WO2016058237A1 - 一种过敏原阳性血清的纯化制备方法 - Google Patents

一种过敏原阳性血清的纯化制备方法 Download PDF

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WO2016058237A1
WO2016058237A1 PCT/CN2014/091114 CN2014091114W WO2016058237A1 WO 2016058237 A1 WO2016058237 A1 WO 2016058237A1 CN 2014091114 W CN2014091114 W CN 2014091114W WO 2016058237 A1 WO2016058237 A1 WO 2016058237A1
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allergen
positive serum
purifying
purification
antiserum
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PCT/CN2014/091114
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English (en)
French (fr)
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王洪
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苏州浩欧博生物医药有限公司
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Priority to US14/906,266 priority Critical patent/US20160231342A1/en
Publication of WO2016058237A1 publication Critical patent/WO2016058237A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G, L chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2496/00—Reference solutions for assays of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the invention belongs to the technical field of in vitro diagnosis, and in particular relates to a method for purifying and preparing an allergen-positive serum.
  • Allergies are an allergic reaction of the body. They are an abnormal reaction of normal substances (allergens). When allergens are exposed to allergies, allergies, allergens, pollen, dust, and allogeneic proteins are allergic. Hundreds of chemical substances, ultraviolet rays, etc. In the process of allergic reactions, allergens play a direct role. Allergens are an external cause of allergic diseases, and the body's immunity is low. The oxidative damage of mast cells and basophils by a large number of free radicals is the internal cause of allergies. . Generally speaking, when an allergen enters the body for the first time, it combines with mast cells or basophils to produce allergic factors such as leukotrienes, prostaglandins, etc., but does not immediately cause allergies. It will last for 2 to 3 days, some months. When the body receives this allergen for the second time, the mast cells will be deformed and allergic factors will be produced, which will lead to a series of allergies.
  • Allergies can be mediated by body fluids (antibodies) or by cellular immune mechanisms.
  • antibodies that produce an allergic reaction are of the IgE class, and these individuals can be classified as having an IgE-mediated allergic reaction.
  • IgE-mediated allergic reaction not all allergic individuals develop IgE-related allergic reactions; in non-IgE-mediated allergic reactions, antibodies can also belong to IgGs, for example, anaphylactic shock caused by immune complexes containing dextran And and now rare serum diseases, they were previously classified as type III allergic reactions.
  • ABPA allergic bronchopulmonary aspergillosis
  • both IgE and IgG antibodies can be detected.
  • Contact allergic dermatitis is a representative of allergic diseases mediated by lymphocytes.
  • IgE testing is still recognized as the mainstream product of allergy testing. Due to the registration of puncture and the improvement of in vitro testing technology, in vitro testing will be the mainstream trend of allergy testing in the future; however, the current academic progress of IgE testing is slow and there is basically no new development, so improve The quality of inspection and the cost of inspection are the main development directions.
  • the publication number is CN103018436A, and the publication date is 2013-4-3.
  • a method for preparing avian infectious bronchitis positive serum by using rabbits after the blood is taken by immunization, only the blood is separated into serum, The serum was subjected to centrifugal filtration to obtain positive serum.
  • this positive serum has a problem in that the use effect is not good.
  • the technical problem to be solved by the present invention is to provide a purification preparation method using allergen-positive serum having excellent effects.
  • the present invention adopts the following technical solutions:
  • a method for purifying an allergen-positive serum comprising the steps of: immunizing a healthy animal with an allergen, obtaining an antiserum after blood collection, and purifying the antiserum to obtain a positive serum, the specific method of purifying for:
  • the anti-serum is affinity-purified to obtain an IgG antibody; after the IgG antibody and the human IgEFc are coupled at a mass ratio of 1:1 to 2, the IgG-IgE Fc linker concentrated solution is obtained by separation and purification, and The concentrated solution of the IgG-IgE Fc conjugate was diluted to a concentration of 0.5 to 1 ⁇ g/ml to obtain the positive serum.
  • the affinity purification is carried out using an agarose affinity medium or using an immunoaffinity chromatography column.
  • the agarose affinity medium is Protein-A sepharose CL-4B.
  • the immunoaffinity chromatography column is an affinity chromatography column in which the allergen is connected to an agarose gel.
  • the antiserum is subjected to ammonium sulfate treatment, and then the affinity purification is performed using the immunoaffinity chromatography column.
  • the human IgE Fc is obtained by dissolving human IgE in papain digestion solution, digesting with papain, and then digesting the reaction with iodoacetamide, and then extracting it with an agarose affinity medium.
  • the IgG antibody and the human IgE Fc are passed through a 2-imine tetrahydrothiophene coupling agent or 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid amber.
  • the coupling is carried out under the conditions of pH 7.2 to 7.4.
  • the concentration of the 2-imine tetrahydrothiophene coupling agent is 9 to 11 mg/ml, and the 4-(N-maleimidomethyl)cyclohexane-1-carboxyl group
  • concentration of the acid succinimide ester coupling agent is 4 to 6 mg/ml.
  • the separation and purification are carried out using a Sephadex 200 gel purification column.
  • the IgG-IgE Fc linker concentrated solution is prepared by using a bovine serum albumin having a mass ratio of 0.4 to 0.6%, a hydroxymethylaminomethane buffer having a pH of 7.5 to 8.5, and a nin to 0.11 mol/L. The dilution described is carried out.
  • the present invention has the following advantages compared with the prior art:
  • IgE Fc to link to IgG in antiserum, which is smaller than the molecular weight of positive serum linked to IgE and IgG, so that cross-reactivity can be avoided as much as possible, and specificity is raised on positive serum. Effectively retains the binding site of the secondary antibody.
  • the method of the invention can prepare the positive serum of various allergens in batches, solves the problem of positive control of the preparation process of the test kit, can also be used as the raw material for the production of the calibrator, and the preparation method is simple and easy.
  • Figure 1 is a diagram showing the isolation and purification of rabbit anti-willow IgG-IgE.
  • allergens allergen lyophilized powder, allergens are beef;
  • adjuvant Freund's complete adjuvant, purchased from sigma company, article number (F5881); Freund's incomplete adjuvant, purchased from sigma company, article number (F5506);
  • animals choose 3 (3 rabbits immunized with each allergen) healthy New Zealand white rabbits 2 months old and weighing 1.5 ⁇ 2.0kg;
  • Preparation of immunogenic allergens Determine the concentration of allergens after lyophilized powder dissolution by BCA protein assay, take 4mg allergen, and dilute it to 600 ⁇ L with PBS, use the three-way device to treat the allergen with complete adjuvant or not.
  • Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent immunization.
  • Animal immunization 3 New Zealand white rabbits to be purchased are cultured in the animal room for 1 week to adapt the animals to the environment; the prepared emulsion is subcutaneously injected into the neck or back with a 1 ml syringe, each New Zealand white rabbits were injected 3 times with 300 ⁇ L per injection; the immune cycle was 14 days.
  • Enzyme-linked immunosorbent assay was used to dilute the allergen (beef) to 10 ⁇ g/ml with a coating buffer, and 100 ⁇ L per well except the negative well.
  • the titer of the last collected antiserum is measured in parallel.
  • the data in column A is the antiserum titer measured after 6 immunizations; the data in column B is the antiserum titer measured after 7 immunizations.
  • the antiserum titer is basically stable after 6 and 7 immunizations of rabbits.
  • the serum gradient evaluated according to the experimental design is 1:100-1:1600000, when the serum is diluted to 1:51200.
  • the OD value is about twice as large as the blank hole, and is judged to be a positive effective price, but when the dilution is further performed, the OD value is substantially equal to the blank value or less than twice the blank control value, and is judged to be an invalid price.
  • A1-A2 and B1-B2 are negative controls.
  • TBS buffer solution 6.06g Tris (50mM), 8.78g NaCl (150mM) and 0.5g sodium azide (0.05%) dissolved in 1L of distilled water, and adjusted to pH 7.4 with HCl;
  • neutralizing buffer solution 121.2g Tris (1M), 87.8g NaCl (1.5M), 0.37g EDTA (1mM) and 5g sodium azide (0.5%) dissolved in 1L of distilled water, and adjusted to pH 8.0 with HCl;
  • Elution buffer solution pH 1.9: 3.75 g of glycine (50 mM) was dissolved in 1 L of distilled water, and pH 1.9 was adjusted with HCl.
  • the prepared antiserum is slowly thawed in ice water or a refrigerator at 4 ° C to avoid protein aggregation. Aggregation that occurs during protein thawing can be dissolved by preheating at 37 °C. Solid sodium azide was added to a concentration of 0.05%, centrifuged at 15000 xg for 5 min at 4 ° C, and the clarified antiserum was removed and filtered through a filter to remove excess lipid.
  • the dissolved antiserum was diluted with a TBS buffer solution at a ratio of 1:5, and then filtered with a filter.
  • the antiserum was applied to the column at a rate of 0.5 ml per minute.
  • the column was continuously applied twice and the sample effluent was retained.
  • the column was washed with TBS buffer solution to A280nm ⁇ 0.008 and then pH 2.7 elution buffer solution was eluted, and all proteins were eluted at a rate of 0.5 ml/min.
  • the eluate was collected using a 1.5 ml EP tube which had been added with 100 ⁇ L of neutralizing buffer solution. After mixing, the pH of the eluate was checked with a pH test paper. If the pH was lower than 7, the neutralization buffer was adjusted to about pH 7.4 to prevent it. Denaturation of antibodies;
  • the content of protein in each tube was measured using a spectrophotometer. If the protein concentration is less than 0.5 mg/ml, 10% glycerol may be added for storage, and the purified IgG antibody is stored at 2 ° C to 8 ° C after being dispensed;
  • the column was washed with TBS buffer solution containing 0.05% sodium azide and stored in a 2 ° C to 8 ° C environment.
  • Papain Digest 0.1 M Tris, 2 mM EDTA, pH 8.0.
  • Papain and Iodoacetamide were purchased from Sigma.
  • Protein-A was purchased from GE.
  • Coupling agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 2-imine tetrahydrothiophene (2-IT) was purchased from TH ERMO, chemical reagents such as trishydroxymethylaminomethane (TRIS) are chemically pure;
  • G-25 gel column and Sephadex 200 gel purification column are GE products.
  • the activated IgG antibody is mixed with activated human IgE Fc, and the reaction is allowed to stand under the condition of pH 7.3 for 20 hours, and the conjugate is purified by Sephadex 200 gel purification column to obtain a concentrated solution of the connector, and stored at 5 ° C for use;
  • the concentrated solution of IgG-IgE Fc conjugate was diluted to 0.5 ⁇ g/ml with TRIS buffer containing 0.5% bovine serum albumin, pH 8.0, and 0.1 mol/L to obtain positive serum of beef allergen.
  • Example 2 The positive serum prepared in Example 1 was measured using a specific allergen IgE antibody test kit produced by Phadia Co., Ltd. The results are shown in Table 2:
  • Phadia allergen IgE test rating ⁇ 0.35 KU / L is negative; 0.35-0.69 is grade 1 positive; 0.7-3.49 is grade 2 positive; 3.5-17.49 is grade 3 positive; 17.5-49.99 is grade 4 positive; 50-99.99 is grade 5 positive; >100KU/L is grade 6 positive.
  • positive serum control samples of 3 batches were positive samples using the Phadia allergen IgE test kit, and were positive for grade 5 positive samples. It can be seen from Table 2 that when the quality control samples were diluted 3 times and 9 times respectively, the measured value of Phadia also showed a similar ratio of 3 times and 9 times. A comprehensive description of our sample preparation is successful and can be used as a sample for quality control of the kit.
  • allergens allergen lyophilized powder, allergens are willows;
  • adjuvant Freund's complete adjuvant, purchased from sigma company, article number (F5881); Freund's incomplete adjuvant, purchased from sigma company, article number (F5506);
  • animals choose 3 (3 rabbits immunized with each allergen) healthy New Zealand white rabbits 2 months old and weighing 1.5 ⁇ 2.0kg;
  • Preparation of immunogenic allergens Determine the concentration of allergens after lyophilized powder dissolution by BCA protein assay.
  • the first immunizations are 0.05mg, 0.15mg and 0.2mg allergens diluted to 300 ⁇ L with PBS, and allergens and Freund's.
  • the complete adjuvant is mixed at a ratio of 0.8:1 (volume ratio).
  • the mixing method is to pipette the mixture 10 times or more, and the mixed product must be immunized immediately; the second and subsequent immunizations are 0.05 mg, 0.15 mg and 0.2.
  • the mg allergen was diluted to 500 ⁇ L with PBS and mixed with Freund's incomplete adjuvant at a ratio of 0.8:1 (volume ratio), and the remaining steps were the same as the first immunization.
  • animal immunization 3 New Zealand white rabbits will be purchased in the animal room for 1 week to adapt the animals to the environment; the prepared milky white mixture, the first immunization with a 1ml syringe to mix 0.5ml for rabbit foot immunization, other immunity Both legs and chest muscles were injected with an immune cycle of 7 days.
  • Antiserum was evaluated by plate chemiluminescence, allergen was diluted to 5 ⁇ g/ml with coating buffer, and 100 ⁇ L of 0.5 ⁇ g allergen was added to each well except the negative well, and coated at 37 ° C for 2 h.
  • the primary antibody is a gradient-diluted antiserum, and the secondary antibody is an AP-labeled goat anti-rabbit IgG.
  • the negative control the serum of the same rabbit taken before the primary antibody is an immunoallergic, and the gradient is diluted as the positive antiserum; Blank control: Primary antibody was incubated with blocking solution containing no serum; serum was diluted 1:200, 1:400, etc. When the antiserum titer reaches 1:100,000, it meets our requirements.
  • both the titer and the titer are greater than 1:100,000.
  • the serum gradient evaluated by the experimental design was 1:200-1:3200000.
  • the 672, 671 and 670 RLU values were greater than about 2 times of the RLU value of the negative control with a dilution gradient of 1:200. It is judged as a positive effective price, but when a large gradient dilution is performed again, the RLU value is substantially equal to the blank value or less than twice the blank control value, and is judged to be an invalid price.
  • Instrument immunoaffinity chromatography column, peristaltic pump, centrifuge tube, centrifuge, filter, chromatography column, spectrophotometer;
  • TBS equilibrium buffer solution 6.06g Tris (50mM), 8.78g NaCl (150mM) and 0.5g sodium azide (0.05%) dissolved in 1L of distilled water, and adjusted to pH 7.4 with HCl;
  • high salt buffer solution 121.2g Tris (1M), 87.8g NaCl (1.5M), 0.37g EDTA (1mM) and 5g sodium azide (0.5%) dissolved in 1L of distilled water, and adjusted to pH 8.0 with HCl;
  • elution buffer solution 3.75g glycine (50mM) dissolved in 1L of distilled water, adjusted to pH 2.6 with HCl;
  • the protein concentration of the supernatant was measured and compared with the concentration and amount of the originally administered allergen to calculate how many antigens were attached to the column.
  • the content of protein in each tube was measured using a spectrophotometer. If the protein concentration is less than 0.5 mg/ml, 10% glycerol may be added for storage, and the purified IgG antibody is stored at 2 ° C to 8 ° C after being dispensed;
  • Papain Digest (Papain Lysate): 0.1 M T ris, 2 mM EDTA (pH 8.0).
  • Coupling agent 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 2-imine tetrahydrothiophene (2-IT) was purchased from TH ERMO, chemical reagents such as trishydroxymethylaminomethane (TRIS) are chemically pure;
  • G-25 gel column and Sephadex 200 gel purification column are GE products.
  • the activated IgG antibody is mixed with the activated human IgE Fc portion, and allowed to stand under the condition of pH 7.3 for 20 hours, and the conjugate is purified by Sephadex 200 gel purification column to obtain a concentrated solution of the connector, and stored at 4 ° C for use;
  • the concentrated solution of IgG-IgE Fc conjugate was diluted to 0.5 ⁇ g/ml with TRIS buffer containing 0.5% bovine serum albumin, pH 8.0, and 0.1 mol/L to obtain positive serum of willow allergen.
  • Example 2 The positive serum prepared in Example 1 was measured using a specific allergen IgE antibody test kit produced by Phadia Co., Ltd. The results are shown in Table 6:
  • Phadia allergen IgE test rating ⁇ 0.35 KU / L is negative; 0.35-0.69 is grade 1 positive; 0.7-3.49 is grade 2 positive; 3.5-17.49 is grade 3 positive; 17.5-49.99 is grade 4 positive; 50-99.99 is grade 5 positive; >100KU/L is grade 6 positive.
  • the positive serum control samples of the three batches were positive samples using the Phadia allergen IgE test kit, and were positive for the 5-6 positive samples.
  • the measured value is also diluted.

Abstract

一种过敏原阳性血清的纯化制备方法，包括用过敏原对健康动物进行免疫、采血后获得抗血清的步骤和对所述的抗血清进行纯化获得阳性血清的步骤。纯化的具体方法为：对抗血清进行亲和纯化得到IgG抗体；将IgG抗体和人IgE Fc按质量比为1：1-2偶联后，经分离纯化获得IgG-IgE Fc连接物浓溶液，将该浓溶液稀释至浓度为0.5-1μg/mL，得到阳性血清。该方法可批量制备各种过敏原的阳性血清，其可作为检测试剂盒制备过程中的阳性质控，也可作为校准品的生产原料，并且制备方法简单易行。

Description

一种过敏原阳性血清的纯化制备方法 技术领域
本发明属于体外诊断技术领域，具体涉及一种过敏原阳性血清的纯化制备方法。
背景技术
过敏是一种机体的变态反应，是人对正常物质(过敏原)的一种不正常的反应，当过敏原接触到过敏体质的人群才会发生过敏，过敏原有花粉、粉尘、异体蛋白、化学物质、紫外线等几百种。在过敏反应的发生过程中，过敏介质起着直接的作用，过敏原是过敏病症发生的外因，而机体免疫能力低下，大量自由基对肥大细胞和嗜碱粒细胞的氧化破坏是过敏发生的内因。一般来讲，当过敏原第一次进入机体时，与肥大细胞或者是嗜碱性粒细胞结合，产生白三烯，前列腺素等等的过敏因子，但并不会立即产生过敏，此特性有些将维持2～3天，有的数月。当机体第二次接受这种过敏原时，肥大细胞才会变形，产生过敏因子，也就产生了一系列的过敏现象。
过敏可以是体液(抗体)或者是细胞免疫机制介导的。在大多数情况下，可产生过敏反应的抗体属于IgE类，这些个体可以归类于患有IgE介导的过敏反应。然而，并非所有的特异反应性个体都会发生与IgE相关的过敏反应；在非IgE介导的过敏反应中，抗体也可以属于IgG类，例如：包含右旋糖苷的免疫复合物导致的过敏性休克和和现在罕见的血清病，它们以前被归于III型过敏反应。在患有过敏性支气管肺曲霉菌病(ABPA)病人中，IgE和IgG抗体都可以被检出。接触性过敏性皮炎是以淋巴细胞为介导的过敏性疾病的代表。IgE检测仍是公认的过敏检测的主流产品，由于点刺注册问题和体外检测技术的提高，体外检测今后将是过敏检测主流趋势；但是目前IgE检测学术进展迟缓，基本没有新的发展，因此提高检测质量和降低检测成本是主要开发方向。
但是，由于过敏诊断试剂的发展缓慢以及前期人们的重视度有限，除了部分过敏原可以购买到商业血清，大部分过敏原的阳性血清还是很难查询到的，临床收集少量可行，大量或者高浓度的血清基本不能收集到，甚至于很多项目很少有合适的阳性血清(如：牛肉、羊肉)。
如公布号为CN103018436A，公布日为2013-4-3的一种用家兔制备鸡传染性支气管炎阳性血清的方法，其在经过免疫取血后，仅将血液分离出血清，对 血清进行离心过滤就得到阳性血清。但是，该阳性血清存在使用效果不好的问题。
发明内容
本发明所要解决的技术问题是提供一种使用效果优良的过敏原阳性血清的纯化制备方法。
为解决以上技术问题，本发明采取如下技术方案：
一种过敏原阳性血清的纯化制备方法，包括用过敏原对健康动物进行免疫、采血后获得抗血清的步骤和对所述的抗血清进行纯化获得阳性血清的步骤，所述的纯化的具体方法为：
对所述的抗血清进行亲和纯化得到IgG抗体；将所述的IgG抗体和人IgEFc按质量比为1:1～2偶联后，经分离纯化获得IgG-IgE Fc连接物浓溶液，将所述的IgG-IgE Fc连接物浓溶液稀释至浓度为0.5～1μg/ml，即得所述的阳性血清。
优选地，采用琼脂糖亲和介质或者采用免疫亲和层析柱进行所述的亲和纯化。
进一步优选地，所述的琼脂糖亲和介质为P rotein-A sepharose CL-4B。
进一步优选地，所述的免疫亲和层析柱为所述的过敏原与琼脂糖凝胶连接成的亲和层析柱。
进一步优选地，所述的抗血清先经过硫酸铵处理后，再采用所述的免疫亲和层析柱进行所述的亲和纯化。
优选地，所述的人IgE Fc是将人源IgE溶解木瓜蛋白酶消化液中，再用木瓜蛋白酶消化，再用碘乙酰胺终止消化反应后，用琼脂糖亲和介质提取获得的。
优选地，所述的IgG抗体和所述的人IgE Fc通过2-亚胺四氢噻吩偶联剂或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯偶联剂活化后，在pH7.2～7.4的条件下进行所述的偶联。
进一步优选地，所述的2-亚胺四氢噻吩偶联剂的浓度为9～11mg/ml，所述的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯偶联剂的浓度为4～6mg/ml。
优选地，采用Sephadex200凝胶纯化柱进行所述的分离纯化。
优选地，采用含质量比为0.4～0.6％的牛血清白蛋白、pH 7.5～8.5、0.09～0.11mol/L的三羟甲基氨基甲烷缓冲液将所述的IgG-IgE Fc连接物浓溶液进行所述的稀释。
由于以上技术方案的实施，本发明与现有技术相比具有如下优点：
我们可以根据市场需要来有计划的生产(从几毫升到几千毫升)，并且可以有效的控制批间差异。这是目前流行的阳性血清的制备方法无法做到的。另外，我们用IgE Fc与抗血清中的IgG连接，相比于用IgE与I gG连接的阳性血清的分子量小，这样就可以尽量避免交叉反应的发生，提高特异性的同时，在阳性血清上有效的保留了二抗的结合位点。
本发明方法可批量制备各种过敏原的阳性血清，解决了检测试剂盒制备过程中的阳性质控问题，也可以作为校准品的生产原料，并且本制备方法简单易行。
附图说明
附图1为兔抗柳树IgG-IgE分离纯化图。
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明，但本发明并不限于以下实施例。实施例中采用的实施条件可以根据具体使用的不同要求做进一步调整，未注明的实施条件为本行业中的常规条件。
实施例1
(一)过敏原的免疫和兔抗血清效价测定
材料与仪器
1、过敏原：过敏原冻干粉，过敏原为牛肉；
2、佐剂：弗氏完全佐剂，购自sigma公司，货号(F5881)；弗氏不完全佐剂，购自sigma公司，货号(F5506)；
3、动物：选3只(每种过敏原免疫3只兔子)2月龄、体重1.5～2.0kg的健康新西兰大白兔；
4、二抗：HRP标记的羊抗兔IgG；
5、耗材：三通器、一次性注射器、移液器等。
免疫步骤
1、免疫过敏原准备：用BCA蛋白质测定法来确定冻干粉溶解后过敏原的浓度，取4mg过敏原，并用PBS将其稀释至600μL，用三通器将过敏原与完全佐剂或不完全佐剂(过敏原V:佐剂V＝6:5)乳化，直至将一滴乳液滴入水中呈现球形而不分散则过敏原准备好。首次免疫用弗氏完全佐剂，后续免疫均用弗氏不完全佐剂。
2、动物免疫：将购买的3只新西兰大白兔在动物房养殖1周，使动物适应环境；将制备好的乳液，用1ml注射器进行颈部或背部皮下免疫注射，每只 新西兰大白兔注射3点，每点注射300μL；免疫周期为14天。
3、抗血清的制备：每只动物免疫前先于一侧耳缘静脉抽取2mL血(作为空白对照)，之后每次免疫14天，下次免疫前进行耳缘静脉取血用于抗血清评价；取血后取下针头，将注射器中的血缓缓转移至离心管中，于4℃冰箱中过夜，取上清即为血清，析出淡黄色抗血清；将抗血清转移至另一管中，血凝块以1500xg离心10min。吸出上清液合并收集到的抗血清管中；抗血清分装保存于－70℃。
4、抗血清效价的测定：采用酶联免疫吸附测定(enzyme linked immunosorbent assay；ELISA)，用包被缓冲液将过敏原(牛肉)稀释至10μg/ml，除阴性孔外每孔加100μL即1μg，4℃包被过夜；阴性对照1包被1μg羊肉过敏原，4℃包被过夜；阴性对照2包被1μg花生过敏原，4℃包被过夜；空白对照1为动物免疫开始前的血清；空白对照2为不含一抗抗体的一抗孵育封闭液；空白对照3为HRP标记的羊抗兔I gG孵育封闭液；其余孔为按1:100、1:200倍比稀释的抗血清，进行效价测定。每次效价测定时平行进行上次收集抗血清的效价测定，免疫前后抗血清效价相近时，则可以终止免疫杀兔取血，如果效价还有明显提高则继续免疫。
以牛肉免疫兔后抗血清评价结果表1：
表1
Figure PCTCN2014091114-appb-000001
Figure PCTCN2014091114-appb-000002
注：A列数据为免疫6次后测定的抗血清效价；B列数据为免疫7次后测定的抗血清效价
从上述表格中可以看出兔免疫6次和7次后，抗血清效价基本趋于稳定，按照实验设计评价的血清梯度为1:100-1:1600000，其中当血清稀释至1：51200时，OD值为空白孔的2倍左右，判断为阳性有效价，但当再进行稀释时OD值基本等于空白值或不到空白对照值的两倍，判断为无效价。A1-A2和B1-B2为阴性对照，从数值可以看出获取的兔抗血清与其他过敏原没有反应性；A3-A5和B3-B5为空白对照，可以看出过敏原与免疫前的兔血清、与二抗均不会产生非特异反应；其他孔的反应为牛肉过敏原和兔抗牛肉过敏原的特异性反应。
(二)抗血清的亲和纯化
材料与仪器
1、Protein-A sepharose CL-4B；蠕动泵；离心管；离心机；过滤器；玻璃柱；分光光度计；
2、TBS缓冲溶液：6.06gTris(50mM)，8.78g NaCl(150mM)以及0.5g叠氮化钠(0.05％)溶于1L蒸馏水中，并用HCl调节pH 7.4；
3、中和缓冲溶液：121.2g Tris(1M),87.8g NaCl(1.5M),0.37g EDTA(1mM)及5g叠氮化钠(0.5％)溶于1L蒸馏水中，并用HCl调节pH 8.0；
4、洗脱缓冲溶液(pH 2.7)：将3.75g甘氨酸(50mM)溶解于1L蒸馏水中，用HCl调节pH 2.7；
5、洗脱缓冲溶液(pH 1.9)：将3.75g甘氨酸(50mM)溶解于1L蒸馏水中，用HCl调节pH 1.9。
操作步骤
1、在真空瓶中将等体积的填料和TBS缓冲溶液混合，搅拌。抽真空约15min 以除去填料中的气泡，否则在柱中形成的气泡影响柱子的容量和分离效果。将Protein-A sepharose CL-4B缓慢加入玻璃柱中，利用泵控制填充速度为1ml/min～2ml/min，避免柱干，利用10倍于床体积并经过预冷的TBS缓冲溶液平衡柱子。
2、将制备得到的抗血清放入冰水或4℃冰箱中缓慢解冻以避免蛋白质的聚集。在蛋白质解冻过程中出现的聚集可通过37℃预热而溶解。加入固体叠氮化钠至浓度为0.05％，4℃，15000xg离心5min，移出澄清的抗血清再经过滤器过滤除去多余的脂。
3、将溶解后的抗血清用TBS缓冲溶液以1：5的比例进行稀释，再用过滤器进行过滤。以每分钟0.5ml的速度将抗血清上到柱上，为保证抗血清与填料的结合，需连续上柱2次并保留上样流出液。用TBS缓冲溶液清洗柱子至A280nm<0.008后加pH 2.7洗脱缓冲溶液，以0.5ml/min的速度洗脱至所有蛋白均流下来。用已经加入100μL中和缓冲溶液的1.5ml EP管分管收集洗脱液，混匀后用pH试纸检查洗脱液的pH，如果pH低于7可利用中和缓冲液调至约pH 7.4以防止抗体的变性；
在柱中加入10ml、pH 1.9洗脱缓冲溶液，按上述方法收集洗脱液至A280nm<0.008；
利用分光光度计测定各管中蛋白质的含量。若蛋白浓度低于0.5mg/ml可加入10％的甘油以便保存，将纯化的IgG抗体分装后在2℃～8℃保存；
用含0.05％叠氮化钠的TBS缓冲溶液清洗柱子后将柱子储存在2℃～8℃环境。
(三)IgG抗体与人IgE Fc偶联
材料与仪器
1、人IgE Fc，由苏州浩欧博生物医药有限公司研制，以磷酸盐缓冲液保存；
2、木瓜蛋白酶消化液(Papain溶解液)：0.1M Tris、2mM EDTA、pH 8.0。
3、Papain、Iodoacetamide购自Sigma公司。Protein-A购自GE公司。
4、偶联剂4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)，2-亚胺四氢噻吩(2-IT)购自TH ERMO公司，三羟甲基氨基甲烷(TRIS)等化学试剂均达到化学纯；
3、G-25凝胶柱和Sephadex200凝胶纯化柱为GE公司产品。
操作步骤
1、将人源IgE溶解在Papain消化液中，再用木瓜蛋白酶消化，再用 Iodoacetamide终止消化反应。然后用Protein-A提取出来Fc.
2、取1mg IgG抗体，并加入3μL 10mg/ml的偶联剂2-IT溶液，室温静置20min，加入10μL 0.1mol/L的甘氨酸溶液，在室温静置5min。用G-25凝胶柱除盐，收集活化后抗体，5℃保存备用；
3、取1.5mg的人IgE Fc溶液，加入5mg/ml的SMCC溶液15μL，室温静置30min，用G-25凝胶柱除盐，收集活化后抗体，5℃保存备用；
4、将上述活化的IgG抗体与活化的人IgE Fc混合，在pH 7.3的条件下静置反应20h，用Sephadex200凝胶纯化柱纯化偶联物，获得连接物浓溶液，5℃保存备用；
5、将IgG-IgE Fc连接物浓溶液用含0.5％牛血清白蛋白、pH 8.0、0.1mol/L的TRIS缓冲液稀释到0.5μg/ml，即得牛肉过敏原的阳性血清。
(四)用Phadia公司生产的特异性过敏原IgE抗体检测盒对实施例1制备得到的阳性血清进行测定，结果如表2：
表2
Figure PCTCN2014091114-appb-000003
注：Phadia过敏原IgE检测定级：<0.35KU/L为阴性；0.35-0.69为1级阳性；0.7-3.49为2级阳性；3.5-17.49为3级阳性；17.5-49.99为4级阳性；50-99.99为5级阳性；>100KU/L为6级阳性。
从表2可见，3批次(LOT131217、LOT140319、LOT140608)的阳性血清质控样本，用Phadia过敏原IgE检测试剂盒测定为阳性样本，并且为5级阳性样本即强阳性。从表格2中可以看到，质控样本分别进行3倍、9倍的稀释时，其Phadia的测定值也呈现类似的3倍、9倍的倍比降低比例。综合说明我们的样本制备成功，可以作为样本用于试剂盒的质控。
实施例2
(一)过敏原的免疫和兔抗血清效价测定
材料与仪器
1、过敏原：过敏原冻干粉，过敏原为柳树；
2、佐剂：弗氏完全佐剂，购自sigma公司，货号(F5881)；弗氏不完全佐剂，购自sigma公司，货号(F5506)；
3、动物：选3只(每种过敏原免疫3只兔子)2月龄、体重1.5～2.0kg的健康新西兰大白兔；
4、二抗：AP标记的羊抗兔IgG；
5、耗材：三通器、一次性注射器、移液器等。
免疫步骤
1、免疫过敏原准备：用BCA蛋白测定法来确定冻干粉溶解后过敏原的浓度，首次免疫分别取0.05mg、0.15mg和0.2mg过敏原用PBS稀释至300μL，将过敏原与弗氏完全佐剂0.8:1(体积比)混合，混匀方式是用移液器吹打混合物10次以上，混匀产物必须马上进行免疫；第二次以后的免疫都是取0.05mg、0.15mg和0.2mg过敏原用PBS稀释至500μL，与弗式不完全佐剂0.8:1(体积比)混合，其余步骤同首次免疫。
2、动物免疫：将购买的3只新西兰大白兔在动物房养殖1周，使动物适应环境；将制备好的乳白色混合物，首次免疫用1ml注射器取混合0.5ml进行兔子的足底免疫，其他免疫均采用进行腿部和胸部肌肉注射，免疫周期为7天。
3、抗血清的制备：每只动物免疫前先于一侧耳缘静脉抽取2mL血(作为空白对照)，之后每进行下一次免疫前进行耳缘静脉取血，用于抗血清评价；另外效价达到要求后采用心脏采血法获取大量免疫血液；取血后通过1000rpm离心15min，收集抗血清；抗血清分装保存于－70℃。
4、抗血清效价的测定：采用板式化学发光评价抗血清，用包被缓冲液将过敏原稀释至5μg/ml，除阴性孔外每孔加100μL即0.5μg过敏原，37℃包被2h；一抗为梯度稀释的抗血清，二抗为AP标记的羊抗兔IgG，其中阴性对照：一抗为免疫过敏原前所取的同一只兔子的血清，与阳性抗血清一样进行梯度稀释；空白对照：一抗孵育不含有任何血清的封闭液；其中血清按1:200、1:400等倍比稀释。抗血清效价达到1：100000时，即达到我们要求。
以柳树免疫兔后抗血清评价结果表3和表4：
表3
Figure PCTCN2014091114-appb-000004
表4
Figure PCTCN2014091114-appb-000005
表5
动物编号 672 671 670
抗血清效价 1：100000 1：200000 1：100000
注：上述数据为免疫4次后测定3只兔子的的抗血清效价；
从上述表格中可以看出兔免疫4次后，均满足和效价大于1：100000，按 照实验设计评价的血清梯度为1:200～1:3200000，其中当血清稀释至1：100000时，672、671和670RLU值大于稀释梯度为1:200的阴性对照的RLU值的2倍左右，判断为阳性有效价，但当再进行大的梯度稀释时RLU值基本等于空白值或不到空白对照值的两倍，判断为无效价。
(二)抗血清的亲和纯化
材料与仪器
1、仪器：免疫亲和层析柱、蠕动泵、离心管、离心机、过滤器、层析柱、分光光度计；
2、TBS平衡缓冲溶液：将6.06g Tris(50mM)、8.78g NaCl(150mM)以及0.5g叠氮化钠(0.05％)溶于1L蒸馏水中，并用HCl调节pH 7.4；
3、高盐缓冲溶液：将121.2g Tris(1M),87.8g NaCl(1.5M),0.37gEDTA(1mM)及5g叠氮化钠(0.5％)溶于1L蒸馏水中，并用HCl调节pH 8.0；
4、洗脱缓冲溶液：将3.75g甘氨酸(50mM)溶解于1L蒸馏水中，用HCl调节pH 2.6；
5、其他材料：0.2M碳酸盐缓冲液(pH 9.5)、1.0mM HCl、1.0M甘氨酸溶液、CNBr-actived Sepharose 4B-Cl(GE)、柱料储存液(0.1MPBS，含1％氨基己酸，pH 7.4)。
操作步骤
1、准备免疫亲和层析柱：将免疫使用的抗原与琼脂糖凝胶连接，做成亲和层析柱。具体为：将特异性的过敏原用0.2M碳酸盐缓冲液(pH 9.5)溶解稀释成1.0mg/ml。再用1.0mM HCL处理活化好的Sepharose 4B-Cl(CNBr-actived Seph arose 4B-Cl)，然后用0.2M碳酸盐缓冲液(pH 9.5)稀释成1.0g/ml。再将这两部分按1：1的比例进行混合，在室温下反应16～20小时。然后离心，收集上清液。测上清液的蛋白浓度，再与最初投放的过敏原的浓度和量进行比较，算出有多少抗原接在柱子上。剩下的Sepharose4B-Cl放入1.0M甘氨酸溶液，溶液的量为Sepharose4B-Cl：1.0M甘氨酸溶液＝1：1，反应4小时后。用0.1M的HCl，0.1M NaOH和2M尿素溶液分别以3倍柱体积依次进行清洗，最后用储存液保存。
2、在容器中将等体积的亲和层析柱与过硫酸铵处理后的动物血清充分混合，轻摇2h。将混合物缓慢加入玻璃柱中，利用泵控制填充速度为1ml/min～2ml/min，避免柱干，利用3～10倍于柱床体积缓冲溶液平衡柱子。
3、用TBS缓冲溶液清洗柱子至吸收值A在280nm<0.008后，再用高盐缓冲液洗去非特异结合蛋白。加pH 2.6洗脱缓冲溶液，以0.5ml/min的速度洗 脱至吸收值A在280nm<0.008。用已经加入100μL中和缓冲溶液的1.5ml EP管分管收集洗脱液，混匀后用pH试纸检查洗脱液的pH，如果pH低于7可利用中和缓冲液调至约pH 7.4以防止抗体的变性；
利用分光光度计测定各管中蛋白质的含量。若蛋白浓度低于0.5mg/ml可加入10％的甘油以便保存，将纯化的IgG抗体分装后在2℃～8℃保存；
用含0.05％叠氮化钠的TBS缓冲溶液清洗柱子后，将柱子储存在2℃～8℃环境。
(三)IgG抗体与人IgE Fc偶联
材料与仪器
1、人IgE Fc，由苏州浩欧博生物医药有限公司研制，以磷酸盐缓冲液保存的；
2、木瓜蛋白酶消化液(Papain溶解液)：0.1M T ris，2mM EDTA(pH 8.0)。
3、Papain，购自Sigma公司；Iodoacetamide,购自Sigma公司；Protein-A购自GE公司；
4、偶联剂4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯(SMCC)，2-亚胺四氢噻吩(2-IT)购自TH ERMO公司，三羟甲基氨基甲烷(TRIS)等化学试剂均达到化学纯；
5、G-25凝胶柱和Sephadex200凝胶纯化柱为GE公司产品。
操作步骤
1、人IgE，纯度为95％，浓度为1mg/ml，先用Papain消化液(也叫溶解液)，pH＝8.0透析，然后加入浓度为Papain：IgE＝100:1(w/w)进行消化30min，再在消化液中加入Iodoacetamide终止反应，使用Protein A将IgE Fc部分纯化出来。
2、取1mg的IgG抗体溶液，加入5mg/ml的SMCC溶液15μL，室温静置30min，用G-25凝胶柱除游离SMCC，收集活化后抗体，4℃保存备用；
3、取1.5mg人IgE Fc部分，加入10mg/ml的偶联剂2-IT溶液3μL，室温静置20min，加入0.1mol/L的甘氨酸溶液10μL，室温静置5min。用G-25凝胶柱除游离的2-IT，收集活化后抗体，4℃保存备用；
4、将上述活化的IgG抗体与活化的人IgE Fc部分混合，在pH 7.3的条件下静置20h，用Sephadex200凝胶纯化柱纯化偶联物，获得连接物浓溶液，4℃保存备用；
5、将IgG-IgE Fc连接物浓溶液用含0.5％牛血清白蛋白、pH 8.0、0.1mol/L的TRIS缓冲液稀释到0.5μg/ml，即得柳树过敏原的阳性血清。
(四)用Phadia公司生产的特异性过敏原IgE抗体检测盒对实施例1制备得到的阳性血清进行测定，结果如表6：
表6
Figure PCTCN2014091114-appb-000006
注：Phadia过敏原IgE检测定级：<0.35KU/L为阴性；0.35-0.69为1级阳性；0.7-3.49为2级阳性；3.5-17.49为3级阳性；17.5-49.99为4级阳性；50-99.99为5级阳性；>100KU/L为6级阳性。
从表6可见，3批次(LOT140105、LOT140327、LOT140507)的阳性血清质控样本，用Phadia过敏原IgE检测试剂盒测定为阳性样本，并且为5-6级阳性样本即强阳性。另外从表格中可以看到，质控样本倍比稀释后，测定值也为倍比稀释的。综合说明我们的样本制备成功，可以作为样本用于试剂盒的质控。
以上对本发明做了详尽的描述，其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施，并不能以此限制本发明的保护范围，凡根据本发明的精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。

Claims (10)

  1. 一种过敏原阳性血清的纯化制备方法，包括用过敏原对健康动物进行免疫、采血后获得抗血清的步骤和对所述的抗血清进行纯化获得阳性血清的步骤，其特征在于：所述的纯化的具体方法为：
    对所述的抗血清进行亲和纯化得到IgG抗体；将所述的IgG抗体和人IgE Fc按质量比为1:1～2偶联后，经分离纯化获得IgG-IgE Fc连接物浓溶液，将所述的IgG-IgE Fc连接物浓溶液稀释至浓度为0.5～1μg/ml，即得所述的阳性血清。
  2. 根据权利要求1所述的过敏原阳性血清的纯化制备方法，其特征在于：采用琼脂糖亲和介质或者采用免疫亲和层析柱进行所述的亲和纯化。
  3. 根据权利要求2所述的过敏原阳性血清的纯化制备方法，其特征在于：所述的琼脂糖亲和介质为Protein-A sepharose CL-4B。
  4. 根据权利要求2所述的过敏原阳性血清的纯化制备方法，其特征在于：所述的免疫亲和层析柱为所述的过敏原与琼脂糖凝胶连接成的亲和层析柱。
  5. 根据权利要求2或4所述的过敏原阳性血清的纯化制备方法，其特征在于：所述的抗血清先经过硫酸铵处理后，再采用所述的免疫亲和层析柱进行所述的亲和纯化。
  6. 根据权利要求1所述的过敏原阳性血清的纯化制备方法，其特征在于：所述的人IgE Fc是将人源IgE溶解木瓜蛋白酶消化液中，再用木瓜蛋白酶消化，再用碘乙酰胺终止消化反应后，用琼脂糖亲和介质提取获得的。
  7. 根据权利要求1所述的过敏原阳性血清的纯化制备方法，其特征在于：所述的IgG抗体和所述的人IgE Fc通过2-亚胺四氢噻吩偶联剂或4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯偶联剂活化后，在pH 7.2～7.4的条件下进行所述的偶联。
  8. 根据权利要求7所述的过敏原阳性血清的纯化制备方法，其特征在于：所述的2-亚胺四氢噻吩偶联剂的浓度为9～11 mg/ml，所述的4-(N-马来酰亚胺基甲基)环己烷-1-羧酸琥珀酰亚胺酯偶联剂的浓度为4～6 mg/ml。
  9. 根据权利要求1所述的过敏原阳性血清的纯化制备方法，其特征在于：采用Sephadex 200凝胶纯化柱进行所述的分离纯化。
  10. 根据权利要求1所述的过敏原阳性血清的纯化制备方法，其特征在于：采用含质量比为0.4～0.6％的牛血清白蛋白、pH 7.5～8.5、0.09～0.11mol/L的三羟甲基氨基甲烷缓冲液将所述的IgG-IgE Fc连接物浓溶液进行所述的稀 释。
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US10872313B2 (en) * 2015-06-02 2020-12-22 ROCA Medical Ltd. Method for repurposing NDC codes in a pharmaceutical database for venom derived allergens involved in venom immunotherapy
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