CN108445227A - A kind of method of anaphylactogen in vitro detection food - Google Patents
A kind of method of anaphylactogen in vitro detection food Download PDFInfo
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- CN108445227A CN108445227A CN201810204457.2A CN201810204457A CN108445227A CN 108445227 A CN108445227 A CN 108445227A CN 201810204457 A CN201810204457 A CN 201810204457A CN 108445227 A CN108445227 A CN 108445227A
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- ileum
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- anaphylactogen
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- 206010070834 Sensitisation Diseases 0.000 claims abstract description 18
- 241000700199 Cavia porcellus Species 0.000 claims abstract description 16
- 230000008313 sensitization Effects 0.000 claims abstract description 16
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- 238000011017 operating method Methods 0.000 claims description 2
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Classifications
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01L—MEASURING FORCE, STRESS, TORQUE, WORK, MECHANICAL POWER, MECHANICAL EFFICIENCY, OR FLUID PRESSURE
- G01L5/00—Apparatus for, or methods of, measuring force, work, mechanical power, or torque, specially adapted for specific purposes
- G01L5/04—Apparatus for, or methods of, measuring force, work, mechanical power, or torque, specially adapted for specific purposes for measuring tension in flexible members, e.g. ropes, cables, wires, threads, belts or bands
- G01L5/047—Specific indicating or recording arrangements, e.g. for remote indication, for indicating overload or underload
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01L—MEASURING FORCE, STRESS, TORQUE, WORK, MECHANICAL POWER, MECHANICAL EFFICIENCY, OR FLUID PRESSURE
- G01L5/00—Apparatus for, or methods of, measuring force, work, mechanical power, or torque, specially adapted for specific purposes
- G01L5/04—Apparatus for, or methods of, measuring force, work, mechanical power, or torque, specially adapted for specific purposes for measuring tension in flexible members, e.g. ropes, cables, wires, threads, belts or bands
- G01L5/10—Apparatus for, or methods of, measuring force, work, mechanical power, or torque, specially adapted for specific purposes for measuring tension in flexible members, e.g. ropes, cables, wires, threads, belts or bands using electrical means
- G01L5/106—Apparatus for, or methods of, measuring force, work, mechanical power, or torque, specially adapted for specific purposes for measuring tension in flexible members, e.g. ropes, cables, wires, threads, belts or bands using electrical means for measuring a reaction force applied on a cantilever beam
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/76—Assays involving albumins other than in routine use for blocking surfaces or for anchoring haptens during immunisation
- G01N2333/77—Ovalbumin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
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Abstract
The invention discloses a kind of methods of anaphylactogen in vitro detection food.From the aspect of the week old of cavy, gender, allergen concentration and detection time four, using guinea pig ileum as research object, receive allergy primary stimuli after-contraction tension using tonotransducer combination physiological signal recorder detection guinea pig ileum, shows that guinea pig ileum receives the sensitization that anaphylactogen in food can be evaluated in the physiological signal value reflected after sensitization primary stimuli.The method of the present invention testing cost is low, reusable, can accurately reflect allergen sensitisation ability, can be used for evaluating the sensitization of raw-food material, reduces the risk of food hypersenstivity.
Description
Technical field
The present invention relates to technical field of food safety detection, specifically, being related to anaphylactogen in a kind of vitro detection food
Method.
Background technology
Food hypersenstivity is one kind of anaphylactia, is likely to become the inducement of certain severe allergic complication.According to prevalence
Disease learns investigation, and allergy incidence is in the trend risen year by year in recent years, and there is 3%~4% adult human hair in developed country every year
The incidence of uncooked food allergy, children and infant are 5%.China's anaphylactia incidence is equally in becoming for rapid growth
Gesture is paid much attention to by hygiene department.Food hypersenstivity invades and harasses allergic human population's health to a great extent, protects consumer
The Tough questions that edible safety has become food safety management department and food enterprise faces.Therefore, food hypersenstivity has been considered as
Important community nutrition hygienic issues, and cause global extensive concern.
Food hypersenstivity be mostly immunoglobulin E (IgE) mediate type Ⅰ hypersensitivity reaction, clinical manifestation be Nausea and vomiting,
The different symptoms such as asthma, diarrhea, peptic ulcer, nettle rash, allergic dermatitis can lead to anaphylactic shock, even when serious
Threat to life.So far, the most effective approach of food hypersenstivity is to avoid contact with food allergen.But it is more due to food component
Cross reaction in sample and production process increases food and there is a possibility that anaphylactogen, food allergy patients must be again
Add carefully, it could be from its evil.Therefore quick Allergic skin test method is established for protecting the safety of consumer very necessary.
Usually, the detection of food allergen is divided into physical examination survey, zoopery and vitro detection.In body detecting method master
If Skin-test, often need intradermal while injecting 30-50 kind antigens, is speculated according to the wheal of skin test and erythematous response loose thin
Born of the same parents discharge the amount of histamine, and then whether speculate patient to certain anaphylactogen allergy, though this method is easy, easy to operate, at low cost,
Specificity is not high, is also easy to produce local pain, and potential danger is larger, Skin-test may cause serious adverse reaction and to patient
Additional pain is brought, and the diagnostic result of this method is sometimes inconsistent with immunochemistry analysis method, often will appear vacation
Positive or false negative.
The external detection method of food allergen mainly has immunochemical test, and (such as IgE inhibits experiment, immunoblotting examination
Test), medium release test (histamine release rate detection, β-hexosaminidase release rate detection) etc..Immunochemical test is mesh
The important means of preceding vitro detection anaphylactogen carries out anaphylodiagnosis by detecting specific IgE content in allergic patients sera.It should
Method only needs a little serum of autopath that can detect a variety of anaphylactogens simultaneously, not by drug influence, and to detected person without danger
Danger, but the result of the immunity test based on human body specific IgE is easily influenced by identical specific IgG, and often tried with skin
The data for testing acquisition are inconsistent.
Still lack a kind of method that rapid in-vitro detects anaphylactogen in food, current detection food allergen sensitization at present
Property the evaluation methods such as BALB/c mouse, BN rat models take it is extremely long, it is of high cost, laboratory hardware facility is required it is high, no
Suitable for as the method for generally detecting anaphylactogen.
Cavy (scientific name：Cavia porcellus) belong to Rodentia Caviidae, there is higher reaction to various stimulations,
Easily cause allergic reaction.Ileum is located at the part of duodenum and jejunum rear in the small intestine of mammal, connects large intestine thereafter；
Even with mesenterium, there is the position that ileum enters caecum in another lateral margin for one lateral margin.When by exogenous stimulation, it can cause smooth in vivo
Flesh is shunk, and vasopermeability increases.Tonotransducer be it is a kind of can a variety of weak biological electric signals of accurate measurement important devices,
Using semiconductor gauge as power-power conversion device, accurate measuring trace quantity tension variation, by mechanical quantity linear transfor at electric energy
Output, signal value is higher to be illustrated to react stronger after receiving stimulation, is suitable for scientific research, medicine inspection, precision height, high sensitivity, dynamic
Effect is good, and compact in structure and convenient to use.Physiological signal instrument is that (including other non-electrical signals pass through transducing by various electric signals
The electric signal that device conversion comes) it is recorded with pen or a kind of instrument for being shown on fluorescent screen, it can record electrocardio, the blood of animal
The electric signals such as pressure, gastrointestinal smooth muscle, skeletal muscle, myocardial contraction or other physical signs.
Invention content
The purpose of the present invention is to provide a kind of methods of anaphylactogen in vitro detection food, to solve existing evaluation method
The problem of allergen sensitisation cannot be accurately reflected.
The present invention shrinks and opens by tonotransducer and the detection of physiological signal recorder using sensitizer stimulation in guinea pigs ileum
Power detects anaphylactogen in food.The present invention selects 3 kinds of albumen representative, sensitization is different, respectively glycinin
(Glycinin, Gly), oralbumin (ovalbumin, OVA), potato acid phosphatase (potato acid
phosphatase,PAP).Glycinin is 11S globulin main components, six aggressiveness being made of 6 subunits interactions
Albumen, molecular mass are about 350kDa, and anaphylactogen scientific name is Gly m 6, are a kind of strong sensibiligen；Oralbumin is chicken
The main allergen of egg accounts for about the 54% of albumen, is a kind of glycoprotein being made of 385 amino acid, and molecular weight is
44.5kDa, anaphylactogen scientific name are Gal d 2, are a kind of moderate sensibiligen, frequently as positive allergy in Evaluation method
It is former；Potato acid phosphatase is a kind of albumen being made of 448 amino acid, no sensitization activity, in Evaluation method
Frequently as negative anaphylactogen.The present invention is not limited thereto 3 kinds of albumen, further include all albumen with sensitization.
The albumen (Gly, OVA, PAP) that the present invention has a different potential sensitizations using 3 kinds to Hartley guinea pig ileums into
It assassinates and swashs, verify the sensitization of 3 kinds of albumen by detecting the shrink tension of Hartley guinea pig ileums, thus establish a kind of use
In the external detection method of evaluation food allergen.
The method of anaphylactogen, includes the following steps in a kind of vitro detection food provided by the invention：
(1) guinea pig ileum is taken, is fixed on tonotransducer, and is positioned in tyrode；
(2) select food to be measured as sensitizer, stimulation in guinea pigs ileum is received with physiological signal recorder detection guinea pig ileum
Contracting power.
The cavy is 5-6 week old cavys.
Preferably, the cavy is Female guinea pigs.
It is highly preferred that the cavy is SPF grades of female Hartley cavys.
The guinea pig ileum is the ileum apart from caecum 1cm or more.Preferably, guinea pig ileum is selected from dead in 3-5 hours
Cavy.
It preferably fetches intestines step in the embodiment of the present invention and is for one：It is cut from ileocecus 1cm, takes out ileum about 8-
10cm is uniformly cut into 1-1.5cm segments and is used to test, be placed in oxygen-saturated tyrode's solution culture dish, mesenterium is removed along intestinal wall,
Then ileum is cut into several segment 1-1.5cm segments, draws tyrode's solution with syringe and rinses intestinal contents well.
The present invention has found that the ileum apart from caecum less than 1cm cannot achieve the technique effect of the present invention by repetition test.
The ileum length 1-1.5cm.
By ileum be fixed on tonotransducer the specific steps are：A bit of intestinal tube (1-1.5cm) is taken, is used at intestinal tube both ends
Line knots, and pays attention to keeping intestinal tube unobstructed, does not make its closing.Intestinal tube one end is lain on bath stay hook, is then placed in 37 DEG C of Maxwells
In bath, the other end of intestinal tube ties up on the cantilever beam of tonotransducer.
The tyrode is well known balanced salt solution, formula：Tyrode's solution prepares 1000ml, contains:NaCl 8.0g、
10%KCl 0.2g, 10%MgSO4.7H2O 0.26g, 5%NaH2PO4.2H2O 0.065g、NaHCO3 1.0g、1M CaCl2
0.2g, glucose 1.0g.
A concentration of 0.01-1.0mg/ml of anaphylactogen, solvent are physiological saline in step (2).
The operating method of step (2) is to stablize initial tension regulation of tonotransducer to 1-3g or so 10-30 minutes
Afterwards, sensitizer is added.
After step (2) sensitizer stimulation in guinea pigs ileum, the mean value and standard of 2-5 minutes tension and initial tension difference are recorded
Difference.
The mean value and standard deviation of 2-5 minutes tension and initial tension difference, stimulus intensity of the reflection guinea pig ileum by sensitizer.
The size of mean value reflects ileum by the post-stimulatory tension variation of sensitizer, and numerical value is directly proportional to sensitization；Standard deviation is reflected in
The fluctuation range that ileum is stimulated by sensitizer within the scope of detection time.
Compared with prior art, the present invention has the advantages that：
(1) testing cost of the present invention is low, and the guinea pig ileum that the present invention uses is derived from cavy, is not necessarily to expensive examination
Agent.
(2) present invention is reusable, and the guinea pig ileum used in the present invention is reusable whithin a period of time, repeatedly
The sensitization for measuring allergic protein is strong and weak.
(3) short the time required to present invention detection, testing result can be instantly available, with the model examination in 4 weeks of conventional animal sensitization
Cycle phase ratio is tested, which substantially shortens.
Description of the drawings
Fig. 1 is the specific design scheme of 1-9 of the embodiment of the present invention, goes out cavy according to the test result analysis of embodiment 1-9
The impact effect that week old, gender, each factor of allergic protein concentration implement the method for the present invention.With SPSS (v24.0) software
The Orthogonal Experiment and Design provided.Variable is allergic protein, the week old of cavy, the gender of cavy, the concentration of allergic protein.
Fig. 2 is the testing result of 11S glycinin stimulation of the 7-8 week old female Hartley cavys to 1mg/ml.
Fig. 3 is the testing result of 11S glycinin stimulation of the 5-6 week old male Hartley guinea pigs to 0.1mg/ml.
Fig. 4 is the testing result of oralbumin stimulation of the 3-4 week old female Hartley cavys to 0.1mg/ml.
Fig. 5 is the testing result of 11S glycinin stimulation of the 3-4 week old female Hartley cavys to 0.01mg/ml.
Fig. 6 is testing result of the 7-8 week old female Hartley cavys to the potato acid phosphatase of 0.1mg/ml.
Fig. 7 is testing result of the 7-8 week old male Hartley guinea pigs to the oralbumin of 0.01mg/ml.
Fig. 8 is testing result of the 5-6 week old female Hartley cavys to the oralbumin of 1mg/ml.
Fig. 9 is testing result of the 3-4 week old male Hartley guinea pigs to the potato acid phosphatase of 1mg/ml.
Figure 10 is testing result of the 5-6 week old female Hartley cavys to the potato acid phosphatase of 0.01mg/ml.
Figure 11 is testing result of the 5-6 week old female Hartley cavys to the 11S glycinins of 1mg/ml.
Figure 12 is testing result of the 5-6 week old female Hartley cavys to the potato acid phosphatase of 1mg/ml.
Specific implementation mode
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention
In the case of essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.
Hartley cavys are purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.；Glycinin (Glycinin, Gly), egg white
Albumin (ovalbumin, OVA), potato acid phosphatase (potato acid phosphatase, PAP) are purchased from Sigma
Company；RM6240C type physiological signal recorders are purchased from Chengdu Instruement Factory；Tonotransducer is electromechanical purchased from Chaoyang District, Beijing City Xinda
Technical research institute.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Detection of the 1 7-8 week old female Hartley cavys of embodiment to the 11S glycinins stimulation of 1.0mg/ml
1) the female Hartley cavys of experimental animal 7-8 week old.
2) the tested albumen of tested material is 11S glycinins.
3) dose of test albumen dosage is 1.0mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the 11S glycinins of 1.0mg/ml are added, record the mean value and mark of 2-5 minutes tension and initial tension difference
It is accurate poor.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 2) tensile force of ileum difference is：0.09±0.04g.
Detection of the 2 5-6 week old male Hartley guinea pigs of embodiment to the 11S glycinins stimulation of 0.1mg/ml
1) male Hartley guinea pigs of experimental animal 5-6 week old.
2) the tested albumen of tested material is 11S glycinins.
3) dose of test albumen dosage is 0.1mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the 11S glycinins of 0.1mg/ml are added, record the mean value and mark of 2-5 minutes tension and initial tension difference
It is accurate poor.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 3) tensile force of ileum difference is：0.23±0.07g.
Detection of the 3 3-4 week old female Hartley cavys of embodiment to the oralbumin stimulation of 0.1mg/ml.
1) the female Hartley cavys of experimental animal 3-4 week old.
2) the tested albumen of tested material is oralbumin.
3) dose of test albumen dosage is 0.1mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the oralbumin of 0.1mg/ml is added, records the mean value and standard of 2-5 minutes tension and initial tension difference
Difference.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 4) tensile force of ileum difference is：0.08±0.04g.
Detection of the 4 3-4 week old female Hartley cavys of embodiment to the 11S glycinins stimulation of 0.01mg/ml
1) the female Hartley cavys of experimental animal 3-4 week old.
2) the tested albumen of tested material is 11S glycinins.
3) dose of test albumen dosage is 0.01mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, be added the 11S glycinins of 0.01mg/ml, record 2-5 minute tension and initial tension difference mean value and
Standard deviation.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 5) tensile force of ileum difference is：-0.08±0.02g.
Detection of the 5 7-8 week old female Hartley cavys of embodiment to the potato acid phosphatase of 0.1mg/ml
1) the female Hartley cavys of experimental animal 7-8 week old.
2) the tested albumen of tested material is potato acid phosphatase.
3) dose of test albumen dosage is 0.1mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the potato acid phosphatase of 0.1mg/ml is added, records the mean value of 2-5 minutes tension and initial tension difference
And standard deviation.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 6) tensile force of ileum difference is：-0.02±0.01g.
Detection of the 6 7-8 week old male Hartley guinea pigs of embodiment to the oralbumin of 0.01mg/ml
1) male Hartley guinea pigs of experimental animal 7-8 week old.
2) the tested albumen of tested material is oralbumin.
3) dose of test albumen dosage is 0.01mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the oralbumin of 0.01mg/ml is added, records the mean value and standard of 2-5 minutes tension and initial tension difference
Difference.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 7) tensile force of ileum difference is：-0.03±0.01g.
Detection of the 7 5-6 week old female Hartley cavys of embodiment to the oralbumin of 1mg/ml
1) the female Hartley cavys of experimental animal 5-6 week old.
2) the tested albumen of tested material is oralbumin.
3) dose of test albumen dosage is 1.0mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the oralbumin of 1.0mg/ml is added, records the mean value and standard of 2-5 minutes tension and initial tension difference
Difference.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 8) tensile force of ileum difference is：0.50±0.06g.
Detection of the 8 3-4 week old male Hartley guinea pigs of embodiment to the potato acid phosphatase of 1.0mg/ml
1) male Hartley guinea pigs of experimental animal 3-4 week old.
2) the tested albumen of tested material is potato acid phosphatase.
3) dose of test albumen dosage is 1.0mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the potato acid phosphatase of 1.0mg/ml is added, records the mean value of 2-5 minutes tension and initial tension difference
And standard deviation.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Fig. 9) tensile force of ileum difference is：0.00±0.01g.
Detection of the 9 5-6 week old female Hartley cavys of embodiment to the potato acid phosphatase of 0.01mg/ml
1) the female Hartley cavys of experimental animal 5-6 week old.
2) the tested albumen of tested material is potato acid phosphatase.
3) dose of test albumen dosage is 0.01mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the potato acid phosphatase of 0.01mg/ml is added, records the mean value of 2-5 minutes tension and initial tension difference
And standard deviation.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Figure 10) tensile force of ileum difference is：0.00±0.01g.
6) interpretation of result (table 1)
Main effect analysis is carried out to orthogonal test using SPSS 24.0, it is found that the effect power of four factors is respectively：Week
Age>Dosage>Allergic protein>Gender.Binding experiment data determine that optimal conditions is：5-6 week old；Female, 1mg/ml.
Effect is examined between 1 main body of table
a.R2=0.949 (R after adjustment2=0.949)
Detection of the 10 5-6 week old female Hartley cavys of embodiment to the 11S glycinins of 1.0mg/ml
1) the female Hartley cavys of experimental animal 5-6 week old.
2) the tested albumen of tested material is 11S glycinins.
3) dose of test albumen dosage is 1.0mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the 11S glycinins of 1.0mg/ml are added, record the mean value and mark of 2-5 minutes tension and initial tension difference
It is accurate poor.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Figure 11) tensile force of ileum difference is：0.77±0.13g.
Detection of the 11 5-6 week old female Hartley cavys of embodiment to the potato acid phosphatase of 1.0mg/ml
1) the female Hartley cavys of experimental animal 5-6 week old.
2) the tested albumen of tested material is potato acid phosphatase.
3) dose of test albumen dosage is 1.0mg/ml.
4) animal is handled
Experimental animal carries out adaptable fed and is tested after 3 days.Free water and feed.
It is cut from ileocecus 1cm, takes out one section of ileum about 10cm or so, be placed in oxygen-saturated tyrode's solution culture dish
In, mesenterium is removed along intestinal wall, ileum is then cut into several segment 1.5cm segments, tyrode's solution is drawn with syringe and holds enteral
Object is rinsed well.
It takes a bit of intestinal tube to be placed in the culture dish for filling tyrode's solution, is knotted with line at intestinal tube both ends, pay attention to keeping intestinal tube
It is unobstructed, do not make its closing.Intestinal tube one end line is lain on bath stay hook, is then placed in 37 DEG C of Magnus' baths.Again by intestinal tube
The other end be bound on the cantilever beam of tonotransducer.
Tension signal is recorded using RM6240C type physiological signal recorders.By initial tension regulation to 1g or so, stablize
After 10-30 minutes, the potato acid phosphatase of 1.0mg/ml is added, records the mean value of 2-5 minutes tension and initial tension difference
And standard deviation.Data processing uses 24.0 statistical analysis softwares of SPSS.Test result is indicated with average ± standard deviation.
5) experimental result (Figure 12) tensile force of ileum difference is：-0.03±0.01g.
6) interpretation of result (table 2-3) uses three group data (embodiment 7, embodiment 10 and of the SPSS to Best experimental condition
Embodiment 11) one-way analysis of variance is carried out, and carry out subsequent two-by-two compare.
2 variance analysis tension difference of table
Post-hoc tests
3 Multiple range test of table
As a result it can be seen that：11S glycinins group, oralbumin group and potato acid phosphatase group are between any two
Significant difference；Its moderate stimulation intensity is：11S glycinins>Oralbumin>Potato acid phosphatase.
Complied fully with the sensitization power of these three substances, it was demonstrated that the evaluation method can be applied to food allergen identification and its
The evaluation of sensitization.
In conclusion evaluation result and three of the guinea pig ileum shrinkage test method established of the present invention to anaphylactogen in food
The sensitization degree of strength of kind of representative allergic protein is consistent, it was demonstrated that the evaluation method can be applied to food allergen identification and
The evaluation of its sensitization.
Although having used general explanation, specific implementation mode and experiment above, the present invention is described in detail,
But some on the basis of the present invention, can be made to it to modify or improve, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of method of anaphylactogen in vitro detection food, which is characterized in that include the following steps：
(1) guinea pig ileum is taken, is fixed on tonotransducer, and is positioned in tyrode；
(2) select food to be measured as sensitizer, stimulation in guinea pigs ileum is shunk with physiological signal recorder detection guinea pig ileum
Power.
2. the method as described in claim 1, which is characterized in that the cavy is 5-6 week old cavys.
3. method as claimed in claim 2, which is characterized in that the cavy is Female guinea pigs.
4. method as claimed in claim 2, which is characterized in that the cavy is SPF grades of female Hartley cavys.
5. the method as described in claim 1, which is characterized in that the guinea pig ileum is the ileum apart from caecum 1cm or more.
6. method as claimed in claim 5, which is characterized in that the ileum length 1-1.5cm.
7. the method as described in claim 1-6 is any, which is characterized in that a concentration of 0.01- of anaphylactogen in step (2)
1.0mg/ml。
8. the method as described in claim 1-6 is any, which is characterized in that the operating method of step (2) is by tonotransducer
Initial tension regulation to 1-3g or so, after stablizing 10-30 minute, addition sensitizer.
9. the method as described in claim 1-6 is any, which is characterized in that after step (2) sensitizer stimulation in guinea pigs ileum, record
The mean value and standard deviation of 2-5 minutes tension and initial tension difference.
10. method as claimed in claim 9, which is characterized in that the mean value and standard deviation of 2-5 minutes tension and initial tension difference
Reflect stimulus intensity of the guinea pig ileum by sensitizer, the size reflection ileum of mean value by the post-stimulatory tension variation of sensitizer,
Numerical value is directly proportional to sensitization；Standard deviation is reflected in the fluctuation range that ileum is stimulated by sensitizer within the scope of detection time.
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