JPH08504187A - Purification method of aqueous extract containing protein active as allergen, extract obtained by this method and use thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to the removal of various low molecular weight components unrelated as allergens from conventional aqueous extracts of plant pollen allergen active proteins. The desorption and subsequent removal of low molecular weight pigments and other compounds normally retained by strong electrostatic and / or hydrophobic forces from allergenic pollen protein extracts prepared by traditional methods has been described. Such depigmented pollen proteins do not impair their allergenic ability and immunological specificity. The present invention allows for the production of fully active allergenic pollen proteins without adsorbed low molecular weight substances that would interfere with safety, diagnostic accuracy and clinical efficacy. Purified pollen proteins provide an improved starting material for attenuated vaccines for chemical derivatization, ie, immunotherapy. The present invention also provides a product suitable for studying the molecular structure of an epitope of an allergen.

Description

The present invention relates to a method for purifying an aqueous extract containing an allergen-active protein, the extract obtained by this method and the use thereof. The present invention comprises an allergen-active protein and also a non-allergenic unwanted compound. It relates to a method for purifying an aqueous extract. Aqueous extracts of pollen grains of herbs, weeds, trees and other plants have been used extensively for in vivo and in vitro diagnosis of hay fever ("hay fever") in predisposed, so-called "atopic" patients since the beginning of this century Has been found. Since first described by Noon and Freeman in 1911, such extracts have also been shown to be useful in injection therapy for long term "desensitization", "desensitization" or "immunotherapy". It has also been used to treat diseases. Based on the observation that the major causative allergen component in pollen extract is a protein with a molecular weight range of 20-70 kD, while components with a molecular weight range of less than 10 kD are considered to be non-allergens, they are used for diagnosis and immunotherapy. In the method for producing pollen extract of the present invention, the aqueous pollen extract is dialyzed or ultrafiltered through a membrane having a nominal cutoff of 5 to 10 kD to remove components assumed to be irrelevant having a molecular size of less than 5 to 10 kD. It is common practice to retain allergenic proteins in the 〜100 kD range to improve the quality of clinically applied allergen extracts. Nevertheless, in the past, many reports have been published in the scientific literature on the potential for allergenicity of low molecular weight dialyzable components of aqueous pollen extracts, ie, substances with a maximum molecular weight of about 10 kD (Moore MB. & Moore). EE., J. Am. Chem. Soc., 1931; 53: 2744; Unger L., Cromwell, HW. & Moore MB., J. Allergy, 1932; 3: 253). Although these studies indicated that low molecular weight components of M <5 kD from pollen extracts actually retained some allergenic activity, their strength on a weight basis was non-dialytic. The coefficient is at least 1000, which is lower than that of the glycoprotein with M> 5 kD. These results, combined with the extremely complex chemical composition of the dialyzable fraction of pollen extract, were unlikely to stimulate these studies to continue. Thus, the state of the art is that low molecular weight components of M <5 kD in pollen extract are irrelevant in terms of allergic and immunological contributions. However, the content and biological activity of the water-soluble flavonoid-glycosides present in almost all pollen extracts remain unknown. It is generally implicitly understood that such compounds, which appear to have a significant effect on cellular functions in humans and animals after parenteral administration, are removed during the dialysis process (Wiermann R., Wollenweber). E. & Rehse, C., Z. Naturforsch., 1981; 36c: 204). However, spectral analysis tests of dialyzed conventional pollen extracts containing M> 10 kD protein for diagnostics and therapeutics have shown that a very high percentage of (flavonoid-) dyes remain strongly adsorbed to the protein. (See comparative data in FIG. 1). The present invention provides a method for purifying an aqueous extract containing an allergen active protein and also a non-allergenic undesirable compound. This method produces a highly purified extract containing a substantially allergen-active protein without the disadvantages (although not always observed) of conventional aqueous extracts containing allergen-active proteins. In a first embodiment, the present invention relates to a method of purifying an aqueous extract containing an allergen-active protein and a further non-allergenic undesirable compound, wherein the non-allergenic compound adhering to the protein is converted to the protein. A method for removing the non-allergenic compound from the protein by using a means for destroying electrostatic force and / or hydrophobic force that causes the non-allergenic compound to adhere to the protein. In particular, the method according to the present invention comprises the following steps: 1) preparing an aqueous extract containing an allergen-active protein and also a non-allergenic undesirable compound as a starting material, 2) converting said starting material into said protein Subjecting the non-allergenic compound attached thereto to removal from the protein using a means for destroying the electrostatic force and / or hydrophobic force that causes the non-allergenic compound to adhere to the protein, Obtaining an aqueous extract substantially free of the attached undesired compounds; and 3) collecting each of the substantially pure extracts. Other embodiments of the present invention will be apparent from the description below. According to the present invention, impurities such as flavonoids and / or glycosides as well as other compounds exemplified below are contained in conventional extracts, but not in "free" form. It was established. The "attachment" of impurities to allergen-active proteins is thought to be based on van der Waals forces, ionic bonds, hydrophobic interactions, and even chemical interactions (covalent bonds). The expression “means of destroying electrostatic, hydrophobic or other physical forces” refers to various chemicals that are effective in removing adsorbed or (strongly) attached compounds (impurities) from allergen-active proteins. About physical and / or physical means. In a preferred embodiment of the method of the present invention, the means for destroying electrostatic forces or other physical forces are a group of chemical means consisting of alkaline substances, including acids, anions and cation exchange substances, salts and electric currents. Is selected from Removal of adsorbed impurities can be performed not only by changing the electrical environment (charge) in the extract used as a starting material, but also by using forces that affect the dielectric constant of the protein in question. Means that affect hydrophobic interactions and Van der Waals forces can be used as well. In the method of the present invention, it is preferable to use a chemical means, particularly an acid, in an amount which is lower than the isoelectric point of the desired allergen-active protein. Preference is given to using acids having a pH value below 3 and preferably in the range from 1.5 to 2.5. These pH values are generally below the isoelectric point of almost all proteins. When an alkali is used, for example, a pH value of 9 to 11, that is, a pH value exceeding a normal isoelectric point can be used. This means that the concentration of acid and alkali is on the order of 0.1 to 0.01 N for (strong) acid and 0.01 to 0.001 N for (strong) alkali. On the other hand, if it is desired to dissociate or desorb low molecular weight compounds with a salt at a neutral pH, for example in the range of pH 6-8, the absolute salt concentration or ionic strength is relevant. Preferably, use is made of monovalent salts such as NaCl, KClI, KCNS, the corresponding bromides or, for example, guanidine hydrochloride. Preferably, the value of the ionic strength is from 2 to 6M, more preferably from 2 to 5M. It should be noted that higher concentrations of guanidine hydrochloride, for example, tend to break the intermolecular chains of the constituent proteins as well as hydrogen bonding of the carrier protein. On the other hand, however, such destruction is not disadvantageous, since in many cases allergenicity and antigenicity are maintained. These immunological properties are thought to be maintained by the epitope with respect to the secondary structure of the protein responsible for this specific activity. In another embodiment of the present invention, the means for destroying electrostatic and physical forces comprises current in the form of electrophoresis. Usually, electrophoresis is performed on aqueous extracts. Normally, electrophoresis should not be performed for too long, to avoid local heat exposure or (complete) denaturation of the protein. In addition, a suitable high voltage should be used between the electrodes used, for example generally 10 to 2000 volts (DC), in particular 200 to 1000 volts (DC). If the voltage is too high, the protein carrier itself may be perturbed by the colloidal chemical electrokinetic potential, resulting in irreversible unfolding of the tertiary or even secondary structure and the formation of a stabilized aqueous layer. May be lost. Of course, the current depends on the salt concentration in the solution, usually in the buffer. The method of the present invention generally removes non-allergenic compounds having a molecular weight of less than 10,000, preferably less than 5,000, but further removes lower molecular weight compounds, for example, those having a molecular weight of less than 1,000, from proteins. It is clear that will give good results as well. However, it is preferred that the flavonoids and / or glycosides are removed. Flavonoids present in this aqueous extract affect arachidonic acid metabolism and interact with macromolecules as phenolic or quinoid derivatives, for example, causing enzyme inactivation by complexing with tannin-like polymers. Strong interactions with proteins are in fact the general properties of polyphenols (including flavonoids), their oxidation products and their polymers. As described above, the interaction of impurities present in the extract used as a starting material in the method of the present invention may be a physical interaction (van der Waals force, ionic bond, hydrophobic interaction) or a chemical interaction [oxidation Covalent force due to the interaction with (bi-) phenol.] Flavonoids and flavonoid glycosides, by themselves, inhibit the release of histidine from mast cells and basophils and significantly modify the normal function of polynuclear leukocytes and neutrophils. These biological activities may significantly affect the results of diagnostic skin and inhalation tests from prior art preparations routinely performed on allergic patients. On the other hand, EP 902005562.8 states that low molecular weight (M <5000) water-soluble, non-adsorbable flavonoid glycosides do not contribute to the binding of IgE antibodies in the serum of specifically sensitized allergic patients. Has been widely proven. Thus, removing impurities such as flavonoids and their glycosides from conventional diagnostic and therapeutic pollen protein vaccines would improve their utility and effectiveness in clinical medicine. Yet another reason for removing adsorbed low molecular weight compounds from plant pollen proteins administered to humans is that many pollen species that produce pollen have low molecular weight (M <1000) non- Flavonoids may contain organic compounds, such as toxic alkaloids, benzoquinones, terpenoids and their derivatives that stimulate mucous membranes, and other aromatic ring compounds. Like flavonoids, such components may resist simple dialysis or ultrafiltration at neutral pH through a 10 kD nominal cutoff membrane due to strong adsorption to proteins by physical forces. The present invention also relates to the extract obtained according to the method of the invention described herein. Such a purified extract guarantees the safety of allergen plant pollen extract intended for diagnosis and treatment of allergic diseases. According to the method of the present invention, a wide variety of naturally occurring substances can be (further) purified. Typical examples are as follows. Pollen extract The following tree pollen, herbaceous pollen and weed pollen: Tree pollen: Epidermal 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; Fox fur: gerbil hair and epithelium; goat hair and dandruff; guinea pig hair and dandruff; hamster hair and epithelium; pig hair and dandruff; horse hair and dandruff; human dandruff; Mouse hair and epithelium; dog hair and epithelium; pyrethrum; rabbit hair and epithelium; rat hair and epithelium. Dust and other extracts: coconut fiber; cotton waste; cotton seed; barley dust; corn dust; house dust; grain milling dust; mattress dust; oat dust; pea dust; rye dust; soy dust; Wheat dust; fern / juniper dust; fir / hemlock dust; rubber dust; mahogany dust; maple dust; oak mixed dust; pine mixed dust; red wood dust; spruce wood dust; walnut wood dust; Spores; flax fiber, seeds; hemp; jute; kapok; karaya gum; lycopodium; rhizome rhizome; pyrethrum; silk; sisal hemp; tobacco; soybean; Insect extract: Ant (black, red); Giant ant; Hari ant; Butterfly; Butterfly; Tobi-kella; Cricket; Cockroach; Abu; Flea antigen; fruit flies; chironomid species; The present invention also relates to the use of the purified extract obtained according to the invention for standardization, diagnostics, synthesis and vaccination purposes. Particular embodiments of the present invention are illustrated by the following operations AC. Operation A Collect pollen grains from botanically defined plants and air-dry at room temperature. Lipids, fatty acids, free flavonoids and other non-polar free organic substances are then removed by continuous extraction from pollen in a Soxhlet apparatus with a water-immiscible organic solvent such as dry diethyl ether or n-hexane. The defatted pollen mass is air-dried again and then with mechanical stirring at 4-20 ° C. with an aqueous solvent, ie a diluting buffer or ammonium bicarbonate solution or distilled water maintaining the pH at 6-8.5. Extract for 2 hours. The mixture is then centrifuged at about 3000-5000 rpm for 30 minutes and the supernatant liquid is collected. The insoluble residue may be discarded or re-extracted once more with the same procedure. The aqueous extract (together) was clarified by filtration through ordinary filter paper, and then dialyzed against distilled water having a pH value of 5.5 to 6.0 or more and 7.5 or less for 18 hours, during which time several distillations were performed. Change the water. The dialysis process uses commercially available membranes (e.g., Visking cellophane dialysis tubing) with a nominal cutoff value of 5-1 OkD or, alternatively, the extract is a suitable membrane with the same cutoff range (e.g., Amicon or It may be diafiltered through a Millipore Ultra or DiaFilter membrane). The undialyzed dialysate solution containing the high molecular weight (HMW) allergenic protein is finally dried to dryness by lyophilization or the solution is further purified to further purify the M> 5-10 kD allergenic protein. Process as is. In this operation, HMW of the lyophilized substance is redissolved in distilled water to a concentration of 0.5 to 1.0% w / v, and the pH of the solution is adjusted by dropping 6N (or higher concentration) hydrochloric acid dropwise. Adjust to pH2. The extract is then dialyzed against 100 volumes of distilled water (pH 6 to 7.5) as an external solution at a temperature of 4 to 20 ° C. for 24 hours. In this process, the external solution is kept under constant stirring by placing a container holding both the external solution and the freely suspended dialysis bag on a magnetic stirrer. After the desorbed dye has passed through the membrane, the pH value of the external solution rises to about 3.5. In the process, it was found that the use of acidified water of pH 2 as the external solution immediately from the beginning did not improve the efficiency of the release of the adsorbed dye. After the separation process, IN-NaOH is added dropwise with stirring to the dialysate liquid inside the dialysis bag or held by the diafiltration membrane to bring the pH to 6.5-7.5. The solution neutralized in this way is finally lyophilized to recover the depigmented allergenic pollen protein, DPP, as the final product. As described in Example III, this procedure revealed that lowering the pH of itself did not significantly affect the antibody recognition site of the allergenic protein. Depending on the nature and species of the pollen grains, the procedure outlined above removes 15-65% w / w of the adsorbed pigment based on the dry weight of the initial allergenic pollen protein preparation (Table 1). . The desorbed dye, which may contain small peptides, is recovered separately when the neutralized external solution is concentrated under reduced pressure on a rotary thin-film distillation apparatus. This method is controlled by the UV absorption spectrum. As shown in FIG. 1, the absorbance at 260-280 nm of the conventional aqueous solution of pollen protein HMW before predialysis at neutral pH but before acid desorption and redialysis shows extremely high values, especially for trees and weeds. High in non-depigmented allergenic proteins of pollen and shrub pollen. Thus, estimating protein content from the extinction coefficient at 260-180 nm often results in overestimation. Removal of the adsorbed dye results in a significant decrease in the extinction coefficient of the major flavonoid absorption range in the 260-280 nm region and 340-360 nm (Table II). The allergen intensity of the final depigmented product DPP is compared to that of the original pollen protein HMW by inhibiting the binding of specific IgE- or IgG-class antibodies in the sera of certain allergic hay fever patients. The IgE-binding ability of the DPP product compared to the HMW preparation may be determined, for example, by chemically coupling the HMW or DPP product to a cellulose disc with cyanogen-bromide or other coupling agents, followed by radioallergen adsorption or enzyme It can be measured quantitatively by the established procedure of the allergen adsorption test. For quantitative evaluation of the reaction with a specific human IgG-antibody, either DPP or HMW protein is physically adsorbed on the surface of the wells of a polystyrene microtiter plate, followed by an established enzyme immunoassay procedure. be able to. Operation B In a variant of the production method, the allergenic protein HMW is prepared from the pollen grains as described in procedure A, but the dialysate solution containing allergenic proteins of M> 10 kD at pH 5.5 to 7.5 is frozen. Does not dry by drying. Instead, 6N hydrochloric acid is added dropwise to the protein solution to bring the pH to 2, and the solution thus acidified is redialized for 6 to 24 hours or freshly passed through a membrane with a nominal cut-off value of M = 10 kD. To ultrafiltration. Next, the dialysate solution that is not dialyzed is collected, adjusted to pH 6.5 to 7.5 with IN-NaOH, and dried by freeze-drying. Operation C Experience gained during the development of operations A and B of the present invention shows that the strong adsorption of flavonoid- or flavonoid-glycoside dyes on pollen proteins is due to electrostatic forces, and the purpose of desorption is to determine the negative carboxyl group of either partner. It has been shown that-and / or phenolic hydroxyl groups can be achieved by discharging at pH values below 3.0. However, such conditions are undesirable in view of the possibility of denaturation or loss of essential constituent protein determinants, and therefore procedures based on removal of adsorbed dye in an electric field in the natural pH range of 6.5 to 7.5. Was developed. In this method, a 0.0IM inorganic buffered salt solution (pH 6.5-7.5), for example, a 2-5% solution of the lyophilized allergenic protein HMW described in Procedure A in a phosphate buffered saline solution is prepared. I do. Depending on the technology equipment selected, an appropriate volume of this solution is then subjected to free electrophoresis to destroy the ionophore that results in protein-dye adsorption. During electrophoresis, the dye migrates rapidly to the anodic compartment and thus migrates slowly and can be separated from protein components retained on the cathodic side. The technical prototype of this operation is shown in Example III. Example I In this experiment, dried pollen of ragweed (Ambrosia eliteator) (commercially available from Beeham Research Laboratories, UK) was defatted with diethyl ether and extracted with distilled water as described in Procedure A. From the dialyzed and freeze-dried HMW ragweed pollen protein preparation, a 25 mg sample was dissolved in 5 ml of distilled water and 6N hydrochloric acid was added dropwise to adjust the pH to 2. Ultraviolet absorption spectra were separately observed at a dilution of 1:20 in 0.01 N hydrochloric acid. The sample was dialyzed for 24 hours against 500 ml of distilled water at room temperature under stirring. After the acid dialysis step, the UV-absorption spectrum of the internal dialysate liquid was again diluted 1: 2 in 0.01N hydrochloric acid and observed. The external solution was concentrated to an initial volume of 5 ml in a thin film evaporator RotaVapor ™, and the UV-absorption spectrum was measured by diluting 1:20 at pH2. The effective removal of the adsorbed flavonoid dye in this experiment is clearly shown by the UV-absorption spectrum recorded in FIG. 2 and the extinction coefficient numbers in Table II. The yield of lyophilized depigmented protein material DPP recovered by Procedure A is listed in Table I. Example II In these experiments, roe (Lolium perene) and duckweed (Dactylis gla merata) are representatives of potent allergenic pollen of grasses, and the rhododendron (Chenopodium album), well known as a representative allergenic weed pollen. ) And mugwort (Artemisi a vulgaris) pollen were subjected to the same procedure as described in Example I. Numerical data on the yield of DPP from HMW and relevant spectral analysis figures are shown in Tables I and II. The results of these experiments indicate that desorption of the flavonoid (-glycoside) pigment from the allergenic pollen protein HMW prepared by the traditional method is not only applied to the weed ragweed pollen described in Example I, but also to other weeds. It is also clear that it is also spread to herb pollen. Example III In these experiments, the same procedure A as described in Examples I and II was used to demonstrate the strong allergenicity of the trees Betula alba, Olea europea and the widespread Mediterranean weed Parietaria judaica. Pollen was treated. The starting allergenic protein HMW and the corresponding depigmented protein DPP were tested for allergenicity in vitro by inhibiting the binding of specific IgE- and IgG-antibodies in the sera of certain allergic patients. For inhibition of binding to specific IgE-antibodies, the established RAST-inhibition method was chosen. In this method, a serum sample of a hay fever patient with the pollen protein to be examined is covalently bound to the pollen protein HMW or the depigmented equivalent DPP using cyano-genbromide or another suitable chemical coupling agent. Incubate with the dried cellulose disc. After capturing the specific IgE-antibody in this incubation phase, the disc is washed in a dilution buffer, followed by incubation with an enzyme-labeled anti-IgE-antibody, and finally an enzyme-specific chromogenic substrate. Color. To assess the IgE-binding capacity of a given allergenic protein, a dilution series of allergen is preincubated with a fixed volume of human serum sample, and the remaining IgE is captured by an allergen-coated cellulose disk. The IgE-binding capacity of the allergenic preparation is read as a 50% inhibition point from a plot of allergen concentration versus IgE-binding, as shown for the example of birch pollen in FIG. In this example, two types of allergen-coated cellulose discs were chemically coupled to the original protein preparation HMW (the dye disc in Table III) and to the protein preparation DPP prepared by Procedure A. A disc coupled to the above (depigmented disc in Table III) was used. As shown in the accumulated data in Table III, the allergenic IgG-E-binding ability of the DPP protein showed a slight but insignificant decrease in the value per μg of the lyophilized substance in the case of birch and Parietaria, while It has to be concluded that in the case of the sample described above, depigmentation actually results in a loss of part of the IgE-binding epitope. In another experiment, it was checked whether maintaining olive HMW protein alone at pH 2 had a denaturing effect on olive pollen proteins. For this purpose, the olive HMW was brought to pH 2 with hydrochloric acid according to procedure A and the solution was left at room temperature for 4 hours. This solution was then readjusted to pH 7.0 without dialysis. This preparation was lyophilized and checked for IgE-binding ability using olive DPP discs by RAST inhibition. The results with certain selected human sera in these assay conditions showed that 50% RAST inhibition was positive for olive. The results were 0.75 μg for HMW, 0.75 μg for olive HMW treated at pH 2 and readjusted to pH 7 without dialysis, and 3.89 μg for olive DPP prepared according to Procedure A. The reduced IgE-binding allergenicity of olive pollen DPP compared to olive HMW is not due to denaturation of the protein at acidic pH, but to other electrostatically adsorbed low allergens that actually serve as antigenic determinants. Considering these points, a comparative study by detailed study of the molecular structure of the antibody-binding allergenic epitope will clarify the usefulness of the depigmented pollen protein because it is due to the removal of organic compounds of high molecular weight. For inhibition of binding to specific IgG antibodies, established enzyme immunoassays were chosen. In this method, a serum sample of a hay fever patient with the pollen protein to be examined is incubated with a dilution series of HMW or DPP allergen. The allergen-serum mixture was then pipetted into the wells of a polystyrene microtiter plate previously coated with HMW or DPP protein by physical adsorption. After 30 minutes at room temperature, the wells are washed with dilution buffer, the specific IgE-antibody captured on the allergen-coated plate is treated with an enzyme-labeled anti-IgG, and then developed with an enzyme-specific chromogenic substrate. And measure. The IgG-binding capacity of the allergenic preparation is evaluated as the 50% inhibition point interpolated from a plot of allergen concentration versus IgG-binding. In the experiments of this Example III, all microtiter wells were coated with DPP-preparation prepared according to Procedure A. The data in Table III shows that the binding capacity of DPP to IgG is slightly increased compared to the HMW product. Example IV In these experiments, isolated ragweed (Ambrosia eliteor) pollen protein, isolated and dialyzed at neutral pH, was depigmented by free electrophoresis according to Procedure C. A 20 mg HMW / ml solution was prepared and 5 ml of this solution was placed in a cellophane dialysis bag (Visking dialysis tubing, nominal cutoff 10000D). Both ends of the bag were closed and mounted in a rotating plastic holder of the electrophoresis apparatus RotaPhor (BioRad, USA, trademark). The void volume of the holder tube was filled with a phosphate buffered saline solution (0.01 M, pH 7.4), and the device was placed in a cold room at 4 ° C. Then, while constantly rotating the dialysis bag and the tube holding the external solution, a DC voltage was applied at a constant power of 12 watts to start electrophoresis. The current dropped from 120 mA at the start of the experiment to 68 mA three hours later. During electrophoresis, the dye is released from the pollen proteins and exits the dialysis bag and moves to the external electrode compartment (cathode). After the electrophoresis, the decolorized protein was recovered from the internal solution of the dialysis gag and dried by freeze-drying. The recovered amount of DPP product was 69 mg from 100 mg of ragweed HMW, and the yield of depigmented material by electrophoresis at neutral pH in operation C was 69%, comparable to the value in operation A (Table I). Was to do.

Claims (1)

[Claims]   1. Allergen-active proteins and further non-allergenic unwanted compounds In the method of purifying an aqueous extract containing The allergenic compound is used as a source of adhesion of the non-allergenic compound to the protein. By using a means to destroy the electrostatic, hydrophobic or other physical forces How to remove   2. Means of destroying electrostatic, hydrophobic or other physical forces include acids and anions Of chemical means consisting of alkalis, salts and electric currents The method of claim 1 selected from the group   3. Acid and alkali chemical means use an amount above the isoelectric point of the protein. Claim 2   4. The means for destroying the electrostatic force is less than pH 3, preferably 1.5 to 2.5. A method according to claim 2 comprising an acid having   5. Claim 2 wherein the means for destroying the electrostatic force comprises a current in the form of electrophoresis. Law   6. The molecular weight of the non-allergen compound is less than 10,000, preferably 5,000 A method according to any of claims 1 to 5, which is less than   7. Are non-allergenic compounds flavonoids and / or their glycosides? A method according to any one of claims 1 to 6, comprising:   8. The following steps:   1) Allergen-active proteins and further non-allergenic undesirable compounds Prepare an aqueous extract containing   2) The starting material is combined with the non-allergen static compound attached to the protein. Electrostatic and hydrophobic forces that cause the non-allergen compound to adhere to the protein And / or remove from said protein using other means of destroying physical forces Upon treatment, the above-mentioned undesirable compounds as attached allergens are substantially eliminated. To obtain an aqueous extract containing no   3) Collect substantially pure extract A method according to any one of claims 1 to 7, comprising each step.   9. Extract obtained by any one of claims 1 to 8   10. Standardization, diagnosis, synthesis and vaccinization of the extract according to claim 9 Use for inoculation purposes