CN106442963B - A kind of method and its kit detecting the substance for causing anaphylactoid reaction - Google Patents
A kind of method and its kit detecting the substance for causing anaphylactoid reaction Download PDFInfo
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Abstract
The present invention relates to drug measurement techniques field, more particularly to a kind of method and its kit detecting the substance for causing anaphylactoid reaction.This method includes：By P815 cell inoculations in culture medium；The biomolecule that determinand is added in the medium and is specifically bound with IP1 and/or cAMP detects the concentration of IP1 and/or cAMP；According to the concentration of IP1 and/or cAMP, confirm whether determinand is the substance for causing anaphylactoid reaction.Detection method result provided by the invention is accurate and reliable, almost the same with traditional β hexosaminidase release rate laboratory test results, but remolding sensitivity β hexosaminidase release rate detection methods want high；The detection method of the present invention is simple and easy to do, quick, overcomes the deficiency of conventional method, can be applied to early stage rapid screening and the identification of the substance for causing anaphylactoid reaction, be with a wide range of applications.
Description
Technical field
The present invention relates to drug measurement techniques field, more particularly to a kind of method detecting the substance for causing anaphylactoid reaction
And its kit.
Background technology
Allergic reaction, that is, allergy is that the one kind occurred between exogenous antigen substance and internal antibody improper is exempted from
Epidemic disease is reacted, and is common one of adverse reaction.It can induce that there are many anaphylactoid substance, such as the albumen with comlete antigen
The macromolecular substances such as matter, polypeptide, polysaccharide；The smaller compound of other molecules can be used as haptens and is combined into vivo protein
Comlete antigen, so as to cause allergic reaction.It is many to be related to anaphylactoid allergin at present, occurrence frequency is higher medicine
Product (Chinese medicine and Western medicine), food, pollen, acarid etc..Acute allergic reaction includes the hypersensitivity of I types and anaphylactoid reaction.It is newest
Investigation shows 77% acute allergic reaction as anaphylactoid reaction, and anaphylactoid reaction incidence is super far above I types quick anti-
It answers.Anaphylactoid reaction is mediated without IgE, and initial contact drug may occur in which allergic symptom, be brought greatly to the life of the mankind
Harm.
In view of the harmfulness of anaphylactoid reaction, the frequent generation especially in field of medicaments, such as the allergy of traditional Chinese medicine injection
Reaction, makes the importance there is a growing awareness that early prevention.Wherein, the early stage of sensibiligen substance, quickly detection was to reduce class
Anaphylactoid important means.
Mast cell is the central hub for inducing anaphylactoid reaction, it directly results in a system in degranulation after by antigenic stimulus
The generation of row allergic symptom.Currently, the method for common vitro detection mast cell degranulation mainly has β-hexosaminidase
Method, trypsinlike enzyme method, histamine method, scanner uni transmission electron microscope etc..These method classics are stablized, but have respective limitation.
In order to detect corresponding index, mast cell loses degranulation under condition of living organism by various chemical fixations and color development treatment
Thus a series of biological informations also cause many detection errors, can not also carry out accurate quantification and dynamic observation.Therefore, how
Accurate detection class anaphylactogen becomes new research direction.
Research finds that during mast cell degranulation, the raising of calcium ion and adenosine cyclophosphate is active medium release
Precondition, this phenomenon was just found in 1973, but really made the live body dynamic monitoring of calcium ion and adenosine cyclophosphate
For evaluation index, the early stage rapid evaluation still nobody for being applied to class anaphylactogen attempts.
It is proposed in the patent of Patent No. CN102154434B, the anaphylaxis of substance is confirmed with calcium current detection method, it can be with
Sensibiligen is screened.But calcium current method is only applicable to the detection of attached cell, if using suspension or half suspension cell, can make
It is untrue to obtain detection data, it is unstable.Meanwhile calcium current detection can only be directed to the chemical combination for quickly being combined and being played a role with receptor
Object is not suitable for the slower compound of reaction.Accordingly, it is desirable to provide a kind of novel detection causes the side of the substance of anaphylactoid reaction
Method and kit.
Invention content
In view of this, the present invention provides a kind of methods and its kit of the substance for detecting and causing anaphylactoid reaction.It should
Detection method result is accurate and reliable, almost the same but sensitive with traditional β-hexosaminidase release rate laboratory test results
Degree is higher than β-hexosaminidase release rate detection method；The detection method of the present invention is simple and easy to do, quick, overcomes tradition
The deficiency of method can be applied to early stage rapid screening and the identification of the substance for causing anaphylactoid reaction, have a wide range of applications valence
Value.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of methods for the substance for detecting and causing anaphylactoid reaction, include the following steps：
Step a：By P815 cell inoculations in culture medium；
Step b：The biomolecule that determinand is added in the medium and is specifically bound with IP1 and/or cAMP, detection
The concentration of IP1 and/or cAMP；
Step c：According to the concentration of IP1 and/or cAMP, confirm whether determinand is the substance for causing anaphylactoid reaction.
In the present invention, it is tested using P815 cells, is come to substitute the detection of calcium current using 1- phosphoinositides (IP1)
Confirm the anaphylaxis of substance.IP1 detection reflections are second messenger's Isosorbide-5-Nitrae, the content of 5- InsP3s (IP3), when in sample
After LiCl is added, IP1 is no longer metabolized, but is accumulated in the cell.As the IP1 generated into the cell increases, free IP1 with it is anti-
Body, which combines, to be increased, and signal gradually weakens.The present invention establish it is a kind of it is simple and easy to do, reliable, by detecting IP1 in mast cell
Reflect the method for its degranulation situation with cAMP variations, and by compared with corresponding with conventional method, using C48/80 as positive drug
Object has carried out practical application to clinical different types of traditional Chinese medicine injection, has absolutely proved the quick of this method, sensitivity and can
By property.The method overcome the deficiencies of conventional method, can be applied to early stage rapid screening and the identification of class anaphylactogen, have extensive
Application value.
In embodiment provided by the invention, biomolecule is antibody in step b.
Preferably, the reagent that the concentration for detecting IP1 and/or cAMP in step b uses is based on the IP1 of HTRF technologies inspection
Test agent and/or cAMP detection reagents.
Preferably, P815 cells are the P815 cells of exponential phase.
Preferably, determinand is drug or mite.
In embodiment provided by the invention, determinand is drug injection.
In embodiment provided by the invention, determinand is traditional Chinese medicine.
In embodiment provided by the invention, determinand is selected from Shenmai injection, panax notoginseng saponins for injection, 'Mailuoning ' injection
Liquid, Floium Ginkgo, tanreqin injection or Reduning injection.
The present invention also provides a kind of kits for the substance for detecting and causing anaphylactoid reaction, including：With IP1 and/or
The biomolecule of cAMP specific bindings.
Preferably, biomolecule is antibody.
In embodiment provided by the invention, with the biomolecule that IP1 and/or cAMP are specifically bound be IP1 antibody and/
Or cAMP antibody.
Preferably, being the biomolecule by label with the IP1 and/or cAMP biomolecule specifically bound.Pass through
Label reads the concentration that signal obtains target substance.
In an embodiment of the present invention, the reagent of the concentration of detection IP1 and/or cAMP is the IP1 inspections based on HTRF technologies
Test agent or cAMP detection reagents.
In the present invention, using IP1 the and cAMP detection kits of HTRF technologies, under condition of living organism dynamic monitoring causing
The variation of P815 intracellular IP1 and cAMP, the standard judged using variation degree as anaphylactoid reaction under quick original material incentive.It should
Method is sensitive, quickly screened to sensibiligen under living cells state, overcomes former methodical deficiency, anti-in class allergy
The early stage that should occur can find potential threat, will be the screening of anaphylactogen anaphylaxis, Allergic skin test, environmental safety monitoring etc.
Field provides new method.
The present invention provides a kind of methods and its kit of the substance for detecting and causing anaphylactoid reaction.This method includes：
By P815 cell inoculations in culture medium；The biology that determinand is added in the medium and is specifically bound with IP1 and/or cAMP
Molecule detects the concentration of IP1 and/or cAMP；According to the concentration of IP1 and/or cAMP, confirm whether determinand is to cause class allergy
The substance of reaction.The present invention at least has one of following advantage：
1, detection method result provided by the invention is accurate and reliable, tests and examines with traditional β-hexosaminidase release rate
It is almost the same to survey result, overcomes the deficiency of conventional method, the early stage that can be applied to the substance for causing anaphylactoid reaction quickly sieves
It looks into and identifies, be with a wide range of applications；
2, the remolding sensitivity β-hexosaminidase release rate detection method of detection method provided by the invention wants high；
3, detection method of the invention is simple and easy to do, quick；
4, detection method is suitable for the detection of attached cell, is also applied for suspension or the detection of half suspension cell；
5, detection method is suitable for the detection of the slower compound of anaphylactoid reaction.
Description of the drawings
Fig. 1 shows the standard curve of IP1；
Fig. 2 shows the standard curve of cAMP.
Specific implementation mode
The invention discloses a kind of method and its kit of the substance for detecting and causing anaphylactoid reaction, people in the art
Member can use for reference present disclosure, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications
Apparent to those skilled in the art, they are considered as being included in the present invention.The method of the present invention and application
Be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope it is right
Method described herein and application are modified or suitably change and combine, to realize and apply the technology of the present invention.
Biomaterial used, examination in the method and its kit provided by the invention for detecting the substance for causing anaphylactoid reaction
Agent, instrument are available on the market.
With reference to embodiment, the present invention is further explained：
Embodiment 1
Experimental method：
1. material
Reagent：RPMI1640 culture mediums (GIBCO)；Fetal calf serum (Gibco), cAMP dynamic2kit (cisbio),
IP1Tb kit (cisbio), 384 orifice plates (Corning), Compound48/80 (Sigma, the U.S.), β-D- aminoglucose osamines
(Sigma), Shenmai injection (Hebei Shineway Pharmaceutical Co., Ltd, lot number：1401192), (Heilongjiang Province is precious for panax notoginseng saponins for injection
Treasured island medicine company limited liability company, lot number：14ka222), MAILUONING ZHUSHEYE (Jinting Pharmaceutical Co., Ltd. Nanjing Nanjing
Pharmaceutical factory, lot number：20140403), Floium Ginkgo (Heilongjiang Province Zhenbao Island medicine company limited liability company, lot number：B20140707),
Tanreqin injection (Shanghai Kaibao Pharmaceutical Co., Ltd, lot number：1502204), Reduning injection (Jiangsu Kang Yuan medicine companies
Limited liability company, lot number：20140722).
Cell：Mouse hypertrophy cell oncocyte P815 cells are purchased from National Laboratory cellular resources shared platform.
Instrument：Carbon dioxide incubator (Thermo, the U.S.), superclean bench (Suzhou purifies, China), calcium current detects work
It stands (FlexStation3, Molecular Devices).
When anaphylactoid reaction occurs for body, the phenomenon that having mast cell degranulation and discharge inflammatory mediator.P815 cells
It is mouse hypertrophy cell oncocyte, there is the response characteristic in body mast cell, frequently as the research object of anaphylactoid reaction.
Compound 48/80 (C48/80) is the polymer that N- methyl-p-methoxies phenyl ethylamine and formaldehyde condensation generate, and has promotion
The effect of mast cell degranulation, often using it as the positive drug of class allergic experiment.
2. method
2.1 cell culture
P815 cells RPMI1640 culture mediums (contain 10% fetal calf serum), in 37 DEG C, saturated humidity, 5%CO2Incubator
Middle culture, 3d passages are primary, and passage ratio is 1:3.
2.2HTRF detects IP1
The P815 cells for taking logarithmic phase to grow adjust cell according to detection reagent cassette method with Stimulate Buffer
Concentration is inoculated in 384 orifice plates, 4900/hole, sets up standard item group (acellular), negative control group (acellular), positive drug separately
Group, injection group and blank control group.Wherein standard item group according to kit illustrate setting 0,10.74,42.97,171.875,
687.5,2750,7 concentration of 11000nM do mark song；The Stimulate Buffer of same volume are added in negative control group.It is positive
Medicine group is with the C48/80 of final concentration of 0,10,20,40,80,160,320,640 μ g/mL；Clinical agent is respectively set in injection group
Amount and 2 times of clinical dosage holes, Shenmai injection SMZSY (final concentration is respectively 13.33ml/L, 26.66ml/L), " Xuesaitong Injection " injection
Liquid XSTZSY (2.22ml/L, 4.44ml/L), MAILUONING ZHUSHEYE MLNZSY (4.44ml/L, 8.88ml/L), Floium Ginkgo injection
Liquid SXNZSY (5.56ml/L, 11.12ml/L), tanreqin injection TRQZSY (4.44ml/L, 8.88ml/L), Redujing Granules injection
Liquid RDNZSY (4.44ml/L, 8.88ml/L)；The Stimulate Buffer of same volume are added in blank control wells, and incubator is incubated
After 1h, the lysis solution of 3 μ L are added in negative control hole, and 3 μ L IP1 reagent working solutions are added in remaining each hole, later, respectively
3 μ L IP1cryptate are added in hole, and after being incubated at room temperature 1h, setting detection parameters, upper machine testing are required according to kit.
Standard curve is made, formula is drawn up, according to formula, calculates the IP1 concentration in each hole.
2.3HTRF detects cAMP
The P815 cells in exponential phase are collected, cell concentration is adjusted, is inoculated in 384 orifice plates, 4900/hole,
Set up standard item group (acellular), negative control group (acellular), positive drug group, injection combination blank control group separately.Positive drug
Group be separately added into C48/80 solution (final concentration of 500 μ g/mL, 250 μ g/mL, 125 μ g/mL, 62.5 μ g/mL, 31.25 μ g/mL,
15.63 μ g/mL, 7.81 μ g/mL), injection group, SMZSY (final concentration is respectively 13.33ml/L, 26.66ml/L), XSTZSY
(2.22ml/L、4.44ml/L)、MLNZSY(4.44ml/L、8.88ml/L)、SXNZSY(5.56ml/L、11.12ml/L)、
Isometric PBS solution is added in TRQZSY (4.44ml/L, 8.88ml/L), RDNZSY (4.44ml/L, 8.88ml/L), control wells,
Isometric cAMP standard items are added in gauge orifice, set and are incubated 30min in incubator, are detected to specifications.
Standard curve is made, formula is drawn up, according to formula, calculates the cAMP concentration in each hole.
2.4 β-hexosaminidase release rates detect
The P815 cells for taking logarithmic phase to grow, by 5 × 105/ ml is inoculated in 96 orifice plates, per 200 μ l of hole, set 37 DEG C, 5%
CO2It is incubated overnight in incubator.300g, 5min are centrifuged, and abandon supernatant, and the various concentration that 100 μ l serum free mediums are prepared is added
C48/80 solution, it is respectively 200 μ g/ml, 100 μ g/ml, 50 μ g/ml to make its final concentration；Each injection group is according to concentrations above
Injection is added, isometric culture solution is added in blank control group, and the Triton X-100 that total enzyme hole is added isometric 0.5% are molten
Liquid, after being incubated 1h in incubator, 1000rpm centrifuges 10min, collects supernatant.Substrate is added in 96 hole elisa Plates per hole
Then 50 μ l of 1mmol/L beta-aminos hexose are added 50 μ l cell conditioned mediums, jiggle mixing, set 37 DEG C, 5%CO2Incubator is incubated
1h is educated, 0.1mol/L Na are added2CO3/NaHCO3200 μ l of buffer solution terminate reaction, the value of detection OD 405nm.According to formula
It calculates：
β-hexosaminidase release rate (%)=(sample well OD- zeroing hole OD)/(total enzyme hole OD- zeroing hole OD) ×
100%.
3. data processing
As a result it usesIt indicates, significant difference is compared with the t methods of inspection between group.
4. result
4.1 IP1 testing results
4.1.1 standard curve
The standard curve of IP1 is shown in Fig. 1.
4.1.2 the influence of C48/80 and variety classes traditional Chinese medicine injection to P815 cell intracellular IP1 concentration
The influence of 1 C48/80 of table and variety classes traditional Chinese medicine injection to P815 cell intracellular IP1 concentration
Note：* compared with Control, p≤0.05, * * are compared with Control, p≤0.01；
The results show that the C48/80 of 40 μ of μ g/mL~640 g/mL, can increase the concentration of P815 cell intracellulars IP1, with
Control groups compare, and difference has statistical significance (p≤0.05 or p≤0.01).Each injection group, the arteries and veins of 2 times of clinical dosages
Network injection for curing significantly increases the concentration of P815 cell intracellular IP1 concentration, and compared with Control groups, difference has statistics
Meaning (p≤0.05).
4.2 cAMP testing results
4.2.1 standard curve
The standard curve of cAMP is shown in Fig. 2.
4.2.2 the influence of C48/80 and variety classes traditional Chinese medicine injection to P815 cell intracellular cAMP concentration
The influence of 2 C48/80 of table and variety classes traditional Chinese medicine injection to P815 cell intracellular cAMP concentration
Note：* compared with Control, P≤0.05, * * are compared with Control, P≤0.01；
The results show that the C48/80 of 7.81 μ of μ g/mL~500 g/mL, can increase the concentration of P815 cell intracellulars cAMP,
Compared with Control groups, difference has statistical significance (p≤0.05 or p≤0.01).Each injection group, 2 times of clinical dosages
Shenmai injection significantly increases the concentration of P815 cell intracellular IP1 concentration, and compared with Control groups, difference has statistics
Meaning (p≤0.05).
4.3 β-hexosaminidase release rate testing result
Influence of the 3 variety classes traditional Chinese medicine injection of table to P815 cell β-hexosaminidase release rates
Note：* compared with Control, p≤0.05, * * are compared with Control, p≤0.01.
The results show that each traditional Chinese medicine injection of various concentration is apparent to P815 cell β-hexosaminidases release rate nothing
It influences.
In application example, the application causes fat de- of maxicell to clinical 6 kinds of traditional Chinese medicine injections with different detection methods
Grain effect is compared Journal of Sex Research.In dosage selection, according to the ratio of dosage and human body blood volume, divide clinical administration agent
Amount and 2 times of clinical dosages.As a result, it has been found that intracellular IP1 and cAMP detection methods and traditional β-hexosaminidase release rate are real
It is almost the same to test testing result, but remolding sensitivity β-hexosaminidase release rate detection method wants high.Conjecture may be by
It is a kind of biochemistry detection method in β-hexosaminidase detection method, carrys out definitive result finally by colorimetric, detection limit may ratio
Higher, less amount of degranulation reaction will not be detected by it.And the detection of intracellular IP1 and cAMP is single due to directly detecting
The variation of cell, and HTRF detections is purpose product accumulated result, therefore sensitivity higher.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of method detecting the substance for causing anaphylactoid reaction, which is characterized in that include the following steps：
Step a：By P815 cell inoculations in culture medium；
Step b：Determinand and the antibody with IP1 specific bindings are added in the culture medium, using based on HTRF technologies
IP1 detection reagents detect the concentration of IP1；
Step c：According to the concentration of IP1, confirm whether the determinand is the substance for causing anaphylactoid reaction.
2. according to the method described in claim 1, it is characterized in that, the P815 cells are the P815 cells of exponential phase.
3. according to the method described in claim 1, it is characterized in that, the determinand is drug or mite.
4. according to the method described in claim 1, it is characterized in that, the determinand is drug injection.
5. according to the method described in claim 1, it is characterized in that, the determinand is traditional Chinese medicine.
6. the method according to any one of claims 1 to 5, it is characterized in that, the determinand be selected from Shenmai injection,
Panax notoginseng saponins for injection, MAILUONING ZHUSHEYE, Floium Ginkgo, tanreqin injection or Reduning injection.
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Citations (3)
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| CN1549721A (en) * | 2001-02-27 | 2004-11-24 | ����Ъ����˷���� �ѧ�о��� | Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation |
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| CN1549721A (en) * | 2001-02-27 | 2004-11-24 | ����Ъ����˷���� �ѧ�о��� | Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation |
| CN102154434A (en) * | 2010-12-13 | 2011-08-17 | 中国中医科学院医学实验中心 | Method for detecting materials causing anaphylactoid reaction |
| CN103344773A (en) * | 2013-06-19 | 2013-10-09 | 天津美德太平洋科技有限公司 | Reagent, method and kit for detection of biological activity of glucagon-like peptide-1 (GLP-1) |
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