CN101012239A - Fenitrothion hapten, artificial antigen, specified antibody and use thereof - Google Patents
Fenitrothion hapten, artificial antigen, specified antibody and use thereof Download PDFInfo
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Abstract
The invention discloses a O, O-dimethyl-O-[-3-methyl-4-amino phenyl] thionic phosphate (FNH2) as fenitrothion hapten, artificial antigen and specific antibody, which is characterized by the following: reducing nitro in the fenitrothion molecular structure into amino under acid condition with zinc powder; reacting to obtain the hapten molecule in connection with carrier protein; diazotizing to couple hapten and protein to obtain immunogen and peridium; using immune animal with immunogen to make the specific antibody.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of fenitrothion 95 haptens, artificial antigen and become specific antibody, the invention still further relates to the purposes of this haptens, artificial antigen, specific antibody with this antigen prepd.
Background technology
Because the continuous expansion of the continuous development of technique in using agriculture chemical and the scale of use, the environmental influence that pesticide residue cause and the chronic and long-time effect of human health is subjected to people's attention and worry day by day, restriction to pesticide residue 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 metabolite thereof, traditional analysing and detecting method mainly relies on gas-chromatography (GC), high performance liquid chromatography (HPLC), mass spectrum physico-chemical analysis means such as (MS), but these analytical procedures 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, and therefore, although it is very short that immunoassay is used for time of pesticide residue analysis, very fast having obtained in the pesticide residue analysis of environmental sample and food samples used widely.
Fenitrothion 95 [fenitrothion, O, O-dimethyl-O-(3-methyl-4-nitrophenyl) thiophosphatephosphorothioate], commodity Sumithion by name etc., be a kind of broad-spectrum organophosphorous pesticide, be widely used in the pest control 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.Fenitrothion 95 to daylight stable, is met the easy decomposition failure of alkali to the higher animal low toxicity under the normal temperature, and the safety interval on fruit, vegetables is 10～15 days.Along with the increase of agricultural chemicals usage quantitys such as fenitrothion 95, especially a large amount of uses on crop and vegetables cause many harm to HUMAN HEALTH, and this has caused people's common concern.Therefore develop a kind of simple trace analysis method quick, that be applicable to the pesticide residue on-site supervision and have important use value.
In pesticide residue analysis, the conventional sense method of fenitrothion 95 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 fenitrothion 95.Yet, because most pesticide molecules belong to small molecules, and micromolecular compound does not generally have immunogenicity, can not produce specific antibody by the direct immunization animal, therefore, haptens that must synthetic outstanding molecule stereo structure specificity position, and connect and compose effectively artificial binding substances with macromolecular carrier could immune animal produces the specific antibody at this target micromolecular compound.The binding substances of this haptens and macromolecular carrier is called artificial antigen.The preparation of artificial antigen need be considered various factors, comprises the structural difference of binding site, combination, kind of carrier and haptens and target analytes, and these factors all may have influence on the character of corresponding antibodies.Therefore, it is that decision produces its specific antibody quality and the key of setting up the immunochemical analyses method.
At present, Shang Weiyou is specially about the argumentation or the works of hapten molecule design and the research of immune recognition mechanism thereof.In general, haptenic molecular designing should be considered the factor of two aspects: the first, and can stimulate body to produce immunne response; The second, can the antibody of generation have the activity of expection, i.e. can its immune identification get a desired effect." ideal " haptens is at first answered the feature chemical structure, particularly stereochemical structure of reflection analysans as much as possible; Secondly, " ideal " haptens should have a handle (or spacerarm) of being convenient to antibody to target structure identification, so that the specificity structure of molecule can be exposed to carrier surface to greatest extent, thereby keep specificity, should consider its length, polarity and position usually for spacerarm; At last, " ideal " haptens should have and link coupled functional groups such as carrier proteins, as-COOH ,-NH2 ,-OH, functional groups such as-SH.In a word, for different target agricultural chemical compounds, its haptenic molecular designing is different, with a kind of agricultural chemicals, haptenic molecular designing also can be designed the haptens of different structure from a plurality of sites, prepares corresponding polyclone or monoclonal antibody respectively.
In the immune analysis method research of fenitrothion 95, the preparation of haptens and artificial antigen also is the same.To haptenic requirement is stable in properties, has the feature structure or the characteristic group of target compound, has the spacerarm of suitable length, and have the certain activity group can with carrier proteins generation linked reaction.This research is intended by the synthetic fenitrothion 95 haptens of design, haptens and carrier protein couplet are obtained artificial antigen, thereby immune animal produces the fenitrothion 95 polyclonal antibody, set up the elisa assay method of fenitrothion 95 on this basis, and be used to prepare the immunoassay kits of fenitrothion 95 residue detection.
Summary of the invention
The fenitrothion 95 haptens that the purpose of this invention is to provide a kind of new texture, and with this haptens and different carrier protein couplets, preparation can be used for the fenitrothion 95 artificial antigen of fenitrothion 95 immunoassay research; Specific antibody with this artificial antigen prepd; And the purposes of this type of haptens, artificial antigen and antibody.
Fenitrothion 95 haptens of the present invention is O, O-dimethyl-O-[-3-methyl-4-aminophenyl] phosphorothionate (being called for short FNH2), its molecular structural formula is:
Above-mentioned fenitrothion 95 haptens and carrier protein couplet are obtained the fenitrothion 95 artificial antigen, and its molecular structural formula is:
Wherein, protein carrier Protein can be bovine serum albumin (BSA), ovalbumin (OVA), albumin rabbit serum (RSA) or human serum albumin (HSA) etc.
Above-mentioned fenitrothion 95 artificial antigen immune animal is obtained mono-clonal or polyclonal antibody with fenitrothion 95 generation specific immune response.
The present invention provides above-mentioned fenitrothion 95 haptenic purposes simultaneously, is the raw material as the antigen system of animal immune.
The present invention provides the purposes of above-mentioned fenitrothion 95 specific antibody simultaneously, is used for detecting the residual quantity of food, agricultural-food and environmental sample fenitrothion 95.
The present invention provides the immunoassay kits of the fenitrothion 95 residual quantity that contains above-mentioned fenitrothion 95 artificial antigen, specific antibody simultaneously.
Based on its substituted-phenyl thiophosphatephosphorothioate of fenitrothion 95 is its specificity functional group, effect through strong reductive agent, make the amidized method of fenitrothion 95, make nitro on the phenyl ring become amino for being connected with carrier proteins, synthesized haptens (chemical name is O, O-dimethyl-O-[-3-methyl-4-aminophenyl] phosphorothionate) (being called for short FNH2).This synthesis of semiantigen is easy, the reservation of maximum possible the structure of original fenitrothion 95, provide assurance for obtaining specific antibody.
The nitro position of the site of the haptenic design of fenitrothion 95 of the present invention in the fenitrothion 95 molecular structure is transformed into amino to the nitro of contraposition on the phenyl ring by chemosynthesis, make its have can with protein link coupled group-NH2.Synthetic fenitrothion 95 hapten compound is the new compound of initiating both at home and abroad among the present invention, the chemical structure that has farthest kept fenitrothion 95, immunizing antigen with this haptens preparation goes immune animal, the tiring of resulting antibody, specificity, avidity are all relatively good, and be all very low with the cross reacting rate of other organophosphorus pesticides.
Description of drawings
Fig. 1 is the typical curve of direct competitive ELISA method fenitrothion 95;
Fig. 2 is the typical curve of indirect competitive ELISA method fenitrothion 95.
Specific implementation method
Describing the present invention below among the embodiment in more detail, is not limitation of the present invention, and wherein ratio and per-cent are not having to be the w/w ratio under the situation of specified otherwise.
The preparation of embodiment 1 fenitrothion 95 haptens, artificial antigen and antibody thereof
1. haptens O, O-dimethyl-O-[3-methyl-4-aminophenyl] phosphorothionate (abbreviating FNH2 as) synthetic
The synthetic of haptens FNH2 is to finish by the amination of fenitrothion 95.Synthetic route is as follows:
Concrete operations are as follows:
Get the about 2.0g of the former medicine of fenitrothion 95, be dissolved in the 20ml ether, with cold 1% the sodium carbonate solution washing of 2X10ml 2 times, tell ether layer, change in the three-necked bottle, add acetate/hydrochloric acid (9: 1) solution 20ml, add the 4g zinc powder, backflow 1.5h filters.With filter residue 30mL CCl
4Wash three times, then with distilled water 30ml washing CCl
4Phase is abandoned water.Filtrate is added 10%NaOH regulate the pH value, have this moment flocks to occur, extract with ether 4 * 25ml to neutrality.With ether and aforementioned CCl
4Merge mutually, add anhydrous sodium sulfate drying and spend the night.The pressure reducing and steaming ether obtains yellow oil.
In yellow oil, add water 10ml, regulate pH to 1-2, divide and remove insolubles,, discard normal hexane with normal hexane 2 * 10ml washing water with 1M HCl.It is 10-11 that water layer is regulated pH with 10%NaOH, uses CHCl
3Water is abandoned in 2 * 20ml extraction.Merge CHCl
3Layer adds the 8g anhydrous sodium sulfate drying and spends the night, and the pressure reducing and steaming solvent obtains the about 1.3g of scarlet oily matter, productive rate about 68%.
The structure of haptens FNH2 product is identified: get above-mentioned synthetic product respectively through IR, MS and
1H-NMR measures its molecular structure.
(a) infrared spectra IR:
cm
-1：3320(m，N-H)，2940(s，C-H)，1620(s，C＝C)，1510(s，P＝S)，1425(s，C-N)，1200(s，P-O).
(b) mass spectroscopy EI:
M/z is: 247[56, M
+], 125[28, M
+-122], 124[62, M
+-123], 123[15, M
+-124], 107[22, M
+-141].
(c) nucleus magnetic resonance (1H-NMR, 300MHz, CDCl
3):
δppm：2.36(s，Ar-CH
3)，3.48(s，2H，NH
2)，3.76(s，3H，CH
3)，3.92(s，3H，CH
3)，8.10-7.55(m，3H，Ar-H).
2. artificial antigen synthesizes and purifying
Synthesizing of artificial antigen, adopt the diazotization method to make haptens pass through amino and carrier proteins generation coupling, synthetic route is as follows:
Concrete operations are as follows:
Get the haptens FNH2 0.05g (about 0.2mmol) for preparing, be dissolved in the 12.5ml 0.08mol/lHCl solution, pour three-necked bottle into and stir, be cooled to 0 ℃ under the ice bath, dropwise add refrigerative NaNO in advance
2Solution (0.07g is dissolved in the 10ml water), ice bath (0-2 ℃) reaction 2h adds urea 0.05g and removes excessive HNO
2
Taking by weighing BSA (perhaps OVA) protein 25 0mg is dissolved in the borate buffer solution of 50ml pH8.7, splash in the above-mentioned azo solution, drip and finish, after keeping 0 ℃ of reaction 2h, with the reaction solution dialysis tubing of packing into, earlier with distill water dialysis 2-4 time, used normal saline dialysis then 3 days, packing is stored in-20 ℃ the refrigerator stand-by.
The mensuration of artificial antigen binding ratio:
Ratio in used haptens, carrier proteins and coupled product of synthetic fenitrothion 95 immunizing antigen reaction, carrying out UV scanning respectively measures, UV scanning result shows, product after the coupling has moved to 220nm and has produced synergistic effect at 276nm and proteic maximum absorption from original 215nm at maximum absorption wavelength, calculates its binding ratio at the absorbance of maximum light absorption value respectively by comparing the three.The result is as follows as calculated: the binding ratio of haptens and BSA is 12: 1, with the binding ratio of OVA be 10: 1.
3. the preparation of antibody
Immune animal prepares antiserum(antisera):
Healthy male new zealand white rabbit is selected in experiment for use, and 3 rabbits of every kind of immunogen immune are numbered 1-3 respectively.Experiment immunization dosage fundamental immunity is 0.5-1.0mg/kg, booster immunization dosage is 1.0-1.5mg/kg, dilutes an amount of BSA binding substances with physiological saline, adds equal-volume Freund's complete adjuvant (adopting Freund's incomplete adjuvant during booster immunization), fully emulsified, emulsion droplet does not disperse in splashing into water.The method that adopts the subcutaneous multi-point injection in back to combine with the leg muscle injection.Carry out booster immunization after three weeks, carry out booster immunization once more every two weeks later on.From immunity for the third time, each immunity back the 8th day, from the rabbit ear edge vein exploitating blood, mensuration is tired.
Purifying antibody:
Sad-the ammonium sulfate salting-out process of general employing also can adopt the albumin A column chromatography.Sad-ammonium sulfate salting-out process is adopted in this experiment.Sad can be under the condition of slant acidity with serum in protein except that IgG all precipitate, have only IgG in the supernatant.Sad adding is different because of the source of antibody, and rabbit anteserum is 75 μ g/ml, and mice serum is 40 μ g/ml, and mouse ascites is 33 μ g/ml.The rate of recovery of this method IgG reaches more than 90%.At last antibody is made lyophilized powder, packing ,-20 ℃ of preservations.
The mensuration of antiserum titre:
After immune programme for children began, from booster immunization for the second time, serum was through suitably tiring with indirect ELISA mensuration after the dilution in the rabbit ear edge vein exploitating blood in the 8th day in immune at every turn back.After the 5th immunity, rabbit has obtained high antibody of tiring, and sero-fast tiring is 1: 12500 (referring to that the OD490nm value equals 1.0).
The assembling of embodiment 2 test kits
The present invention is applied to prepare the immunoassay kits that is suitable for the fenitrothion 95 residue detection with the artificial antigen and the antibody of preparation, this test kit comprises box body, intravital 96 holes of box/40 hole enzyme plate and detection reagent, in every hole of enzyme plate, by the coating buffer bag by can with anti-fenitrothion 95 antibodies specific bonded envelope antigen, and seal with 2%～5% gelatin.
Reagent comprises in the box: anti-fenitrothion 95 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 washings (diluent), substrate diluent, anti-fenitrothion 95 antibody (being applicable to indirect ELISA), fenitrothion 95 standardized solution, horseradish peroxidase-labeled or horseradish peroxidase-labeled, and compound method is as follows:
Washings (reaction diluent) 40mL, proportion of composing is potassium primary phosphate 0.1g, Sodium phosphate dibasic 4g, Repone K 0.1g, tween 20 3mL, distilled water 1000mL, is normal 15～30 times of concentrated solutions that use;
Substrate diluent 50mL is formulated as follows: citric acid 3g, Sodium phosphate dibasic 1g, distilled water 1000mL are normal 5～10 times of concentrated solutions that use;
Enzyme substrates is 30% hydrogen peroxide 15mL, developer is 3,3 ', 5,5 '-tetramethyl benzidine (TMB) solution 15mL or O-Phenylene Diamine (OPD) pressed powder 20mg;
Anti-fenitrothion 95 antibody (IgG) 400mL, working concentration are 1: 1000 (indirect elisa method); Described anti-fenitrothion 95 antibody (IgG) can carry out specificity bonded immunoglobulin (Ig) (IgG) with fenitrothion 95 molecule, fenitrothion 95 coating antigen for what produce after as immunogen injection rabbit by haptens and bovine serum albumin synthetic conjugate;
Goat anti-rabbit antibody (indirect elisa method) the 200 μ L of anti-fenitrothion 95 rabbit antibody of horseradish peroxidase-labeled (directly ELISA method) or horseradish peroxidase-labeled, 800～1500 times of concentrated solutions during for normal the use; The fenitrothion 95 antibody of above-mentioned horseradish peroxidase-labeled is that anti-fenitrothion 95 antibody (IgG) combines the mixture that forms with horseradish peroxidase (HRP);
Reaction terminating liquid 30mL is 2mol/L sulfuric acid;
6 bottles in fenitrothion 95 different concns series (0.1,0.5,2,10,50,100mg/L) standardized solution, 1～4mL/ bottle, methanol constant volume, during use with 10 times of PBST dilutions.
The preparation of embodiment 3 enzyme labelled antibodies (improvement sodium periodate method)
Take by weighing 5～10mg horseradish peroxidase HRP and be dissolved in the 1L distilled water, add the 0.1mol/L NaIO that 0.2～0.4mL newly joins
4Solution, lucifuge stirs 15～30min under the room temperature.Above-mentioned solution is packed in the dialysis tubing, and with the acetate buffer solution dialysis of 1mmol/LpH 4.4,4 ℃ are spent the night.Carbonate buffer solution with pH 9.5 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.Reaction solution is packed in the dialysis tubing, and with the PBS dialysis of 0.15mol/L pH7.4,4 ℃ are spent the night.Under agitation dropwise add equal-volume saturated ammonium sulphate solution, put 4 ℃ 1～2 hour.3000rpm centrifugal half an hour, abandon supernatant liquor.Throw out is washed and secondary with the semi-saturation ammoniumsulphate soln, and last throw out is dissolved among the PBS of a small amount of 0.15mol/L pH7.4.Above-mentioned solution is packed in the dialysis tubing, with the PBS damping fluid dialysis of 0.15mol/L pH7.4, remove (detecting with Nai Shi reagent) behind the ammonium ion, centrifugal, supernatant liquor is enzyme conjugates, after the packing of equivalent glycerine, respectively at-4 ℃ ,-20 ℃ preservations.
The preparation of embodiment 4 coated elisa plates
Envelope antigen (FEN-OVA) (contains 1～2g yellow soda ash and 2～4g sodium bicarbonate with the carbonate buffer solution of pH 9.6 0.05mol/L, distilled water 1L) is diluted to 0.5～4 μ g/mL, add 100 μ L in every hole of enzyme 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 thermostat containers and sealing 0.4～1 hour, with PBST washing 3 times, pat dry after drying and preserve.
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, united extraction liquid concentrates, and is settled to 10mL with the PBST dilution then, carries out elisa assay.
Vegetable sample: get and take by weighing 10g after vegetable sample is pulverized with pulverizer, with 20～40mL acetone extraction three times, united extraction liquid concentrates, and is settled to 10mL with PBST, takes a sample and carries out elisa assay.
Blood: get blood of human body, directly analyze after adding the anti-freezing element with the ELISA method.
Liquid of gastric lavage (2% sodium hydrogen carbonate 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 liquor is analyzed with the ELISA method.
Embodiment 6
The test kit operating process is as follows:
1) direct competitive ELISA method: take out an enzyme plate that is coated with the fenitrothion 95 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 solution 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 color reaction in 15～25 minutes; Add 50 μ L reaction terminating liquids, after mixing, measure OD
450nmPerhaps OD
490nmValue.
2) indirect competitive ELISA method: take out an enzyme plate that is coated with the fenitrothion 95 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 solution 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 color 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 4) ODmax is not dosing, the light absorption value when ODx is agricultural chemicals X, ODmin
Light absorption value for the blank hole.
5) the standard specimen value of Ji Suaning plots the semilog coordinate system graphic representation (Fig. 1,2) of a corresponding fenitrothion 95 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.
The test of embodiment 7 preservation perives
Test kit being positioned over 4 ℃ and-20 ℃ of preservations, getting 0,20,40,60,120,180,240,300 and the test 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.Preservation period measurement result such as following table:
Table 1. direct competitive ELISA method test kit preservation period test-results
| 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 test kit preservation period test-results
| 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, test kit can be preserved more than 12 months under 4 ℃ at least.
The fenitrothion 95 standardized 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, fenitrothion 95 are in 0.01～10mg/L scope, and log (B/B0) is linear with the logarithmic value of fenitrothion 95 concentration, relation conefficient 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, fenitrothion 95 is in 0.005～10mg/L scope, log (B/B0) is linear with the logarithmic value of fenitrothion 95 concentration, relation conefficient is r2=0.9845, detects to be limited to 0.005mg/L.
Embodiment 9 accuracy tests and precision test
Get the fenitrothion 95 standard specimen of three different concns, add in the blank sample, each concentration is established 6 repetitions, measures.The result of the test kit rate of recovery is as follows, and water is 96.8%～106.7%, and vegetables are 110.8%～117.5%.The variation coefficient of water determination all is lower than 5.5%, and the variation coefficient of vegetables all is lower than 11.5%.
The specificity test of antibody
The specificity of antibody cross reactivity commonly used is as the major criterion of estimating, and the cross reaction between the compound antigen of promptly resulting antibody and other different structures is more little, and the specificity of antibody is just good more.
Target compound and analogue thereof are done serial dilution, respectively with the antibody that obtains, press the same method production standard curve of production standard curve, and the consumption when on curve, drawing the dosage of inhibiting rate 50% and analogue inhibiting rate 50%, calculate the cross reacting rate of each analogue then.
Select fenitrothion 95 analogue such as parathion-methyl, thiophos, Tiguvon, 3-methyl-4-nitro
Phenol etc., reactions steps are with the test kit operating process, obtain concentration in the inhibition of various agricultural chemicals, calculate the cross reactivity of agricultural chemicals to fenitrothion 95 with following formula again.Cross reacting rate is more little, and the specificity of reaction is 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 fenitrothion 95 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 test kit is described is good, can guarantee the reliability to fenitrothion 95 determined result of residue in the sample.
The test of table 3 test kit specificity
| The compound title | Concentration in the inhibition (μ g/mL) | Cross reacting rate (%) |
| Fenitrothion 95 | 0.0136 | / |
| Parathion-methyl | 8.94 | 0.152 |
| Thiophos | 1560 | ＜0.001 |
| Tiguvon | 3200 | ＜0.001 |
| 3-methyl-4-nitro-phenol | 2.38 | 0.571 |
Claims (9)
1, a kind of fenitrothion 95 haptens is characterized in that molecular structure is:
2, a kind of fenitrothion 95 artificial antigen is characterized in that molecular structure is:
Wherein, Protein is a carrier proteins.
3, artificial antigen as claimed in claim 2, wherein said carrier proteins are BSA, OVA, RSA or HSA.
4, a kind of fenitrothion 95 specific antibody, it is to adopt claim 2 or 3 described artificial antigen immune animals to obtain.
5, the ELISA detection kit that contains the described specific antibody of claim 4.
6, the described haptens of claim 1 is as the application in the raw material of the antigen system of animal immune.
7, the application of the described specific antibody of claim 4 in the fenitrothion 95 residual quantity of test sample.
8, application as claimed in claim 7 is characterized in that, described sample is food, agricultural-food or environmental sample.
9, application as claimed in claim 8 is characterized in that, described environmental sample is pedotheque or water sample.
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| CN105424934A (en) * | 2015-10-30 | 2016-03-23 | 山东博科生物产业有限公司 | N-acetyl-beta-D glucosidase reagent and detection method |
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| CN106706924A (en) * | 2016-12-08 | 2017-05-24 | 申联生物医药（上海）股份有限公司 | Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1677108A (en) * | 2004-04-02 | 2005-10-05 | 韩丽君 | Enzyme-linked immunosorbentassay reagent kit for analyzing snout-killing sulfur-phosphous residual |
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2007
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| CN102229672A (en) * | 2011-05-23 | 2011-11-02 | 重庆大学 | Single-chain antibody against fenitrothion and preparation method thereof |
| CN104914101A (en) * | 2015-06-04 | 2015-09-16 | 大同市城区北关社区卫生服务中心 | ELISA detection method for vincristine |
| CN104914101B (en) * | 2015-06-04 | 2018-06-19 | 大同市城区北关社区卫生服务中心 | A kind of ELISA detection method of vincristine |
| CN105424934A (en) * | 2015-10-30 | 2016-03-23 | 山东博科生物产业有限公司 | N-acetyl-beta-D glucosidase reagent and detection method |
| 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 |
| CN106706924A (en) * | 2016-12-08 | 2017-05-24 | 申联生物医药（上海）股份有限公司 | Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine |
| CN108931640A (en) * | 2018-09-27 | 2018-12-04 | 北京勤邦生物技术有限公司 | A kind of test strips and its application detecting organophosphorus pesticide |
| CN108931640B (en) * | 2018-09-27 | 2022-10-18 | 北京勤邦生物技术有限公司 | Test strip for detecting organophosphorus pesticide and application thereof |
| CN113388037A (en) * | 2021-04-14 | 2021-09-14 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
| CN113388037B (en) * | 2021-04-14 | 2022-04-29 | 华南农业大学 | Preparation and application of specific recognition fenitrothion nano antibody |
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