CN114159555A - Antigen protein freeze-dried small ball and preparation method thereof - Google Patents
Antigen protein freeze-dried small ball and preparation method thereof Download PDFInfo
- Publication number
- CN114159555A CN114159555A CN202111495288.0A CN202111495288A CN114159555A CN 114159555 A CN114159555 A CN 114159555A CN 202111495288 A CN202111495288 A CN 202111495288A CN 114159555 A CN114159555 A CN 114159555A
- Authority
- CN
- China
- Prior art keywords
- freeze
- antigen protein
- temperature
- solution
- drying
- 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
- 102000036639 antigens Human genes 0.000 title claims abstract description 195
- 108091007433 antigens Proteins 0.000 title claims abstract description 195
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 238000004108 freeze drying Methods 0.000 claims abstract description 86
- 239000000243 solution Substances 0.000 claims abstract description 84
- 239000007788 liquid Substances 0.000 claims abstract description 78
- 239000008188 pellet Substances 0.000 claims abstract description 68
- 239000000427 antigen Substances 0.000 claims abstract description 43
- 239000012460 protein solution Substances 0.000 claims abstract description 32
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 31
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 31
- 230000001681 protective effect Effects 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 16
- 239000011324 bead Substances 0.000 claims description 66
- 238000000034 method Methods 0.000 claims description 41
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- 238000001035 drying Methods 0.000 claims description 23
- 230000000890 antigenic effect Effects 0.000 claims description 21
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
- 230000008569 process Effects 0.000 claims description 18
- 238000007710 freezing Methods 0.000 claims description 11
- 230000008014 freezing Effects 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 229910052751 metal Inorganic materials 0.000 claims description 8
- 239000002184 metal Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 150000001298 alcohols Chemical class 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 230000000975 bioactive effect Effects 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 150000001720 carbohydrates Chemical class 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000004094 surface-active agent Substances 0.000 claims description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 29
- 238000001514 detection method Methods 0.000 abstract description 22
- 238000003860 storage Methods 0.000 abstract description 8
- 238000012546 transfer Methods 0.000 abstract description 6
- 238000005070 sampling Methods 0.000 abstract description 5
- 238000002347 injection Methods 0.000 description 33
- 239000007924 injection Substances 0.000 description 33
- 239000004005 microsphere Substances 0.000 description 24
- 230000000694 effects Effects 0.000 description 17
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 238000012360 testing method Methods 0.000 description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000006148 magnetic separator Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 230000010355 oscillation Effects 0.000 description 6
- 239000003223 protective agent Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 5
- 239000004471 Glycine Substances 0.000 description 5
- 229930195725 Mannitol Natural products 0.000 description 5
- 229920001213 Polysorbate 20 Polymers 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000018044 dehydration Effects 0.000 description 5
- 238000006297 dehydration reaction Methods 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 239000000594 mannitol Substances 0.000 description 5
- 235000010355 mannitol Nutrition 0.000 description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 5
- 238000003825 pressing Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 4
- 239000002202 Polyethylene glycol Substances 0.000 description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 229920001223 polyethylene glycol Polymers 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 101000687905 Homo sapiens Transcription factor SOX-2 Proteins 0.000 description 3
- 102100024270 Transcription factor SOX-2 Human genes 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 241000283074 Equus asinus Species 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000004067 bulking agent Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- -1 for example Proteins 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Inorganic Chemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides an antigen protein freeze-dried pellet, which comprises a freeze-dried protective solution and an antigen protein solution, wherein the antigen protein freeze-dried pellet is obtained by mixing the freeze-dried protective solution and the antigen protein solution and then freeze-drying, the volume ratio of the freeze-dried protective solution to the antigen protein solution is 5:1-9:1, the antigen protein freeze-dried pellet is in a spherical structure, so that the antigen protein freeze-dried pellet can be directly mixed with a sample to be tested for use, the deviation of a detection result caused by sampling deviation in a liquid state is avoided, the antigen protein freeze-dried pellet is in a pellet shape and is not adhered to a tube wall, transfer can be realized, particularly when a plurality of different antigen proteins are mixed for use, the antigen protein freeze-dried pellets can be rapidly combined for use according to actual requirements, and the pellet shape is more beneficial to transportation and storage. The invention also provides a preparation method of the antigen protein freeze-dried pellet, the preparation process is simple and convenient, the spherical original protein freeze-dried pellet is ensured to be prepared, and the stability and the storage life are improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antigen protein freeze-dried pellet and a preparation method thereof.
Background
Antigen is a substance that can stimulate the body to produce immune response or specific immune response, and can combine with the immune response product antibody and sensitized lymphocytes in vivo and in vitro to produce immune effect or specific reaction.
At present, in the domestic market, the antigen protein freeze-dried product is mainly prepared into powder and is less in preparation into pellets. The antigenic proteins lyophilized to powder have the following drawbacks: (1) the test tube is easy to adhere to the tube wall, and incomplete redissolution can occur during redissolution, so that the result test is inaccurate; (2) a plurality of antigen proteins are mixed and freeze-dried into powder, and in the freeze-dried powder, the difference of the amount of each antigen protein is large, so that the detection of a reagent is inconvenient.
Chinese patent with publication number CN111110638A discloses a freeze-dried microsphere preparation coupled with protein, a preparation method and a storage mode thereof, wherein the freeze-dried microsphere preparation comprises a freeze-drying protective agent and microspheres coupled with protein; the product is obtained by mixing microspheres coupled with antibodies or antigens and a freeze-drying protective agent and then freeze-drying, wherein the proteins in the microspheres coupled with proteins are antibodies or antigens, for example, SOX2 antigen protein (GenScript company) with a c-Myc label at the N end is coupled with MagPlex magnetic microspheres fluorescently encoded by Luminex company, and the preparation process comprises the following steps: (1) activating the microspheres and taking out
The microspheres are subjected to vortex oscillation and ultrasonic treatment for 20s, so that the microspheres are fully mixed, uniformly mixed and resuspended, and then an appropriate volume of microsphere suspension is sucked into a centrifugal tube and placed in the centrifugal tube30s-60s on a magnetic separator, remove the supernatant and add 500. mu.L of activation buffer (O.1M NaH)2P04pH 6.2), placing the microspheres on a magnetic separator for separating the microspheres after vortex oscillation and ultrasonic resuspension, repeating the step for 1-2 times, adding 480 mu L of activation buffer solution into the cleaned microspheres, adding 10 mu L of NHS and 10 mu L of EDC once after vortex oscillation and ultrasonic mixing, and incubating for 20-30min at 300rpm in a dark place at room temperature on a rotary mixer; (2) coupling SOX2 protein to microspheres, placing the activated microspheres on a magnetic separator for separation for 60s, removing the supernatant, repeating the step for 2 times, adding 500 mu L of diluted SOX2 fusion protein (diluted to 10 mu g/mL by coupling buffer) into the activated microspheres, performing vortex oscillation and ultrasonic treatment, placing on a rotary mixer for 300rpm dark room temperature incubation for 2h, placing on the magnetic separator for 60s, and removing the supernatant; (3) washing the microspheres, adding 500 μ L of washing solution (10mM PBS, 0.05% Tween-20) into the centrifuge tube, performing vortex oscillation and ultrasonic resuspension on the coupled microspheres, and placing on a magnetic separator for 60s to remove supernatant; (4) and (3) sealing the microspheres, adding 500 mu L of sealing buffer solution (10mM PBS, 5% donkey serum and 0.03% Prolin300) into the cleaned microspheres, performing vortex oscillation and ultrasonic treatment, performing dark incubation for 30min at 300rpm on a rotary mixer at room temperature, placing the microspheres on a magnetic separator for 60s, removing supernatant, adding 500 mu L of washing solution, cleaning, and removing the washing solution on the magnetic separator. The microsphere freeze-dried preparation coupled with the antibody or the antigen exists in a powder form, and a freeze-dried sample is easily adhered to the inner wall of a container of a freeze-dried product and cannot be subjected to solid state transfer.
Therefore, there is a need to provide a novel lyophilized pellet of antigenic protein and a method for preparing the same to solve the above-mentioned problems in the prior art.
Disclosure of Invention
The invention aims to provide an antigen protein freeze-dried pellet and a preparation method thereof, which are used for overcoming the defects of adhesion of a powder preparation to a tube wall, incomplete redissolution and uneven mixing of multiple antigens.
In order to achieve the purpose, the antigen protein freeze-dried pellet comprises a freeze-dried protective solution and an antigen protein solution, wherein the antigen protein freeze-dried pellet is obtained by mixing the freeze-dried protective solution and the antigen protein solution and then freeze-drying the mixture, and the volume ratio of the freeze-dried protective solution to the antigen protein solution is 5:1-9: 1.
The antigen protein freeze-dried pellet has the beneficial effects that: the antigen protein freeze-dried globule is obtained by mixing the freeze-dried protective solution and the antigen protein solution and then freeze-drying, the two components are both solutions, the even mixing is easier, and the process is simpler and more convenient than the process of coupling the antibody or the antigen microspheres; the volume ratio of the freeze-drying protection solution to the antigen protein solution is 5:1-9:1, so that the freeze-drying protection solution can be fully mixed with the antigen protein solution, the antigen protein can be fully coated and protected by the freeze-drying protection solution after freeze-drying, the antigen protein freeze-drying globules with a globular structure are obtained, the antigen protein freeze-drying globules can be directly mixed with a sample to be tested for use, the deviation of a detection result caused by sampling deviation in a liquid state is avoided, the antigen protein freeze-drying globules are in a globule shape and are not adhered to the tube wall, solid transfer can be realized, particularly when a plurality of different antigen proteins are mixed for use, the antigen protein freeze-drying globules can be rapidly combined for use according to actual requirements, meanwhile, the change from the liquid state to the solid state is realized by the globule shape, and the transportation and the storage are facilitated.
Preferably, the diameter of the antigen protein freeze-dried bead is 2.5-3.0 mm. The beneficial effects are that: the small balls cannot be adhered to the inner wall of the container, so that solid transfer can be realized, and the problems of incomplete redissolution and uneven mixing of multiple antigens caused by adhesion of a powder preparation to the wall of the container are avoided; the spherical shape can be directly mixed with a sample to be tested for use, so that the deviation of a detection result caused by sampling deviation of a liquid state is avoided; the preparation process is simpler and more convenient than that of the microspheres coupled with the antibody or the antigen.
Preferably, the freeze-drying protection solution is a mixed solution, and comprises alcohols, amino acids, high molecular compounds, bioactive proteins, surfactants, saccharides, salt substances and a buffer system. The beneficial effects are that: the freeze-drying protective solution is suitable for different antigen proteins, and has good universality.
Preferably, the lyoprotectant solution comprises:
preferably, the pH value of the freeze-drying protection solution is 7.2-7.4. The beneficial effects are that: effectively ensuring the stability of the antigen protein.
Preferably, the invention also provides a preparation method of the antigen protein freeze-dried pellet, which comprises the following steps:
s1: providing an antigen protein solution and preparing a freeze-drying protection solution;
s2: uniformly mixing the freeze-drying protection solution and the antigen protein solution according to the volume ratio of 5:1-9:1 to prepare an antigen protein freeze-drying original solution;
s3: dropping the antigen protein freeze-drying original solution into liquid nitrogen through a bead freezing device, so that the antigen protein freeze-drying original solution in a liquid drop shape is frozen in the liquid nitrogen to form antigen protein frozen beads with spherical structures;
s4: and preparing the antigen protein frozen beads into antigen protein freeze-dried beads by a freeze-drying process.
The preparation method of the antigen protein freeze-dried pellet has the beneficial effects that: by S1: providing an antigen protein solution and formulating a lyoprotectant solution, S2: uniformly mixing the freeze-drying protection solution and the antigen protein solution according to the volume ratio of 5:1-9:1 to prepare an antigen protein freeze-drying original solution, wherein both components are solutions, and are easy to uniformly mix and ensure that the freeze-drying protection solution can be sufficiently mixed with the antigen protein solution, so that the freeze-drying protection solution can fully coat and protect the antigen protein after freeze-drying; by S3: the antigen protein freeze-drying original solution is dripped into liquid nitrogen through a bead freezing device, so that the formation of the antigen protein freeze-drying original solution in a droplet shape is facilitated, and antigen protein frozen beads with spherical structures are frozen in the liquid nitrogen; by S4: preparing the antigen protein frozen beads into antigen protein freeze-dried pellets by a freeze-drying process, enabling the antigen protein freeze-dried pellets to be in a spherical structure, namely transferring the antigen protein freeze-dried pellets into a freeze-drying machine with set parameters for freeze-drying, dehydrating the frozen pellets in the freeze-drying machine and keeping the spherical state, thereby forming the antigen protein freeze-dried pellets, ensuring that the spherical antigen protein freeze-dried pellets are prepared, and improving the stability and the storage life; the preparation process is simple and convenient, the prepared antigen protein freeze-dried pellets can be directly mixed with a sample to be tested for use, the deviation of a detection result caused by sampling deviation in a liquid state is avoided, the antigen protein freeze-dried pellets are in a pellet shape and are not adhered to the tube wall, transfer can be realized, especially when a plurality of different antigen proteins are mixed for use, the antigen protein freeze-dried pellets can be quickly combined for use according to actual requirements, detection is convenient, the problem that powdery antigen proteins are adhered to the tube wall and are incompletely redissolved, meanwhile, the pellet shape realizes the conversion from the liquid state to the solid state, and the transportation and storage are facilitated.
Preferably, the step of preparing the antigen protein frozen beads into antigen protein lyophilized beads by a freeze-drying process in step S4 specifically includes:
s40: starting a freeze dryer, precooling the freeze dryer to a precooling temperature, and placing the antigen protein frozen beads in the freeze dryer;
s41: setting a temperature of a shelf in the freeze dryer for a first stage drying;
s42: adjusting the temperature of the shelf for second stage drying;
s43: adjusting the temperature of the shelf to perform a third stage drying. The beneficial effects are that: drying by stages is beneficial to ensuring the stability of the antigen protein.
Preferably, the step of preparing the lyophilization protection solution in the step S1 includes: and preparing the freeze-drying protective solution according to a preset formula, and adjusting the pH value of the freeze-drying protective solution to 7.2-7.4 by adopting 0.01mol/L hydrochloric acid solution or 0.01mol/L sodium hydroxide solution. The beneficial effects are that: the method is favorable for guaranteeing the stability of the antigen protein, is suitable for being used as freeze-drying protective solutions of different antigen proteins, and has good universality.
Preferably, the parameters of the bead freezing device in the step S3 are set as follows: the advancing speed is 40-60mm/s, the withdrawing speed is 250-320mm/s, and the dropping volume is 9-15 mu L. The beneficial effects are that: the liquid drops can be frozen quickly under the action of liquid nitrogen to form the globular antigen protein frozen beads, and the stability of the antigen protein is guaranteed.
Preferably, in the step S3, the dropping needle of the bead freezing device is disposed above a metal cup containing liquid nitrogen, the outer diameter of the dropping needle is 1.1-1.3mm, the distance between the liquid outlet of the dropping needle and the liquid nitrogen plane in the metal cup is 5-15cm, and the liquid outlet interval of the dropping needle is 10-20 seconds. The beneficial effects are that: the liquid drops can be frozen quickly under the action of liquid nitrogen to form the globular antigen protein frozen beads, and the stability of the antigen protein is guaranteed.
Preferably, the pre-cooling temperature in the step S40 is less than-20 ℃. The beneficial effects are that: avoid destroying the performance of the antigen protein and is beneficial to ensuring the stability of the antigen protein.
Preferably, the step of setting the temperature of the shelf in the freeze dryer to perform the first-stage drying in step S41 specifically includes:
s411: setting the temperature of the shelf to-45 ℃ and maintaining the temperature for 4 h;
s412: setting the temperature of the shelf to-35 ℃, and keeping the temperature for 8-12h after vacuumizing the freeze dryer. The beneficial effects are that: avoid destroying the performance of the antigen protein and is beneficial to ensuring the stability of the antigen protein.
Preferably, the step of adjusting the temperature of the shelf to perform the second stage drying in step S42 specifically includes:
s421: setting the temperature of the shelf to-30 ℃ and keeping the temperature for 8-12 h;
s422: setting the temperature of the shelf to-25 ℃ and keeping the temperature for 8-12 h;
s423: setting the temperature of the shelf to-20 ℃ and keeping the temperature for 1-2 h;
s424: setting the temperature of the shelf to-10 ℃ and keeping the temperature for 1-2 h;
s425: the temperature of the shelf is set to 0 ℃ and maintained for 1-8 h. The beneficial effects are that: avoid destroying the performance of the antigen protein and is beneficial to ensuring the stability of the antigen protein.
Preferably, the step of adjusting the temperature of the shelf to perform the third stage drying in step S43 specifically includes:
s431: setting the temperature of the shelf to 10 ℃ and keeping the temperature for 1-2 h;
s432: the temperature of the shelf is set to 25 ℃ and maintained for 2-8 h. The beneficial effects are that: avoid destroying the performance of the antigen protein and is beneficial to ensuring the stability of the antigen protein.
Preferably, the vacuum degree formed by the vacuum pumping of the freeze dryer in the step S412 is less than 20 pa. The beneficial effects are that: avoid destroying the performance of the antigen protein and is beneficial to ensuring the stability of the antigen protein.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing lyophilized beads of antigenic protein according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a frozen bead apparatus according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of comparison results obtained by respectively detecting the homemade lyophilized pellet prepared by the method for preparing the antigen protein lyophilized pellet and the BD reagent pellet with a detection reagent in a kit for human isolated whole blood samples.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. As used herein, the word "comprising" and similar words are intended to mean that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.
In order to overcome the problems in the prior art, the embodiment of the invention provides the antigen protein freeze-dried pellet and the preparation method thereof, so that the antigen protein freeze-dried pellet can be directly mixed with a sample to be tested for use, the deviation of a test result caused by liquid state sampling deviation is avoided, the antigen protein freeze-dried pellet is in a pellet shape and is not adhered to a tube wall, the transfer can be realized, particularly, when a plurality of different antigen proteins are mixed for use, the antigen protein freeze-dried pellet can be quickly combined for use according to actual requirements, the detection is convenient, the problem that powdery antigen protein is adhered to the tube wall and is incompletely redissolved when the antigen protein freeze-dried pellet is redissolved, and meanwhile, the pellet shape realizes the conversion from liquid state to solid state, and is more favorable for transportation and storage.
The test sample of the present invention is essentially an isolated whole blood sample of a human.
In some embodiments of the present invention, the antigen protein lyophilized pellet includes a lyophilization protection solution and an antigen protein solution, the antigen protein lyophilized pellet is obtained by mixing the lyophilization protection solution and the antigen protein solution and then lyophilizing, a volume ratio of the lyophilization protection solution to the antigen protein solution is 5:1-9:1, and the shape of the prepared antigen protein lyophilized pellet is a spherical structure. Make antigen albumen freeze-drying bobble can with receive this direct mixed use of sample, avoided liquid because of the sample deviation, the deviation of the testing result that leads to, and antigen albumen freeze-drying bobble is the bobble form, it is not sticky with the pipe wall, can realize shifting, especially when the mixed use of a plurality of different antigen albumen, can make up the use fast according to the actual demand, be convenient for detect, powdered antigen albumen is glued and is linked the pipe wall, the incomplete problem of redissolution can appear during the redissolution, the bobble form has realized by liquid to solid-state conversion simultaneously, more do benefit to transportation and save.
In some embodiments of the invention, the antigenic protein lyophilized pellet has a diameter of 2.5-3.0 mm.
In some embodiments of the invention, the freeze-drying protection solution is a mixed solution, and comprises alcohols, amino acids, high molecular compounds, bioactive proteins, surfactants, saccharides, salt substances and a buffer system, so that the stability of the antigen proteins is guaranteed, and the freeze-drying protection solution of the components is suitable for different antigen proteins and has good universality.
In some embodiments of the present invention, the antigen protein solution is prepared by conventional methods in the art, and the antigen refers to a substance that can stimulate the body to generate a specific or immune response, and can be combined with the immune response product antibody and sensitized lymphocytes in vitro and in vivo to generate an immune effect or a specific reaction.
In some embodiments of the invention, the antigenic protein solution is a common cytokine, IL-2, IL-6, IL-10, TNF- α, or the like.
The reagents and starting materials used in the present invention are commercially available.
In some embodiments of the invention, the pH value of the freeze-drying protection solution is 7.2-7.4, which effectively ensures the stability of the antigen protein.
FIG. 1 is a schematic flow chart of a method for preparing lyophilized beads of antigenic protein according to an embodiment of the present invention.
In some embodiments of the present invention, referring to fig. 1, the method for preparing the antigen protein lyophilized pellet comprises the following steps:
s1: providing an antigen protein solution and preparing a freeze-drying protection solution;
s2: uniformly mixing the freeze-drying protection solution and the antigen protein solution according to the volume ratio of 5:1-9:1 to prepare an antigen protein freeze-drying original solution;
s3: dropping the antigen protein freeze-drying original solution into liquid nitrogen through a bead freezing device, so that the antigen protein freeze-drying original solution in a liquid drop shape is frozen in the liquid nitrogen to form antigen protein frozen beads with spherical structures;
s4: and preparing the antigen protein frozen beads into antigen protein freeze-dried beads by a freeze-drying process, so that the antigen protein freeze-dried beads are in a spherical structure.
In some embodiments of the present invention, after the step S4 is executed, the method further includes the steps of: drying, taking out, filling into a penicillin bottle, and sealing with a cover.
In some embodiments of the present invention, the step of preparing the lyoprotectant solution in step S1 includes: the freeze-drying protection solution is prepared according to a preset formula, and the pH value of the freeze-drying protection solution is adjusted to 7.2-7.4 by adopting 0.01mol/L hydrochloric acid solution or 0.01mol/L sodium hydroxide solution, so that the stability of the antigen protein is ensured, and the freeze-drying protection solution is suitable for being used as freeze-drying protection solutions of different antigen proteins, and has good universality.
In some embodiments of the present invention, the predetermined formulation, i.e., the lyoprotectant solution, includes:
specifically, the mannitol and the glycine are commonly used bulking agents for freeze-dried protein preparations and have the characteristic of no toxicity. Mannitol is used as a good excipient, has no hygroscopicity, is quick to freeze, can prevent the effective components from sublimating along with water vapor in the freeze-drying process, and enables the effective components to be formed; the glycine can increase collapse temperature of frozen products, stabilize protein, and is a good excipient
The polyethylene glycol plays a role of a low-temperature protective agent and a dehydration protective agent, and simultaneously plays a role of accelerating reaction and improving the sensitivity of the reagent in immune reaction;
the bovine serum albumin plays a role in protein protection, plays a role in a filler in the dehydration and drying process, and can play a role in controlling background and eliminating false positive in an immunoreagent;
the Tween 20 is used for reducing freezing and dehydration deformation caused by ice water interfacial tension in the freeze-drying process, playing the roles of a wetting agent and a refolding agent on active components in the rehydration process, and reducing non-specific reaction in the immunoreaction process;
the trehalose or sucrose plays a role of low-temperature protection in the freezing process and plays a role of a dehydration protective agent in the drying and dehydration process;
the fetal calf serum is used for providing binding protein, can identify vitamins, lipids, metals, other hormones and the like, can combine or modulate the activity of substances combined by the vitamins, the lipids, the metals and the other hormones, and has the function of a pH buffer solution;
the sodium chloride is used for preventing the reagent from keeping a certain ionic strength in the freeze-drying process and the storage process, and is beneficial to the stability of antigen protein after redissolution;
the phosphate buffer solution has the function of keeping the pH value of the solution stable.
Examples 1-5 preparation of lyoprotectant solutions: weighing the mannitol, the glycine, the polyethylene glycol, the tween 20, the trehalose, the sodium chloride and the phosphate buffer according to the components and the content in the table 1, adding the weighed mannitol, the glycine, the polyethylene glycol, the tween 20, the trehalose, the sodium chloride and the phosphate buffer into 800mL of purified water, stirring and dissolving, weighing the bovine serum albumin and the fetal bovine serum, adding the bovine serum into the solution, uniformly mixing, using the purified water, fixing the volume of the prepared solution to 1L, and finally filtering the solution by using a 0.22 mu m filter membrane. In Table 1, "/" means that the component is not contained.
TABLE 1 composition and content of lyoprotectant solution
| Example 1 | Example 2 | Example 3 | Example 4 | Example 5 | |
| Mannitol (g/L) | 10 | 20 | 30 | 40 | 50 |
| Glycine (g/L) | 1 | 5 | 10 | 15 | 20 |
| Polyethylene glycol (g/L) | 10 | 25 | 50 | 75 | 100 |
| Bovine serum albumin (g/L) | 10 | 25 | 50 | 75 | 100 |
| Tween 20(mL/L) | 0.5 | 1.5 | 2.75 | 3.5 | 5 |
| Trehalose (g/L) | 10 | / | 15 | / | 30 |
| Sucrose (g/L) | / | 10 | / | 30 | / |
| Fetal bovine serum (mL/L) | 5 | 15 | 27.5 | 35 | 50 |
| Sodium chloride (g/L) | 5 | 10 | 12.5 | 15 | 20 |
| Phosphate buffer (mol/L) | 0.01 | 0.025 | 0.05 | 0.075 | 0.1 |
| pH | 7.4±0.05 | 7.4±0.05 | 7.4±0.05 | 7.4±0.05 | 7.4±0.05 |
Effect examples 1 to 5: the lyophilized protectant prepared according to the embodiments 1-5 is respectively mixed with the antigen protein solution according to the volume ratio of 5:1, and then lyophilized to obtain 5 groups of antigen protein lyophilized beads containing lyophilized protectants with different formulas. Among them, the antigen protein solution in effect examples 1 to 5 was prepared by the method comprising: antigen protein is prepared into antigen protein solution with the concentration of 4-10 mug/mL by using protein diluent (0.01mol/L phosphate buffer solution, the pH value is 7.2 +/-0.2 and 1 percent of bovine serum albumin is contained). The 5 groups of lyophilized beads of the antigen protein containing different lyoprotectants were then analyzed for accelerated stability by accelerated stability assay at 37 ℃ under high temperature conditions.
Specifically, the accelerated stability test method comprises the following steps: effect examples 1-5 the home-made antigen protein lyophilized beads that were just lyophilized were tested 3 each, and the test results, i.e., lyophilized bead test value (ng/bead), were recorded. At least 12 of the home-made lyophilized beads of the antigen protein as just lyophilized in effect examples 1-5 were then placed in a 37 ℃ incubator. Then, 3 of the antigen protein lyophilized beads of effect examples 1 to 5 were each taken out from a 37 ℃ incubator on day 3, day 5, day 7, and day 9, respectively, and the detection results were recorded. The specific test results are shown in Table 2. The reagents and starting materials used in Table 2 are commercially available.
Wherein, CV in table 2 refers to the coefficient of variation of the current experimental result of this embodiment, the mean refers to the detection mean of the current experimental result of this embodiment, and the P-point ratio refers to the ratio of the detection mean of the current experimental result of this embodiment to the detection mean of the 0 th day of high-temperature acceleration at 37 ℃.
TABLE 2 Effect of freeze-drying preservation Effect of antigen protein lyophilized pellets of examples 1 to 5
Referring to table 2, it can be seen that the P-spot ratios of the antigen protein lyophilized beads of effect examples 1-5 on days 3, 5, 7 and 9 stored at a high temperature of 37 ℃ are decreased, but the P-spot ratios of the antigen protein lyophilized beads of effect examples 1-5 on day 9 are all greater than 92%. It is demonstrated that the stability of the sample is still kept above 92% activity on day 9 under the accelerated stability examination at 37 ℃, and the stability of the antigen protein freeze-dried beads prepared by the freeze-drying protective agent with different formulations in examples 1-5 is not greatly different.
In some embodiments of the present invention, the parameters of the frozen bead device in the step S3 are set as follows: the forward speed is 40-60mm/s, the backward speed is 250-320mm/s, and the drop volume is 9-15 mu L, which is favorable for the drop to be rapidly frozen under the action of liquid nitrogen to form a small spherical antigen protein frozen bead and is favorable for improving the stability of antigen protein.
In some embodiments of the present invention, the dropping needle of the bead freezing device in step S3 is disposed above a metal cup containing liquid nitrogen, the outer diameter of the dropping needle is 1.1-1.3mm, the distance between the liquid outlet of the dropping needle and the liquid nitrogen plane in the metal cup is 5-15cm, and the liquid outlet interval of the dropping needle is 10-20 seconds, which is beneficial to rapidly freezing the liquid drop under the action of the liquid nitrogen to form a globular antigen protein frozen bead, and is beneficial to improving the stability of the antigen protein.
FIG. 2 is a schematic structural diagram of a frozen bead apparatus according to an embodiment of the present invention.
In some embodiments of the invention, the frozen bead apparatus comprises two freeze-drying stations, a robot, a control panel, and control buttons. Referring to fig. 2, the two freeze-drying stations are a first freeze-drying station 1 and a second freeze-drying station 2, and a switch can be selected according to requirements. The manipulator 3 is fixed in horizontal guide post 4, manipulator 3 with horizontal guide post 4 swing joint and perpendicular setting, manipulator 3 can move along the X axle direction on the horizontal guide post 4 to and remove along the Z axle direction, the bottom of manipulator 3 is provided with annotates liquid needle 6. The X-axis direction is along the length direction of the transverse guide post 4, and the Z-axis direction is perpendicular to the X-axis direction. The control buttons include an emergency stop button 7, a reset button 8, a start button 9, a power switch 10 and a power indicator light 11. The control panel 5 is provided with a plurality of functional modules.
Specifically, the functional module includes:
the target cycle number module is used for setting cycle number of liquid injection, one cycle of liquid injection is respectively completed on the first freeze-drying station and the second freeze-drying station, and multiple cycles can be set if multiple times of liquid injection are needed;
a current cycle number module for displaying the currently completed cycle number;
the cycle delay module is used for setting the delay time between adjacent cycles of the multiple cycles;
the automatic starting module is used for pressing the starting button 9 until the starting button turns green, the automatic starting module in the control panel 5 is pressed, and the liquid injection needle in the frozen bead equipment can automatically move to perform liquid injection under the driving of the manipulator;
the automatic stopping module can stop the frozen bead equipment when the automatic operation needs to be suspended or the emergency stopping button 7 is pressed in an emergency, and the automatic stopping module is reset or the emergency stopping button 7 is released and then the automatic starting module is pressed to start again after the treatment is finished;
the X-axis picture module is used for entering an X-axis position parameter setting picture;
the Z-axis picture module is used for entering a Z-axis position parameter setting picture;
the liquid injection picture module is used for entering a liquid injection pump picture parameter setting picture;
and the alarm picture module is used for entering an alarm recording picture, displaying the alarm recording picture in a touch screen of the control panel 5 through a scroll bar during real-time alarm, and resetting by pressing a panel reset button, wherein if the alarm recording picture is not reset, the power supply needs to be restarted.
The X-axis picture module includes:
a first current position unit, configured to set a current position of a transverse guide column 4 of the manipulator 3 in the X-axis direction;
the first inching speed unit is used for setting the moving speed of the manipulator 3 moving left and right on the transverse guide post 4;
a first automatic speed unit for setting the speed of the manipulator 3 when walking on the transverse guide post 4 and automatically producing;
the first waiting position unit is used for setting the position of the manipulator 3 after finishing the action, so that the best waiting position for next liquid injection is convenient;
the first liquid injection unit is used for arranging the position of the manipulator 3 for liquid injection at the first freeze-drying station 1;
and the second liquid injection unit is used for arranging the position of the manipulator 3 for injecting liquid in the second freeze-drying station 2.
The manipulator is moved manually to find a proper liquid injection position, and adaptive parameters are input into corresponding modules and units of the control panel according to the displayed numerical value of the current position.
The Z-axis picture module includes:
a second current position unit, configured to set a current position of the manipulator 3 in the Z-axis direction;
a second jog speed unit for setting a moving speed at which the manipulator 3 moves up and down in the Z-axis direction;
a second automatic speed unit for setting a speed of the manipulator 3 when it is moved in the Z-axis direction and automatically produced;
the second waiting unit is used for setting the manipulator 3 to move in the X-axis direction so that the liquid injection needle 6 does not touch the height of the freeze-drying station;
and the third liquid injection unit is used for setting the height of the liquid injection needle 6 extending into the first freeze-drying station 1 and the second freeze-drying station 2, and is set according to the product process.
The liquid injection picture module comprises:
the liquid injection amount setting unit is used for inputting the required liquid injection amount according to the actual requirement, for example, 15UL needs to be injected, the liquid injection amount is input by 15, the liquid injection is started by pressing down, the liquid injection is carried out once, the injected amount is weighed, and the amount is reduced or increased and can be modified through liquid amount compensation;
the advancing speed setting unit is used for setting the advancing speed of the liquid injection pump during liquid injection;
the backward speed setting unit is used for setting the backward speed of the liquid injection pump during liquid injection;
the retraction distance setting unit is used for setting the retraction distance of the liquid injection needle under the condition of liquid hanging, starting the retraction function and setting the retraction distance according to the actual condition;
the standby time delay setting unit is used for pressing a standby mode under the condition that equipment is not produced, the pump can run according to set time, and a liquid injection valve in the standby mode is closed and cannot inject liquid onto the platform;
the liquid injection frequency setting unit is used for setting the operation liquid injection frequency of the metering pump and setting the specific number of turns;
the current frequency display unit displays the current operating liquid injection frequency of the metering pump, only makes reference, and cannot set;
and the liquid injection starting unit is used for debugging parameters under the current picture, starting the liquid injection by pressing the liquid injection pump once.
In some embodiments of the present invention, the step of preparing the antigen protein frozen beads into antigen protein lyophilized beads by a freeze-drying process in step S4 specifically includes:
s40: starting a freeze dryer, precooling the freeze dryer to a precooling temperature, and placing the antigen protein frozen beads in the freeze dryer;
s41: setting a temperature of a shelf in the freeze dryer for a first stage drying;
s42: adjusting the temperature of the shelf for second stage drying;
s43: adjusting the temperature of the shelf to perform a third stage drying. Drying by stages is beneficial to improving the stability of the antigen protein.
In some embodiments of the present invention, it is preferable that the pre-cooling temperature in the step S40 is less than-20 ℃. Avoid destroying the performance of the antigen protein and is beneficial to improving the stability of the antigen protein.
In some embodiments of the present invention, the step of setting the temperature of the shelf in the freeze dryer to perform the first stage drying in step S41 specifically includes:
s411: setting the temperature of the shelf to-45 ℃ and maintaining the temperature for 4 h;
s412: setting the temperature of the shelf to-35 ℃, and keeping the temperature for 8-12h after vacuumizing the freeze dryer. Avoid destroying the performance of the antigen protein and is beneficial to improving the stability of the antigen protein.
In some possible embodiments of the present invention, in the step S412, the vacuum degree formed by vacuumizing the freeze dryer is less than 20pa, so as to avoid damaging the performance of the antigen protein, and to facilitate improvement of the stability of the antigen protein.
In some embodiments of the present invention, the step of adjusting the temperature of the shelf to perform the second stage drying in step S42 specifically includes:
s421: setting the temperature of the shelf to-30 ℃ and keeping the temperature for 8-12 h;
s422: setting the temperature of the shelf to-25 ℃ and keeping the temperature for 8-12 h;
s423: setting the temperature of the shelf to-20 ℃ and keeping the temperature for 1-2 h;
s424: setting the temperature of the shelf to-10 ℃ and keeping the temperature for 1-2 h;
s425: the temperature of the shelf is set to 0 ℃ and maintained for 1-8 h. Avoid destroying the performance of the antigen protein and is beneficial to improving the stability of the antigen protein.
In some embodiments of the present invention, the step of adjusting the temperature of the shelf to perform the third stage drying in step S43 specifically includes:
s431: setting the temperature of the shelf to 10 ℃ and keeping the temperature for 1-2 h;
s432: the temperature of the shelf is set to 25 ℃ and maintained for 2-8 h. Avoid destroying the performance of the antigen protein and is beneficial to improving the stability of the antigen protein.
Examples 6 to 10 are preparations of antigen protein lyophilized beads, in which the antigen protein lyophilized beads are obtained by uniformly mixing the lyoprotectant solution prepared according to the components and contents of the lyoprotectant solution shown in example 1 in table 1 and the antigen protein solution in the effect example in the volume ratio shown in table 3, and then lyophilizing the mixture.
TABLE 3 volume ratio of lyoprotectant solution to antigenic protein solution
The 5 groups of lyophilized pellets of antigenic proteins prepared in examples 6-10 were analyzed for accelerated stability by accelerated stability test at 37 ℃ under high temperature conditions. The results of the experiments are shown in Table 4. The accelerated stability test method performed on the antigen protein lyophilized beads prepared in examples 6 to 10 is the same as the accelerated stability test method performed on the antigen protein lyophilized beads prepared in effect examples 1 to 5, and is not described herein again.
TABLE 4 Freeze-drying preservation Effect of antigen protein lyophilized pellets of examples 6 to 10
Referring to table 4, it can be seen that the P-point ratios of the antigen protein lyophilized beads prepared in examples 6 to 10 decreased on days 3, 5, 7 and 9 when they were stored at 37 ℃, but the P-point ratios of the antigen protein lyophilized beads prepared in examples 6 to 10 were all greater than 91% on day 9. It is demonstrated that the stability of the sample is still maintained at more than 91% activity on the 9 th day under the accelerated stability examination at 37 ℃, and the volume ratio of the freeze-drying protection solution to the antigen protein solution in examples 6-10 is different, and the stability of the prepared antigen protein freeze-dried pellets is not greatly different.
FIG. 3 is a schematic diagram of comparison results obtained by respectively detecting the homemade lyophilized pellet prepared by the method for preparing the antigen protein lyophilized pellet and the BD reagent pellet with a detection reagent in a kit for human isolated whole blood samples.
The lyophilized pellets of the antigenic protein prepared in the effect example 1 and the comparative example were subjected to a control experiment. The Kit is a Human Th1/Th2 Cytoke ine Kit II with the cargo number of 551809, wherein the antigen protein freeze-dried beads of the original standard cytokine in the Kit of American BD company are used as a comparative example.
Among them, BD corporation (Becton, Dickinson and Company), a chinese translation is bidi medical instruments ltd, which is one of the largest medical technology companies in the world that produce and sell medical devices, medical systems and reagents.
Comparative example: the method comprises the steps of detecting an original standard substance in a kit of a BD company and a detection reagent in the kit to human whole blood samples in vitro, testing 120 samples and recording detection results. Experimental example: the antigen protein freeze-dried beads of the effect example 1 in the invention are used for replacing the original standard product in the kit of the BD company, the antigen protein freeze-dried beads and the detection reagent in the kit are used for detecting the whole blood sample of human in vitro, 120 samples are tested, and the detection result is recorded. The specific detection method is a conventional means in the field and is not described herein.
The test results of the comparative example and the test results of the experimental example were used to prepare a calibration curve, and the comparison results are shown in fig. 3. Referring to fig. 3, as can be seen from fig. 3, the slope of the "home-made lyophilized pellet" in the embodiment 1 of the present invention is close to "1" as compared with the original standard "BD reagent pellet" in the kit of BD company, and the correlation coefficient r reaches 0.9991, the result of the t test shows that the detection results of the two reagents are consistent, i.e., the detection performance of the lyophilized pellet of the embodiment of the present invention is consistent with that of the control.
The invention also provides an application of the antigen protein freeze-dried globule, and the antigen protein freeze-dried globule is applied to any one of a calibrator of a quantitative detection kit, a quality control product of the quantitative detection kit, preparation of a calibration curve of the quantitative detection kit and quality control of a reagent.
Although the embodiments of the present invention have been described in detail hereinabove, it is apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention as described herein is capable of other embodiments and of being practiced or of being carried out in various ways.
Claims (15)
1. The antigen protein freeze-dried pellet is characterized by comprising a freeze-dried protective solution and an antigen protein solution, wherein the antigen protein freeze-dried pellet is obtained by mixing the freeze-dried protective solution and the antigen protein solution and then freeze-drying the mixture, and the volume ratio of the freeze-dried protective solution to the antigen protein solution is 5:1-9: 1.
2. The lyophilized antigenic protein pellet as claimed in claim 1, wherein said antigenic protein pellet has a diameter of 2.5-3.0 mm.
3. The lyophilized antigenic protein pellet as claimed in claim 1, wherein the lyophilized protective solution is a mixed solution comprising alcohols, amino acids, high molecular compounds, bioactive proteins, surfactants, saccharides, salts and buffer systems.
4. The antigenic protein lyophilized pellet of claim 3, wherein said lyoprotectant solution comprises:
5. the lyophilized pellet of claim 3, wherein the pH of the lyoprotectant solution is 7.2-7.4.
6. The preparation method of the antigen protein freeze-dried pellet is characterized by comprising the following steps:
s1: providing an antigen protein solution and preparing a freeze-drying protection solution;
s2: uniformly mixing the freeze-drying protection solution and the antigen protein solution according to the volume ratio of 5:1-9:1 to prepare an antigen protein freeze-drying original solution;
s3: dropping the antigen protein freeze-drying original solution into liquid nitrogen through a bead freezing device, so that the antigen protein freeze-drying original solution in a liquid drop shape is frozen in the liquid nitrogen to form antigen protein frozen beads with spherical structures;
s4: and preparing the antigen protein frozen beads into antigen protein freeze-dried beads by a freeze-drying process.
7. The method for preparing lyophilized antigenic protein beads according to claim 6, wherein the step of preparing the frozen antigenic protein beads into lyophilized antigenic protein beads by a freeze-drying process in step S4 specifically comprises:
s40: starting a freeze dryer, precooling the freeze dryer to a precooling temperature, and placing the antigen protein frozen beads in the freeze dryer;
s41: setting a temperature of a shelf in the freeze dryer for a first stage drying;
s42: adjusting the temperature of the shelf for second stage drying;
s43: adjusting the temperature of the shelf to perform a third stage drying.
8. The method for preparing lyophilized beads of antigenic protein according to claim 6, wherein the step of preparing the lyoprotectant solution in step S1 comprises: and preparing the freeze-drying protective solution according to a preset formula, and adjusting the pH value of the freeze-drying protective solution to 7.2-7.4 by adopting 0.01mol/L hydrochloric acid solution or 0.01mol/L sodium hydroxide solution.
9. The method for preparing antigen protein lyophilized pellet as claimed in claim 6, wherein the parameters of the frozen bead device in step S3 are set as follows: the advancing speed is 40-60mm/s, the withdrawing speed is 250-320mm/s, and the dropping volume is 9-15 mu L.
10. The method for preparing lyophilized antigenic protein beads according to claim 6, wherein the dropping needle of the freezing device in step S3 is disposed above the metal cup containing liquid nitrogen, the outer diameter of the dropping needle is 1.1-1.3mm, the distance between the liquid outlet of the dropping needle and the liquid nitrogen plane in the metal cup is 5-15cm, and the time interval between the liquid outlet of the dropping needle is 10-20 seconds.
11. The method for preparing lyophilized beads of antigenic protein according to claim 7, wherein said pre-cooling temperature in step S40 is less than-20 ℃.
12. The method for preparing an antigenic protein lyophilized pellet as claimed in claim 11, wherein the step of setting the temperature of the shelf in the lyophilizer to perform the first stage drying in step S41 specifically comprises:
s411: setting the temperature of the shelf to-45 ℃ and maintaining the temperature for 4 h;
s412: setting the temperature of the shelf to-35 ℃, and keeping the temperature for 8-12h after vacuumizing the freeze dryer.
13. The method for preparing an antigen protein lyophilized pellet as claimed in claim 12, wherein the step of adjusting the temperature of the shelf to perform the second stage drying in step S42 specifically comprises:
s421: setting the temperature of the shelf to-30 ℃ and keeping the temperature for 8-12 h;
s422: setting the temperature of the shelf to-25 ℃ and keeping the temperature for 8-12 h;
s423: setting the temperature of the shelf to-20 ℃ and keeping the temperature for 1-2 h;
s424: setting the temperature of the shelf to-10 ℃ and keeping the temperature for 1-2 h;
s425: the temperature of the shelf is set to 0 ℃ and maintained for 1-8 h.
14. The method for preparing an antigen protein lyophilized pellet as claimed in claim 13, wherein the step of adjusting the temperature of the shelf to perform the third stage drying in step S43 specifically comprises:
s431: setting the temperature of the shelf to 10 ℃ and keeping the temperature for 1-2 h;
s432: the temperature of the shelf is set to 25 ℃ and maintained for 2-8 h.
15. The method for preparing lyophilized beads of antigenic protein as claimed in claim 12, wherein the vacuum degree of said lyophilizer in step S412 is less than 20 pa.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111495288.0A CN114159555A (en) | 2021-12-08 | 2021-12-08 | Antigen protein freeze-dried small ball and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111495288.0A CN114159555A (en) | 2021-12-08 | 2021-12-08 | Antigen protein freeze-dried small ball and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114159555A true CN114159555A (en) | 2022-03-11 |
Family
ID=80484576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111495288.0A Pending CN114159555A (en) | 2021-12-08 | 2021-12-08 | Antigen protein freeze-dried small ball and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114159555A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116087503A (en) * | 2023-01-08 | 2023-05-09 | 杭州联科生物技术股份有限公司 | Method for realizing ELISA simple operation |
| CN117159480A (en) * | 2023-11-01 | 2023-12-05 | 江西赛基生物技术有限公司 | Recombinant human IFN-gamma protein freeze-dried pellet and preparation method and application thereof |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1163102A (en) * | 1996-03-07 | 1997-10-29 | 阿克佐诺贝尔公司 | Container with freeze-dried vaccine component |
| US20030059766A1 (en) * | 1999-07-14 | 2003-03-27 | Susan Goertz | Preparation of spheres for diagnostic tests |
| CN1409758A (en) * | 1999-12-14 | 2003-04-09 | 热生物之星公司 | Stabilizing diluent for polypeptides and antigens |
| CN1798575A (en) * | 2003-04-04 | 2006-07-05 | 健泰科生物技术公司 | High concentration antibody and protein formulations |
| WO2013041542A1 (en) * | 2011-09-19 | 2013-03-28 | Alf Lamprecht | Pharmaceutical formulations comprising spherolyophilisates of biological molecules |
| CN104428004A (en) * | 2012-07-10 | 2015-03-18 | 英特维特国际股份有限公司 | A method to produce a medicinal product comprising a biologically active protein and the resulting product |
| CN104958759A (en) * | 2015-06-24 | 2015-10-07 | 天津康希诺生物技术有限公司 | Multivalent meningococcus preparation box, vaccine preparation and preparation method thereof |
| CN110452972A (en) * | 2018-05-08 | 2019-11-15 | 北京中科生仪科技有限公司 | A kind of freeze-drying microballoon of nucleic acid amplification reaction reagent and preparation method thereof |
| CN111110638A (en) * | 2020-01-10 | 2020-05-08 | 广州市丹蓝生物科技有限公司 | Protein-coupled microsphere freeze-dried preparation and preparation method and storage mode thereof |
| CN111351318A (en) * | 2020-03-07 | 2020-06-30 | 苏州德沃生物技术有限公司 | Freeze-drying method of antibody coupling latex |
| CN112074294A (en) * | 2018-04-30 | 2020-12-11 | 默沙东公司 | Method for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugate from freeze-dried spheres |
-
2021
- 2021-12-08 CN CN202111495288.0A patent/CN114159555A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1163102A (en) * | 1996-03-07 | 1997-10-29 | 阿克佐诺贝尔公司 | Container with freeze-dried vaccine component |
| US20030059766A1 (en) * | 1999-07-14 | 2003-03-27 | Susan Goertz | Preparation of spheres for diagnostic tests |
| CN1409758A (en) * | 1999-12-14 | 2003-04-09 | 热生物之星公司 | Stabilizing diluent for polypeptides and antigens |
| CN1798575A (en) * | 2003-04-04 | 2006-07-05 | 健泰科生物技术公司 | High concentration antibody and protein formulations |
| WO2013041542A1 (en) * | 2011-09-19 | 2013-03-28 | Alf Lamprecht | Pharmaceutical formulations comprising spherolyophilisates of biological molecules |
| CN104428004A (en) * | 2012-07-10 | 2015-03-18 | 英特维特国际股份有限公司 | A method to produce a medicinal product comprising a biologically active protein and the resulting product |
| CN104958759A (en) * | 2015-06-24 | 2015-10-07 | 天津康希诺生物技术有限公司 | Multivalent meningococcus preparation box, vaccine preparation and preparation method thereof |
| CN112074294A (en) * | 2018-04-30 | 2020-12-11 | 默沙东公司 | Method for producing streptococcus pneumoniae capsular polysaccharide carrier protein conjugate from freeze-dried spheres |
| CN110452972A (en) * | 2018-05-08 | 2019-11-15 | 北京中科生仪科技有限公司 | A kind of freeze-drying microballoon of nucleic acid amplification reaction reagent and preparation method thereof |
| CN111110638A (en) * | 2020-01-10 | 2020-05-08 | 广州市丹蓝生物科技有限公司 | Protein-coupled microsphere freeze-dried preparation and preparation method and storage mode thereof |
| CN111351318A (en) * | 2020-03-07 | 2020-06-30 | 苏州德沃生物技术有限公司 | Freeze-drying method of antibody coupling latex |
Non-Patent Citations (3)
| Title |
|---|
| 刘微，等: "幽门螺杆菌全菌蛋白抗原壳聚糖微球体外释放特性", vol. 15, no. 38, pages 7085 - 7089 * |
| 王朝瑾，等: "《水生产物流通与加工贮藏技术》", 上海科学技术出版社, pages: 67 - 72 * |
| 王梅，等: "鼠疫F1抗原血球冻干保护剂的筛选研究", vol. 36, no. 5, pages 409 - 411 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116087503A (en) * | 2023-01-08 | 2023-05-09 | 杭州联科生物技术股份有限公司 | Method for realizing ELISA simple operation |
| CN117159480A (en) * | 2023-11-01 | 2023-12-05 | 江西赛基生物技术有限公司 | Recombinant human IFN-gamma protein freeze-dried pellet and preparation method and application thereof |
| CN117159480B (en) * | 2023-11-01 | 2024-03-01 | 江西赛基生物技术有限公司 | Recombinant human IFN-gamma protein freeze-dried pellet and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114159555A (en) | Antigen protein freeze-dried small ball and preparation method thereof | |
| US6890512B2 (en) | Methods of preventing aggregation of various substances upon rehydration or thawing and compositions obtained thereby | |
| CN111965344B (en) | Freeze-dried magnetic particle chemiluminescence immunoassay kit and preparation method thereof | |
| CN104428675B (en) | For detection property immediately (POCT) immunochromatographiassays assays lyophilizing conjugate structure body, comprise its immunity detection reagent, and use the method that this test kit is analyzed | |
| Young et al. | Radioimmunoassay of secretin in human serum | |
| CN104204802A (en) | Immunochromatography detection method | |
| JPH05500854A (en) | Improved immunoassays containing lyophilized reaction reagent mixtures | |
| CN104215753B (en) | A kind of concentration CO2 combining power, CO2 CP detectable lyophilizing microsphere and preparation method thereof | |
| CN213023169U (en) | Single-person freeze-dried chemiluminescence reagent immunological reagent tube | |
| CN111110638A (en) | Protein-coupled microsphere freeze-dried preparation and preparation method and storage mode thereof | |
| CN103764028B (en) | Solidification controlling agent and the device comprising solidification controlling agent | |
| EP0193208A2 (en) | Lyophilization of reagent-coated particles | |
| CN101411864A (en) | Thymopentin dry powder formulation and preparation method thereof | |
| CN102539766A (en) | Beta-bungarotoxin detection kit and preparation method thereof | |
| US3562384A (en) | Immunological indicator and test system | |
| US4100268A (en) | Preparation of gentamicin sensitized particles for agglutination tests | |
| CN114167049A (en) | Freeze-dried pellet and preparation method thereof | |
| CN114152739A (en) | Fluorescent antibody freeze-dried pellet and preparation method thereof | |
| CN115541869B (en) | Latex microsphere reagent freeze-drying process and application thereof in immunoturbidimetric reagent | |
| CA3037383C (en) | Stable pooled breastmilk antibodies for oral delivery | |
| CN102707073A (en) | Enzyme linked immunosorbent assay kit for detecting metandienone and using method of enzyme linked immunosorbent assay kit | |
| CN116183903A (en) | Freeze-drying protective agent composition for AlphaLISA detection and preparation method thereof | |
| CN113092767B (en) | Limulus reagent freeze-dried microsphere and preparation method and application thereof | |
| CA1153314A (en) | Method of obtaining a lyophilized product | |
| CN103592435A (en) | Magnetic-particle chemiluminescence kit used for detecting monensin and applications of the kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |