WO2004037862A1 - Special igy of resist allergic and its preparation - Google Patents

Special igy of resist allergic and its preparation Download PDF

Info

Publication number
WO2004037862A1
WO2004037862A1 PCT/CN2003/000333 CN0300333W WO2004037862A1 WO 2004037862 A1 WO2004037862 A1 WO 2004037862A1 CN 0300333 W CN0300333 W CN 0300333W WO 2004037862 A1 WO2004037862 A1 WO 2004037862A1
Authority
WO
WIPO (PCT)
Prior art keywords
igy
specific
allergy
weight
preparation
Prior art date
Application number
PCT/CN2003/000333
Other languages
French (fr)
Chinese (zh)
Inventor
Shing Paau
Rongjian Yang
Original Assignee
Jason Medical Group (Far East) Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jason Medical Group (Far East) Limited filed Critical Jason Medical Group (Far East) Limited
Priority to AU2003242074A priority Critical patent/AU2003242074A1/en
Publication of WO2004037862A1 publication Critical patent/WO2004037862A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • 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.
  • 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.
  • 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 ⁇ 2 -receptor agonists is the bane of increasing asthma mortality in previous years.
  • 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.
  • 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.
  • 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.
  • 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.
  • cockroach antigen ingredients or cockroach extracts such as "black-breasted big cockroach extract”
  • 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.
  • Allergens can be made using pet sebaceous gland secretions, saliva or urine egg extracts.
  • these pets' representative allergen proteins can be directly used as the raw material of the antigen.
  • the main allergen protein for cats is Fel dl
  • the allergen protein for dog saliva is Ca dl.
  • 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.
  • 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;
  • the same immunization technology can also be applied to immunize laying ducks with various single or composite allergen antigens and IgE antigens;
  • 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.
  • 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;
  • 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.
  • dry powder inhalers 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.
  • 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.
  • a booster must be added during filling. Frequently used boosters are Chlorofluorocarbon (CFC) or nitrogen (N 2 ).
  • CFC Chlorofluorocarbon
  • N 2 nitrogen
  • 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".
  • 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.
  • the invention 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.
  • Example 1 Preparation of dust mite specific IgY and detection titer
  • 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
  • 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;
  • 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;
  • 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.
  • 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.
  • 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;
  • Example 2 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.
  • 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;
  • 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;
  • 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;
  • 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;
  • the anti-dust mite-specific IgY prepared by the method of the present invention has a high antibody binding titer against dust mite allergens.
  • 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.
  • it also has "antigen crossover” on the same genus. "Other dust mites (such as” house dust mites ”) can also be suppressed.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 3 people had hyposmia
  • 2 people had hyposmia.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Pulmonology (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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

抗过敏特异性 IgY及其制剂  Anti-allergy specific IgY and its preparation
技术领域  Technical field
本发明涉及一种抗体及其制备方法，特别是涉及一种抗各种过敏性疾病 的复合 IgY及其制备方法。  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.
背景技术  Background technique
过敏症"是一种最为常见的多发病， 对人类健康构成极大危害。 全世界 不知有多少人终生饱尝这种疾病的折磨，甚至造成残废或猝死。在印度， "过 敏症"发病率占人口总数高达 11一 12% ;在美国仅花粉过敏症的人群发病率 就高达 15%，每年要耗资 6亿美元治疗这种疾病；而在中国，据不完全统计， 各种过敏性疾病的患者有上亿人之多。有报道显示， 全世界每年仅用于治疗 支气管哮喘的医疗费用就超过 30亿美元， 由于这类疾病而导致的误工、 误 学及早亡所造成的损失更是无法计算。  "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 .
过敏性疾病，是一种由外界抗原物质作用于体内发生的不正常的免疫反 应。 临床表现如支气管哮喘和支气管炎、 过敏性鼻炎、 过敏性咽喉炎、 过敏 性皮肤炎等， 症状顽固， 治疗起来相当困难。 通常采用激素类和拟肾上腺素 等化学药物治疗。 令人遗憾的是， 目前广泛应用的治疗方案及其药物都仅仅 是局部抗炎或短暂纾缓症状的、 不完善的治标手段， 而不是 "病因治疗"。 更重要的是， 所带来的副作用都相当强烈。 如全身应用皮质激素会引致中枢 神经***和肌肉骨骼以及内分泌方面的不良反应，诱发口咽部和食道真菌感 染， 甚至易患致命性疱疹病毒感染； 对小儿危害更大， 会使小儿生长受抑、 影响小儿骨骼和性腺等方面的发育。 采用拟肾上腺素能药和 2—受体激动 剂， 还会引起高血压、 心律失常等心血管疾病， 出现骨骼肌震颤 (如手抖）， 甚至造成患者肾上腺皮质功能不全或衰竭； 严重时会引起心脏停搏而死亡。 国际医学界根据长期观察和统计分析揭示，过度使用沙丁胺醇和 β 2—受体激 动剂等是前几年哮喘死亡率不断增加的祸根。 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.
鉴于局部抗炎和平喘等治疗方法存在的严重缺陷，针对过敏性疾病是由 于某种 "过敏原"导致人体发生 "特异反应"而引起的机理， 医学界研究开 发了一种特异性免疫方法（ "脱敏疗法"）， 通过反复注射特异性过敏原刺激 机体产生 "封闭抗体"， 和再次进入机体的 "过敏原"结合为复合物而被网 状内皮***清除。 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.
这种方法被称为 "过敏症" 的 "最新疗法"， 但是在实施中却存在着许 多困难和危险性。 不但起效慢、 疗程长 (长达 2— 5年)而难于坚持； 而且， 注射后容易出现乏力、 关节痛、 荨麻疹等不良反应， 还会诱发支气管痉挛和 过敏性休克等严重不良反应， 甚至造成死亡。 因此， 严重影响了这种本来是 先进的 "最新疗法" 的推广应用。  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".
目前的现状是， 虽然近几十年来全世界有关过敏性疾病的治疗药物和设 备不断问世， 但是， 过敏性疾病的发病率， 不仅未能下降， 甚至还出现哮喘 的患病率和死亡率反而逐年增加的反常现象。残酷的现实表明， 至今全世界 尚未找到一种根治过敏性疾病特别是哮喘的安全可靠的方法。 因此， 研究开 发一种从病发根源上入手、 副作用又小的有效治疗方法和药物， 对于使千百 万患者彻底摆脱过敏性疾病的煎熬、 保障人民身心健康、 提高广大国民健康 素质， 无疑具有十分巨大的意义。  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.
技术内容  Technical content
本发明的目的在于提供一种对哮喘等过敏性疾病安全、特效、 使用方便 的抗体制剂及其制备方法。  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.
本发明目的是通过如下技术方案实现的：  The objective of the present invention is achieved through the following technical solutions:
1、 筛选出引致过敏性疾病的过敏原  1. Screen out allergens that cause allergic diseases
A、 花粉症过敏原： 蒿属花粉、 豚草花粉、 狗尾草、 梯牧草、 黑麦、 树 木花粉如法国梧桐、 枫、 杨、 榆、 柏、 构、 桑、 柳、 松、 悬铃木、 桦、 银杏 等。  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.
同时也可依据对多数过敏症患者的 "特异性皮肤试验"结果的统计分析， 来筛选决定本地区主要的致敏花粉种类。 再根据这些致敏花粉的 "抗原交叉 性"， 进行分类组合， 确定有代表性的几种花粉过敏原。  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.
也可根据各地最常见的有代表性的致敏花粉所鉴定出的过敏原蛋白质 或抗原成份， 如豚草、 艾草为 Hrt VI (学名）、 鸭茅为 Arab al (学名）、 垂枝 桦木为 Dac gl (学名）、 扁柏为 Bet VI (学名）、 杉木为 Cry IC (学名）等等有 代表性的、最常见的过敏原蛋白质或抗原成份作为这部分致敏花粉的代表性 过敏原。 还可以根据筛选出的致敏花粉种类，选用已商品化的若干种致敏花粉浸 出液或萃取剂作为有代表性的花粉过敏原。 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、 霉菌过敏原  c, mold allergens
将空气中最常见、 孢子播散数量最大和致敏性最强的真菌进行分离和筛 选， 保留纯菌种， 进行实验室培养， 可制成各种真菌过敏原浸出液。 然后， 根据这些有代表性的致敏真菌的 "抗原交叉性"进行分类组合。  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、 蟑螂过敏原  D. Cockroach Allergens
采用已商品化的蟑螂抗原成份或蟑螂浸出液 (如 "黑胸大蠊"萃取液）， 也可用当地蟑螂自制浸出液， 或将蟑螂剪去翅膀和脚后， 放入高速匀浆机搅 成碎片， 作为制备抗原的基础原材料。  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、 宠物过敏原  E. Pet Allergens
可采用宠物的皮脂腺分泌物、 唾液或尿蛋的萃取液制作过敏原。  Allergens can be made using pet sebaceous gland secretions, saliva or urine egg extracts.
也可选用这些宠物有代表性过敏原蛋白质直接作抗原的原材料。如猫的 主要过敏原蛋白质为 Fel dl， 狗唾液的过敏原蛋白质为 Ca dl。  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、 在筛选和分类组合各种过敏原的基础上， 分别制备单一或复合的过 敏原抗原。  2. Based on the screening and classification of various allergens, single or composite allergen antigens are prepared separately.
将各组别单一或混合的过敏原提取液或制剂， 按 1一 10 : 1— 10比例加入 福氏佐剂， 再放入高速匀浆机中， 高速搅拌成乳状浆液， 即制成单一或复合 过敏原抗原；  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
也可以直接将花粉、 尘螨、 真菌、 蟑螂碎片、 宠物皮毛屑等加入福氏佐 剂混合后放入高速匀浆机中， 以 10000〜30000rpm转速进行高速粉碎匀化， 形成乳浆液； 制得含全菌和菌体成份或含全部致敏蛋白质成份的复合抗原； 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、 制备免疫球蛋白 IgE抗原 3. Preparation of immunoglobulin IgE antigen
可以从人体特别是过敏症患者的血清中提取 IgE，也可购买现成的 IgE 免疫球蛋白作为抗原原料。  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.
将 IgE免疫球蛋白按 1一 10 : 1— 10比例配以福氏佐剂，放入高速匀浆机 中， 以 10000〜30000rpm转速进行高速粉碎匀化， 即制成 IgE抗原；  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、制备免疫蛋：应用本公司已申请的专利技术 (专利申请号： 00101270. 3 和 02121244. 9)， 用各种单一或复合过敏原抗原以及 IgE抗原， 分别对产蛋 鸡进行免疫； 每隔二周再强化注射一次， 一个月共免疫三次； 第一次免疫注 射 20天后， 检取鸡所产免疫蛋， 并对应不同的抗原， 对所检取的免疫蛋进 行编码标记。 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.
也可应用上述同样的免疫技术， 用各种单一或复合过敏原抗原以及 IgE 抗原， 分别对产蛋鸭进行免疫；  The same immunization technology can also be applied to immunize laying ducks with various single or composite allergen antigens and IgE antigens;
5、抗各种过敏原特异性 IgY粗提物和抗 IgE特异性 IgY粗提物的制备: 取编码标记过的免疫蛋蛋黄， 搅拌均匀； 按蛋黄体积的 4-6倍加入蒸馏 水稀释并混合均匀， 用 NaOH溶液调整 pH至于 5. 5-6. 0之间；  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;
将调整好 pH值的稀释液充分搅拌， 然后将其冷却至 2-6 °C， 静置 12 - 24 小时； 将稀释液加入高速离心分离机中， 离心分离 20分钟； 将分离所得的 上清再加入超滤器中进行超滤并浓缩 10-20倍，按 0. 1〜0. 3 %的比例将海藻 酸钠加入浓缩后的浆料中， 充分搅拌； 再加入高速离心机中离心， 取上清， 去除脂蛋白； 将离心分离后的浆料加入超滤机， 过超微膜， 进行过滤除菌， 即得；  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
也可将过滤除菌后的产品用冷冻干燥机进行冷冻干燥， 即制得一系列抗 过敏原特异性 IgY粗提物或抗 IgE特异性 IgY粗提物干粉成品；  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.
最后把所制备的各种不同抗原的 IgY全部进行编码标记；  Finally, all the IgY of the different antigens are encoded and labeled;
6、 抗各种过敏原特异性 IgY和抗 IgE特异性 IgY的纯化：  6. Purification of anti-allergen-specific IgY and anti-IgE-specific IgY:
将以上工艺所制取的抗各种过敏原特异性 IgY和抗 IgE特异性 IgY粗提 物分别过离子交换柱和凝胶交换柱， 即得纯 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、 制备抗某一种特定过敏原的特异性复合 IgY:  7.Preparation of specific composite IgY against a specific allergen:
不同抗原制得的纯 IgY， 同样按照以上述各自不同的编码标记严格标记 以便区分，将一种抗过敏特异性 IgY或多种抗过敏特异性 IgY混合物与抗 IgE 特异性 IgY按 1一 10 : 0— 10的比例混合均匀，然后加入一定比例蒸馏水或生 理盐水配制成溶剂；  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;
也可将抗 IgE特异性 IgY干粉和抗某一种过敏原的特异性 IgY或多种抗 过敏特异性 IgY混合物干粉按 1一 10 : 0— 10比例混合直接制成粉剂；  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;
上述多种抗过敏特异性 IgY混合物是选用多种抗相应的不同过敏原的特 异性 IgY, 先将它们按 1— 10 : 1— 10 (2种）或 1一 10 : 1— 10 : 1— 10 (3种）或 1 一 10： 1— 10： 1— 10： 1— 10 (4种)或依此类推的比例均匀混合制成； 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;
本发明制备的一系列抗过敏原特异性复合 IgY溶剂或粉剂直接加入各种 雾化器 (Nebulizers)中， 经雾化器雾化形成雾状微粒， 供患者吸入气管、 肺 部或者鼻咽部就可产生有效的治疗和预防作用。这种雾化器可以是喷射式气 动雾化器 (有电动泵式、 压缩气泵式、脚踏气泵或压缩氧气等形式)或者超声 波雾化器， 也可以是其他任何形式的雾化装置。  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.
本发明制备的一系列抗过敏原特异性复合 IgY干粉也可直接装入多种干 粉吸入器 (DryPowder inhalers)内供患者使用。 干粉吸入器有单剂量和多 剂量两种， 单剂量型干粉吸入器又分成旋转式吸入器 (Spinhaler)和转动式 吸入器 (Rotahaler)两种， 多剂量型干粉吸入器又分碟式吸入器 (DisKhaler) 和涡流式吸入器 (Turbuhaler)两种。患者只要操作这种干粉吸入器就可很方 便地将抗过敏原复合 IgY组合制剂吸入气管、 肺部或者吸入鼻咽部。  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.
同样， 也可将所制取的一系列抗过敏原复合 IgY溶剂或者粉剂加水 (蒸 馏水或生理盐水等)稀释后加入各种喷雾罐中制成各种喷雾剂。 为使喷雾药 物微粒足够细小， 灌装时必须加入助推剂， 常用的助推剂为氟里昂 (Chlorofluorocarbon, CFC)或者氮气 (N2)等。把本发明制备的抗过敏原复合 IgY以一定压力保存于喷雾罐中， 制成一种轻便的压力式定量吸入产品。 当 手压驱动定量活瓣时， 就可喷出充分雾化的 IgY溶液。 患者只要含住这种喷 雾罐的出口， 配合掀压喷头吸气， 或者借助 "储雾罐"吸气， 就可以将这种 抗过敏原复合 IgY吸入气管或肺部。 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".
对于过敏性鼻炎和咽喉炎患者， 喷雾剂在灌装时可加 "助推剂"， 也可不 加 "助推剂"， 只需将所制得的抗过敏原复合 IgY加水稀释在常压条件下灌 入罐内， 装上普通定量喷头就可制成喷雾剂。 使用时， 只要将这种常压定量 喷雾罐出口对准鼻子往鼻子内喷入雾状液体即可。  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.
本发明在深入研究哮喘等过敏疾病的免疫学发生机制的基础上，准确地 抓住外因致敏原和内因特异性 IgE两大最关键的影响因素， 并且创造性地应 用被动免疫技术，制备一系列抗过敏原特异性 IgY和一种抗 IgE特异性 IgY。 一方面清除和抑制了进入气道的致敏原， 另一方面又直接抑制并减少了特异 性 IgE水平有效地阻止了过敏症状的发生。 同时， 本发明所制备的抗过敏原 特异性复合 IgY和抗 IgE特异性 IgY是一种独特的纯天然生物制剂，将这种 天然成份直接喷施于呼吸道， 而不是注射或口服进入人体循环***， 作用直 接， 安全无害， 见效又快。 由于这是一种相当于食品的天然物质， 甘甜可口， 即使长期经常喷雾吸入体内， 也不产生任何毒副作用和不良反应。 哮喘等过 敏症患者以及有过敏体质的人只要随身带一支用本发明的成份制作的轻巧 喷雾剂， 经常喷一喷， 就既可有效抑制症状发作， 又可完全预防新的过敏现 象的出现， 达到既治疗又预防的双重目的。 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.
实施例 1 : 制备抗粉尘螨特异性 IgY及检测效价  Example 1: Preparation of dust mite specific IgY and detection titer
将市面上购得的现成的粉尘螨注射液按 1 : 1比例与福氏佐剂混匀，加入 高速匀桨机， 以 20000rpm转速进行高速匀化， 制得乳状匀质抗原；  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;
用所制得的粉尘螨抗原对良种蛋鸡进行免疫，采用皮下注射和翅静脉注 射相结合的强化免疫法， 每只鸡每次注射量达到 l-2ml 抗原， 相当于约 5000-10000只尘螨； 每隔二周再强化注射一次， 一个月内共免疫 3次； 第一 次免疫注射 20天后， 检取鸡所产免疫蛋；  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;
用打蛋机将检致的免疫蛋打碎， 用蛋黄筛筛滤去蛋清， 留下蛋黄， 搅拌 均匀； 按蛋黄体积的 4倍加入蒸馏水， 进行稀释并混合均勾； 用 NaOH溶液 调整 pH至 5. 5 ;  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;
将调整好 pH值的稀释液进一步充分搅拌均匀，然后将其冷却至 2°C ,静 置 12小时； 将稀释液加入高速离心分离机中， 以 8000- 12000rpm转速高速 离心分离 20分钟； 将分离所得的上清再入超滤器中进行超滤并浓缩 15倍， 按 0. 2 %的比例海藻酸加入浓缩后的桨料中， 充分搅拌； 再加入高速离心机 离心， 取上清， 去除脂蛋白； 将离心分离后的浆料加入超滤机， 过超微膜， 进行过滤除菌； 将过滤除菌后的产品用冷冻干燥机进行冷冻干燥， 即制得抗 粉尘螨特异性 IgY粗提物干粉成品；过离子交接柱和凝胶交接柱把粗提物纯 化， 即得抗粉尘螨特异性纯 IgY;  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;
， 实施例 2: 免疫产蛋鸭， 制备鸭抗尘螨特异性 IgY Example 2: Immunization of laying ducks and preparation of duck dust mite-specific IgY
将市面上购得的现成的粉尘螨注射液按 3 : 2比例与福氏佐剂混匀，加入 高速匀浆机， 以 20000rpm转速进行高速匀化， 制得乳状匀质抗原；  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;
用所制得的粉尘螨抗原对良种蛋鸡进行免疫，采用皮下注射和翅静脉注 射相结合的强化免疫法， 每只鸡每次注射量达到 l-2ml 抗原， 相当于约 5000-10000只尘螨； 每隔二周再强化注射一次， 一个月内共免疫 3次； 第一 次免疫注射 20天后， 检取鸡所产免疫蛋； 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;
用打蛋机将检致的免疫蛋打碎， 用蛋黄筛筛滤去蛋清， 留下蛋黄， 搅拌 均匀； 按蛋黄体积的 5倍加入蒸馏水， 进行稀释并混合均匀； 用 NaOH溶液 调整 pH至 6. 0;  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;
将调整好 pH值的稀释液进一步充分搅拌均匀，然后将其冷却至 4Ό，静 置 12小时； 将稀释液加入高速离心分离机中， 以 8000- 12000rpm转速高速 离心分离 20分钟； 将分离所得的上清再入超滤器中进行超滤并浓缩 18倍， 按 0. 3%的比例海藻酸加入浓缩后的浆料中， 充分搅拌； 再加入高速离心机 离心， 取上清， 去除脂蛋白； 将离心分离后的浆料加入超滤机， 过超微膜， 进行过滤除菌； 将过滤除菌后的产品用冷冻干燥机进行冷冻干燥， 即制得抗 粉尘螨特异性 IgY粗提物干粉成品；过离子交接柱和凝胶交接柱把粗提物纯 化， 即得抗粉尘螨特异性纯 IgY;  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;
实施例 3:制备抗蒿草花粉、豚草花粉、藜草花粉和藻草花粉特异性 IgY 分别用蒿草花粉、 豚草花粉、 藜草花粉和藻草花粉制备抗原， 首先将所 采集的四种花粉分别用高速勾浆机以 lOOOOrpm转速高速粉碎， 然后按 5 : 4 比例分别加入福氏佐剂， 再在高速浆机中以 20000rpm转速高速匀化， 制得 四种乳状花粉蛋白抗原；  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;
用所制得的蒿草花粉等抗原分别对良种蛋鸡进行免疫， 同样采用皮下注 射和翅静脉注射相结合的强化免疫法， 每只鸡每次注射量达 1. 5ml抗原， 相 当于约 50-100 u g花粉蛋白质； 每隔二周再强化注射一次， 一个月内共免疫 3次； 第一次免疫注射后 20天后， 分别检取鸡所产免疫蛋；  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;
用打蛋机将检致的免疫蛋打碎， 用蛋黄筛筛滤去蛋清， 留下蛋黄， 搅拌 均匀； 按蛋黄体积的 6倍加入蒸馏水， 进行稀释并混合均勾； 用 NaOH溶液 调整 pH至 6. 0;  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;
将调整好 pH值的稀释液进一步充分搅拌均勾，然后将其冷却至 4°C，静 置 12小时； 将稀释液加入高速离心分离机中， 以 8000-12000rpm转速高速 离心分离 20分钟； 将分离所得的上清再入超滤器中进行超滤并浓缩 16倍， 按 0. 3%的比例海藻酸加入浓缩后的浆料中， 充分搅拌； 再加入高速离心机 离心， 取上清， 去除脂蛋白； 将离心分离后的浆料加入超滤机， 过超微膜， 进行过滤除菌； 将过滤除菌后的产品用冷冻干燥机进行冷冻干燥， 即制得抗 蒿草花粉、 豚草花粉、 藜草花粉和藻草花粉特异性 IgY粗提物干粉成品； 过 离子交接柱和凝胶交接柱把粗提物纯化， 即得抗蒿草花粉、 豚草花粉、 藜草 花粉和藻草花粉特异性纯 IgY。 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.
实施例 4: 制备抗真菌过敏原特异性 IgY  Example 4: Preparation of antifungal allergen-specific IgY
分别用烟曲霉菌、土曲霉菌、棒曲霉菌、黑曲霉菌、黄曲霉菌制作抗原: 将以上 4种霉菌按常规试验室培养后分别以 6 : 7比例加入福氏佐剂，然后放 入高速匀浆机以 20000rpm转速高速粉碎匀化， 制得 4种抗原；  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;
用所制得的烟曲霉菌、 土曲霉菌、 棒曲霉菌、 黑曲霉菌、 黄曲霉菌抗原 分别对良种蛋鸡进行免疫， 同样采用皮下注射和翅静脉注射相结合的强化免 疫法， 每只鸡每次注射量达 1. 5ml抗原， 相当于约 50-100 抗原蛋白质； 每隔二周再强化注射一次， 一个月内共免疫 3次； 第一次免疫注射后 20天 后， 分别检取鸡所产免疫蛋；  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;
用打蛋机将检致的免疫蛋打碎， 用蛋黄筛筛滤去蛋清， 留下蛋黄， 搅拌 均匀； 按蛋黄体积的 6倍加入蒸馏水， 进行稀释并混合均勾； 用 NaOH溶液 调整 pH至 6. 0;  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;
将调整好 pH值的稀释液进一步充分搅拌均匀，然后将其冷却至 4°C，静 置 12小时； 将稀释液加入高速离心分离机中， 以 8000- 12000rpm转速高速 离心分离 20分钟； 将分离所得的上清再入超滤器中进行超滤并浓缩 16倍， 按 0. 3%的比例海藻酸加入浓縮后的浆料中， 充分搅拌； 再加入高速离心机 离心， 取上清， 去除脂蛋白； 将离心分离后的浆料加入超滤机， 过超微膜， 进行过滤除菌； 将过滤除菌后的产品用冷冻干燥机进行冷冻干燥， 即制得抗 烟曲霉菌、 土曲霉菌、 棒曲霉菌、 黑曲霉菌、 黄曲霉菌特异性 IgY粗提物干 粉成品； 过离子交接柱和凝胶交接柱把粗提物纯化， 即得抗真菌过敏原 特异性纯 IgY。  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.
实施例 5: 制备抗宠物过敏的特异性 IgY  Example 5: Preparation of specific IgY against pet allergies
取猫唾液和尿液按 1 : 1比例混合， 取混合物按 7 : 8比例加入福氏佐剂， 放入高速匀浆机以 20000rpm转速高速混匀， 制得乳状猫蛋白抗原； 用所制得的乳状猫蛋白抗原分别对良种蛋鸡进行免疫， 同样采用皮下注 射和翅静脉注射相结合的强化免疫法， 每只鸡每次注射量达 2ml抗原， 相当 于约 50-100 g抗原蛋白质； 每隔二周再强化注射一次， 一个月内共免疫 3 次； 第一次免疫注射后 20天后， 分别检取鸡所产免疫蛋； 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;
用打蛋机将检致的免疫蛋打碎， 用蛋黄筛筛滤去蛋清， 留下蛋黄， 搅拌 均匀； 按蛋黄体积的 6倍加入蒸馏水， 进行稀释并混合均匀； 用 NaOH溶液 调整 pH至 6. 0;  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;
将调整好 pH值的稀释液进一步充分搅拌均匀，然后将其冷却至 4°C，静 置 12小时； 将稀释液加入高速离心分离机中， 以 8000- 12000rpm转速高速 离心分离 20分钟； 将分离所得的上清再入超滤器中进行超滤并浓缩 16倍， 按 0. 3%的比例海藻酸加入浓縮后的浆料中， 充分搅拌； 再加入高速离心机 离心， 取上清， 去除脂蛋白； 将离心分离后的浆料加入超滤机， 过超微膜， 进行过滤除菌； 即制得抗宠物过敏的特异性 IgY粗提物成品； ' 过离子交接柱和凝胶交接柱把粗提物纯化， 即得抗宠物过敏的特异性纯 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。 IgY.
实施例 6: 制备抗蟑螂过敏特异性 IgY  Example 6: Preparation of anti-cockroach allergy-specific IgY
收集 30-50只的蟑螂， 剪去其翅膀和脚， 将其躯干集中先放入高速匀浆 机以 lOOOOrpm转速进行粉碎； 然后按蟑螂碎屑的重量， 以 6 : 8的重量比加 入福氏佐剂， 再在高速匀桨机中以 20000rpm转速高速粉碎匀化， 制得乳状 蟑螂蛋白抗原；  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;
用所制得的抗原， 按照实施例 1同样的方法免疫良种鸡， 并用同样方法 制备抗蟑螂过敏原特异性 IgY。  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.
实施例 7: 抗 IgE特异性 IgY的制备  Example 7: Preparation of anti-IgE-specific IgY
将从市面上'采购的免疫球蛋白 IgE按 3 : 5比例与福氏佐剂混合，然后加 入高速匀浆机以 lOOOOrpm转辣速高速匀化， 制得 IgE抗原；  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;
用所制得的 IgE抗原， 按照实验例 1所述方法免疫良种鸡， 并按实验例 1相同方法制备得抗 IgE特异性 IgY。  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.
实施例 8: 抗哮喘组合雾化剂的制备 抗粉尘螨特异性 IgY干粉 100g和抗蒿草花粉特异性 IgYlOOg以及抗工 gE 特异性 IgY干粉 100g加入小型搅拌机中充分混和， 得到抗哮喘特异性复合 IgY300g; 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;
按以下配方调配 30升抗哮喘复合 IgY组合溶液  Prepare 30 liters of anti-asthma compound IgY combination solution according to the following formula
抗哮喘特异性复合 IgY 1 %  Anti-asthma specific IgY 1%
阿斯巴甜 0. 12 %  Aspartame 0.12%
香橙香精 1. 30%  Orange Flavor 1. 30%
水蜜桃香精 0. 10%  Peach flavor 0. 10%
生理盐水 加至 30升  Saline to 30 liters
先调制生理盐水 25升， 然后加入阿斯巴甜和香精， 搅拌均匀后， 再边 搅拌边缓缓加入复合 IgY; 接着， 加水至 30升， 充分搅拌均匀； 测量溶液的 pH值，用 1丽 aOH溶液调节 pH至 6. 5-7. 5间；然后采用无菌玻璃瓶灌装 1500 支抗哮喘组合喷雾剂， 每支装 20ml。  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.
实施例 9: 制备抗过敏手揿定量压力喷雾罐  Example 9: Preparation of anti-allergic hand-cushion quantitative pressure spray can
抗粉尘螨特异性 IgY干粉 100g和抗蟑螂过敏原特异性 IgY干粉 100g以 及抗 IgE特异性 IgY干粉 100g加入小型搅拌机中充分混和， 制成抗过敏特 异性复合 IgY干粉 300g; 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;
按以下配方调配 200升抗过敏复合 IgY组合溶液；  Prepare 200 liters of anti-allergy compound IgY combination solution according to the following formula;
0. 15 %  0. 15%
0. 12%  0. 12%
0. 30%  0. 30%
0. 10%  0. 10%
0. 70 %  0. 70%
加至 200升  Up to 200 liters
先取蒸馏水 180升， 然后分别加入阿斯巴甜、 香精， 搅拌均匀， 再边搅 拌边缓缓加入复合 IgY; 接着加蒸馏水至 200升， 充分搅拌均匀， 测量溶液 pH值， 根据所测的 pH值用 IN NaOH溶液调节 pH至 6. 5-7. 5间；  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;
溶液调配好后， 必须在压力罐生产线上灌装手动定量压力喷雾罐 20000 支，每支喷雾罐灌足 10ml后，必须再充入压力氮气或者氟里昂作为助推剂 (抛 射剂）， 装上定量阀门， 密封， 即得。 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.
实施例 10: 制备抗过敏定量常压喷雾罐  Example 10: Preparation of anti-allergic quantitative atmospheric pressure spray cans
抗真菌过敏原特异性纯干粉 IgYlOOg, 抗宠物过敏的特异性纯 IgY干粉 lOOg, 抗粉尘螨特异性 IgY干粉 lOOg加入小型搅拌机中充分混和， 制成抗 过敏特异性复合 IgY干粉 300g;  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;
按以下配方调配 200升抗过敏复合 IgY组合溶液；  Prepare 200 liters of anti-allergy compound IgY combination solution according to the following formula;
0. 15 %  0. 15%
0. 12%  0. 12%
0. 30%  0. 30%
0. 10%  0. 10%
0. 70%  0. 70%
加至 200升  Up to 200 liters
先取蒸馏水 180升， 然后分别加入阿斯巴甜、 香精， 搅拌均匀， 再边撹 拌边缓缓加入复合 IgY; 接着加蒸馏水至 200升， 充分搅拌均勾， 测量溶液 pH值， 根据所测的 pH值用 IN NaOH溶液调节 pH至 6. 5-7. 5间；  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;
灌装手揿定量常压喷雾罐 20000支， 每支 10ml， 每支灌装满 10ml。 实验例 1: 分别用粉尘螨和屋尘螨加入福氏佐剂混勾制作粉尘螨抗原和 屋尘螨抗原。 以 ELISA法 (酶联免疫吸附实验)检测所制得的抗粉尘螨特异性 IgY对这两种抗原的结合效价。 结果如下：  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:
Figure imgf000012_0001
从检测结果可以看出，用本发明的方法所制备的抗粉尘螨特异性 IgY对 粉尘螨过敏原有很高的抗体结合效价。 同时， 本发明的抗原制作方法和强化 免疫方式， 所制得的抗粉尘螨 IgY具有一定的广谱性， 除了对所免疫的粉尘 螨有很强的抑制作用外，对同属的有"抗原交叉"的其他尘螨 (如"屋尘螨"） 同样可以抑制。 试验结果提示我们： 只要用一种有代表性的尘螨 (如 "粉尘螨"）制作抗 原免疫母鸡，所制备的抗粉尘螨特异性 IgY对常见的几种尘螨都有抑制作用。 这样， 就可以精简抗尘螨特异性工 gY的品种， 降低成本。
Figure imgf000012_0001
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.
实验例 2: 分别用粉尘螨和屋尘螨加入福氏佐剂混匀制作粉尘螨抗原和 屋尘螨抗原。 以 ELISA、法检测所制得的鸭抗粉尘螨 IgY对这两种抗原的结合 效价。  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.
结果如下：  The results are as follows:
Figure imgf000013_0001
检测结果说明采用本发明的方法免疫产蛋鸭，所制备的鸭抗粉尘螨特异 性 IgY完全可以达到与免疫鸡一样的效果。所得的鸭抗粉尘螨特异性 IgY照 样有很高的结合效价并具有一定的广谱性。
Figure imgf000013_0001
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.
实验例 3: 以 ELISA法分别检测所制备的抗蒿草花粉特异性 IgY对这四 种花粉抗原的结合效价。 结果如下表：  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:
Figure imgf000013_0002
从表中可以看出，采用本发明的方法免疫母鸡所制得的抗蒿草花粉特异 性工 gY对免疫的过敏原蒿草花粉的抗体结合效价很高， 同时具有一定的广谱 性； 对于有 "抗原交叉"的同属莠草类花粉都同样有一定的抑制作用。 这样 就提示我们，用本发明的方法制备的抗蒿草花粉特异性 Υ可用于凡是对莠 草类花粉过敏的患者作预防和治疗之用。
Figure imgf000013_0002
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.
由于同属同科的植物花粉之间有抗原交叉， 因此， 本草类植物花粉也只 需以有代表性的几种树花粉 (如法国梧桐花粉、 柳树花粉、 松树花粉等)制备 几种抗相对应花粉的特异性 IgY， 每种抗某属花粉特异性 IgY对同一属树花 粉引致的过敏症都可通用。 禾木植物花粉也只需以有代表性的狗尾草、梯牧 草、 黑麦等作抗原分别制备抗狗尾草等花粉的特异性 IgY， 对各自同一属的 有 "抗原交叉" 的禾木植物花粉过敏症都可适用。  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.
实验例 4: 以 ELISA法分别检测所制得的抗烟曲霉菌特异性 IgY对这 5 种抗原的抗体结合效价。 结果如下表所示-  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 −
Figure imgf000014_0001
从表中看出，采用本发明的抗原制作方法和免疫方式所制得的抗真菌特 异性 IgY抗体结合效价相当高，对同属的其他霉菌的结合效价也很高；从而, 进一步验证了这五种霉菌之间有很高的抗原交叉性。本发明制备的抗霉菌特 异性 IgY广谱性也很强， 只要制备抗某种霉菌的特异性 IgY, 对凡有 "抗原 交叉性" 的其他霉菌引发的过敏症也都会有很好的作用。
Figure imgf000014_0001
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".
实验例 5: 以 ELISA法， 用所制取的检测抗原捡测抗猫过敏特异性 IgY 对该猫过敏原的抗体结合效价， 检测结果效价为 1 : 128。 证明按本发明的方 法制得的抗猫过敏特异性 IgY对猫过敏原抑制效果相当理想。  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.
实验例 6: 以 ELISA法，采用本例（1)项中所列方法制作的蟑螂蛋白抗原 作检测抗原，检测所制备的抗蟑螂过敏特异性 IgY对蟑螂过敏原的抗体结合 效价， 检测结果效价为 1 : 256。 验证了按本发明的方法制备的抗宠物过敏特 异性 IgY效果十分理想。 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.
实验例 7:以 ELISA法，采用本例（1)项中所制得的 IgE抗原作检测抗原， 检测所制备的抗 IgE特异性 IgY对 IgE的抗体结合效价， 检测结果效价为 1 : 512。 实验证明， 采用本发明的方法制备的抗 IgE特异性 IgY具有很高的 活性， 完全达到预期的效果。  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.
实验例 8: 哮喘患者使用抗哮喘组合雾化剂的临床观察  Experimental Example 8: Clinical observation of anti-asthma combination nebulizer for asthma patients
(1)在患中度哮喘的住院成年患者中， 挑选经过 "皮肤点刺试验"诊断 为对尘螨过敏或莠草类花粉过敏， 或二者皆过敏的患者共计 20名。  (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)采用本发明制备的 "抗哮喘组合雾化剂"， 药液瓶打开后， 把药液倒 入 PARI型压缩雾化气泵中， 经雾化供患者吸入。 每天吸二次， 每次持续吸 入 5-10分钟。  (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)每天观察试验患者的症状变化情况。  (3) Observe the symptoms of the test patients daily.
(4)经吸用 "抗哮喘组合雾化剂" 2周后， 15名患者哮喘音基本消失， 喘息症状减轻。 有 5名患者夜间仍有阵发性喘息， 给予补充吸入 "抗哮喘组 合雾化剂"， 吸入 10分钟后， 喘息即停止。  (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)连续吸入 2个月后， 其中 17名患者的喘息症状消失， 另 3名患者喘 息症状也明显改善， 由每日有发作， 变为每周 1-2次偶发性发作。  (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.
实验例 9: 过敏性鼻炎患者使用手揿常压喷雾剂型的 "抗过敏特异性 IgY" 的临床观察结果- Experimental Example 9: Clinical Observation Results of "Antiallergic Specific IgY" in Patients with Allergic Rhinitis Using a Hand-held Normal Pressure Spray Form-
(1)通过 "皮肤点刺试验"诊断， 选择对尘螨或蟑螂过敏的慢性鼻炎患 者 50名作临床试验。 (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)将 50名患者随时分为二组， 治疗组使用抗过敏 "常压定量喷雾罐"， 对照组使用装有安慰剂的 "常压定量喷雾罐"。  (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.
治疗前， 2组患者都患有较严重的过敏性鼻炎持续鼻塞鼻痒和阵发性喷 嚏， 流大量浆液性鼻涕， 嗅觉减退。 治疗组有 3人嗅觉减退， 对照组 2人嗅 觉减退。  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)两组患者分别使用手揿喷雾罐往鼻部喷入雾状溶剂， 每天喷 3次。 观察并严格记录两组患者症状。 (4)治疗 2周后， 治疗组 25人中有 19人鼻塞和鼻痒症状基本消除， 鼻 涕量明显减少； 有 4人症状有改善， 有 2人没有明显感觉。 (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.
对照组中 25人， 症状基本维持不变。  In the control group, the symptoms remained basically unchanged.
(5)连续治疗 12周后， 治疗组 25人中， 有 22人鼻塞和鼻痒症状完全消 除， 流鼻涕情况也基本恢复正常； 原嗅觉减退的 3人已恢复正常嗅觉： 有 2 人症状明显减轻， 有 1人无改善。 对照组中 25人， 症状反复， 没有改善。  (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

权 利 要 求 Rights request
1、 一种抗过敏特异性 IgY， 其特征在于该抗过敏特异性 IgY的制备方法包括下列步 骤：  1. An anti-allergy-specific IgY, characterized in that the preparation method of the anti-allergy-specific IgY includes the following steps:
筛选出引致过敏性疾病的过敏原， 在筛选和分类组合各种过敏原的基础上， 分别制 备单一或复合的过敏原抗原； 制备免疫球蛋白 IgE抗原； 制备免疫蛋， 制备抗各种过敏 原特异性 IgY粗提物， 制备抗 IgE特异性 IgY粗提物； 抗各种过敏原特异性 IgY粗提物 和抗 IgE特异性 IgY粗提物的纯化； 制备抗某一种或多种特定过敏原的特异性复合 IgY。  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、 如权利要求 1所述的抗过敏特异性 IgY, 其特征在于该抗过敏特异性 IgY的制备 方法包括下列步骤： 2. The anti-allergic specific IgY according to claim 1, wherein the preparation method of the anti-allergic specific IgY comprises the following steps:
筛选出引致过敏性疾病的过敏原； 在筛选和分类组合各种过敏原的基础上， 分别制 备单一或复合的过敏原抗原， 将各组别单一或混合的过敏原提取液或制剂， 按 1一 10: 1 一 10比例加入福氏佐剂， 再置入高速匀浆机中， 高速搅拌形成油包水乳液， 即制成单一 或复合过敏原抗原； .  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;
制备免疫球蛋白 IgE抗原：可以从人体特别是过敏症患者的血清中提取 IgE，或购买 现成的 IgE免疫球蛋白作为抗原原料， 将: [gE免疫球蛋白按 1一 10: 1— 10比例配以福氏 佐剂， 置入高速勾浆机中， 高速匀化， 即制成 IgE抗原；  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;
制备免疫蛋： 用各种单一或复合过敏原抗原或 IgE抗原， 分别对产蛋鸡进行免疫， 每隔二周再强化注射一次，一个月计免疫三次，第一次免疫 20天后，检取鸡所产免疫蛋， 并对相应不同的抗原， 对所检取的免疫蛋进行编码标记；  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;
抗各种过敏原特异性 IgY粗提物的制备： 流动水逐个洗净免疫蛋， 再用酒精擦洗消 毒； 用打蛋机击破蛋， 蛋黄筛取出蛋黄， 搅打蛋黄均勾， 按蛋黄液量加入 4一 6倍蒸馏水 稀释， 搅拌均匀， 用 1. (^的¾;1调 11至5. 5—6. 0， 2—6°C静置 12— 24小时， 虹吸得 取上清液， 称 A液； 将以上沉淀液以 8000rpm离心 20分种， 得取上清液， 称 B液； 将 A 液和 B液混和均匀，超滤， 得到超滤浓缩液，加入 2. 0%海藻酸钠液， 至终浓度为 1. 0%。， 搅拌直至浑浊沉淀，加入 2. 0%氯化钙液， 至终浓度 1. 0%。，搅拌均匀， 2— 6Ό静置过夜， 8000rpra离心 30分种， 取上清液， 进而通过细菌过滤器除菌， 得到无菌滤液， 冷冻'干燥， 即为 IgY粗提物， 最后把所制备的抗各种不同抗原的 IgY粗提物进行编码标记；  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. ;
抗各种过敏原特异性 IgY粗提物和抗 IgE特异性 IgY粗提物的纯化： 将以上工艺所 制取的抗各种过敏原特异性 IgY和抗 IgE特异性 IgY粗提物分别先后过离子交换柱和凝 胶层析柱， 即得纯 IgY;  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;
制备抗某一种或多种特定过敏原的特异性复合 IgY: 不同抗原制得的纯 IgY， 同样按 照上述各自不同的编码标记严格标记以便区分， 将一种抗过敏特异性 IgY或多种抗过敏 特异性 IgY混合物与抗 IgE特异性 IgY按 1一 10: 1— 10的比例混合均匀， 然后加配制成 溶剂， 即得。  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、 如权利要求 1或 2所述的抗过敏特异性 IgY, 其特征在于该抗过敏特异性 IgY的 制备方法中引致过敏性疾病的过敏原可以是任何蛋白质过敏原。 4、 如权利要求 3所述的抗过敏特异性 IgY, 其特征在于蛋白质过敏原是指花粉过敏 原， 尘螨过敏原， 霉菌过敏原， 蟑螂过敏原， 宠物过敏原。 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、 如权利要求 1、 2或 3所述的抗过敏特异性 IgY， 其特征在于该抗过敏特异性 IgY 的制备方法中也可以直接将花粉、 尘螨、 霉菌、 蟑螂碎片、 宠物皮毛屑等加入福氏佐剂 混合后， 置入高速匀浆机中， 以 10000〜30000rpm进行高速粉碎匀化， 形成袖包水液， 制得含全菌和菌体成份或含全部致敏蛋白质成份的复合抗原。 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、 如权利要求 1、 2或 3所述的抗过敏特异性 IgY， 其特征在于该抗过敏特异性 IgY 的制备方法中也可应用同样的免疫技术， 用各种单一或复合过敏原抗原以及 IgE抗原， 分别对产蛋鸭进行免疫。 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、 如权利要求 1、 2或 3所述的抗过敏特异性 IgY， 其特征在于该抗过敏特异性 IgY 的制备方法中也可将抗 IgE特异性 IgY干粉和抗某一种过敏原的特异性 IgY或多种抗过 敏特异性 IgY混合物千粉按 1一 10 : 1— 10比例混合直接制成粉剂。 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、 如权利要求 6所述的抗过敏特异性 IgY， 其特征在于该抗过敏特异性 IgY的制备 方法中上述多种抗过敏特异性 IgY混合物是选用多种抗相应的不同过敏原的特异性 IgY, 先将它们按 1一 10: 1— 10或 1— 10: 1— 10: 1— 10或 1一 10 : 1— 10 : 1— 10: 1— 10依此类推 的比例均匀混合制成。 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、 如权利要求 1或 2所述的抗过敏特异性 IgY， 其特征在于该抗过敏特异性 IgY可 以制成临床可接受的剂型。 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、 一种抗粉尘螨特异性 IgY ， 其特征在于该抗粉尘螨特异性 IgY的制备方法包括 下列步骤： 10. A dust mite-resistant IgY, which is characterized in that the preparation method of the dust mite-resistant IgY includes the following steps:
将市面上购得的现成的粉尘螨注射液按 1 : 1比例与福氏佐剂混匀， 置于高速匀浆机， 以 20000rPm进行高速匀化， 制得乳状匀质抗原； 用所制得的粉尘螨抗原对良种蛋鸡进行 免疫， 釆用皮下注射和翅静脉注射相结合的免疫法， 每只鸡每次注射 1- 2ml抗原， 相当 于约 5000-10000只尘螨； 每隔二周再强化注射一次， 一个月内计免疫 3次； 第一次免疫 20天后， 捡取鸡所产免疫蛋； 用酒精擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄筛取出蛋 黄， 搅打蛋黄均匀， 按蛋黄液量加入 4一 6倍蒸镏水稀释， 搅拌均匀， 用 0. 1N的 Hcl调 整 pH5. 5-6. 0，2-6°C， 静置过夜， 虹吸得取上清液， 称 A液； 以上沉淀液以 8000rpm离心 20分钟， 得取上清液， 称 B液； 将 A液和 B液混和均匀， 超滤， 得取超滤浓缩液， 加入 2. 0%海藻酸钠液， 至终浓度为 1. 0%。， 搅拌， 加入 2. 0%氯化钙液， 至终浓度 1. 0%。， 搅 拌均匀， 2— 6°C静置过夜， 8000rmp离心 30分钟， 取上清液， 细菌过滤器过滤， 得到无 菌滤液，冷冻干燥，即为抗粉尘螨特异性 IgY粗提物；将以上 IgY粗提取物溶解于 PH7. 0、 0. 01MPB,经 DEAE— SephadexA50柱层析和 S印 hadexG200凝胶柱层析，得到纯抗粉尘螨特 异性 IgY。 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、 一种抗粉尘螨特异性鸭 IgY ， 其特征在于该抗粉尘螨特异性鸭 IgY的制备方法 包括下列步骤： 11. A dust mite-specific duck IgY, characterized in that the preparation method of the dust mite-specific duck IgY includes the following steps:
将粉尘螨注射液按 3 : 2比例与福氏佐剂混匀， 置于高速勾桨机， 以 20000rpm进行高 速匀化， 制得乳状匀质抗原； 用所制得的粉尘螨抗原对良种蛋鸭进行免疫， 采用皮下注 射和翅静脉注射相结合的免疫法， 每只鸭每次注射 1- 2ml抗原， 相当于约 5000- 10000只 尘螨； 每隔二周再注射一次， 计免疫 3次； 第一次免疫注射 20天后，检取鸭所产免疫蛋; 用酒精擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄筛取出蛋黄， 搅打蛋黄均匀， 按蛋黄液 量加入 4一 6倍蒸馏水稀释， 搅拌均匀， 用 0. 1N的 Hcl调整 ρΗ5. 5-6. 0, 2- 6Ό静置过夜， 虹吸得取上清液， 称 A液； 以上沉淀液以 8000rpm离心 20分钟， 得取上清液， 称 B液； 将 A液和 B液混和均匀， 超滤， 得取超滤浓缩液， 加入 2. 0%海藻酸钠液， 至终浓度为 1. 0%0,搅拌，加入 2. 0%氯化钙液，至终浓度 1. 0%0,搅拌均匀， 2— 6°C静置过夜， 8000rmp 离心 30分钟， 取上清液， 细菌过滤器过滤， 得到无菌滤液， 冷冻干燥， 即为抗粉尘螨特 异性鸭 IgY; 将以上 IgY粗提取物溶解于 pH7. 0、 0. 01MPB,经 DEAE—S印 hadexA50柱层析 和 SephadexG200凝胶柱层析， 得到纯抗粉尘螨特特异性鸭 IgY。 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、 抗蒿草花粉、 抗豚草花粉、抗藜草花粉和抗藻草花粉四种特异性 IgY， 其特征在 于该四种特异性 IgY的制备方法包括下列步骤： 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:
分别用蒿草花粉、 豚草花粉、 藜草花粉和藻草花粉制备抗原， 首先将所釆集的四种 花粉分别用高速匀浆机以 lOOOOrpm转速高速粉碎， 然后按 5 : 4比例分别加入福氏佐剂， 再在高速匀浆机中以 20000rpm高速匀化， 分别制得四种乳状花粉蛋白抗原； 用所制得的 蒿草花粉等抗原分别对良种蛋鸡进行免疫， 同样采用皮下注射和翅静脉注射相结合的免 疫法， 每只鸡每次注射 1. 5ml抗原， 相当于约 50- 100 花粉蛋白质； 每隔二周再强化 注射一次， 一个月内计免疫 3次； 第一次免疫 20天后， 分别检取鸡所产免疫蛋； 用酒精 擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄筛取出蛋黄， 搅打蛋黄均匀， 按蛋黄液量加入 4 一 6倍蒸馏水， 静置过夜， 虹吸得取上清液， 称 A液； 以上沉淀液以 8000rpm离心 20分 钟， 得取上清液， 称 B液； 将 A液和 B液混和均匀， 超滤， 得到超滤浓缩液， 加入 2. 0 %海藻酸钠液， 至终浓度为 1. 0%。， 搅拌， 加入 2. 0%氯化钙液， 至终浓度 1. 0%。， 搅拌 均勾， 2— 6Ό静置过夜， 8000rmp离心 30分钟， 取上清液， 细菌过滤器过滤， 得到无菌 滤液， 冷冻千燥， 分别制得抗蒿草花粉、 抗豚草花粉、 抗藜草花粉和抗藻草花粉特异性 IgY粗提物干粉； 将以上 IgY粗提取物分别溶解于 pH7. 0、 0. 01MPB,分别先后经 DEAE— SephadexA50柱层析和 SephadexG200凝胶柱层析， 把粗提物纯化， 即得抗蒿草花粉、 抗 豚草花粉、 抗藜草花粉和抗藻草花粉四种特异性纯 IgY。  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、 一种抗霉菌过敏原特异性 IgY ， 其特征在于该特异性 IgY的制备方法包括下列 步骤- 用烟曲霉菌、 土曲霉菌、 棒曲霉菌、 黑曲霉菌、 黄曲霉菌混合制作抗原： 将以上 4 种霉菌分别按常规培养后， 制取菌液； 各菌液以 1: 1： 1： 1比例混合， 制成混合菌液； 将此混合菌液按 6 : 7比例加入福氏佐剂， 然后置高速匀浆机以 20000rpm高速匀化， 制得 4种霉菌混合抗原；用此混合抗原对良种蛋鸡进行免疫， 同样采用皮下注射和翅静脉注射 相结合的免疫法， 每只鸡每次注射 1. 0ml抗原， 相当于约 200 y g蛋白质抗原； 每隔二周 再强化注射一次， 一个月内计免疫 3次； 第一次免疫 20天后， 检取鸡所产免疫蛋； 用酒 精擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄筛取出蛋黄， 搅打蛋黄均勾， 按蛋黄液量加 入 4一 6倍蒸馏水静置过夜， 虹吸得取上清液， 称 A液； 以上沉淀液以 8000rpm离心 20 分钟， 得取上清液， 称 B液； 将 A液和 B液混和均匀， 超滤， 得到超滤浓缩液， 加入 2. 0 %海藻酸钠液， 至终浓度为 1. 0%。， 搅拌， 加入 2. 0%氯化钙液， 至终浓度 1. 0%。， 搅拌 均匀， 2— 6°C静置过夜， 8000rmp离心 30分钟， 取上清液， 细菌过滤器过滤， 得到无菌 滤液， 冷冻干燥， 即制得抗烟曲霉菌、 抗土曲霉菌、 抗棒曲霉菌、 抗黑曲霉菌、 抗黄曲 霉菌特异性 IgY粗提物干粉； 将以上 IgY粗提取物溶解于 PH7. 0、 0. 01MPB,经 DEAE— SephadexA50柱层析和 SephadexG200凝胶柱层析， 即得抗霉菌过敏原特异性纯 IgY。 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、 一种抗宠物过敏的特异性 IgY , 其特征在于该特异性 IgY的制备方法包括下列 步骤- 取猫唾液和尿液按 1 : 1比例混合， 取混合物按 7 : 8比例加入福氏佐剂， 置入髙速匀 浆机以 20000rpm转速高速混勾， 制得乳状猫抗原； 用所制得的乳状猫抗原对良种蛋鸡进 行免疫，同样采用皮下注射和翅静脉注射相结合的免疫法，每只鸡每次注射 1一 2ml抗原， 相当于约 50- lOO w g蛋白质抗原； 每隔二周再强化注射一次， 一个月内计免疫 3次； 第 一次免疫 20天后， 检取鸡所产免疫蛋； 用酒精擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄 筛取出蛋黄， 搅打蛋黄均匀， 按蛋黄液量加入 4一 6倍蒸馏水， 静置过夜， 虹吸得取上清 液， 称 A液； 以上沉淀液以 8000rpni离心 20分钟， 得取上清液， 称 B液； 将 A液和 B液 混和均匀， 超滤， 得到超滤浓缩液， 加入 2. 0%海藻酸钠液， 至终浓度为 1. 0%。， 搅拌， 加入 2. 0%氯化钙液， 至终浓度 1. 0%。， 搅拌均匀， 2— 6°C静置过夜， 8000rrap离心 30 分钟， 取上清液， 细菌过滤器过滤， 得到无菌滤液， 冷冻干燥， 即制得抗宠物过敏的特 异性 IgY粗提物成品；将以上 IgY粗提取物溶解于 IW. 0,0. 01MPB,经 DEAE—S印 hadexA50 柱层析和 SephadeXG200凝胶柱层析， 即得抗宠物过敏的特异性纯 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、一种抗蟑螂过敏特异性 IgY，其特征在于该特异性 IgY的制备方法包括下列步骤： 收集 30- 50只的蟑螂， 剪去其翅膀， 将其躯千集中置入高速匀浆机以 lOOOOrpm进行 粉碎； 然后按蟑螂碎屑的重量， 以 6 :8 的重量比加入福氏佐剂，再在高速匀浆机中以 20000rpm高速粉碎匀化，制得乳状蟑螂蛋白抗原；用所制得的嶂螂抗原对良种蛋鸡进行免 疫， 采用皮下注射和翅静脉注射相结合的强化免疫法， 每只鸡每次注射 1- 2ml抗原， 相 当于 50— 100微克蛋白质； 每隔二周再强化注射一次， 一个月内计免疫 3次； 第一次免 疫 20天后， 检取鸡所产免疫蛋； 用酒精擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄筛取出 蛋黄， 搅打蛋黄均匀， 按蛋黄液量加入 4一 6倍蒸馏水稀释， 搅拌均匀， 用 0. 1N的 Hcl 调整 pH5. 5-6. 0， 2-6 °C， 静置过夜， 虹吸得取上清液， 称 A液； 以上沉淀液以 8000rpm 离心 20分钟， 得取上清液， 称 B液； 将 A液和 B液混和均勾， 超滤， 得取超滤浓缩液， 加入 2. 0%海藻酸钠液， 至终浓度为 1. 0%, 搅拌， 加入 2. 0%氯化钙液， 至终浓度 1. 0 %o , 搅拌均匀， 2— 6°C静置过夜， 8000rmp离心 30分钟， 取上清液， 细菌过滤器过滤， 得到无菌滤液， 冷冻干燥， 即为抗蟑螂过敏特异性 IgY粗提物； 将以上 IgY粗提物溶解 于 ΡΗ7· 0、 0. 01MPB,经 DEAE— SephadexA50柱层析和 SephadexG200凝胶柱层析， 得抗蟑 螂过敏特异性纯 IgY。 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、 一种抗 IgE特异性 IgY， 其特征在于该特异性 IgY的制备方法包括下列步骤： 将免疫球蛋白 IgE按 3 : 5比例与福氏佐剂混合，然后加入高速匀浆机以 lOOOOrpm高 速匀化， 制得 IgE抗原； 用所制得的 IgE抗原对良种蛋鸡进行免疫， 采用皮下注射和翅 静脉注射相结合的免疫法， 每只鸡每次注射 1- 2ml抗原， 相当于 50— 100微克蛋白质； 每隔二周再强化注射一次， 一个月内计免疫 3次； 第一次免疫 20天后， 检取鸡所产免疫 蛋； 用酒精擦洗消毒免疫蛋， 用打蛋机击破蛋， 蛋黄筛取出蛋黄， 搅打蛋黄均匀， 按蛋 黄液量加入 4一6倍蒸馏水稀释， 搅拌均匀， 用 0. 1N的 Hcl调整 pH5. 5-6. 0， 2- 6°C， 静置 过夜， 虹吸得取上清液， 称 A液； 以上沉淀液以 8000rPm离心 20分钟， 得取上清液， 称 B液； 将 A液和 B液混和均勾， 超滤， 得取超滤浓缩液， 加入 2. 0%海藻酸钠液， 至终浓 度为 1. 0%。， 搅拌， 加入 2. 0%氯化钙液， 至终浓度 1. 0%。， 搅拌均勾， 2— 6Ό静置过夜， 8000rmp离心 30分钟， 取上清液， 细菌过滤器过滤， 得到无菌滤液， 冷冻千燥， 即为抗 IgE 特异性 IgY 粗提取物； 将以上 IgY 粗提取物溶解于 PH7. 0、 0. 01MPB,经 DEAE— SephadexA50柱层析和 S印 hadexG200凝胶柱层析， 得抗 IgE特异性纯 IgY。 一种抗哮喘特异性复合 IgY， 其特征在于该抗哮喘特异性复合 IgY由 抗粉尘螨特异性 IgY干粉 1重量份 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
抗蒿草花粉特异性 IgY 1重量份  Anti-Artemisia pollen-specific IgY 1 part by weight
抗 IgE特异性 IgY干粉 1重量份，充分混和制成。  Anti-IgE specific IgY dry powder 1 part by weight, fully mixed.
18、 一种抗哮喘 IgY制剂， 其特征在于该制剂中每升含有抗哮喘特异性复合 IgY8— 12克。 18. An anti-asthma IgY preparation, characterized in that the preparation contains 8-12 grams of anti-asthma-specific complex IgY per liter.
19、 一种抗哮喘喷雾剂的制备方法， 其特征在于该方法为： 19. A method for preparing an anti-asthma spray, characterized in that the method is:
抗粉尘螨特异性 IgY干粉 100重量份， 抗蒿草花粉特异性 IgYlOO重量份， 抗 IgE特 异性 IgY干粉 100重量份加入小型搅拌机中充分混和，得到抗哮喘特异性复合 IgY300重 量份；  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;
按以下配方调配 30升抗哮喘复合 IgY组合溶液  Prepare 30 liters of anti-asthma compound IgY combination solution according to the following formula
抗哮喘特异性复合 IgY 300重量份  Anti-asthma specific complex IgY 300 parts by weight
阿斯巴甜 36重量份  Aspartame 36 parts by weight
香橙香精 90重量份  Orange Flavor 90 parts by weight
水蜜桃香精 30重量份  Peach flavor 30 parts by weight
生理盐水 加至 30000重量份  Normal saline to 30,000 parts by weight
先调制生理盐水 25000重量份， 然后加入阿斯巴 和香精， 搅拌均勾后， 再边搅拌 边缓缓加入复合 IgY; 接着， 加生理盐水至 30体积份， 充分搅拌均匀； 测量溶液的 pH 值， 用 1. 0蘭&011溶液调1)11至6. 5-7. 5; 经细菌过滤除菌， 最后采用无菌瓶灌装抗哮喘复 合喷雾剂。 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、 一种抗过敏特异性复合 IgY， 其特征在于该抗过敏特异性复合 IgY由 20. An anti-allergy specific complex IgY, characterized in that the anti-allergy specific complex IgY consists of
抗豚草特异性 IgY干粉 1重量份、 Anti-ragweed-specific IgY dry powder 1 part by weight,
抗蟑螂过敏原特异性 IgY干粉 1重量份、 Anti-cockroach allergen-specific IgY dry powder 1 part by weight,
抗 IgE特异性 IgY干粉 1重量份，充分混和制成。 Anti-IgE specific IgY dry powder 1 part by weight, fully mixed.
21、如权利要求 20所述的抗过敏复合 IgY制剂， 其特征在于该制剂中每 200升含有 抗过敏特异性复合 IgY300克左右。 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、 一种抗过敏喷雾剂的制备方法， 其特征在于该方法为： 22. A method for preparing an anti-allergic spray, characterized in that the method is:
抗豚草特异性 IgY干粉 100重量份， 抗蟑螂过敏原特异性 IgY干粉 100重量份， 抗 IgE特异性 IgY干粉 100重量份加入小型搅拌机中充分混和，制成抗过敏特异性复合 IgY 干粉 300重量份；  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
按以下配方调配 200000重量份抗过敏复合 IgY组合溶液；  Prepare 200,000 parts by weight of anti-allergic compound IgY combination solution according to the following formula;
抗过敏特异性复合 IgY 300重量份  Anti-allergy specific complex IgY 300 parts by weight
阿斯巴甜 240重量份  Aspartame 240 parts by weight
香橙香精 600重量份  Orange flavor 600 parts by weight
水蜜桃香精 200重 M份  Peach flavor 200 weight M servings
薄荷香精 1400重量份  1400 parts by weight
蒸馏水 加至 200000重量份  Distilled water to 200,000 parts by weight
先取蒸馏水 180000重量份， 然后分别加入阿斯巴甜、 香精， 搅拌均匀， 再边搅拌边 缓缓加入复合 IgY; 接着加蒸馏水至 2000Q0重量份， 充分搅拌均匀， 测量溶液 pH值， 根 据所测的 pH值用 1. (^ 011溶液调 1{至6. 5-7. 5; 溶液调配好后， 经细菌过滤除菌， 最 后在压力罐生产线上无菌灌装手动定量压力喷雾罐。每支喷雾罐灌足 10ml后， 再充入压 力氮气或者氟里昂等作为助推剂 (抛射剂）， 装上定量阀门， 密封， 即得。 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、 一种抗过敏特异性复合 IgY, 其特征在于该抗过敏特异性复合 IgY由 23.An anti-allergy specific complex IgY, characterized in that the anti-allergy specific complex IgY consists of
抗霉菌过敏原特异性 IgY干粉 1重量份、  Anti-fungus allergen-specific IgY dry powder 1 part by weight,
抗宠物过敏特异性纯 IgY干粉 ' 1重量份、  Anti-Pet Allergy Specific Pure IgY Dry Powder '' 1 part by weight,
抗 IgE特异性 IgY干粉 1重量份， 充分混和制成。  Anti-IgE specific IgY dry powder 1 part by weight, fully mixed.
24、 如权利要求 23所述的抗过敏复合 IgY制剂， 其特征在于该制剂中每 200升含有 抗过敏特异性复合 IgY300克左右。 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、 一种抗过敏喷雾剂的制备方法， 其特征在于该方法为： 25. A method for preparing an anti-allergic spray, characterized in that the method is:
抗霉菌过敏原特异性纯 IgY干粉 100重量份， 抗宠物过敏特异性纯 IgY干粉 100重 量份，， 抗 IgE特异性 IgY干粉 100重量份， 加入小型搅拌机中充分混和， 制成抗过敏特 异性复合 IgY干粉 300重量份，； 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;
按以下配方调配 200000重量份抗过敏复合 IgY组合溶液；  Prepare 200,000 parts by weight of anti-allergic compound IgY combination solution according to the following formula;
抗过敏特异性复合 IgY 300重量份，  300 parts by weight of anti-allergy specific complex IgY,
阿斯巴甜 240重量份，  240 parts by weight of aspartame,
香橙香精 600重量份，  Orange flavor 600 parts by weight,
水蜜桃香精 200重量份，  200 parts by weight of peach flavor,
薄荷香精 1400重量份，  1400 parts by weight of mint flavor,
蒸馏水 加至 200000重量份  Distilled water to 200,000 parts by weight
先取蒸馏水 180000重量份， 然后分别加入阿斯巴甜、 香精， 搅拌均匀， 再边搅拌边 缓缓加入复合 IgY; 接着加蒸馏水至 200000重量份， 充分搅拌均匀， 测量溶液 pH值， 根 据所测的 pH值用 1. 0N NaOH溶液调 PH6. 5- 7. 5; 经细菌过滤除菌， 最后无菌灌装手揿定 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、如权利要求 2所述的抗过敏特异性 IgY，其特征在于该抗过敏特异性 IgY的制备 方法中单一或复合的过敏原抗原与福氏佐剂的比例可以是 3 : 2。 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、如权利要求 2所述的抗过敏特异性 IgY，其特征在于该抗过敏特异性 IgY的制备 方法中单一或复合的过敏原抗原与福氏佐剂的比例可以是 3 : 5。 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、如权利要求 2所述的抗过敏特异性 IgY,其特征在于该抗过敏特异性 IgY的制备 方法中单一或复合的过敏原抗原与福氏佐剂的比例可以是 5 : 4。 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.
29、如权利要求 2所述的抗过敏特异性 IgY，其特征在于该抗过敏特异性 IgY的制备 方法中单一或复合的过敏原抗原与福氏佐剂的比例可以是 6 : 7。 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、如权利要求 2所述的抗过敏特异性 IgY，其特征在于该抗过敏特异性 IgY的制备 方法中单一或复合的过敏原抗原与福氏佐剂的比例可以是 7 : 8。 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、如权利要求 2所述的抗过敏特异性 IgY，其特征在于该抗过敏特异性 IgY的制备 方法中单一或复合的过敏原抗原与福氏佐剂的比例可以是 6 : 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.
PCT/CN2003/000333 2002-10-25 2003-05-08 Special igy of resist allergic and its preparation WO2004037862A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003242074A AU2003242074A1 (en) 2002-10-25 2003-05-08 Special igy of resist allergic and its preparation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN02146026.4 2002-10-25
CNB021460264A CN1286527C (en) 2002-10-25 2002-10-25 Anti irritability Specificity Igy and its preparation

Publications (1)

Publication Number Publication Date
WO2004037862A1 true WO2004037862A1 (en) 2004-05-06

Family

ID=32111546

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2003/000333 WO2004037862A1 (en) 2002-10-25 2003-05-08 Special igy of resist allergic and its preparation

Country Status (3)

Country Link
CN (1) CN1286527C (en)
AU (1) AU2003242074A1 (en)
WO (1) WO2004037862A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103913578B (en) * 2014-03-11 2016-01-20 安徽润敏江生物科技有限公司 A kind of method of quality control of cockroach allergen preparation
CN105541999A (en) * 2016-01-29 2016-05-04 西北农林科技大学 Formula of degreasing liquid in IgY preparation process and preparation method of formula
CN107893090B (en) * 2017-10-20 2020-05-15 国家***第三海洋研究所 Application of fermentation compound of aspergillus terreus H768 in preparation of antiallergic drugs

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1250056A (en) * 1999-09-08 2000-04-12 中德联合研究院 Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application
CN1354187A (en) * 2001-12-02 2002-06-19 重庆和润实业(集团)有限公司 Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1250056A (en) * 1999-09-08 2000-04-12 中德联合研究院 Pyloric bacillary spirillum resisting yolk immunoglobulin product and its application
CN1354187A (en) * 2001-12-02 2002-06-19 重庆和润实业(集团)有限公司 Hen egg yolk antibody for resisting pyloric helicobacterium cytotoxin, its preparation and application

Also Published As

Publication number Publication date
CN1513558A (en) 2004-07-21
AU2003242074A1 (en) 2004-05-13
CN1286527C (en) 2006-11-29

Similar Documents

Publication Publication Date Title
US9375444B2 (en) Composition for prevention and treatment of allergic and/or inflammatory diseases
US9006401B2 (en) Methods of treatment using thymus-derived compositions
CA3175596A1 (en) Traditional chinese medicine for dispersing lung qi and detoxicating
WO2007068154A1 (en) A method for preparing specific igy against mutant avian influenza virus and the preparation tηereof
CN102258780B (en) Lyophilized mite allergen vaccine and preparation method thereof
TW201322991A (en) Herbal composition and method for treatment of airway inflammation using the same
O'Hehir et al. Sublingual allergen immunotherapy: Immunological mechanisms and prospects for refined vaccine preparations
WO2004037862A1 (en) Special igy of resist allergic and its preparation
WO2008124870A1 (en) Delivery of flu antibodies to surfaces in contact with air
US20040142047A1 (en) Korean acanthopanax senticosus extract, protein extract, crude protein-polysaccharide which were extracted from korean acanthopanax senticosus, and immunoregulating compositions comprising the same and use thereof
CN1318449C (en) Anti SARS specificity IgY and combination preparation thereof
CN101555281A (en) Preparation method of anti-influenza A H1N1 virus specific IgY and related formulation thereof
US20060251672A1 (en) Composition and methods of treating allergies with reishi extract
CN117224518B (en) Application of sofalcone in preparation of medicine for preventing/treating allergic asthma
WO2015073958A1 (en) Ephedra alata extracts and methods of use thereof
US20230026406A1 (en) Therapeutic fractions and proteins from asthma-protective farm dust
CN103784712A (en) Health care product capable of repairing lung injuries caused by environmental pollution and preparation method of health care product
CN113041271B (en) Composition with preventing and antiallergic effects
KR102331377B1 (en) Pharmaceutical composition for prevention or treatment of respiratory diseases induced by particulate matter comprising extract of red ginseng, and a method for manufacturing the same
CN115814027B (en) Pharmaceutical composition for preventing respiratory tract infection diseases, preparation and application thereof
CN116712480B (en) Pharmaceutical composition for preventing influenza, preparation and application thereof
RU2800051C2 (en) Method of treatment and/or prevention of asthma, asthma aggression, allergic asthmatic disorders and/or respiratory disorders associated with microbiota
CN1657541A (en) Process for preparating specificity complex IgY of anti coxsackie virus myocarditis and its compound preparation
RU2497531C2 (en) Method of treating gastrointestinal diseases in calves
WO2022107081A1 (en) Methods of treating and preventing infections of coronavirus and other indications, and compositions for use with same

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP