CN102898410B - Method for preparing anti-coagulation raticide bromadiolone half antigen and complete antigen - Google Patents
Method for preparing anti-coagulation raticide bromadiolone half antigen and complete antigen Download PDFInfo
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Abstract
The invention relates to a method for preparing an anti-coagulation raticide bromadiolone half antigen and a complete antigen. The method comprises the following steps of: (1) adding bromadiolone and anhydrous potassium carbonate into 1,4-dioxane respectively, reacting while stirring, adding succinic anhydride, heating and refluxing; (2) pouring a mixture obtained by reacting in the step (1) into water, and precipitating a greyish-white solid out; (3) filtering, and recrystallizing with ethanol to obtain a bromadiolone half antigen; (4) adding the bromadiolone half antigen, dicyclohexylcarbodiimide and BSA (Bovine Serum Albumin) into dimethylsulfoxide, adding a phosphate buffer solution of which the pH is 6-7, and stirring; and (5) dialyzing, and performing freeze drying to obtain a bromadiolone complete antigen. The preparation method provided by the invention is easy, convenient and rapid, can be applied to immune animals, can be used for preparing a monoclonal antibody of bromadiolone and establishing a rapid detection method of anti-coagulation raticide bromadiolone, and is widely applied in the fields of forensic science, clinical medicine and the like.
Description
Technical field
The present invention relates to the preparation method of haptens and complete antigen in immunological technique field, particularly the preparation method of a kind of anticoagulation raticide bromadiolone haptens and complete antigen.
Background technology
Bromadiolone is a kind of slow rodenticide, is coumarin kind compound, utilizes this compounds can destroy thrombogen in mouse body, cause each internal organs and mucous membrane large-area hemorrhage in body, finally reach the object of killing mouse, therefore copper is called anticoagulation class rodenticide.In recent years along with the acute rodenticide such as Tetramine, monofluoroacetamide is disabled, the substitute is the anticoagulation rodenticide of these a new generations.
Although this type of rodenticide has good deratization effect, along with widespread use in recent years, the case caused because of misuse, suicide etc. and event happen occasionally.This just requires that judicial identification of public security organs department is detected the various sample relating to anticoagulation class rodenticide.Set up the detection method of these anticoagulation rodenticides in biological sample clinically, have very large meaning to the clinical diagnosis and treatment coordinating toxic patient.
Immunity is organism identification and gets rid of antigen foreign matter and reach the balance of safeguarding own physiological and stable.Various immunological technique development based on antigen antibody reaction rapidly, be widely used in qualitative, the quantitative and location in medical science and biological study field, because of its high degree of specificity and susceptibility (10-12 ~ 10-9g level can be reached), be particularly suited for the analytical study of the complex systems such as biological specimen.
Bromadiolone belongs to small-molecule substance (molecular weight is less than 1000Da), lack t cell epitope and directly cannot produce specific antibody by induced animal body, but after protein macromolecule carrier conjugation, the proliferation and growth of indirect induction B cell can be carried out by its t cell epitope, produce specific antibody.The conjugate of small molecules and protein carrier is called complete antigen.If small molecules self is with the active function groups (as amino, carboxyl etc.) for coupling, then linked reaction can directly be carried out, otherwise just must carry out derivatize to poisonous substance molecule, connect and supply the active function groups of linked reaction to synthesize artificial antigen, this step is also called haptenic synthesis.The synthesis of haptens and artificial antigen is most important for setting up its immunoassay, can many factors all prepare high-quality antibody by directly having influence on, as the coupling ratio etc. selecting suitable intermediate, active function groups to introduce the selection in site, the separation and purification of haptens product, the length of coupling arm, haptens and carrier molecule.Some poisonous substances, because of many, the product separation purification difficult of synthesis step in hapten synthesis process, even cannot prepare gratifying antibody sometimes.The haptenic preparation method of relevant bromadiolone yet there are no pertinent literature report.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to directly to produce specific antibody by induced animal body for bromadiolone as small molecules poisonous substance, the preparation method of a kind of anticoagulation raticide bromadiolone haptens and complete antigen is provided.
Technical scheme of the present invention is achieved in that the haptenic preparation method of a kind of anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined in Isosorbide-5-Nitrae-dioxane respectively, under agitation react; Add Succinic anhydried again, reflux; React as follows:
(2) mixture be obtained by reacting in step (1) is poured into water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone in step (1), is stir 5-20h under the condition of 15-40 DEG C in temperature; Reflux 1-6h.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone in step (1), is stir 10h under the condition of 25-27 DEG C in temperature; Reflux 4h.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone, in step (2), the temperature of water is 0 DEG C.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone, bromadiolone is 1:0.5 ~ 1.5 with the ratio of the amount of substance of Anhydrous potassium carbonate, and the concentration of bromadiolone in Isosorbide-5-Nitrae-dioxane is 0.2 ~ 0.7mol/L.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone, bromadiolone is 1:1.5 ~ 6 with the ratio of the amount of substance of Succinic anhydried.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone, the concentration of bromadiolone in Isosorbide-5-Nitrae-dioxane is 0.3mol/L.
The preparation method of above-mentioned anticoagulation raticide bromadiolone complete antigen, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined in Isosorbide-5-Nitrae-dioxane respectively, under agitation react; Add Succinic anhydried again, reflux; ;
(2) mixture be obtained by reacting in step (1) is poured into water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens;
(4) respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA are joined in methyl-sulphoxide, and then add the phosphate buffer solution that pH is 6 ~ 7, stir;
(5) dialyse, after lyophilize, make bromadiolone complete antigen.
The preparation method of above-mentioned anticoagulation raticide bromadiolone complete antigen, in step (4): the mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.5 ~ 2, the mass ratio of bromadiolone haptens and BSA is 1.5 ~ 4:1, the phosphate buffer solution 2 ~ 2.5 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 1 ~ 3 milliliter, need pH to be 6 ~ 7.
The preparation method of above-mentioned anticoagulation raticide bromadiolone complete antigen, in step (4): stir under the condition of 15 ~ 30 DEG C.
The invention has the beneficial effects as follows: preparation method of the present invention is easy, quick, the antigen obtained can be used for immune animal, prepare the monoclonal antibody of bromadiolone, and then set up the method for quick of anticoagulation raticide bromadiolone, have broad application prospects, to be widely used in the field such as forensic science, clinical medicine, to meet the demand of unit centering drug case (thing) the part quick tests such as public security system, Disease Control and Prevention Center and hospital.
Establish the haptenic preparation method of bromadiolone, can be used for immune animal, prepare the monoclonal antibody of bromadiolone, and then set up the method for quick of anticoagulation raticide bromadiolone, be widely used in the field such as forensic science, clinical medicine.Solve bromadiolone and directly can not produce the problem of specific antibody by induced animal body as small molecules poisonous substance, by preparing haptens and complete antigen, and then Dispersal risk.
Embodiment
Embodiment 1
The haptenic preparation method of the present embodiment anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:0.5 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.7mol/L, is to stir 20h under the condition of 15 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:1.5, reflux 6h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured in 15 DEG C of water, have pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens, and the haptenic productive rate of bromadiolone is 78%.
Embodiment 2
The haptenic preparation method of the present embodiment anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:0.5 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.7mol/L, is to stir 20h under the condition of 15 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:1.5, reflux 6h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured in 5 DEG C of water, have pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens, and the haptenic productive rate of bromadiolone is 75%.
Embodiment 3
The haptenic preparation method of the present embodiment anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:0.5 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.7mol/L, is to stir 20h under the condition of 15 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:1.5, reflux 6h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured in mixture of ice and water, have pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens, and the haptenic productive rate of bromadiolone is 83%.
Embodiment 4
The haptenic preparation method of the present embodiment anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:1.5 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.7mol/L, is to stir 20h under the condition of 40 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:6, reflux 6h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured into water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens, and the haptenic productive rate of bromadiolone is 85%.
Embodiment 5
The haptenic preparation method of the present embodiment anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:1.2 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.3mol/L, is to stir 10h under the condition of 25 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:3, reflux 4h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured in mixture of ice and water, have pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens, and the haptenic productive rate of bromadiolone is 90%.
Embodiment 6
The haptenic preparation method of the present embodiment anticoagulation raticide bromadiolone, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:0.85 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.3mol/L, is to stir 10h under the condition of 27 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:4, reflux 4h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured into water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens, and the haptenic productive rate of bromadiolone is 93%.
Embodiment 7
The bromadiolone haptens of embodiment 1 is adopted to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined in methyl-sulphoxide that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.5, the mass ratio of bromadiolone haptens and BSA is 1.5:1, the phosphate buffer solution 2 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 1 milliliter, need pH to be 6], and then add the phosphate buffer solution that pH is 6, stir under the condition of 15 DEG C; Dialysis, makes bromadiolone complete antigen after lyophilize, calculate the productive rate of bromadiolone complete antigen for 85% with bromadiolone haptens.
Ultra-violet analysis, infrared analysis and flight mass spectrum analysis are carried out to bromadiolone complete antigen prepared by the present embodiment, result shows: there is the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also the haptenic absorption peak of bromadiolone is there is in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows that bromadiolone haptens and BSA there occurs linked reaction (ratio of amount of substance is bromadiolone haptens: BSA=15:1), generates bromadiolone haptens BSA binding substances.
Embodiment 8
The bromadiolone haptens of embodiment 2 is adopted to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined in methyl-sulphoxide that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:2, the mass ratio of bromadiolone haptens and BSA is 4:1, the phosphate buffer solution 2.5 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 1 ~ 3 milliliter, need pH to be 7], and then add the phosphate buffer solution that pH is 7, stir under the condition of 30 DEG C; Dialysis, makes bromadiolone complete antigen after lyophilize, calculate the productive rate of bromadiolone complete antigen for 88% with bromadiolone haptens.
Ultra-violet analysis, infrared analysis and flight mass spectrum analysis are carried out to bromadiolone complete antigen prepared by the present embodiment, result shows: there is the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also the haptenic absorption peak of bromadiolone is there is in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows that bromadiolone haptens and BSA there occurs linked reaction (ratio of amount of substance is bromadiolone haptens: BSA=15:1), generates bromadiolone haptens BSA binding substances.
Embodiment 9
The bromadiolone haptens of embodiment 3 is adopted to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined in methyl-sulphoxide that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.3, the mass ratio of bromadiolone haptens and BSA is 2:1, the phosphate buffer solution 2.2 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 2 milliliters, need pH to be 7], and then add the phosphate buffer solution that pH is 7, stir under the condition of 20 DEG C; Dialysis, makes bromadiolone complete antigen after lyophilize, calculate the productive rate of bromadiolone complete antigen for 92% with bromadiolone haptens.
Ultra-violet analysis, infrared analysis and flight mass spectrum analysis are carried out to bromadiolone complete antigen prepared by the present embodiment, result shows: there is the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also the haptenic absorption peak of bromadiolone is there is in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows that bromadiolone haptens and BSA there occurs linked reaction (ratio of amount of substance is bromadiolone haptens: BSA=15:1), generates bromadiolone haptens BSA binding substances.
Embodiment 10
The bromadiolone haptens of embodiment 4 is adopted to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined in methyl-sulphoxide that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.8, the mass ratio of bromadiolone haptens and BSA is 2.6:1, the phosphate buffer solution 2.5 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 1.5 milliliters, need pH to be 6], and then add the phosphate buffer solution that pH is 6, stir under the condition of 26 DEG C; Dialysis, makes bromadiolone complete antigen after lyophilize, calculate the productive rate of bromadiolone complete antigen for 93% with bromadiolone haptens.
Ultra-violet analysis, infrared analysis and flight mass spectrum analysis are carried out to bromadiolone complete antigen prepared by the present embodiment, result shows: there is the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also the haptenic absorption peak of bromadiolone is there is in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows that bromadiolone haptens and BSA there occurs linked reaction (ratio of amount of substance is bromadiolone haptens: BSA=15:1), generates bromadiolone haptens BSA binding substances.
Embodiment 11
The bromadiolone haptens of embodiment 5 is adopted to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined in methyl-sulphoxide that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.7, the mass ratio of bromadiolone haptens and BSA is 3.5:1, the phosphate buffer solution 2.5 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 2 milliliters, need pH to be 7], and then add the phosphate buffer solution that pH is 7, stir under the condition of 19 DEG C; Dialysis, makes bromadiolone complete antigen after lyophilize, calculate the productive rate of bromadiolone complete antigen for 94% with bromadiolone haptens.
Ultra-violet analysis, infrared analysis and flight mass spectrum analysis are carried out to bromadiolone complete antigen prepared by the present embodiment, result shows: there is the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also the haptenic absorption peak of bromadiolone is there is in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows that bromadiolone haptens and BSA there occurs linked reaction (ratio of amount of substance is bromadiolone haptens: BSA=15:1), generates bromadiolone haptens BSA binding substances.
Embodiment 12
The bromadiolone haptens of embodiment 6 is adopted to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined in methyl-sulphoxide that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.6, the mass ratio of bromadiolone haptens and BSA is 3.8:1, the phosphate buffer solution 2.5 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 2.6 milliliters, need pH to be 6.5], and then add the phosphate buffer solution that pH is 6.5, stir under the condition of 30 DEG C; Dialysis, makes bromadiolone complete antigen after lyophilize, calculate the productive rate of bromadiolone complete antigen for 90% with bromadiolone haptens.
Ultra-violet analysis, infrared analysis and flight mass spectrum analysis are carried out to bromadiolone complete antigen prepared by the present embodiment, result shows: there is the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also the haptenic absorption peak of bromadiolone is there is in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows that bromadiolone haptens and BSA there occurs linked reaction (ratio of amount of substance is bromadiolone haptens: BSA=15:1), generates bromadiolone haptens BSA binding substances.
In above-described embodiment 1-6, bromadiolone and Succinic anhydried react and prepare the following institute of the haptenic reaction formula of bromadiolone
Above-described embodiment is only for the invention example is clearly described, and the restriction not to the invention embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And thus the apparent change of amplifying out or variation be still among the protection domain of the invention claim.
Claims (2)
1. the haptenic preparation method of anticoagulation raticide bromadiolone, is characterized in that, comprise the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:0.85 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.3mol/L, is to stir 10h under the condition of 27 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:4, reflux 4h with the ratio of the amount of substance of Succinic anhydried; React as follows:
(2) mixture be obtained by reacting in step (1) is poured into water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens.
2. the preparation method of anticoagulation raticide bromadiolone complete antigen, is characterized in that, comprise the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined 1 respectively, in 4-dioxane, bromadiolone is 1:1.2 with the ratio of the amount of substance of Anhydrous potassium carbonate, and bromadiolone is 1, concentration in 4-dioxane is 0.3mol/L, is to stir 10h under the condition of 25 DEG C to react in temperature; Add Succinic anhydried again, bromadiolone is 1:3, reflux 4h with the ratio of the amount of substance of Succinic anhydried;
(2) mixture be obtained by reacting in step (1) is poured in mixture of ice and water, have pale solid to separate out;
(3) filter, ethyl alcohol recrystallization, obtains bromadiolone haptens;
(4) respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA are joined in methyl-sulphoxide, the mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.7, the mass ratio of bromadiolone haptens and BSA is 3.5:1, the phosphate buffer solution 2.5 milliliters that every 100 milligrams of bromadiolone haptens need methyl-sulphoxide 2 milliliters, need pH to be 7, and then add the phosphate buffer solution that pH is 7, stir under the condition of 19 DEG C;
(5) dialyse, after lyophilize, make bromadiolone complete antigen.
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| CN109824645B (en) * | 2019-02-28 | 2020-10-27 | 中国农业大学 | Warfarin hapten and artificial antigen as well as preparation method and application thereof |
| CN109897025B (en) * | 2019-02-28 | 2021-01-01 | 中国农业大学 | Anticoagulant raticide hapten and artificial antigen as well as preparation method and application thereof |
| CN110950961B (en) * | 2019-12-11 | 2021-02-09 | 中国农业大学 | Bromadiolone nano antibody and application thereof |
| CN111289753B (en) * | 2020-04-13 | 2023-04-11 | 北京维德维康生物技术有限公司 | Enzyme linked immunosorbent assay kit for detecting coumarin and indandione rodenticide and preparation and application thereof |
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