CN101633697A - Matched monoclonal antibodies established in accordance with small-molecule carbaryl, and application thereof - Google Patents
Matched monoclonal antibodies established in accordance with small-molecule carbaryl, and application thereof Download PDFInfo
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- CN101633697A CN101633697A CN200810117185A CN200810117185A CN101633697A CN 101633697 A CN101633697 A CN 101633697A CN 200810117185 A CN200810117185 A CN 200810117185A CN 200810117185 A CN200810117185 A CN 200810117185A CN 101633697 A CN101633697 A CN 101633697A
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Abstract
The invention provides matched monoclonal antibodies established in accordance with small-molecule carbaryl, and application thereof. By analyzing the molecular structure of carbaryl, two methods, namely an active ester method and a succinic anhydride method are used to form different space arms and expose different antigen sites; macromolecular proteins are connected to form carbaryl small-molecule artificial immunogen; monoclonal antibodies aiming at different antigenic determinants are prepared by utilizing hybridoma technology; and two matched antibodies I and II are screened out in a double-antibody sandwich way. The matched monoclonal antibodies can be used for the double-antibody sandwich method construction of colloidal gold immuno-chromatography, wherein one antibody called I is used to coat test lines, and the other antibody called II is used to label colloidal gold. Fast immune detection products prepared from the carbaryl matched monoclonal antibodies provided by the invention have the advantages of strong specificity, high sensitivity, easy storage, no need for operation of professional technicians, easily read results and the like.
Description
Technical field
The present invention relates to a kind ofly set up the pairing monoclonal antibody method, and be applied to the preparation of carbaryl colloidal gold immunochromatographimethod double antibodies sandwich method test strip at the small molecules carbaryl.
Background technology
In the rapid expansible of world population today, reasonably use agricultural chemicals can improve grain yield, but excessive use can cause serious environmental to pollute, and cause many genetic diseasess.In recent years, owing to agricultural chemicals residual poisoning that causes that exceeds standard in food happens occasionally.Therefore, in the hot issue that this whole world of food safety is paid close attention to, how detecting agricultural chemicals problem residual in the agricultural byproducts quickly and accurately just becomes the problem of the most important thing.
Carbaryl is a small-molecule substance, and molecular weight 201.13 is a kind of new agriculture pesticides.Its desinsection selectivity is strong, and effect is fast, therefore uses wider.But be stored in the plant not easy-clear of residual hazard, contact this type of agricultural chemicals the people is poisoned.Characteristics of incidence is fast, violent, rescues untimely meeting and causes death.Toxicity symptom is hydrostomia, feels sick, vomits, suffers from abdominal pain, and heartbeat is slow, blood pressure drops, profuse sweating, the urinary incontinence, expiratory dyspnea, myosis, pale complexion etc., serious causing death.
National standard according to the People's Republic of China (PRC), and with reference to other countries or geographic examination criteria, the analytical procedure that detects carbaryl residue mainly is that liquid chromatography is replenished the GC method, and other detection method mainly is dependence, vapor-phase chromatography (GC) or gas-chromatography and mass spectrum (MS) coupling method (GC/MS).These method ubiquities the sample pretreatment process complexity, sensitivity be subjected to sample purification, step such as concentrate influence very big, consuming time, test set costliness, and require to have technical professional and long analytical cycle.
To detect cost in order reducing, to reduce operation easier, using immune colloid gold method detection agricultural chemicals carbaryl residue is a kind of preferably technology.According to traditional definition, small-molecule substance can only adopt the colloid gold immune competition law to detect, as patent of invention 200410071941.0 test paper strip for quick detecting pesticide sevin and preparation method thereof and application, patent of invention 200510013371.4 Collaurum marking rapid detection sevin test paper strips and preparation method thereof and application, the colloid gold test paper of 200520008931.2 1 kinds of rapid detection carbaryl residues of utility model patent, immunity competition law technology obviously reduces small molecules detection cost, operation is also obviously simple, but its susceptibility is lower than double antibodies sandwich method, and the small molecules low for detectability can't detect.Though and the double antibodies sandwich method is the detection technique of using always, not being reported is applied to the small molecules detection with it, and pertinent literature and methodology are set forth and thought that all the small-molecule substance molecular weight is little, simple in structure, therefore are difficult to while and two macromole antibody and combine.
The present invention modifies different spacerarms with different methods, exposes different antigen sites, thereby immunity obtains paired carbaryl monoclonal antibody through screening meticulously, carrying out double antibodies sandwich method colloid gold immune detects, advantages such as this method has high specificity, and is highly sensitive, and difference between batch is little.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of and set up the pairing monoclonal antibody method at the small molecules carbaryl, and be applied to the tachysynthesis testing product, and use the double antibodies sandwich method to detect the small molecules carbaryl, obtain higher susceptibility, good specificity.
For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:
By the analyzing carbaryl molecular structure, form different spacerarms with two kinds of methods of active ester method and succinyl oxide method, expose different antigen sites, connect high molecular weight protein again, form carbaryl small molecules artificial immunogen, utilize the monoclonal antibody of hybridoma technology preparation at different antigenic determinants, filter out two kinds of antibody I of paired, II by the double-antibody sandwich mode afterwards, the pairing monoclonal antibody can be used for the structure of Radioactive colloidal gold double antibodies sandwich method, wherein a strain antibody is called I and is used for bag by test wire, and another strain kind antibody is called II and is used for colloid gold label.
Radioactive colloidal gold double antibodies sandwich ratio juris is: the detection zone bag on nitrocellulose filter is by carbaryl monoclonal antibody I, and wrap by the sheep anti mouse polyclonal antibody check plot.When in the determinand carbaryl of being being arranged, antigenic substance in the determinand will form the Ag-Ab-Au mixture with the carbaryl monoclonal antibody II of mark Radioactive colloidal gold, because capillary chromatography effect, mixture is along the film Tape movement, can form the double antibodies sandwich immunocomplex with the carbaryl monoclonal antibody I that is coated on the nitrocellulose filter, at this moment an obvious colour band (T line) can be seen by the detection line place, the carbaryl monoclonal antibody II of remaining mark Radioactive colloidal gold can continue to move, because it derives from mouse, therefore can combine with the sheep anti mouse polyclonal antibody and form an obvious colour band (C line), therefore in the time of on detecting test paper, can seeing two lines (C line and T line), contain carbaryl in the proof sample, the result is positive, illustrates that the agricultural chemicals carbaryl exceeds standard in the detected sample; And when not having tested carbaryl in the test substance, just can not (T line) form the double antibodies sandwich immunocomplex at the detection line place, and can a colour band appear at C line place only, and the result is negative, illustrates that the agricultural chemicals carbaryl does not exceed standard in the detected sample.
Owing to adopt technique scheme, the double antibodies sandwich tachysynthesis testing product that employing carbaryl pairing monoclonal antibody provided by the present invention is made has high specificity, and is highly sensitive, easily stores, need not the technical skill personnel operation, and advantage such as readability as a result.
Embodiment
Embodiment one: small molecules agricultural chemicals carbaryl is immunogenic synthetic
The haptens that can synthesize different brachiums by two kinds of different reactions:
Reaction 1: naphthoic acid and excessive acyl chlorides under the solvent existence condition back flow reaction 2-10 hour, cooling dropwise adds excess of ammonia base acid solution (this solution is for being made into the aqueous solution by alkali and amino acid), stirring reaction 2-10 hour, can obtain product, the purified haptens I that obtains.
Reaction 2: naphthols and excessive slightly acid anhydrides lucifuge back flow reaction 5-20 hour under the condition that solvent exists obtains product, the purified haptens II that obtains.
Haptens I, II are connected with high molecular weight protein respectively, obtain synthetic immunogen I, II.
Embodiment two: the carbaryl MONOCLONAL ANTIBODIES SPECIFIC FOR
With synthetic immunogen I, II immunity BALB/c mouse, set up the knurl strain with the SP2/0 cytogamy and screen, get oncocyte and be injected in the BALB/c mouse abdominal cavity, make it produce ascites.Extract the screening of mouse ascites purifying, obtain monoclonal antibody I, II at the different antigenic determinants of carbaryl.
Embodiment three: the preparation of carbaryl colloidal gold immunochromatographimethod double antibodies sandwich method test strip
1. the preparation of colloidal gold solution: get distilled water and add an amount of hydrochloro-auric acid magnetic agitation and be warmed to 90～95 ℃, add an amount of Citric Acid three and receive and continue heated and stirred to seething with excitement 5 minutes, it is standby to keep in Dark Place after the cooling.
2. combining of gold solution and carbaryl monoclonal antibody II: on the Radioactive colloidal gold liquid adsorbing fiber material with carbaryl monoclonal antibody II mark, dry back is standby.
3. the preparation of film: film-making machine system film, utilize computer control transmission speed, guarantee that the carbaryl monoclonal antibody I solution amount of bag quilt on the per unit film equates.
4. the making of test strip:
Test paper base plate middle part is a nitrocellulose filter, a test wire and a sheep anti mouse polyclonal antibody control line are arranged on the nitrocellulose filter, in base plate one end termination is water accepting layer, the other end termination is the sample liquid-adsorption layer, the nitrocellulose filter two ends overlap mutually with monoclonal antibody gold mark pad with water accepting layer respectively and are connected (the overlapping connection portion is in 1-2 millimeter scope), are pressed with the sample liquid-adsorption layer on monoclonal antibody gold mark pad.Be cut into the film bar of 3mm with slitting shear machine, be colloidal gold colloidal gold detection test paper strip.
5. using method: the sample liquid-adsorption layer end of test paper is put into vegetables soak juice, take out test paper after 1 minute and keep flat, because kapillary and syphonic effect sample will move observations in the time of 5 minutes along test strip water accepting layer end.
6. the result judges: the vegetable pesticide residue examination criteria according to country's promulgation is formulated boundary value, carbaryl concentration value beyond the mark in the sample, the response line color is red, i.e. positive when colour developing district is two red line, otherwise negative when having only a red control line.
Claims (4)
1. the pairing monoclonal antibody and the application thereof of setting up at the small molecules carbaryl, it is characterized in that at the small molecules carbaryl, adopt two kinds of methods of active ester method and succinyl oxide method to form different spacerarms, expose different antigen sites, connect high molecular weight protein again, form carbaryl small molecules artificial immunogen I, II, utilize the monoclonal antibody of hybridoma technology preparation at different antigenic determinants, filter out two kinds of antibody I of paired by the double-antibody sandwich mode afterwards, II, the pairing monoclonal antibody can be used for the structure of Radioactive colloidal gold double antibodies sandwich method, wherein a strain antibody is called I and is used for bag by test wire, and another strain kind antibody is called II and is used for colloid gold label.
2. pairing monoclonal antibody and application thereof of setting up according to claim 1 at the small molecules carbaryl, it is characterized in that the macromole that connects comprise any can with micromolecular all or part of structure in conjunction with producing immunogenic molecule.
3. pairing monoclonal antibody and application thereof of setting up according to claim 1 at the small molecules carbaryl, it is characterized in that adopting carbaryl small molecules artificial immunogen I, II immune mouse respectively, obtaining can be simultaneously and small molecules carbaryl bonded, promptly at two kinds of carbaryl monoclonal antibody I, the II of the different antigenic determinants of small molecules carbaryl.
4. pairing monoclonal antibody and application thereof of setting up at the small molecules carbaryl according to claim 1 is characterized in that obtaining the preparation that paired carbaryl monoclonal antibody can be used for carbaryl double antibodies sandwich immuno-chromatographic test paper strip.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200810117185A CN101633697A (en) | 2008-07-25 | 2008-07-25 | Matched monoclonal antibodies established in accordance with small-molecule carbaryl, and application thereof |
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| CN200810117185A CN101633697A (en) | 2008-07-25 | 2008-07-25 | Matched monoclonal antibodies established in accordance with small-molecule carbaryl, and application thereof |
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| CN101633697A true CN101633697A (en) | 2010-01-27 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486858A (en) * | 2014-10-11 | 2016-04-13 | 江苏维赛科技生物发展有限公司 | Carbaryl colloidal gold test paper strip preparation method and usage method thereof |
| CN106636010A (en) * | 2017-01-13 | 2017-05-10 | 中国农业科学院油料作物研究所 | Hybridoma cell strain Jnw1D2 and anti-carbaryl monoclonal antibody generated by same |
| CN108398412A (en) * | 2018-03-28 | 2018-08-14 | 韶关学院 | Double-antibody sandwich Immune competition method detects the quantum dot immune chromatography detection card and detection method of brufen |
| CN113816877A (en) * | 2021-11-22 | 2021-12-21 | 信达安检测技术(天津)有限公司 | New carbaryl hapten and complete antigen as well as preparation and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2847294Y (en) * | 2005-03-24 | 2006-12-13 | 万积成 | Colloidal gold test paper for quick detecting carbaryl residue |
-
2008
- 2008-07-25 CN CN200810117185A patent/CN101633697A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2847294Y (en) * | 2005-03-24 | 2006-12-13 | 万积成 | Colloidal gold test paper for quick detecting carbaryl residue |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105486858A (en) * | 2014-10-11 | 2016-04-13 | 江苏维赛科技生物发展有限公司 | Carbaryl colloidal gold test paper strip preparation method and usage method thereof |
| CN106636010A (en) * | 2017-01-13 | 2017-05-10 | 中国农业科学院油料作物研究所 | Hybridoma cell strain Jnw1D2 and anti-carbaryl monoclonal antibody generated by same |
| CN108398412A (en) * | 2018-03-28 | 2018-08-14 | 韶关学院 | Double-antibody sandwich Immune competition method detects the quantum dot immune chromatography detection card and detection method of brufen |
| CN113816877A (en) * | 2021-11-22 | 2021-12-21 | 信达安检测技术(天津)有限公司 | New carbaryl hapten and complete antigen as well as preparation and application thereof |
| CN113816877B (en) * | 2021-11-22 | 2022-02-15 | 信达安检测技术(天津)有限公司 | New carbaryl hapten and complete antigen as well as preparation and application thereof |
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Application publication date: 20100127 |