WO2004037862A1

WO2004037862A1 - Special igy of resist allergic and its preparation

Info

Publication number: WO2004037862A1
Application number: PCT/CN2003/000333
Authority: WO
Inventors: Shing Paau; Rongjian Yang
Original assignee: Jason Medical Group (Far East) Limited
Priority date: 2002-10-25
Filing date: 2003-05-08
Publication date: 2004-05-06
Also published as: AU2003242074A1; CN1286527C; CN1513558A

Abstract

The present invention relates to a composite IgY for resist kainds of irritability disease and its preparing method, the preparing method of the special IgY for resist allergic include: Screen out the allergen that bring on the irritability disease, base on the screening and classing combination, preparation single or composite allergen antigen respectively; preparation immounoglobulin IgE antigen; preparation immune egg, preparation special IgY crude product of resist kinds of allergens and purification the IgY crude product of resist IgE; preparation special composite IgY resist of one or more special allergen. The present invention eliminate and resistant the allergens of entering into tracheal, and decrease the special IgE level, prevent the allergic symptom occur effectively. At the same time, the present invention adopt pure nature biology preparation, safety, take effect soon, have not any toxin or side effect and badness responses.

Description

Anti-allergy specific IgY and its preparation

Technical field

The invention relates to an antibody and a preparation method thereof, in particular to a composite IgY against various allergic diseases and a preparation method thereof.

Background technique

"Allergy" is one of the most common diseases that poses great harm to human health. I don't know how many people around the world have been suffering from this disease for life, even causing disability or sudden death. In India, the incidence of "allergy" accounts for The total population is as high as 11-12%; in the United States, the incidence of pollen allergy is as high as 15%, and it costs 600 million US dollars to treat this disease each year; and in China, according to incomplete statistics, patients with various allergic diseases There are hundreds of millions of people. It is reported that the world ’s annual medical costs for the treatment of bronchial asthma alone exceed 3 billion U.S. dollars, and the loss caused by missed work, misunderstanding and premature death due to these diseases is incalculable .

Allergic diseases are abnormal immune reactions that occur in the body by external antigen substances. Clinical manifestations such as bronchial asthma and bronchitis, allergic rhinitis, allergic laryngitis, allergic dermatitis, etc., are stubborn symptoms and it is quite difficult to treat. It is usually treated with chemicals such as hormones and epinephrine. It is regrettable that the currently widely used treatment schemes and their drugs are only local anti-inflammatory or short-term relief of symptoms and imperfect symptoms, not "cause treatment". What's more, the side effects are quite strong. For example, systemic application of corticosteroids can cause adverse reactions in the central nervous system, musculoskeletal, and endocrine systems, induce fungal infections in the oropharynx and esophagus, and even be susceptible to fatal herpes virus infections. It is more harmful to children and can inhibit children's growth. Affect the development of children's bones and gonads. Adrenergic drugs and ₂ -receptor agonists can also cause cardiovascular diseases such as hypertension and arrhythmia, skeletal muscle tremor (such as hand tremor), and even cause patients with adrenal insufficiency or failure; in severe cases, Causes cardiac arrest and dies. Based on long-term observations and statistical analysis, the international medical community has revealed that excessive use of salbutamol and β ₂ -receptor agonists is the bane of increasing asthma mortality in previous years.

In view of the serious defects of local anti-inflammatory and asthma treatment methods, the medical community has developed a specific immune method to address the mechanism caused by "specific reactions" caused by certain "allergens" in the human body ( ("Desensitization therapy"), which stimulates the body to produce "blocking antibodies" by repeated injections of specific allergens, and is combined with the "allergens" that reenter the body to form a complex and is netted Clearance of the endothelial system.

This method is called the "latest treatment" for "allergies", but there are many difficulties and dangers in its implementation. Not only slow onset, long course of treatment (up to 2-5 years) and difficult to adhere to; moreover, after the injection, it is prone to adverse reactions such as fatigue, joint pain, urticaria, and serious adverse reactions such as bronchospasm and anaphylactic shock. It even caused death. Therefore, it has seriously affected the promotion and application of this advanced "latest therapy".

The current status quo is that, despite the continuous development of drugs and equipment for the treatment of allergic diseases in the world in recent decades, the incidence of allergic diseases has not only failed to decline, but even the prevalence and mortality of asthma. Anomalies that increase year by year. The harsh reality shows that, to date, no safe and reliable method has been found to cure allergic diseases, especially asthma. Therefore, research and development of an effective treatment method and medicine starting from the root cause of the disease with small side effects will undoubtedly relieve millions of patients from the suffering of allergic diseases, protect the physical and mental health of the people, and improve the quality of health of the people. Very significant.

Technical content

An object of the present invention is to provide an antibody preparation that is safe, specific, and easy to use for allergic diseases such as asthma and a method for preparing the same.

The objective of the present invention is achieved through the following technical solutions:

1. Screen out allergens that cause allergic diseases

A, hay fever allergens: Artemisia pollen, ragweed pollen, ragtail, Timothy grass, rye, tree pollen such as Platanus, Maple, Poplar, Elm, Cypress, Gooseberry, Mulberry, Willow, Pine, Plane Wood, Birch, Ginkgo Wait.

At the same time, it is also possible to screen and determine the main allergenic pollen species in the region based on statistical analysis of the results of the "specific skin test" of most allergic patients. Based on the "antigenic cross" of these allergenic pollens, they were classified and combined to determine the representative pollen allergens.

It can also be based on allergen protein or antigen components identified by the most common representative allergenic pollen in various places, such as ragweed, wormwood as Hrt VI (scientific name), Dactylis as Arab al (scientific name), weeping birch It is Dac gl (scientific name), Hinoki cypress is Bet VI (scientific name), Cunninghamia lanceolata is Cry IC (scientific name), etc. The representative and most common allergen protein or antigen component is used as the representative allergen of this allergenic pollen. According to the type of allergenic pollen screened out, several commercially available allergenic pollen extracts or extractants can be selected as representative pollen allergens.

c, mold allergens

Isolation and screening of the most common fungi in the air, the largest number of spores spreading, and the most sensitive allergens. Pure strains are retained and cultured in the laboratory to prepare various fungal allergen extracts. Then, according to the "antigen cross" of these representative allergenic fungi, they are classified and combined.

D. Cockroach Allergens

Use commercial cockroach antigen ingredients or cockroach extracts (such as "black-breasted big cockroach extract"), or use local cockroach homemade extracts, or cut off the wings and feet of the cockroaches and put them into a high-speed homogenizer to stir them into pieces. As a basic raw material for the preparation of antigens.

E. Pet Allergens

Allergens can be made using pet sebaceous gland secretions, saliva or urine egg extracts.

Alternatively, these pets' representative allergen proteins can be directly used as the raw material of the antigen. For example, the main allergen protein for cats is Fel dl, and the allergen protein for dog saliva is Ca dl.

2. Based on the screening and classification of various allergens, single or composite allergen antigens are prepared separately.

The single or mixed allergen extracts or preparations of each group are added with Freund's adjuvant at a ratio of 1-10: 1-10, and then placed in a high-speed homogenizer and stirred at high speed to form a milky slurry. Complex allergen antigen

You can also directly add pollen, dust mites, fungi, cockroach fragments, pet dander, etc. with Freund's adjuvant and mix it into a high-speed homogenizer, and pulverize and homogenize at a high speed of 10,000 to 30,000 rpm to form a emulsion. Compound antigens containing whole bacteria and bacteria components or containing allergenic protein components;

3. Preparation of immunoglobulin IgE antigen

IgE can be extracted from the serum of humans, especially patients with allergies, and off-the-shelf IgE immunoglobulins can also be purchased as antigen raw materials.

IgE immunoglobulin is formulated with Freund's adjuvant at a ratio of 1-10: 1-10, placed in a high-speed homogenizer, and pulverized and homogenized at a high speed at 10,000 to 30,000 rpm to prepare an IgE antigen;

4. Preparation of immune eggs: applying the patented technology (patent application numbers: 00101270. 3 and 02121244. 9) that our company has applied, using various single or composite allergen antigens and IgE antigens to lay eggs separately Chickens are immunized; booster injections are given every two weeks, three times a month; 20 days after the first immunization, the immune eggs produced by the chickens are detected, and corresponding antigens are coded to encode the detected immune eggs mark.

The same immunization technology can also be applied to immunize laying ducks with various single or composite allergen antigens and IgE antigens;

5. Preparation of anti-allergen-specific crude IgY extract and anti-IgE-specific crude IgY extract: Take the coded labeled egg yolk and mix well; add 4-6 times the volume of the yolk to dilute and mix with distilled water Uniform, adjust the pH to between 5. 5-6. 0 with NaOH solution;

Fully stir the adjusted pH value, then cool it to 2-6 ° C, and let it stand for 12-24 hours; add the diluted solution to a high-speed centrifuge, centrifuge for 20 minutes; separate the supernatant Then add ultrafiltration to ultrafiltration and concentrate 10-20 times, add sodium alginate to the concentrated slurry at a ratio of 0.1 ~ 0.3%, stir well; then add it to a high-speed centrifuge to centrifuge, Take the supernatant to remove lipoprotein; add the centrifuged slurry to an ultrafiltration machine, pass through an ultra-micro membrane, and filter and sterilize it to obtain

The filtered and sterilized product can also be freeze-dried with a freeze dryer to obtain a series of anti-allergen-specific IgY crude extracts or anti-IgE-specific IgY crude extracts.

Finally, all the IgY of the different antigens are encoded and labeled;

6. Purification of anti-allergen-specific IgY and anti-IgE-specific IgY:

The anti-allergen-specific IgY and anti-IgE-specific IgY crude extracts prepared by the above process were passed through an ion exchange column and a gel exchange column, respectively, to obtain pure IgY;

7.Preparation of specific composite IgY against a specific allergen:

Pure IgY prepared from different antigens are also strictly labeled according to their respective different coding marks to distinguish them. One anti-allergy-specific IgY or multiple anti-allergy-specific IgY mixtures and anti-IgE-specific IgY are 1 to 10: Mix in a proportion of 0-10, then add a certain proportion of distilled water or physiological saline to prepare a solvent;

Anti-IgE specific IgY dry powder and specific anti-allergen-specific IgY or multiple anti-allergic specific IgY mixture dry powder can also be mixed directly in the ratio of 1-10: 0-10;

The above-mentioned multiple anti-allergy specific IgY mixtures are selected from a variety of specific anti-allergen-specific IgYs, and they are firstly pressed according to 1-10: 1-10 (2 types) or 1-10: 1-10: 1- 10 (3 types) or 1 1: 10: 1—10: 1—10: 1—10 (4 types) or the like and made by uniform mixing;

A series of anti-allergen-specific composite IgY solvents or powders prepared by the present invention are directly added to various nebulizers, and atomized by the nebulizer to form mist-like particles for patients to inhale the trachea, lungs or nasopharynx Can produce effective therapeutic and preventive effects. This atomizer can be a jet pneumatic atomizer (including electric pump type, compression air pump type, foot air pump or compressed oxygen) or an ultrasonic wave atomizer, or any other type of atomizer.

The series of anti-allergen-specific composite IgY dry powder prepared by the present invention can also be directly filled into a variety of Dry Powder inhalers for use by patients. There are two types of dry powder inhalers: single-dose dry powder inhalers are divided into two types: rotary inhalers (Spinhaler) and rotary inhalers (Rotahaler). (DisKhaler) and vortex inhaler (Turbuhaler). The patient can easily inhale the anti-allergen complex IgY combination into the trachea, lungs or nasopharynx by operating the dry powder inhaler.

Similarly, a series of anti-allergen compound IgY solvents or powders prepared can be diluted with water (distilled water or physiological saline, etc.) and then added to various spray tanks to prepare various sprays. In order to make the spray drug particles sufficiently fine, a booster must be added during filling. Frequently used boosters are Chlorofluorocarbon (CFC) or nitrogen (N ₂ ). The anti-allergen compound IgY prepared by the present invention is stored in a spray tank at a certain pressure to make a portable pressure-type quantitative inhalation product. When hand pressure drives the quantitative valve, a fully atomized IgY solution can be sprayed. Patients can inhale this anti-allergen compound IgY into the trachea or lungs by simply holding the outlet of the spray can, inhaling with the pressure spray nozzle, or inhaling through the "mist tank".

For patients with allergic rhinitis and pharyngitis, the spray can be added with "booster" or without "booster", and the prepared anti-allergen compound IgY can be diluted with water at normal pressure. It can be poured into a tank and put into a regular spray nozzle to make a spray. When using, just point the outlet of this constant-pressure fixed-quantity spray tank towards the nose and spray the misty liquid into the nose.

Based on the in-depth study of the immunological mechanism of allergic diseases such as asthma, the invention accurately grasps the two most critical influencing factors of external allergens and internal specific IgE, and creatively applies passive immune technology to prepare a series of Anti-allergen-specific IgY and an anti-IgE-specific IgY. On the one hand, it clears and inhibits allergens entering the airway, on the other hand, it directly inhibits and reduces specific IgE levels, which effectively prevents the occurrence of allergic symptoms. Meanwhile, the anti-allergen prepared by the present invention Specific composite IgY and anti-IgE specific IgY is a unique pure natural biological preparation. This natural ingredient is directly sprayed into the respiratory tract, instead of being injected or taken into the human circulatory system. The effect is direct, safe and harmless. fast. Since this is a natural substance equivalent to food, it is sweet and delicious, and even if it is often sprayed and inhaled for a long time, it does not produce any toxic side effects and adverse reactions. Patients with allergies such as asthma and people with allergies can bring a lightweight spray made with the ingredients of the present invention, and often spray it, which can effectively suppress the onset of symptoms and completely prevent the emergence of new allergies To achieve the dual purpose of both treatment and prevention.

Example 1: Preparation of dust mite specific IgY and detection titer

Mix the commercially available powder mite injection with Freund's adjuvant at a 1: 1 ratio, add it to a high-speed homogenizer, and perform high-speed homogenization at 20000 rpm to obtain a milky homogenous antigen;

Use the prepared dust mite antigen to immunize the breeding layer hens, and use the combination of subcutaneous injection and wing vein injection to strengthen the immune method. Mites; booster injections every two weeks, a total of 3 immunizations within a month; 20 days after the first immunization, the immune eggs produced by the chickens are retrieved;

Break the detected immune eggs with an eggbeater, filter the egg whites with an yolk sieve, leave the yolks, and stir well; add distilled water at 4 times the volume of the yolks, dilute and mix them; adjust the pH to NaOH solution 5. 5;

Stir the pH-adjusted dilution well, and then cool it down to 2 ° C and let it stand for 12 hours. Add the dilution to a high-speed centrifuge and centrifuge at 8000-12000rpm for 20 minutes; The obtained supernatant was further subjected to ultrafiltration in an ultrafilter and concentrated 15 times, and alginic acid was added to the concentrated paddle material at a ratio of 0.2%, and the mixture was thoroughly stirred; and then added to a high-speed centrifuge for centrifugation, and the supernatant was removed and removed. Lipoprotein; adding the centrifuged slurry to an ultrafiltration machine, passing through an ultra-micro membrane, and filtering and sterilizing; lyophilizing the filtered and sterilized product using a freeze dryer to obtain a dust mite-specific IgY crude Extraction of dry powder finished product; purification of crude extract by ion transfer column and gel transfer column, to obtain dust-mite-resistant pure IgY;

Example 2: Immunization of laying ducks and preparation of duck dust mite-specific IgY

Mix the commercially available powder mite injection with Freund's adjuvant at a ratio of 3: 2, add it to a high-speed homogenizer, and perform high-speed homogenization at 20000 rpm to obtain a milky homogenous antigen;

Immunized elite breeding hens with the prepared dust mite antigen by subcutaneous injection and intravenous injection in wings The combined method of booster immunization with each shot reaches 1 to 2 ml of antigen per injection, which is equivalent to about 5000 to 10,000 dust mites. The booster injection is performed every two weeks, and a total of 3 immunizations are made within one month. 20 days after the second immunization, the immune eggs produced by the chickens were retrieved;

Break the detected immune eggs with an eggbeater, filter the egg whites with an yolk sieve, leave the yolks, and stir well; add distilled water at 5 times the volume of the yolks, dilute and mix well; adjust the pH to 6 with NaOH solution . 0;

Stir the adjusted pH value sufficiently, and then cool it down to 4 ° C and let it stand for 12 hours. Add the dilution to a high-speed centrifuge and centrifuge at 8000-12000rpm for 20 minutes at high speed. The supernatant was put into an ultrafilter for ultrafiltration and concentrated 18 times, alginic acid was added to the concentrated slurry at a ratio of 0.3%, and the mixture was thoroughly stirred; and then added to a high-speed centrifuge to centrifuge, take the supernatant, and remove lipoproteins. The slurry after centrifugation is added to an ultrafiltration machine, passed through an ultra-micro membrane, and filtered and sterilized; the filtered and sterilized product is freeze-dried with a freeze dryer to obtain a dust mite-specific crude IgY extract Finished dry powder product; the crude extract was purified by ion transfer column and gel transfer column to obtain dust-mite-resistant pure IgY;

Example 3: Preparation of specific anti-Artemisia pollen, ragweed pollen, quinoa pollen and algal grass pollen-specific IgY. Antigen was prepared from wormwood pollen, ragweed pollen, quinoa pollen and algae pollen, respectively. First, the four collected pollen were collected. Use high-speed pulper to pulverize at a high speed of 1000 rpm, then add Freund's adjuvant at a ratio of 5: 4, and then homogenize at high speed in a high-speed pulper at 20,000 rpm to obtain four milky pollen protein antigens;

Immunized elite breeding hens with the prepared wormwood and other pollen antigens, as well as the enhanced immune method using a combination of subcutaneous injection and wing vein injection, each chicken injection amount reached 1.5ml antigen, equivalent to about 50 -100 ug of pollen protein; booster injections every two weeks, a total of 3 immunizations within a month; 20 days after the first immunization injection, the immune eggs produced by the chickens were separately detected;

Break the detected immune eggs with an eggbeater, filter the egg whites with an yolk sieve, leave the yolks, and stir well; add distilled water at 6 times the volume of the yolks, dilute and mix them; adjust the pH with NaOH solution to 6. 0;

The diluted diluent with adjusted pH value is further stirred and homogenized, then it is cooled to 4 ° C and left for 12 hours; the diluent is added to a high-speed centrifugal separator and centrifuged at 8000-12000rpm for 20 minutes at high speed; The separated supernatant was put into an ultrafilter for ultrafiltration and concentrated 16 times. Alginic acid was added to the concentrated slurry at a ratio of 0.3%, and the mixture was thoroughly stirred; then added to a high-speed centrifuge to centrifuge, the supernatant was removed to remove lipoproteins; the centrifuged slurry was added to an ultrafiltration machine, and ultramicronized Membrane, filtering and sterilizing; freeze-drying the filtered and sterilized product with a freeze dryer to obtain a dried IgY crude extract of anti-Artemisia pollen, ragweed pollen, quinoa pollen and algae pollen specific dry powder; The crude extract was purified by an ion transfer column and a gel transfer column to obtain pure IgY specific to Artemisia selengensis pollen, ragweed, pollen, and algae pollen.

Example 4: Preparation of antifungal allergen-specific IgY

Use Aspergillus fumigatus, Aspergillus terreus, Aspergillus flavus, Aspergillus niger, Aspergillus flavus to make antigens: After the above four kinds of molds are cultured in a conventional laboratory, the Freund's adjuvant is added in a 6: 7 ratio, and then The high-speed homogenizer pulverized and homogenized at a high speed of 20000 rpm to obtain 4 kinds of antigens;

The prepared breeding hens were immunized with the prepared Aspergillus fumigatus, Aspergillus terreus, Aspergillus flavus, Aspergillus niger, and Aspergillus flavus antigens, respectively, and the same method was used to strengthen the immune system by combining subcutaneous injection and wing vein injection. Each injection of chicken reaches 1.5 ml of antigen, which is equivalent to about 50-100 antigen protein. Intensified injection every two weeks, a total of 3 immunizations within a month; 20 days after the first immunization injection, chickens were seized separately Produced immune eggs;

Stir the pH-adjusted dilution well, and then cool it down to 4 ° C and let it stand for 12 hours. Add the dilution to a high-speed centrifuge and centrifuge at 8000-12000rpm for 20 minutes; The obtained supernatant was further subjected to ultrafiltration and concentrated 16 times in an ultrafilter, and alginic acid was added to the concentrated slurry in a ratio of 0.3%, and the mixture was thoroughly stirred; and then added to a high-speed centrifuge to centrifuge, and the supernatant was taken. Removal of lipoproteins; adding the centrifuged slurry to an ultrafiltration machine, passing through an ultra-micro membrane, and filtering and sterilizing; lyophilizing the filtered and sterilized product with a freeze dryer to obtain anti-Aspergillus fumigatus, soil Aspergillus, Aspergillus, Aspergillus niger, Aspergillus flavus specific IgY crude extract dry powder finished product; the crude extract was purified by ion transfer column and gel transfer column to obtain anti-fungal allergen-specific pure IgY.

Example 5: Preparation of specific IgY against pet allergies

Take cat saliva and urine at a ratio of 1: 1, add the mixture at a ratio of 7: 8 and add Freund's adjuvant. Put into a high-speed homogenizer and mix at a high speed of 20,000 rpm to prepare milky cat protein antigens; use the prepared milky cat protein antigens to immunize the breeding hens, and use the combination of subcutaneous injection and wing vein injection to strengthen the immunity Method, each chicken injected 2ml of antigen each time, equivalent to about 50-100 g of antigen protein; every two weeks booster injection, a total of 3 times a month immunization; 20 days after the first immunization injection, respectively Seize the immune eggs produced by chickens;

Break the detected immune eggs with an eggbeater, filter the egg whites with an yolk sieve, leave the yolks, and stir well; add distilled water at 6 times the volume of the yolks, dilute and mix well; adjust the pH to 6 with NaOH solution . 0;

Stir the pH-adjusted dilution well, and then cool it down to 4 ° C and let it stand for 12 hours. Add the dilution to a high-speed centrifuge and centrifuge at 8000-12000rpm for 20 minutes; The obtained supernatant was further subjected to ultrafiltration and concentrated 16 times in an ultrafilter, and alginic acid was added to the concentrated slurry in a ratio of 0.3%, and the mixture was thoroughly stirred; and then added to a high-speed centrifuge to centrifuge, and the supernatant was taken. Removal of lipoproteins; adding the centrifuged slurry to an ultrafiltration machine, passing through an ultra-micromembrane, and filtering and sterilizing; that is, a finished product of specific IgY crude extracts resistant to pet allergy was prepared; The crude extract is purified by a column to obtain specific pure anti-allergic

IgY.

Example 6: Preparation of anti-cockroach allergy-specific IgY

Collect 30-50 cockroaches, cut off their wings and feet, and focus their torso into a high-speed homogenizer to pulverize at 1000 rpm. Then add Fowler's at a weight ratio of 6: 8 according to the weight of the cockroach debris. The adjuvant is pulverized and homogenized at a high speed of 20,000 rpm in a high-speed homogenizer to obtain a milky cockroach protein antigen;

Using the obtained antigen, immunizing breeder chickens in the same manner as in Example 1 and preparing an anti-cockroach allergen-specific IgY in the same manner.

Example 7: Preparation of anti-IgE-specific IgY

The immunoglobulin IgE purchased from the market is mixed with Freund's adjuvant at a ratio of 3: 5, and then added to a high-speed homogenizer and homogenized at a high speed of 1,000 rpm to obtain an IgE antigen;

Using the prepared IgE antigen, immunize breeder chickens according to the method described in Experimental Example 1, and prepare anti-IgE-specific IgY in the same manner as in Experimental Example 1.

Example 8: Preparation of an anti-asthma combination nebulizer Dust mite anti-specific IgY powder and 100g humilis pollen-specific anti-anti-workers IgYlOOg _g E and specific IgY was added 100g dry thoroughly mixed in a small mixer, to obtain an anti-asthmatic specific complex _IgY300g;

Prepare 30 liters of anti-asthma compound IgY combination solution according to the following formula

Anti-asthma specific IgY 1%

Aspartame 0.12%

Orange Flavor 1. 30%

Peach flavor 0. 10%

Saline to 30 liters

First prepare 25 liters of normal saline, then add aspartame and flavor, stir well, and then slowly add compound IgY while stirring; then, add water to 30 liters, and stir well; measure the pH of the solution, use 1 Li The aOH solution adjusts the pH to 6. 5-7. 5; then 1500 anti-asthma combination sprays are filled in sterile glass bottles, each containing 20 ml.

Example 9: Preparation of anti-allergic hand-cushion quantitative pressure spray can

Anti-dust mite-specific IgY dry powder 100g and anti-cockroach allergen-specific IgY dry powder 100g and anti-IgE-specific IgY dry powder 100g are added to a small mixer and fully mixed to make an anti-allergy-specific composite IgY dry powder 300 _g;

Prepare 200 liters of anti-allergy compound IgY combination solution according to the following formula;

0. 15%

0. 12%

0. 30%

0. 10%

0. 70%

Up to 200 liters

First take 180 liters of distilled water, then add aspartame and flavor separately, stir well, and then slowly add compound IgY while stirring; then add distilled water to 200 liters, stir well, measure the pH value of the solution, according to the measured pH value 5-7. 5 之间。 With IN NaOH solution to adjust the pH to 6. 5-7. 5;

After the solution is prepared, 20,000 manual quantitative pressure spray tanks must be filled on the pressure tank production line. After each spray tank is filled with 10ml, it must be filled with pressurized nitrogen or Freon as a booster (polishing Injection), fitted with a quantitative valve, sealed, that is.

Example 10: Preparation of anti-allergic quantitative atmospheric pressure spray cans

Antifungal allergen-specific pure dry powder IgYlOOg, anti-pet allergy-specific pure IgY dry powder lOOg, anti-dust mite-specific IgY dry powder lOOg is added to a small mixer and fully mixed to make an anti-allergy specific composite IgY dry powder 300g;

0. 15%

0. 12%

0. 30%

0. 10%

0. 70%

Up to 200 liters

First take 180 liters of distilled water, then add aspartame and flavor separately, stir well, and then slowly add compound IgY while mixing; then add distilled water to 200 liters, stir well, and measure the pH of the solution. According to the measured pH 5-7. 5 之间。 IN NaOH solution to adjust the pH to 6. 5-7. 5 between;

Filling a hand-held quantitative atmospheric pressure spray can of 20,000 pieces, each 10ml, each filled with 10ml. Experimental example 1: The dust mite antigen and the house dust mite antigen were respectively mixed with F. und. Adjuvant to prepare dust mite antigen and house dust mite antigen. The binding titer of the anti-dust mite-specific IgY to these two antigens was measured by ELISA (enzyme-linked immunosorbent assay). The results are as follows:

It can be seen from the test results that the anti-dust mite-specific IgY prepared by the method of the present invention has a high antibody binding titer against dust mite allergens. At the same time, the anti-dust mite IgY prepared by the antigen preparation method and the enhanced immunity method of the present invention has a certain broad spectrum. In addition to having a strong inhibitory effect on the immunized dust mite, it also has "antigen crossover" on the same genus. "Other dust mites (such as" house dust mites ") can also be suppressed. The test results suggest that: As long as a representative dust mite (such as "dust mite") is used to make an antigen-immunized hen, the anti-dust mite-specific IgY prepared can inhibit several common dust mites. In this way, the variety of dust mite-specific gY can be simplified, and the cost can be reduced.

Experimental example 2: Dust mite and house dust mite were added and mixed with Freund's adjuvant to prepare dust mite antigen and house dust mite antigen. The binding titer of the duck anti-dust mite IgY to these two antigens was detected by ELISA and method.

The results are as follows:

The test results show that using the method of the present invention to immunize laying ducks, the duck-specific dust mite-specific IgY prepared can completely achieve the same effect as the immunized chickens. The obtained duck specific dust mite-specific IgY still has a high binding titer and has a certain broad spectrum.

Experimental example 3: ELISA method was used to detect the binding potency of the anti-Artemisia pollen-specific IgY to these four pollen antigens. The results are as follows:

It can be seen from the table that the specific anti-artemisia pollen-specific gY prepared by immunizing hens by the method of the present invention has a high antibody binding titer against the allergen Artemisia selengensis pollen and has a certain broad spectrum It also has a certain inhibitory effect on pollen belonging to the same genus Yarrow that has "antigen cross". This reminds us that the pollen-resistant specific wormwood prepared by the method of the present invention can be used for all kinds of Patients with grass pollen allergy are used for prevention and treatment.

Because there is an antigen cross between the pollens of plants belonging to the same family, the herbaceous plant pollen also only needs to prepare several types of tree pollen (such as sycamore pollen, willow pollen, pine pollen, etc.) Pollen-specific IgY, each kind of anti-pollen-specific IgY can be universal to allergies caused by pollen of the same genus. Pollens of grasses also need to use representative dogtail, Timothy grass, rye, etc. as antigens to prepare specific IgY against pollen and other pollen, and they are allergic to the pollen of grasses that have "antigen crossover" in the same genus. Are applicable.

Experimental Example 4: The antibody-binding titers of the anti-Aspergillus fumigatus-specific IgY to these five antigens were measured by ELISA. The results are shown in the following table −

As can be seen from the table, the anti-fungal specific IgY antibody prepared by using the antigen preparation method and the immunization method of the present invention has a relatively high binding titer and high binding titer to other molds of the same genus; thus, further verification There is a high degree of antigenic cross between these five species. The broad-spectrum specificity of the anti-mold IgY prepared by the present invention is also very strong. As long as the specific IgY prepared against a certain mold is prepared, it will also have a good effect on allergies caused by other molds with "antigen cross".

Experimental example 5: The ELISA method was used to detect the anti-cat allergy-specific IgY antibody-binding titer of the cat with the prepared detection antigen. The titer of the test result was 1: 128. It was proved that the anti-cat allergy-specific IgY prepared according to the method of the present invention has quite satisfactory cat allergen suppression effect.

Experimental Example 6: The ELISA method using the cockroach protein antigen prepared in the method listed in item (1) of this example as the detection antigen, and the prepared anti-cockroach allergy-specific IgY antibody binding titer against the cockroach allergen was detected. The potency is 1: 256. Validated anti-pet allergic characteristics prepared by the method of the present invention The effect of the opposite sex IgY is very ideal.

Experimental Example 7: Using the ELISA method, the IgE antigen prepared in item (1) of this example was used as the detection antigen, and the prepared antibody-binding titer of the anti-IgE-specific IgY to IgE was detected, and the titer of the test result was 1: 512. The experiment proves that the anti-IgE-specific IgY prepared by the method of the present invention has high activity and completely achieves the expected effect.

Experimental Example 8: Clinical observation of anti-asthma combination nebulizer for asthma patients

(1) Among the adult hospitalized patients with moderate asthma, a total of 20 patients who were diagnosed as allergic to dust mite or hayweed pollen, or both, were diagnosed through the "skin prick test".

(2) With the "anti-asthma combination atomizer" prepared by the present invention, after the medicine liquid bottle is opened, the medicine liquid is poured into a PARI-type compression atomizing air pump, and the patient is inhaled after being atomized. Inhale twice daily for 5-10 minutes.

(3) Observe the symptoms of the test patients daily.

(4) Two weeks after inhaling the "anti-asthma combination nebulizer", asthma sounds of 15 patients basically disappeared, and wheezing symptoms were alleviated. Five patients still had paroxysmal wheezing at night and were given supplemental inhalation "anti-asthma combination nebulizer". After 10 minutes of inhalation, wheezing stopped.

(5) After 2 months of continuous inhalation, the wheezing symptoms of 17 patients disappeared, and the wheezing symptoms of the other 3 patients also improved significantly, from daily seizures to 1-2 sporadic seizures per week.

Experimental Example 9: Clinical Observation Results of "Antiallergic Specific IgY" in Patients with Allergic Rhinitis Using a Hand-held Normal Pressure Spray Form-

(1) 50 patients with chronic rhinitis who were allergic to dust mites or cockroaches were selected for clinical trials through the "skin prick test" diagnosis.

(2) The 50 patients were divided into two groups at any time. The treatment group used an anti-allergic "normal-pressure quantitative spray can" and the control group used a "normal-pressure quantitative spray can" equipped with a placebo.

Before treatment, both groups of patients had severe allergic rhinitis, persistent nasal congestion, nasal itching, paroxysmal sneezing, a large amount of serous snot, and a loss of smell. In the treatment group, 3 people had hyposmia, and in the control group, 2 people had hyposmia.

(3) The two groups of patients sprayed the misty solvent into the nose with a hand spray can, and sprayed three times a day. Observe and strictly record the symptoms of the two groups of patients. (4) After 2 weeks of treatment, 19 of the 25 patients in the treatment group had basically eliminated the symptoms of nasal congestion and nasal itching, and the amount of nasal discharge was significantly reduced; 4 had improved symptoms and 2 had no obvious feeling.

In the control group, the symptoms remained basically unchanged.

(5) After 12 weeks of continuous treatment, 22 of the 25 patients in the treatment group completely eliminated the symptoms of nasal congestion and nasal itching, and the runny nose basically returned to normal; 3 of the original patients with reduced sense of smell had recovered to normal sense of smell: 2 patients had obvious symptoms Alleviated, 1 person did not improve. In the control group, 25 patients had repeated symptoms without improvement.

Claims

Rights request

1. An anti-allergy-specific IgY, characterized in that the preparation method of the anti-allergy-specific IgY includes the following steps:

Screening of allergens that cause allergic diseases, based on screening and classification of various allergens, preparation of single or composite allergen antigens; preparation of immunoglobulin IgE antigens; preparation of immune eggs, preparation of anti-allergens Specific IgY crude extracts to prepare anti-IgE-specific IgY crude extracts; purification of various allergen-specific IgY crude extracts and anti-IgE-specific IgY crude extracts; to prepare one or more specific allergies IgY specific for progeny.

2. The anti-allergic specific IgY according to claim 1, wherein the preparation method of the anti-allergic specific IgY comprises the following steps:

Screen out allergens that cause allergic diseases; based on screening and classification of various allergens, prepare single or composite allergen antigens separately, and use single or mixed allergen extracts or preparations for each group, press 1 Add 10: 1 to 10 ratio Freund's adjuvant, then put it into a high-speed homogenizer, stir at high speed to form a water-in-oil emulsion, that is, make a single or composite allergen antigen;

Preparation of immunoglobulin IgE antigen: IgE can be extracted from the serum of humans, especially allergic patients, or purchased ready-made IgE immunoglobulins as antigen raw materials, and [gE immunoglobulins are prepared in a ratio of 1 to 10: 1-10 The Freund's adjuvant is placed in a high-speed pulper and homogenized at high speed to prepare an IgE antigen;

Preparation of immunized eggs: Immunize laying hens with various single or complex allergen antigens or IgE antigens, and then intensify the injection every two weeks, and immunize three times a month. After 20 days of the first immunization, the chickens are seized Produced immune eggs, and coded and labeled the detected immune eggs with corresponding different antigens;

Preparation of anti-allergen-specific crude IgY extracts: Wash the immune eggs one by one with running water, then scrub and disinfect with alcohol; break the eggs with an eggbeater, remove the yolks from the yolk sieve, and beat the yolks. Add 4 to 6 times of distilled water to dilute, stir well, and adjust 11 to 5. 5-6. 0, 2-6 ° C, leave it at 12-6 hours for 1-24 hours, siphon to get the supernatant, Weigh solution A; centrifuge the above precipitation at 8000 rpm for 20 minutes to obtain the supernatant, call solution B; mix solution A and solution B uniformly, ultrafiltration to obtain an ultrafiltration concentrate, add 2.0% alginic acid Sodium solution, to a final concentration of 1.0%., Stir until turbid precipitation, add 2.0% calcium chloride solution, to a final concentration of 1.0%., Stir well, let stand 2-6 2 overnight, centrifuge at 8000rpra 30 Separate the seeds, take the supernatant, and then sterilize through a bacterial filter to obtain a sterile filtrate, freeze-dried, that is, the crude IgY extract, and finally encode the prepared crude IgY extracts against various antigens. ;

Purification of various anti-allergen-specific crude IgY and anti-IgE-specific crude IgY extracts: The anti-allergen-specific IgY and anti-IgE-specific IgY crude extracts prepared by the above process were sequentially passed through Ion exchange column and gel chromatography column to obtain pure IgY;

Preparation of specific complex IgY against one or more specific allergens: Pure IgY made from different antigens, also strictly labeled according to the above-mentioned respective different coding marks to distinguish one anti-allergy-specific IgY or multiple antibodies The allergy-specific IgY mixture and the anti-IgE-specific IgY are mixed uniformly at a ratio of 1-10: 1-10, and then added to prepare a solvent to obtain.

3. The anti-allergy-specific IgY according to claim 1 or 2, characterized in that the allergen causing the allergic disease in the preparation method of the anti-allergy-specific IgY can be any protein allergen. 4. The anti-allergic specific IgY according to claim 3, wherein the protein allergens are pollen allergens, dust mite allergens, mold allergens, cockroach allergens, and pet allergens.

5. The anti-allergy-specific IgY according to claim 1, 2 or 3, characterized in that in the preparation method of the anti-allergy-specific IgY, pollen, dust mites, molds, cockroach fragments, pet fur, etc. After adding Freund's adjuvant and mixing, put it into a high-speed homogenizer, and perform high-speed pulverization and homogenization at 10,000 to 30,000 rpm to form a water-in-sleeve solution. A compound containing whole bacteria and bacterial body components or containing allergenic protein components is prepared. antigen.

6. The anti-allergy-specific IgY according to claim 1, 2 or 3, characterized in that the same immune technology can also be applied in the preparation method of the anti-allergy-specific IgY, using various single or composite allergen antigens and IgE antigen was used to immunize laying ducks.

7. The anti-allergy-specific IgY according to claim 1, 2 or 3, characterized in that in the preparation method of the anti-allergy-specific IgY, anti-IgE-specific IgY dry powder and specificity against an allergen can also be used. A thousand powders of sexual IgY or a variety of anti-allergic specific IgY mixtures are mixed directly at a ratio of 1-10: 1-10 to make powders.

8. The anti-allergy-specific IgY according to claim 6, characterized in that in the preparation method of the anti-allergy-specific IgY, the above-mentioned multiple anti-allergy-specific IgY mixtures are selected from a variety of specificities against corresponding different allergens. IgY, first mix them uniformly in the ratio of 1-10: 1-10 or 1-10: 1-10: 1-10 or 1-10: 1-10: 1-10: 1-10 and so on. .

9. The anti-allergic specific IgY according to claim 1 or 2, characterized in that the anti-allergic specific IgY can be made into a clinically acceptable dosage form.

10. A dust mite-resistant IgY, which is characterized in that the preparation method of the dust mite-resistant IgY includes the following steps:

Mix the commercially available powder mite injection with Freund's adjuvant at a ratio of 1: 1, place it in a high-speed homogenizer, and perform high-speed homogenization at 20,000 r _P m to obtain a milky homogenous antigen; The prepared dust mite antigen is used to immunize the breeding layer hens. 釆 Immunization method combining subcutaneous injection and wing vein injection. Each chicken is injected with 1-2 ml of antigen each time, which is equivalent to about 5000-10000 dust mites. Intensive injections are given once every two weeks, and the immunization is counted 3 times within a month. 20 days after the first immunization, the immune eggs produced by the chickens are picked up. The immune eggs are scrubbed and disinfected with alcohol, the eggs are broken with an eggbeater, and the yolks are taken out to remove the yolks. Stir Beat egg yolks evenly, add 4 to 6 times distilled water according to the amount of egg yolk to dilute, stir well, adjust the pH to 5. 5-6. 0, 2-6 ° C with 0.1N Hcl, let stand overnight, siphon to get Clear solution, called solution A; The above precipitate was centrifuged at 8000 rpm for 20 minutes to obtain the supernatant, which was called solution B; the solution A and solution B were mixed uniformly, and ultrafiltration was performed to obtain an ultrafiltration concentrate, and added 2.0% 0%。 Alginate solution to a final concentration of 1.0%. 0%。 Stir, add 2.0% calcium chloride solution to a final concentration of 1.0%. Stir well, stand still at 2-6 ° C overnight, centrifuge at 8000rmp for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, and freeze-dry, that is, the anti-dust mite specific IgY crude extract; The crude IgY extract was dissolved in _PH 7.0, 0.01 MPB, and subjected to DEAE—Sephadex A50 column chromatography and Sindhadex G200 gel column chromatography to obtain pure dust mite resistance. Heterosexual IgY.

11. A dust mite-specific duck IgY, characterized in that the preparation method of the dust mite-specific duck IgY includes the following steps:

Mix the dust mite injection with Freund's adjuvant at a ratio of 3: 2, put it in a high-speed hook paddle machine, and homogenize at 20000 rpm at high speed to prepare a milky homogenous antigen; use the prepared dust mite antigen to fine breed eggs Ducks are immunized, using a combination of subcutaneous injection and wing vein injection. Each duck is injected with 1-2 ml of antigen each time, which is equivalent to about 5000-10,000 dust mites. Re-injected every two weeks, and the immunization is counted 3 times. ; 20 days after the first immunization, check the immune eggs produced by ducks; scrub and disinfect the immune eggs with alcohol, break the eggs with an eggbeater, remove the yolks from the yolk sieve, beat the yolks evenly, and add 4 to 6 times the amount of yolk. Dilute with distilled water, stir well, adjust ρΗ 5. 5-6. 0, 2- 6Ό with 0.1N Hcl and let stand overnight, siphon to get the supernatant, which is called liquid A; The above precipitate is centrifuged at 8000 rpm for 20 minutes. Supernatant, called B liquid; Mix A liquid and B liquid evenly, ultrafiltration to obtain ultrafiltration concentrate, add 2.0% sodium alginate solution to a final concentration of 1.0% ₀ , stir, add 2. 0% calcium chloride solution, to a final concentration of 1.0% ₀ , stir well, let stand at 2-6 ° C overnight, 8 Centrifuge at 000rmp for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, and freeze-dry it, which is dust-mite specific duck IgY; the above crude IgY extract was dissolved in pH 7.0, 0.01 MPB, and DEAE-Sind hadex A50 column chromatography and Sephadex G200 gel column chromatography were used to obtain pure duck dust mite-specific duck IgY.

12. Four specific IgYs that are resistant to wormwood, ragweed, pollen, and algae pollen, which are characterized in that the preparation method of the four specific IgYs includes the following steps:

Antigens were prepared from wormwood pollen, ragweed pollen, quinoa pollen and algal grass pollen, respectively. Firstly, the four pollen collected were pulverized at a high speed with a high-speed homogenizer at 1000 rpm, and then added to Fuchs at a ratio of 5: 4. The adjuvant was then homogenized in a high-speed homogenizer at 20000 rpm to obtain four types of milky pollen protein antigens. Immunized elite breeding hens with the prepared wormwood and other pollen antigens. Subcutaneous injection and wings were also used. Combined with the intravenous immunization method, each chicken was injected with 1.5ml antigen each time, which is equivalent to about 50-100 pollen protein. The injection was boosted every two weeks, and the immunization was counted 3 times within one month. The first immunization was 20 After a few days, the immune eggs produced by the chickens were separately examined; the immune eggs were scrubbed and disinfected with alcohol, the eggs were broken with an eggbeater, the yolks were taken out from the yolk sieve, and the yolks were evenly mixed. The supernatant was siphoned and called A liquid; the above precipitate was centrifuged at 8000 rpm for 20 minutes, and the supernatant was taken as B liquid. The A and B liquids were mixed uniformly, and ultrafiltration was used to obtain To the ultrafiltration concentrate, add 2.0% sodium alginate to a final concentration of 1.0%. Stir and add 2.0% calcium chloride solution to a final concentration of 1.0%. Stir and stir, let stand for 2-6 minutes overnight, centrifuge at 8000rmp for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, freeze and dry, and prepare anti-Artemisia pollen, anti-ragweed pollen, anti- Dry powder of quinoa pollen and algae-resistant pollen-specific IgY crude extract; The above crude IgY extract was dissolved in pH 7.0 and 0.01 MPB, respectively, and subjected to DEAE-Sephadex A50 column chromatography and Sephadex G200 gel column chromatography, respectively. The crude extract is purified to obtain four kinds of specific pure IgY, which are resistant to Artemisia pollen, ragweed-resistant pollen, quinoa-resistant pollen, and algae-resistant pollen.

13. An anti-fungus allergen-specific IgY, which is characterized in that the preparation method of the specific IgY includes the following steps-preparing an antigen by mixing Aspergillus fumigatus, Aspergillus terreus, Aspergillus niger, Aspergillus niger, and Aspergillus flavus: The above four kinds of molds are respectively cultured according to a conventional method to prepare a bacterial solution; each bacterial solution is mixed at a ratio of 1: 1: 1: 1 and a mixed bacterial solution is prepared; This mixed bacterial solution was added with Freund's adjuvant at a ratio of 6: 7, and then placed in a high-speed homogenizer at 20000 rpm to homogenize at high speed to obtain 4 kinds of mixed mold antigens. The mixed antigens were used to immunize elite breeding hens, and the same was applied subcutaneously. The combined immunization method of injection and wing vein injection, each chicken injects 1.0ml antigen each time, which is equivalent to about 200 yg of protein antigen; the injection is boosted once every two weeks, and the immunization is counted 3 times within one month; the first time After 20 days of immunization, check the immune eggs produced by the chickens; scrub and disinfect the immune eggs with alcohol, break the eggs with an eggbeater, remove the egg yolks from the yolk sieve, beat the yolks, and add 4-6 times distilled water according to the amount of egg yolk to stand overnight The siphon obtained the supernatant, which was called A liquid; the above precipitate was centrifuged at 8000 rpm for 20 minutes to obtain the supernatant, which was called B liquid; the A and B liquids were mixed uniformly, and ultrafiltration was performed to obtain an ultrafiltration concentrated liquid, which was added 0%。 0% sodium alginate solution to a final concentration of 1.0%. 0%。 Stir, add 2.0% calcium chloride solution to a final concentration of 1.0%. Stir well, stand still at 2-6 ° C overnight, centrifuge at 8000rmp for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, and freeze-dry to obtain Aspergillus fumigatus, Aspergillus terreus, and Corynebacterium, Aspergillus niger, Aspergillus flavus-resistant IgY crude extract dry powder; The above crude IgY extract was dissolved in pH 7.0, 0.01 MPB, and subjected to DEAE-Sephadex A50 column chromatography and Sephadex G200 gel column layer Analysis to obtain anti-fungus allergen-specific pure IgY.

14. A specific IgY for anti-allergic pets, characterized in that the preparation method of the specific IgY includes the following steps-taking cat saliva and urine in a 1: 1 ratio, and adding the mixture in a ratio of 7: 8 to Freund's Agent, put it into a homogenizer and mix at a high speed of 20000 rp _{m to} prepare a milky cat antigen; use the prepared milky cat antigen to immunize the breeding hens, and also use a combination of subcutaneous injection and wing vein injection to immunize Method, each chicken is injected with 1-2 ml of antigen each time, which is equivalent to about 50-100 wg protein antigen; the injection is boosted every two weeks, and the immunization is counted 3 times within a month; 20 days after the first immunization, the chickens are seized Immune eggs produced; Scrub and disinfect the immune eggs with alcohol, break the eggs with an eggbeater, remove the yolks from the yolk sieve, beat the yolks evenly, add 4 to 6 times distilled water according to the yolk volume, and let stand overnight. Siphon the supernatant. Weigh A liquid; the above precipitate was centrifuged at 8000 rpni for 20 minutes to obtain the supernatant, called liquid B; mix liquid A and liquid B uniformly, ultrafiltration to obtain ultrafiltration concentrated solution, add 2.0% sodium alginate 0% to a final concentration of 1.0% 0%。 Stir, add 2.0% calcium chloride solution to a final concentration of 1.0%. Stir well, stand still at 2-6 ° C overnight, centrifuge at 8000rrap for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, and freeze-dry to obtain a specific IgY crude extract that is resistant to pet allergy. ; The above crude IgY extract was dissolved in IW. 0. 01MPB, and subjected to DEAE-SindhadexA50 column chromatography and Sephad _eX G200 gel column chromatography to obtain specific pure IgY against pet allergy.

15. An anti-cockroach allergy-specific IgY, characterized in that the preparation method of the specific IgY includes the following steps: Collect 30-50 cockroaches, cut off their wings, and place their trunks in a high-speed homogenizer to lOOOOrpm for pulverization; then add Freund's adjuvant at a weight ratio of 6: 8 according to the weight of the cockroach debris, and then pulverize and homogenize at 20000rpm at high speed in a high-speed homogenizer to obtain a milky cockroach protein antigen; The dung beetle antigen was used to immunize the breeding layer hens. The combination of subcutaneous injection and wing vein injection was used to strengthen the immune method. Each chicken was injected with 1-2 ml of antigen each time, which is equivalent to 50-100 micrograms of protein. Immunize 3 times a month after injection. Check the immune eggs produced by chickens 20 days after the first immunization. Scrub and disinfect the immune eggs with alcohol. Break the eggs with an eggbeater. Remove the egg yolk from the yolk sieve. Beat the egg yolks evenly. Add 4 to 6 times of distilled water according to the amount of egg yolk to dilute, stir well, adjust the pH to 5. 5-6. 0, 2-6 ° C with 0.1N Hcl, leave it overnight, and siphon to get the supernatant, which is called A liquid ; The above precipitation at 8000rpm Centrifuge for 20 minutes to obtain the supernatant, which is called liquid B; mix liquid A and liquid B, and carry out ultrafiltration to obtain an ultrafiltration concentrate, add 2.0% sodium alginate solution to a final concentration of 1. 0%, stirring, adding 2.0% calcium chloride solution to a final concentration of 1.0 % o, stir well, stand still at 2-6 ° C overnight, centrifuge at 8000rmp for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, and freeze-dry it, which is a crude extract of anti-cockroach allergy specific IgY; The above crude IgY extract was dissolved in pH 7.0, 0.01 MPB, and subjected to DEAE-Sephadex A50 column chromatography and Sephadex G200 gel column chromatography to obtain anti-cockroach allergy-specific pure IgY.

16. An anti-IgE specific IgY, characterized in that the preparation method of the specific IgY includes the following steps: mixing immunoglobulin IgE with Freund's adjuvant at a ratio of 3: 5, and then adding it to a high-speed homogenizer at a high speed of 1000 rpm IgE antigen was prepared by homogenization; immunized breeder hens were immunized with the prepared IgE antigen, and a combination of subcutaneous injection and wing vein injection was used. Each chicken was injected with 1-2 ml of antigen each time, which is equivalent to 50- 100 micrograms of protein; booster injections every two weeks, the immunization is counted 3 times within a month; 20 days after the first immunization, the immune eggs produced by the chickens are detected; the immune eggs are scrubbed and disinfected with alcohol, and the eggs are broken with a beater. Take the egg yolk out of the egg yolk sieve, beat the egg yolk evenly, add 4 to 6 times distilled water to dilute according to the amount of egg yolk, stir well, adjust the pH to 5. 5-6. 0, 2- 6 ° C with 0.1N Hcl, and let stand overnight. the supernatant was siphoned too, said liquid A; above the precipitate was centrifuged at 8000r _P m for 20 minutes to obtain the supernatant, said solution B; solution A and the solution B were mixed hook, ultrafiltration, ultrafiltration have taken Solution, adding 2.0% sodium alginate solution to a final concentration of 1. 0%. 0%。 Stir, add 2.0% calcium chloride solution to a final concentration of 1.0%. Stir and stir, let stand for 2-6 minutes overnight, centrifuge at 8000rmp for 30 minutes, take the supernatant, filter with a bacterial filter to obtain a sterile filtrate, and freeze dry, which is the crude anti-IgE specific IgY extract; The crude extract was dissolved in pH 7.0 and 0.01 MPB, and subjected to DEAE-Sephadex A50 column chromatography and Sindhadex G200 gel column chromatography to obtain anti-IgE specific pure IgY. An anti-asthma-specific compound IgY, characterized in that the anti-asthma-specific compound IgY is composed of 1 part by weight of anti-dust mite-specific IgY dry powder

Anti-Artemisia pollen-specific IgY 1 part by weight

Anti-IgE specific IgY dry powder 1 part by weight, fully mixed.

18. An anti-asthma IgY preparation, characterized in that the preparation contains 8-12 grams of anti-asthma-specific complex IgY per liter.

19. A method for preparing an anti-asthma spray, characterized in that the method is:

100 parts by weight of dust-resistant mite-specific IgY dry powder, 100 grams by weight of anti-Artemisia pollen specific IgY, and 100 parts by weight of anti-IgE specific IgY dry powder were added to a small mixer to be fully mixed to obtain anti-asthma-specific compound IgY300 parts by weight;

Anti-asthma specific complex IgY 300 parts by weight

Aspartame 36 parts by weight

Orange Flavor 90 parts by weight

Peach flavor 30 parts by weight

Normal saline to 30,000 parts by weight

First prepare 25,000 parts by weight of normal saline, then add aspartame and essence, stir and mix, then slowly add compound IgY while stirring; then, add normal saline to 30 parts by volume, and stir well; measure the pH of the solution Value, use 1.0 blue & 011 solution to adjust ₁ ) 11 to 6. 5-7. 5 _; sterilize by bacterial filtration, and finally fill the anti-asthma compound spray with a sterile bottle.

20. An anti-allergy specific complex IgY, characterized in that the anti-allergy specific complex IgY consists of

Anti-ragweed-specific IgY dry powder 1 part by weight,

Anti-cockroach allergen-specific IgY dry powder 1 part by weight,

Anti-IgE specific IgY dry powder 1 part by weight, fully mixed.

21. The anti-allergic composite IgY preparation according to claim 20, characterized in that the preparation contains about 300 grams of anti-allergic specific complex IgY per 200 liters.

22. A method for preparing an anti-allergic spray, characterized in that the method is:

100 parts by weight of anti-ragweed specific IgY dry powder, 100 parts by weight of anti-cockroach allergen-specific IgY dry powder, 100 parts by weight of anti-IgE specific IgY dry powder are added to a small mixer and fully mixed to make 300 grams of anti-allergy specific composite IgY dry powder Share

Prepare 200,000 parts by weight of anti-allergic compound IgY combination solution according to the following formula;

Anti-allergy specific complex IgY 300 parts by weight

Aspartame 240 parts by weight

Orange flavor 600 parts by weight

Peach flavor 200 weight M servings

1400 parts by weight

Distilled water to 200,000 parts by weight

First take 180,000 parts by weight of distilled water, then add aspartame and flavor separately, stir well, and then slowly add compound IgY while stirring; then add distilled water to 2000Q0 parts by weight, stir well, and measure the pH of the solution. According to the measured The pH value is adjusted with 1. (^ 011 solution 1 {to 6. 5-7. 5 _; after the solution is prepared, it is sterilized by bacterial filtration, and finally the manual quantitative pressure spray tank is aseptically filled on the pressure tank production line. Each After the spray tank is filled with 10ml, it is filled with pressurized nitrogen or Freon as a booster (propellant), a quantitative valve is installed, and the seal is obtained.

23.An anti-allergy specific complex IgY, characterized in that the anti-allergy specific complex IgY consists of

Anti-fungus allergen-specific IgY dry powder 1 part by weight,

Anti-Pet Allergy Specific Pure IgY Dry Powder '' 1 part by weight,

Anti-IgE specific IgY dry powder 1 part by weight, fully mixed.

24. The anti-allergic composite IgY preparation according to claim 23, characterized in that the preparation contains about 300 grams of anti-allergic specific composite IgY per 200 liters.

25. A method for preparing an anti-allergic spray, characterized in that the method is:

100 parts by weight of anti-myco allergen-specific pure IgY dry powder, 100 parts by weight of anti-pet allergy-specific pure IgY dry powder, 100 parts by weight of anti-IgE-specific dry IgY powder, mixed in a small mixer to make anti-allergic special 300 parts by weight of anisotropic composite IgY dry powder;

300 parts by weight of anti-allergy specific complex IgY,

240 parts by weight of aspartame,

Orange flavor 600 parts by weight,

200 parts by weight of peach flavor,

1400 parts by weight of mint flavor,

Distilled water to 200,000 parts by weight

First take 180,000 parts by weight of distilled water, then add aspartame and flavor separately, stir well, and then slowly add compound IgY while stirring; then add distilled water to 200,000 parts by weight, stir well, and measure the pH of the solution. According to the measured The pH value was adjusted with 1.0 N NaOH solution to _P H6. 5- 7. 5; sterilized by bacterial filtration, and finally aseptically filled by hand.

26. The anti-allergy-specific IgY according to claim 2, characterized in that the ratio of the single or composite allergen antigen to Freund's adjuvant in the preparation method of the anti-allergy-specific IgY can be 3: 2.

27. The anti-allergy-specific IgY according to claim 2, characterized in that the ratio of the single or composite allergen antigen to Freund's adjuvant in the preparation method of the anti-allergy-specific IgY can be 3: 5.

28. The anti-allergy-specific IgY according to claim 2, characterized in that the ratio of the single or composite allergen antigen to Freund's adjuvant in the preparation method of the anti-allergy-specific IgY can be 5: 4.

The anti-allergy-specific IgY according to claim 2, characterized in that the ratio of the single or composite allergen antigen to Freund's adjuvant in the method for preparing the anti-allergy-specific IgY can be 6: 7.

30. The anti-allergy-specific IgY according to claim 2, characterized in that the ratio of the single or composite allergen antigen to Freund's adjuvant in the preparation method of the anti-allergy-specific IgY can be 7: 8.

31. The anti-allergy-specific IgY according to claim 2, characterized in that the ratio of the single or composite allergen antigen to Freund's adjuvant in the preparation method of the anti-allergy-specific IgY can be 6: 8.