CN100357426C - Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation - Google Patents
Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation Download PDFInfo
- Publication number
- CN100357426C CN100357426C CNB2005100933531A CN200510093353A CN100357426C CN 100357426 C CN100357426 C CN 100357426C CN B2005100933531 A CNB2005100933531 A CN B2005100933531A CN 200510093353 A CN200510093353 A CN 200510093353A CN 100357426 C CN100357426 C CN 100357426C
- Authority
- CN
- China
- Prior art keywords
- dermatophagoides pteronyssinus
- der
- anaphylactogen
- allergen
- pbs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 57
- 241000238740 Dermatophagoides pteronyssinus Species 0.000 title claims description 79
- 239000013566 allergen Substances 0.000 title abstract description 15
- 238000002360 preparation method Methods 0.000 title description 18
- 238000003745 diagnosis Methods 0.000 title description 8
- 239000007788 liquid Substances 0.000 claims description 28
- 238000004108 freeze drying Methods 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000004033 plastic Substances 0.000 claims description 4
- 229920003023 plastic Polymers 0.000 claims description 4
- 235000008957 cocaer Nutrition 0.000 claims description 3
- ZPUCINDJVBIVPJ-LJISPDSOSA-N cocaine Chemical compound O([C@H]1C[C@@H]2CC[C@@H](N2C)[C@H]1C(=O)OC)C(=O)C1=CC=CC=C1 ZPUCINDJVBIVPJ-LJISPDSOSA-N 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000005238 degreasing Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000007667 floating Methods 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 244000022782 cocaer Species 0.000 claims 1
- 210000002966 serum Anatomy 0.000 abstract description 34
- 230000000172 allergic effect Effects 0.000 abstract description 4
- 208000010668 atopic eczema Diseases 0.000 abstract description 3
- 241000238710 Dermatophagoides Species 0.000 abstract description 2
- 238000012258 culturing Methods 0.000 abstract 2
- 238000005259 measurement Methods 0.000 abstract 1
- 238000010790 dilution Methods 0.000 description 22
- 239000012895 dilution Substances 0.000 description 22
- 239000000243 solution Substances 0.000 description 20
- 238000012360 testing method Methods 0.000 description 20
- 206010020751 Hypersensitivity Diseases 0.000 description 19
- 208000026935 allergic disease Diseases 0.000 description 18
- 230000007815 allergy Effects 0.000 description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 16
- 239000000126 substance Substances 0.000 description 15
- 230000005764 inhibitory process Effects 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000000586 desensitisation Methods 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 7
- 238000011047 acute toxicity test Methods 0.000 description 7
- 201000010099 disease Diseases 0.000 description 7
- 239000000428 dust Substances 0.000 description 7
- 235000011187 glycerol Nutrition 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 230000002009 allergenic effect Effects 0.000 description 6
- 230000002052 anaphylactic effect Effects 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 102000005936 beta-Galactosidase Human genes 0.000 description 4
- 108010005774 beta-Galactosidase Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000013016 damping Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 208000001840 Dandruff Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 235000004252 protein component Nutrition 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- GHCZTIFQWKKGSB-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;phosphoric acid Chemical compound OP(O)(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O GHCZTIFQWKKGSB-UHFFFAOYSA-N 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 2
- 241001313855 Bletilla Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000238557 Decapoda Species 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 241000735552 Erythroxylum Species 0.000 description 2
- 241001203952 Feia Species 0.000 description 2
- 235000019733 Fish meal Nutrition 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 239000004698 Polyethylene Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 206010039085 Rhinitis allergic Diseases 0.000 description 2
- 206010070834 Sensitisation Diseases 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 201000009961 allergic asthma Diseases 0.000 description 2
- 201000010105 allergic rhinitis Diseases 0.000 description 2
- 208000006673 asthma Diseases 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000006287 biotinylation Effects 0.000 description 2
- 238000007413 biotinylation Methods 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000004467 fishmeal Substances 0.000 description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000007654 immersion Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 206010025482 malaise Diseases 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- -1 polyethylene Polymers 0.000 description 2
- 229920000573 polyethylene Polymers 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000008313 sensitization Effects 0.000 description 2
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- YUDPTGPSBJVHCN-DZQJYWQESA-N 4-methylumbelliferyl beta-D-galactoside Chemical compound C1=CC=2C(C)=CC(=O)OC=2C=C1O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O YUDPTGPSBJVHCN-DZQJYWQESA-N 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 108010061629 Dermatophagoides pteronyssinus antigen p 1 Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 206010021138 Hypovolaemic shock Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229930003451 Vitamin B1 Natural products 0.000 description 1
- 229930003779 Vitamin B12 Natural products 0.000 description 1
- 229930003471 Vitamin B2 Natural products 0.000 description 1
- LXNHXLLTXMVWPM-UHFFFAOYSA-N Vitamin B6 Natural products CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229960000304 folic acid Drugs 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- BUKHSQBUKZIMLB-UHFFFAOYSA-L potassium;sodium;dichloride Chemical compound [Na+].[Cl-].[Cl-].[K+] BUKHSQBUKZIMLB-UHFFFAOYSA-L 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 230000001235 sensitizing effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 206010040560 shock Diseases 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000010374 vitamin B1 Nutrition 0.000 description 1
- 239000011691 vitamin B1 Substances 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 239000011726 vitamin B6 Substances 0.000 description 1
- 235000019158 vitamin B6 Nutrition 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000005282 vitamin D3 Nutrition 0.000 description 1
- 239000011647 vitamin D3 Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention discloses a method for culturing and collecting Dermatophagoides pterronyssinus (Der p for short), extracting an allergen of Der p and measuring the content of a first component of the allergen of the Der p (Der pl) in a Der P extracting solution. A double-sanwich monoclonal antibody method is used for measuring the content of the Der pl in the Der p extracting solution. In addition, the present invention also discloses the establishment of a standard serum base of Der p allergic patients and the measurement of the total bioactivity of the allergen of the Der p extracting solution. The method for culturing and collecting the Der p and extracting the allergen of the Der p and the method for measuring the Derpl content in the Der p extracting solution both have the characteristics of convenient operation and high speed.
Description
Invention field
The invention belongs to the allergy medicine preparation field, especially the extracting method that relates to cultivation, collection method and the dermatophagoides pteronyssinus anaphylactogen of dermatophagoides pteronyssinus (Dermatophagoides pterronyssinus be called for short Der p), the foundation etc. that the present invention also discloses Der p1 Determination on content in the dermatophagoides pteronyssinus extracting solution and set up dermatophagoides pteronyssinus allergy patient standard serum storehouse.
Background of invention
Anaphylactic disease has become influences the healthy major issue of people.Wherein the dirt mite is most important indoor anaphylactogen, and it is a kind of ubiquitous biology, and naked eyes generally be cannot see, mainly be present in the room, as: carpet, pillow, mattress, sofa, curtain.Dirt mite viability is strong, is food with the human body scurf mainly, eliminates also so very difficulty of dust mite allergen.In worldwide, the mite of advantage is dermatophagoides pteronyssinus and dust mite in the room, and main allergic protein component is first component (Der p1).At present, suffer from the dust mite allergy disease, be the sixth-largest chronic disease, sickness rate 20-40% the U.S. 5,000 ten thousand people that have an appointment; Also be ascendant trend year by year in worldwide, 4.8 hundred million people are ill in China, sickness rate 37% (the statistics eighties).
The anaphylactogen preparation can be used in the body of anaphylactic disease, external specific diagnosis and desensitization treatment, is the basis of diagnosis and treatment anaphylactic disease, the good and bad health that directly influences hundreds of millions anaphylactic disease patients of its quality.1998, WHO quited approve of the curative effect and the security of desensitization treatment, pointed out that desensitization treatment is unique methods of treatment that influences the anaphylactic disease nature process, can effectively stop allergic rhinitis to develop into allergic asthma.The development of stdn anaphylactogen preparation and use are the inexorable trends of anaphylactic disease diagnosis and treatment.
At present, external anaphylactogen crude preparation by using is the crude extract that the anaphylactogen raw material is made after extraction, dialysis and sterile filtration, comprise whole allergenic components, it has following shortcoming: 1) be unit with W/V, main sensitization component content instability, biological value and effective constituent that can not the actual response anaphylactogen; 2) criticize between effective constituent variation can reach 1000 times, the accuracy and the security of influence diagnosis and treatment; 3) no quality control standard is unfavorable for pharmaceuticals administration, and the research data between various countries, the geographic product can't compare.Simultaneously, as a sci-tech product that added value is higher, stdn anaphylactogen preparation has huge market, promise well.Entering of external high-quality product, to bring enormous impact to the national anaphylactogen manufacture of China: 1, increase national medical expense and people burden, 2, because regional disparity is different with geographical environment, most external anaphylactogen products are unsuitable for the irritated crowd of China, the external reagent of undue dependence, to make treatment and diagnosis limitation, 3, have a problem of biological safety.
The inventor has formed the present invention through years of researches.Cultural method for the dirt mite has multiple both at home and abroad: the use dried small shrimp or the fish meal that have; The end user's scurf that has; The somebody uses milk powder.These methods all contain a large amount of sensitizing substances.For the allergy patient danger of height is arranged, and form false positive reaction easily.Cultural method of the present invention has been avoided above-mentioned shortcoming, and the risk when having reduced allergy patient's skin test and desensitization treatment has improved the security of clinical treatment greatly.The present invention has also reduced the false positive (promptly having improved the specificity to the dermatophagoides pteronyssinus anaphylodiagnosis greatly) of dermatophagoides pteronyssinus pricking method test when avoiding above-mentioned false positive reaction.The method of collecting the dirt mite both at home and abroad has the lucifuge method, climbs dish method and saturated salt water law.The dirt mite purity that aforesaid method is collected is not high, and saturated brine of the present invention has improved the purity that the dirt mite is collected greatly in conjunction with sieving network method.In addition, the present invention uses proteins extraction and analytical technology, has determined best dermatophagoides pteronyssinus anaphylactogen leaching process; Prepare polyclonal antibody, adopted crossed immunoelectrophoresis to detect the total moiety of anaphylactogen then; Set up dermatophagoides pteronyssinus allergy patient specific IgE antibody storehouse; Iso-electric point and molecular weight according to main sensitization component (first component), adopt molecular sieve and ion exchange technique to carry out initial gross separation, obtain the main anaphylactogen component of relative purifying, adopt high pressure liquid chromatography-mass spectrometry instrument, obtained the main anaphylactogen component of purifying; Verified the aminoacid sequence of main allergic protein with analytical technique of mass spectrum, suppressed the main allergenic components immunocompetence that purifying has been verified in experiment and dermatophagoides pteronyssinus allergy patient specific IgE antibody storehouse with radioimmunity absorption; Prepare monoclonal antibody with the control dermatophagoides pteronyssinus anaphylactogen quality of the pharmaceutical preparations with main allergenic components; In addition, the contriver utilizes the how anti-of preparation voluntarily and the monoclonal antibody control dermatophagoides pteronyssinus anaphylactogen quality of the pharmaceutical preparations, has set up the internal reference product and the internal reference standard of stdn dermatophagoides pteronyssinus anaphylactogen preparation.
Summary of the invention
One object of the present invention is to provide a kind of cultivation and collection method of dermatophagoides pteronyssinus.
Another object of the present invention provides a kind of extracting method of dermatophagoides pteronyssinus anaphylactogen.
A further object of the present invention is for providing a kind of method of measuring Der p1 content in the dermatophagoides pteronyssinus extracting solution.
The objective of the invention is to be achieved through the following technical solutions:
1. the cultivation of dermatophagoides pteronyssinus and collection:
At 20-25 ℃, 70-75% relative humidity is with ventilative plastic culture flask culture dermatophagoides pteronyssinus with cover.Feed is a yeast powder 30%, bean powder: 35.2%, and Semen Maydis powder 3.2%, sugar: 35.2%, lard: 3.7%, VITAMIN and salt prescription (/ 100g feed): Vit A 904.579 IU, VitD 201.729 IU, Vit B1:2.001mg, Vit B2:7.525mg, Vit B6:1.189mg, Vit B12:0.945mg, Vit C:60.519mg, Vit K3:10.490mg, Vit D3:0.755mg, nicotinic acid 9.888mg, vitamin H: 0.040mg, folic acid: 0.312mg, calcium pantothenate: 8.875mg, inositol: 12.104mg, NaCl:103.477mg, FeSO
4: 63.701mg, ZnSO
4: 4.711mg, MnSO
4: 1.705mg, Vit E:10.11mg.Do not contain animal proteinums such as fish meal, dried small shrimp, people's scurf.Collection method: utilize saturated aqueous common salt floating method and 50-200 eye mesh screen to collect or utilize the lucifuge method to collect, the dermatophagoides pteronyssinus that freeze-drying is collected obtains the raw material of dermatophagoides pteronyssinus anaphylactogen stdn preparation.
2. the extracting method of dermatophagoides pteronyssinus anaphylactogen:
Freeze dried dermatophagoides pteronyssinus through the acetone degreasing, is used the dissolving of Coca ' s liquid, the extraction of 1: 10 to 1: 20 ratio (W/V).When extracting earlier 4 ℃ ultrasonic 2-3 time, each 2-3 minute, and then 4 ℃ extracted 4-24 hour, 4 ℃, centrifugal 15 minutes of 10000rpm got supernatant liquor and obtains dermatophagoides pteronyssinus extracting solution stoste through filter paper and the filtration of 0.2 μ m filter.
3. Der p1 Determination on content (double fastener heart monoclonal antibody method) in the dermatophagoides pteronyssinus extracting solution:
To be dissolved in the PBS solution through the anti-Der P1 monoclonal antibody of HPLC purifying.Use NaHCO
3Damping fluid dilutes Der P1 monoclonal antibody to 2 μ g/ml in proportion.Bag is by polyethylene board, overnight incubation.With containing Tween-20, the PBS damping fluid (PBS-T) of pH7.4 is washed plate.
Add the PBS-T that contains B S A, incubated at room.PBS-T washes plate.
The anaphylactogen standard substance or the sample that add dilution.Incubated at room.
Use anaphylactogen standard substance (S T-DP1) proportional diluted to prepare Der P1 typical curve.A1 and B1 hole at enzyme plate add PBS-T, and then add Der P1 standard substance.Mixing shifts some liquid then and enters next PBS-T, forms a plurality of dilution gradients.PBS-T should only be contained as blank in A11, B11, A12, B12 hole.
The conventional dilution of dust or anaphylactogen sample.Wash plate with PBS-T, add the monoclonal antibody of the anti-Der P1 of biotinylation of dilution then.Incubated at room.
Wash plate with PBS-T, add the Streptomycin sulphate ovum protein-peroxidase (S5512 of SigMA company) of dilution then.And then use PBS-T to dilute in proportion.Incubated at room half an hour.
Wash plate with PBS-T, add colour developing then, also will add the H of dilution in proportion during colour developing with Citric Acid phosphoric acid buffer dissolved AB T S
2O
2When the 405nm optical density value is read plate during at 2.0-4.0.
4. set up dermatophagoides pteronyssinus allergy patient standard serum storehouse:
Dermatophagoides pteronyssinus moderate allergy patient's standard: allergic rhinitis is arranged or/and the allergic asthma symptom, it is the 3-4 level that the RAST test (adsorption test of anaphylactogen radioactivity) of the immunoCAP of Sweden Pharmacia Corporation detects the irritated grade of dermatophagoides pteronyssinus (d1).The ultimate principle that RAST detects (RAST FEIA) is: will be coated with the reaction of allergenic solid phase carrier and positive serum, as containing in the serum at the allergenic specific IgE of this kind, then can form immune complex, after hatching and washing, enzyme-added mark anti-IgE antibodies is hatched again, promptly forms the anti-IgE mixture of antigen-IgE-, add substrate after the flushing, this mixture and substrate-function produce fluorescent substance.The content of specific IgE is high more, and the fluorescent absorption value is high more.
Dermatophagoides pteronyssinus moderate allergy patient's collection: according to dermatophagoides pteronyssinus moderate allergy patient's standard, at signature informed consent postscript, collect 20-30 dermatophagoides pteronyssinus moderate allergy patient serum, every example is gathered 20ml serum, after the balanced mix dilution, with contain 0.5% bovine serum albumin bletilla, 0.05% sodium azide solution dilution to light absorption value at 10-30KU/L, the packing of 2ml plastic centrifuge tube, serum is preserved in freeze-drying, is dermatophagoides pteronyssinus allergy patient standard serum storehouse.
5. the total biological activity determination of dermatophagoides pteronyssinus extracting solution anaphylactogen:
By the anaphylactogen gross activity of RAST inhibition test Detection and Extraction liquid, adopt freeze-drying redissolution method to guarantee the constant relatively of anaphylactogen gross activity, the total bioactive unit of anaphylactogen represents with anaphylactogen unit (AU).
Adopt the CAP system of Pharmacia, carry out dermatophagoides pteronyssinus anaphylactogen radioactivity absorption inhibition test (RAST inhibition is called for short the RAST inhibition test).The change and forming on above-mentioned RAST FEIA basis of RAST inhibition test.Be allergy patient's sero-reaction with a series of different certain allergens of measuring with corresponding positive serum earlier promptly, carry out above-mentioned RAST step again,, cause the fluorescent absorption value to descend because allergen has suppressed the specific IgE of some amount in the positive serum.Allergen content is high more, and strong more to the restraining effect of specific IgE, the fluorescent absorption value is low more.
The RAST inhibition test is that it can not reflect the antigenicity of one-component in the anaphylactogen to the analysis of the overall biological activity of dermatophagoides pteronyssinus anaphylactogen (being antigenicity).In the relevant test of dermatophagoides pteronyssinus, the serum pond generally is made up of 10 above patients' serum equivalent.
The RAST inhibition test can adopt each detection system of the method for exempting from of putting, enzyme linked immunosorbent assay, fluorescence enzyme linked immunosorbent assay to measure.Because the CAP system has higher sensitivity and specific degree, and is easy and simple to handle, so the present invention adopts the external anaphylactogen detection system of the UniCAP of Sweden Pharmacia Corporation to carry out.
6. the mensuration of dermatophagoides pteronyssinus allergenic components:
Adopt SDS-PAGE to detect the protein component of dermatophagoides pteronyssinus extracting solution, Western Blotting detects the main anaphylactogen composition of dermatophagoides pteronyssinus, wherein, one anti-is the standard serum in the standard serum storehouse, and the result shows and contains 24KD and two kinds of protein components of 14KD at least.
7. the preparation of dermatophagoides pteronyssinus anaphylactogen pricking method liquid:
After obtaining qualified dermatophagoides pteronyssinus extracting solution, the glycerine that adds equivalent in 1: 1 ratio, 0.4% phenol, the content that employing freeze-drying redissolution method is guaranteed Der p1 in the extracting solution is divided in then and promptly makes dermatophagoides pteronyssinus pricking method liquid in the pricking method liquid bottle at 100-200 μ g/ml.
8. the preparation of dermatophagoides pteronyssinus allergen desensitization preparation: after obtaining qualified dermatophagoides pteronyssinus extracting solution,, be prepared into the dermatophagoides pteronyssinus desensitization preparation of a series of different concns by different Dilution ratio adding Coca ' s liquid and 0.4% phenol.The total volume of every bottle of dermatophagoides pteronyssinus desensitization preparation can be different, to reach the purpose that makes things convenient for patient and saving.In the dermatophagoides pteronyssinus desensitization preparation, the maximum concentration content of Der p 1 is 20 μ g/ml.
9. dermatophagoides pteronyssinus anaphylactogen pricking method liquid acute toxicity test: before glycerol adding, carry out the acute toxicity test of dermatophagoides pteronyssinus anaphylactogen pricking method liquid, get 56 week-8 weeks, body weight is in the BALB/c mouse of 17-22 gram, injection 0.5ml dermatophagoides pteronyssinus anaphylactogen pricking method liquid stoste in every mouse peritoneal, observed for 1 week, according to whether having dead mouse to judge the toxicity of anaphylactogen pricking method liquid.
Description of drawings
Fig. 1: the enzyme plate synoptic diagram, 1%BSA PBS-T is only contained as blank in A11 wherein, B11, A12, B12 hole.A1 to A10, B1 to B10 hole is the multiple hole of the standard curve determination of Der p1 standard substance, and its content is from 250 to 0.5ng/ml proportional diluted.
Embodiment 1: Der p1 Determination on content in the dermatophagoides pteronyssinus anaphylactogen extracting solution
Through the anti-Der P1 monoclonal antibody 5H8 of HPLC purifying is that concentration with the 2mg/ml storing solution is dissolved in the PBS solution.With the NaHCO3 damping fluid of 50mM pH9.6, by 1: the 1000 anti-Der P1 of dilution proportion monoclonal antibody 5H8 (i.e. 10 μ l/10ml) Der P1 monoclonal antibody to 2 μ g/ml.Bag is by polyethylene board, 4 ℃ of overnight incubation.With containing 0.05%Tween-20, the PBS damping fluid (PBS-T) of pH7.4 is washed plate 3 times.
Add the PBS-T100 μ l/ hole contain 1%B S A, incubated at room half an hour.PBS T washes plate 3 times.
The anaphylactogen standard substance or the sample that add the dilution of 100 μ l/ holes.Incubated at room 1 hour.
Use anaphylactogen standard substance (S T-DP1) proportional diluted to prepare Der P1 typical curve.The dilution range of Der P1 standard substance is 0.5-250ng/ml.A1 and B1 hole at enzyme plate add the PBS-T of 180 μ l 1%B S A, and then add 20 μ l Der P1 standard substance.Mixing shifts 100 μ l liquid then and enters the PBS-T that the next one contains 100 μ l 1%B S A, forms 10 dilution gradients.1%B S A PBS-T should only be contained as blank in A11, B11, A12, B12 hole.
The conventional 2 times of dilutions of dust or anaphylactogen sample were from 1: 10 to 1: 80.
Der P1 standard substance contain 2500ng/ml Der P1, and through with the contrast standardization of the dermatophagoides pteronyssinus reference standard product (N I B S C 82/518) of WHO/IUIS.The Der P1 content that the dermatophagoides pteronyssinus reference standard product of WHO/IUIS (N I B S C 82/518) contain is 12.5 μ g/ml.
Embodiment 2: the total bioactive mensuration of anaphylactogen in the dermatophagoides pteronyssinus anaphylactogen extracting solution
1). the foundation in dermatophagoides pteronyssinus anaphylactogen serum pond
Standard according to dermatophagoides pteronyssinus moderate allergy patient, at signature informed consent postscript, collect 30 dermatophagoides pteronyssinus moderate allergy patient serum, every example is gathered 20ml serum, after the balanced mix dilution, with contain 0.5% bovine serum albumin bletilla, 0.05% sodium azide solution dilution to light absorption value at 10-30KU/L, the packing of 2ml plastic centrifuge tube, serum is preserved in freeze-drying, is dermatophagoides pteronyssinus allergy patient standard serum storehouse.
2). reagent: 1 * PBS:
| Final concentration | Concrete amount (1L) | |
| NaCl KCl Na 2HPO 4·7H 2O KH 2PO 4 | 137mM 2.7mM 4.3mM 1.4mM | 8g 0.2g 1.15g 0.2g |
3). material
A). allergen to be checked is the dermatophagoides pteronyssinus anaphylactogen that Trizol extracts, and mixes with dehydrated alcohol to be placed on-40 ℃ of refrigerators preservations, and 10000g * 10min gets precipitation and redissolves with 1 * PBS original volume
B). dermatophagoides pteronyssinus allergen CAP, dust mite allergen CAP
C). anti-IgE CAP: identical with above-mentioned CAP, but the cellulose grain bonded is not an allergen, but the sheep anti human IgE, for the drawing standard curve
D) .IgE standard substance: have 6 concentration, be respectively 0.35,0.7,3.5,17.5,50 and 100KUa/L.
E). the mouse anti human IgE (ELIAS secondary antibody) of enzyme labelling, used enzyme are beta-galactosidase enzymes (β-galactosidase)
F). substrate is 4-methyl umbrella osmanthus-beta galactose glycosides (4-Methylumbelliferyl-β-D-galactoside)
G). stop buffer is that sodium carbonate solution, washing fluid are PBS solution
4). instrument
Adopt full-automatic UniCAP 100 System of Pharmacia, function software is UniCAP100version1.61/009.
5). working method
A). the dilution of dermatophagoides pteronyssinus immersion liquid: original liquid concentration is 8.9mg/ml, dilutes to be 0.0089mg/ml 0.089mg/ml, 0.89mg/ml and 8.9mg/ml, i.e. 10-3,10-2,10-1 and stoste respectively with 1 * PBS.
B). the inhibition of positive serum: get 7 of Eppendoff pipes, positive serum 20 μ 1 that every pipe all adds after the redissolution add dermatophagoides pteronyssinus immersion liquid 160 μ l respectively,, under 4 ℃ condition, hatched 12 hours
C). get negative serum 20 μ l simultaneously, add 1 * PBS160 μ l, by above-mentioned same processing, as negative control
D). input test sample project: comprise 12 the anti-IgECAP and dermatophagoides pteronyssinus and the dust mite sIgECAP that are used for the production standard curve.
E). add serum sample and IgE standard substance (every kind of concentration is all double).
F). add reagent: the sheep anti human IgE (ELIAS secondary antibody) of enzyme labelling, beta-galactosidase enzymes (β-galactosidase), substrate: 4-methyl umbrella osmanthus-beta galactose glycosides (4-Methylunbelliferryl-β-galactosidase), stop buffer, washing fluid and attached liquid thereof.
G). the fluorescence absorbance of each test sample book of computer automation testing.
H). the absorbancy and the corresponding concentration of IgE standard substance are depicted as typical curve on semilogarithmic paper.
I). read corresponding sIgE content according to the serum fluorescence absorbance that records on typical curve, above-mentioned two steps are finished by computer.
J). the method for the RAST that provides by CAP System is to positive serum, and the positive serum after the inhibition, negative serum carry out dermatophagoides pteronyssinus sIgE to be measured, and writes down its light absorption value.
K). use IgE standard substance preparation standard curve simultaneously
6. data processing
A). deduct the absorbance of negative control respectively with the positive serum absorbance after positive serum (positive serum OD value is the OD value of positive serum 20 μ l+PBS60 μ l mixed solutions) and the inhibition
B). to use a). income value is the maximum absorbance value divided by the serum absorbance that does not suppress with allergen
C). deduct b with 100%). gained ratio is the percentage (being called for short RAST inhibition test percentage) that the absorption of anaphylactogen radioactivity suppresses
D). the logarithm of used allergic effect commercial weight is made independent variable(s) when handling with serum sample, and RAST inhibition test percentage is a dependent variable, does straight-line regression on semilogarithmic paper, and RAST is suppressed curve carry out the t check analysis.
RAST inhibition test percentage will reach more than 50%.
Embodiment 3: the acute toxicity test of dermatophagoides pteronyssinus anaphylactogen pricking method liquid
Before and after glycerol adding, carry out the acute toxicity test of dermatophagoides pteronyssinus anaphylactogen pricking method liquid respectively, get 56 week-8 weeks, body weight is in the BALB/c mouse of 17-22 gram, injection 0.5ml dermatophagoides pteronyssinus anaphylactogen pricking method liquid stoste in every mouse peritoneal, observed for 1 week, according to whether having dead mouse to judge the toxicity of anaphylactogen pricking method liquid.The acute toxicity test of dermatophagoides pteronyssinus anaphylactogen pricking method liquid shows: the mouse all if qualified dermatophagoides pteronyssinus pricking method liquid carries out acute toxicity test after adding glycerine is all dead; Its reason may be oozed environment for glycerine causes occurring in the mouse peritoneal height, causes the mouse Q volume of blood to reduce, dewater, and produces hypovolemic shock and death.The pathological biopsy of these mouse peritoneals and pathological section are not found other organic disease.If carry out acute toxicity test before the adding glycerine then qualified dermatophagoides pteronyssinus pricking method liquid does not have dead mouse.
Embodiment 4: the sensitivity and the specific degree of dermatophagoides pteronyssinus anaphylactogen stdn pricking method liquid.
Carry out the clinical pricking method test of the dermatophagoides pteronyssinus anaphylactogen stdn pricking method liquid of 1000 examples in transformation reactions section of BJ Union Hospital.The each patient accepts the pricking method test and whether has dermatophagoides pteronyssinus allergy with diagnosis at signature informed consent postscript.Used select pricker be Tsing-Hua University produce select pricker (the accurate font size of medical device: No. the 1010015th, Soviet Union's salt pencil tool (standard) word 2004).Clinical pricking method test through the dermatophagoides pteronyssinus anaphylactogen stdn pricking method liquid of 500 examples shows: with at present, internationally recognized dermatophagoides pteronyssinus external detection method hypersensitive relatively, the sensitivity of this stdn pricking method liquid is 91%, the specific degree degree is 92%.
Claims (2)
1. method of extracting the dermatophagoides pteronyssinus anaphylactogen, it comprises step:
A). at 20-25 ℃, under the 70-75% relative humidity condition, with ventilative plastic culture flask culture dermatophagoides pteronyssinus with cover;
B). utilize saturated aqueous common salt floating method and 50-200 eye mesh screen to collect freeze-drying;
C). the freeze-drying dermatophagoides pteronyssinus is carried out the acetone degreasing, use the dissolving of Coca ' s liquid, the extraction of 1: 10 to 1: 20 ratio (W/V).
2. according to the method for claim 1, it is characterized in that: during extraction earlier 4 ℃ ultrasonic 2-3 time, each 2-3 minute, and then 4 ℃ extracted 4-24 hour, centrifugal 15 minutes of 4 ℃ of following 10000rpm got supernatant liquor through filter paper and the filtration of 0.2 μ m filter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100933531A CN100357426C (en) | 2005-08-26 | 2005-08-26 | Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100933531A CN100357426C (en) | 2005-08-26 | 2005-08-26 | Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1763174A CN1763174A (en) | 2006-04-26 |
| CN100357426C true CN100357426C (en) | 2007-12-26 |
Family
ID=36747497
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100933531A Expired - Fee Related CN100357426C (en) | 2005-08-26 | 2005-08-26 | Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100357426C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101628119B (en) * | 2009-08-21 | 2012-07-04 | 北京新华联协和药业有限责任公司 | Dermatophagoides pteronyssinus (Der p) allergen diagnostic reagent and preparation method thereof |
| CN113831400A (en) * | 2021-07-29 | 2021-12-24 | 沈阳医学院 | Separation and purification method of dust mite allergenic protein Der p 1 |
-
2005
- 2005-08-26 CN CNB2005100933531A patent/CN100357426C/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 变应原粉尘螨的培养 李文全 等.Acta Acad Med Weifang,Vol.26 No.2. 2004 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1763174A (en) | 2006-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bittencourt et al. | Paracoccidioidomycosis mortality in the State of Paraná, Brazil, 1980/1998 | |
| CN103454412A (en) | Liquid phase chip for detecting allergen specific antibody and preparation method of liquid phase chip | |
| Pradhan et al. | WHO (World Health Organization) guidelines for standardization of herbal drugs | |
| CN101581726B (en) | New-generation brucellosis antibody competition enzyme-linked immunosorbent adsorption test detection kit | |
| CN101271110A (en) | Immunity colloidal gold test paper strip for detecting and staphylococcal enterotoxin A and its production method | |
| CN101592661B (en) | Brucellosis antibody competitive enzyme-linked immunosorbent assay reagent kit | |
| CN103940925A (en) | Method for rapidly detecting sulfonamide antibiotics by high performance liquid chromatography and application | |
| CN107466319A (en) | GM hybridomas, monoclonal antibody, kit and preparation method and application | |
| CN100357426C (en) | Culture and collection of dermatophagoides pteronyssinus and method for preparing its allergen diagnosis and treatment preparation | |
| Trucksess et al. | Comparison of two immunochemical methods with thin-layer chromatographic methods for determination of aflatoxins | |
| Asevedo et al. | Palynological analysis of dental calculus from Pleistocene proboscideans of southern Brazil: a new approach for paleodiet and paleoenvironmental reconstructions | |
| CN109632980B (en) | Method for simultaneously detecting uridine and ergosterol and application thereof | |
| CN101592660A (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit | |
| Sun et al. | Analysis of gallic acid and ellagic acid in leaves of Elaeagnus angustifolia L. from different habitats and times in Xinjiang by HPLC with cluster analysis | |
| CN109692291A (en) | Lung power cough mixture medicinal extract and application thereof and method of quality control | |
| CN106645748B (en) | A kind of exception lymphatic temperament type impotence disease mammalian serum and testosterone histological difference albumen system and screening technique and application | |
| Abu El‐Enin et al. | Preparation of chemically stable allergen‐specific sublingual immunotherapy from Egyptian allergens | |
| CN106770037A (en) | A kind of discrimination method of datura flower medicinal material | |
| CN106546754A (en) | Yimusake table acts on abnormal mucus cross-examination with sexual impotence Syndrome model target point protein and its screening technique | |
| Mendoza-Morales et al. | Neosporosis in sheep: A systematic review and meta-analysis of global seroprevalence and related risk factors | |
| CN106994145A (en) | Purposes for treating or slowing down autoimmune disease, its complication and/or the medical composition of ephritis and its active ingredient | |
| Mbassa et al. | The comparative haematology of cross-bred and indigenous East African goats of Tanzania and breeds reared in Denmark | |
| CN103575912A (en) | Method for detecting allergen in crocus sativus preparation | |
| CN103288964A (en) | Albendazole-2-amino sulfone residue detection monoclonal antibody as well as preparation method and application thereof | |
| CN101988925B (en) | ELISA (Enzyme Linked Immunosorbent Assay) reagent kit for detecting Hum j3 specific IgE antibodies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071226 |