CN106199007B - Protein protective agent - Google Patents

Protein protective agent Download PDF

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Publication number
CN106199007B
CN106199007B CN201610628713.1A CN201610628713A CN106199007B CN 106199007 B CN106199007 B CN 106199007B CN 201610628713 A CN201610628713 A CN 201610628713A CN 106199007 B CN106199007 B CN 106199007B
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protein
mgcl
protective agent
tea polyphenols
pvp
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CN106199007A (en
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崔大伟
刘艳
李米川
徐红
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YANTAI PROTGEN BIOTECHNOLOGY DEVELOPMENT Co Ltd
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YANTAI PROTGEN BIOTECHNOLOGY DEVELOPMENT Co Ltd
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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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates

Abstract

The present invention provides a series of protein protection liquid, can effective protection Hsp90 α albumen activity, improve the stability of Hsp90 α, the consistency under the concentration of nanogram level.The protein protection liquid adds other protective agent compositions, described other protective agent compositions to be selected from AMP, liquaemin, MgCl on the basis of traditional protein is protectant2, PVP (PVP40), Tea Polyphenols, cyclodextrin and K2SO4。

Description

Protein protective agent
Technical field
The present invention relates to biological technical field, and in particular to the protein protective agent of tumor markers Hsp90 α.
Background technology
Heat shock protein72 (90 α of Heat shock protein, Hsp90 α) is that content is the most in eukaryotic kytoplasm One of abundant chaperone, accounts for the 1-2% of total protein content in normal cell.In tumour cell, the content of Hsp90 α May be up to 2-7% (Whitesell, the L.and S.L.Lindquist., 2005, Nat Rev of intracellular protein total amount Cancer.5 (10), 761-772), the stability and auxiliary to GAP-associated protein GAP prime factor during raising tumour occurrence and development It is all required which functions.Research shows that Hsp90 α are expressed in the surface height of many tumour cells, including small cell carcinoma, Melanoma, and hepatoma cell strain (Ferrarini et al., 1992, Int.J.Cancer.51,613-19).Separately there is article Report, the Hsp90 α being secreted in serum it is related to the clinical stages of non-small cell lung cancer (Xu et al., 2007, J.Cancer Mol.3,107-112)。
2013, China scientist had found first and proves that the Hsp90 α in blood plasma are a brand-new tumor markerses, Hsp90 α immue quantitative detection reagent boxes (ELISA) of independent research obtains national 3rd class (highest class in April, 2013 Not) medicine equipment certificate, and passed through European Union's CE certifications, it is approved into Chinese and EU market.This is that people Hsp90 α are found Over 24 years, the world is first granted for clinical product, for the state of illness monitoring and therapeutic evaluation level, reality that improve tumor patient Existing tumor individual therapy has important impetus.
In Hsp90 α immue quantitative detection reagent boxes, Hsp90 α albumen is antigen standard, and its quality directly decides reagent Every detection performance of box, thus it is high to quality requirement.But, Hsp90 alpha protein molecules be highly prone to physically or chemically because Element impact and occurred conformation and its property change, such as：Generation denaturation.Once protein denaturation, along with 1) stability Drop；2) forfeiture of biologically active；3) exposure of some side-chain radicals；4) change of physicochemical properties；5) biochemical property Change.Also, protein concentration is lower, its stability is poorer, is more susceptible to denaturation.Therefore, pharmaceutical grade protein and examining Disconnected reagent research and development and industrialization field, the protein example that preparation structure is stable, activity is high is the important core for ensureing product quality Heart technology.
The space conformation flexibility of Hsp90 α albumen is extremely strong, is full of variety, and its crystal structure is not yet obtained so far.The albumen is managed Change unique, unstable (see Fig. 1), and its stability is significantly reduced as temperature is raised, even if at normal temperatures also can be very It is fast to form aggressiveness, (Minami et al., 1993), the more mutability particularly under conditions of protein concentration is low and lose work Property.At present, the shelf-life of Hsp90 α immue quantitative detection reagent boxes used in clinic be also only 12 months, mainly receive Hsp90 α albumen The restriction of antigen standard stability.
Industrially there are various methods to prevent protein denaturation, such as：Freeze Drying Technique, addition protein protectant etc.. Conventional protein protectant has polyol, carbohydrate, protein, polymer, amino acid, surfactant, amine etc..
The protective agent that polyol is used as albumen is long-standing, common are glycerine, mannitol, sorbierite, poly- second Glycol etc..
Amino acid is the common protein protectant of a class, protects egg by the speed and degree of reduction buffer salt crystallization White matter freezes medicine, mitigates its activity and loses.For example, in refrigerating process, glycine can be by suppressing phosphate-buffered salt crystallization Caused pH value changes and prevents pharmaceutical grade protein denaturation.The conventional amino acid for doing protein protectant has glycine, alanine, dried meat Propylhomoserin, glutamic acid, arginine, phenylalanine etc..
Some protein may also used as protective agent, such as：Bovine serum albumin(BSA) is exactly a classics, excellent protein protection Agent.1% bovine serum albumin(BSA) can make the activity of rabbit muscle lactic dehydrogenase not suffer a loss in freezing.
Additionally, the surfactant such as Tween 80, dodecyl naphthenic acid sodium also can be to the protein in freezing and dry run Play certain protective effect.
As the stabiliser of protein is extremely complex, it is difficult to be realized to protein with single protective agent in practical operation Effective protection, some protective agents even can the different shape of protein or under the conditions of play antipodal effect (Chang et al.,1993,J.Pharm Res.10(10),1478-1483).Therefore, in order to strengthen protected effect, it usually needs various Protective agent collocation is used.But due to protein properties it is different, it is difficult to being directed to different protein sets up general protection agent prescription, So the more excellent protein protective agent of exploitation remains an emphasis of the field concern of biological medicine research and development now.There are no in the past The report of Hsp90 alpha protein long term storages.
The content of the invention
The present invention provides protein protective agent, and the protein protection liquid adds other on the basis of traditional protein is protectant Protective agent composition, described other protective agent compositions are selected from AMP (AMP), liquaemin, MgCl2, PVP (PVP40), Tea Polyphenols, cyclodextrin and K2SO4.The present invention protein protective agent can effective protection Hsp90 α albumen activity, carry The stability of high Hsp90 α, the consistency under the concentration of nanogram level.
According to an aspect of the present invention, there is provided protein protective agent, which contains acid-base modifier, surfactant, protein The protective agent of type and other protective agents, or be made up of them；Wherein described other protective agents are selected from liquaemin, MgCl2、 AMP, PVP40, Tea Polyphenols, cyclodextrin and K2SO4One or more composition.
In some preferred embodiments, described other protective agents are liquaemins, and albumen in the protein protective agent The weight ratio of the protective agent and liquaemin of matter type is 5:0.01-1.
In some preferred embodiments, described other protective agents are Tea Polyphenols, and albumen in the protein protective agent The weight ratio of the protective agent and Tea Polyphenols of matter type is 5:0.1-5.
In some preferred embodiments, described other protective agents are cyclodextrin, and albumen in the protein protective agent The weight ratio of the protective agent and cyclodextrin of matter type is 5:0.5-5.
In other preferred embodiments, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and The protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in the protein protective agent2Weight ratio be：5:1-20: 0.1-5:0.69-3.47:0.48。
In other preferred embodiments, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in the protein protective agent, liquaemin, PVP40, Tea Polyphenols, AMP, MgCl2And K2SO4Weight ratio be：5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48:0.87-8.71.
In other preferred embodiments, described other protective agents are PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4 And the protective agent of protein types, PVP40, Tea Polyphenols, AMP, MgCl in cyclodextrin, and the protein protective agent2、K2SO4And ring The weight ratio of dextrin is：5:1-20:0.1-5:0.69-3.47:0.48:0.87-8.71:0.5-5.
In other preferred embodiments, described other protective agents be liquaemin, PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and the protein protective agent, liquaemin, PVP40, Tea Polyphenols, AMP、MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48:0.87- 8.71:0.5-5。
In a more preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and it is described The protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in protein protective agent2Weight ratio be：5:1:0.1: 0.69:0.48。
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in the protein protective agent2Weight ratio be：5:20: 5:3.47:0.48。
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in the protein protective agent2Weight ratio be：5:10: 0.5:1.74:0.48。
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in the protein protective agent, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4Weight ratio be：5:0.01:1:0.1:0.69:0.48:0.87.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in the protein protective agent, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4Weight ratio be：5:0.1:20:5:3.47:0.48:8.71.
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types, PVP 40, Tea Polyphenols, AMP, MgCl in cyclodextrin, and the protein protective agent2、 K2SO4Weight ratio with cyclodextrin is：5:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types, PVP 40, Tea Polyphenols, AMP, MgCl in cyclodextrin, and the protein protective agent2、 K2SO4Weight ratio with cyclodextrin is：5:20:5:3.47:0.48:8.71:5.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and the protein protective agent, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.01:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and the protein protective agent, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.1:20:5:3.47:0.48:8.71:5.
In the most preferred embodiment, described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types in cyclodextrin, and the protein protective agent, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.1:20:5:1.74:0.48:0.87:0.5.
Preferably, the amount of acid-base modifier contained in the protein protective agent is to make protein protective agent shape in water Into containing protein types 0.5% concentration of protective agent solution when the solution pH value be for about 6.5 amount.
In the protein protective agent of the present invention, the content of surfactant can be any appropriate amount, appropriate surface-active The amount of agent can be determined according to routine techniques by those skilled in the art.Preferably, in the protein protective agent, surface-active Protectant envelope-bulk to weight ratio of agent and protein types is 2:5(ml:G), the ratio between surfactant and other compositions Can calculate in this ratio.
Foregoing proteins protective agent can be any appropriate form.In some embodiments, protein protection of the invention Agent is the form of solution, i.e. protein protection liquid, and the protein protection liquid contains acid-base modifier, surfactant, 0.5% egg The protective agent of white matter type and other protective agents, or be made up of them, its pH value is 6.5.
In some embodiments, described other protective agents are that 0.01-1mg/ml liquaemins, 0.01%～0.5% tea are more Phenol or 0.05%-0.5% cyclodextrin.
In other embodiments, described other protective agents are selected from：
1) 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP and 5mM MgCl2；
2) 0.01-0.1mg/ml liquaemins, 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP、5mM MgCl2With 5mM-50mM K2SO4；
3) 0.1%-2%PVP40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP, 5mM MgCl2、5mM-50mM K2SO4With 0.05%-0.5% cyclodextrin；Or
4) 0.01-0.1mg/ml liquaemins, 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP、5mM MgCl2、5mM-50mM K2SO4With 0.05%-0.5% cyclodextrin.
In a more preferred embodiment, described other protective agents are selected from：
1) 0.1%PVP 40,0.01% Tea Polyphenols, 2mM AMP and 5mM MgCl2；
2) 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP and 5mM MgCl2；
3) 1%PVP 40,0.05% Tea Polyphenols, 5mM AMP and 5mM MgCl2；
4) 0.01mg/ml liquaemins, 0.1%PVP 40,0.01% Tea Polyphenols, 2mM AMP, 5mM MgCl2And 5mM K2SO4；
5) 0.1mg/ml liquaemins, 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2With 50mM K2SO4；
6) 0.1%PVP40,0.01% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4With 0.05% cyclodextrin；
7) 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4With 0.5% cyclodextrin；
8) 0.01mg/ml liquaemins, 0.1%PVP40,0.01% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4 With 0.05% cyclodextrin；Or
9) 0.1mg/ml liquaemins, 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4With 0.5% cyclodextrin.
In particularly preferred embodiments, described other protective agents be 0.1mg/ml liquaemins, 2%PVP40,0.5% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4With 0.05% cyclodextrin.
Preferably, in the protein protection liquid, the content of surfactant is 0.2% (percent by volume).
The protein protective agent of the present invention is used to protect Hsp90 α, can be used as the freeze drying protectant of Hsp90 α.
According to a further aspect in the invention, there is provided any of the above-described kind of protein protective agent answering in freeze-drying Hsp90 α With.
According to a further aspect in the invention, there is provided any of the above-described kind of protein protective agent is in the quantitative calibration for preparing Hsp90 α Application in product, quality-control product or immue quantitative detection reagent box.
According to another aspect of the present invention, there is provided Hsp90 α protect composition, and which contains Hsp90 α and any of the above-described kind Protein protective agent.
Specifically, the Hsp90 α protections composition contains Hsp90 α, acid-base modifier, surfactant, protein The protective agent of type and other protective agents, or be made up of them；Wherein described other protective agents are selected from liquaemin, MgCl2、 AMP, PVP40, Tea Polyphenols, cyclodextrin and K2SO4One or more composition.
In some preferred embodiments, described other protective agents are liquaemins, and Hsp90 α protection compositions The weight ratio of the protective agent and liquaemin of middle protein types is 5:0.01-1.
In some preferred embodiments, described other protective agents are Tea Polyphenols, and Hsp90 α protection compositions The weight ratio of the protective agent and Tea Polyphenols of middle protein types is 5:0.1-5.
In some preferred embodiments, described other protective agents are cyclodextrin, and Hsp90 α protection compositions The weight ratio of the protective agent and cyclodextrin of middle protein types is 5:0.5-5.
In other preferred embodiments, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and The protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in the Hsp90 α protections composition2Weight ratio be： 5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48。
In other preferred embodiments, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in Hsp90 α protection compositions, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2And K2SO4Weight ratio be：5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48:0.87-8.71.
In other preferred embodiments, described other protective agents are PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4 And the protective agent of protein types, PVP40, Tea Polyphenols, AMP, MgCl in cyclodextrin, and Hsp90 α protection compositions2、 K2SO4Weight ratio with cyclodextrin is：5:1-20:0.1-5:0.69-3.47:0.48:0.87-8.71:0.5-5.
In other preferred embodiments, described other protective agents be liquaemin, PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types, liquaemin, PVP40, tea in cyclodextrin, and Hsp90 α protection compositions Polyphenol, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48: 0.87-8.71:0.5-5。
In a more preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and it is described The protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in Hsp90 α protection compositions2Weight ratio be：5:1: 0.1:0.69:0.48。
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in Hsp90 α protection composition2Weight ratio It is：5:20:5:3.47:0.48.
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, Tea Polyphenols, AMP and MgCl in Hsp90 α protection composition2Weight ratio It is：5:10:0.5:1.74:0.48.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in Hsp90 α protection compositions, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2And K2SO4Weight ratio be：5:0.01:1:0.1:0.69:0.48:0.87.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in Hsp90 α protection compositions, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2And K2SO4Weight ratio be：5:0.1:20:5:3.47:0.48:8.71.
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types in cyclodextrin, and Hsp90 α protection compositions, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, described other protective agents are PVP 40, Tea Polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types in cyclodextrin, and Hsp90 α protection compositions, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:20:5:3.47:0.48:8.71:5.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and Hsp90 α protection compositions, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.01:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and Hsp90 α protection compositions, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.1:20:5:3.47:0.48:8.71:5.
In the most preferred embodiment, described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types in cyclodextrin, and Hsp90 α protection compositions, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.1:20:5:1.74:0.48:0.87:0.5.
Preferably, the amount of acid-base modifier contained in the Hsp90 α protections composition is to make the Hsp90 α protection groups When compound forms the solution of 0.5% concentration of protective agent containing protein types in water, the pH value of the solution is for about 6.5 amount.
In the Hsp90 α protection compositions of the present invention, the content of surfactant can be any appropriate amount, appropriate table The amount of face activating agent can be determined according to routine techniques by those skilled in the art.Preferably, combine in Hsp90 α protections In thing, protectant envelope-bulk to weight ratio of surfactant and protein types is 2:5(ml:G), surfactant and other into / ratio can calculate in this ratio.
Preferably, in Hsp90 α protection compositions, the weight between Hsp90 α and the protective agent of protein types Than being 2x10-5-4x10-4:Weight ratio between 5, Hsp90 α and other each compositions can be calculated in this ratio.
The Hsp90 α protection compositions of the present invention can be the form of Hsp90 α freeze-dried powders.
According to another aspect of the present invention, there is provided Hsp90 α solution, wherein containing Hsp90 α, acid-base modifier, surface Activating agent, the protective agent of 0.5% protein types and other protective agents are made up of them, and its pH value is 6.5.
In some embodiments, described other protective agents are that 0.01-1mg/ml liquaemins, 0.01%～0.5% tea are more Phenol or 0.05%-0.5% cyclodextrin.
In other embodiments, described other protective agents are selected from：
1) 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP and 5mM MgCl2；
2) 0.01-0.1mg/ml liquaemins, 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP、5mM MgCl2With 5mM-50mM K2SO4；
3) 0.1%-2%PVP40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP, 5mM MgCl2、5mM-50mM K2SO4With 0.05%-0.5% cyclodextrin；Or
4) 0.01-0.1mg/ml liquaemins, 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP、5mM MgCl2、5mM-50mM K2SO4With 0.05%-0.5% cyclodextrin.
In a more preferred embodiment, described other protective agents are selected from：
1) 0.1%PVP 40,0.01% Tea Polyphenols, 2mM AMP and 5mM MgCl2；
2) 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP and 5mM MgCl2；
3) 1%PVP 40,0.05% Tea Polyphenols, 5mM AMP and 5mM MgCl2；
4) 0.01mg/ml liquaemins, 0.1%PVP 40,0.01% Tea Polyphenols, 2mM AMP, 5mM MgCl2And 5mM K2SO4；
5) 0.1mg/ml liquaemins, 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2With 50mM K2SO4；
6) 0.1%PVP40,0.01% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4With 0.05% cyclodextrin；
7) 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4With 0.5% cyclodextrin；
8) 0.01mg/ml liquaemins, 0.1%PVP40,0.01% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4 With 0.05% cyclodextrin；Or
9) 0.1mg/ml liquaemins, 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4With 0.5% cyclodextrin.
In particularly preferred embodiments, described other protective agents be 0.1mg/ml liquaemins, 2%PVP40,0.5% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4With 0.05% cyclodextrin.
Preferably, in the Hsp90 α solution, the content of surfactant is 0.2% (percent by volume).
In preferred embodiments, in the Hsp90 α solution, the concentration of Hsp90 α is 20-400ng/ml, preferably 20ng/ml, 50ng/ml, 100ng/ml, 200ng/ml and/or 400ng/ml.
According to a further aspect in the invention, there is provided for detecting the kit of Hsp90 α, wherein comprising any of the above-described hatching egg White protective agent, any of the above-described kind of Hsp90 α freeze-dried powder or any of the above-described kind of Hsp90 α solution.
The protective agent of heretofore described protein types can be bovine serum albumin, human serum albumin, chicken serum Albumin, casein or casein-sodium or its combination.
Heretofore described surfactant can be Tween 80, polysorbate40 or Triton X-100 or its combination.
Heretofore described acid-base modifier can include PB.
The Hsp90 α of nanogram level concentration can meet following index after the protective agent using the present invention：
1) stability：37 DEG C of inspection accelerates the dried frozen aquatic products of 12 days, and which is compared with the sample of 2-8 DEG C of storage, its detected value Reduce less than 15%.
The lyophilized calibration objects of the Hsp90 α after accelerating are detected using Hsp90 α ELISA methods kit, its performance should meet following Index：
1) minimum detectability≤1.96ng/ml；
2) calibration curve is linear：
In 20-400ng/ml, coefficient R 2 should be not less than 0.980 to the range of linearity；
3) quality-control product measured value：
The quality-control product measured value of high, middle concentration should be in the range of 85.0%-the 115.0% of labelled amount, the Quality Control of low concentration Product should be in the range of 80.0%-the 120.0% of labelled amount.
The Hsp90 α dried frozen aquatic products performance indications prepared using the protein protection liquid of the present invention meet requirements above, its dried frozen aquatic products The term of validity by original 37 DEG C of accelerated stability terms of validity 6 days (equivalent to conventional condition of storage (2-8 DEG C) term of validity 12 Month), 37 DEG C of accelerated stability terms of validity 12 days are extended to, conventional storage is extended to into 24 months, so as to Hsp90 can be greatly improved The use term of validity of α detection kits.When using single protective agent composition, Hsp90 α protein stabilized phases, part can be also improved Stationary phase can be by 20 months.
The protein protection liquid of the present invention can be used to prepare the quantitative calibration product of Hsp90 α, quality-control product, immue quantitative detection reagent box Or detection method.Quantitative calibration product, quality-control product, immue quantitative detection reagent box or the detection method can be used to determine that individuality whether there is Whether tumour, especially malignant tumour and tumour shift；By the Hsp90 alpha levels in detection blood plasma, serum or body fluid Tumor screening is carried out to people at highest risk；By the Hsp90 alpha levels in detection blood plasma, serum or body fluid to tumor patient Prognosis is judged；Judge the operation to tumour patient, put by detecting the Hsp90 alpha levels in blood plasma, serum or body fluid Treat or whether drug therapy is effective and/or determines when stop treatment.
Description of the drawings
Fig. 1 is the stability of Hsp90 under different temperatures.
Specific embodiment
Term as used herein " protective agent ", " protein protective agent " or " protein protectant " can be in protein quilt The protective agent shielded when lyophilized, which may refer to the protein protective agent of single component, or by Multiple components group Into mixture.Common protein protective agent can be polyol, carbohydrate, protein, polymer, amino acid, surface Activating agent, amine etc.." protective agents of protein types " specifically described herein refer to such protein protective agent, its constituent It is protein, such as bovine serum albumin, human serum albumin, chicken serum albumin, casein, casein-sodium etc..
" protein protection liquid " as herein described or " protein protection liquid " can also be referred to as " frozen-dried protective liquid ", and it is right to refer to The protection liquid that protein adds when carrying out lyophilized, for the activity and stability of Protein requirement.Frozen-dried protective liquid is usually slow Liquid is rushed, wherein usually containing the protective agent and surfactant that can be shielded when protein is lyophilized.
" surfactant " as herein described can be Tween 80, polysorbate40, Triton X-100, dodecyl sodium sulfate etc..
" Hsp90 α " as herein described is Heat shock protein72 (90 α of Heat shock protein, Hsp90 α).
" AMP " as herein described is AMP.
" acid-base modifier " as herein described can be selected from buffer, acid or alkali, including but not limited to NaOH, hydrogen-oxygen Change potassium, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, hydrochloric acid, nitric acid, sulfuric acid or its combination.
Percentage composition mentioned herein, unless otherwise indicated, when solute is solid under normal conditions, is quality/body Product concentration, when solute is liquid under normal conditions, is volume/volume concentration." under normal conditions " refers at normal temperatures and pressures.
In order to the present invention is explained in greater detail, embodiments of the invention are given below, these embodiments merely for the sake of Explain and descriptive purpose, shall not be construed as limitation of the scope of the invention.
Embodiment 1 adds impact of the single component protective agent to Hsp90 α protein stabilities
Purpose：The new Hsp90 α protein protective agents of screening, and determine its concentration scope and condition.
Reagent：Phosphate, polysorbas20 and Tea Polyphenols are purchased from Chemical Reagent Co., Ltd., Sinopharm Group, bovine serum albumin purchase From sigma companies, liquaemin is purchased from the extensive and profound in meaning star biotechnology responsibility Co., Ltd in Beijing purchased from glad brilliant biology, cyclodextrin.Hsp90 α finished products kit is purchased from Yantai Pu Luoji biotechnologies Development Co., Ltd, lot number 201602002,201604001, the reagent The calibration object of box is Hsp90 α dried frozen aquatic productses.
Method：
1st, base soln is prepared, using purified water, wherein containing 0.318% phosphate (sodium dihydrogen phosphate), 0.5% cow's serum Albumin and 0.2% polysorbas20, adjust acid-base value to pH6.5, and this solution is the basic solution of later stage test.
2nd, prepare the frozen-dried protective liquid of the liquaemin containing certain content, cyclodextrin or Tea Polyphenols using base soln respectively, The content of liquaemin, cyclodextrin or Tea Polyphenols is：
1) liquaemin：0.01、0.03、0.06、0.1、1、10mg/ml；
2) Tea Polyphenols：0.01%th, 0.05%, 0.1%, 0.5%, 1%；
3) cyclodextrin：0.05%th, 0.1%, 0.5%, 1%.
3rd, the frozen-dried protective liquid of the liquaemin containing certain content, cyclodextrin or the Tea Polyphenols obtained using step 2 is matched somebody with somebody respectively The Hsp90 α eggs of 20ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml in system correspondence Hsp90 α finished product kits White solution, and be dispensed in the cillin bottle of 2ml specifications by per bottle of 400 μ l.
4th, the bottled Hsp90 α protein solutions containing protectant XiLin will be prepared to be freezed, step of freeze drying and Hsp90 α Finished product kit Hsp90 α albumen step of freeze drying is consistent.
5th, the Hsp90 α albumen after will be lyophilized adds aluminium-plastic cap to be tamping, and taking half quantity carries out 37 DEG C of Acceleration study (foundations： During the term of validity of external diagnosis reagent industry is calculated, 37 DEG C accelerate deposited 2 months equivalent to 2-8 DEG C in real time within 1 day), second half is put Put 2-8 DEG C of preservation.
6th, after the Hsp90 α albumen dried frozen aquatic products acceleration time reaches, detected using Hsp90 α kits.Contrast does not add Testing result after speed and acceleration, the detected value after acceleration should be reduced less than 15%.
Detection operating procedure is as follows：
1) take out kit to balance 30 minutes at 37 DEG C, add 475mL deionized waters to add after mixing concentrated cleaning solution It is stand-by in board-washing machine；
2) dissolving of 0.4ml analytes dilution is added to mix the dried frozen aquatic products not accelerated and accelerate；
3) by the lath of desirable number as the dried frozen aquatic products on grillage, added after 50 μ l dissolvings；
4) in each hole 50 μ L are added to survey Heat shock protein72 mark thing liquid, gently vibration is mixed；
5) shrouding film covers microwell plate, incubates 60 minutes at 37 DEG C；
6) board-washing：Reactant liquor is got rid of, is added 300 μ L washing lotion board-washings per hole, or board-washing is carried out using board-washing machine, altogether board-washing 6 times, It is last to buckle dry on blotting paper；
7) each 50 μ L of developer Α, B liquid are sequentially added in every hole, gently vibration is mixed, 37 DEG C incubate 20 minutes；
8) 50 μ L terminate liquid color development stoppings are added in every hole；
9) in 10 minutes after addition reaction terminating liquid, OD values are read under 450nm/620nm wavelength, wherein 450nm is inspection Wavelength is surveyed, 620nm is reference wavelength, for removing ambient interferences.
As a result：
After testing, the concentration that disclosure satisfy that 10 days results of accelerated stability is：0.01-1mg/ml liquaemins, 0.01%～ 0.5% Tea Polyphenols, 0.05%-0.5% cyclodextrin.Result of the test is as shown in Table 1 and Table 2.
Table 1 is accelerated 10 days for 37 DEG C using different protective agents
Table 2 is accelerated 10 days for 37 DEG C using different protective agents
More than 2 protective agent of embodiment is used in combination the impact to Hsp90 α protein stabilities
Purpose：The new Hsp90 α protein protective agent combination formulas of research and development, and determine its concentration scope and condition.
Reagent：Phosphate, polysorbas20, MgCl2, Tea Polyphenols and K2SO4Purchased from Chemical Reagent Co., Ltd., Sinopharm Group, ox Serum albumin and AMP are purchased from sigma companies, and liquaemin is purchased from glad brilliant biology, and PVP40 is purchased from purchased from glad brilliant biology, cyclodextrin The extensive and profound in meaning star biotechnology responsibility Co., Ltd in Beijing.Hsp90 α finished products kit is limited purchased from Yantai Pu Luoji biotechnology development Company, lot number 201602002,201604001, the calibration object of the kit is Hsp90 α dried frozen aquatic productses.
Method:
Basic experiment method and steps is with the method and step in embodiment 1, and employs multiple protections in the present embodiment Agent is in combination using formula.
Protective agent combination point four big class, it is all kinds of in be respectively provided with different concentration combinations:
1) 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP, 5mM MgCl2；
2) 0.01-0.1mg/ml liquaemins, 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP、5mM MgCl2、5mM-50mM K2SO4；
3) 0.1%-2%PVP40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP, 5mM MgCl2、5mM-50mM K2SO4, 0.05%-0.5% cyclodextrin；
4) 0.01-0.1mg/ml liquaemins, 0.1%-2%PVP 40,0.01%-0.5% Tea Polyphenols, 2mM-10mM AMP、5mM MgCl2、5mM-50mM K2SO4, 0.05%-0.5% cyclodextrin；
As a result：
After testing, meet the accelerated stability result of the test of 12 days as shown in table 3, table 4 and table 5.
Protective agent combined arrangement：
1) 0.1%PVP 40,0.01% Tea Polyphenols, 2mM AMP, 5mM MgCl2
2) 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2
3) 1%PVP 40,0.05% Tea Polyphenols, 5mM AMP, 5mM MgCl2
4) 0.01mg/ml liquaemins, 0.1%PVP 40,0.01% Tea Polyphenols, 2mM AMP, 5mM MgCl2、5mM K2SO4
5) 0.1mg/ml liquaemins, 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4
6) 0.1%PVP40,0.01% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4, 0.05% cyclodextrin
7) 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4, 0.5% cyclodextrin
8) 0.01mg/ml liquaemins, 0.1%PVP40,0.01% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4、 0.05% cyclodextrin
9) 0.1mg/ml liquaemins, 2%PVP 40,0.5% Tea Polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4、 0.5% cyclodextrin
Table 3 combines 37 DEG C using different protective agents and accelerates 12 days
Table 4 combines 37 DEG C using different protective agents and accelerates 12 days
Table 5 combines 37 DEG C using different protective agents and accelerates 12 days
The optimum effect combination formula for obtaining：0.1mg/ml liquaemins, 2%PVP40,0.5% Tea Polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4, 0.05% cyclodextrin.
37 DEG C of acceleration for stabilization testing results of combination (OD450/630 values) is as shown in table 6 below：
6 optimum effect 37 DEG C of acceleration for stabilization testing results of combination formula of table
Embodiment 3 carries out the detection of Hsp90 α kits performance indications using optimum effect protective agent combination formula
Purpose：By 12 days 37 DEG C of Acceleration studies, the optimum effect protective agent combination formula of test was to Hsp90 α albumen and reagent Box detects the impact of performance.
Method：Detected according to the operating procedure in embodiment 1, wherein, using the Hsp90 α dried frozen aquatic productses after acceleration as Calibration object curve, while adding quality-control product to carry out the detection of kit performance.
As a result：The lyophilized calibration objects of Hsp90 α using optimum effect combination formula are still stable after accelerating 12 days, kit Property indices result is as shown in table 7 below：
7 kit property indices structure of table
Wherein, minimum detectability≤1.96ng/ml；Coefficient R2More than 0.980；The quality-control product of high, middle concentration is determined Value should be in the range of 85.0%-the 115.0% of labelled amount, and the quality-control product of low concentration should be 80.0%-the 120.0% of labelled amount In the range of.Actually detected value meets relevant national standard.

Claims (9)

1. protein protective agent, which contains acid-base modifier, surfactant, the protective agent of protein types and other protective agents； Wherein
1) described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and protein types in the protein protective agent Protective agent, PVP 40, Tea Polyphenols, AMP and MgCl2Weight ratio be：5:1-20:0.1-5:0.69-3.47:0.48；
2) described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and in the protein protective agent The protective agent of protein types, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4Weight ratio be：5:0.01-0.1: 1-20:0.1-5:0.69-3.47:0.48:0.87-8.71；
3) described other protective agents are PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4And in cyclodextrin, and the protein protective agent The protective agent of protein types, PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:1-20:0.1- 5:0.69-3.47:0.48:0.87-8.71:0.5-5；Or
4) described other protective agents are liquaemin, PVP40, Tea Polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and albumen guarantor The protective agent of protein types, liquaemin, PVP40, Tea Polyphenols, AMP, MgCl in shield agent2、K2SO4With the weight ratio of cyclodextrin It is：5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48:0.87-8.71:0.5-5.
2. protein protective agent according to claim 1, wherein
1) described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and protein types in the protein protective agent Protective agent, PVP 40, Tea Polyphenols, AMP and MgCl2Weight ratio be：5:1:0.1:0.69:0.48；
2) described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and protein types in the protein protective agent Protective agent, PVP 40, Tea Polyphenols, AMP and MgCl2Weight ratio be：5:20:5:3.47:0.48；
3) described other protective agents are PVP 40, Tea Polyphenols, AMP and MgCl2, and protein types in the protein protective agent Protective agent, PVP 40, Tea Polyphenols, AMP and MgCl2Weight ratio be：5:10:0.5:1.74:0.48；
4) described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and in the protein protective agent The protective agent of protein types, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4Weight ratio be：5:0.01:1: 0.1:0.69:0.48:0.87；
5) described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4, and in the protein protective agent The protective agent of protein types, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2And K2SO4Weight ratio be：5:0.1:20:5: 3.47:0.48:8.71；
6) described other protective agents are PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And in cyclodextrin, and the protein protective agent The protective agent of protein types, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:1:0.1: 1.74:0.48:0.87:0.5；
7) described other protective agents are PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And in cyclodextrin, and the protein protective agent The protective agent of protein types, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4Weight ratio with cyclodextrin is：5:20:5: 3.47:0.48:8.71:5；
8) described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and the albumen The protective agent of protein types, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl in protective agent2、K2SO4With the weight of cyclodextrin Than being：5:0.01:1:0.1:1.74:0.48:0.87:0.5；Or
9) described other protective agents are liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and the albumen The protective agent of protein types, liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl in protective agent2、K2SO4With the weight of cyclodextrin Than being：5:0.1:20:5:3.47:0.48:8.71:5.
3. protein protective agent according to claim 1, wherein described other protective agents be liquaemin, PVP 40, Tea Polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and the protein protective agent, liquaemin, PVP 40, Tea Polyphenols, AMP、MgCl2、K2SO4Weight ratio with cyclodextrin is：5:0.1:20:5:1.74:0.48:0.87:0.5.
4. the protein protective agent according to any one of claim 1-3, which is the form of liquid.
5. application of the protein protective agent of any one of claim 1-4 in freeze-drying Hsp90 α.
6. the protein protective agent of any one of claim 1-4 is preparing quantitative calibration product, quality-control product or the quantitative determination of Hsp90 α Application in kit.
7.Hsp90 α protect composition, and which contains the protein protective agent of Hsp90 α and any one of claim 1-4.
8. Hsp90 α according to claim 7 protect composition, and which is solution or freeze-dried powder.
9. it is used for detecting the kit of Hsp90 α, wherein the protein protective agent comprising any one of claim 1-4 or claim 7 Or 8 Hsp90 α protect composition.
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