CN1677108A - Enzyme-linked immunosorbentassay reagent kit for analyzing snout-killing sulfur-phosphous residual - Google Patents
Enzyme-linked immunosorbentassay reagent kit for analyzing snout-killing sulfur-phosphous residual Download PDFInfo
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- CN1677108A CN1677108A CN 200410030740 CN200410030740A CN1677108A CN 1677108 A CN1677108 A CN 1677108A CN 200410030740 CN200410030740 CN 200410030740 CN 200410030740 A CN200410030740 A CN 200410030740A CN 1677108 A CN1677108 A CN 1677108A
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Abstract
The invention discloses a enzymoimmunoassy absorption test agent box adapted on fenitrothion remain analysis which includes a box, 96 hole or 40 hole enzyme labeling board or test tube set in the box and the antigen in the box. In each hole on the enzyme labeling board, envelope antigen which is able to unite with fenitrothion antigen, is enveloped by parisarc liquor, and then sealed with celluloid of 2 percents to 5 percents. Antigens in the box consists of washing solution, basal material dilution, fenitrothion standard solution, fenitrothion antigen (indirect ELISA), sheep rabbit antigen labeled with horse radish peroxidase (indirect ELISA), or fenitrothion rabbit antigen labeled with horse radish peroxidase (direct ELISA), basal material, coloration matter and reaction termination liquor. The invention can be used on fast detecting of fenitrothion remain in water, soil, vegetable, fruits and poisoned sample. The pre-process of the sample is simple and can detecting groups of samples at the same time. The sample detecting cost is lower than traditional detecting method.
Description
(1) technical field
The invention belongs to a kind of enzyme-linked immunosorbent assay kit of analyzing at residual fenifrothion that is applicable to, this kit is applicable to that mainly the fenifrothion in environmental sample such as in batches water sample of fast measuring, soil and the food samples such as poisoning sample and vegetables is residual, belongs to the remains of pesticide detection range.
(2) background technology
Because the continuous expansion of the continuous development of technique in using agriculture chemical and the scale of use, the environmental impact that residues of pesticides cause and the chronic and long-term effect of human health is subjected to people's attention and worry day by day, restriction to residues of pesticides in personal consumption and foreign trade is also more and more stricter, and all many-sides such as analyzing and testing object, kind, quantity, scope, index have been proposed new requirement and higher standard.Retention analysis context of detection at agricultural chemicals and metabolin thereof, traditional analyzing detecting method mainly relies on gas chromatography (GC), high performance liquid chromatography (HPLC), mass spectrum physicochemical analysis means such as (MS), but these analytical approachs all need the pre-treatment work of very complicated usually, so workload is big, instrument is expensive in the testing and need those skilled in the art and long analytical cycle.Therefore people urgently wishes to have a kind of simple, fast, sensitive and cheap detection technique can carry out large batch of detection application in field, market or laboratory.Immunoassay has really possessed these advantages, thus very short although immunoassay is used for time of pesticide residue analysis, still be used for the analysis of environmental sample and food samples residues of pesticides very soon.
Fenifrothion [fenitrothion, O, O-dimethyl-O-(3-methyl-4-nitrobenzophenone) thiophosphate], commodity Folithion by name etc., be a kind of broad-spectrum organophosphorous pesticide, be widely used in the control of insect of paddy, barley, corn and economic class crop, also can prevent and treat the various pests on cotton, vegetables, tealeaves, the fruit tree, rice borer there is special efficacy, also can be exclusively used in the grain storage pest of raw grain and seed grain.Fenifrothion to daylight stable, is met the easy decomposition failure of alkali to the higher mammal low toxicity under the normal temperature, and the safety interval on fruit, vegetables is 10～15 days.Along with the increase of agricultural chemicals use amounts such as fenifrothion, especially a large amount of uses on crop and vegetables cause many harm to health, and this has caused people's common concern.Therefore develop a kind of simple trace analysis method quick, that be applicable to the residues of pesticides on-site supervision and have important use value.
In pesticide residue analysis, the conventional sense method of fenifrothion is a vapor-phase chromatography, but pre-treatment steps such as this method need carry out loaded down with trivial details extraction, purification to sample, concentrate, and this method needs expensive chromatographic apparatus, complicated operating process and consuming time longer 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 fenifrothion.
(3) summary of the invention
[problem that will solve]
The purpose of this invention is to provide a kind of residual enzyme-linked immunosorbent assay kit of fenifrothion that detects;
Another object of the present invention provides a kind of kit that high specific, high sensitivity, method of operating simply fast, also can be used for the residual fenifrothion of a large amount of sample fast detecting that has.
[technical scheme]
The present invention is a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis, and it is based on immune response and enzymatic reaction, can detect the residual of fenifrothion in the food such as water sample, soil, poisoning sample, vegetables.The present invention is the enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion 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, in every hole of ELISA Plate, by the coating buffer bag by the envelope antigen that can combine with anti-fenifrothion antibody specificity, and seal with 2%～5% gelatin, reagent comprises cleansing solution (dilution) in the box, the substrate dilution, anti-fenifrothion antibody (being applicable to indirect ELISA), the fenifrothion standard solution, the anti-fenifrothion rabbit antibody (being applicable to direct competitive ELISA) of goat anti-rabbit antibody of horseradish peroxidase-labeled (being applicable to indirect competitive ELISA) or horseradish peroxidase-labeled, substrate, substance that show color and reaction terminating liquid, wherein:
Envelope antigen (FEN-OVA) (contains 1～2g sodium carbonate and 2～4g sodium bicarbonate with the carbonate buffer solution of pH9.6 0.05mol/L, distilled water 1L, promptly bag is cushioned liquid) be diluted to 0.5～4.0 μ g/mL, and put and be added on 96 holes/40 hole ELISA Plate, 100 μ L/ holes, and sealed; Wherein envelope antigen is haptens (I) O-methyl-O-(3-methyl 4-nitrobenzophenone-N-(2-carboxyethyl) thio-phosphamide or haptens (II) O, compound of O-dimethyl-O-(3-methyl-4-(6-carboxylic hexyl acylamino-) phenyl) thiophosphate and ovalbumin;
One bottle of cleansing solution (reaction dilution), 40～80mL/ bottle, proportion of composing is 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 500～1000mL, is normal 15～30 times of concentrates that use;
One bottle of substrate dilution, 30～50mL/ bottle is formulated as follows: citric acid 3～6g, sodium hydrogen phosphate 1～3g, distilled water 1000mL are normal 5～10 times of concentrates that use;
Zymolyte is 30% hydrogen peroxide, and 10～15mL/ bottle, developer are 3,3 ', 5,5 '-tetramethyl benzidine (TMB) solution, and 4～6 of 10～15mL/ bottle or o-phenylenediamine (OPD) pressed powders, 10～20mg/ props up;
4 bottles of anti-fenifrothion antibody (IgG), 100mL/ bottle, working concentration are 1: 500～1000 (indirect elisa methods); Described anti-fenifrothion antibody (IgG) can carry out the immunoglobulin (Ig) (IgG) that specificity combines with fenifrothion molecule, fenifrothion coating antigen for what produce after as immunogene injection rabbit by the synthetic conjugate of haptens and bovine serum albumin(BSA);
One bottle of the goat anti-rabbit antibody (indirect elisa method) of anti-fenifrothion rabbit antibody of horseradish peroxidase-labeled (directly ELISA method) or horseradish peroxidase-labeled, 200～400 μ L/ bottles, 800～1500 times of concentrates during for normal the use; The fenifrothion antibody of above-mentioned horseradish peroxidase-labeled is that anti-fenifrothion antibody (IgG) combines the compound that forms with horseradish peroxidase (HRP);
One bottle of reaction terminating liquid, 30～50mL/ bottle is 2mol/L sulfuric acid;
6 bottles of fenifrothion variable concentrations series (0.1,0.5,2,10,50,100mg/L) standard solution, 1～4mL/ bottle, methanol constant volume, during use with 10 times of PBST dilutions.
[kit measurement principle]
At first the compound that pesticide molecule and macromolecular carrier (as protein) coupling are prepared is adsorbed on the solid phase carrier as envelope antigen, add agricultural chemicals to be measured and anti-fenifrothion antibody (indirect elisa method) or enzyme then and mark anti-fenifrothion antibody (directly ELISA method), solid-phase coating is former, agricultural chemicals to be measured and anti-fenifrothion antibody (or enzyme is marked anti-fenifrothion antibody) being at war with property association reaction, pesticide concentration to be measured is many, the antibody that then is bonded on the solid phase antigen is few, otherwise the antibody (or enzyme labelled antibody) that is combined in solid phase antigen is many, adding substrate solution after the reaction again chromogenic reaction occurs and is measured (indirect elisa method, add substrate solution before also need add ELIAS secondary antibody again).Amount one timing when antibody or enzyme labelled antibody, pesticide volume to be measured in the sample is many more, the enzyme labelled antibody that combines with solid phase antigen is just few more, the color development habituation, and inhibiting rate increases, otherwise, color development increased response then, inhibiting rate lowers, thereby according to the typical curve of known quantity agricultural chemicals and the inhibiting rate of testing sample, 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.
[beneficial effect]
Advantage of the present invention is to be used for the residual detections of food fenifrothion 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.Kit adopts strong specificity, high-titer antibody, improves the sensitivity, accuracy, the precision that detect.The storage life of kit was above 12 months.The present invention is simply quick, is applicable to the trace analysis method of residues of pesticides on-site supervision, has important application value.
(4) description of drawings
Fig. 1 is the typical curve of direct competitive ELISA method fenifrothion;
Fig. 2 is the typical curve of indirect competitive ELISA method fenifrothion.
(5) embodiment
Describing the present invention below among the embodiment in more detail, is not limitation of the present invention, and wherein ratio and number percent are not having to be the w/w ratio under the situation of specified otherwise.
Product of the present invention comprises 96 holes/40 hole ELISA Plate and the detectable in the box body, box body, in every hole of ELISA Plate,, and is sealed with 2%～5% gelatin by the envelope antigen that can combine with anti-fenifrothion antibody specificity by the coating buffer bag.
Reagent comprises in the box: anti-fenifrothion rabbit antibody (being applicable to direct competitive ELISA), substrate, substance that show color and the reaction terminating liquid of the goat anti-rabbit antibody (being applicable to indirect competitive ELISA) of cleansing solution (dilution), substrate dilution, anti-fenifrothion antibody (being applicable to indirect ELISA), fenifrothion standard solution, horseradish peroxidase-labeled or horseradish peroxidase-labeled, and compound method is as follows:
Cleansing solution (reaction dilution) 40mL, proportion of composing is potassium dihydrogen phosphate 0.1g, sodium hydrogen phosphate 4g, potassium chloride 0.1g, Tween-20 3mL, distilled water 1000mL, is normal 15～30 times of concentrates that use;
Substrate dilution 50mL is formulated as follows: citric acid 3g, sodium hydrogen phosphate 1g, distilled water 1000mL are normal 5～10 times of concentrates that use;
Zymolyte is 30% hydrogen peroxide 15mL, developer is 3,3 ', 5,5 '-tetramethyl benzidine (TMB) solution 15mL or o-phenylenediamine (OPD) pressed powder 20mg;
Anti-fenifrothion antibody (IgG) 400mL, working concentration are 1: 1000 (indirect elisa method); Described anti-fenifrothion antibody (IgG) can carry out the immunoglobulin (Ig) (IgG) that specificity combines with fenifrothion molecule, fenifrothion coating antigen for what produce after as immunogene injection rabbit by the synthetic conjugate of haptens and bovine serum albumin(BSA);
Goat anti-rabbit antibody (indirect elisa method) the 200 μ L of anti-fenifrothion rabbit antibody of horseradish peroxidase-labeled (directly ELISA method) or horseradish peroxidase-labeled, 800～1500 times of concentrates during for normal the use; The fenifrothion antibody of above-mentioned horseradish peroxidase-labeled is that anti-fenifrothion antibody (IgG) combines the compound that forms with horseradish peroxidase (HRP);
Reaction terminating liquid 30mL is 2mol/L sulfuric acid;
6 bottles of fenifrothion variable concentrations series (0.1,0.5,2,10,50,100mg/L) standard solution, 1～4mL/ bottle, methanol constant volume, during use with 10 times of PBST dilutions.
The preparation of enzyme labelled antibody (improvement sodium periodate method), concrete operations are as follows:
Take by weighing 5～10mg horseradish peroxidase HRp and be dissolved in the 1L distilled water, add the 0.1mol/L NalO4 solution that 0.2～04mL newly joins, lucifuge stirs 15～30min under the room temperature.Above-mentioned solution is packed in the bag filter, and with the acetate buffer solution dialysis of 1mmol/L pH4.4,4 ℃ are spent the night.Carbonate buffer solution with pH9.5 makes the pH of the HRP solution of above-mentioned hydroformylation be elevated to 9.0～9.5, adds the 0.01mol/L carbonate buffer solution that 1～2mL contains 10～20mg antibody purification then immediately, and the room temperature lucifuge stirred 2～3 hours.Add the 4mg/mL NaBH4 solution that 0.1～0.2mL newly joins, mixing, put again 4 ℃ 2～3 hours.Reactant liquor is packed in the bag filter, and with the PBS dialysis of 0.15mol/L pH7.4,4 ℃ 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 and secondary with the semi-saturation ammonium sulfate, and last sediment is dissolved among the PBS of a small amount of 0.15mol/L pH7.4.Above-mentioned solution is packed in the bag filter, with the PBS damping fluid dialysis of 0.15mol/L pH7.4, remove (detecting with Nai Shi reagent) behind the ammonium ion, centrifugal, supernatant is enzyme conjugates, after the packing of equivalent glycerine, respectively at-4 ℃ ,-20 ℃ preservations.
The preparation of coated elisa plate
Envelope antigen (FEN-OVA) (contains 1～2g sodium carbonate and 2～4g sodium bicarbonate with the carbonate buffer solution of pH9.6 0.05mol/L, distilled water 1L) is 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 2 hours, coating buffer inclines, with PBST washing 3 times, pat dry, in every hole, add the gelatin of 150 μ L 2%～5% then, after putting into 37 ℃ of constant temperature ovens and sealing 0.4～1 hour, with PBST washing 3 times, pat dry the back kept dry.
The pre-treatment of test sample:
Water sample: can take a sample after the filtration and carry out elisa assay.
Soil sample: get the 10g soil sample 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 is pulverized with comminutor,, merge extract, concentrate, be settled to 10mL, take a sample and carry out elisa assay with PBST with 20～40mL acetone extraction three times.
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): get the 10mL liquid of gastric lavage, regulating the pH value with rare HCl is that available ELISA method is analyzed after neutrality.
Vomitus: sample thief grinds, and the centrifuging and taking supernatant is analyzed with the ELISA method.
The kit operating process is as follows:
1) direct competitive ELISA method: take out an ELISA Plate that is coated with the fenifrothion envelope antigen, 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 washing 2～6 times, pat dry with 200 μ L to guarantee to remove fully the liquid in the hole; Every then hole adds the mixed liquor of 100 μ L substrate solutions and substance that show color, and it is even slightly to shake, and hatches 37 ℃ of dark places and to carry out chromogenic reaction in 15～25 minutes; Add 50 μ L reaction terminating liquids, after mixing, measure 0D
450nmPerhaps OD
490nmValue.
2) indirect competitive ELISA method: take out an ELISA Plate that is coated with the fenifrothion envelope antigen, 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 50 μ L and dilute good 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 washing 2～6 times, pat dry with 200 μ L to guarantee to remove fully the liquid in the hole; Add 100 μ L and dilute good 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 washing 2～6 times, pat dry with 200 μ L to guarantee to remove fully the liquid in the hole; Every then hole adds the mixed liquor of 100 μ L substrate solutions and substance that show color, and it is even slightly to shake, and hatches 37 ℃ of dark places and to carry out chromogenic reaction in 15～25 minutes; Add 50 μ L reaction terminating liquids, after mixing, measure OD
450nmPerhaps OD
490nmValue.
3) with the light absorption value and the inhibiting rate in each hole of mean value calculation of the standard specimen that obtained and sample light absorption value
Light absorption value when ODmax is not dosing, the light absorption value when ODx is agricultural chemicals X, ODmin are the light absorption value in blank hole.The standard specimen value of calculating plots the semilog coordinate system curve map of a corresponding fenifrothion concentration (mg/L), 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.
Embodiment 6
The storage life test
Kit being positioned over 4 ℃ and-20 ℃ of preservations, getting 0,20,40,60,120,180,240,300 and the kit of 360d respectively, serves as to measure concentration with optimum antibody antigen working concentration, carries out standard model and detects, and detects effect to measure it.Storage life measurement result such as following table:
Table 1. direct competitive ELISA method kit storage life test findings
| Time (d) | ??0 | ??20 | ??40 | ??60 | ??120 | ??180 | ??240 | ??300 | ??360 |
| ?OD 492nm? ?(4℃) | ? ??1.069 | ? ??1.067 | ? ??1.066 | ? ??1.064 | ? ??1.063 | ? ??1.062 | ? ??1.062 | ? ??1.060 | ? ??1.058 |
| ?OD 492nm? ?(-20℃) | ? ??1.067 | ? ??1.064 | ? ??1.063 | ? ??1.062 | ? ??1.062 | ? ??1.060 | ? ??1.060 | ? ??1.059 | ? ??1.057 |
Table 2. indirect competitive ELISA method kit storage life test findings
| Time (d) | ??0 | ??20 | ?40 | ??60 | ??120 | ??180 | ?240 | ??300 | ??360 |
| ?OD 492nm?(4℃) | ??1.116 | ??1.115 | ?1.113 | ??1.112 | ??1.110 | ??1.108 | ?1.106 | ??1.104 | ??1.100 |
| ?OD 492nm?(-20℃) | ??1.120 | ??1.120 | ?1.115 | ??1.114 | ??1.112 | ??1.109 | ?1.108 | ??1.105 | ??1.102 |
Above result as can be seen, kit can be preserved more than 12 months under 4 ℃ at least.
Embodiment 7
The kit sensitivity determination
The fenifrothion standard solution is diluted to series concentration, analyze with direct ELISA method, obtain with lower curve (Fig. 1), by Tu Kede, directly ELISA method regression equation is: y=44.92+16.44x, fenifrothion are in 0.01～10mg/L scope, and log (B/BO) is linear with the logarithm value of fenifrothion concentration, related coefficient is r2=0.9431, detects to be limited to 0.01mg/L.Obtain with lower curve (Fig. 2) with the indirect elisa method analysis, by Tu Kede, the regression equation of indirect elisa method is: y=63.34+9.82x, fenifrothion is in 0.005～10mg/L scope, log (B/BO) is linear with the logarithm value of fenifrothion concentration, related coefficient is r2=0.9845, detects to be limited to 0.005mg/L.
Accuracy test and precision test
Get the fenifrothion standard specimen of three variable concentrations, add in the blank sample, each concentration is established 6 repetitions, measures.The result of the kit recovery is as follows, and water is 96.8%～106.7%, and vegetables are 110.8%～117.5%.The Variation Lines number average of water determination is lower than 5.5%, and the Variation Lines number average of vegetables is lower than 11.5%.
Embodiment 9
The test of kit specificity
Select fenifrothion analog such as parathion-methyl, parathion, Entex, 3-methyl-4-nitrophenol etc., reactions steps is with the kit operating process, obtains concentration in the inhibition of various agricultural chemicals, calculates the cross reactivity of agricultural chemicals to fenifrothion with following formula again.Cross reacting rate is more little, reaction
Specificity strong more.Cross reacting rate can be calculated as follows:
This test determination the results are shown in Table 3.As known from Table 3, the indirect elisa method inhibiting rate reaches at 50% o'clock, and the fenifrothion desired concn is 13 μ g/L, and other several agricultural chemicals desired concns are all greater than 800 μ g/L.The specificity that kit is described is good, can guarantee the reliability to fenifrothion determined result of residue in the sample.
The test of table 3 kit specificity
| The compound title | Concentration in the inhibition (μ g/mL) | Cross reacting rate (%) |
| Fenifrothion | 0.0136 | / |
| Parathion-methyl | 8.94 | 0.152 |
| Parathion | 1560 | ＜0.001 |
| Entex | 3200 | ＜0.001 |
| 3-methyl-4-nitro-phenol | 2.38 | 0.571 |
Claims (9)
1. enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis, it comprises box body, be located at 96 holes/40 hole ELISA Plate and/or the test tube 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-fenifrothion antibody generation specificity association reaction, and seal with 2%～5% gelatin, reagent comprises cleansing solution in the box, the substrate dilution, anti-fenifrothion antibody, the fenifrothion standard solution, the anti-fenifrothion rabbit antibody of the goat anti-rabbit antibody of horseradish peroxidase-labeled or horseradish peroxidase-labeled, substrate and reaction terminating liquid.
2. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1, it is characterized in that said envelope antigen is haptens (I) O-methyl-O-(3-methyl 4-nitrobenzophenone-N-(2-carboxyethyl) thio-phosphamide or haptens (II) O, compound of O-dimethyl-O-(3-methyl-4-(6-carboxylic hexyl acylamino-) phenyl) thiophosphate and ovalbumin.
3. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1 is characterized in that anti-fenifrothion antibody is for injecting the immunoglobulin (Ig) (IgG) that can combine with fenifrothion molecule, fenifrothion coating antigen specificity that produces behind the rabbit by fenifrothion haptens and the synthetic conjugate of bovine serum albumin(BSA) as immunogene.
4. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1 is characterized in that it is that fenifrothion antibody (IgG) combines the compound that forms with horseradish peroxidase (HRP) that enzyme is marked anti-fenifrothion antibody.
5. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1 is characterized in that said cleansing solution proportion of composing is potassium dihydrogen phosphate 0.1～0.3 gram, sodium hydrogen phosphate 2～4 grams, potassium chloride 0.1～0.3 gram, polysorbas20 0.5～3mL, distilled water 500～1000mL.
6. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1 is characterized in that said substrate dilution proportion of composing is citric acid 3～6 grams, sodium hydrogen phosphate 1～3 gram, distilled water 500～1000mL.
7. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1 is characterized in that said substrate is 30% hydrogen peroxide, 10～15mL and o-phenylenediamine (OPD) pressed powder 40～120mg.
8. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1 is characterized in that said reaction terminating liquid is a 2mol/L sulfuric acid.
9. a kind of enzyme-linked immunosorbent assay kit that is applicable to the fenifrothion retention analysis according to claim 1, it is characterized in that said carbonate buffer solution as coating buffer, compound method is: 1～2g sodium carbonate and 2～4g sodium bicarbonate, distilled water 1L, pH=9.6.
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|---|---|---|---|
| CN 200410030740 CN1677108A (en) | 2004-04-02 | 2004-04-02 | Enzyme-linked immunosorbentassay reagent kit for analyzing snout-killing sulfur-phosphous residual |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012239B (en) * | 2007-01-26 | 2010-11-10 | 中国农业大学 | Fenitrothion hapten, artificial antigen, specified antibody and use thereof |
| CN104974257A (en) * | 2015-04-28 | 2015-10-14 | 江苏省农业科学院 | Hybridoma cell strain 4F2, monoclonal antibody produced therefrom and application of monoclonal antibody |
| CN106290830A (en) * | 2016-07-26 | 2017-01-04 | 大连民族大学 | A kind of based on up-conversion fluorescence nanoparticle quickly immune chromatography test paper detecting fenifrothion and preparation method thereof |
| CN107807237A (en) * | 2017-12-13 | 2018-03-16 | 百奥森（江苏）食品安全科技有限公司 | A kind of food inspection kit for being used to detect organophosphor |
-
2004
- 2004-04-02 CN CN 200410030740 patent/CN1677108A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101012239B (en) * | 2007-01-26 | 2010-11-10 | 中国农业大学 | Fenitrothion hapten, artificial antigen, specified antibody and use thereof |
| CN104974257A (en) * | 2015-04-28 | 2015-10-14 | 江苏省农业科学院 | Hybridoma cell strain 4F2, monoclonal antibody produced therefrom and application of monoclonal antibody |
| CN106290830A (en) * | 2016-07-26 | 2017-01-04 | 大连民族大学 | A kind of based on up-conversion fluorescence nanoparticle quickly immune chromatography test paper detecting fenifrothion and preparation method thereof |
| CN107807237A (en) * | 2017-12-13 | 2018-03-16 | 百奥森（江苏）食品安全科技有限公司 | A kind of food inspection kit for being used to detect organophosphor |
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