CN106199007A - Protein protective agent - Google Patents

Protein protective agent Download PDF

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Publication number
CN106199007A
CN106199007A CN201610628713.1A CN201610628713A CN106199007A CN 106199007 A CN106199007 A CN 106199007A CN 201610628713 A CN201610628713 A CN 201610628713A CN 106199007 A CN106199007 A CN 106199007A
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protective agent
protein
mgcl
tea polyphenols
pvp
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CN106199007B (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

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  • Proteomics, Peptides & Aminoacids (AREA)
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Abstract

The present invention provides a series of protein protection liquid, can effectively protect the activity of Hsp90 α albumen, improves the stability of Hsp90 α, invariance under the concentration of nanogram level.Described protein protection liquid, on the basis of traditional protein is protectant, adds other protective agent composition, and other protective agent composition described is selected from AMP, heparin sodium, MgCl2, PVP (PVP40), tea polyphenols, cyclodextrin and K2SO4。

Description

Protein protective agent
Technical field
The present invention relates to biological technical field, be specifically related to the protein protective agent of tumor markers Hsp90 α.
Background technology
Heat shock protein72 (Heat shock protein 90 α, Hsp90 α) is that in eukaryotic cell kytoplasm content is the most One of abundant chaperone, accounts for the 1-2% of total protein content in normal cell.In tumor 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), to the stability and the auxiliary that improve associated protein prime factor in tumor generation and evolution Its functionating is all required.Research display, Hsp90 α is at the surface high expressed of many tumor 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 relevant to the clinical stages of nonsmall-cell lung cancer (Xu et al., 2007, J.Cancer Mol.3,107-112)。
2013, China scientist found first and the Hsp90 α that proves in blood plasma is a brand-new tumor markers, Hsp90 α immue quantitative detection reagent box (euzymelinked immunosorbent assay (ELISA)) of independent research obtains country's the 3rd class (the highest class in April, 2013 Not) medical apparatus and instruments certificate, and passed through European Union's CE certification, it is approved to enter Chinese and EU market.This is that people Hsp90 α is found Over 24 years, the world is first granted for clinical product, for improving state of illness monitoring and therapeutic evaluation level, the reality of tumor patient Existing tumor individual therapy has important impetus.
In Hsp90 α immue quantitative detection reagent box, Hsp90 α albumen is antigen standard, and its quality directly decides reagent Every detection performance of box, therefore high to prescription.But, Hsp90 alpha protein molecule be highly prone to physically or chemically because of The impact of element and the change of occurred conformation and its character, such as: degeneration occurs.Protein once degeneration, along with 1) under stability Fall；2) bioactive forfeiture；3) exposure of some side-chain radicals；4) change of physicochemical properties；5) biochemical property Change.Further, protein concentration is the lowest, and its stability is the poorest, is more susceptible to degeneration.Therefore, at pharmaceutical grade protein and examining Disconnected reagent research and development and industrialization field, preparation Stability Analysis of Structures, the high protein example of activity are the important core of products quality guarantee Heart technology.
The space conformation flexibility of Hsp90 α albumen is extremely strong, is full of variety, and its crystal structure the most not yet obtains.This albumen is managed Change unique, unstable (see Fig. 1), and its stability raises along with temperature and significantly reduces, even if the most also can be very Form aggressiveness soon, (Minami et al., 1993), particularly under conditions of protein concentration is low more changeableness and lose work Property.At present, the shelf-life of the Hsp90 α immue quantitative detection reagent box used in clinic is also only 12 months, mainly by Hsp90 α albumen The restriction of antigen standard stability.
Industrial have multiple method to prevent protein denaturation, such as: Freeze Drying Technique, addition protein protectant etc.. Conventional protein protectant has polyol, saccharide, protein, polymer, aminoacid, surfactant, amine etc..
The protective agent that polyol is used as albumen is long-standing, common are glycerol, mannitol, sorbitol, poly-second Glycol etc..
Aminoacid is the protein protectant that a class is common, protects egg by the speed and degree reducing buffer salt crystallization White matter lyophilizing medicine, alleviates its activity and loses.Such as, in refrigerating process, glycine can be by suppression phosphoric acid buffer salt-pepper noise Caused pH value changes and stops pharmaceutical grade protein degeneration.The conventional aminoacid 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 is exactly classics, excellent protein protection Agent.1% bovine serum albumin can make the activity of rabbit muscle lactic acid dehydrogenase not suffer a loss when 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.
Owing to the stabiliser of protein is extremely complex, practical operation is difficult to realize protein with single protective agent Effectively protection, some protective agent 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 multiple Protective agent collocation uses.But owing to protein properties is different, it is difficult to set up general protective agent formula for different protein, So developing more excellent protein protective agent to remain the emphasis that the field of biological medicine research and development now is paid close attention to.There are no in the past The report of Hsp90 alpha protein long term storage.
Summary of the invention
The present invention provides protein protective agent, described protein protection liquid on the basis of traditional protein is protectant, to add other Protective agent composition, other protective agent composition described is selected from adenosine phosphate (AMP), heparin sodium, MgCl2, PVP (PVP40), tea polyphenols, cyclodextrin and K2SO4.The protein protective agent of the present invention can effectively protect the activity of Hsp90 α albumen, carries The stability of high Hsp90 α, invariance under the concentration of nanogram level.
According to an aspect of the present invention, it is provided that protein protective agent, it contains acid-base modifier, surfactant, protein The protective agent of type and other protective agent, or be made up of them；Other protective agent wherein said is selected from heparin sodium, MgCl2、 AMP, PVP40, tea polyphenols, cyclodextrin and K2SO4One or more compositions.
In some preferred embodiments, other protective agent described is heparin sodium, and albumen in described protein protective agent The protective agent of matter type and the weight ratio of heparin sodium are 5:0.01-1.
In some preferred embodiments, other protective agent described is tea polyphenols, and albumen in described protein protective agent The protective agent of matter type and the weight ratio of tea polyphenols are 5:0.1-5.
In some preferred embodiments, other protective agent described is cyclodextrin, and albumen in described protein protective agent The protective agent of matter type and the weight ratio of cyclodextrin are 5:0.5-5.
In other preferred embodiments, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and The protective agent of protein types, PVP 40, tea polyphenols, AMP and MgCl in described protein protective agent2Weight ratio be: 5:1-20: 0.1-5:0.69-3.47:0.48。
In other preferred embodiments, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in described protein protective agent, heparin sodium, 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, other protective agent described is PVP40, tea polyphenols, AMP, MgCl2、K2SO4 And the protective agent of protein types, PVP40, tea polyphenols, AMP, MgCl in cyclodextrin, and described 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, other protective agent described be heparin sodium, PVP40, tea polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and described protein protective agent, heparin sodium, PVP40, tea polyphenols, AMP、MgCl2、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。
In a more preferred embodiment, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and 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, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, tea polyphenols, AMP and MgCl in described protein protective agent2Weight ratio be: 5:20: 5:3.47:0.48。
In another further preferred embodiment, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, tea polyphenols, AMP and MgCl in described protein protective agent2Weight ratio be: 5:10: 0.5:1.74:0.48。
In another further preferred embodiment, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in described protein protective agent, heparin sodium, 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, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and the protective agent of protein types in described protein protective agent, heparin sodium, 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, other protective agent described is PVP 40, tea polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types, PVP 40, tea polyphenols, AMP, MgCl in cyclodextrin, and described protein protective agent2、 K2SO4With the weight ratio of cyclodextrin it is: 5:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, other protective agent described is PVP 40, tea polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types, PVP 40, tea polyphenols, AMP, MgCl in cyclodextrin, and described protein protective agent2、 K2SO4With the weight ratio of cyclodextrin it is: 5:20:5:3.47:0.48:8.71:5.
In another further preferred embodiment, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and described protein protective agent, heparin sodium, PVP 40, tea polyphenols, AMP、MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.01:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and described protein protective agent, heparin sodium, PVP 40, tea polyphenols, AMP、MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.1:20:5:3.47:0.48:8.71:5.
In the most preferred embodiment, other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、 K2SO4And the protective agent of protein types in cyclodextrin, and described protein protective agent, heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.1:20:5:1.74:0.48:0.87:0.5.
Preferably, the amount of contained in described protein protective agent acid-base modifier is to make described protein protective agent shape in water When becoming the solution of protective agent 0.5% concentration containing protein types, the pH value of this solution is about the amount of 6.5.
In the protein protective agent of the present invention, the content of surfactant can be any suitable amount, suitable surface activity The amount of agent can be determined according to routine techniques by those skilled in the art.Preferably, in described protein protective agent, surface activity Protectant envelope-bulk to weight ratio of agent and protein types is 2:5 (ml:g), the ratio between surfactant and other composition Can calculate in this ratio.
Foregoing proteins protective agent can be to be any suitable form.In some embodiments, the protein protection of the present invention Agent is the form of solution, i.e. protein protection liquid, and described protein protection liquid contains acid-base modifier, surfactant, the egg of 0.5% The protective agent of white matter type and other protective agent, or be made up of them, its pH value is 6.5.
In some embodiments, other protective agent described be 0.01-1mg/ml heparin sodium, 0.01%～0.5% tea many Phenol or 0.05%-0.5% cyclodextrin.
In other embodiments, other protective agent described is 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 heparin sodium, 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 heparin sodium, 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, other protective agent described is 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 heparin sodium, 0.1%PVP 40,0.01% tea polyphenols, 2mM AMP, 5mM MgCl2And 5mM K2SO4；
5) 0.1mg/ml heparin sodium, 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 heparin sodium, 0.1%PVP40,0.01% tea polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4 With 0.05% cyclodextrin；Or
9) 0.1mg/ml heparin sodium, 2%PVP 40,0.5% tea polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4With 0.5% cyclodextrin.
In particularly preferred embodiments, other protective agent described be 0.1mg/ml heparin sodium, 2%PVP40,0.5% Tea polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4With 0.05% cyclodextrin.
Preferably, in described protein protection liquid, the content of surfactant is 0.2% (percent by volume).
The protein protective agent of the present invention is used for protecting Hsp90 α, can be as the freeze drying protectant of Hsp90 α.
According to a further aspect in the invention, it is provided that any of the above-described kind of protein protective agent answering in lyophilization Hsp90 α With.
According to a further aspect in the invention, it is provided that any of the above-described kind of protein protective agent is at the quantitative calibration preparing Hsp90 α Application in product, quality-control product or immue quantitative detection reagent box.
According to another aspect of the present invention, it is provided that Hsp90 α protects compositions, it contains Hsp90 α and any of the above-described kind Protein protective agent.
Specifically, described Hsp90 α protection compositions contains Hsp90 α, acid-base modifier, surfactant, protein The protective agent of type and other protective agent, or be made up of them；Other protective agent wherein said is selected from heparin sodium, MgCl2、 AMP, PVP40, tea polyphenols, cyclodextrin and K2SO4One or more compositions.
In some preferred embodiments, other protective agent described is heparin sodium, and described Hsp90 α protects compositions The protective agent of middle protein types and the weight ratio of heparin sodium are 5:0.01-1.
In some preferred embodiments, other protective agent described is tea polyphenols, and described Hsp90 α protects compositions The protective agent of middle protein types and the weight ratio of tea polyphenols are 5:0.1-5.
In some preferred embodiments, other protective agent described is cyclodextrin, and described Hsp90 α protects compositions The protective agent of middle protein types and the weight ratio of cyclodextrin are 5:0.5-5.
In other preferred embodiments, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and The protective agent of protein types, PVP 40, tea polyphenols, AMP and MgCl in described Hsp90 α protection compositions2Weight ratio be: 5:0.01-0.1:1-20:0.1-5:0.69-3.47:0.48。
In other preferred embodiments, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, 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, other protective agent described is PVP40, tea polyphenols, AMP, MgCl2、K2SO4 And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, PVP40, tea polyphenols, AMP, MgCl2、 K2SO4With the weight ratio of cyclodextrin it is: 5:1-20:0.1-5:0.69-3.47:0.48:0.87-8.71:0.5-5.
In other preferred embodiments, other protective agent described be heparin sodium, PVP40, tea polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, PVP40, tea Polyphenol, AMP, MgCl2、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。
In a more preferred embodiment, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and 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, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, tea polyphenols, AMP and MgCl in described Hsp90 α protection compositions2Weight ratio It is: 5:20:5:3.47:0.48.
In another further preferred embodiment, other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, And the protective agent of protein types, PVP 40, tea polyphenols, AMP and MgCl in described Hsp90 α protection compositions2Weight ratio It is: 5:10:0.5:1.74:0.48.
In another further preferred embodiment, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, 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, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, 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, other protective agent described is PVP 40, tea polyphenols, AMP, MgCl2、 K2SO4And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, other protective agent described is PVP 40, tea polyphenols, AMP, MgCl2、 K2SO4And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:20:5:3.47:0.48:8.71:5.
In another further preferred embodiment, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, PVP 40, Tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.01:1:0.1:1.74:0.48:0.87:0.5.
In another further preferred embodiment, other protective agent described be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, PVP 40, Tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.1:20:5:3.47:0.48:8.71:5.
In the most preferred embodiment, other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、 K2SO4And cyclodextrin, and described Hsp90 α protection compositions in the protective agent of protein types, heparin sodium, PVP 40, tea polyphenols, AMP、MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.1:20:5:1.74:0.48:0.87:0.5.
Preferably, the amount of contained in described Hsp90 α protection compositions acid-base modifier is to make described Hsp90 α protection group When compound forms the solution of protective agent 0.5% concentration containing protein types in water, the pH value of this solution is about the amount of 6.5.
In the Hsp90 α protection compositions of the present invention, the content of surfactant can be any suitable amount, suitable table The amount of face activating agent can be determined according to routine techniques by those skilled in the art.Preferably, in described Hsp90 α protection combination In thing, protectant envelope-bulk to weight ratio of surfactant and protein types is 2:5 (ml:g), and surfactant becomes with other / ratio can calculate in this ratio.
Preferably, in described Hsp90 α protection compositions, the weight between Hsp90 α and the protective agent of protein types Ratio is 2x10-5-4x10-4: the weight ratio between 5, Hsp90 α and other each composition can be calculated in this ratio.
The Hsp90 α protection compositions of the present invention can be with the form being Hsp90 α lyophilized powder.
According to another aspect of the present invention, it is provided that Hsp90 α solution, Hsp90 α, acid-base modifier, surface are wherein contained Activating agent, protective agent and other protective agent of protein types of 0.5% or be made up of them, its pH value is 6.5.
In some embodiments, other protective agent described be 0.01-1mg/ml heparin sodium, 0.01%～0.5% tea many Phenol or 0.05%-0.5% cyclodextrin.
In other embodiments, other protective agent described is 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 heparin sodium, 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 heparin sodium, 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, other protective agent described is 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 heparin sodium, 0.1%PVP 40,0.01% tea polyphenols, 2mM AMP, 5mM MgCl2And 5mM K2SO4；
5) 0.1mg/ml heparin sodium, 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 heparin sodium, 0.1%PVP40,0.01% tea polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4 With 0.05% cyclodextrin；Or
9) 0.1mg/ml heparin sodium, 2%PVP 40,0.5% tea polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4With 0.5% cyclodextrin.
In particularly preferred embodiments, other protective agent described be 0.1mg/ml heparin sodium, 2%PVP40,0.5% Tea polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4With 0.05% cyclodextrin.
Preferably, in described Hsp90 α solution, the content of surfactant is 0.2% (percent by volume).
In preferred embodiments, in described Hsp90 α solution, the concentration of Hsp90 α is 20-400ng/ml, is preferably 20ng/ml, 50ng/ml, 100ng/ml, 200ng/ml and/or 400ng/ml.
According to a further aspect in the invention, it is provided that for detecting the test kit of Hsp90 α, wherein comprise any of the above-described hatching egg White protective agent, any of the above-described kind of Hsp90 α lyophilized 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 sidium or a combination thereof.
Heretofore described surfactant can be Tween 80, polysorbate40 or Triton X-100 or a combination thereof.
Heretofore described acid-base modifier can comprise phosphate buffer.
The Hsp90 α of nanogram level concentration, after the protective agent using the present invention, can meet following index:
1) stability: check 37 DEG C of dried frozen aquatic productses accelerating 12 days, it compares with the 2-8 DEG C of sample deposited, its detected value Reduce less than 15%.
Hsp90 α lyophilizing calibration object after using the detection of Hsp90 α ELISA method test kit to accelerate, below its performance should meet Index:
1) minimum detectability≤1.96ng/ml；
2) standard curve is linear:
The range of linearity is when 20 400ng/ml, and coefficient R 2 should be not less than 0.980；
3) quality-control product measured value:
The quality-control product measured value of concentration high, middle 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 using the protein protection liquid of the present invention to prepare meet requirements above, its dried frozen aquatic products Effect duration (be equivalent to conventional condition of storage (2-8 DEG C) effect duration 12 by original 6 days 37 DEG C of accelerated stability effect duration Month), extend to 12 days 37 DEG C of accelerated stability effect duration, conventional storage is extended to 24 months, thus Hsp90 can be greatly improved The use effect duration of α detection kit.When using single protective agent composition, it is possible to improve Hsp90 α protein stabilized phase, part Stable phase can be by 20 months.
The protein protection liquid of the present invention can be used for preparing the quantitative calibration product of Hsp90 α, quality-control product, immue quantitative detection reagent box Or detection method.These quantitative calibration product, quality-control product, immue quantitative detection reagent box or detection method can be used for determining whether individuality exists Whether tumor, especially malignant tumor and tumor shift；By the Hsp90 alpha levels in detection blood plasma, serum or body fluid High-risk group is carried out tumor screening；By the Hsp90 alpha levels in detection blood plasma, serum or body fluid to tumor patient Prognosis judges；Judge the operation to tumour patient by the Hsp90 alpha levels in detection blood plasma, serum or body fluid, put Treat or Drug therapy the most effectively and/or determines when stop treatment.
Accompanying drawing explanation
Fig. 1 is the stability of Hsp90 under different temperatures.
Detailed description of the invention
Term as used herein " protective agent ", " protein protective agent " or " protein protectant " is can be at protein quilt The protective agent shielded during lyophilizing, it may refer to the protein protective agent of single component, it is also possible to is by Multiple components group The mixture become.Common protein protective agent can be polyol, saccharide, protein, polymer, aminoacid, surface Activating agent, amine etc.." protective agent of protein types " specifically described herein refers to such protein protective agent, its constituent It is protein, such as bovine serum albumin, human serum albumin, chicken serum albumin, casein, casein sidium 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 Protein carries out the protection liquid of interpolation during lyophilizing, for activity and the stability of Protein requirement.Frozen-dried protective liquid is the most slow Rush liquid, wherein usually contain the protective agent and surfactant that can shield 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 (Heat shock protein 90 α, Hsp90 α).
" AMP " as herein described is adenosine phosphate.
" acid-base modifier " as herein described can be selected from buffer agent, acid or alkali, includes but not limited to sodium hydroxide, hydrogen-oxygen Change potassium, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, hydrochloric acid, nitric acid, sulphuric acid or a combination thereof.
Percentage composition mentioned herein, unless otherwise indicated, when solute is solid under normal conditions, for quality/body Volume concentrations, when solute is liquid under normal conditions, for volume/volume concentration.Described " 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 the impact on Hsp90 α protein stability of the single component protective agent
Purpose: screen new Hsp90 α protein protective agent, and determine its concentration scope and condition.
Reagent: phosphate, polysorbas20 and tea polyphenols are purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and bovine serum albumin is purchased From sigma company, heparin sodium is purchased from glad brilliant biological, and cyclodextrin is purchased from Beijing extensive and profound in meaning star biotechnology responsibility company limited.Hsp90 α finished product test kit is purchased from Yantai Pu Luoji biotechnology Development Co., Ltd, lot number 201602002,201604001, this reagent The calibration object of box is Hsp90 α dried frozen aquatic products.
Method:
1, preparation base soln, uses purified water, wherein containing 0.318% phosphate (sodium dihydrogen phosphate), 0.5% Ox blood serum Albumin and 0.2% polysorbas20, regulation acid-base value is to pH6.5, and this solution is the basic solution of later stage test.
2, base soln is used to prepare the frozen-dried protective liquid of the heparin sodium containing certain content, cyclodextrin or tea polyphenols respectively, The content of heparin sodium, cyclodextrin or tea polyphenols is:
1) heparin sodium: 0.01,0.03,0.06,0.1,1,10mg/ml；
2) tea polyphenols: 0.01%, 0.05%, 0.1%, 0.5%, 1%；
3) cyclodextrin: 0.05%, 0.1%, 0.5%, 1%.
3, the frozen-dried protective liquid using the heparin sodium containing certain content, cyclodextrin or the tea polyphenols of step 2 acquisition is joined respectively The Hsp90 α egg of 20ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 400ng/ml in the corresponding Hsp90 α finished product test kit of system White solution, and be dispensed in the cillin bottle of 2ml specification by every bottle of 400 μ l.
4, the bottled Hsp90 α protein solution containing protectant XiLin prepared is carried out lyophilizing, step of freeze drying and Hsp90 α Finished product test kit Hsp90 α albumen step of freeze drying is consistent.
5, the Hsp90 α albumen after lyophilizing is added aluminium-plastic cap to be tamping, take half quantity carry out 37 DEG C of Acceleration study (foundation: During the effect duration of external diagnosis reagent industry calculates, 37 DEG C are accelerated within 1 day, to be equivalent to 2-8 DEG C and deposit in real time 2 months), second half is put Put 2-8 DEG C of preservation.
6, after the Hsp90 α albumen dried frozen aquatic products acceleration time reaches, Hsp90 α test kit is used to detect.Contrast does not adds Testing result after speed and acceleration, the detected value after acceleration should reduce less than 15%.
Detection operating procedure is as follows:
1) take out test kit to balance 30 minutes at 37 DEG C, add after concentrated cleaning solution is added the mixing of 475mL deionized water Wash in trigger stand-by；
2) dried frozen aquatic products not accelerating and accelerating is added 0.4ml analyte diluent and dissolve mixing；
3) by the lath of desirable number as on grillage, the dried frozen aquatic products after 50 μ l dissolve is added；
4) in each hole, add 50 μ L survey Heat shock protein72 label liquid, mixing of vibrating gently；
5) shrouding film covers microwell plate, incubation 60 minutes at 37 DEG C；
6) washing plate: get rid of reactant liquor, every hole adds 300 μ L washing liquids and washes plate, or use is washed trigger and carried out washing plate, washes plate altogether 6 times, Last button in absorbent paper is done；
7) every hole is sequentially added into each 50 μ L of developer Α, B liquid, mixing of vibrating gently, 37 DEG C of incubations 20 minutes；
8) every hole adds 50 μ L stop buffer color development stopping；
9) after addition reaction terminating liquid in 10 minutes, reading OD value under 450nm/620nm wavelength, wherein 450nm is inspection Surveying wavelength, 620nm is reference wavelength, is used for removing ambient interferences.
Result:
After testing, it is possible to the concentration meeting 10 days results of accelerated stability is: 0.01-1mg/ml heparin sodium, 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 uses different protective agent 37 DEG C to accelerate 10 days
Table 2 uses different protective agent 37 DEG C to accelerate 10 days
More than 2 protective agent of embodiment is used in combination the impact on Hsp90 α protein stability
Purpose: research and develop new Hsp90 α protein protective agent combination formula, and determine its concentration scope and condition.
Reagent: phosphate, polysorbas20, MgCl2, tea polyphenols and K2SO4Purchased from Chemical Reagent Co., Ltd., Sinopharm Group, cattle Serum albumin and AMP are purchased from sigma company, and heparin sodium is purchased from glad brilliant biological, and PVP40 is purchased from glad brilliant biological, and cyclodextrin is purchased from Beijing extensive and profound in meaning star biotechnology responsibility company limited.Hsp90 α finished product test kit is limited purchased from the development of Yantai Pu Luoji biotechnology Company, lot number 201602002,201604001, the calibration object of this test kit is Hsp90 α dried frozen aquatic products.
Method:
Basic experiment method and steps is with the method step in embodiment 1, and have employed multiple protection in the present embodiment Agent uses formula in combination.
Protective agent combination point four big classes, each apoplexy due to endogenous wind is respectively provided with different concentration combination:
1) 0.1%-2%PVP 40,0.01%-0.5% tea polyphenols, 2mM-10mM AMP, 5mM MgCl2；
2) 0.01-0.1mg/ml heparin sodium, 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 heparin sodium, 0.1%-2%PVP 40,0.01%-0.5% tea polyphenols, 2mM-10mM AMP、5mM MgCl2、5mM-50mM K2SO4, 0.05%-0.5% cyclodextrin；
Result:
After testing, the accelerated stability result of the test of 12 days is met 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 heparin sodium, 0.1%PVP 40,0.01% tea polyphenols, 2mM AMP, 5mM MgCl2、5mM K2SO4
5) 0.1mg/ml heparin sodium, 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 heparin sodium, 0.1%PVP40,0.01% tea polyphenols, 5mM AMP, 5mM MgCl2、5mM K2SO4、 0.05% cyclodextrin
9) 0.1mg/ml heparin sodium, 2%PVP 40,0.5% tea polyphenols, 10mM AMP, 5mM MgCl2、50mM K2SO4、 0.5% cyclodextrin
Table 3 uses different protective agent to combine 37 DEG C of acceleration 12 days
Table 4 uses different protective agent to combine 37 DEG C of acceleration 12 days
Table 5 uses different protective agent to combine 37 DEG C of acceleration 12 days
The optimum effect combination formula obtained: 0.1mg/ml heparin sodium, 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 this combination (OD450/630 value) is as shown in table 6 below:
Table 6 optimum effect 37 DEG C of acceleration for stabilization testing results of combination formula
Embodiment 3 uses optimum effect protective agent combination formula to carry out the detection of Hsp90 α test kit performance indications
Purpose: by 12 days 37 DEG C of Acceleration study, test optimum effect protective agent combination formula was to Hsp90 α albumen and reagent The impact of box detection performance.
Method: detect according to the operating procedure in embodiment 1, wherein, using accelerate after Hsp90 α dried frozen aquatic products as Calibration object curve, is simultaneously introduced quality-control product and carries out the detection of test kit performance.
Result: the Hsp90 α lyophilizing calibration object using optimum effect combination formula is still stable after accelerating 12 days, test kit Property indices result is as shown in table 7 below:
Table 7 test kit property indices structure
Wherein, minimum detectability≤1.96ng/ml；Coefficient R2More than 0.980；The quality-control product of concentration high, middle measures 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 all meets relevant national standard.

Claims (10)

1. protein protective agent, it contains acid-base modifier, surfactant, the protective agent of protein types and other protective agent； Wherein
1) other protective agent described is heparin sodium, and the protective agent of protein types and the weight of heparin sodium in described protein protective agent Amount ratio is 5:0.01-1；
2) other protective agent described is tea polyphenols, and the protective agent of protein types and the weight of tea polyphenols in described protein protective agent Amount ratio is 5:0.1-5；Or
3) other protective agent described is cyclodextrin, and the protective agent of protein types and the weight of cyclodextrin in described protein protective agent Amount ratio is 5:0.5-5.
2. protein protective agent, it contains acid-base modifier, surfactant, the protective agent of protein types and other protective agent； Wherein
1) other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and protein types in described 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) other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and in described protein protective agent The protective agent of protein types, heparin sodium, 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) other protective agent described is PVP40, tea polyphenols, AMP, MgCl2、K2SO4And in cyclodextrin, and described protein protective agent The protective agent of protein types, PVP40, tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:1-20:0.1- 5:0.69-3.47:0.48:0.87-8.71:0.5-5；Or
4) other protective agent described is heparin sodium, PVP40, tea polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and described albumen guarantor Protect the protective agent of protein types, heparin sodium, PVP40, tea polyphenols, AMP, MgCl in agent2、K2SO4Weight ratio with 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.
Protein protective agent the most according to claim 2, wherein
1) other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and protein types in described protein protective agent Protective agent, PVP 40, tea polyphenols, AMP and MgCl2Weight ratio be: 5:1:0.1:0.69:0.48；
2) other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and protein types in described protein protective agent Protective agent, PVP 40, tea polyphenols, AMP and MgCl2Weight ratio be: 5:20:5:3.47:0.48；
3) other protective agent described is PVP 40, tea polyphenols, AMP and MgCl2, and protein types in described protein protective agent Protective agent, PVP 40, tea polyphenols, AMP and MgCl2Weight ratio be: 5:10:0.5:1.74:0.48；
4) other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and in described protein protective agent The protective agent of protein types, heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4Weight ratio be: 5:0.01:1: 0.1:0.69:0.48:0.87；
5) other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4, and in described protein protective agent The protective agent of protein types, heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2And K2SO4Weight ratio be: 5:0.1:20:5: 3.47:0.48:8.71；
6) other protective agent described is PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And in cyclodextrin, and described protein protective agent The protective agent of protein types, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:1:0.1: 1.74:0.48:0.87:0.5；
7) other protective agent described is PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And in cyclodextrin, and described protein protective agent The protective agent of protein types, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:20:5: 3.47:0.48:8.71:5；
8) other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and described albumen The protective agent of protein types, heparin sodium, PVP 40, tea polyphenols, AMP, MgCl in protective agent2、K2SO4Weight with cyclodextrin Ratio is: 5:0.01:1:0.1:1.74:0.48:0.87:0.5；Or
9) other protective agent described is heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And cyclodextrin, and described albumen The protective agent of protein types, heparin sodium, PVP 40, tea polyphenols, AMP, MgCl in protective agent2、K2SO4Weight with cyclodextrin Ratio is: 5:0.1:20:5:3.47:0.48:8.71:5.
Protein protective agent the most according to claim 2, other protective agent wherein said be heparin sodium, PVP 40, tea polyphenols, AMP, MgCl2、K2SO4And the protective agent of protein types in cyclodextrin, and described protein protective agent, heparin sodium, PVP 40, tea polyphenols, AMP、MgCl2、K2SO4With the weight ratio of cyclodextrin it is: 5:0.1:20:5:1.74:0.48:0.87:0.5.
5., according to the protein protective agent of any one of claim 1-4, it is the form of liquid.
6. the protein protective agent of any one of claim 1-5 application in lyophilization Hsp90 α.
7. the protein protective agent of any one of claim 1-5 is preparing quantitative calibration product, quality-control product or the detection by quantitative of Hsp90 α Application in test kit.
8.Hsp90 α protects compositions, and it contains Hsp90 α and the protein protective agent of any one of claim 1-5.
Hsp90 α the most according to claim 8 protects compositions, and it is solution or lyophilized powder.
10., for detecting the test kit of Hsp90 α, wherein comprise protein protective agent or the claim of any one of claim 1-5 The Hsp90 α of 8 or 9 protects compositions.
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