CN104977401A - Purifying preparation method of allergen positive serum - Google Patents

Abstract

The invention relates to a purifying preparation method of allergen positive serum, wherein the method includes the operations of obtaining antiserum through immunity and blood collection to healthy animals with an allergen, and performing purification to the antiserum to obtain the positive serum. The purification particularly includes the following steps: 1) performing affinity purification to the antiserum to prepare an IgG antibody; 2) coupling the IgG antibody with human IgEFc according to the mass ratio of 1:1-2, and performing separation and purification to obtain an IgG-IgEFc junctional complex concentrated solution; and 3) diluting the IgG-IgEFc junctional complex concentrated solution to 0.5-1 [mu]g/ml. By means of the method, batched preparation of the positive serums of various allergens and purifying preparation of other various positive serums are achieved, thereby solving a problem of positive quality control of a preparation process of a detection kit. The allergen positive serum also can be used as a production raw material of a calibrator. In addition, the preparation method is easy to carry out.

Description

A kind of method for preparing purified of anaphylactogen positive serum

Technical field

The invention belongs to vitro diagnostic techniques field, be specifically related to a kind of method for preparing purified of anaphylactogen positive serum.

Background technology

Allergy is a kind of allergic reaction of body, the one abnormal reaction of people to koinomatter (anaphylactogen), just can there is allergy in the crowd touching allergic constitution when anaphylactogen, the hundreds of kinds such as irritated original pollen, dust, foreign protein, chemical substance, ultraviolet.In anaphylactoid generating process, Anaphylactic mediator plays direct effect, and anaphylactogen is the external cause that allergic condition occurs, and body immunity is low, and the Oxidative demage of a large amount of radical pair mast cell and basophil is the irritated internal cause occurred.In general, when anaphylactogen first time enters body, is combined with mast cell or basophilic granulocyte, generation leukotriene, the allergenic factor of prostaglandin etc., but allergy can't be produced immediately, this characteristic some by maintenance 2 ~ 3 days, some several months.When body second time accepts this anaphylactogen, mast cell just can be out of shape, and produces allergenic factor, also just creates a series of allergic phenomena.

Allergy can be body fluid (antibody) or cellular immune mechanism mediation.In most of the cases, can produce anaphylactoid antibody and belong to IgE class, these individualities can range the allergic reaction suffering from IgE mediation.But, and all can there is the allergic reaction relevant to IgE in the atopy of not all individuality; In the allergic reaction that non-IgE mediates, antibody also can belong to IgG class, such as: the anaphylactic shock that the immune complex comprising dextran causes and and now rare serum sickness, they were attributed to type III allergic reaction in the past.Suffering from allergic bronchopulmonary aspergillosis (ABPA) patient, IgE and IgG antibody can be detected.Contact hyper sensitization dermatitis is the representative of the anaphylactia taking lymphocyte as mediation.IgE detects the main product being still generally acknowledged allergy and detecting, and due to the raising of pricking method registration problem and vitro detection technology, vitro detection will be irritated detection main trend from now on; But IgE detection science makes slow progress at present, does not substantially have new development, therefore improving Detection job and reducing testing cost is main developing way.

But, due to anaphylodiagnosis reagent slower development and early stage people attention degree limited, except part anaphylactogen can buy business serum, the positive serum of major part anaphylactogen is still difficult to inquire, clinical collection is feasible on a small quantity, serum that is a large amount of or high concentration can not be collected substantially, and a lot of project seldom has suitable positive serum (as: beef, mutton) even.

If publication No. is CN103018436A, date of publication is a kind of method preparing infectious bronchitis of chicken positive serum with rabbit of 2013-4-3, and blood is only isolated serum by it after getting blood through immunity, carries out centrifugal filtration just obtain positive serum to serum.But there is the bad problem of result of use in this positive serum.

Summary of the invention

Technical matters to be solved by this invention is to provide the method for preparing purified of the excellent anaphylactogen positive serum of a kind of result of use.

For solving above technical matters, the present invention takes following technical scheme:

A method for preparing purified for anaphylactogen positive serum, comprise and carry out immunity, the rear step obtaining sero-fast step and described antiserum is carried out to purifying acquisition positive serum of blood sampling with anaphylactogen to healthy animal, the concrete grammar of described purifying is:

Affinity purification is carried out to described antiserum and obtains IgG antibody; Be after the coupling of 1:1 ~ 2 in mass ratio by described IgG antibody and people IgEFc, obtain IgG-IgE Fc connector strong solution through separation and purification, it is 0.5 ~ 1 μ g/ml that described IgG-IgE Fc connector strong solution is diluted to concentration, obtains described positive serum.

Preferably, adopt agarose compatible medium or adopt immune affinity chromatographic column to carry out described affinity purification.

Further preferably, described agarose compatible medium is Protein-A sepharose CL-4B.

Further preferably, described immune affinity chromatographic column is the affinity column that described anaphylactogen and Ago-Gel connect into.

Further preferably, described antiserum first after ammonium persulfate process, then adopts described immune affinity chromatographic column to carry out described affinity purification.

Preferably, people source IgE dissolves in papain digestion liquid by described people IgE Fc, then uses papain digestion, then after stopping digestion reaction with iodoacetamide, extracts obtain with agarose compatible medium.

Preferably, described IgG antibody and described people IgE Fc, by after 2-imines thiophane coupling agent or the activation of 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester coupling agent, carry out described coupling under the condition of pH 7.2 ~ 7.4.

Further preferably, the concentration of described 2-imines thiophane coupling agent is 9 ~ 11mg/ml, and the concentration of described 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester coupling agent is 4 ~ 6mg/ml.

Preferably, Sephadex 200 gel-purified post is adopted to carry out described separation and purification.

Preferably, adopt containing mass ratio be 0.4 ~ 0.6% bovine serum albumin(BSA), pH 7.5 ~ 8.5,0.09 ~ 0.11mol/L TRIS buffer described IgG-IgE Fc connector strong solution is carried out described dilution.

Due to the enforcement of above technical scheme, the present invention compared with prior art tool has the following advantages:

We can carry out planned production (from several milliliters to several thousand milliliters) according to market demand, and effectively can control differences between batches.This is that the preparation method of popular at present positive serum cannot accomplish.In addition, we are connected with the IgG in antiserum with IgE Fc, little compared to the molecular weight of the positive serum connected with IgE and IgG, so just can avoid the generation of cross reaction as far as possible, the while of improving specific, positive serum effectively remains two anti-binding sites.

The inventive method can prepare the positive serum of various anaphylactogen in batches, solves the positive quality control problem in detection kit preparation process, also can as the raw materials for production of calibration object, and this preparation method is simple.

Accompanying drawing explanation

Accompanying drawing 1 is rabbit anti-willow IgG-IgE separation and purification figure.

Embodiment

Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following examples.The implementation condition adopted in embodiment can require to do further adjustment according to the concrete difference used, and not marked implementation condition is the normal condition in the industry.

Embodiment 1

(1) immunity of anaphylactogen and rabbit anti-serum titration

Material and instrument

1, anaphylactogen: anaphylactogen freeze-dried powder, anaphylactogen is beef;

2, adjuvant: Freund's complete adjuvant, purchased from sigma company, article No. (F5881); Incomplete Freund's adjuvant, purchased from sigma company, article No. (F5506);

3, animal: the healthy new zealand white rabbit selecting 3 (often kind of anaphylactogen immunity 3 rabbits) 2 monthly ages, body weight 1.5 ~ 2.0kg;

4, two resist: the goat anti-rabbit igg of HRP mark;

5, consumptive material: three-way device, disposable syringe, pipettor etc.

Immunity step

1, immune anaphylactogen prepares: determine that freeze-dried powder dissolves the concentration of rear anaphylactogen with BCA protein determination, get 4mg anaphylactogen, and be diluted to 600 μ L with PBS, with three-way device by anaphylactogen and Freund's complete adjuvant or Freund's incomplete adjuvant (anaphylactogen V: adjuvant V=6:5) emulsification, until spherically not disperse presenting in an emulsion instillation water, anaphylactogen is ready to.First immunisation Freund's complete adjuvant, follow-up immunization all uses incomplete Freund's adjuvant.

2, animal immune: 3 new zealand white rabbits bought are cultivated 1 week at Animal House, animal is conformed; By the emulsion prepared, carry out neck or dorsal sc inoculation with 1ml syringe, every new zealand white rabbit injects 3 points, often some injection 300 μ L; The immunity cycle is 14 days.

3, sero-fast preparation: extract 2mL blood (as blank) prior to side auricular vein before every animal immune, each immunity afterwards 14 days, carries out auricular vein and gets blood for antiserum evaluation before next immunity; Take off syringe needle after getting blood, the blood in syringe is slowly transferred in centrifuge tube, in 4 DEG C of refrigerator overnight, get supernatant and be serum, separate out faint yellow antiserum; Be transferred to by antiserum in another pipe, blood clot is with the centrifugal 10min of 1500xg.Sucking-off supernatant merges in the antiserum pipe collected; Antiserum packing is stored in-70 DEG C.

4, the mensuration of antiserum titre: adopt enzyme linked immunosorbent assay (ELISA) (enzyme linkedimmunosorbent assay; ELISA), be buffered liquid with bag and anaphylactogen (beef) is diluted to 10 μ g/ml, except negative hole, every hole adds 100 μ L i.e. 1 μ g, and 4 DEG C of bags are spent the night; Negative control 1 wraps by 1 μ g mutton anaphylactogen, and 4 DEG C of bags are spent the night; Negative control 2 wraps by 1 μ g Peanut Allergen, and 4 DEG C of bags are spent the night; Blank 1 for animal immune start before serum; Blank 2 is do not hatch confining liquid containing the primary antibodie of an antiantibody; The goat anti-rabbit igg that blank 3 marks for HRP hatches confining liquid; All the other holes are the antiserum by 1:100,1:200 doubling dilution, carry out titration.During each titration, parallel carrying out collected sero-fast titration last time, when antiserum titre is close before and after immunity, then can stops immunity and kill rabbit and get blood, also be significantly improved, continue immunity if tired.

With antiserum evaluation result table 1 after beef immunize rabbit:

Table 1

Note: A column data is the antiserum titre that immunity measures for 6 times afterwards; B column data is the antiserum titre that immunity measures for 7 times afterwards

Can find out from above table rabbit immunity 6 times and 7 times after, antiserum titre tends towards stability substantially, be 1:100-1:1600000 according to the serum gradient of experimental design evaluation, wherein when serum-dilution is to 1:51200, OD value is about 2 times of blank well, be judged as that the positive is tired, but OD value equaling blank value or the twice less than blank value substantially when diluting again, being judged as without tiring.A1-A2 and B1-B2 is negative control, can find out that the rabbit anti-serum of acquisition and other anaphylactogens do not have reactivity from numerical value; A3-A5 and B3-B5 is blank, can find out the rabbit anteserum before anaphylactogen and immunity, anti-all can not produce non-specific responding with two; The reaction in other holes is the specific reaction of the anti-beef anaphylactogen of beef anaphylactogen and rabbit.

(2) sero-fast affinity purification

Material and instrument

1, Protein-A sepharose CL-4B; Peristaltic pump; Centrifuge tube; Hydro-extractor; Filtrator; Glass column; Spectrophotometer;

2, TBS buffer solution: 6.06g Tris (50mM), 8.78g NaCl (150mM) and 0.5g sodium azide (0.05%) are dissolved in 1L distilled water, and regulate pH 7.4 with HCl;

3, neutralization buffer solution: 121.2g Tris (1M), 87.8g NaCl (1.5M), 0.37g EDTA (1mM) and 5g sodium azide (0.5%) are dissolved in 1L distilled water, and regulate pH 8.0 with HCl;

4, elution buffer solution (pH 2.7): be dissolved in 1L distilled water by 3.75g glycocoll (50mM), regulates pH 2.7 with HCl;

5, elution buffer solution (pH 1.9): be dissolved in 1L distilled water by 3.75g glycocoll (50mM), regulates pH 1.9 with HCl.

Operation steps

1, in Dewar bottle, isopyknic filler and TBS buffer solution are mixed, stir.Vacuumize about 15min to remove the bubble in filler, otherwise the capacity of the aeration pillar formed in post and separating effect.Protein-A sepharose CL-4B is slowly added in glass column, utilize pump control filling speed be 1ml/min ~ 2ml/min, avoid post to do, utilize 10 times to bed volume and through precooling TBS buffer solution balance pillar.

2, the antiserum prepared is put into the gathering that frozen water or 4 DEG C of refrigerators slowly thaw to avoid protein.The gathering occurred in protein course of defrosting is dissolved by 37 DEG C of preheatings.Adding solid sodium azide to concentration is 0.05%, 4 DEG C, the centrifugal 5min of 15000xg, and the antiserum shifting out clarification is filtered the unnecessary fat of removing again.

3, the antiserum TBS buffer solution after dissolving is diluted with the ratio of 1:5, then filter with filtrator.With the speed of 0.5ml per minute by antiserum on post, for ensureing the combination of antiserum and filler, need continuous upper prop 2 times also retains loading efflux.Add pH 2.7 elution buffer solution with after TBS buffer solution cleaning pillar to A 280nm<0.008, be eluted to all albumen with the speed of 0.5ml/min and all flow down.Be in charge of collection eluent with the 1.5ml EP pipe adding 100 μ L neutralization buffer solution, check the pH of eluent after mixing with pH test paper, if pH can utilize neutralization buffer to be adjusted to about pH 7.4 to prevent the sex change of antibody lower than 7;

In post, add 10ml, pH 1.9 elution buffer solution, collect eluent extremely as stated above 280nm<0.008;

Utilize the content of protein in each pipe of spectrophotometric determination.If protein concentration can add the glycerine of 10% to preserve lower than 0.5mg/ml, by after the IgG antibody packing of purifying 2 DEG C ~ 8 DEG C preservations;

With containing after the TBS buffer solution cleaning pillar of 0.05% sodium azide, pillar is stored in 2 DEG C ~ 8 DEG C environment.

(3) IgG antibody and people IgE Fc coupling

Material and instrument

1, people IgE Fc, is developed by Suzhou Haooubo Biopharmaceutical Co., Ltd., preserves with phosphate buffer;

2, papain digestion liquid (Papain lysate): 0.1M Tris, 2mM EDTA, pH 8.0.

3, Papain, Iodoacetamide available from Sigma.Protein-A is purchased from GE company.

4, coupling agent 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC), 2-imines thiophane (2-IT) is purchased from THERMO company, and the chemical reagent such as trishydroxymethylaminomethane (TRIS) all reach chemical pure;

3, G-25 gel column and Sephadex 200 gel-purified post are GE Products.

Operation steps

1, people source IgE is dissolved in Papain digestive juice, then uses papain digestion, then stop digestion reaction with Iodoacetamide.Then Fc. is extracted with Protein-A

2, get 1mg IgG antibody, and add the coupling agent 2-IT solution of 3 μ L 10mg/ml, room temperature leaves standstill 20min, adds the glycine solution of 10 μ L 0.1mol/L, leaves standstill 5min in room temperature.With G-25 gel column desalination, collect the rear antibody of activation, 5 DEG C save backup;

3, get the people IgE Fc solution of 1.5mg, add the SMCC solution 15 μ L of 5mg/ml, room temperature leaves standstill 30min, and with G-25 gel column desalination, collect the rear antibody of activation, 5 DEG C save backup;

4, the IgG antibody of above-mentioned activation mixed with the people IgE Fc of activation, under the condition of pH 7.3, leave standstill reaction 20h, purify conjugate with Sephadex 200 gel-purified post, obtain connector strong solution, 5 DEG C save backup;

5, the TRIS damping fluid of IgG-IgE Fc connector strong solution containing 0.5% bovine serum albumin(BSA), pH 8.0,0.1mol/L is diluted to 0.5 μ g/ml, obtains the positive serum of beef anaphylactogen.

(4) detect box by the specific allergen IgE antibody that Phadia company produces to measure the positive serum that embodiment 1 prepares, result is as table 2:

Table 2

Note: Phadia anaphylactogen IgE detects deciding grade and level: <0.35KU/L is negative; 0.35-0.69 is 1 grade of positive; 0.7-3.49 is 2 grades of positives; 3.5-17.49 be 3 grades of positives; 17.5-49.99 be 4 grades of positives; 50-99.99 is 5 grades of positives; >100KU/L is 6 grades of positives.

As seen from Table 2, the positive serum Quality Control sample of 3 batches (LOT131217, LOT140319, LOT140608), is determined as positive sample by Phadia anaphylactogen IgE detection kit, and is 5 grades of positive sample and strong positive.Can see from form 2, when Quality Control sample carries out the dilution of 3 times, 9 times respectively, the measured value of its Phadia also presents the similar multiple proportions of 3 times, 9 times and reduces ratio.Our sample of comprehensive explanation is successfully prepared, and can be used for the Quality Control of kit as sample.

Embodiment 2

(1) immunity of anaphylactogen and rabbit anti-serum titration

Material and instrument

1, anaphylactogen: anaphylactogen freeze-dried powder, anaphylactogen is willow;

2, adjuvant: Freund's complete adjuvant, purchased from sigma company, article No. (F5881); Incomplete Freund's adjuvant, purchased from sigma company, article No. (F5506);

3, animal: the healthy new zealand white rabbit selecting 3 (often kind of anaphylactogen immunity 3 rabbits) 2 monthly ages, body weight 1.5 ~ 2.0kg;

4, two resist: the goat anti-rabbit igg of AP mark;

5, consumptive material: three-way device, disposable syringe, pipettor etc.

Immunity step

1, immune anaphylactogen prepares: determine that freeze-dried powder dissolves the concentration of rear anaphylactogen with BCA albuminometry, first immunisation is got 0.05mg, 0.15mg and 0.2mg anaphylactogen PBS respectively and is diluted to 300 μ L, anaphylactogen is mixed with Freund's complete adjuvant 0.8:1 (volume ratio), blending manner is that mixing product must carry out immunity at once with pipettor piping and druming potpourri more than 10 times; The later immunity of second time is all get 0.05mg, 0.15mg and 0.2mg anaphylactogen PBS to be diluted to 500 μ L, mixes, the same first immunisation of all the other steps with not formula Freund's incomplete adjuvant 0.8:1 (volume ratio).

2, animal immune: 3 new zealand white rabbits bought are cultivated 1 week at Animal House, animal is conformed; By the opalescent mixture prepared, first immunisation 1ml syringe gets the foot-pad immunization that mixing 0.5ml carries out rabbit, and other immunity all adopt carries out leg and chest muscle injection, and the immunity cycle is 7 days.

3, sero-fast preparation: extract 2mL blood (as blank) prior to side auricular vein before every animal immune, carry out auricular vein before often carrying out immunity next time afterwards and get blood, for antiserum evaluation; Tiring in addition after reaching requirement adopts Culling heart blood method to obtain a large amount of immune blood; Get by the centrifugal 15min of 1000rpm after blood, collect antiserum; Antiserum packing is stored in-70 DEG C.

4, the mensuration of antiserum titre: adopt board-like chemiluminescence to evaluate antiserum, be buffered liquid with bag and anaphylactogen is diluted to 5 μ g/ml, every hole adds 100 μ L i.e. 0.5 μ g anaphylactogen except negative hole, 37 DEG C of bags are by 2h; Primary antibodie is the antiserum of gradient dilution, and two resist the goat anti-rabbit igg for AP mark, wherein negative control: the serum of same rabbit of primary antibodie for getting before immune anaphylactogen, equally with positive antiserum carries out gradient dilution; Blank: primary antibodie hatches the confining liquid not containing any serum; Wherein the doubling dilutions such as 1:200,1:400 pressed by serum.When antiserum titre reaches 1:100000, namely reach our requirement.

With antiserum evaluation result table 3 and table 4 after willow immunize rabbit:

Table 3

Table 4

Table 5

Number of animals	672	671	670
				Antiserum titre	1：100000	1：200000	1：100000

Note: above-mentioned data be immunity 4 times afterwards measure 3 rabbits antiserum titre;

Can find out from above table rabbit immunity 4 times after, all meet and tire and be greater than 1:100000, be 1:200 ~ 1:3200000 according to the serum gradient of experimental design evaluation, wherein when serum-dilution is to 1:100000,672,671 and 670RLU value to be greater than dilution gradient be about 2 times of the RLU value of the negative control of 1:200, be judged as that the positive is tired, but RLU value equaling blank value or the twice less than blank value substantially when carrying out large gradient dilution again, being judged as without tiring.

(2) sero-fast affinity purification

Material and instrument

1, instrument: immune affinity chromatographic column, peristaltic pump, centrifuge tube, hydro-extractor, filtrator, chromatographic column, spectrophotometer;

2, TBS equilibrating buffer: 6.06g Tris (50mM), 8.78g NaCl (150mM) and 0.5g sodium azide (0.05%) are dissolved in 1L distilled water, and regulate pH 7.4 with HCl;

3, high salt buffer solution: by 121.2g Tris (1M), 87.8g NaCl (1.5M), 0.37gEDTA (1mM) and 5g sodium azide (0.5%) are dissolved in 1L distilled water, and regulate pH 8.0 with HCl;

4, elution buffer solution: be dissolved in 1L distilled water by 3.75g glycocoll (50mM), regulates pH 2.6 with HCl;

5, other materials: 0.2M carbonate buffer solution (pH 9.5), 1.0mM HCl, 1.0M glycine solution, CNBr-actived Sepharose 4B-Cl (GE), post material storage liquid (0.1MPBS, containing 1% aminocaproic acid, pH 7.4).

Operation steps

1, immune affinity chromatographic column is prepared: be connected with Ago-Gel by the antigen that immunity uses, make affinity column.Be specially: specific anaphylactogen 0.2M carbonate buffer solution (pH 9.5) dissolved dilution is become 1.0mg/ml.Again with the Sepharose 4B-Cl (CNBr-actived Seph arose 4B-Cl) that 1.0mM HCL process has activated, 0.2M carbonate buffer solution (pH 9.5) is then used to be diluted to 1.0g/ml.Again the ratio of these two parts in 1:1 is mixed, at room temperature react 16 ~ 20 hours.Then centrifugal, collect supernatant.Survey the protein concentration of supernatant, then compare with the concentration of the anaphylactogen thrown at first and amount, calculated how much antigen and be connected on pillar.Remaining Sepharose4B-Cl puts into 1.0M glycine solution, and the amount of solution is Sepharose4B-Cl:1.0M glycine solution=1:1, reacts after 4 hours.Clean successively with 3 times of column volumes respectively with the HCl of 0.1M, 0.1M NaOH and 2M urea liquid, finally preserve by storage liquid.

2, in a reservoir isopyknic affinity column is fully mixed with the animal blood serum after ammonium persulfate process, jog 2h.Slowly added in glass column by potpourri, utilizing pump to control filling speed is 1ml/min ~ 2ml/min, avoids post to do, and utilizes 3 ~ 10 times to bed volume buffer solution balance pillar.

3, clean pillar to absorption value A after 280nm<0.008 with TBS buffer solution, then wash away non-specific binding albumen with high-salt buffer.Add pH 2.6 elution buffer solution, be eluted to absorption value A at 280nm<0.008 with the speed of 0.5ml/min.Be in charge of collection eluent with the 1.5ml EP pipe adding 100 μ L neutralization buffer solution, check the pH of eluent after mixing with pH test paper, if pH can utilize neutralization buffer to be adjusted to about pH 7.4 to prevent the sex change of antibody lower than 7;

Utilize the content of protein in each pipe of spectrophotometric determination.If protein concentration can add the glycerine of 10% to preserve lower than 0.5mg/ml, by after the IgG antibody packing of purifying 2 DEG C ~ 8 DEG C preservations;

With containing after the TBS buffer solution cleaning pillar of 0.05% sodium azide, pillar is stored in 2 DEG C ~ 8 DEG C environment.

(3) IgG antibody and people IgE Fc coupling

Material and instrument

1, people IgE Fc, is developed by Suzhou Haooubo Biopharmaceutical Co., Ltd., preserves with phosphate buffer;

2, papain digestion liquid (Papain lysate): 0.1M Tris, 2mM EDTA (pH 8.0).

3, Papain, available from Sigma; Iodoacetamide, available from Sigma; Protein-A is purchased from GE company;

4, coupling agent 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC), 2-imines thiophane (2-IT) is purchased from THERMO company, and the chemical reagent such as trishydroxymethylaminomethane (TRIS) all reach chemical pure;

5, G-25 gel column and Sephadex 200 gel-purified post are GE Products.

Operation steps

1, people IgE, purity is 95%, concentration is 1mg/ml, first use Papain digestive juice (being also lysate), pH=8.0 dialyses, then adding concentration is that Papain:IgE=100:1 (w/w) carries out digestion 30min, then in digestive juice, add Iodoacetamide cessation reaction, uses Protein A by IgEFc partial purification out.

2, get the IgG antibody solution of 1mg, add the SMCC solution 15 μ L of 5mg/ml, room temperature leaves standstill 30min, and with G-25 gel column except free SMCC, collect the rear antibody of activation, 4 DEG C save backup;

3, get 1.5mg people IgE Fc part, add the coupling agent 2-IT solution 3 μ L of 10mg/ml, room temperature leaves standstill 20min, and add the glycine solution 10 μ L of 0.1mol/L, room temperature leaves standstill 5min.With G-25 gel column except free 2-IT, collect the rear antibody of activation, 4 DEG C save backup;

4, the people IgE Fc of the IgG antibody of above-mentioned activation and activation part mix, under the condition of pH 7.3, leave standstill 20h, purify conjugate with Sephadex 200 gel-purified post, acquisition connector strong solution, 4 DEG C save backup;

5, the TRIS damping fluid of IgG-IgE Fc connector strong solution containing 0.5% bovine serum albumin(BSA), pH 8.0,0.1mol/L is diluted to 0.5 μ g/ml, obtains the positive serum of willow anaphylactogen.

(4) detect box by the specific allergen IgE antibody that Phadia company produces to measure the positive serum that embodiment 1 prepares, result is as table 6:

Table 6

Note: Phadia anaphylactogen IgE detects deciding grade and level: <0.35KU/L is negative; 0.35-0.69 is 1 grade of positive; 0.7-3.49 is 2 grades of positives; 3.5-17.49 be 3 grades of positives; 17.5-49.99 be 4 grades of positives; 50-99.99 is 5 grades of positives; >100KU/L is 6 grades of positives.

As seen from Table 6, the positive serum Quality Control sample of 3 batches (LOT140105, LOT140327, LOT140507), is determined as positive sample by Phadia anaphylactogen IgE detection kit, and is 5-6 level positive sample and strong positive.Can see from form in addition, after Quality Control sample doubling dilution, measured value is also doubling dilution.Our sample of comprehensive explanation is successfully prepared, and can be used for the Quality Control of kit as sample.

Above to invention has been detailed description; its object is to allow the personage being familiar with this art can understand content of the present invention and be implemented; can not limit the scope of the invention with this; the equivalence change that all Spirit Essences according to the present invention are done or modification, all should be encompassed in protection scope of the present invention.

Claims (10)

1. the method for preparing purified of an anaphylactogen positive serum, comprise, with anaphylactogen, immunity, the rear step obtaining sero-fast step and described antiserum is carried out to purifying acquisition positive serum of blood sampling carried out to healthy animal, it is characterized in that: the concrete grammar of described purifying is:

Affinity purification is carried out to described antiserum and obtains IgG antibody; Be after the coupling of 1:1 ~ 2 in mass ratio by described IgG antibody and people IgE Fc, IgG-IgE Fc connector strong solution is obtained through separation and purification, it is 0.5 ~ 1 μ g/ml that described IgG-IgE Fc connector strong solution is diluted to concentration, obtains described positive serum.

2. the method for preparing purified of anaphylactogen positive serum according to claim 1, is characterized in that: adopt agarose compatible medium or adopt immune affinity chromatographic column to carry out described affinity purification.

3. the method for preparing purified of anaphylactogen positive serum according to claim 2, is characterized in that: described agarose compatible medium is Protein-A sepharose CL-4B.

4. the method for preparing purified of anaphylactogen positive serum according to claim 2, is characterized in that: described immune affinity chromatographic column is the affinity column that described anaphylactogen and Ago-Gel connect into.

5. the method for preparing purified of the anaphylactogen positive serum according to claim 2 or 4, is characterized in that: described antiserum first after ammonium persulfate process, then adopts described immune affinity chromatographic column to carry out described affinity purification.

6. the method for preparing purified of anaphylactogen positive serum according to claim 1, it is characterized in that: people source IgE dissolves in papain digestion liquid by described people IgE Fc, use papain digestion again, to stop after digestion reaction with iodoacetamide again, extract with agarose compatible medium and obtain.

7. the method for preparing purified of anaphylactogen positive serum according to claim 1, it is characterized in that: described IgG antibody and described people IgE Fc, by after 2-imines thiophane coupling agent or the activation of 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester coupling agent, carry out described coupling under the condition of pH 7.2 ~ 7.4.

8. the method for preparing purified of anaphylactogen positive serum according to claim 7, it is characterized in that: the concentration of described 2-imines thiophane coupling agent is 9 ~ 11 mg/ml, the concentration of described 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester coupling agent is 4 ~ 6 mg/ml.

9. the method for preparing purified of anaphylactogen positive serum according to claim 1, is characterized in that: adopt Sephadex 200 gel-purified post to carry out described separation and purification.

10. the method for preparing purified of anaphylactogen positive serum according to claim 1, is characterized in that: adopt containing mass ratio be 0.4 ~ 0.6% bovine serum albumin(BSA), pH 7.5 ~ 8.5,0.09 ~ 0.11mol/L TRIS buffer described IgG-IgE Fc connector strong solution is carried out described dilution.