JP3397791B2 - Purification method of aqueous extract containing protein active as allergen, extract obtained by this method and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to allergen active proteins and further non-allergenic proteins.
Of an aqueous extract containing undesired rugenic compounds
Purification method. Aqueous pollen grains of herbs, weeds, trees and other plants
Extracts have been a predisposing patient since the beginning of this century, the so-called
Hay fever ("hay fever") in "atopic" patients
Widespread application for in vivo and in vitro diagnostics found
Has been. First written in 1911 by Noon and Freeman
Since being featured, such extracts have also been used for long-term
Injection for "sensitization", "desensitization" or "immunotherapy"
With therapeutic application, it has also been used to treat this disease
It was. The major causative allergen component in pollen extracts is a molecule
Based on the observation that the amount of protein is 20-70 kD,
On the other hand, components in the molecular weight range of less than 10 kD are non-allergens.
Therefore, pollen for diagnosis and immunotherapy
In the method of manufacturing the extract, the molecular size less than 5-10 kD
Aqueous pollen extract to remove components that are supposed to be
Dialysis or ultrafiltration through a membrane with a nominal cutoff of 5-10 kD
It is filtered and the allergenicity in the molecular size range of 10 to 100 kD
Allergen exhaustion that retains protein quality and is applied clinically
It is a common practice to improve the quality of video. Nevertheless, in the past, low molecular weight dialysis of aqueous pollen extracts
Possible components, that is, the distribution of substances with an upper limit molecular weight of about 10 kD
Many reports on the potential for rugenogenicity have been published in the scientific literature.
It was represented (Moore MB. & Moore EE., J.Am.Chem.S
oc., 1931; 53: 2744; Unger L., Cromwell, HW. & Moore M
B., J. Allergy, 1932; 3: 253). In these studies, low M <5kD from pollen extract
The molecular weight component actually leaves some allergen activity
Although they were instructed to
The strength of the non-dialysable M> 5kD glycoprotein is
The coefficient is at least 1000, which is lower than the case.
These results show that the dialysable fraction of pollen extract is extremely complex.
Coupled with the crude chemical composition, these studies were allowed to continue.
There was little inspiration. Therefore,
The level of performance technology is low molecular weight M> 5kD in pollen extract.
Minutes are irrelevant in terms of allergic and immunological contributions
That is. However, present in almost all pollen extracts
Water-soluble flavonoids-Clicoside content and organisms
The biological activity remains unclear. Non-currency
Significant effects on human and animal cell function after oral administration
Such compounds, which are believed to give
Is usually understood implicitly (Wi
ermann R., Wollenweber E. & Rehse, C., Z.Naturforsc
h., 1981; 36c: 204). However, for diagnosis and treatment
, Conventional dialyzed pollen containing M> 10kD protein
In the spectral analysis test of the extract, a very high rate (fla
Bonoid-) pigment remains strongly adsorbed to proteins
(See comparative data in FIG. 1). The present invention is directed to allergen active proteins and to non-allelic proteins.
Of an aqueous extract containing undesired rugenic compounds
Provide a purification method. According to this method, allergen activity is
Disadvantages of conventional aqueous extracts containing soluble proteins (always recognized
Not as) virtually as an allergen
Highly purified extract containing active protein
Generate. In a first embodiment, the present invention provides an allergen activity
Of non-allergenic proteins and even non-allergenicity
A method for purifying an aqueous extract containing a compound
The non-allergenic compound attached to the protein is
Sources of attachment of the above non-allergenic compounds to the proteins
A means of destroying the causative electrostatic and / or hydrophobic forces
By removing from the above proteins by using
is there. In particular, the method according to the invention comprises the following steps:
1) Allergen-active protein and non-allergenicity
Starting material is an aqueous extract containing unwanted compounds of
2) The above starting material is attached to the above protein
The above-mentioned non-allergenic compound is added to the above-mentioned protein.
Electrostatic forces and / or damage that can cause the attachment of allergenic compounds
Or from the above proteins using a means of disrupting hydrophobicity
In addition to the removal of the adhered undesirable
An aqueous extract containing substantially no compound is obtained, and 3) each step of collecting the substantially pure extract. Other embodiments of the present invention will be apparent from the description below.
U. According to the invention flavonoids and / or glycos
Impurities such as sid as well as other compounds exemplified below
Things are contained in ordinary extracts, but these are
It was established that it was not included in the “off” type. A
The “adhesion” of impurities to the allergen active protein is
Nderwaals force, ionic bond, hydrophobic interaction, further
Is based on chemical interactions (covalent bonds)
Conceivable. "Means to destroy electrostatic, hydrophobic or other physical forces"
Expression was adsorbed from allergen active protein
Or removes (strongly) attached compounds (impurities)
Various chemical and / or physical hands that are effective in
Regarding steps. In a preferred embodiment of the method of the present invention, electrostatic force
Or other means for destroying physical forces are acid, anion.
Alkaline substances including salts and cation exchange substances, salts
It is selected from the group of chemical means consisting of electric current and electric current. Removal of adsorbed impurities is used as a starting material
Due to changes in the electrical environment (charge) in the extract
Not only the force that affects the dielectric constant of the protein in question
It can also be implemented by use. Hydrophobic interactions and
Means affecting the Nderwaals force can be used as well. In the method of the present invention, chemical means, especially acid, is used.
Of allergen active protein below the isoelectric point
PH value less than 3, preferably in the range of 1.5 to 2.5
The use of acids having a pH value of is preferred. These pH values are
Generally, below the isoelectric point of almost all proteins
The When using alkali, for example, 9 to 11,
However, pH values above the normal isoelectric point can be used. This is because the concentration of acid and alkali is 0 for (strong) acid.
1 to 0.01N, 0.01 to 0.001N for (strong) alkali
It means that. On the other hand, salts at neutral pH, for example in the range of pH 6-8,
If dissociation or desorption of small molecules of
The relative salt concentration or ionic strength is involved. Preferably
Is a monovalent salt, such as NaCl, KCl, KCNS, the corresponding bromide,
Or for example guanidine hydrochloride is used. Preferred
The ionic strength value is 2 to 6 M, and 2 to 5 M is more preferable.
Yes. For example, the concentration of guanidine hydrochloride is higher than this
And, at the same time as hydrogen bonding of carrier protein, constituent protein
Note that the chain of the intermolecular chains of
Should be. However, on the other hand, such destruction
Are often allergenic and antigenic
There is no disadvantage. These immunological properties are
Regarding the secondary structure of proteins responsible for specific activity
Is considered to be maintained by the epitope.
The In another embodiment of the invention, electrostatic forces and physical
The force breakdown means consists of electric current in the form of electrophoresis. Normal
Electrophoresis is performed on aqueous extracts. Usually
Electrophoresis is performed by exposing the protein to local heat or (complete).
Do not run too long to avoid (all) denaturation
Absent. Furthermore, between the electrodes used, a suitable high voltage,
For example, generally 10 to 2000 volts (DC), especially 2
You should use 100-1000 volts (DC). High voltage
If too much, the protein carrier itself becomes colloidal chemical electrokinetic.
It may be perturbed by potentials, resulting in tertiary structure or
Or irreversible unfolding of secondary structure
The stabilized water layer may be lost due to. Mochiro
The current depends on the degree of hydrochloric acid in the solution, usually in the buffer.
The According to the method of the present invention, the molecular weight is usually less than 10,000,
Preferably, non-allergenic compounds having a molecular weight of less than 5000 are excluded.
However, the molecular weight is lower than that of 1000, for example.
Equally good results can be obtained by removing the full compound from the protein.
It will be obvious that the result will be obtained. However, flavonoids and / or glycosi
Is preferably removed. The flavonoids present in this aqueous extract are arachi
Affects donic acid metabolism and is a phenol or quinoid derivative
Interact with macromolecules as, for example, tannin-like
Causes enzyme inactivation by forming a complex with a polymer
Scrub. Strong interactions with proteins are actually
Enols (including flavonoids), their oxidation products
And the general properties of those polymers. Above
As used as a starting material in the method of the invention.
The interaction of impurities present in the extract is a physical interaction.
Application (van der Waals force, ionic bond, hydrophobic interaction
Or chemical interaction [oxidized (bi-) pheno
Covalent force due to the interaction with Flavono
Id and flavonoid glycosides are molecules of their own
Of histidine from mast cells and basophils
Release and impair normal function of polymorphonuclear leukocytes and neutrophils
Will be modified. These biological activities are
According to prior art preparations that are routinely carried out by
Can significantly affect diagnostic skin and inhalation test results.
It is possible to get it. On the other hand, European Patent No. 90200562.8
Is a low molecular weight (M <5000) water-soluble, non-adsorbing flavo
Allergy patients specifically sensitized with nucleoside glycosides
Does not contribute to IgE antibody binding in the serum
Widely proven. Therefore, it is commonly used
Flavo from diagnostic and therapeutic pollen protein vaccines
Removes impurities such as noids and their glycosides
What to do and their usefulness in clinical medicine
It improves the efficacy. Adsorbed from plant pollen proteins administered to humans
Yet another reason to remove low molecular weight compounds is to use pollen
Many pollen species they produce are low in content that can be harmful to humans.
Offspring (M <1000) non-flavonoid organic compounds, even
Toxic alkaloids, benzoquinones, terpenoids and
And their derivatives that stimulate the mucosa and other aromatic rings
May contain structural compounds. Similar to flavonoids
In addition, such components also have a strong effect on proteins due to physical force.
By solid adsorption, through a membrane with a nominal cutoff of 10 kD
A field that resists simple dialysis or ultrafiltration at neutral pH.
There is a match. The invention also relates to the method of the invention described herein.
Therefore, it relates to the extract obtained. Such purified
The extract is intended for the diagnosis and treatment of allergic diseases
Guarantees the safety of allergen plant pollen extract
is there. According to the method of the present invention, a wide variety of naturally occurring
The substance can be (further) purified. A typical example is
It is as follows. Pollen extract Tree pollen, herbaceous pollen and weed pollen for: Tree pollen: Herbaceous and weed pollen: Epidermis and glandular components Camel hair and dandruff; cattle hair and dandruff; cat
Hair and Dandruff; Deer Hair and Dandruff; Chicken Feathers;
Duck feathers; goose feathers; ohm feathers; pigeon feathers
Feathers; turkey feathers; fox fur; gerbil hair
And epithelium; goat hair and dandruff; guinea pig hair and
Dandruff; hamster hair and epithelium; pig hair and hair
Horsehair and dandruff; Human dandruff; Monkey hair and
Dandruff; mouse hair and epithelium; dog hair and epithelium;
Moss chrysanthemum; rabbit hair and epithelium; rat hair and epithelium. Dust and other extracts: coconut fiber; cotton waste; cotton seeds; barley dust;
ー Sorghum dust; House dust; Grain milling dust; Ma
Tress dust; oat dust; pea dust;
Rye dust; soybean dust; furniture dust; wheat dust;
Fern / Juniper dust; Fir / Tsuga dust; Rubber
Wood dust; Mahogany wood dust; Maple wood dust; Oh
Black mixed material dust; pine mixed material dust; red material dust; spruce wood
Dust; walnut dust; fern spores; flax fibers, seeds
Child; Asa; Jute; Kapok; Karaya gum; Hijageno
Vine; rhizome of iris; insect chrysanthemum; silk; sisal hemp;
Tobacco; soybeans; castor beans. Insect extract: ants (black, red); giant ants;
C; trichomes; crickets; cockroaches; fly; flea antigen;
Fruit fly; chironomid species; mite (D. farinae, D. pteronyssimu
s, Lepidaglyphus spp.); The present invention also provides a purified extract obtained by the present invention.
For standardization, diagnosis, synthesis, and vaccination
Regarding the use of. A particular embodiment of the present invention will be described by the following operations AC.
Here is an example. Operation A Collect pollen grains from botanically determined plants and
Air dry. Lipids, fatty acids, free flavonoids and others
Of the non-polar free organic material, followed by a water-immiscible organic solvent.
For example, with dry diethyl ether or n-hexane
Continuously extract and remove pollen from a soxhlet machine.
The defatted pollen mass is air dried again and then mechanically agitated.
However, at 4 to 20 ° C, use an aqueous solvent or dilution buffer.
Is an ammonium hydrogen carbonate solution or the pH value is maintained at 6 to 8.5.
Extract for 2 hours with the distilled water. The mixture is then about 30
Centrifuge at 00-5000 rpm for 30 minutes to collect the supernatant liquid
The Discard insoluble residue or repeat the procedure
You may re-extract twice. Aqueous extract (combined) normal
It is clarified by filtering through filter paper, and then the pH value is 5.5 to 6.0 or more.
Dialyze for 18 hours against distilled water with a pH value below 7.5,
Change the distilled water several times in between. Nominal cut for dialysis process
Commercially available membranes with off values of 5-10 kD (eg Visking cellophane).
Dialysis tubing) or, alternatively,
Kiss is a suitable membrane with the same cutoff range (eg Amico
n or Millipore Ultra or DiaFilter membrane)
It may be diafiltered. High molecular weight (HMW) allergenicity
Undialysed dialysate solution containing protein was reconstituted.
Later, M> 5-10kD allergenic proteins were further refined.
To produce, it is either lyophilized to dryness or thawed.
Process the liquid as it is. In this procedure, HMW of freeze-dried substance was added to distilled water at 0.5%.
Redissolve to a concentration of ~ 1.0% w / v and adjust the pH of the solution to 6N (or
Adjust the pH to 2 by adding hydrochloric acid (higher concentration). Exhaust
Then, 100 volume of distilled water (pH 6-7.5) is used as the external liquid.
Again, redialyze at a temperature of 4 to 20 ° C for 24 hours. In the process
The external fluid is both the external fluid and the free floating dialysis bag.
By placing the container holding the on the magnetic stirrer,
Keep under constant stirring. Completed passage of desorbed dye through the membrane
After that, the pH value of the external solution rises to about 3.5. This
Immediately from the beginning, use acidified water of pH 2 as the external solution.
When used, the efficiency of release of adsorbed dye is not improved
It was revealed. Inside the dialysis bag after the separation process
Dialysate liquid retained in part or by diafiltration membranes
1N-NaOH was added dropwise to the mixture under stirring to adjust the pH to 6.5-7.5.
Do. The solution thus neutralized is finally freeze-dried.
And the final product is depigmented allergenic pollen protein
Quality, DPP is collected. As described in Example III, this operation
And the decrease in the pH of itself is due to the allergenic protein
It was revealed that it does not have a significant effect on the antibody recognition site.
It was. Depending on the nature and species of the pollen grain, the procedure outlined above
Is the first allergenic pollen protein preparation
15 to 65% w / w of the adsorbed dye with respect to the dry weight of
(Table 1). Desorbed, which may include small peptides
The neutralized external liquid was removed by a rotary thin-film distillation system under reduced pressure.
It is collected separately when concentrated in the storage. This method is controlled by the UV absorption spectrum
The As shown in Fig. 1, acid dialysis was performed after predialysis at neutral pH.
Aqueous solution of pollen protein HMW before and redialysis
The absorption value at 260 to 280 nm is extremely high.
Non-depigmenting allergenicity of pollens on trees, weeds and shrubs
The quality is high. Therefore, the absorption at 260-180 nm
When the protein content is evaluated from the coefficient, it is often found that
This will result in a great evaluation. Removes adsorbed dye
Then, the flavonos in the 260-280 nm region and 340-360 nm
Significant decrease in extinction coefficient in the main absorption range of id
(Table II). Determine the allergen intensity of the final depigmented product DPP to a specific
Specific IgE in serum of patients with allergic pollinosis-also
Inhibits the binding of IgG-class antibody to the first pollen target.
Compare with the case of high quality HMW. For HMW preparation
The IgE-binding capacities of the DPP products compared were, for example, HMW and
Applies the DPP product to a cellulose disk with cyanogen broth.
Chemically coupled with amide or other coupling material
Followed by radiant allergen adsorption or enzyme allele
Quantitatively measured by the established operation of gen adsorption test
Wear. For specific human IgG-antibody reaction and quantitative evaluation,
Polystyrene mimics either DPP or HMW proteins.
Physically adsorbed on the surface of the wells of the clot titer plate.
Then perform the operation of the established enzyme immunoassay.
Can be. Operation B In a modification of the manufacturing method, as described in Operation A, pollen is used.
Preparation of allergenic protein HMW from granules, M> 10k
PH 5.5 to 7.5 containing D allergenic protein
The dialysate solution is not dried by freeze-drying. in addition
Instead, add 6N hydrochloric acid dropwise to the protein solution as it is.
PH to 2 and the thus acidified solution to 6
~ 24 hours redialysis or M = 10kD nominal cut-off membrane
Subject to new ultrafiltration. Then, not dialyzed
The product solution was collected and adjusted to pH 6.5-7.5 with 1N-NaOH,
Dry by lyophilization. Operation C The experience gained in developing operations A and B of the present invention is:
Flavonoids to pollen proteins-or flavonoids
-The strong adsorption of glycoside dyes is due to electrostatic force,
The purpose of desorption is the negative carboxy of either partner.
A hydroxyl group and / or a phenolic hydroxyl group to 3.0
What can be achieved by discharging at the following pH values
Proved. However, such conditions are essential
Consider the possibility of denaturation or loss of constituent protein determinants
This is not desirable, so it is not possible to use the voltage in the natural pH range 6.5 to 7.5.
An operation based on the removal of adsorbed dyes in the field was developed. In this method, the freeze-dried allele described in operation A is used.
0.01 M inorganic buffered salt solution of the genic protein HMW (pH 6.5 to 7.
5), eg make a 2-5% solution in phosphate buffered saline
Do. Depending on the technical equipment chosen, the appropriate solution of this solution
The volume is then subjected to free electrophoresis and protein-dye
Destroys the ionic force that causes adsorption. Color during electrophoresis
The element migrates rapidly to the anode compartment and therefore
Protein constituents that move slowly and are retained on the cathode side
Can be separated from. The technical prototype of this operation is shown in Example III.
I will. Example I In this experiment, ragweed (Ambrosia elatior)
Dried pollen (from Beecham Research Laboratories, UK
Degrease (commercially available product) with diethyl ether,
Extracted with distilled water as described. Dialyzed and freeze-dried
From HMW Ragweed Pollen Protein Preparation, 25
Dissolve the mg sample in 5 ml of distilled water and add 6N hydrochloric acid dropwise.
In addition, the pH was set to 2. UV absorption spectrum is 0.01N hydrochloric acid
It was diluted 1:20 and observed separately. Keep the sample under agitation
Dialyzed for 24 hours against distilled water with a total volume of 500 ml at high temperature
did. After the acid dialysis step, the UV-absorbance of the internal dialysate liquid
The vector was again diluted 1: 2 in 0.01N hydrochloric acid and observed. Outside
The liquid is the first in the thin-film evaporator RotaVapor ™.
Concentrate to a volume of 5 ml and make the UV-absorption spectrum 1:20 at pH 2.
It was diluted and measured. In this experiment, the adsorbed flavonoids
The effective removal of the dye was confirmed by the UV recorded in Figure 2.
-Obvious from absorption spectra and extinction coefficient figures in Table II
Are shown in. Freeze-drying removal recovered by operation A
The yields of chromoprotein material DPP are listed in Table I. Example II In these experiments, strong allergenic flowers of grasses were used.
Typical examples of flour are dory wheat (Lolium perene) and mosquitoes.
Mogaya (Dactylis glamerata), and typical
The red lizard (Ch
enopodium album) and mugwort (Artemisia vulgaris)
The same procedure as described in Example I was applied to pollen of HMW.
Et al. For numerical data on the yield of DPP and related
Numerical values for the kutor analysis are shown in Tables I and II. These experiments
The results show that allergenic pollen tampers prepared by the traditional method
Removal of flavonoid (-glycoside) pigments from humic HMW
The garment is applied to the weed ragweed pollen described in Example I.
Not only spread to other weeds and herbaceous pollen
It is clear. Example III In these experiments, the same procedure A as described in Examples I and II was used.
By tree birch (Betula alba), olives
(Olea europea) and a widespread Mediterranean weed
Powerful allergy of Pietaria judaica
Pollen was treated. Raw material allergenic protein HM
W and the corresponding depigmenting protein DPP were identified as specific alleles.
Binding of specific IgE- and IgG-antibodies in the serum of ghee patients
The in vitro allergenicity was examined by inhibition. Inhibition of binding to specific IgE-antibodies has been established.
The RAST-inhibition method was selected. This method should be considered
Serum samples from patients with hay fever caused by pollen proteins
Cyano Powdered Protein HMW or Depigmented Equivalent DPP
Gen bromide or other suitable chemical coupling agent
Cellulose disks covalently bound using
Incubate with. This incubation
Digest the disc after capturing specific IgE-antibody in phase
Wash with buffer and then with enzyme-labeled anti-IgE-antibody
Incubation step followed by enzyme-specific
Color is developed by the chromogenic substrate. Given allergen
To evaluate the IgE-binding ability of a sex protein,
Pre-incubate a dilution series with a fixed volume of human serum sample.
After beating, the remaining IgE is treated with allergen-coated cellulosics.
Capture with a Sudisk. Allergenic prepare
Fig. 3 shows the IgE-binding ability of the sion as an example of birch pollen.
As can be seen from the plot of allergen concentration vs. IgE-binding,
Read as 0% inhibition point. In this example, two types of allergic
-Coated cellulose disc or first protein
Chemical to Preparation HMW (Dye Disc in Table III)
The disk coupled to the
Chemically to the prepared protein preparation DPP
Coupled discs
) Was used. As shown in the aggregated data in Table III, the DPP
The allergenic IgG-E-binding ability of the protein is
And in the case of Parietaria, per 1 μg of lyophilized substance
The value of is not significant but shows a slight downward trend, while
In the case of olive samples, IgE-
We have to conclude that it results in the loss of some of the binding epitopes.
Absent. In a separate experiment, olive HMW protein
H2 retention denatures into olive pollen protein
We checked whether or not the effect was shown. For this purpose,
The HMW of the tube was adjusted to pH 2 with hydrochloric acid according to procedure A, and this solution was stored in the chamber.
It was left warm for 4 hours. This solution is then dialyzed.
The pH was readjusted to 7.0. This preparation
Lyophilize and use olive DPP discs with RAST inhibition
When the IgE-binding ability was checked by
The results for a particular selected human serum under the
% RAST inhibition treated with 0.75 μg, pH 2 for olive HMW
0.75 for olive HMW readjusted to pH 7 without dialysis
μg, 3.89 for olive DPP prepared according to procedure A
The result was μg. IgE-binding of olive pollen DPP
Allergenicity is reduced compared to Olive HMW
Is not due to protein denaturation at acidic pH.
And other electrostatically adsorbed substances that actually act as antigenic determinants.
It is due to the removal of low molecular weight organic compounds
Considering these points, antibody-binding allergenic epitopes
Comparative study by the detailed study of the molecular structure of
Clarify the usefulness of proteins. Inhibition of binding to specific IgG antibodies has been established.
Different enzyme immunoassays were selected. In this method,
Serum Samples of Pollinator Patients Due to Pollen Proteins
The HMW or DPP allergen dilution series and incubation.
To The allergen-serum mixture was then pre-physical
Police coated with HMW or DPP protein by adsorption
Pipette into the wells of a microtiter plate.
Was added in. After 30 minutes at room temperature, the wells are diluted with buffer.
Uniquely washed with liquid and captured on allergen-coated plates
The specific IgE-antibody was treated with enzyme-labeled anti-IgG, and then the enzyme-
The color is measured with a specific chromogenic substrate. That
The IgG-binding ability of the rugenic preparation is
50% inhibition point interpolated from the plot of IgG concentration vs. IgG-binding
Evaluate as. In this Example III experiment,
All Tarwells were DPP-pre-prepared according to Procedure A.
Covered with paration. Data in Table III are for IgG
The binding capacity of DPP is slightly higher than that of HMW products
Which indicates that. Example IV In these experiments, isolated and dialyzed at neutral pH
Raw pollen protein of Tacta (Ambrosia elatior)
Was destained by free electrophoresis according to procedure C. 20 m
Prepare a solution of gHMW / ml and dialyzate 5 ml of this solution with cellophane.
Bag (Visking dialysis tube, nominal cut-off 10000
D). Close both ends of the bag and place the electrophoresis device (R
Rotating plastic holder from otaPhor (BioRad, USA, trademark)
I attached it to the dar. The void volume of the holder tube is phosphoric acid
Fill with buffer-saline solution (0.01M, pH7.4), the device is 4 ℃
It was placed in the cold room at. Then store the dialysis bag and the external solution.
While continuously rotating the tube that you have,
A DC voltage was applied at constant power to start electrophoresis. Electric
The current drops from 120mA at the beginning of the experiment to 68mA after 3 hours
It was. During the course of electrophoresis, pigment is released from pollen proteins.
Electrode compartment for external fluid
Move to (cathode). Depigmented tamper after migration
The protein is recovered from the internal solution of dialysis gag and freeze-dried.
Therefore, the recovered amount of DPP product was 100 mg of ragweed.
69 mg from HMW, electrophoresed at neutral pH by procedure C
The yield of depigmented substance was 69%,
It was comparable to (Table I).

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Claims (7)

(57) [Claims] 1. A method for purifying an aqueous extract containing an allergen-active protein and a non-allergenic undesirable compound, wherein the non-allergenic compound attached to the protein is attached to the protein. A method of eliminating electrostatic force, hydrophobic force or other physical force that is the cause of, by means of destroying electrostatic force, hydrophobic force or other physical force,
A method for purifying the above-mentioned aqueous extract, which comprises treating the aqueous extract with an acid, an alkali or a salt, or exposing the aqueous extract to an electric current. 2. The method according to claim 1, wherein the acid or alkali is used in an amount exceeding the isoelectric point of the protein. 3. The method of claim 1 wherein the means for destroying electrostatic forces comprises an acid having a pH value of less than 3, preferably a pH value of 1.5 to 2.5. 4. The method of claim 1 wherein the means for disrupting electrostatic forces comprises electrical current in the form of electrophoresis. 5. The method according to claim 1, wherein the non-allergen compound has a molecular weight of less than 10,000, preferably less than 5,000. 6. The method according to claim 1, wherein the non-allergenic compound comprises flavonoids and / or their glycosides. 7. An extract obtained according to any one of claims 1 to 6.