detecting the activity of recombinant human acidic fibroblast growth factor (rh-aFGF) in rh-aFGF gel:
(1) Standard substance solution preparation: taking recombinant human acid fibroblast growth factor standard, re-dissolving under aseptic condition according to instructions, and diluting to 40IU per 1 ml. In the sample dilution plate, 4-fold serial dilutions were made for a total of 8 dilution gradients, and the diluted standard solution in the sample dilution plate was transferred to a 96-well cell culture plate, 50 μl per well, 2 multiplex wells per sample.
(2) Sample solution preparation: adding 1.5g rh-aFGF gel into 9mL DMEM test culture solution containing 1.5% bovine serum and 20ug/mL heparin sodium, mixing well, and adding 1.5mL CaCl 0.15mol/mL 2 The solution was stirred and centrifuged in a constant temperature high speed centrifuge (temperature 4 ℃ C., centrifugal force 3000g,10 min) for a centrifuge tube. The supernatant was taken and diluted to 40IU per 1ml with test medium. In the sample dilution plate, 4-fold serial dilutions were made for a total of 8 dilutions, and the diluted samples in the sample dilution plate were transferred to a 96-well cell culture plate at 50 μl per well, 2 multiplex wells per sample.
(3) The test solution was allowed to stand at a temperature of 10℃for 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours and 36 hours, respectively, and then immediately subjected to the test in the following step (4).
(4) Activity measurement: the NIH3T3 cell strain is taken and cultured in a complete culture solution under the condition of 37 ℃ and 5% CO2, the cell concentration is controlled to be 1.0X105-5.0X105 cells/ml, and the biological activity is measured 24-36 hours after passage. Discarding culture solution in culture flask, digesting and collecting cells, and preparing into 1.0X10 with complete culture solution 5 ～2.0×10 5 Cell suspensions of individual cells/ml were seeded into 96-well cell culture plates at 50 μl per well. After the completion of the culture at 37℃under 5% CO2 for 66-72 hours, 20. Mu.L of MTT solution was added to each well, and the mixture was left to stand at 37℃under 5% CO2 overnight. 10% SDS (sodium dodecyl sulfate) 100 μl was added to each well, the mixture was mixed and put into an enzyme-labeled instrument, absorbance was measured at 570nm with 630nm as a reference wavelength, and the measurement result was recorded.
(5) And (3) calculating results: the test data are processed by a computer program or a linear regression calculation method, and the result is calculated according to the following formula:
biological activity of test sample (IU/ml) =pr×ds×es/(dr×er), where Pr is standard biological activity, unit IU/ml; ds is the pre-dilution of the test sample; dr is the standard pre-dilution; es is the dilution multiple of the half-effective amount of the sample corresponding to the standard sample; er is half-effect dilution of the standard substance; the test data are shown in Table 1:
example 2: taking the activity of rh-bFGF in recombinant human basic fibroblast growth factor (rh-bFGF) gel as an example, the recombinant human basic fibroblast growth factor (rh-bFGF) gel is taken as a detection object, the corresponding recombinant human acidic fibroblast growth factor standard is adopted as a standard, the preparation of the detection standard, the preparation of a test solution, the activity test and the calculation method of the result are the same as those of example 1, and the detection data are shown in Table 1.
Comparative example 1: taking the activity of rh-aFGF in recombinant human acidic fibroblast growth factor (rh-aFGF) gel as an example, the preparation, activity test and result calculation methods of the detection standard are the same as those of example 1, except that the DMEM test culture solution used for preparing the test solution does not contain fetal bovine serum and heparin sodium, and the detection data are shown in Table 1.
Comparative example 2: taking the activity of rh-bFGF in recombinant human basic fibroblast growth factor (rh-bFGF) gel as an example, the preparation, activity test and result calculation methods of the detection standard are the same as those of example 2, except that the DMEM test culture solution used for preparing the test solution does not contain fetal bovine serum and heparin sodium, and the detection data are shown in Table 1.
TABLE 1
The data show that the addition of the fetal bovine serum and the heparin sodium solution can effectively avoid the reduction of the biological activity of the protein caused by the electrolyte solution; when a large number of samples need to be tested, the addition of the fetal bovine serum and the heparin sodium solution can avoid the distortion of the activity detection result caused by overlong waiting time of the samples to be detected, and the detection accuracy is improved. In addition, the present invention shows that even if a certain amount of fetal bovine serum and heparin sodium solution are added, the queuing time of the diluted sample needs to be controlled, preferably within 24 hours.
The above description should not be taken as limiting the practice of the invention to these descriptions, but it will be understood by those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and the invention is defined by the appended claims.
Claims (10)
1. The method for detecting the biological activity of the protein in the medicine gel is characterized by comprising the following steps of: (1) diluting the drug gel with DMEM test medium; (2) Adding a protein release agent to completely release the drug protein from the gel; (3) detecting the biological activity of the protein in the drug.
2. The method for detecting the biological activity of a protein in a pharmaceutical gel according to claim 1, wherein the DMEM test culture broth comprises fetal bovine serum and heparin sodium.
3. Method for the detection of the biological activity of a protein in a pharmaceutical gel according to claim 2, characterized in that said DMEM test broth is selected from DMEM test broths comprising 1-2% fetal bovine serum and 10-30ug/ml heparin sodium.
4. The method for detecting the biological activity of the protein in the pharmaceutical gel according to claim 1, wherein 1ml to 8ml of DMEM test culture solution is added to each gram of the gel to be detected.
5. The method for detecting biological activity of protein in pharmaceutical gel according to claim 1, wherein the releasing agent is prepared by dissolving calcium salt in deionized water or purified water.
6. The method for detecting the biological activity of the protein in the pharmaceutical gel according to claim 1, wherein 0.2ml to 1.0ml of release agent is added to each gram of the gel to be detected.
7. The method for detecting biological activity of protein in pharmaceutical gel according to claim 5, wherein the calcium salt is selected from water-soluble calcium salt, preferably one or more of calcium chloride, calcium sulfate, calcium nitrate and calcium bicarbonate.
8. The method for detecting biological activity of protein in pharmaceutical gel according to claim 5, wherein the concentration of calcium ions in the aqueous solution containing calcium ions is in the range of: 0.015mol/L to 0.15mol/L.
9. The method for detecting the biological activity of a protein in a pharmaceutical gel according to claim 1, wherein the method is applicable to the detection of the biological activity of a protein in a gel drug using carbomers as gel matrices.
10. Method for the detection of the biological activity of a protein in a pharmaceutical gel according to claim 1, characterized in that it is suitable for the detection of the biological activity of a protein in a protein gel, preferably said protein being selected from the group consisting of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF).
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