CN101289463A - Competitive hapten of ethyl alkron, competitive antigen and uses thereof - Google Patents
Competitive hapten of ethyl alkron, competitive antigen and uses thereof Download PDFInfo
- Publication number
- CN101289463A CN101289463A CNA2008100621548A CN200810062154A CN101289463A CN 101289463 A CN101289463 A CN 101289463A CN A2008100621548 A CNA2008100621548 A CN A2008100621548A CN 200810062154 A CN200810062154 A CN 200810062154A CN 101289463 A CN101289463 A CN 101289463A
- Authority
- CN
- China
- Prior art keywords
- competition
- ethyl parathion
- ethyl
- haptens
- parathion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an ethyl parathion competition hapten, and the molecular structural formula thereof is showed on the right; wherein, n is equal to 1 to 6, and R is an atom or a group which exists on a benzene ring in the form of one, two or the combination of each two. The invention also provides an ethyl parathion competition source prepared by the ethyl parathion competition hapten, and a heterogeny ELISA analysis method established by the ethyl parathion competition source and immunogen PB-BSA at the same time, which can be used for detecting the residue of the ethyl parathion in foods, agricultural products and environmental samples.
Description
Technical field
The present invention relates to former structure of a kind of ethyl parathion competition haptens and competition and uses thereof, belong to agricultural chemicals immunochemical technique field.
Background technology
Molecular weight is less than 1000 daltonian small molecules noxious chemical such as agricultural chemicals and meta-bolitess thereof, and its traditional residue analysis method mainly is to rely on gas-chromatography (GC), liquid chromatography (HPLC), gas chromatography mass spectrometry (GC/MS) or LC-MS physico-chemical analysis means such as (HPLC/MS).But shortcomings such as pre-treatment step is loaded down with trivial details, solvent-oil ratio is big, length consuming time that instrument analytical method has are difficult to satisfy the needs that a large amount of samples carried out low-cost rapid screening.Therefore people urgently wishes to have a kind of simple, fast, sensitivity and inexpensive detection technique can carry out large batch of shaker test in the open air with in the laboratory; The immunochemical analyses technology has one of the trace analysis of using value and development potentiality technology most just because of possessing these advantages, having become.This technology has become a brand-new field of pesticide residue Study on Trace at present, by the Analytical Techniques of Pesticide Residues of the row current preferential research in position, development and utilization, this technology is recommended to many countries by the World Food Programme (FAO), and U.S. EPA is classified immuno analytical method and gas-chromatography, liquid chromatography as a column technology of pesticide residue analysis jointly.
Immune analysis method detects principle according to it can be divided into competition law and non-competing method again.Competition law is owing to have the strong advantage of highly sensitive and anti-matrix interference ability than non-competing method, and is most widely used.The competitive immunoassay method can be divided into homoimmune analysis (HomologousAssays) and alloimmunization analysis (Heterologous Assays) method again according to the similarities and differences of its coating antigen or enzyme mark haptens structure and immune haptens structure.Homology is analyzed, and promptly uses with a kind of artificial antigen and corresponding antibody of haptens preparation and carries out immunoassay, also is present method the most frequently used in the immunochemical analyses method of agricultural chemicals and veterinary drug and in the kit developing.The homology analysis is connected with different proteins (BSA, OVA, KLH etc.) through different coupling methods because coating antigen that uses in immune haptens and the immunoassay or enzyme mark haptens all adopt with a kind of haptens, and haptens synthesizes comparatively small amt, and is time saving and energy saving.But a little less than the immunity from interference of homology analysis to matrix effect, the detection Chang Yin of actual environment sample there is the matrix influence and method sensitivity to occur low.And, maximum " bottleneck " problem that immunochemical analyses method development at present runs into be exactly in the homology analysis small molecules agricultural chemicals and antigen to compare the competitive power of antibody low, and sensitivity often occurs when being applied to the actual environment sample analysis and descend greatly because of matrix effect, finally cause immunization method and instrument detecting ability that certain gap is arranged, cause the application in practice of immunochemical analyses method to be restricted.This also is that puzzlement agricultural chemicals Fast Detection Technique is carried out the most key problem that commercialization conversion (as products such as exploitation quick detection kit, gold test strips) is faced.Analysis of heterogensis then can increase the competitive power of small molecules agricultural chemicals antagonist, and Enhancement Method improves the sensitivity of method greatly to the freedom from jamming of matrix effect.In addition, analysis of heterogensis also has raising method specificity, increases the antibody purposes, passes through to change advantages such as coating antigen and the many method for detecting residue of immune haptenic package development.
Analysis of heterogensis is meant and uses the immunoassay that carries out with prepared coating antigen of different haptens or labelled antigen respectively with a kind of antibody of different haptens gained of analyte.Wherein, adopting the haptens different with immune haptens promptly is the competition haptens, comprises that bag is by haptens and enzyme mark haptens.At present, pay attention on the analysis of heterogensis measuring method of superiority that proves analysis of heterogensis and the different agricultural chemicals of development, competition haptens Research on rational design also being belonged to the starting stage more than the analysis of heterogensis technical development.Different with immune haptens design is, thereby the purpose of immunity haptens design is the antibody that can obtain high specific to the at utmost similar haptens of target compound in order to design, and the haptenic design requirements of best competition be not only will with the target compound dissmilarity, also need simultaneously and immune haptens dissmilarity, therefore need to satisfy the requirement of two aspects, and dissimilar design space is bigger, and design difficulty also increases thereupon.
Ethyl parathion is the malicious organophosphate insecticides of a kind of widely used height, is mainly used in the various pests on the farm crop such as control paddy rice, cotton, corn.The ethyl parathion toxicity grading belongs to hypertoxic type, and " three cause " effect is arranged.Route of entry is divided into: suck, eat, absorb through skin.Eat the food that polluted by ethyl parathion (comprise melon and fruit, vegetables, dairy products, grain, fishery products and the fowl poultry that dies like a rat etc.) by mistake, very big to human health damage, acute poisoning shows as the inhibition cholinesterase activity, causes the nervous physiology dysfunction; Chronic moderate symptom is not generally not clearly, clinical manifestation mostly is neurasthenia syndrome, muscarinic and nicotinic signs and symptoms can appear in part patient, the infringement of sight functions such as that small number of patients also can have is ametropia, constriction of visual field, dyschromatopsia, can have nerve-electromyogram(EMG) to change and electroencephalogram unusual.Because ethyl parathion toxicity is higher, be inhibited on vegetables, the fruit in recent years and used, but in the supervision of foreign trade, food and the environmental safety of agricultural-food detected, a class height such as ethyl parathion poisoned the important pollutent that compound remains conventional sense.And, ethyl parathion and other agricultural chemicals composite also increasing; Therefore press for can the rapid detection agricultural-food in the method for ethyl parathion residual quantity, guaranteeing the healthy and safe of the people, and the smooth outlet of agricultural products in China.
Traditional ethyl parathion detection method has gas-chromatography (GC), liquid chromatography (HPLC) or mass spectrum (MS) etc., sample pretreatment process complexity, the analytical cycle that workload is big, instrument is expensive, requirement has those skilled in the art and grows, and the trace target analytes lose easily, be difficult to satisfy the needs of great amount of samples field quick detection.Immunoassay provides a new analyzing and testing approach for the quick retention analysis of ethyl parathion.Forefathers have and set up the residue detection that the homoimmune analytical procedure is applied to ethyl parathion, but because the immunogen structure changes too much antigenic determinant, and cause low, the poor specificity of affinity of antibody, and homology is analyzed anti-matrix interference ability, the homoimmune analytical procedure that causes setting up is difficult to be applied to the detection of actual environment sample and agricultural-food, and this also is the major cause that does not have sophisticated ethyl parathion commercial kit to emerge.And the advantage of alloimmunization analytical procedure (the especially advantage of its anti-matrix interference effect) makes it to become the fine means that address this problem.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of competition haptens of the alloimmunization analytical procedure that can set up highly sensitive, high specific and competes formerly, and its purposes also is provided.
In order to solve the problems of the technologies described above, the invention provides a kind of ethyl parathion competition haptens, its molecular structural formula is:
N=1～6 wherein, R is present in atom or group on the phenyl ring for the form with 1,2 or combination between any two.That is to say: such atom or group are positioned on the phenyl ring with the form of ortho position, contraposition or a position.
Compete haptenic improvement as ethyl parathion of the present invention: atom is F, Cl or Br; Group is OCH
3, CH
3, C
2H
5, NO
2Or C (CH
3)
3
The present invention also provides a kind of simultaneously and has utilized the made ethyl parathion competition of above-mentioned ethyl parathion competition haptens former, and its molecular structural formula is:
The present invention also provides a kind of allos elisa assay method that above-mentioned ethyl parathion competition is former and immunogen PB-BSA is set up of utilizing simultaneously, it is characterized in that: the residual quantity that is used for detecting food, agricultural-food and environmental sample ethyl parathion.
The present invention be directed to the weak point that homology is analyzed in the prior art, it is former to have designed a kind of ethyl parathion competition haptens and the competition of corresponding ethyl parathion, and it can be used as the raw material of the antigen system of animal immune.
Ethyl parathion competition haptens of the present invention is starting raw material with the phosphorus thiochloride, generate O-ethylenebis dithiocarbamate phosphinylidyne dichloro with the dehydrated alcohol reaction earlier, with phenol compound formation reaction intermediate thiophosphoryl one chlorine, last and amino acid reaction generates the competition haptens again.Reaction formula is as follows:
N=1～6 wherein; R represents F, Cl, Br, OCH
3, CH
3, C
2H
5, NO
2, C (CH
3)
3Deng atom and group exist with the form of 1,2 or combination between any two and phenyl ring on (ortho position, contraposition, a position).
The ethyl parathion competition haptens that the present invention proposes, mainly be to change at the antigenic determinant position, thereby reduce antibody with competition the avidity between former, in the increase immunoassay antibody to the recognition capability and the efficient of ethyl parathion, thereby the sensitivity of raising method.The elisa assay method of the relevant ethyl parathion of report is to the lowest detectable limit (IC of standard specimen at present
10) mostly be between the 2-50ng/mL greatly, but it is few that the ELISA method of setting up is applied to the report of actual agricultural-food and environmental sample detection, minimum detectable concentration mostly is between the 100ng/mL-1000ng/mL greatly in the existing report, and at present various countries mostly are 10ng/mL greatly to the residual limit standard of ethyl parathion, therefore present existing ELISA method can't be adapted to the requirement of extensive rapid screening, can't bring into play the fast and convenient advantage of ELISA.The applicant is in patent of invention " ethyl parathion artificial semiantigen, artificial antigen, Antibody Preparation method and uses thereof the " (application number: 200610052531.0) inform that lowest detection is limited to 6ng/mL of 2006.07.19 application.
And the allos ELISA method that the present invention sets up is to the lowest detectable limit (IC of ethyl parathion standard specimen
10) can reach 0.1ng/mL (0.0001mg/kg), all very low with the cross reacting rate of other organophosphorus pesticide, to the residual minimum detectable concentration of ethyl parathion in agricultural-food and the environmental sample is 1ng/mL (water, soil), 5ng/mL (cucumber, rice, corn), illustration method sensitivity and specificity height, and can be applied to the Detecting Pesticide of actual agricultural-food and environmental sample.
In sum, the present invention can be used for the ethyl parathion residue of trace in the rapid detection sample, and this method is highly sensitive, high specificity, sample pre-treatments simple, be convenient to carry out advantage such as on-site supervision, can complement one another with ordinary method; And lay the foundation for setting up the allos competitive immunoassay method that can be applied to ethyl parathion retention analysis in agricultural-food and the environmental sample.
Embodiment
Embodiment 1, competition haptens H1's is synthetic:
1), phosphorus thiochloride 80.0g (0.46mol) is packed in the there-necked flask, magnetic agitation, begin slowly to drip dehydrated alcohol 79.35mL (1.38mol) under the ice bath-5 ℃ the time, finish, continuation was reacted 1 hour down at 0 ℃, 0 ℃ of frozen water washed product is crossed anhydrous sodium sulphate, promptly gets product O-ethylenebis dithiocarbamate phosphinylidyne dichloro.
2), press triethylamine: O-ethylenebis dithiocarbamate phosphinylidyne dichloro: 2, the feed ratio of 6-syringol=6.7: 1.3: 1 (mol ratio), in there-necked flask, drop into triethylamine 20g after; 6.55g is dissolved in the 20mL acetonitrile with O-ethylenebis dithiocarbamate phosphinylidyne dichloro, gets solution I; With 2,6-syringol 1.17g is dissolved in the 15mL acetonitrile, gets solution II; Solution I and solution II are changed in the there-necked flask together over to stirring reaction 1h under the room temperature.The B suction filtration is crossed anhydrous sodium sulphate, concentrates the volatilization acetonitrile, gets faint yellow oily thing.Get product O-ethyl-O-4-(2, the 6-syringol) thiophosphoryl one chlorine through purification by silica gel column chromatography.
3), press sodium hydroxide: aminobutyric acid: O-ethyl-O-4-(2, the 6-syringol) feed ratio of thiophosphoryl one chlorine=2.5: 1.2: 1 (mol ratio), in there-necked flask, drop into O-ethyl-O-4-(2, the 6-syringol) thiophosphoryl one chlorine 4.16g and be dissolved in the 15mL dioxane.1.33g sodium hydroxide, 1.50g aminobutyric acid add the mixed aqueous solution that is made into 10mL behind the water.Ice bath stirs and slowly drips this mixed aqueous solution down to there-necked flask.After dropwising, be warming up to 20 ℃, reacted 4 hours.After reaction finishes, with about 0.1mol/L dilute hydrochloric acid conditioned reaction liquid pH value to 4.0, use chloroform 3 * 20mL to extract, the combined chloroform extracting solution is crossed anhydrous sodium sulphate, concentrates the volatilization chloroform, gets faint yellow oily thing.Get product H1 through purification by silica gel column chromatography, structure as follows:
Get above-mentioned synthetic product respectively through ESI,
1H-NMR and IR measure its molecular structure.
1H-NMR result is as follows: δ 6.89 (1H, s, ArH), 6.61 (2H, d, J=7.2, ArH), 6.96 (1H, d, J=4.4, ArH), 4.12 (2H, m, CH
2OP), 3.89 (6H, s, 2 * OCH
3), 3.19 (1H, m, NH), 2.65 (2H, m, CH
2NH), 2.24 (2H, m, CH
2COOH), 1.81 (2H, m, CH
2CH
2CH
2), 1.18 (3H, t, J=6.8, CH
3).
From
1H-NMR result as can be known, institute's synthetic product is a target compound.
Embodiment 2, competition haptens H2's is synthetic:
1) phosphorus thiochloride 80.0g (0.46mol) is packed in the there-necked flask, magnetic agitation, begin slowly to drip dehydrated alcohol 79.35mL (1.38mol) under the ice bath-5 ℃ the time, finish, continuation was reacted 1 hour down at 0 ℃, 0 ℃ of frozen water washed product is crossed anhydrous sodium sulphate, promptly gets product O-ethylenebis dithiocarbamate phosphinylidyne dichloro.
2) press triethylamine: O-ethylenebis dithiocarbamate phosphinylidyne dichloro: the feed ratio (mol ratio) of p-ethyl phenol=6.7: 1.3: 1, in there-necked flask, drop into triethylamine 20g after, 6.03g is dissolved in the 20mL acetonitrile with O-ethylenebis dithiocarbamate phosphinylidyne dichloro, solution I; P-ethyl phenol 1.78g is dissolved in the 15mL acetonitrile, gets solution II; Solution I and solution II are changed in the there-necked flask together over to stirring reaction 1h under the room temperature.The B suction filtration is crossed anhydrous sodium sulphate, concentrates the volatilization acetonitrile, gets faint yellow oily thing.Get product O-ethyl-O-4-p-ethyl phenol thiophosphoryl one chlorine through purification by silica gel column chromatography.
3) press sodium hydroxide: aminobutyric acid: the feed ratio of O-ethyl-O-4-p-ethyl phenol thiophosphoryl one chlorine=2.5: 1.2: 1 (mol ratio), drop into O-ethyl-O-4-p-ethyl phenol thiophosphoryl one chlorine 3.98g and be dissolved in 20mL methyl alcohol in there-necked flask.1.56g sodium hydroxide, 1.24g aminobutyric acid and be made into the mixed aqueous solution of 10mL after water mixes.Ice bath stirs down and slowly drips in this mixed aqueous solution to three mouthful reaction flask.After dropwising, be warming up to 30 ℃, reacted 2.5 hours.After reaction finishes, with about 0.1mol/L dilute hydrochloric acid conditioned reaction liquid pH value to 4.0, use chloroform 3 * 20mL to extract, the combined chloroform extracting solution is crossed anhydrous sodium sulphate, concentrates the volatilization chloroform, gets faint yellow oily thing.Get product H2 through purification by silica gel column chromatography, structure as follows:
Get above-mentioned synthetic product respectively through ESI,
1H-NMR and IR measure its molecular structure.
1H-NMR result is as follows: and δ 7.15 (4H, m, ArH), 4.20 (2H, m, CH
2OP), 4.06 (1H, m, NH), 3.15 (2H, m, CH
2NH), 2.61 (2H, m, CH
2COOH), 2.46 (2H, q, J=7.2 and 7.2, Ar-CH
2), 1.88 (2H, m, CH
2CH
2CH
2), 1.34 (3H, t, J=8.4, Ar-CH
2CH
3) 1.25 (3H, t, J=6.8, CH
3).
From
1H-NMR result as can be known, institute's synthetic product is a target compound.
Embodiment 3, the former H1-OVA's of competition is synthetic:
Compete the former synthetic mixed anhydride method of utilizing.0.25mmol haptens H1 (embodiment 1 gained) is dissolved in the N of 1mL, in the dinethylformamide, adds positive Tributylamine of 60 μ L and 30 μ L butyl chlorocarbonates, reacted under the room temperature 1～2 hour; Then reaction solution 100 μ L～800 μ L are joined in ovalbumin (OVA) carbonate buffer solution of 5mL10mg/mL, room temperature reaction is 2 hours under magnetic agitation, the dialysis tubing of packing into then, use distill water dialysis 3 times earlier, then with the PBS buffered soln of 0.01mol/L dialysis 3～5d, packing is stored in-20 ℃ the refrigerator.The structural formula of competing former H1-OVA is:
The ratio of immunity haptens H1 and OVA reactant and product is got reactant and product respectively and is carried out ultraviolet (200nm～400nm) scan when synthetic.Immune as calculated haptens is 5～10: 1 with the combination of proteins ratio.
Embodiment 4, the former H2-OVA's of competition is synthetic:
Compete the former synthetic mixed anhydride method of utilizing.0.25mmol haptens H2 (embodiment 2 gained) is dissolved in the N of 1mL, in the dinethylformamide, add positive Tributylamine of 60 μ L and 30 μ L butyl chlorocarbonates, reacted under the room temperature 1～2 hour, reaction solution 100 μ L～800 μ L join in ovalbumin (OVA) carbonate buffer solution of 5mL10mg/mL, room temperature reaction is 1 hour under magnetic agitation, the dialysis tubing of packing into then, use distill water dialysis 3 times earlier, then with the PBS buffered soln of 0.01mol/L dialysis 3～5d, packing is stored in-20 ℃ the refrigerator.The structural formula of competing former H2-OVA is:
The ratio of immunity haptens H2 and OVA reactant and product is got reactant and product respectively and is carried out ultraviolet (200nm～400nm) scan when synthetic.Immune as calculated haptens is 3～8: 1 with the combination of proteins ratio.
Embodiment 5, ethyl parathion allos ELISA set up and identify:
Utilize that immunogen PB-BSA immune rabbit obtains how anti-(particular content please see application number for details: 200610052531.0 patent of invention " ethyl parathion artificial semiantigen, artificial antigen, Antibody Preparation method and uses thereof "), to the former foundation of competition of design allos ELISA method at ethyl parathion.The method of setting up has very high specificity and sensitivity when ethyl parathion is residual in samples such as check and analysis vegetables and fruit, low with the cross reacting rate of similar compound, working method is simply quick simultaneously, do not need complicated pre-treatment process, once can detect gross sample simultaneously, with low cost, to operator require low, be convenient to carry out on-site supervision, can complement one another with ordinary method.
5.1 determining of optimum antibody working concentration and envelope antigen complex concentration
Select antibody and the former H1-OVA of competition square formation volumetry, dilute antibody and solid phase antigen coating buffer simultaneously, the ELISA reaction pattern is the indirect competition method.Under same competition primordial covering liquid concentration, along with the dilution of antibody, the OD value of gained is on a declining curve; Under same antibody dilution concentration, along with the decline of coating buffer concentration, gained OD value is also on a declining curve equally.Antibody when selecting OD value 1.0 left and right sides usually and bag are competed original content as working concentration.From experiment as can be known, when antibody concentration is 4.0 μ g/mL, the OD value approximated 1.0 when the former H1-OVA of bag quilt competition was 0.25 μ g/mL.
5.2 the foundation of allos elisa assay method standard curve
5.2.1 bag quilt
1) preparation of envelope antigen solution
From cryogenic refrigerator, take out H1-OVA coupled complex (embodiment 3 gained), after making it to thaw fully, be diluted to relevant work concentration, i.e. 0.25 μ g/mL.
2) the bag quilt of micro-reaction plate
The antigen coated liquid 100 μ L/ holes that 96 hole polystyrene micro-reaction plates are joined above adding, incubation 2h in 37 ℃ of incubators.
5.2.2 sealing
Take out bag by good micro-reaction plate, get rid of coating buffer, with PBST (0.01MPBS that contains 0.05% tween 20, pH7.4) after the washing 3 times, every hole adds 2.0% skimmed milk 300 μ L, incubation 0.5h in 37 ℃ of incubators washs 3 times with PBST.
5.2.3 some plate
1) standardized solution of preparation ethyl parathion
Get the ethyl parathion standard specimen, be mixed with the standardized solution of 100mg/L, therefrom take out 1mL with methyl alcohol, (0.01M, pH7.4) solution make it into 10mg/L solution to add 9mL PBS, become 156.25,78.13,39.06 with the PBS solution dilution that contains 10% methyl alcohol again, 19.53,9.77,4.88,2.44,1.22,0.61,0.30,0.15,0.07ng/mL.
2) preparation of ethyl parathion antibody diluent
From refrigerator, take out the anti-PB-BSA antibody that has been mixed with proper concn, be diluted to working concentration 4.0 μ g/mL with PBS.
3) some plate
Take out the plate that has wrapped quilt, every hole adds the ethyl parathion reference liquid 50 μ L of a series of concentration, adds antibody diluent 50 μ L again, the blank hole adds PBS 50 μ L and antibody diluent 50 μ L, use the micro oscillator mixing, put into 37 ℃ of incubator incubation 1h, use PBST solution washing 3 times.
5.2.4 add ELIAS secondary antibody
Every hole adds the goat-anti rabbit horseradish peroxidase PBS solution 100 μ L through dilution in 1: 1000, puts into 37 ℃ of incubators 1 hour, uses PBST solution washing 3 times.
5.2.5 colour developing
Every hole adds substrate OPD-superoxol 100 μ L, and incubation 15min in 37 ℃ of incubators uses 2M H
28O
4(50 μ L/ hole) termination reaction.At the light absorption value of measuring on the microplate reader under the 490nm wavelength.Drawing ethyl parathion then and suppress immunoreactive canonical plotting, wherein is that the logarithm of ordinate zou, ethyl parathion concentration is an X-coordinate with the inhibiting rate, and obtains inhibited reaction 50% and 10% required concentration IC respectively
50And IC
10Inhibiting rate is calculated as follows:
In the formula: the light absorption value when ODmax is not dosing, OD
XLight absorption value when being x for ethyl parathion concentration, ODmin are the light absorption value in blank hole.
5.3 the affinity of allos elisa assay method
The graticule that part is competed the allos elisa assay method of haptens foundation the results are shown in Table 1.
Table 1, part are competed the allos ELISA result that haptens is set up
Typical curve equation with the immunogen PB-BSA and the allos elisa assay method of the former H1-OVA foundation of competition is: y=10.012Ln (x)+32.768, IC
50Be 5.60ng/mL, IC
10Be 0.10ng/mL.And the typical curve equation of the homology elisa assay method of setting up by PB-OVA is: y=10.192Ln (x)+5.1192IC
50Be 81.64ng/mL, IC
10Be 1.61ng/mL.Clearly, the analysis of heterogensis method is higher than the homology analysis to the detection sensitivity of ethyl parathion, with IC
10Meter, analysis of heterogensis has improved 16.1 times than the method sensitivity of homology analysis.
5.4 the specificity of allos elisa assay method
Allos elisa assay method to immunogen PB-BSA and the former H1-OVA foundation of competition is carried out the cross reacting rate experiment.Choosing the cross reaction thing is: parathion-methyl, paraoxon, diazinon, amino thiophos, 4-nitrophenols, O, 12 kinds of organophosphorus insecticides such as O-diethyl sulfo-phosphoryl one chlorine, triazophos, Tiguvon, Sumithion, Chlorpyrifos 94, chlorpyrifos_methyl and intermediate, carry out cross reacting rate mensuration, drawn concentration (IC in the inhibition of various medicines
50), then with the IC of this method to ethyl parathion
50Compare, cross reacting rate is low more, illustrates that the specificity of this method is high more.Being calculated as of cross reacting rate: CR (%)=[IC
50(thiophos)/IC
50(other compound)] * 100%.The cross reacting rate experimental result of 10 kinds of compounds sees Table 2.The result shows, the allos competitive ELISA method of setting up to the CR of other analog substantially less than 0.05%, only parathion-methyl is 7.8%, paraoxon is 2.5%, illustrate that these organophosphorus pesticides and intermediate do not have to disturb the specificity height that this method is measured ethyl parathion substantially to the mensuration of ethyl parathion.
Table 2, cross reacting rate result
| Compound | IC 50(ng/mL) | CR(％) |
| Ethyl parathion | 4.79 | 100 |
| Parathion-methyl | 61.45 | 7.8 |
| Paraoxon | 191.68 | 2.5 |
| Diazinon | ＞10000 | ＜0.05 |
| Amino thiophos | ＞10000 | ＜0.05 |
| The 4-nitrophenols | ＞10000 | ＜0.05 |
| O, O-diethyl sulfo-phosphoryl one chlorine | ＞10000 | ＜0.05 |
| Triazophos | ＞10000 | ＜0.05 |
| Tiguvon | ＞10000 | ＜0.05 |
| Sumithion | 684.29 | 0.7 |
| Chlorpyrifos 94 | ＞10000 | ＜0.05 |
| Chlorpyrifos_methyl | ＞10000 | ＜0.05 |
5.5 allos elisa assay method detects the ability of actual sample
To the allos elisa assay method of immunogen PB-BSA and the former H1-OVA foundation of competition, the ethyl parathion that carries out in water, soil, cucumber, rice and 5 kinds of samples of corn adds rate of recovery experiment.Sample pretreatment process is as follows:
Water sample: draw 10mL and filter in advance or the centrifugal water sample, accurately add the agricultural chemicals mark liquid 0.1mL with the methyl alcohol preparation, mixing is directly used in ELISA and detects.
Pedotheque: pedotheque is air-dry, cross 60 order testing sieves, after getting sieve down sample dividing sample by quartering, take by weighing the 2g sample, extract 1min with the concussion of 2mL acetonitrile, it is centrifugal that (4000rpm 5min), draws the 1mL supernatant liquor, nitrogen dries up, add a certain amount of PBS (containing 10% methyl alcohol), vortex concussion 1min is used for ELISA and detects.
Cucumber sample: take by weighing the sample that 10g smashs to pieces in advance, accurately add agricultural chemicals mark liquid 0.1mL, accurately add the concussion of 10mL acetonitrile and extract 1min with the methyl alcohol preparation.Add 1g sodium-chlor and 4g sal epsom, jolting 1min, it is centrifugal that (4000rpm 5min), draws the 1mL supernatant liquor, and nitrogen dries up, and adds a certain amount of PBS (containing 10% methyl alcohol), and vortex concussion 1min is used for ELISA and detects.
Rice and corn sample: take by weighing the sample that 5g pulverizes in advance, accurately add agricultural chemicals mark liquid 0.1mL, add 3mL water with the methyl alcohol preparation, stir into even wet-milling shape, leave standstill 30min, accurately add the concussion of 10mL acetonitrile and extract 1min, add 1g sodium-chlor and 4g sal epsom, jolting 1min, centrifugal (4000rpm, 5min), draw the 1mL supernatant liquor, nitrogen dries up, and adds a certain amount of PBS (containing 10% methyl alcohol), vortex concussion 1min is used for ELISA and detects.
The ELISA detection method is with the operation of typical curve.Wrapped the sample liquid 50 μ L/ holes that added serial known interpolation concentration by good plate, added the antibody-solutions 50 μ L/ holes that prepare again, compared the hole, used the micro oscillator mixing with the sample liquid of not adding agricultural chemicals, 37 ℃ of incubations 1 hour, remaining step is the same.
The interpolation rate of recovery of 5 kinds of samples the results are shown in Table 3.In water sample and the pedotheque, adding class is 1,5, and during 10ng/mL, sample recovery rate is respectively at 75.37%-92.37%, between the 82.59%-102.95%; In cucumber, rice and the corn sample, adding class is 5,10, and during 20ng/mL, sample recovery rate is respectively 78.19%-95.09%, between 88.30%-107.76% and the 74.23%-101.91%.From adding the rate of recovery (Recovery), standard deviation (SE) and the variation coefficient (CV) result, the ELISA method of foundation all meets the requirement of Detecting Pesticide criterion to the detectivity of PA in these 5 kinds of samples.
The interpolation rate of recovery result of table 3,5 kinds of samples
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (4)
1, a kind of ethyl parathion competition haptens is characterized in that molecular structural formula is:
2, ethyl parathion competition haptens according to claim 1, it is characterized in that: described atom is F, Cl or Br; Described group is OCH
3, CH
3, C
2H
5, NO
2Or C (CH
3)
3
3, a kind ofly utilize the made ethyl parathion competition of the described ethyl parathion of claim 1 competition haptens former, it is characterized in that molecular structural formula is:
4, a kind of described ethyl parathion of claim 3 that utilizes is competed allos elisa assay method former and that immunogen PB-BSA is set up, it is characterized in that: the residual quantity that is used for detecting food, agricultural-food and environmental sample ethyl parathion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100621548A CN101289463B (en) | 2008-06-03 | 2008-06-03 | Competitive hapten of ethyl alkron, competitive antigen and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100621548A CN101289463B (en) | 2008-06-03 | 2008-06-03 | Competitive hapten of ethyl alkron, competitive antigen and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101289463A true CN101289463A (en) | 2008-10-22 |
| CN101289463B CN101289463B (en) | 2012-04-25 |
Family
ID=40033931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008100621548A Expired - Fee Related CN101289463B (en) | 2008-06-03 | 2008-06-03 | Competitive hapten of ethyl alkron, competitive antigen and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101289463B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105181949A (en) * | 2015-09-22 | 2015-12-23 | 浙江大学 | Rapid test method for 3 kinds of pesticide residue such as imidacloprid in tea and test paper strip used in rapid test method |
| CN105273003A (en) * | 2015-10-12 | 2016-01-27 | 深圳市检验检疫科学研究院 | Isocarbophos semiantigen, and preparation method and application thereof |
-
2008
- 2008-06-03 CN CN2008100621548A patent/CN101289463B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105181949A (en) * | 2015-09-22 | 2015-12-23 | 浙江大学 | Rapid test method for 3 kinds of pesticide residue such as imidacloprid in tea and test paper strip used in rapid test method |
| CN105181949B (en) * | 2015-09-22 | 2017-09-15 | 浙江大学 | Rapid test method for 3 kinds of pesticide residue such as imidacloprid in tea and test paper strip used in rapid test method |
| CN105273003A (en) * | 2015-10-12 | 2016-01-27 | 深圳市检验检疫科学研究院 | Isocarbophos semiantigen, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101289463B (en) | 2012-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107907687A (en) | A kind of dicofol haptens preparation method and applications | |
| CN101434652A (en) | Pyrethroid pesticide artificial antigen, antibody and preparation thereof | |
| CN102516148B (en) | Half antigen and antigen of tenuazonic acid and preparation method and application thereof | |
| CN111187346B (en) | Colloidal gold test strip for detecting fipronil and metabolites thereof and preparation method thereof | |
| CN101245032A (en) | Leuco malachite green hapten, produced antibody and application of the antibody | |
| CN102585005B (en) | Monoclonal antibody and ELLSA (Enzyme Linked Immunosorbent Assay) method for detection of methyl-3-quinoxaline-2-carboxylic acid (MQCA), and kit | |
| CN101799472A (en) | Diethylstilbestrol detection kit and detection method | |
| CN102768278B (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting beta-receptor stimulant | |
| CN101788560A (en) | Enzyme-linked immunoassay kit for detecting residual chloramphenicol and detection method thereof | |
| CN105807055A (en) | Test strip for detecting quinclorac and preparation method and application of test strip | |
| CN109265401A (en) | A kind of preparation method and application of iprodione haptens and antigen | |
| CN109265404A (en) | A kind of preparation method and application of carbendazim haptens and antigen | |
| CN101100456A (en) | Fipronil artificial hapten, synthetic method for the same, and its antigen, antibody and use | |
| CN102766208A (en) | Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin | |
| CN109061147A (en) | A kind of test strips and its preparation method and application detecting pendimethalin | |
| CN105624119B (en) | The general monoclonal antibody of anti-capsaicine, Dihydrocapsaicin, the synthetic capsaicin of hybridoma cell strain YQQD8 and its generation | |
| CN101289463B (en) | Competitive hapten of ethyl alkron, competitive antigen and uses thereof | |
| CN103575887A (en) | Test paper card for detecting aflatoxin B1 and application of test paper card | |
| CN104610079B (en) | A kind of salbutamol hapten derivant, salbutamol artificial antigen and its preparation method and application | |
| CN101135683B (en) | Bifenthrin antigen, antibody and uses thereof | |
| CN102372737A (en) | Preparation method and application of parathion-methyl artificial hapten, artificial antigen and specific antibody | |
| CN102507930A (en) | Kit for quickly detecting trace imidacloprid residue | |
| CN108059620A (en) | Trimethoprim class drug haptens, the monoclonal antibody for detecting trimethoprim class drug and its application | |
| CN107389927A (en) | A kind of time-resolved fluorescence test strips for detecting Profenofos and its application | |
| CN102778564B (en) | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| EE01 | Entry into force of recordation of patent licensing contract |
Application publication date: 20081022 Assignee: Ningbo NewCon Co., Ltd. Assignor: Zhejiang University Contract record no.: 2014330000338 Denomination of invention: Competitive hapten of ethyl alkron, competitive antigen and uses thereof Granted publication date: 20120425 License type: Common License Record date: 20140825 |
|
| LICC | Enforcement, change and cancellation of record of contracts on the licence for exploitation of a patent or utility model | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120425 Termination date: 20200603 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |