CN107860907A - Application of the albumen of Pru p 3 in wormwood artemisia pollen correlation peach allergy detection kit is prepared - Google Patents
Application of the albumen of Pru p 3 in wormwood artemisia pollen correlation peach allergy detection kit is prepared Download PDFInfo
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- CN107860907A CN107860907A CN201711065498.XA CN201711065498A CN107860907A CN 107860907 A CN107860907 A CN 107860907A CN 201711065498 A CN201711065498 A CN 201711065498A CN 107860907 A CN107860907 A CN 107860907A
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- peach
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
Abstract
The invention provides application of the albumen of Pru p 3 in wormwood artemisia pollen correlation peach allergy detection kit is prepared.The kit prepared using the albumen of Pru p 3, joint Basohil activation tests (basophil activation test, BAT) it is used to predict wormwood artemisia pollen correlation peach allergic reaction, its prediction effect is better than the Detection results that clinical the most frequently used external detection method at present is peach and its component sIgE, peach sensitization and peach autopath caused by it can effectively distinguish cross reaction.The detection that the kit prepared using the albumen of Pru p 3 is carried out is the optimal external detection method occurred for predicting the allergic reaction of wormwood artemisia pollen correlation peach, has optimal sensitivity and specificity.
Description
Technical field
The present invention relates to detection reagent preparation field, and in particular to the albumen of Pru p 3 is preparing wormwood artemisia pollen correlation peach allergy
Application in detection kit.
Background technology
The goldstandard of food hypersenstivity diagnosis is double-blind placebo-controlled control Food provocative test, but the inspection needs to cure in training
It is raw to instruct lower progress, higher is required to hospital equipment and operator quality, has time-consuming, cost high and severe allergy occurs
The characteristics of reacting risk, therefore clinically not conventional use.At present, clinically diagnosis food hypersenstivity is based primarily upon feed sensitivity
Medical history and Skin-test/Specificity IgE (specific IgE, the sIgE) detection of acute allergic reaction occur after food.
Skin-test, because being influenceed by operator's technology and agents useful for same, specificity and sensitiveness are limited, and it is anaphylactoid generation to be present
Risk, make its application in clinic significantly limited.Serum sIgE detections are easy to operate, safety, are widely used to clinic, wrap
Radio-allergo-sorbent test, EUSA and CAP total IgEs (CAP system) are included, wherein
CAP system are the goldstandards of detection serum sIgE antibody generally acknowledged at present.But the sIgE of this method detection is in serum
Free antibody, only when this antibody is combined with basophilic granulocyte and (or) mast cell, while run into anaphylactogen formed it is " anti-
Former bridge " competence exertion biological effect, i.e. serum sIgE contents can not reflect the true pathogenic effects of this antibody in vivo.Cause
This, the sIgE positives can only reflect that body is in sensitization, can not effectively assess the risk of allergic reaction generation.In addition, blood
Clear sIgE detections are vulnerable to the interference of cross reaction, and specificity is also affected.
In recent years, application of the component detection in food hypersenstivity increasingly attracts attention.Because it is without using the change of routine
Answer former crude extract, but it is pure cause the allergen protein of sensitization, therefore improve the specificity of Allergic skin test.Abroad,
The peanut allergy incidence of disease is higher, and research of the component detection in the disease is more.Research finds that Ara h 2 are the main sensitization eggs of peanut
In vain, prompt the component sensitization it to feed peanut and allergic symptom easily occurs.Although component is detected in peanut allergy diagnosis very
It is valuable, but it is still limited at present to the data of other food allergens components.
Wormwood artemisia pollen is the northern area of China summer and autumn most important pollen.Except with typical seasonal rhinitis and/or
Outside SOA, diet diversity can occur after the wormwood artemisia pollinosis patients diet's veterinary antibiotics of part.Wormwood artemisia pollen concerned foodstuff
In allergy, peach is the most common food that causes allergic reaction.Due to the cross reaction between wormwood artemisia pollen and food allergens, a lot
The concerned foodstuff sIgE positives, i.e. food sensitization can occur in wormwood artemisia pollinosis patient's body, but feed concerned foodstuff without clinic
Allergic symptom.Detect serum food sIgE, can not effective district share the meal the patient of thing sensitization and real allergy.It is in addition, of the invention
Inventor early-stage Study find PLTP (lipid transfer protein, LTP) be participate in wormwood artemisia pollen phase
Close the intersection allergen of diet diversity.Due to Art v 3 (wormwood artemisia pollen LTP) and the main albumen of Allergen Pru p 3 of peach
There is cross reaction in (peach LTP), for assessing wormwood artemisia pollinosis patient the anaphylactoid meaning of peach occurs for the detection Pru p 3sIgE positives
Justice will decrease.Therefore, for clinician, there is an urgent need to a kind of not only safety but also accurately and reliably vitro detection reagent
And method, come that the related diet diversity of wormwood artemisia pollen can occur for aid forecasting which wormwood artemisia pollinosis patient.
Basophilic granulocyte and mast cell are the important effect cells for participating in I type hypersensitivity, and its surface expression is high close
With the IgE of power Fc receptor seqs (Fc ε RI).Anaphylactogen and basophilic granulocyte and mast cell surface in sensitization
IgE is combined, and activates basophilic granulocyte by bridge linking effect and mast cell, cell degranulation simultaneously discharge histamine, leukotriene etc.
Inflammatory mediator, so as to cause allergic symptom.In recent years, people are detected using flow cytometry (flowcytometry, FCM)
(basophil activation are tested in the change of basophil after birth surface active mark, i.e. Basohil activation
Test, BAT), the research report in wormwood artemisia pollen concerned foodstuff allergy on BAT is there is no at present.
The content of the invention
It is an object of the invention to provide the albumen of Pru p 3 answering in wormwood artemisia pollen correlation peach allergy detection kit is prepared
With the kit of preparation can be used for peach sensitization and peach autopath caused by distinguishing cross reaction.
To achieve the above object, the present invention use following technical scheme for：
Application of the albumen of Pru p 3 in wormwood artemisia pollen correlation peach allergy detection kit is prepared.
Wormwood artemisia pollen correlation peach allergy detection kit as described above, including the main albumen of allergic protein Pru p 3 of peach.
Wormwood artemisia pollen correlation peach allergy detection kit as described above, the working concentration of the albumen of Pru p 3 for 10~
50ng/ml。
Wormwood artemisia pollen correlation peach allergy detection kit as described above, in addition to stimulate buffer solution, stimulate Quality Control, be anti-
CD63-FITC and anti-CCR3-PE premix reagent, erythrocyte cracked liquid.
Wormwood artemisia pollen correlation peach allergy detection kit as described above, it is preferable that in the stimulation buffer solution containing interleukin-
3, the working concentration of the interleukin-3 is 3~6ng/ml, and the buffer solution is PBS；
Resist in the stimulation Quality Control containing working concentration for the monoclonal of the anti-high-affinity IgE acceptors of 0.5~12.5ug/ml
Body, as positive control；
The anti-CD 6 3-FITC and anti-CCR3-PE premix reagent moderate resistance CD63-FITC concentration is 0.1~1.0mg/
Ml, the anti-CCR3-PE concentration are 5~30 μ g/ml；
Contain NH in the erythrocyte cracked liquid4Cl 100~150mmol/L, Tris 10~30mmol/L, pH7.0~
7.4。
Wormwood artemisia pollen correlation peach allergy detection kit as described above, it is preferable that the working concentration of the albumen of Pru p 3
For 25ng/ml, the working concentration of the interleukin-3 is 4.5ng/ml；
Monoclonal antibody containing the anti-high-affinity IgE acceptors that working concentration is 2.5 μ g/ml in the stimulation Quality Control；
The concentration of the anti-CD 6 3-FITC is 0.5mg/ml, and the concentration of the anti-CCR3-PE is 10 μ g/ml；
The NH4Cl is 139.6mmol/L, and the Tris is 16.96mmol/L, pH7.2.
The beneficial effects of the present invention are：
The kit prepared in the present invention using the albumen of Pru p 3, joint Basohil activation experiment (BAT) are used for
Predict wormwood artemisia pollen correlation peach allergic reaction, its prediction effect better than the most frequently used external detection method clinical at present, i.e., peach and its
Component sIgE Detection results, peach sensitization and peach autopath caused by it can effectively distinguish cross reaction.Using the eggs of Pru p 3
The detection that the kit prepared in vain is carried out is the optimal external detection method occurred for predicting the allergic reaction of wormwood artemisia pollen correlation peach,
With optimal sensitivity and specificity.
Brief description of the drawings
Fig. 1 is that cell divides group on FCS/SSC histograms in embodiment 2.
Fig. 2 is selection basophilic granulocyte CCR3pos/SSClow。
The basophilic granulocyte of wormwood artemisia pollen correlation peach allergy group is lived when Fig. 3 is in embodiment 2 using Pru p 3 as stimulant
The result of change.
The result of the Basohil activation of peach sensitization group when Fig. 4 is in embodiment 2 using Pru p 3 as stimulant.
The knot of the Basohil activation of Normal group when Fig. 5 is in embodiment 2 using Pru p 3 as stimulant
Fruit.
Fig. 6 is the ROC curve in embodiment 2.
Embodiment
Stimulant of the present invention using the main allergic protein Pru p 3 of peach as activation basophilic granulocyte, it was demonstrated that Pru p 3
Peach sensitization and peach autopath caused by albumen joint BAT can distinguish cross reaction are anti-in prediction wormwood artemisia pollen correlation peach allergy
Specificity and sensitiveness in answering detect better than peach sIgE and component.
Two Specific markers commonly used in BAT clinical researches at present are CD63 and CD203c.Wherein, CD63 is also known as
Lysosomal associated membrane glycoprotein 3 (LAMP-3), it is a four cross-film superfamily albumen (transmembrane 4super
Family, TM4SF), various kinds of cell surface can be expressed in, such as monocyte, blood platelet and basophilic granulocyte.When basophilla grain
When cell is in quiescent condition, CD63 is only expressed in intracellular particle, is seldom expressed in film surface；And in state of activation, it is thermophilic
Transposition occurs for alkaline granulocyte degranulation, and CD63 is expressed in cell membrane surface with cell membrane fusion and height, turns into the basophilic of activation
The Specific marker of property granulocyte.What the present invention detected is basophilic granulocyte surface C D63 expressions, calculates CD63 sun
The percentage of the basophilic granulocyte of property is as Basohil activation rate.
Following examples are used to further illustrate the present invention, but should not be construed as limiting the invention.Without departing substantially from this
On the premise of spirit and essence, modification or replacement made for the present invention, scope of the invention is belonged to.
Embodiment 1
The heparin anti-coagulating of subject is gathered, taking 50ul patient whole blood's samples to sequentially add 100ul stimulates buffer solution, 50ul
Corresponding stimulant, after mixing, 20ul fluorescence antibodies reagent (anti-CD 6 3-FITC and anti-CCR3-PE premixs reagent) is added, softly
Mixing, 37 DEG C of water-baths are incubated 15 minutes.The erythrocyte cracked liquid of 2ml preheatings (18-28 DEG C), soft mixing, in 18-28 are added afterwards
Lucifuge is incubated 5-10 minutes under the conditions of DEG C.Centrifugal force 300g, centrifuge 5 minutes, discarding supernatant, resuspension cell, on flow cytometer
Machine testing.50000-100000 leucocyte is collected, it is white thin on forward angle light scatter (FCS)/side scatter (SSC) histogram
Born of the same parents are divided into three groups (lymphocyte, monocyte and granulocytes), in side scatter low value (SSClow) region finds whole basophilics
Property granulocyte CCR3posGroup, collect basophilic granulocyte quantity>300.Calculate basophilla grain of the CD63 positive cells in acquisition
Percentage in cell, as Basohil activation rate.
For every kind of allergen, the optium concentration for determining Basohil activation is very important.Therefore, this hair
Five concentration gradients (1ng/ml-10 μ g/ that stimulant is 10 times of thinner ratios of peach allergen (i.e. peach total protein) are have selected in bright
Ml) stimulated in vitro basophilic granulocyte.During using peach total protein as stimulant, Basohil activation ratio is in concentration dependant
Relation with increase, plateau is entered when allergic effect original content increases to 100ng/ml.Therefore in the present invention, peach allergen Activated in Vitro is thermophilic
The optium concentration of alkaline granulocyte is 100ng/ml.The present inventor have selected stimulant as 3 albumen of Pru p, 5 times of dilutions
Three concentration gradient (1ng/ml-25ng/ml) stimulated in vitro basophilic granulocytes of ratio, it is 25ng/ml to determine optium concentration.
The present inventor compares 3 groups of peach allergy, peach sensitization and normal control patient's basophilic granulocytes in Pru p
Activation levels under the stimulation of 3 albumen.Research shows, with the albumen of Pru p 3 (25ng/ml) stimulated in vitro basophilic granulocyte, peach
Autopath's Basohil activation degree is apparently higher than peach sensitized recipients and normal healthy controls (P<0.01).The invention of the present invention
People determines using Pru p 3 as stimulant that Basohil activation Shuai≤15% is as prediction wormwood artemisia pollen phase through many experiments
Close the cutoff values that peach allergic reaction occurs.It is peach and its component Pru p with clinical the most frequently used external detection method at present
3sIgE testing result compares, and the BAT using Pru p 3 as stimulant predicts that peach allergic reaction occurs to have highest accurate
Exactness, it is external detection method optimal at present.
In addition, the peripheral blood that BAT can take less than 0.1ml detects 150-2000 basophilic granulocyte in allergenic effect
Under Activation, therefore specimen amount is few needed for clinical detection, and detection time is short.Meanwhile BAT is extremely approx simulated in vitro
The pathogenic process of I type hypersensitivity, i.e., excite lower basophilic granulocyte that activation, degranulation occurs in anaphylactogen, and using stream
Formula cell art, the basophilic granulocyte quantity of quantitative analysis activation, there is important value to prediction anaphylactia.
Embodiment 2
The anaphylactoid detection of wormwood artemisia pollen correlation peach is predicted for the albumen of the Pru p 3 joint BAT established in embodiment 1,
Specifically detected in the present embodiment, select wormwood artemisia pollen correlation peach allergy (n=28), peach sensitization (n=23) and normal control
(n=8) 3 groups of patients, collection heparin anti-coagulating are stand-by.Inclusion criteria is as follows：Wormwood artemisia pollen correlation peach autopath：Wormwood artemisia pollinosis disease
Shape, at the same have clearly feed peach occur in 2 hours whole skin wheal sample fash, mucous membrane of mouth oedema, expiratory dyspnea, nausea,
Stomachache, or even the medical history of anaphylactic shock, peach sIgE >=3.5kUA/L and/or peach skin prick test wheal diameter >=
3mm；Peach sensitized recipients：Wormwood artemisia pollinosis symptom, while peach sIgE is positive, but peach is fed without Clinical allergy symptoms；Normal control：Nothing
Anaphylactia medical history.The BAT allergen stimulants of use：Peach total protein (extracting method is as follows)；The main sensitization of peach of purifying
Albumen Pru p 3 (are purchased from Alpco companies of the U.S.).In each reaction system, using stimulate buffer solution (containing interleukin-3) as
Negative control, with the monoclonal antibody (anti-FC ε RI, purchased from Alpco companies of the U.S.) of anti-high-affinity IgE acceptors as sun
Property control.
Pru p 3 combine the BAT prediction anaphylactoid detections of wormwood artemisia pollen correlation peach, comprise the following steps that：
(1) heparin anti-coagulating is gathered, mixes blood sample.In the sample tube of each patient add 100ul stimulate buffer solution and
50 μ l patient whole blood's samples, do 4 Duplicate Samples and be used for step (2).
(2) each patient treats to be separately added into the 50 corresponding stimulants of μ l in test tube：Stimulation buffer solution (negative control),
Anti-FC ε RI (positive control), Pru p 3 (working concentration 25ng/ml), peach allergen (i.e. peach total protein 100ng/ml), gently
Soft mixing.Wherein, peach allergen is as a comparison case.
(3) 20ul fluorescence antibodies reagent (anti-CD 6 3-FITC and anti-CCR3- are separately added into the above-mentioned pipe added with stimulant
PE premixes reagent) softly mixing, lid is covered, 37 DEG C of water-baths are incubated 15 minutes.
(4) erythrocyte cracked liquid of 2ml preheatings (18-28 DEG C), soft mixing, under the conditions of 18-28 DEG C are often added in pipe
Lucifuge is incubated 5-10 minutes.
(5) centrifugal force 300g, centrifuge 5 minutes, abandon supernatant.
(6) cell is resuspended in 600-800 μ l PBSs, machine testing on flow cytometer, is collected into data use
FloMax software analysis.
Wherein, peach total protein is obtained using following extracting method：
1. configure protein extract：Sequentially added in 10mmol/L PBS (PH 7) 2% crospovidone,
2mmol/L disodium ethylene diamine tetraacetates, 10mmol/L second diyl dithiocarbamates.
2. fresh peach skin is taken, by 4：1 (wt/vol) is added in above-mentioned protein extract, and 4 DEG C of temperature persistently stirs 30 points
Clock.Centrifugal force 15000g, 4 DEG C of temperature centrifuge 30 minutes.
3. the supernatant after centrifugation passes through 0.2 μm of membrane filtration.
4. the peach protein allergen immersion liquid after filtering uses PBS 24h.
It is 325 μ g/ml that 5.Bradford methods, which detect its protein concentration,；
Stimulate buffer solution：Containing interleukin-3, working concentration 4.5ng/ml；
Peach allergen (100ng/ml)：Using PBS solution；
Pru p 3(25ng/ml)：Using PBS solution；
Fluorescence antibody reagent：Anti-CD 6 3-FITC and anti-CCR3-PE premixs reagent moderate resistance CD63-FITC concentration is
0.5mg/ml, anti-CCR3-PE concentration are 10ug/ml, solution PBS, PH 7.4；
Erythrocyte cracked liquid：Containing NH4Cl 139.6mmol/L, Tris 16.96mmol/L, PH7.2；
Testing result is as follows：By taking wormwood artemisia pollen correlation peach allergy group as an example, 50000-100000 leucocyte is 1. collected, preceding
Leucocyte is divided into three groups (lymphocyte, monocyte and grain is thin on to angle scattering (FCS)/side scatter (SSC) histogram
Born of the same parents), as shown in Figure 1.
2. door 1 (R1) is set, in side scatter low value (SSClow) region finds whole basophilic granulocyte CCR3pos
Group, as a result as shown in Fig. 2 the basophilic granulocyte quantity collected>300.
3. calculating CD63 positive cells, (FITC highlights fluorescence；Q2) hundred in the basophilic granulocyte obtained in R1 settings
Divide ratio, Basohil activation rate when shown in Fig. 3 is using Pru p 3 as stimulant.
By taking peach sensitization group as an example, step with it is above-mentioned 1. -3., as a result as shown in figure 4, the group is used as stimulant using Pru p 3
When Basohil activation rate (<15%).
Exemplified by healthy group, step with it is above-mentioned 1. -3., as shown in figure 5, being Normal group with Pru p 3 for healthy group
Basohil activation rate during as stimulant (<15%).
Comparative example：
Using ImmunoCAP methods (goldstandard of generally acknowledged sIgE detections at present) to above-mentioned 3 groups of patient peaches and its main cause
Quick albumen Pru p 3sIgE levels are detected and (can use Phadia, Thermo Fisher Scientific reagents are carried out),
SIgE >=0.35kUA/L is the positive.Peach sIgE, Pru p 3sIgE refer to three groups of patient peaches and Pru p 3sIgE detection water respectively
Flat, BAT- peaches, BAT-Pru p 3 refer to using peach total protein and Pru p 3 respectively is used as stimulant Basohil activation rate, knot
Fruit is shown in Table 1.Data are represented with median (scope) in table 1, the results showed that above-mentioned four kinds of detection methods are respectively adopted, peach allergy is suffered from
The testing result of person is above peach sensitized recipients (P<0.01), illustrate that BAT testing results using Pru p 3 as stimulant can be with
Distinguish peach sensitized recipients and peach autopath.
The BAT of table 1 can distinguish wormwood artemisia pollen correlation peach allergy and peach sensitization
Above-mentioned four kinds of Allergic skin test methods (peach sIgE, Pru p 3sIgE, BAT- peaches are further analyzed using ROC curve
To BAT-Pru p 3) acted in the related peach anaphylodiagnosis of wormwood artemisia pollen.TG-AUC (AUC) shows the detection method pre-
The value surveyed in peach allergic reaction.As a result show, the BAT curves of stimulant are used as using the main allergic protein Pru p 3 of peach
Lower area is maximum (see Fig. 6).Statistical analysis is carried out using SPSS 17.0 (SPSS I, Chicago, IL, USA), determines Pru
Optimal cutoff values of the p 3 under 25ng/ml concentration, occurred with the prediction peach allergic reaction of this index, its susceptibility is 95%, special
Different degree 95%, positive predicted value and negative predicted value are 92%；BAT using 100ng/ml peaches total protein as stimulant is sensitive
Degree is high, but specificity is relatively low；The specificity of peach and Pru p 3sIgE diagnosis is minimum, only 67% (result is specifically shown in Table 2).Cause
This, BAT using Pru p 3 as stimulant predicts that peach allergic reaction occurs to have the highest degree of accuracy, is current optimal
External detection method.
The value of the peach of table 2, Pru p 3sIgE and BAT in the related peach allergic reaction of prediction wormwood artemisia pollen
Above experimental result is shown, with the albumen stimulated in vitro basophilic granulocytes of Pru p 3, peach autopath's basophilla grain
Cell activation degree is apparently higher than peach sensitized recipients and normal healthy controls (P<0.01).Peach and its main cause are compared using ROC curve
Quick albumen Pru p 3sIgE detections, BAT are acted in wormwood artemisia pollen correlation peach anaphylodiagnosis, and ROC curve is as shown in fig. 6, with peach master
Want allergic protein Pru p 3 maximum as the BAT TG-AUCs of stimulant.Determine the albumen of Pru p 3 under 25ng/ml concentration
Optimal cutoff values, occurred with the prediction peach allergic reaction of this index, its sensitivity and specificity are all remarkably higher than current clinic
The most frequently used external detection method, i.e. peach and its component Pru p 3sIgE detect.Therefore, the albumen of Pru p 3 joint BAT is mesh
The preceding prediction anaphylactoid optimal external detection method of wormwood artemisia pollen correlation peach.
Claims (6)
- Application of the albumen of 1.Pru p 3 in wormwood artemisia pollen correlation peach allergy detection kit is prepared.
- 2. a kind of wormwood artemisia pollen correlation peach allergy detection kit, it is characterised in that including the albumen of Pru p 3.
- 3. wormwood artemisia pollen correlation peach allergy detection kit as claimed in claim 2, it is characterised in that the albumen of Pru p 3 Working concentration be 10~50ng/ml, its buffer solution is PBS.
- 4. wormwood artemisia pollen correlation peach allergy detection kit as claimed in claim 2, in addition to stimulate buffer solution, stimulate Quality Control, Anti-CD 6 3-FITC and anti-CCR3-PE premix reagent, erythrocyte cracked liquid.
- 5. wormwood artemisia pollen correlation peach allergy detection kit as claimed in claim 4, described stimulate in buffer solution contains working concentration For 3~6ng/ml interleukin-3s；Containing the monoclonal antibody that working concentration is the anti-high-affinity IgE acceptors of 0.5~12.5 μ g/ml in the stimulation Quality Control, make For positive control；The anti-CD 6 3-FITC and anti-CCR3-PE premix reagent moderate resistance CD63-FITC concentration for 0.1~ 1.0mg/ml, anti-CCR3-PE concentration are 5~30 μ g/ml；Contain NH in the erythrocyte cracked liquid4Cl 10~30mmol/L of 100~150mmol/L, Tris, pH7.0~7.4.
- 6. wormwood artemisia pollen correlation peach allergy detection kit as claimed in claim 5, it is characterised in that the albumen of Pru p 3 Working concentration be 25ng/ml, the working concentration of the interleukin-3 is 4.5ng/ml；Monoclonal antibody containing the anti-high-affinity IgE acceptors that working concentration is 2.5 μ g/ml in the stimulation Quality Control；The concentration of the anti-CD 6 3-FITC is 0.5mg/ml, and the concentration of the anti-CCR3-PE is 10 μ g/ml；The NH4Cl is 139.6mmol/L, and the Tris is 16.96mmol/L, pH7.2.
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| US20100221756A1 (en) * | 2007-09-11 | 2010-09-02 | Buhlmann Laboratories Ag | Allergy test based on flow cytometric analysis |
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