CN1431503A - Test kit of enzyme linked immuno sorbent assay applicable to methylparathion retention analysis - Google Patents
Test kit of enzyme linked immuno sorbent assay applicable to methylparathion retention analysis Download PDFInfo
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- CN1431503A CN1431503A CN 03114896 CN03114896A CN1431503A CN 1431503 A CN1431503 A CN 1431503A CN 03114896 CN03114896 CN 03114896 CN 03114896 A CN03114896 A CN 03114896A CN 1431503 A CN1431503 A CN 1431503A
- Authority
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- China
- Prior art keywords
- parathion
- methyl
- applicable
- enzyme
- linked immunosorbent
- 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.)
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Links
- RLBIQVVOMOPOHC-UHFFFAOYSA-N parathion-methyl Chemical compound COP(=S)(OC)OC1=CC=C([N+]([O-])=O)C=C1 RLBIQVVOMOPOHC-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000012360 testing method Methods 0.000 title claims abstract description 18
- 238000002965 ELISA Methods 0.000 title claims description 31
- 238000004458 analytical method Methods 0.000 title claims description 23
- 230000014759 maintenance of location Effects 0.000 title claims description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 239000000427 antigen Substances 0.000 claims abstract description 15
- 102000036639 antigens Human genes 0.000 claims abstract description 15
- 108091007433 antigens Proteins 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 12
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims abstract description 9
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 238000008157 ELISA kit Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- 238000010790 dilution Methods 0.000 claims description 11
- 239000012895 dilution Substances 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical group OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 241000283707 Capra Species 0.000 claims description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 4
- 238000000576 coating method Methods 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 4
- 235000019800 disodium phosphate Nutrition 0.000 claims description 4
- 235000020183 skimmed milk Nutrition 0.000 claims description 4
- 239000012086 standard solution Substances 0.000 claims description 4
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 3
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 2
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 2
- 239000001103 potassium chloride Substances 0.000 claims description 2
- 235000011164 potassium chloride Nutrition 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims 2
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical group C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 claims 1
- 108010058846 Ovalbumin Proteins 0.000 claims 1
- 125000004202 aminomethyl group Chemical group [H]N([H])C([H])([H])* 0.000 claims 1
- 229940078916 carbamide peroxide Drugs 0.000 claims 1
- 229940092253 ovalbumin Drugs 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 33
- 102000004190 Enzymes Human genes 0.000 abstract description 11
- 108090000790 Enzymes Proteins 0.000 abstract description 11
- 239000000843 powder Substances 0.000 abstract description 2
- 239000012530 fluid Substances 0.000 abstract 3
- 238000004140 cleaning Methods 0.000 abstract 1
- 239000008267 milk Substances 0.000 abstract 1
- 210000004080 milk Anatomy 0.000 abstract 1
- 235000013336 milk Nutrition 0.000 abstract 1
- 238000007781 pre-processing Methods 0.000 abstract 1
- 230000002860 competitive effect Effects 0.000 description 12
- 238000001514 detection method Methods 0.000 description 12
- 239000003905 agrochemical Substances 0.000 description 10
- 239000000575 pesticide Substances 0.000 description 9
- 235000013311 vegetables Nutrition 0.000 description 9
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 239000002689 soil Substances 0.000 description 8
- 230000002401 inhibitory effect Effects 0.000 description 7
- 231100000572 poisoning Toxicity 0.000 description 7
- 230000000607 poisoning effect Effects 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- BAFQDKPJKOLXFZ-UHFFFAOYSA-N Paraoxon-methyl Chemical compound COP(=O)(OC)OC1=CC=C([N+]([O-])=O)C=C1 BAFQDKPJKOLXFZ-UHFFFAOYSA-N 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000003018 immunoassay Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- WYMSBXTXOHUIGT-UHFFFAOYSA-N paraoxon Chemical compound CCOP(=O)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 WYMSBXTXOHUIGT-UHFFFAOYSA-N 0.000 description 3
- 229960004623 paraoxon Drugs 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 238000003811 acetone extraction Methods 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 238000004454 trace mineral analysis Methods 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical group CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000872931 Myoporum sandwicense Species 0.000 description 1
- 230000000895 acaricidal effect Effects 0.000 description 1
- 239000000642 acaricide Substances 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000010794 food waste Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000026781 habituation Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000007037 hydroformylation reaction Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000003986 organophosphate insecticide Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000000447 pesticide residue Substances 0.000 description 1
- 238000005064 physico chemical analysis method Methods 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 231100000004 severe toxicity Toxicity 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The test kit includes the box body, the 96-eyelets enzyme target/test tube in the box, and the reagent in the box. In each eyelet of the enzyme target, the peridium fluid coats the peridium antigen, which generates the specificity fixatino reaction with anti metacide antibody and the 1.0-3.0% defatted milk powder used to carry out the closing. The reagent includes the cleaning solution, the diluentt, and standard metacide fluid, the anti metacide antibody, the horseradish peroxidase marked rabbit antibody of anti metacide, the substrate, the coloration matter and the reaction termination fluid. The invention provides the fast testing, the simple procedure for preprocessing the sample, not time-consuming, and capable of testing in batch mode.
Description
Technical field
The present invention relates to a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis, mainly be applicable to the parathion-methyl in environmental sample such as in batches water sample of this kit fast measuring, soil and the food samples such as poisoning sample and vegetables residual.
Background technology
The traditional residue analysis method of agricultural chemicals and metabolin thereof mainly is to rely on gas chromatography (GC), high performance liquid chromatography (HPLC), mass spectrum physicochemical analysis means such as (MS), but because agricultural chemicals uses scale constantly to enlarge, residues of pesticides cause the chronic and long-term effect of environmental impact and human health to be subjected to people's concern and worry day by day, restriction to residues of pesticides is also more and more stricter, to the assay determination object, kind, quantity, scope, aspects such as index have all proposed new requirement and higher standard, but traditional common very complicated of physico-chemical analysis method, the sample pretreatment process complexity, workload is big, the instrument costliness, and require to have those skilled in the art and long analytical cycle.Therefore people urgently wish to have a profit simple, and fast, sensitive and cheap detection technique can be carried out large batch of shaker test in the open air with in the laboratory.Immunoassay is just possessing these advantages, thus very short although immunoassay is used for the time of pesticide residue analysis, be used for the analysis of environmental sample and food residues of pesticides very soon.
Parathion-methyl (Parathion-methyl, O, O-dimethyl-O-p-nitrophenyl phosphorothionate), from 1949 by Bayer Leverkusen develop and spread after promptly as a kind of broad spectrum activity efficient insecticide, acaricide, worldwide be widely used in the control of insect of industrial crops such as grain, cotton.But because parathion-methyl is equivalent to 1/3 of parathion to people and animals' toxicity, but still belong to severe toxicity, and interior absorption is arranged, limited its use.However, in recent years,, still of common occurrence because of the report of methyl parathion poisoning.Especially a large amount of uses on crop and vegetables cause great harm to health, and this has caused people's attention.Therefore, develop a kind of simple fast, be applicable to that the trace analysis method of residues of pesticides on-site supervision has important practical significance.
Detecting parathion-methyl residual quantity conventional method is vapor-phase chromatography etc.Yet the sensitivity of this method be subjected to sample purification, step such as concentrate influence very big, moreover this method needs expensive instrument, and process is loaded down with trivial details, is not suitable for the detection and the analysis of batch samples.Immunoassay provides a new analyzing and testing approach for the residue detection of parathion-methyl.The report of at present existing parathion-methyl immune analysis method, but immune analysis method will be applied in the reality, also need it is developed into kit.This does not at home and abroad appear in the newspapers as yet.
Summary of the invention
The purpose of this invention is to provide that a kind of to have high specific, high sensitivity, pin-point accuracy, pinpoint accuracy, method of operating simply quick, and can be used for the enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis of batch samples fast detecting.
It comprises box body, is located at the 96 holes/40 hole ELISA Plate in the box body and is located at the interior reagent of box body, it is characterized in that, in every hole of ELISA Plate, by the coating buffer bag by can with the envelope antigen of anti-parathion-methyl antibody specificity association reaction, and seal with 1.0～3.0% skimmed milk powers, reagent comprises cleansing solution, substrate dilution, parathion-methyl standard solution, horseradish peroxidase-labeled goat anti-rabbit antibody or the anti-parathion-methyl rabbit of horseradish peroxidase-labeled antibody, substrate, substance that show color and reaction terminating liquid in the box.
Advantage of the present invention is to be used for the residual detections of food parathion-methyl such as water sample, soil, poisoning sample, vegetables, and the pre-treatment process of sample is simple, and is consuming time few, can detect sample in batches simultaneously, and the sample detection cost is far below traditional detection method.The antibody that kit adopts strong specificity, height to tire improves the sensitivity, accuracy, the precision that detect.The storage life of kit was above 6 months.The present invention is simply quick, is applicable to that the trace analysis method of residues of pesticides on-site supervision has important practical significance.
Description of drawings
Fig. 1 is a direct competitive ELISA method parathion-methyl typical curve;
Fig. 2 is an indirect competitive ELISA method parathion-methyl typical curve.
Embodiment
The present invention is a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis, and it is based on immune response and enzymatic reaction, can detect the residual of parathion-methyl in the food such as water sample, soil, poisoning sample, vegetables.A kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis, it comprises box body, be located at the 96 holes/40 hole ELISA Plate/test tubes in the box body and be located at the interior reagent of box body, it is characterized in that, in every hole of ELISA Plate, by the coating buffer bag by can with the envelope antigen of anti-parathion-methyl antibody specificity association reaction, and seal with 1.0～3.0% skimmed milk powers, reagent comprises cleansing solution (dilution) in the box, the substrate dilution, the parathion-methyl standard solution, horseradish peroxidase-labeled goat anti-rabbit antibody (being applicable to the indirect competitive ELISA method), the anti-parathion-methyl rabbit of horseradish peroxidase-labeled antibody (being applicable to direct competitive ELISA method), 30% hydrogen peroxide or 0.75% hydrogen peroxide urea, substance that show color and reaction terminating liquid, wherein: (1) envelope antigen (M1605-OVA) is used pH9.6,0.05mol/L carbonate buffer solution (contain 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L) is diluted to 0.5～4 μ g/mL, (2) cleansing solution (dilution) is one bottle, 40～80mL/ bottle, contain sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 0.1～0.3g, Tween-20 0.5～3mL, distilled water is normal 15～30 times of concentrates that use; (3) the substrate dilution is one bottle, and 30～50mL/ bottle is formulated as follows: citric acid 3～6g, and sodium hydrogen phosphate 1～3g, distilled water is normal 5～10 times of concentrates that use; (4) zymolyte is the hydrogen peroxide urea of 30% hydrogen peroxide or 0.75%, 10～15mL/ bottle; (5) substance that show color is 3,3 ', 5,5 '-tetramethyl benzidine (TMB) solution, and 4～6 of 10～15mL/ bottle or o-phenylenediamine pressed powders, 10～20mg/ props up; (6) horseradish peroxidase-labeled anti-parathion-methyl rabbit antibody or horseradish peroxidase-labeled goat anti-rabbit antibody are each one bottle, and 200～400 μ L/ bottles are 800～1500 times of concentrates of normal use; (7) reaction terminating liquid is one bottle, and 30～50mL/ bottle is 2mol/L sulfuric acid; (8) parathion-methyl variable concentrations series (0.1,0.5,2,10,50,100mg/L) titer is 6 bottles, 1～4mL/ bottle, methanol constant volume, during use with 10 times of PBST dilutions.The specificity of this kit is good, with the cross reacting rate of methyl paraoxon be 25%, with the cross reacting rate of parathion be 3%, with the cross reacting rate of paraoxon be＞3%.The lowest detection of indirect competitive ELISA method is limited to 0.005mg/L, and linear detection range is 0.005～10mg/L, sample detection batch in, batch between, whole Variation Lines number average is lower than between 8.00%, the recovery all is higher than 89.62%; The lowest detection of direct competitive ELISA method is limited to 0.01mg/L, linear detection range is 0.01～10mg/L, sample detection batch in, batch between, whole Variation Lines number average is lower than 8.0%, water, soil, the vegetables recovery all are higher than 90.24%, and the recovery of poisoning sample (qualitative analysis) is higher than 57.78%.Kit can be preserved more than 6 months under 4 ℃ or 20 ℃ at least.
Embodiment 1
Its measuring principle is, at first the compound that pesticide molecule and macromolecular carrier (as protein) coupling are made is adsorbed on the solid phase carrier as envelope antigen, add agricultural chemicals to be measured and enzyme labelled antibody then, agricultural chemicals on the solid phase antigen, the reaction that is at war with of agricultural chemicals to be measured and enzyme labelled antibody, pesticide concentration to be measured is many, the enzyme labelled antibody that then is bonded on the solid phase antigen is few, otherwise the enzyme labelled antibody that is combined in solid phase antigen is many, adding substrate in reaction back develops the color and is measured, when one timing of enzyme labelled antibody amount, the pesticide volume to be measured of adding is many more, and the enzyme labelled antibody that combines with solid phase antigen is just few more, the color development habituation, inhibiting rate increases, otherwise, color development increased response then, inhibiting rate lowers, thereby according to the standard lines of known quantity agricultural chemicals and the inhibiting rate of sample to be checked, mapping promptly gets typical curve according to the relation of the semilog between inhibiting rate and the pesticide concentration again, and extrapolates the concentration of agricultural chemicals to be measured.
Embodiment 2
The preparation of enzyme labelled antibody (adopting improvement sodium periodate method)
Concrete operations are as follows: claim that 5～10mgHRP is dissolved in the 1mL distilled water, add the 0.1mol/L NaIO that 0.2～0.4mL newly joins in last liquid
4Solution, lucifuge stirred 15～30 minutes under the room temperature.Above-mentioned solution is packed in the bag filter, and with the acetate buffer dialysis of 1mmol/L pH4.4,4 ℃ are spent the night.Add 20～40 μ l0.2mol/L pH9.5 carbonate buffer solutions, make the pH of above hydroformylation HRP be elevated to 9.0～9.5, add the 0.01mol/L carbonate buffer solution that 1～2ml contains 10～20mg antibody purification then immediately, the room temperature lucifuge stirred 2～3 hours gently.Add the 4mg/mL NaBH that 0.1～0.2mL newly joins
4Liquid, mixing, put again 4 ℃ 2～3 hours.Reactant liquor is packed in the bag filter, and with 0.15mol/L pH7.4 PBS dialysis, 4 ℃ are spent the night.Under agitation dropwise add equal-volume saturated ammonium sulfate solution, put 4 ℃ 1～2 hour.3000rpm centrifugal half an hour, abandon supernatant.Sediment is washed secondary with the semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15mol/LpH7.4.Above-mentioned solution is packed in the bag filter,, remove (detecting) behind the ammonium ion with Nai Shi reagent to the PBS buffer saline dialysis of 0.15mol/L pH7.4,10,000～12, centrifugal 30 minutes of 000rpm, supernatant is enzyme conjugates, after the packing of equivalent glycerine, respectively at-4 ℃ ,-20 ℃ preservations.Measure through direct ELISA method (E-Ab method), tiring is 4000.
Embodiment 3
The preparation of coated elisa plate
Envelope antigen (M1605-OVA) is used pH9.6,0.05mol/L carbonate buffer solution (contain 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L) be diluted to 0.5～4 μ g/mL, add 100 μ L in every hole of ELISA Plate, 4 ℃ down bag spent the night or 37 ℃ of bags by 2h, coating buffer inclines, with PBST washing 3 times, pat dry, in every hole, add 150 μ L1.0～3.0% skimmed milk power then, put into 37 ℃ of incubators and wash 3 times with PBST after 0.4～1 hour, pat dry the back kept dry.
Embodiment 4
The pre-treatment of test sample:
Water sample: can take a sample after the filtration and carry out elisa assay.
Soil sample: get 10g soil with 20～40mL acetone extraction three times, merge extract, concentrate, be settled to 10mL with the PBST dilution then, carry out elisa assay.
Vegetable sample: get and take by weighing 10g after vegetable sample rubs with comminutor, 20～40mL acetone extraction three times merges extract, concentrates, and is settled to 10mL with PBST, and elisa assay is carried out in sampling.
Blood: get blood of human body, directly analyze after adding the anti-freezing element with the ELISA method.
Liquid of gastric lavage (2% sodium bicarbonate solution): getting the 10mL liquid of gastric lavage, is that available ELISA method is analyzed with rare HCl adjust pH after neutrality.
Vomitus: sample thief grinds, and the centrifuging and taking supernatant is analyzed with the ELISA method.
Embodiment 5
The kit operating process is as follows:
1) direct competitive ELISA method: take out and be coated with parathion-methyl envelope antigen ELISA Plate, return to after the room temperature standby; The sample that adds 50 μ L standard specimens or handle well is in hole separately, and standard specimen and sample are done 2～4 repetitions; Add the enzyme labelled antibody of 50 μ L dilution, hatched 1～2 hour for 37 ℃; Pour out the liquid in the hole, microwell plate is upside down on the thieving paper pats,, dilute good PBST with 200 μ L and wash 2～6 times, pat dry to guarantee to remove fully the liquid in the hole; Every then hole adds the mixed liquid of 100 μ L substrate solutions and substance that show color, and it is even slightly to shake, and hatches 15～25 minutes 37 ℃ of dark places; Add 50 μ L reaction terminating liquids, after mixing, measure OD
450nmValue or OD
490nmValue.
2) indirect competitive ELISA method: take out and be coated with parathion-methyl envelope antigen ELISA Plate, return to after the room temperature standby; The sample that adds 50 μ L standard specimens or handle well is in hole separately, and standard specimen and sample are done 2～4 repetitions; Add the antibody of 50 μ L dilution, hatched 1～2 hour for 37 ℃; Pour out the liquid in the hole, microwell plate is upside down on the thieving paper pats,, dilute good PBST with 200 μ L and wash 2～6 times to guarantee to remove fully the liquid in the hole; Add 100 μ L and diluted ELIAS secondary antibody, hatched 1～2 hour for 37 ℃; Pour out the liquid in the hole, microwell plate is upside down on the thieving paper pats,, dilute good PBST with 200 μ L and wash 2～6 times to guarantee to remove fully the liquid in the hole; Every then hole adds the mixed liquid of 100 μ L substrate solutions and substance that show color, and it is even slightly to shake, and hatches 15～25 minutes 37 ℃ of dark places; Add 50 μ L reaction terminating liquids, after mixing, measure OD
450nmValue or OD
490nmValue.
Inhibiting rate with the light absorption value in each hole of mean value calculation of the standard specimen that obtained and sample light absorption value
OD
MaxLight absorption value during for not dosing, OD
xLight absorption value during for agricultural chemicals x, OD
MinLight absorption value for the blank hole
It is the semilog coordinate system curve map of a corresponding parathion-methyl concentration (mg/L) that the standard specimen value of calculating plots, and the calibration curve of direct competitive ELISA method is linear in 0.01～10mg/L scope; The calibration curve of indirect competitive ELISA method is linear in 0.005～10mg/L scope, and counter sample concentration can be read from calibration curve, also can obtain linear equation according to the concentration and the inhibiting rate of standard specimen, obtains the concentration of counter sample then.
The test of embodiment 6 storage lives
Kit being positioned over 4 ℃ and-20 ℃ of preservations, getting 0,10,20,30,60,90,120,150 and the kit of 180d respectively, serves as to measure concentration with optimum antibody antigen working concentration, carries out standard model and detects to measure it and detect effect.Storage life measurement result such as following table:
Table 1. direct competitive ELISA method kit storage life test findings
Table1.The?Validity?of?the?Direct?ELISA?kits
| Time (d) | 0 | ?10 | ?20 | ?30 | ?60 | ?90 | ?120 | ?150 | ?180 |
| ?OD 450nm?(4℃) | 1.078 | ?1.073 | ?1.073 | ?1.072 | ?1.071 | ?1.068 | ?1.063 | ?1.055 | ?1.043 |
| ?OD 450nm?(20℃) | 1.079 | ?1.074 | ?1.074 | ?1.076 | ?1.072 | ?1.074 | ?1.073 | ?1.072 | ?1.069 |
Table 2. indirect competitive ELISA method kit storage life test findings
Table2.The?Validity?of?the?Indirect?ELISA?kits
Above result as can be seen, kit can be preserved more than 6 months under 4 ℃ at least.
| Time (d) | 0 | ?10 | ?20 | ?30 | ?60 | ?90 | ?120 | ?150 | ?180 |
| ?OD 450nm?(4℃) | 1.143 | ?1.142 | ?1.139 | ?1.138 | ?1.137 | ?1.136 | ?1.133 | ?1.125 | ?1.120 |
| ?OD 450nm?(20℃) | 1.144 | ?1.142 | ?1.140 | ?1.140 | ?1.140 | ?1.141 | ?1.139 | ?1.140 | ?1.137 |
Embodiment 7
The kit sensitivity determination
The parathion-methyl standard solution is diluted to series concentration, obtain respectively with lower curve (Fig. 1) with the analysis of direct ELISA method, by Tu Kede, directly the ELISA method is: y=5.034+0.6588x, parathion-methyl in 0.01mg/L～10mg/L scope, Logit (B/B
0) with the logarithm value significant linear of parathion-methyl concentration relation, related coefficient is r
2=0.9965, detect and be limited to 0.01mg/L.Obtain respectively with lower curve (Fig. 2) with the indirect elisa method analysis, by Tu Kede, indirect elisa method is: y=5.2062+0.6766x, parathion-methyl in 0.005mg/L～10mg/L scope, Logit (B/B
0) with the logarithm value significant linear of parathion-methyl concentration relation, related coefficient is r
2=0.9935, detect and be limited to 0.005mg/L.
Embodiment 8
The test of accuracy test precision
Get the parathion-methyl standard specimen of three concentration, add in the sample, each concentration is established 6 repetitions, measures.The result of the kit recovery is as follows, and water is 95.30%～115.24%, and soil is 93.97%～98.92%, and vegetables are 91.24%～97.24%, and poisoning sample (qualitative reaction) is 56.78%～96.24%.The Variation Lines number average of water sample is lower than 7%, and the Variation Lines number average of soil is lower than 8%, and the Variation Lines number average of vegetables is lower than 6%, and it is 9% that the Variation Lines number average of poisoning sample is lower than.Embodiment 9
The test of kit specificity
The analog of selection parathion-methyl such as methyl paraoxon, parathion, paraoxon, reactions steps is operated with kit, obtains concentration in the various agricultural chemicals inhibition.Calculate the cross reactivity of agricultural chemicals with following formula again to parathion-methyl.Cross reacting rate is littler, and the specificity of reaction is stronger.Cross reacting rate is bigger, and cross reacting rate can be calculated as follows,
This test determination the results are shown in Table 3.Can know that from table 3 the indirect elisa method inhibiting rate reaches at 50% o'clock, the parathion-methyl desired concn is 30ug/L, and other several organophosphorus insecticide desired concns are 120～1000ug/L.The specificity that this kit is described is good, can guarantee the reliability to parathion-methyl determined result of residue in the sample.
The test of table 3. kit specificity
Table3.Recognition?of?Several?Compounds?by?ELISA?kits
| The compound title | Concentration in the inhibition (mg/L) | Cross reaction percent (%) |
| Parathion-methyl | ????0.8 | ????/ |
| Methyl paraoxon | ????3.9 | ????25 |
| Parathion | ????33.4 | ????3 |
| Paraoxon | ????＞33.4 | ????＞3 |
Claims (8)
1. enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis, it comprises box body, be located at the 96 holes/40 hole ELISA Plate/test tubes in the box body and be located at the interior reagent of box body, it is characterized in that, in every hole of ELISA Plate, by the coating buffer bag by can with the envelope antigen of anti-parathion-methyl antibody specificity association reaction, and seal with 1.0～3.0% skimmed milk powers, reagent comprises cleansing solution in the box, the substrate dilution, the parathion-methyl standard solution, the anti-parathion-methyl rabbit of horseradish peroxidase-labeled goat anti-rabbit antibody or horseradish peroxidase-labeled antibody, substrate, substance that show color and reaction terminating liquid.
2. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said envelope antigen is the compound of amino methyl parathion and ovalbumin.
3. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said cleansing solution contains sodium chloride 7～9g, potassium dihydrogen phosphate 0.1～0.3g, sodium hydrogen phosphate 2～4g, potassium chloride 0.1～0.3g, Tween-20 0.5～3mL, distilled water.
4. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said substrate dilution contains citric acid 3～6g, sodium hydrogen phosphate 1～3g, distilled water.
5. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said substrate is hydrogen peroxide or carbamide peroxide.
6. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said substance that show color is tetramethyl benzidine or o-phenylenediamine.
7. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said reaction terminating liquid is sulfuric acid or hydrochloric acid.
8. a kind of enzyme-linked immunosorbent assay kit that is applicable to the parathion-methyl retention analysis according to claim 1 is characterized in that said carbonate buffer solution, contains 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03114896 CN1431503A (en) | 2003-01-13 | 2003-01-13 | Test kit of enzyme linked immuno sorbent assay applicable to methylparathion retention analysis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03114896 CN1431503A (en) | 2003-01-13 | 2003-01-13 | Test kit of enzyme linked immuno sorbent assay applicable to methylparathion retention analysis |
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| Publication Number | Publication Date |
|---|---|
| CN1431503A true CN1431503A (en) | 2003-07-23 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477120A (en) * | 2008-12-18 | 2009-07-08 | 孙家隆 | Enzyme linked immunosorbent assay reagent kit for simultaneously analyzing chlorpyrifos and methyl parathion |
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2003
- 2003-01-13 CN CN 03114896 patent/CN1431503A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477120A (en) * | 2008-12-18 | 2009-07-08 | 孙家隆 | Enzyme linked immunosorbent assay reagent kit for simultaneously analyzing chlorpyrifos and methyl parathion |
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