CN105486869A - Enzyme linked immunosorbent assay kit for detecting methimazole residues in animal-derived food and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting methimazole residues in animal-derived food and application thereof Download PDFInfo
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- CN105486869A CN105486869A CN201410523551.6A CN201410523551A CN105486869A CN 105486869 A CN105486869 A CN 105486869A CN 201410523551 A CN201410523551 A CN 201410523551A CN 105486869 A CN105486869 A CN 105486869A
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- PMRYVIKBURPHAH-UHFFFAOYSA-N methimazole Chemical group CN1C=CNC1=S PMRYVIKBURPHAH-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 241001465754 Metazoa Species 0.000 title claims abstract description 12
- 235000013305 food Nutrition 0.000 title claims abstract description 11
- 238000008157 ELISA kit Methods 0.000 title abstract 2
- 102000004190 Enzymes Human genes 0.000 claims abstract description 33
- 108090000790 Enzymes Proteins 0.000 claims abstract description 33
- 239000007788 liquid Substances 0.000 claims abstract description 21
- 239000000758 substrate Substances 0.000 claims abstract description 15
- 229960002178 thiamazole Drugs 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 17
- 239000000427 antigen Substances 0.000 claims description 15
- 102000036639 antigens Human genes 0.000 claims description 15
- 108091007433 antigens Proteins 0.000 claims description 15
- 239000003550 marker Substances 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 11
- 238000012360 testing method Methods 0.000 claims description 11
- 239000012530 fluid Substances 0.000 claims description 9
- 230000001900 immune effect Effects 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 8
- 238000002372 labelling Methods 0.000 claims description 8
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 7
- 229940005654 nitrite ion Drugs 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000008363 phosphate buffer Substances 0.000 claims description 6
- 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 description 4
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- AQLJVWUFPCUVLO-UHFFFAOYSA-N urea hydrogen peroxide Chemical compound OO.NC(N)=O AQLJVWUFPCUVLO-UHFFFAOYSA-N 0.000 claims description 4
- 108010058846 Ovalbumin Proteins 0.000 claims description 3
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 229920000053 polysorbate 80 Polymers 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 230000002335 preservative effect Effects 0.000 claims description 3
- 238000002203 pretreatment Methods 0.000 claims description 3
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 3
- 229960004906 thiomersal Drugs 0.000 claims description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical group NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 claims description 2
- 239000000470 constituent Substances 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 14
- 238000001514 detection method Methods 0.000 abstract description 11
- 239000011248 coating agent Substances 0.000 abstract description 8
- 238000000576 coating method Methods 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 238000005406 washing Methods 0.000 abstract description 2
- 238000003556 assay Methods 0.000 abstract 2
- 230000035945 sensitivity Effects 0.000 abstract 1
- 239000012224 working solution Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 20
- 238000002965 ELISA Methods 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 10
- 239000012086 standard solution Substances 0.000 description 9
- 239000003814 drug Substances 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 241001494479 Pecora Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 229960003387 progesterone Drugs 0.000 description 3
- 239000000186 progesterone Substances 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- 239000004411 aluminium Substances 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 description 1
- 206010020850 Hyperthyroidism Diseases 0.000 description 1
- HWGBHCRJGXAGEU-UHFFFAOYSA-N Methylthiouracil Chemical compound CC1=CC(=O)NC(=S)N1 HWGBHCRJGXAGEU-UHFFFAOYSA-N 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- ZFCIUHIFXPAOHS-UHFFFAOYSA-N Phenylthiouracil Chemical compound O=C1C=CNC(=S)N1C1=CC=CC=C1 ZFCIUHIFXPAOHS-UHFFFAOYSA-N 0.000 description 1
- KNAHARQHSZJURB-UHFFFAOYSA-N Propylthiouracile Chemical compound CCCC1=CC(=O)NC(=S)N1 KNAHARQHSZJURB-UHFFFAOYSA-N 0.000 description 1
- 150000008065 acid anhydrides Chemical class 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 150000008064 anhydrides Chemical class 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 229960002545 methylthiouracil Drugs 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229960002662 propylthiouracil Drugs 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- ZEMGGZBWXRYJHK-UHFFFAOYSA-N thiouracil Chemical compound O=C1C=CNC(=S)N1 ZEMGGZBWXRYJHK-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to an enzyme linked immunosorbent assay kit for detecting methimazole residues in animal-derived food. The kit comprises the following components according to proportion: a coating plate, a standard substance, an enzyme-labeled substrate working solution, a substrate solution A, a substrate solution B, a terminating solution, a concentrated washing liquid and a concentrated extract. The kit sensitivity is 0.3 ppb, and the precision is as follows: intra assay variation coefficient (CV%) is 5%, and inter assay variation coefficient (CV%) is 10%; the accuracy is shown by recovery rate, and the recovery rate is in the range of 80-110%. IC 50 range is between 0.3 and 0.5 ppb, and the linear correlation coefficient |r| is not less than 0.9900, and the kit has the advantages of wide usage scope, convenient usage and accurate detection.
Description
Technical field
The present invention relates to enzyme linked immunosorbent detection technology, be specifically related to a kind of enzyme linked immunological kit detecting methimazole, it is specially adapted to the detection of methimazole residues in animal-derived food.
Technical background
Methimazole is the conventional medicine of hyperthyroidism, the endocrine in animal body can be changed, animal is after the feeding feed containing methimazole, and what have in various degree in urine, chicken and tissue is residual, and forbidden drugs in the food of zoo is clearly listed in by the countries such as European Union.At present, hormonal substance is forbidden using in aquaculture by most countries, and must not detect in food.But the domestic detection residual to forbidden drugs such as methimazoles in livestock products is almost blank, and remain monitoring regulation and the requirement about importer according to China to Animal by-product, studying various detection method becomes the task of top priority.
At present, detect the method mainly high performance liquid chromatography (HPLC) of methimazole residues amount, due to the instrument and equipment of complexity and loaded down with trivial details process and the high professional qualification requirement to reviewer, be not suitable for the examination of on-site supervision and great amount of samples.
Summary of the invention
The object of the invention is to provide a kind of for above-mentioned deficiency and detect enzyme linked immunological kit for methimazole residues in animal-derived food, the screening of its applicable on-the-spot batch samples simple to operate.
Kit of the present invention, it contains:
(1) ELISA Plate (coating antigen is antigen, antibody or antiantibody) of coating antigen is coated with;
(2) enzyme marker (for ENR-HRP, enzymic-labelled antibody or enzyme labeling antiantibody);
(3) methimazole antibody working fluid (when envelope antigen in ELISA Plate and enzyme marker be enzyme labeling antiantibody or ELISA Plate are wrapped by antiantibody and enzyme marker is enzyme-labelled antigen time contain);
(4) methimazole standard solution;
(5) substrate nitrite ion;
(6) stop buffer;
(7) concentrated cleaning solution;
(8) the concentrated liquid that redissolves;
The enzyme linked immunological kit of detection methimazole residues amount provided by the invention, comprise ELISA Plate and the enzyme marker working fluid of methimazole specific antibody working fluid and pre-coated coating antigen, described enzyme marker is the antiantibody of enzyme labeling, enzyme labeling methimazole haptens or enzyme labeling methimazole specific antibody; Described antiantibody is sheep anti mouse antiantibody or goat-anti rabbit antiantibody.
The marker enzyme of described enzyme marker is horseradish peroxidase, and the sheep anti mouse antiantibody of enzyme labeling adopts mixed acid anhydride or carbodiimide method that marker enzyme and antiantibody are carried out coupling and obtains.
Described methimazole specific antibody is methimazole monoclonal antibody, they obtain as immunogene with the conjugate that methimazole haptens and carrier protein adopt mixed anhydride method to obtain, the preferred methimazole mouse monoclonal antibody of described methimazole monoclonal antibody.
In order to more convenient on-site supervision and great amount of samples examination, described kit also comprises methimazole standard solution, developer, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves.
The preferred concentrated cleaning solution of described cleansing solution is pH7.1 ~ 17.7, and containing 0.8% ~ 1.2% Tween 80 and 0.03 ~ 10.05% thiomersal preservative, 0.02 ~ 10.04mol/L phosphate buffer, described number percent is percent weight in volume.
When the marker enzyme of enzyme marker is horseradish peroxidase, substrate nitrite ion A is hydrogen peroxide or urea peroxide, and substrate nitrite ion B is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1 ~ 2mol/L sulfuric acid or hydrochloride buffer;
Described redissolution liquid is for containing 0.08 ~ 10.12% polysorbas20,3 ~ 18% ovalbumins, 0.2 ~ 0.5mol/L phosphate buffer, and described number percent is percent weight in volume.
In the present invention, the preparation process of ELISA Plate is for be diluted to 0.2 ~ 0.3 μ g/ml with damping fluid by coating antigen, every hole adds 100 μ L, 37 DEG C of incubation 2h or 4 DEG C spend the night, and incline coating buffer, wash twice with the cleansing solution after dilution, each 30s, pat dry, in every hole, then add 150 ~ 200 μ L confining liquids, 37 DEG C of incubation 1 ~ 2h, the liquid in hole that inclines pats dry, and preserves after dry with the vacuum seal of aluminium film.
The building-up process of antigen in the present invention:
1. haptenic synthesis
Methimazole haptens by progesterone and succinic anhydride by being obtained by reacting
2. the preparation of methimazole antibody
Adopt carbodiimide method to carry out coupling methimazole haptens and carrier protein and obtain immunogene
Methimazole standard solution in kit of the present invention; Mark product solution 6 bottles is 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L.
Cleaning Principle of the present invention is:
When on capillary strip during pre-coated methimazole antigen, after adding sample solution or standard solution, add ELIAS secondary antibody immediately, add methimazole specific antibody solution again, methimazole residual in sample and ELISA Plate are wrapped the methimazole antigenic competition progesterone specific antibody of quilt, enzyme labeling antiantibody carries out amplification, develop the color with nitrite ion, sample light absorption value becomes negative correlation with the content of methimazole, and comparing with typical curve can the concentration range of methimazole residues amount in judgement sample roughly.
Present invention also offers a kind of method applying methimazole residues in above-mentioned enzyme linked immunological kit detection animal-derived food, it comprises step:
(1) sample pre-treatments;
(2) detect with kit;
(3) testing result is analyzed.
Sample pretreatment mainly in order to obtain methimazole solution from sample, thus for follow-up detection.The method of Sample pretreatment:
Take 2.0+ (-) 0.5 beef in 50ml polystyrene centrifuge tube, add 5 ~ 8ml ethyl acetate, oscillator vibrates 10min, the centrifugal 5min of more than 3000r, gets supernatant 1 ~ 3ml, to the glass test tube that 10ml is clean, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up, adding 1 ~ 3ml sample redissolution liquid, with vortex instrument vortex 1min, getting 50 μ l for analyzing.
When detecting with kit in the present invention, when coating antigen is methimazole antigen, in ELISA Plate micropore, add standard solution or sample solution, add ELIAS secondary antibody immediately, then add antibody working fluid, after incubation, washing pats dry, colour developing, stops, measures absorbance by microplate reader.Interpretation of result process in the present invention for: be multiplied by 100% with the absorbance values (B) of the standard solution of each concentration obtained again divided by the absorbance (B0) of first standard solution (0 standard), i.e. percentage absorbance, computing formula is:
Percentage absorbance (%)=(B/B0) × 100%
With the semilog value of the concentration of methimazole standard items (μ g/L) for X-axis, percentage absorbance is Y-axis, drawing standard curve map, with the percentage absorbance in same way calculation sample, the concentration of corresponding each sample then can read the residual quantity of methimazole in sample from typical curve.
In the present invention, the analysis of testing result also can adopt regression equation method, calculates the concentration of sample solution.
In the present invention, the analysis of testing result can also utilize computer software, and this method is more convenient for the express-analysis of a large amount of sample, and whole testing process only needs can complete less than 1h.
The enzyme linked immunological kit that the present invention detects methimazole residues in animal-derived food mainly adopts indirect competitive ELISA method qualitative or quantitatively detect the residual quantity of the methimazole in sample, require low to sample pre-treatments, sample pretreatment process is simple, can detect batch samples fast, main agents provides with the form of working fluid, and the method for inspection is convenient and easy simultaneously, there is specificity high, highly sensitive, degree of accuracy is high, accuracy high.Enzyme linked immunological kit structure of the present invention is simple, and easy to use, carrying convenience, detection method efficiently and accurately, is suitable for batch samples selective mechanisms.Kit of the present invention plays a significant role in the detection of methimazole residues.
Accompanying drawing explanation
Fig. 1 methimazole chemical structure of general formula.
The typical curve of Fig. 2 methimazole kit.
Embodiment
The present invention is set forth further below in conjunction with concrete enforcement step example.
1. the preparation of ELISA Plate
Be buffered liquid with bag and methimazole antigen diluent is become 0.20 μ g/mL, every hole adds 100 μ l, 37 DEG C of incubations two hours, incline coating buffer, washes twice with the concentrated cleaning solution of dilution, each 30s, pat dry, and then every hole adds 200 μ l confining liquids, 37 DEG C of incubation 2h, incline liquid in hole, preserves after dry with the vacuum seal of aluminium film.
2. set up the enzyme linked immunological kit detecting methimazole residues, make it comprise following component:
(1) bag is by the ELISA Plate of methimazole antigen
(2) with the sheep anti mouse antiantibody of horseradish peroxidase-labeled
(3) methimazole antibody working fluid
(4) methimazole standard items 6 bottles, concentration is respectively 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L;
(5) substrate nitrite ion is made up of A liquid and B liquid, and substrate colour developing A liquid is urea peroxide, and substrate colour developing B liquid is tetramethyl benzidine
(6) stop buffer is 2mol/L hydrochloric acid
(7) concentrated cleaning solution is for being pH7.1 ~ 17.7, containing 0.8% ~ 1.2% Tween 80 and 0.03 ~ 10.05% thiomersal preservative, and 0.02 ~ 10.04mol/L phosphate buffer, described number percent is percent weight in volume;
(8) the concentrated liquid that redissolves is for containing 0.08 ~ 10.12% polysorbas20,3 ~ 18% ovalbumins, 0.2 ~ 0.5mol/L phosphate buffer, and described number percent is percent weight in volume.
3. the detection of methimazole residues in sample:
(1) 2.0+ (-) 0.5 chicken is taken in 50ml polystyrene centrifuge tube, add 5 ~ 8ml ethyl acetate, oscillator vibrates 10min, the centrifugal 5min of more than 3000r, gets supernatant 1 ~ 3ml, to the glass test tube that 10ml is clean, flow down in 50 ~ 60 DEG C of water-bath nitrogen and dry up, adding 1 ~ 3ml sample redissolution liquid, with vortex instrument vortex 1min, getting 50 μ l for analyzing.
(2) detect with kit:
Methimazole standard solution or sample solution 50 μ l is added in the ELISA Plate micropore being coated with methimazole antigen, add ELIAS secondary antibody 50 μ l immediately, add methimazole antibody working fluid 50 μ l again, with cover plate film shrouding, 30min is reacted in 37 DEG C of constant temperature ovens, pour out liquid in hole, every hole adds 250 μ l cleansing solutions, liquid in hole is poured out after 30s, repeatable operation like this washes plate 5 times, pat dry with thieving paper, every hole adds substrate colour developing A liquid urea peroxide, substrate colour developing B liquid tetramethyl benzidine, to vibrate gently mixing, 37 DEG C of constant temperature oven colour developing 15min, every hole adds 2mol/L stop buffer hydrochloric acid 50 μ l, to vibrate gently mixing, 450nm place is set in by microplate reader, measure every hole absorbance (OD value).
The typical curve painted as shown in Figure 2, IC50=1.9ng/ml
4. standard items precision test:
Respectively the ELISA Plate prepared from three different time periods respectively extract a collection of ELISA Plate out, often criticize each extraction 10 kits, 20 micropores extracted out by every plate, measure the absorbance of 2.7 μ g/L standard solution, calculate the coefficient of variation.
Table 1 standard repeatability test (CV%)
Can be found out by above-mentioned test findings, often criticize each 10 the standard items coefficient of variation of kit between 4.6% ~ 11.2%, meet the regulation that precision is less than or equal to 25%.
5. sample accuracy experiment:
The progesterone standard items getting two concentration are respectively 10 μ g/kg, and 20 μ g/kg, carry out interpolation recovery experiment to sample respectively, each concentration do 4 parallel, respectively accuracy in computation.
The accuracy of table 2 kit
Result shows that the TIANZHU XINGNAO Capsul of beef sample is between 82.0% ~ 89.4%.
7. cross reacting rate experiment
Select the drug monitoring cross reacting rate having similar structures and similar functions with methimazole, obtain its inhibition concentration of 50% respectively by the typical curve of various medicine, calculate kit to the cross reacting rate of other drug with lower examination.Cross reacting rate (%)=(causing the analog degree of the concentration of 50% suppression methimazole/cause 50% suppression) × 100%.
The specificity of table 3 kit:
Medicine name cross reacting rate (%)
Methimazole 100%
Deracil <2.5%
Methylthiouracil <1%
Propylthiouracil <0.5%
Phenyl thiouracil <0.5%
8. kit preservation condition is 2 ~ 8 DEG C, and through the mensuration of 6 months, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, methimazole added practical measurement value all in normal range.Consider in transport and use procedure, have improper preservation condition and occur, placed 6 days under 37 DEG C of preservation conditions by kit, carry out accelerated aging test, result shows that this kit indices meets the requirements completely.Consider that the freezing situation of kit occurs, kit is put into-20 DEG C of refrigerator freezings 5 days, the bright kit indices of measurement result page table is completely normal, and can draw from above result, kit can preserve 6 months at 2 ~ 8 DEG C.
Claims (5)
1. detect an enzyme linked immunological kit for methimazole residues in animal-derived food, it is characterized in that: its constituent and ratio as follows:
Comprise by plate, calibration object, enzyme marker working fluid, substrate solution A, substrate solution B, stop buffer, concentrated cleaning solution, concentrated extracting solution; Described bag is be coated with methimazole antigen at the bottom of hole by plate, and described enzyme marker is enzyme labeling methimazole antibody.
2. kit as described in right 1, is characterized in that described methimazole antibody is monoclonal antibody.
3. kit as claimed in claim 1 or 2 is characterized in that the marker enzyme of enzyme marker is horseradish peroxidase, substrate nitrite ion A is hydrogen peroxide or urea peroxide, substrate nitrite ion B is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1 ~ 2mol/L sulfuric acid or hydrochloride buffer.
4. kit as claimed in claim 1 or 2, it is characterized in that: concentrated cleaning solution is pH7.1 ~ 7.7, containing 0.8% ~ 1.2% Tween 80 and 0.03 ~ 0.05% thiomersal preservative, 0.02 ~ 0.04mol/L phosphate buffer: the concentrated liquid that redissolves is for containing 0.08 ~ 0.12% polysorbas20,3 ~ 8% ovalbumins, 0.2 ~ 0.5mol/L phosphate buffer, the concentration of methimazole standard items is respectively 0 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L, 24.3 μ g/L, described number percent is percent weight in volume.
5. examine a method for methimazole residues in animal-derived food, comprise step:
(1) sample pre-treatments;
(2) detect with claim 1 kit;
(3) testing result is analyzed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094815A (en) * | 2020-09-23 | 2020-12-18 | 绿城农科检测技术有限公司 | Hybridoma cell strain secreting methimazole monoclonal antibody, monoclonal antibody and application |
| CN114813596A (en) * | 2022-04-21 | 2022-07-29 | 兰州大学 | Astragalus polysaccharide-functionalized silver nanoparticle colorimetric sensor, preparation method thereof, and application in detecting methimazole |
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