CN102288749B - Dipterex bionic enzyme linked immunosorbent detection method - Google Patents
Dipterex bionic enzyme linked immunosorbent detection method Download PDFInfo
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- NFACJZMKEDPNKN-UHFFFAOYSA-N trichlorfon Chemical compound COP(=O)(OC)C(O)C(Cl)(Cl)Cl NFACJZMKEDPNKN-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 238000001514 detection method Methods 0.000 title claims abstract description 28
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 15
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 15
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 13
- 239000003547 immunosorbent Substances 0.000 title abstract description 6
- 229960001952 metrifonate Drugs 0.000 claims abstract description 44
- 229920000344 molecularly imprinted polymer Polymers 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 32
- 239000000758 substrate Substances 0.000 claims description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000002835 absorbance Methods 0.000 claims description 15
- 230000002401 inhibitory effect Effects 0.000 claims description 15
- 238000010790 dilution Methods 0.000 claims description 14
- 239000012895 dilution Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000000890 antigenic effect Effects 0.000 claims description 6
- 238000009739 binding Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 claims description 5
- 239000012491 analyte Substances 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- OZAIFHULBGXAKX-VAWYXSNFSA-N AIBN Substances N#CC(C)(C)\N=N\C(C)(C)C#N OZAIFHULBGXAKX-VAWYXSNFSA-N 0.000 claims description 3
- 229920001353 Dextrin Polymers 0.000 claims description 3
- 239000004375 Dextrin Substances 0.000 claims description 3
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical class [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Natural products NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 3
- 239000004202 carbamide Substances 0.000 claims description 3
- 235000013877 carbamide Nutrition 0.000 claims description 3
- -1 carbamide peroxides Chemical class 0.000 claims description 3
- 235000019425 dextrin Nutrition 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 230000009977 dual effect Effects 0.000 claims description 3
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Chemical compound CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 235000011091 sodium acetates Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 239000012086 standard solution Substances 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- PSYGHMBJXWRQFD-UHFFFAOYSA-N 2-(2-sulfanylacetyl)oxyethyl 2-sulfanylacetate Chemical compound SCC(=O)OCCOC(=O)CS PSYGHMBJXWRQFD-UHFFFAOYSA-N 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 2
- 239000003431 cross linking reagent Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000000178 monomer Substances 0.000 claims description 2
- 230000001900 immune effect Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 17
- 235000013305 food Nutrition 0.000 abstract description 5
- 238000002965 ELISA Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 231100000703 Maximum Residue Limit Toxicity 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 238000003018 immunoassay Methods 0.000 abstract description 2
- 238000011896 sensitive detection Methods 0.000 abstract description 2
- 230000003592 biomimetic effect Effects 0.000 abstract 1
- 239000000284 extract Substances 0.000 description 6
- 240000006108 Allium ampeloprasum Species 0.000 description 3
- 235000005254 Allium ampeloprasum Nutrition 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical class C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 206010001497 Agitation Diseases 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- ZCHPKWUIAASXPV-UHFFFAOYSA-N acetic acid;methanol Chemical compound OC.CC(O)=O ZCHPKWUIAASXPV-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 206010007269 Carcinogenicity Diseases 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000007670 carcinogenicity Effects 0.000 description 1
- 231100000260 carcinogenicity Toxicity 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000010813 internal standard method Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 230000007886 mutagenicity Effects 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
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Abstract
本发明涉及一种敌百虫仿生酶联免疫吸附检测方法，是以敌百虫分子印迹聚合物膜作为人工抗体代替生物抗体，利用酶联免疫吸附测定原理，建立对敌百虫具有高灵敏度的仿生酶联免疫吸附检测方法。该方法的最低检出限为7μgL-1，大大低于最高残留限量值，且大大缩短了分析时间（比传统生物免疫分析缩短1.0h），适用于快速检测敌百虫。本发明制备的仿生抗体可以重复使用50次以上，成本大大降低；并且前处理简单，灵敏度高，实验操作简单，适用于各种食品中敌百虫的快速检测。
The invention relates to a bionic enzyme-linked immunosorbent detection method for trichlorfon, which uses trichlorfon molecularly imprinted polymer film as an artificial antibody instead of a biological antibody, and uses the principle of enzyme-linked immunosorbent assay to establish a highly sensitive detection method for trichlorfon Bionic enzyme-linked immunosorbent assay method. The minimum detection limit of this method is 7μgL -1 , which is much lower than the maximum residue limit, and the analysis time is greatly shortened (1.0h shorter than the traditional biological immunoassay), which is suitable for rapid detection of trichlorfon. The biomimetic antibody prepared by the invention can be reused more than 50 times, and the cost is greatly reduced; and the pretreatment is simple, the sensitivity is high, and the experimental operation is simple, and is suitable for rapid detection of trichlorfon in various foods.
Description
Technical field
The present invention relates to a kind of Dipterex bionic enzyme linked immunosorbent detection method, belong to technical field of food safety detection, the rapid sensitive detection method of particularly a kind of metrifonate.
Background technology
Metrifonate（Trichlorphon, O, O- dimethyl-(2,2,2- tri- chloro- 1- hydroxyethyls) phosphate）It is a kind of organophosphor spectrum insecticide, nineteen fifty-two is synthesized by Bayer A.G and starts production.It has efficiently, low toxicity, low-residual, good water solubility the features such as, therefore be widely used in terms of agricultural, gardening, herding, health.Also just because of it is widely used, abuse phenomenon is on the rise, and infringement and carcinogenicity and mutagenicity of the metrifonate to animal nervous system also increasingly cause the attention of people.Metrifonate maximum residue limit must not be higher than 0.1 mg Kg in GB 16319-1996 regulation food-1。
Qualitative, quantitative detection is mostly carried out using AAS, liquid chromatography or liquid phase, the method for internal standard method for gas chromatography about metrifonate detection method in food both at home and abroad at present, but equipment price used in above-mentioned several detection methods is expensive and relatively low in metrifonate content（Trace）When be difficult detection, it usually needs complex sample pretreatment technology.And although ELISA has high sensitivity and low detection limits, there is biological antibody long preparation period in it, the problems such as preserving improper easy in inactivation.Molecularly imprinted polymer is used as bionic antibody, not only there is high selectivity to metrifonate, and imprinted polymer short preparation period, in the absence of deactivation prob, therefore it is applied to ELISA as artificial antibody, bionical immuno absorbence detection technique is set up, it is significant for detecting the metrifonate in agricultural product and food.
The content of the invention
It is an object of the invention to the defect such as overcome detection time length, instrument price costliness, poor accuracy, pre-treatment that traditional metrifonate detection method is present loaded down with trivial details, there is provided a kind of method for quick of metrifonate.
The present invention is adopted the technical scheme that：
A kind of Dipterex bionic enzyme linked immunosorbent detection method, is completed according to the following steps：
1）By metrifonate（It is dissolved in acetonitrile and water）, function monomer（Methacrylic acid, MAA）And crosslinking agent（GDMA, EGDMA）According to 1:2:2 molar ratio and a certain amount of azodiisobutyronitrile（AIBN）Fully mix, then add the above-mentioned mixed liquor polymerisation 18h of 200 μ L in 96 hole elisa Plates per hole；With 1:9（v/v）Glacial acetic acid-methanol solution continuously elutes 8h to remove metrifonate, then is washed till neutrality with 100% methanol, and molecular imprinted polymer membrane is obtained after drying；
2）Using molecular imprinted polymer membrane as bionic antibody, the row enzyme mark hole of 96 hole elisa Plates the 1st is set to blank group, only adds 200 μ LPBS solution per hole（5 times of PBS：Na2HPO4·12H2O：68.8 g, NaH2PO4：6.9 g, NaCl：45 g, plus distilled water is to 1L, pH value 6.7）；2nd row enzyme mark hole is set to control group, only adds 200 μ L1 per hole:3000（v/v）Horseradish peroxidase mark antigenic dilution；3-8 row enzyme marks hole sequentially adds 100 μ L standard specimen gradient dilution liquid（5 times of dilutions）, 100 μ L1 are added immediately after:3000 horseradish peroxidase mark antigenic dilutions；
3）Above-mentioned 96 hole elisa Plates are incubated at room temperature after 1 h and use phosphate Tween buffer PBST（5 times of PBS：200 mL, 10% Tween-20：5mL, plus distilled water are settled to 1L.）Board-washing five times.150 μ L substrate solutions are added in every hole again（Substrate A：8.2 g anhydrous sodium acetates, 2.5 g beta-schardinger dextrins, 150 mg carbamide peroxides, plus distilled water adjust pH to 5.0,4 DEG C of preservations to 1000 mL；Substrate B：The tetramethyl benzidines of 50 mg 3,3,5,5- are dissolved in 5 mL dimethyl sulfoxide (DMSO)s, and room temperature keeps in dark place.Using first 15 minutes, 14.6 mL substrate As and 0.45 mL substrate Bs were mixed into substrate solution）, 30 min are reacted at room temperature, and the mol L of 50 μ L 1.25 are added per hole-1Sulfuric acid, terminating reaction；
4）In dual wavelength mode（450-650 nm）It is lower to read 96 hole elisa Plates 1-8 row absorbances A with ELIASA；
Inhibiting rate value of the various concentrations metrifonate to antigen-antibody binding reaction is calculated according to the following formula：
IC%=×100
In formula：
Inhibiting rate of IC% --- the metrifonate to antigen-antibody binding reaction；
AControl--- control wells mean absorbance values；
ASample--- the mean absorbance values of metrifonate titer or sample extracting solution（3-8 rows）；
ABlank--- the mean absorbance values of blank well.
5）Using concentration of standard solution logarithm value as abscissa, inhibiting rate is ordinate, draws standard curve；
6）Analyte is crushed, by weight volume ratio（g/mL）1：10 add PBS solution ultrasonic extraction 3 times, and 0.45 μm of membrane filtration obtains sample extracting solution.Sample extracting solution is replaced into standard specimen gradient dilution liquid, repeat step 2-4 operations calculate metrifonate content in analyte inhibiting rate and analyte according to absorbance by standard curve.
Advantages and positive effects of the present invention are：
1. the metrifonate adsorption functional material that the present invention is provided has higher selectivity, biological antibody can be replaced to be applied to Enzyme-multiplied immune technique；The adsorption functional material is prepared by chemical method, the ability with higher stability, longer service life and stronger anti-adverse environment, the shortcomings of overcoming traditional biological Antibody preparation cycle length, easy in inactivation, high cost.
2. molecular imprinting technology and Enzyme-multiplied immune technique are combined by the present invention, foundation has highly sensitive bionic enzyme linked immunosorbent detection to metrifonate.The minimum detectability of this method is 7 μ g L-1, MRL value is significantly less than, detection needs are disclosure satisfy that.And substantially reduce analysis time（Shorten 1.0 h than traditional biological immunoassay）, it is adaptable to quick detection metrifonate.
3. bionic antibody prepared by the present invention may be reused more than 50 times, cost is substantially reduced；And pre-treatment is simple, sensitivity is high, and experimental implementation is simple, it is adaptable to the quick detection of metrifonate in various food.
Comparison of the invention with other existing detection methods
| Detection method | Liquid-mass chromatography detection method | Gas phase detection method | ||
| Sample-pretreating method | PBS is extracted, filtering, it is not necessary to other pre-treatments. | Solvent Extract methods, SPE or other method are enriched with. | Solvent Extract methods, nitrogen is redissolved after blowing with acetone. | |
| Minimum detection | 7μg L | -1 | 10 |
10 μg Kg-1 |
Brief description of the drawings
Fig. 1 is metrifonate BELISA standard curves
As shown in Figure 1, the sensitivity of this method（Inhibiting rate 50 is worth）For 7 mg L-1, minimum detection limit（Inhibiting rate 15 is worth）For 7 μ g L-1.
Embodiment
With reference to embodiment, the present invention is further described；Following embodiments are illustrative, are not limited, it is impossible to limit protection scope of the present invention with following embodiments.
The present invention is combined metrifonate molecular imprinted polymer membrane as bionic antibody and Enzyme-multiplied immune technique, so that setting up has highly sensitive method for quick to metrifonate.Its specific embodiment is：
1）1 mmol (257.45 mg) metrifonate is dissolved in 3 mL acetonitriles and 2 mL water, then adds 2 mmol（86.09 mg）2 mmol are added after the min of MAA magnetic agitations 30（198.2 mg）EGDMA and 0.03g azodiisobutyronitriles（AIBN）, the h of magnetic agitation 1.The above-mentioned mixed liquor polymerisation 18h of 200 μ L are added in 96 hole elisa Plates per hole, reaction solution are discarded, with 1:9（v/v）Glacial acetic acid-methanol solution continuously elutes 8h to remove metrifonate, then is washed till with 100% methanol neutrality, and 37 DEG C of dry 2h obtain molecular imprinted polymer membrane（Bionic antibody）.
2. the row enzyme mark hole of 96 hole elisa Plates the 1st is set into blank group, only add 200 μ LPBS solution per hole（5 times of PBS：Na2HPO4·12H2O：68.8 g, NaH2PO4：6.9 g, NaCl：45 g, plus distilled water is to 1L, pH value 6.7.）；2nd）Row enzyme mark hole is set to control group, only adds 200 μ L1 per hole:3000（v/v）Horseradish peroxidase mark antigenic dilution.3-8 row enzyme marks hole sequentially adds 100 μ L standard specimen gradient dilution liquid（Concentration is respectively 50000,10000,2000,400,80 and 16 μ g L-1, diluted with PBS）, 100 μ L1 are added immediately after:3000 horseradish peroxidase mark antigenic dilutions (3-8 rows).
3）Above-mentioned 96 hole elisa Plates are incubated at room temperature after 1 h and use phosphate Tween buffer PBST（5 times of PBS：200 mL, 10% Tween-20：5mL, plus distilled water are settled to 1L.）Board-washing five times.96 hole elisa Plates add 150 μ L substrate solutions per hole（Substrate A：8.2 g anhydrous sodium acetates, 2.5 g beta-schardinger dextrins, 150 mg carbamide peroxides, plus distilled water adjust pH to 5.0,4 DEG C of preservations to 1000 mL；Substrate B：The tetramethyl benzidines of 50 mg 3,3,5,5- are dissolved in 5 mL dimethyl sulfoxide (DMSO)s, and room temperature keeps in dark place.Using first 15 minutes, 14.6 mL substrate As and 0.45 mL substrate Bs were mixed into substrate solution）, 30 min are reacted at room temperature.The mol L of 50 μ L 1.25 are added per hole-1Sulfuric acid, terminating reaction.
4）In dual wavelength mode（450-650 nm）It is lower to read 96 hole elisa Plates 1-8 row absorbances A with ELIASA.
Inhibiting rate value of the various concentrations metrifonate to antigen-antibody binding reaction is calculated according to the following formula：
IC%=×100
In formula：
Inhibiting rate of IC% --- the metrifonate to antigen-antibody binding reaction；
AControl--- control wells mean absorbance values；
ASample--- the mean absorbance values of metrifonate titer or sample extracting solution（3-8 rows）；
ABlank--- the mean absorbance values of blank well.
5）Using concentration of standard solution logarithm value as abscissa, inhibiting rate is ordinate, draws standard curve.
6）The g of leek 2 accurately is weighed, is shredded, 20 mL PBS solutions ultrasonic extraction is added 3 times, extract solution is settled to 100 mL, 0.45 μm of membrane filtration.
7）The sample extracting solution that step 6 is obtained replaces standard specimen gradient dilution liquid（Sample extracting solution concentration is respectively 50000,10000,2000,400,80 and 16 μ g L-1, diluted with PBS）, repeat step 2-4 operation obtains the absorbance of sample extracting solution, and it is 29.06% to calculate leek extracts inhibiting rate according to above-mentioned formula.
8）Bring above-mentioned leek extracts inhibiting rate into metrifonate BELISA standard curves and obtain metrifonate extract concentration for 140 μ g L-1, it is 7.0 μ g g to calculate metrifonate content in surveyed leek-1（140 μg L-1×100 mL/ 2 g）.
Claims (1)
1. a kind of bionical immunological adsorption detection method of metrifonate, it is characterised in that comprise the following steps：
1）Metrifonate is dissolved in after acetonitrile and water with function monomer methacrylic acid MAA and crosslinking agent GDMA EGDMA according to 1:2:2 molar ratio and azodiisobutyronitrile AIBN is fully mixed, and then adds the above-mentioned mixed liquor polymerisation 18h of 200 μ L in 96 hole elisa Plates per hole；Use v/v1:9 glacial acetic acids-methanol solution continuously elutes 8h to remove metrifonate, then is washed till neutrality with 100% methanol, and molecular imprinted polymer membrane is obtained after drying；
2）Using molecular imprinted polymer membrane as bionic antibody, the row enzyme mark hole of 96 hole elisa Plates the 1st is set to blank group, only adds 200 μ LPBS solution per hole；2nd row enzyme mark hole is set to control group, only adds 200 μ L1 per hole:3000v/v horseradish peroxidase mark antigenic dilutions；3-8 row enzyme marks hole sequentially adds 100 μ L standard specimen gradient dilution liquid, and described standard specimen gradient dilution liquid concentration is respectively 50000,10000,2000,400,80 and 16 μ g L-1, diluted with PBS, 100 μ L1 added immediately after:3000 horseradish peroxidase mark antigenic dilutions；First by Na2HPO4·12H2O：68.8g, NaH2PO4：6.9g, NaCl：45g, plus distilled water are configured to 5 times of PBS to 1L, then 5 times of PBS are diluted into 5 times obtain described PBS solution；
3）Above-mentioned 96 hole elisa Plates are incubated at room temperature after 1h and use phosphate Tween buffer PBST, board-washing five times；150 μ L substrate solutions are added in every hole again, 30min is reacted at room temperature, 50 μ L1.25mol L are added per hole-1Sulfuric acid, terminating reaction；Described substrate solution is made up of substrate A and substrate B；Described substrate A is：8.2g anhydrous sodium acetates, 2.5g beta-schardinger dextrins, 150mg carbamide peroxides, plus distilled water adjust pH to 5.0,4 DEG C of preservations to 1000mL；Described substrate B is：50mg3,3,5,5- tetramethyl benzidines are dissolved in 5mL dimethyl sulfoxide (DMSO)s, and room temperature keeps in dark place；Using first 15 minutes, 14.6mL substrate As and 0.45mL substrate Bs were mixed into described substrate solution；Use step 2）Described in 5 times of PBS：200mL, 10% Tween-20：5mL, plus distilled water are settled to 1L and obtain described PBST solution；
4）Under dual wavelength mode 450-650nm 96 hole elisa Plates 1-8 row absorbances A are read with ELIASA；
Inhibiting rate value of the various concentrations metrifonate to antigen-antibody binding reaction is calculated according to the following formula：
In formula：
Inhibiting rate of IC% --- the metrifonate to antigen-antibody binding reaction；
AControl--- control wells mean absorbance values；
ASample--- the mean absorbance values of metrifonate titer or sample extracting solution；
ABlank--- the mean absorbance values of blank well；
5）Using concentration of standard solution logarithm value as abscissa, inhibiting rate is ordinate, draws standard curve；
6）Analyte is crushed, by weight volume ratio g/mL1：10 add PBS solution ultrasonic extraction 3 times, and 0.45 μm of membrane filtration obtains sample extracting solution；Repeat step 2）--4）Operation, by step 2）Middle sample extracting solution replaces standard specimen gradient dilution liquid, and described sample extracting solution concentration is respectively 50000,10000,2000,400,80 and 16 μ g L-1, diluted with PBS；Metrifonate content in sample extracting solution inhibiting rate and analyte is calculated by standard curve according to absorbance.
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| CN104048953B (en) * | 2013-03-11 | 2016-08-31 | 华中科技大学 | The method for quick of a kind of trace levels of endotoxin and kit |
| CN103278622A (en) * | 2013-04-27 | 2013-09-04 | 大连海洋大学 | Preparation method of 96-well enzyme label plate chloramphenicol molecularly imprinted polymer film |
| CN103399146A (en) * | 2013-08-14 | 2013-11-20 | 山东农业大学 | Enzyme-labeled bionic-immunoassay method of acrylamide |
| CN104330552B (en) * | 2014-11-20 | 2016-09-21 | 山东农业大学 | A kind of enzyme labelling bionical immunoassay detection metrifonate and the method for orthene |
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