CN101413943B - Method for detecting melamine and specific enzyme-linked immunologic reagent kit - Google Patents
Method for detecting melamine and specific enzyme-linked immunologic reagent kit Download PDFInfo
- Publication number
- CN101413943B CN101413943B CN200810227894A CN200810227894A CN101413943B CN 101413943 B CN101413943 B CN 101413943B CN 200810227894 A CN200810227894 A CN 200810227894A CN 200810227894 A CN200810227894 A CN 200810227894A CN 101413943 B CN101413943 B CN 101413943B
- Authority
- CN
- China
- Prior art keywords
- melamine
- liquid
- sample
- kit
- enzyme
- 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
- JDSHMPZPIAZGSV-UHFFFAOYSA-N melamine Chemical compound NC1=NC(N)=NC(N)=N1 JDSHMPZPIAZGSV-UHFFFAOYSA-N 0.000 title claims abstract description 152
- 229920000877 Melamine resin Polymers 0.000 title claims abstract description 147
- 238000000034 method Methods 0.000 title claims abstract description 34
- 102000004190 Enzymes Human genes 0.000 title claims description 62
- 108090000790 Enzymes Proteins 0.000 title claims description 62
- 230000001900 immune effect Effects 0.000 title claims description 9
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 4
- 239000007788 liquid Substances 0.000 claims abstract description 119
- 238000002203 pretreatment Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 57
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 42
- 239000000427 antigen Substances 0.000 claims description 37
- 102000036639 antigens Human genes 0.000 claims description 37
- 108091007433 antigens Proteins 0.000 claims description 37
- 239000011248 coating agent Substances 0.000 claims description 35
- 238000000576 coating method Methods 0.000 claims description 35
- 238000002372 labelling Methods 0.000 claims description 30
- 239000008363 phosphate buffer Substances 0.000 claims description 30
- 235000013336 milk Nutrition 0.000 claims description 26
- 210000004080 milk Anatomy 0.000 claims description 26
- 238000004140 cleaning Methods 0.000 claims description 25
- 239000008267 milk Substances 0.000 claims description 25
- 102000014914 Carrier Proteins Human genes 0.000 claims description 22
- 108010078791 Carrier Proteins Proteins 0.000 claims description 22
- 239000012086 standard solution Substances 0.000 claims description 22
- 238000002156 mixing Methods 0.000 claims description 19
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 18
- 239000000758 substrate Substances 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000003814 drug Substances 0.000 claims description 15
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 210000004408 hybridoma Anatomy 0.000 claims description 13
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000012141 concentrate Substances 0.000 claims description 9
- 230000008878 coupling Effects 0.000 claims description 9
- 238000010168 coupling process Methods 0.000 claims description 9
- 238000005859 coupling reaction Methods 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 8
- 230000028327 secretion Effects 0.000 claims description 8
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 8
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims description 7
- 241001494479 Pecora Species 0.000 claims description 7
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 235000008476 powdered milk Nutrition 0.000 claims description 6
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 5
- 239000008351 acetate buffer Substances 0.000 claims description 5
- 238000004458 analytical method Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000005070 sampling Methods 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- NFJCQBGAUBIGKV-UHFFFAOYSA-N nitro dihydrogen phosphate Chemical compound OP(O)(=O)O[N+]([O-])=O NFJCQBGAUBIGKV-UHFFFAOYSA-N 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 abstract description 19
- 238000012216 screening Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000008157 ELISA kit Methods 0.000 abstract 3
- 239000000523 sample Substances 0.000 description 55
- 239000000203 mixture Substances 0.000 description 32
- 239000012530 fluid Substances 0.000 description 26
- 238000002360 preparation method Methods 0.000 description 25
- 238000002965 ELISA Methods 0.000 description 22
- 238000002835 absorbance Methods 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 229940098773 bovine serum albumin Drugs 0.000 description 9
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 239000012488 sample solution Substances 0.000 description 8
- 150000001718 carbodiimides Chemical class 0.000 description 7
- 238000004321 preservation Methods 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 239000006143 cell culture medium Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000013016 damping Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- FTOAOBMCPZCFFF-UHFFFAOYSA-N 5,5-diethylbarbituric acid Chemical compound CCC1(CC)C(=O)NC(=O)NC1=O FTOAOBMCPZCFFF-UHFFFAOYSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- 239000000376 reactant Substances 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- WBJINCZRORDGAQ-UHFFFAOYSA-N ethyl formate Chemical compound CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 239000013067 intermediate product Substances 0.000 description 3
- ZFSLODLOARCGLH-UHFFFAOYSA-N isocyanuric acid Chemical compound OC1=NC(O)=NC(O)=N1 ZFSLODLOARCGLH-UHFFFAOYSA-N 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- JDXQWYKOKYUQDN-UHFFFAOYSA-N 3-hydroxypyrrolidine-2,5-dione Chemical compound OC1CC(=O)NC1=O JDXQWYKOKYUQDN-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 208000006568 Urinary Bladder Calculi Diseases 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229960002319 barbital Drugs 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- OTNVGWMVOULBFZ-UHFFFAOYSA-N sodium;hydrochloride Chemical compound [Na].Cl OTNVGWMVOULBFZ-UHFFFAOYSA-N 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical class OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108010058846 Ovalbumin Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 239000004411 aluminium Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 108060003552 hemocyanin Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229940092253 ovalbumin Drugs 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000009967 tasteless effect Effects 0.000 description 1
- KSGWLNWYCHAFIX-UHFFFAOYSA-N triazine-4,5-diamine Chemical compound NC1=CN=NN=C1N KSGWLNWYCHAFIX-UHFFFAOYSA-N 0.000 description 1
- -1 triazines nitrogen heterocyclic ring organic compound Chemical class 0.000 description 1
- 210000001635 urinary tract Anatomy 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for detecting melamine and a special enzyme-linked immunosorbent assay kit thereof. The enzyme-linked immunosorbent assay kit for detecting the melamine comprises a hapten and a specific antibody of the melamine; and the specific antibody is the polyclonal antibody or the monoclonal antibody of the melamine. The kit adopts the high specific monoclonal antibody of the melamine, thereby ensuring the reliability of test results; the test results show that the kit is characterized by high specificity, high sensitivity, high precision and high accuracy; main reagents of the kit all adopt the form of working liquid, thereby having convenient use and low cost. The method for detecting the melamine by using the kit has simple operation, low requirements on the pre-treatment of samples and can rapidly detect large quantities of samples. Therefore, the use of the method for detecting by using the enzyme-linked immunosorbent assay kit can monitor on-site and be applicable to qualitative and quantitative screening of a large number of samples.
Description
Technical field
The present invention relates to a kind of method and special ELISA reagent kit thereof that detects melamine.
Background technology
Melamine is a kind of triazines nitrogen heterocyclic ring organic compound, and its structural formula is as shown in Figure 1.Its nitrogen content is up to 66%, and white monocrystalline Oblique Crystal is tasteless.Because of it is easy to buy and produces, cost is low, and intrinsic illegal manufacturer is to the deficiency of Kjeldahl, and it is made an addition in food and the feed to increase percent protein, and obtains interests.Though melamine belongs to low toxicity compounds, it can metabolism be a cyanuric acid in animal body, and melamine and cyanuric acid react and accumulate, and can cause vesical calculus and the urinary tract pathology.Tumor of bladder can appear in the vesical calculus long-time stimulus.Therefore, be necessary to set up the method for high, the highly sensitive detection melamine of a kind of degree of accuracy in practice.
At present; The conventional sense method of melamine residual amount mainly contains liquid chromatography/mass spectrometry method (LC/MS), high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrum (LC/MSMS) etc.; But these methods exist the instrument and equipment complicacy, testing process is loaded down with trivial details and to the demanding shortcoming of reviewer's technical ability, be not suitable for the examination of on-site supervision and a large amount of samples.
Summary of the invention
An object of the present invention is to provide a kind of enzyme linked immunological kit that detects melamine.
The enzyme linked immunological kit of detection melamine provided by the present invention comprises the specific antibody of haptens and melamine; Said specific antibody is the polyclonal antibody or the monoclonal antibody of said melamine; Said haptenic structural formula is following:
Wherein, the specific antibody of said haptens and melamine can exist with following any form:
1) said haptens and carrier protein are carried out coupling, obtain the conjugate of haptens and carrier protein, as coating antigen, said specific antibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said specific antibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
Said kit can also not only comprise the specific antibody of haptens and melamine but also comprise antiantibody; Said antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody; Wherein, said haptens and antiantibody exist with following any form:
1) said haptens and carrier protein are carried out coupling, obtain the conjugate of haptens and carrier protein, as coating antigen, said antiantibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said antiantibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
Said melamine polyclonal antibody or melamine monoclonal antibody all are that the conjugate with said melamine hapten and carrier protein obtains as immunogene; Said carrier protein can be thyroprotein, mouse haemocyanin, human albumin, bovine serum albumin, rabbit anteserum albumen, hemocyanin or ovalbumin etc.
Said monoclonal antibody is to be the antibody that secretion produces to melamine medicine monoclonal hybridoma strain C-3-4 of CGMCC No.2716 by preserving number.
Said melamine polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody.
For more convenient on-site supervision and great amount of samples examination, said kit also comprises melamine standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrates redissolution liquid;
Wherein, said concentrated cleaning solution can be 7.8-8.5 for the pH value, to contain final concentration in said concentrated cleaning solution be the 0.02-0.05% sodium azide, the final concentration in said concentrated cleaning solution is the phosphate buffer of 1.0-2.0% Tween-20,0.1-0.3mol/L; Said concentrated redissolution liquid can be that DMSO, the pH of 5-10% is the phosphate buffer of 6.5-6.7,0.1-0.2mol/L for containing final concentration in said concentrated redissolution liquid.Above-mentioned percentage composition is the quality percentage composition.
Used marker enzyme is horseradish peroxidase or alkaline phosphatase in the said enzyme labeling; When marker enzyme is horseradish peroxidase; Said colour developing liquid is made up of colour developing liquid A liquid and colour developing liquid B liquid; Colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1-2mol/L sulfuric acid or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer was the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
Said concentrated cleaning solution can be 8.2 for the pH value specifically, contain final concentration in said concentrated cleaning solution is 0.03% sodium azide, the final concentration in said concentrated cleaning solution is 2.0% Tween-20,0.2mol/L phosphate buffer; Said concentrated redissolution liquid can be that 8% DMSO, pH are 6.6, the 0.2mol/L phosphate buffer for containing final concentration in said concentrated redissolution liquid specifically; Said substrate colour developing liquid A liquid is urea peroxide, and said substrate colour developing liquid B liquid is tetramethyl benzidine; Said stop buffer is the hydrochloric acid of 2mol/L.Said percentage composition is the quality percentage composition.
Melamine is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.Adopt carbodiimide method to carry out coupling melamine hapten and thyroprotein and obtain immunogene; Melamine hapten and carrier protein to combine ratio to cross low or too high all unfavorable to immunity, haptens is that 23-28:1 is proper with the mol ratio that combines of thyroprotein.When the preparation coating antigen, the mole proportioning of melamine hapten and said carrier protein is that 26:1 is proper.Melamine hapten is through obtaining melamine and bromo-acetic acid tert-butyl through chemical reaction.
When making is coated with the ELISA Plate of coating antigen; The said damping fluid that encapsulates can be 9.0-9.6,0.1mol/L carbonate buffer solution for the pH value, and used confining liquid can be that bovine serum albumin(BSA), the pH value of 10% (quality percentage composition) is 8.4-8.8,0.1mol/L barbital sodium-hydrochloride buffer for containing final concentration in said confining liquid.
Another object of the present invention provides a kind of method that detects melamine.
The method of detection melamine provided by the present invention may further comprise the steps:
1) sample pre-treatments
When said sample was milk sample, said sample-pretreating method was: with said concentrated redissolution liquid and milk mixing, the volume ratio of wherein said milk and said concentrated redissolution liquid is 1:8-10, and the sample of getting behind the said mixing is used for analyzing;
When said sample is powdered milk sample; Said sample-pretreating method is: with acetate buffer solution ultrasonic dissolution milk powder; The proportioning of wherein said acetate buffer solution and said milk powder is 4-6ml:0.5-1.5g; The centrifuging and taking supernatant, with volume ratio and the said concentrated redissolution liquid mixing of said supernatant with 1:8-10, sampling is used for analyzing;
When said sample was feed, said sample-pretreating method was: with acetonitrile and feed mixing, the proportioning of wherein said acetonitrile and said feed is 8-12ml:0.5-1.5g, ultrasonic Extraction 8-15min then, centrifuging and taking supernatant; Said supernatant is dried up again, add normal hexane, mixing adds said concentrated redissolution liquid again, and the volume ratio of wherein said supernatant, said normal hexane and said concentrated redissolution liquid is 1:1:1, centrifuging and taking lower floor water; With said lower floor water and the said concentrated redissolution liquid volume ratio mixing with 1:8-10, sampling is used for analyzing;
2) utilize above-mentioned arbitrary described enzyme linked immunological kit to detect 1) in resulting sample.
By preserving number is that the monoclonal antibody of the melamine that melamine medicine monoclonal hybridoma strain C-3-4 secretion is produced of CGMCC No.2716 belongs to protection scope of the present invention.
Preserving number be CGMCC No.2716 melamine medicine monoclonal hybridoma strain C-3-4 is also belonged to protection scope of the present invention.
The enzyme linked immunological kit of detection melamine of the present invention mainly adopts the residual quantity of melamine in the qualitative or detection by quantitative sample of indirect competitive ELISA method.Adopt the melamine monoclonal antibody of high specific in this kit, guaranteed the reliability of testing result.Experimental result shows that this kit has specificity height, highly sensitive, characteristics such as degree of accuracy is high, accuracy height; The main agents of this kit all adopts the working fluid form, and is easy to use, with low cost.Detect the method for melamine with kit of the present invention, easy and simple to handle, simplified the step of traditional detection method, shortened the time of detecting, the pre-treatment of sample is required low, fast detecting batch samples simultaneously.Therefore, the method for utilizing enzyme linked immunological kit of the present invention to detect can be carried out the qualitative and quantitative examination of on-site supervision and suitable a large amount of samples, will in melamine detection, play a significant role.
Description of drawings
Fig. 1 is the structural formula of melamine.
Fig. 2 is the intermediate product I that produces in melamine and the bromo-acetic acid tert-butyl reaction.
Fig. 3 gets haptens for intermediate product I through hydrolysis reaction.
Fig. 4 is that haptens and carrier protein couplet thing, enzyme labeling thing are the typical curve of the kit of enzyme mark antiantibody for coating antigen.
Embodiment
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Embodiment 1, be that coating antigen, ELIAS secondary antibody are the preparation and the use of the kit of enzyme labeling thing with haptens and carrier protein couplet thing
One, be that coating antigen, ELIAS secondary antibody are that the detection principle of kit of enzyme labeling thing is following with melamine hapten and carrier protein couplet thing:
When the coating antigen on the ELISA Plate capillary strip is melamine hapten and carrier protein couplet thing; After in the ELISA Plate micropore, adding standard solution or sample solution; Add the melamine specific antibody; Melamine coupled antigen competition melamine specific antibody on residual melamine and the ELISA Plate adds enzyme mark antiantibody again and carries out amplification, with the colour developing of colour developing liquid in the sample; The content of sample light absorption value and melamine is negative correlation, relatively can draw the residual content of melamine in the sample with typical curve.Simultaneously also can be according to the depth of color on the ELISA Plate, through with the more rough judgement sample of series concentration melamine standard solution color in the concentration range of melamine.
Two, be that coating antigen, ELIAS secondary antibody are that the kit of enzyme labeling thing generally can comprise as follows with melamine hapten and carrier protein couplet thing:
1, be coated with the ELISA Plate of melamine hapten and carrier protein couplet thing, the concentration of coating antigen can be 0.15-0.25 μ g/ml.
2, enzyme mark antiantibody working fluid: ELIAS secondary antibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody with horseradish peroxidase-labeled; The dilution of ELIAS secondary antibody is that to contain final concentration in dilution be that lowlenthal serum, the pH of 6-8% (quality percentage composition) is 9.1-9.6,0.2-0.3mol/L phosphate buffer, and enzyme mark antiantibody working fluid dilutability is 1:500.
3, melamine specific antibody working fluid: can be melamine polyclonal antibody or melamine monoclonal antibody working fluid; With 3000 times of melamine monoclonal antibody dilutions, obtain the specific antibody working fluid with dilution, said dilution is that the pH value is the carbonate buffer solution of 9.3-9.7,0.2mol/L.
4, (Beijing Ai Jieer Science and Technology Ltd., CAS:108-78-1): 6 bottles, concentration is respectively 0 μ g/L to the melamine standard solution, 10 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L.The solution of preparation standard items is to contain that final concentration is that DMSO, the pH of 5-10% (quality percentage composition) is the phosphate buffer of 6.5-6.7,0.1-0.2mol/L in the solution of preparation standard items.
5, substrate colour developing liquid: be made up of A liquid and B liquid, substrate colour developing liquid A liquid is urea peroxide or hydrogen peroxide, 7ml/ bottle, 1 bottle; Substrate colour developing liquid B liquid is tetramethyl benzidine or o-phenylenediamine, 7ml/ bottle, 1 bottle.
6, stop buffer: 1-2mol/L hydrochloric acid or sulfuric acid.
7, concentrated cleaning solution: the final concentration that the pH value is 7.8-8.5, contain in cleansing solution is that 0.02-0.05% (quality percentage composition) sodium azide and the final concentration in cleansing solution are the Tween-20 of 1.0-2.0% (quality percentage composition), the phosphate buffer of 0.1-0.3mol/L; The 40ml/ bottle, 1 bottle.
8, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that DMSO, the pH of 5-10% (quality percentage composition) is the phosphate buffer of 6.5-6.7,0.1-0.2mol/L; The 200ml/ bottle, 1 bottle.
Three, be that coating antigen, ELIAS secondary antibody are the concrete composition and the preparation thereof of the kit of enzyme labeling thing with melamine hapten and carrier protein couplet thing:
(1) forms
1, be coated with the ELISA Plate of melamine hapten and bovine serum albumin(BSA) conjugate, the concentration of coating antigen can be 0.20 μ g/ml.
2, enzyme mark antiantibody working fluid: with the sheep anti mouse antiantibody of horseradish peroxidase-labeled; Enzyme mark antiantibody working fluid dilutability is 1:3000; The dilution of ELIAS secondary antibody is that lowlenthal serum, the pH of 7% (quality percentage composition) is 9.4, the 0.3mol/L phosphate buffer for final concentration in dilution, and enzyme mark antiantibody working fluid dilutability is 1:500.
3, melamine monoclonal antibody working fluid: monoclonal antibody is to be CGMCC No.2716 melamine medicine monoclonal hybridoma strain C-3-4 secretion is produced by preserving number; Melamine monoclonal antibody working fluid prepares according to following method: with dilution the melamine monoclonal antibody is diluted 3000 times; Obtain the monoclonal antibody working fluid, said dilution is that the pH value is 9.6, the phosphate buffer of 0.2mol/L.
4, the melamine standard solution (Beijing Ai Jieer Science and Technology Ltd., CAS:108-78-1): 6 bottles, concentration is respectively 0 μ g/L; 10 μ g/L; 30 μ g/L, 90 μ g/L, 270 μ g/L; 810 μ g/L, the solution of preparation standard items is that DMSO, the pH of 8% (quality percentage composition) is 6.6, the phosphate buffer of 0.2mol/L for final concentration in the solution of preparation standard items.
5, substrate colour developing liquid: be made up of A liquid and B liquid, substrate colour developing liquid A liquid is urea peroxide, 7ml/ bottle, 1 bottle; Substrate colour developing liquid B liquid is tetramethyl benzidine, 7ml/ bottle, 1 bottle.
6, stop buffer: 2mol/L hydrochloric acid.
7, concentrated cleaning solution: the pH value is 8.2, and the final concentration that contains in cleansing solution is that 0.03% (quality percentage composition) sodium azide, the final concentration in cleansing solution are 2.0% (quality percentage composition) Tween-20,0.2mol/L phosphate buffer; The 40ml/ bottle, 1 bottle.
8, concentrate to redissolve liquid: containing redissolving final concentration in the liquid is that 8% (quality percentage composition) DMSO, pH are 6.6, the 0.2mol/L phosphate buffer; The 200ml/ bottle, 1 bottle.
(2) preparation
1, is coated with the preparation of the ELISA Plate of haptens and bovine serum albumin(BSA) conjugate
(1) haptenic preparation
1) gets 0.5g (3.97mmol) melamine in the 50ml of drying round-bottomed flask; Add 35ml DMF stirring and dissolving, get 0.44g (7.93mmol) KOH, 0.12g anhydrous Na I adds in the above-mentioned solution, drip the 0.646ml bromo-acetic acid tert-butyl; After fully mixing, 65 ℃ of reactions of heat temperature raising 10h.Reaction is cooled to room temperature after finishing, and thin up is used ethyl acetate extraction, and drying is revolved steaming, gets yellow oil, and ethyl acetate: sherwood oil=2:1 crosses post and gets product I (as shown in Figure 2);
2) get 0.32g intermediate product I and use the 5ml dissolve with methanol, add the 5ml trifluoroacetic acid again, stirring at room 20h.Revolve after reaction is accomplished to steam to remove and desolvate, get grease,, in solution, drip sherwood oil with methyl alcohol and the dissolving of ethyl acetate 1:1 mixed solvent; Until the muddy appearance of adularescent, heating makes it to become clarification, places and cools off; Separate out white precipitate, vacuum filtration gets haptens product (as shown in Figure 3).
(2) preparation of coating antigen
Haptens and bovine serum albumin(BSA) are obtained coating antigen through the carbodiimide method coupling, and concrete steps are following:
Haptens 10mg, 40mg carbodiimides (DEC) and the 15mg N monohydroxy succinimide (NHS) of step (1) preparation fully are dissolved among the 1mL DMF, under heating condition, stir 24h, obtain reactant liquor I liquid; Take by weighing BSA 20mg, make it fully to be dissolved among the 3.0mL PBS (pH 8.2), obtain reactant liquor II liquid (the mole proportioning of haptens and bovine serum albumin(BSA) is 26:1); I liquid is slowly joined in the II liquid, and under room temperature, stir 5h, with 0.01mol/L PBS damping fluid dialysis 3d; Change dislysate every day 2 times, to remove unreacted small-molecule substance, with the centrifugal 30min of the speed of 12000rpm; Collect supernatant; Obtain the conjugate of haptens and bovine serum albumin(BSA), packing, subsequent use in-20 ℃ of preservations.
(3) be coated with the preparation of the ELISA Plate of coating antigen
With encapsulating damping fluid coating antigen is diluted to 0.15-0.25 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h; 4 ℃ are spent the night again, and the coating buffer that inclines is with cleansing solution washing 2 times; Each 30 seconds, clap and do, in every hole, add 150-200 μ l confining liquid then; 37 ℃ of incubation 1-2h, liquid is clapped and is done in the hole of inclining, and preserve with the vacuum seal of aluminium film dry back.
Wherein, the used damping fluid that encapsulates is that the pH value is 9.5, the 0.1mol/L carbonate buffer solution; Used confining liquid is that to contain in confining liquid final concentration be that bovine serum albumin(BSA), the pH value of 8% (quality percentage composition) is 8.6, barbital sodium-hydrochloride buffer of 0.1mol/L.
2, melamine MONOCLONAL ANTIBODIES SPECIFIC FOR
(1) immunogenic preparation
Melamine is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.
Adopt carbodiimide method to carry out coupling the above-mentioned haptens that obtains and thyroprotein and obtain immunogene, concrete steps are following:
Haptens 10mg, 40mg carbodiimides (DEC) and the 15mg N monohydroxy succinimide (NHS) of above-mentioned preparation fully are dissolved among the 1mL DMF, under heating condition, stir 24h, obtain reactant liquor I liquid; Take by weighing thyroprotein 25mg, make it fully to be dissolved among the 3.0mL PBS (pH 8.2), obtain reactant liquor II liquid; I liquid is slowly joined in the II liquid, and under room temperature, stir 5h, the mol ratio of haptens and thyroprotein is 26:1; With 0.01mol/L PBS damping fluid dialysis 3d, change dislysate every day 2 times, to remove unreacted small-molecule substance; With the centrifugal 30min of the speed of 12000rpm, collect supernatant, obtain the conjugate of haptens and thyroprotein; Packing, subsequent use in-20 ℃ of preservations.
(2) preparation monoclonal antibody
A. animal immune
The immunogene that step (1) is obtained is injected in the Balb/c mouse body, and immunizing dose is 150 μ g/, makes it produce antiserum.
B. Fusion of Cells and cloning
Get immune Balb/c mouse boosting cell; Merge in 9:1 (quantitative proportion) ratio and SP2/0 myeloma cell; Screening obtain stably excreting melamine monoclonal antibody to the strain of melamine medicine monoclonal hybridoma, with this cell line called after C-3-4, this cell line has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 20th, 2008 and (has been called for short CGMCC; Address: Datun Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.2716.
C. cell cryopreservation and recovery
C-3-4 processes 1 * 10 with cryopreserving liquid with hybridoma
9The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
D. MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying
Increment cultivation: hybridoma CGMCC No.2716 is placed cell culture medium, under 37 ℃ of conditions, cultivate, the nutrient solution that obtains is carried out purifying, obtain monoclonal antibody ,-20 ℃ of preservations with sad-saturated ammonium sulfate method.
Said cell culture medium is in the RPMI-1640 nutrient culture media, to add calf serum and soda mint; Making the final concentration of calf serum in cell culture medium is 20% (quality percentage composition), and making the final concentration of soda mint in cell culture medium is 0.2% (quality percentage composition); The pH of said cell culture medium is 7.4.
3, melamine Polyclonal Antibody Preparation
Adopting new zealand white rabbit as immune animal, is immunogene with haptens and thyroprotein conjugate, and immunizing dose is 1.5mg/kg; Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent; The subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3-4 week adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once; Immunity is 5 times altogether, does not add adjuvant for the last time.Serum antibody titer is measured in last immunity blood sampling after 10 days, and heart is taken a blood sample, and obtains the polyclonal antibody of purifying with ammonium sulfate precipitation.
4, horseradish peroxidase-labeled antiantibody:
The sheep anti mouse of used horseradish peroxidase-labeled, goat-anti rabbit antiantibody are all available from U.S. JACKSON company.
Four, be the application of the enzyme linked immunological kit of coating antigen with haptens and carrier protein couplet thing
Kit of the present invention can be used for detecting the melamine of samples such as milk powder, milk, feed.
1, tissue sample pre-treatment
Milk sample:
Draw 100 μ l fresh milks, add 900 μ l redissolution working fluid, mixing is got 50 μ l and is used for analyzing.
Powdered milk sample:
Take by weighing 1g milk powder, add 5ml 0.02M acetate buffer solution ultrasonic dissolution, centrifugal 5min more than the 4000g draws supernatant 100 μ l and adds 900 μ l redissolution working fluid mixing, gets 50 μ l and is used for analyzing.
The feed sample:
Take by weighing the 1g feed, add the 10ml acetonitrile, ultrasonic Extraction 10min, centrifugal 5min more than the 4000g; Draw the 1ml supernatant and dry up, add the 1ml normal hexane, whirling motion 30s in 45 ℃ of water-baths; Add 1ml redissolution working fluid, whirling motion 30s, centrifugal 5min more than the 4000g; Remove the upper strata normal hexane, take off layer water 100 μ l and add 900 μ l redissolution working fluid mixing, get 50 μ l and be used for analyzing.
2, detect
In the ELISA Plate micropore that is coated with the melamine coupled antigen, add melamine standard solution or sample solution 50 μ l, add melamine monoclonal antibody working fluid 50 μ l again, with cover plate mould shrouding; React 30min in 37 ℃ of constant temperature ovens; Pour out liquid in the hole, every hole adds 250 μ l cleansing solutions, pours out liquid in the hole behind the 30s; So repetitive operation is washed plate 5 times altogether, claps with thieving paper and does; Add the sheep anti mouse antiantibody working fluid 100 μ l of horseradish peroxidase-labeled, react 30min in 37 ℃ of constant temperature ovens, pour out liquid in the hole, repeat to wash the plate step; Every hole adds substrate colour developing liquid A liquid urea peroxide; Substrate colour developing liquid B liquid tetramethyl benzidine (TMB), the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 15min; Every hole adds 2mol/L stop buffer sulfuric acid 50 μ l; The mixing that vibrates gently at the 450nm place, is measured every hole absorbance (OD value) with the ELIASA wavelength set.
3, testing result analysis
The absorbance mean value (B) of standard solution of using each concentration that is obtained is divided by the absorbance (B of first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.
With melamine standard items concentration (μ g/L) value is the X axle, and the percentage absorbance is the Y axle, drawing standard curve map (Fig. 4).With the percentage absorbance of same way calculation sample solution, the concentration of corresponding each sample, the residual quantity that then can read melamine the sample from typical curve.The analysis of testing result also can be adopted regression equation method among the present invention, calculates sample solution concentration.The analysis of testing result can also utilize computer professional software among the present invention, the be more convenient for express-analysis of a large amount of samples of this method, and whole testing process only needed to accomplish in 75 minutes.
Five, kit sensitivity, precision, accuracy and storage life detect
(1) standard items precision test:
From embodiment 1, respectively extract 10 kits in the kit of the different batches (01 batch, 02 batch, 03 batch) of different time sections preparation in the step 3; From the ELISA Plate of each kit, respectively extract 20 micropores out; Measure the absorbance (OD value) of 270 μ g/L melamine standard solution, calculate the coefficient of variation.
Experimental result is as shown in table 1, and the coefficient of variation of standard items absorbance meets precision and is less than or equal to 20% regulation between 3.5%-9.4%.
Table 1, the repeatable tests of standard items (CV%)
(2) sample precision and accuracy test
1, sample precision test:
In the milk powder that does not contain melamine, milk, feed sample, add the melamine standard items, making the final concentration of melamine in sample is 2mg/kg, carries out sample pre-treatments more respectively according to the method described above.Respectively extract 3 kits in three batches the kit (01 batch, 03,06 batch) of the different time sections preparation from embodiment 1 described in the step 3, experimentize, each sample repeats 5 times, calculates the coefficient of variation (CV%) respectively.The result is (numerical value in each table is 5 mean values that repeat) shown in table 2, table 3 and table 4.
The result shows the Variation Lines number average of milk powder, milk, feed sample between 6.2%-15.2%, has met " Ministry of Agriculture's file " farming doctor [2005] No. 17 annex 2 kits and has put on record with reference to the 4th precision standard in the judgment criteria.
Table 2, the repeatable test of milk sample
Table 3, the repeatable test of powdered milk sample
Table 4, the repeatable test of feed sample
2, sample accuracy test
In the milk that does not contain melamine, milk powder, feed sample, add the melamine standard solution; In milk, powdered milk sample, make its final concentration be respectively 1mg/kg (L) and 2mg/kg (L); Make its final concentration be respectively 2mg/kg (L) and 4mg/kg (L) in the feed sample, handle according to the sample-pretreating method described in the embodiment 1 then; Detect with the kit described in the step 3 among the embodiment 1 again, each concentration do 4 parallel, accuracy in computation (accuracy=measured value/interpolation value) respectively.The result is as shown in table 5.The result shows that milk sample adds the recovery between 68.7%-97.6%, and powdered milk sample adds the recovery between 62.7%-96.8%, and the feed sample adds the recovery between 62.5%-89.6%.
The accuracy of table 5, kit
(3) cross reacting rate test:
Select to have 3 kinds of drug monitoring cross reacting rates of similar structures and similar functions with melamine.Typical curve through various medicines obtains its 50% inhibition concentration respectively.With kit in the computes step 3 to the cross reacting rate of other medicines.Kit is big more for the cross reacting rate of melamine, and then its specificity to this drug test is just good more.Replication 3 times, results averaged.
Cross reacting rate (%)=(cause 50% suppress melamine the similar substrate concentration of melamine that suppresses of concentration/cause 50%) * 100%
Experimental result is as shown in table 6, shows, kit of the present invention is good to the specificity of melamine, and kit promptly of the present invention can detect melamine.
The specificity of table 6, kit
| Medicine name | Cross reacting rate (%) |
| Melamine | 100 |
| Cyanuric acid | <1 |
| Triazine | <1 |
| Triazinediamine | <1 |
(4) kit storage life test
The kit preservation condition is 2-8 ℃, and through 6 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, melamine added the practical measurement value all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and kit the condition held of 37 ℃ of preservations 6 days, is carried out accelerated deterioration and tests, and the result shows that this kit each item index meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measure the result and show that also kit each item index is normal fully.Can draw kit from above result can preserve more than 6 months at 2-8 ℃ at least.
(5) LDL of kit
Get the negative milk sample that does not contain melamine, carry out 20 times respectively with kit in the step 3 and detect, the mean value of measuring the result adds the LDL of 3 times of standard deviations as kit.
The result is as shown in table 7, shows that the lowest detection of kit is limited to 99.7 μ g/kg.
Table 7, negative milk sample are measured statistical form (μ g/kg) as a result
Embodiment 2, the kit that is used to detect melamine can also have following several kinds:
One, coating antigen is a specific antibody, and the enzyme labeling thing is that enzyme is marked haptenic kit
(1) principle of work of this kit is:
When on capillary strip, encapsulating melamine specific antibody described in the embodiment 1 in advance, behind adding sample solution or the standard solution, add enzyme labeling haptens solution again.Melamine in the sample or melamine standard items and enzyme-labelled antigen competition are coated on the melamine specific antibody on the ELISA Plate; With the colour developing of colour developing liquid; The content of melamine becomes negative correlation in sample light absorption value and the sample, relatively can draw the content of melamine in the sample with typical curve.Simultaneously also can be according to the shade on the ELISA Plate, through with the more rough judgement sample of the melamine standard solution color of series concentration in the concentration range of melamine.
(2) consisting of of this kit:
(1) be coated with the ELISA Plate of coating antigen: coating antigen is the melamine monoclonal antibody, is that the monoclonal hybridoma strain C-3-4 secretion to the melamine medicine of CGMCC No.2716 produces by preserving number; The concentration of coating antigen can be 0.15-0.25 μ g/ml.
(2) enzyme labeling thing: the working fluid of enzyme mark melamine hapten; Marker enzyme is a horseradish peroxidase.
(3) (Beijing Ai Jieer Science and Technology Ltd., CAS:108-78-1): 6 bottles, concentration is respectively 0 μ g/L to the melamine standard solution, 10 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L.The solution of preparation standard items: final concentration is that DMSO, the pH of 10% (quality percentage composition) are the phosphate buffer of 6.5-6.7,0.2mol/L in the solution of said preparation standard items.
(4) substrate colour developing liquid: be made up of colour developing liquid A liquid and colour developing liquid B liquid, colour developing liquid A liquid is hydrogen peroxide, and colour developing liquid B liquid is o-phenylenediamine.
(5) stop buffer is the 1-2mol/L sulfuric acid solution.
(6) concentrated cleaning solution: the final concentration that the pH value is 8.5, contain in concentrated cleaning solution is that 0.05% (quality percentage composition) sodium azide, the final concentration in concentrated cleaning solution are the phosphate buffer of 2.0% (quality percentage composition) Tween-20,0.3mol/L; The 40ml/ bottle, 1 bottle.
(7) concentrate to redissolve liquid: containing concentrating the final concentration that redissolves in the liquid is that DMSO, the pH of 10% (quality percentage composition) is 6.7, the phosphate buffer of 0.2mol/L; The 200ml/ bottle, 1 bottle.
Two, coating antigen is that melamine hapten and carrier protein couplet thing, enzyme labeling thing are the kit of enzyme mark melamine specific antibody
(1) principle of work
When on capillary strip, encapsulating melamine hapten and carrier protein couplet thing in advance, behind adding sample solution or the standard solution, add melamine specific antibody described in the enzyme mark embodiment 1 again.The melamine hapten competition melamine specific antibody that encapsulates on melamine in the sample or melamine standard items and the ELISA Plate; With the colour developing of colour developing liquid; The content of melamine becomes negative correlation in sample light absorption value and the sample, relatively can draw the content of melamine in the sample with typical curve.Simultaneously also can be according to the shade on the ELISA Plate, through with the more rough judgement sample of series concentration melamine standard solution color in the concentration range of melamine.
(2) composition of this kit
(1) be coated with the ELISA Plate of coating antigen: coating antigen is melamine hapten and bovine serum albumin(BSA) conjugate.
(2) enzyme labeling thing: enzyme mark specific antibody working fluid, marker enzyme is an alkaline phosphatase; Specific antibody is a monoclonal antibody, is produced by the monoclonal hybridoma strain C-3-4 secretion to the melamine medicine that by preserving number is CGMCC No.2716.
(3) (Beijing Ai Jieer Science and Technology Ltd., CAS:108-78-1): 6 bottles, concentration is respectively 0 μ g/L to the melamine standard solution, 10 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L.The solution of preparation standard items: DMSO, the pH that contains final concentration in the solution of said preparation standard items and be 5% (quality percentage composition) is 6.5, the phosphate buffer of 0.1mol/L.
(4) developer is nitro phosphate buffer (a 4-nitrophenols phosphate buffer).
(5) stop buffer is the 1mol/L sodium hydroxide solution.
(6) concentrated cleaning solution: the final concentration that the pH value is 7.8, contain in said concentrated cleaning solution is that 0.02% (quality percentage composition) sodium azide, the final concentration in said concentrated cleaning solution are the phosphate buffer of 1.0% (quality percentage composition) Tween-20,0.1mol/L; The 40ml/ bottle, 1 bottle.
(7) concentrate to redissolve liquid: DMSO, the pH that contains final concentration in said concentrated redissolution liquid and be 5% (quality percentage composition) is 6.5, the phosphate buffer of 0.1mol/L; The 200ml/ bottle, 1 bottle.
Three, coating antigen is an antiantibody, and the enzyme labeling thing is an enzyme mark melamine hapten
(1) principle of work
When on capillary strip, encapsulating antiantibody in advance, after adding melamine specific antibody is hatched, add sample solution or standard solution, add enzyme mark melamine hapten again.Melamine in the sample or melamine standard items and enzyme mark melamine hapten competition melamine specific antibody; With the colour developing of colour developing liquid; The content of melamine becomes negative correlation in sample absorbance and the sample, relatively can draw the content of melamine in the sample with typical curve.Simultaneously also can be according to the shade on the ELISA Plate, through with the more rough judgement sample of series concentration melamine standard solution color in the concentration range of melamine.
(2) kit is formed as follows:
(1) be coated with the ELISA Plate of coating antigen: coating antigen is sheep anti mouse antiantibody or goat-anti rabbit antiantibody; The concentration of coating antigen can be 0.15-0.25 μ g/ml.
(2) enzyme labeling thing: melamine hapten described in the embodiment 1 of horseradish peroxidase-labeled;
(3) specific antibody working fluid: monoclonal antibody: be to be the monoclonal hybridoma strain C-3-4 secretion generation of CGMCC No.2716 by preserving number to the melamine medicine.
(4) (Beijing Ai Jieer Science and Technology Ltd., CAS:108-78-1): 6 bottles, concentration is respectively 0 μ g/L to the melamine standard solution, 10 μ g/L, 30 μ g/L, 90 μ g/L, 270 μ g/L, 810 μ g/L.The solution of preparation standard items: DMSO, the pH that contains final concentration in the solution of said preparation standard items and be 7% (quality percentage composition) is 6.6, the phosphate buffer of 0.15mol/L.
(5) substrate colour developing liquid: be made up of A liquid and B liquid, substrate colour developing liquid A liquid is urea peroxide, 7ml/ bottle, 1 bottle; Substrate colour developing liquid B liquid is o-phenylenediamine, 7ml/ bottle, 1 bottle.
(6) concentrated cleaning solution: the pH value is 8.0, and the final concentration that contains in said concentrated cleaning solution is that 0.04% (quality percentage composition) sodium azide, the final concentration in said concentrated cleaning solution are the phosphate buffer of 1.5% (quality percentage composition) Tween-20,0.15mol/L; The 40ml/ bottle, 1 bottle.
(7) concentrate to redissolve liquid: DMSO, the pH that contains final concentration in said concentrated redissolution liquid and be 8% (quality percentage composition) is 6.6, the phosphate buffer of 0.15mol/L; The 200ml/ bottle, 1 bottle.
(8) stop buffer: stop buffer is 1-2mol/L sulfuric acid or hydrochloric acid solution.
Claims (10)
1. enzyme linked immunological kit that detects melamine comprises the specific antibody of haptens and melamine; Said specific antibody is the monoclonal antibody of said melamine; Said haptenic structural formula is following:
Said monoclonal antibody is to be the antibody that secretion produces to melamine medicine monoclonal hybridoma strain C-3-4 of CGMCC No.2716 by preserving number.
2. kit according to claim 1 is characterized in that: the spy of said haptens and melamine
Heterogenetic antibody exists with following any form:
1) said haptens and carrier protein are carried out coupling, obtain the conjugate of haptens and carrier protein, as coating antigen, said specific antibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said specific antibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
3. kit according to claim 1 is characterized in that: said kit also comprises antiantibody; Said antiantibody is the sheep anti mouse antiantibody;
Said haptens and antiantibody exist with following any form:
1) said haptens and carrier protein are carried out coupling, obtain the conjugate of haptens and carrier protein, as coating antigen, said antiantibody carries out after the enzyme labeling as the enzyme labeling thing with it;
2) said antiantibody is a coating antigen, and said haptens carries out after the enzyme labeling as the enzyme labeling thing.
4. according to arbitrary described kit among the claim 1-3, it is characterized in that: said kit also comprises melamine standard solution, colour developing liquid, stop buffer, concentrated cleaning solution, concentrates redissolution liquid;
Said concentrated cleaning solution is that the pH value is 7.8-8.5, to contain quality final concentration in said concentrated cleaning solution be the 0.02-0.05% sodium azide, the quality final concentration in said concentrated cleaning solution is the phosphate buffer of 1.0-2.0% Tween-20,0.1-0.3mol/L; Said concentrated redissolution liquid is that to contain quality final concentration in said concentrated redissolution liquid be that DMSO, the pH of 5-10% is the phosphate buffer of 6.5-6.7,0.1-0.2mol/L.
5. according to claim 2 or 3 described kits, it is characterized in that: used marker enzyme is horseradish peroxidase or alkaline phosphatase in the said enzyme labeling; When marker enzyme is horseradish peroxidase; Said colour developing liquid is made up of colour developing liquid A liquid and colour developing liquid B liquid; Colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1-2mol/L sulfuric acid or hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer was the nitro phosphate buffer, and stop buffer is the 1-2mol/L sodium hydroxide solution.
6. kit according to claim 4 is characterized in that: said concentrated cleaning solution is that the pH value is 8.2, to contain quality final concentration in said concentrated cleaning solution be that 0.03% sodium azide, the quality final concentration in said concentrated cleaning solution are 2.0% Tween-20,0.2mol/L phosphate buffer; Said concentrated redissolution liquid is that to contain quality final concentration in said concentrated redissolution liquid be that 8% DMSO, pH are 6.6, the 0.2mol/L phosphate buffer.
7. kit according to claim 5 is characterized in that: said substrate colour developing liquid A liquid is urea peroxide, and said substrate colour developing liquid B liquid is tetramethyl benzidine; Said stop buffer is the hydrochloric acid of 2mol/L.
8. method that detects melamine may further comprise the steps:
1) sample pre-treatments
When said sample was milk sample, said sample-pretreating method was: will concentrate and redissolve liquid and milk mixing, the volume ratio of wherein said milk and said concentrated redissolution liquid is 1: 8-10, and the sample of getting behind the said mixing is used for analysis;
When said sample is powdered milk sample; Said sample-pretreating method is: with acetate buffer solution ultrasonic dissolution milk powder; The proportioning of wherein said acetate buffer solution and said milk powder is 4-6ml: 0.5-1.5g; The centrifuging and taking supernatant, with said supernatant with 1: the volume ratio of 8-10 with concentrate to redissolve the liquid mixing, sampling is used for analyzing;
When said sample was feed, said sample-pretreating method was: with acetonitrile and feed mixing, the proportioning of wherein said acetonitrile and said feed is 8-12ml: 0.5-1.5g, ultrasonic Extraction 8-15min then, centrifuging and taking supernatant; Said supernatant is dried up again, add normal hexane, mixing adds the concentrated liquid that redissolves again, and the volume ratio of wherein said supernatant, said normal hexane and said concentrated redissolution liquid is 1: 1: 1, centrifuging and taking lower floor water; With said lower floor water and said concentrated redissolution liquid with 1: the volume ratio mixing of 8-10, sampling are used for analyzing;
2) utilize that arbitrary described enzyme linked immunological kit detects 1 among the claim 1-6) in resulting sample.
9. by preserving number the monoclonal antibody of the melamine that secretion produces to melamine medicine monoclonal hybridoma strain C-3-4 of CGMCC No.2716.
Preserving number be CGMCC No.2716 to melamine medicine monoclonal hybridoma strain C-3-4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810227894A CN101413943B (en) | 2008-12-02 | 2008-12-02 | Method for detecting melamine and specific enzyme-linked immunologic reagent kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810227894A CN101413943B (en) | 2008-12-02 | 2008-12-02 | Method for detecting melamine and specific enzyme-linked immunologic reagent kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101413943A CN101413943A (en) | 2009-04-22 |
| CN101413943B true CN101413943B (en) | 2012-09-05 |
Family
ID=40594576
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810227894A Expired - Fee Related CN101413943B (en) | 2008-12-02 | 2008-12-02 | Method for detecting melamine and specific enzyme-linked immunologic reagent kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101413943B (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643454B (en) * | 2009-05-27 | 2011-05-18 | 北京维德维康生物技术有限公司 | Melamine hapten and antigen as well as preparation method and application thereof |
| CN101643453B (en) * | 2009-05-27 | 2011-05-18 | 北京维德维康生物技术有限公司 | Melamine hapten and antigen as well as preparation method and application thereof |
| CN101705210B (en) * | 2009-11-27 | 2011-10-05 | 北京生命科学研究所 | Anti-melamine monoclonal antibody and application thereof |
| CN102565400A (en) * | 2010-12-15 | 2012-07-11 | 北京勤邦生物技术有限公司 | Magnetic granule chemiluminescence kit for detecting melamine and application of magnetic granule chemiluminescence kit |
| CN102321177B (en) * | 2011-09-23 | 2013-05-01 | 福州大学 | Anti-melamine single-chain antibody and application thereof |
| CN102435729A (en) * | 2011-12-02 | 2012-05-02 | 杭州迪恩科技有限公司 | One-step enzyme-linked immunoassay method for quickly detecting melamine and kit thereof |
| CN102798711B (en) * | 2012-08-08 | 2015-01-07 | 广州市质量监督检测研究院 | Fluorescent brightening agent VBL detection kit and preparation method thereof |
| CN103145633B (en) * | 2013-02-06 | 2015-12-02 | 天津科技大学 | Novel melamine antigen and antibody and application |
| CN103342747B (en) * | 2013-07-16 | 2015-10-07 | 江苏省家禽科学研究所 | For the artificial antigen and antibody and preparation method thereof of melamine residual immunoassay |
| CN105277536A (en) * | 2015-03-31 | 2016-01-27 | 贵州勤邦食品安全科学技术有限公司 | Chemiluminescence ELISA kit for detecting melamine in milk |
| CN115650927A (en) * | 2022-09-29 | 2023-01-31 | 深圳市绿诗源生物技术有限公司 | Novel melamine hapten, complete antigen, antibody and preparation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101013130A (en) * | 2007-02-13 | 2007-08-08 | 北京望尔康泰生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furantoin metabolite and uses thereof |
| CN101183105A (en) * | 2007-12-13 | 2008-05-21 | 中国农业科学院农业质量标准与检测技术研究所 | ELISA reagent kit for detecting melamine |
| CN101206223A (en) * | 2007-12-13 | 2008-06-25 | 中国农业科学院农业质量标准与检测技术研究所 | Direct racing method ELISA reagent box for detecting melamine |
-
2008
- 2008-12-02 CN CN200810227894A patent/CN101413943B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101013130A (en) * | 2007-02-13 | 2007-08-08 | 北京望尔康泰生物技术有限公司 | Enzyme linked immunosorbent reagent casing for detecting furantoin metabolite and uses thereof |
| CN101183105A (en) * | 2007-12-13 | 2008-05-21 | 中国农业科学院农业质量标准与检测技术研究所 | ELISA reagent kit for detecting melamine |
| CN101206223A (en) * | 2007-12-13 | 2008-06-25 | 中国农业科学院农业质量标准与检测技术研究所 | Direct racing method ELISA reagent box for detecting melamine |
Non-Patent Citations (1)
| Title |
|---|
| 陈锡龙等.使用酶联免疫吸附法测定饲料中三聚氰胺的研究.《饲料工业》.2008,第29卷(第18期),53-55. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101413943A (en) | 2009-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101413943B (en) | Method for detecting melamine and specific enzyme-linked immunologic reagent kit | |
| CN101256188B (en) | ELISA kit for detecting lincomycin medicine as well as usage thereof | |
| CN101571539B (en) | Elisa kit for detecting cephalo-type medicine and application thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| CN102080066B (en) | Method for detecting T-2 toxin and special reagent kit thereof | |
| CN101776685B (en) | Enzyme linked immunosorbent assay kit for detecting trimethoprim medicament and application thereof | |
| CN101839918B (en) | Method for detecting spiramycin and special enzyme-linked immunoassay kit thereof | |
| CN103323593B (en) | A kind of test paper and application thereof detecting fluoroquinolones | |
| CN104655847A (en) | Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof | |
| CN101358967B (en) | Method for detecting chlorpromazine and special ELISA kit thereof | |
| CN103575889A (en) | Test strip and method for detecting vancomycin | |
| CN102080067B (en) | Method for detecting deoxynivalenol and special reagent kit thereof | |
| CN101349693A (en) | Fluorobenzene niekau series medicament fast detecting reagent kit and uses thereof | |
| CN102608316B (en) | Kit or test strip for detecting quinoxaline compound | |
| CN101571540A (en) | Enzyme-linked immunosorbent kit for inspecting porcine immunoglobulin G and application thereof | |
| CN100489530C (en) | Method for assaying sulfaquinoxaline and special enzyme-linked immune reagent kit | |
| CN101315378A (en) | Method and special ELISA reagent kit for detecting nitryl imidazole medicament | |
| CN101526528A (en) | Furaltadone metabolite detection kit | |
| CN101782579B (en) | Enzyme-linked immunoassay kit for detecting tiamulin medicaments, and application thereof | |
| CN100533148C (en) | Method for detecting tylosin and special enzyme-linked immune reagent kit thereof | |
| CN100582778C (en) | Method for detecting Ivermectin and special enzyme-linked immune reagent kit thereof | |
| CN103364553A (en) | Enzyme linked immunosorbent assay kit for detecting nitroimidazole drugs and application thereof | |
| CN100489532C (en) | Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof | |
| CN102565399A (en) | Method for detecting hydrocortisone and special enzyme-linked immunosorbent assay kit thereof | |
| CN103304495B (en) | A kind of olaquindox metabolite hapten preparation method and applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Free format text: FORMER OWNER: BEIJING WANGER BIOTECHNOLOGY CO., LTD. Effective date: 20120605 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20120605 Address after: 102206 Beijing city Changping Huilongguan international information industry base, four High Street No. 8 Applicant after: Beijing Wanger Biotechnology Co.,Ltd. Address before: 102206 Beijing city Changping Huilongguan international information industry base, four High Street No. 8 Applicant before: Beijing Wanger Kangtai Biotechnology Co.,Ltd. Co-applicant before: Beijing Wanger Biotechnology Co.,Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: GUIZHOU QINBANG FOOD SAFETY SCIENCE AND TECHNOLOGY Free format text: FORMER OWNER: BEIJING WANGER BIOTECHNOLOGY CO., LTD. Effective date: 20130605 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 102206 CHANGPING, BEIJING TO: 550009 GUIYANG, GUIZHOU PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20130605 Address after: 550009, Guizhou province Guiyang Xiaohe Industrial Park standard factory building No. two, building 1, building 1-2 Patentee after: GUIZHOU KWINBON SCIENCE AND TECHNOLOGY FOR FOOD SAFETY Co.,Ltd. Address before: 102206 Beijing city Changping Huilongguan international information industry base, four High Street No. 8 Patentee before: Beijing Wanger Biotechnology Co.,Ltd. |
|
| DD01 | Delivery of document by public notice |
Addressee: GUIZHOU KWINBON SCIENCE AND TECHNOLOGY FOR FOOD SAFETY Co.,Ltd. Document name: Notification to Pay the Fees |
|
| DD01 | Delivery of document by public notice | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120905 Termination date: 20211202 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |