CN102898410A - 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 the 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 the interior thrombogen of mouse body, causes each internal organs and mucous membrane large-area hemorrhage in the body, reaches at last the purpose of killing mouse, therefore copper is called anticoagulation class rodenticide.Along with the acute rodenticides such as Tetramine, monofluoroacetamide are disabled, the substitute is these anticoagulation rodenticides of new generation in recent years.
Although this type of rodenticide has good deratization effect, along with widespread use in recent years, the case and the event that cause because of misuse, suicide etc. happen occasionally.This just requires judicial identification of public security organs department that the various samples that relate to anticoagulation class rodenticide are detected.Set up clinically the detection method of these anticoagulation rodenticides in the biological sample, clinical diagnosis and the treatment that cooperates toxic patient had very large meaning.
Immunity is organism identification and gets rid of the antigen foreign matter and reach and safeguard self physiological equilibrium and stable.Various immunological technique development take antigen antibody reaction as the basis rapidly, qualitative, the quantitative and location that is widely used in medical science and biological study field, because of its high degree of specificity and susceptibility (can reach 10-12～10-9g level), 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 direct induced animal body generation specific antibody, but after the coupling of protein macromolecule carrier, can come by its t cell epitope propagation and the differentiation of indirect induction B cell, 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 (such as amino, carboxyl etc.) for coupling, then linked reaction can directly be carried out, otherwise just must carry out derivatize to the poisonous substance molecule, connect the active function groups of confession linked reaction with synthetic artificial antigen, this step is called again haptenic synthetic.Synthesizing of haptens and artificial antigen is most important for setting up its immunoassay, can many factors all will directly have influence on prepare high-quality antibody, as selects suitable intermediate, active function groups to introduce the coupling ratio etc. of length, haptens and the carrier molecule of the separation and purification of the selection in site, haptens product, coupling arm.Some poisonous substances are many because of synthesis step in the haptens building-up process, the product separation purification difficult, sometimes even can't prepare gratifying antibody.The haptenic preparation method of relevant bromadiolone yet there are no the pertinent literature report.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to for bromadiolone as the small molecules poisonous substance directly the induced animal body produce specific antibody, 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 respectively in Isosorbide-5-Nitrae-dioxane, under agitation react; Add again Succinic anhydried, reflux; React as follows:
(2) mixture that reaction in the step (1) is obtained is poured into water, and has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the bromadiolone haptens.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone in step (1), is to stir 5-20h under 15-40 ℃ the condition in temperature; Reflux 1-6h.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone in step (1), is to stir 10h under 25-27 ℃ the condition in temperature; Reflux 4h.
The haptenic preparation method of above-mentioned anticoagulation raticide bromadiolone, in step (2), the temperature of water is 0 ℃.
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, 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 respectively in Isosorbide-5-Nitrae-dioxane, under agitation react; Add again Succinic anhydried, reflux;
(2) mixture that reaction in the step (1) is obtained is poured into water, and has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the bromadiolone haptens;
(4) respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA are joined in the methyl-sulphoxide, and then to add pH be 6～7 phosphate buffer solution, stir;
(5) the bromadiolone complete antigen is made in dialysis after the lyophilize.
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, and per 100 milligrams of bromadiolone haptens need 1～3 milliliter of methyl-sulphoxide, need pH is 2～2.5 milliliters of 6～7 phosphate buffer solutions.
The preparation method of above-mentioned anticoagulation raticide bromadiolone complete antigen is in the step (4): stir under 15～30 ℃ condition.
The invention has the beneficial effects as follows: preparation method of the present invention is easy, quick, the antigen that obtains can be used for immune animal, the monoclonal antibody of preparation bromadiolone, and then set up the method for quick of anticoagulation raticide bromadiolone, have broad application prospects, to be widely used in the fields such as forensic science, clinical medicine, to satisfy the demand of unit centering drug case (thing) the part quick tests such as public security system, Disease Control and Prevention Center and hospital.
Set up the haptenic preparation method of bromadiolone, can be used for immune animal, prepared the monoclonal antibody of bromadiolone, and then set up the method for quick of anticoagulation raticide bromadiolone, be widely used in the fields such as forensic science, clinical medicine.Solved bromadiolone as the small molecules poisonous substance directly the induced animal body produce the problem of specific antibody, by preparation 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 respectively 1, in the 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 the 4-dioxane is 0.7mol/L, in temperature is to stir 20h under 15 ℃ the condition to react; Add Succinic anhydried, bromadiolone is 1:1.5 with the ratio of the amount of substance of Succinic anhydried, reflux 6h again;
(2) mixture that reaction in the step (1) is obtained is poured in 15 ℃ of water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the 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 respectively 1, in the 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 the 4-dioxane is 0.7mol/L, in temperature is to stir 20h under 15 ℃ the condition to react; Add Succinic anhydried, bromadiolone is 1:1.5 with the ratio of the amount of substance of Succinic anhydried, reflux 6h again;
(2) mixture that reaction in the step (1) is obtained is poured in 5 ℃ of water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the 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 respectively 1, in the 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 the 4-dioxane is 0.7mol/L, in temperature is to stir 20h under 15 ℃ the condition to react; Add Succinic anhydried, bromadiolone is 1:1.5 with the ratio of the amount of substance of Succinic anhydried, reflux 6h again;
(2) mixture that reaction in the step (1) is obtained is poured in the mixture of ice and water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the 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 respectively 1, in the 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 the 4-dioxane is 0.7mol/L, in temperature is to stir 20h under 40 ℃ the condition to react; Add Succinic anhydried, bromadiolone is 1:6 with the ratio of the amount of substance of Succinic anhydried, reflux 6h again;
(2) mixture that reaction in the step (1) is obtained is poured into water, and has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the 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 respectively 1, in the 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 the 4-dioxane is 0.3mol/L, in temperature is to stir 10h under 25 ℃ the condition to react; Add Succinic anhydried, bromadiolone is 1:3 with the ratio of the amount of substance of Succinic anhydried, reflux 4h again;
(2) mixture that reaction in the step (1) is obtained is poured in the mixture of ice and water, has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the 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 respectively 1, in the 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 the 4-dioxane is 0.3mol/L, in temperature is to stir 10h under 27 ℃ the condition to react; Add Succinic anhydried, bromadiolone is 1:4 with the ratio of the amount of substance of Succinic anhydried, reflux 4h again;
(2) mixture that reaction in the step (1) is obtained is poured into water, and has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the bromadiolone haptens, and the haptenic productive rate of bromadiolone is 93%.
Embodiment 7
Adopt the bromadiolone haptens of embodiment 1 to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.5 in the methyl-sulphoxide, the mass ratio of bromadiolone haptens and BSA is 1.5:1, per 100 milligrams of bromadiolone haptens need 1 milliliter of methyl-sulphoxide, need pH is 2 milliliters of 6 phosphate buffer solutions], and then to add pH be 6 phosphate buffer solution, stirs under 15 ℃ condition; The bromadiolone complete antigen is made in dialysis after the lyophilize, calculate the productive rate of bromadiolone complete antigen as 85% take the bromadiolone haptens.
Bromadiolone complete antigen to the present embodiment preparation carries out ultra-violet analysis, infrared analysis and flight mass spectrum analysis, the result shows: have the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also there is the haptenic absorption peak of bromadiolone in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows and bromadiolone haptens and BSA linked reaction has occured (ratio of amount of substance is the bromadiolone haptens: BSA=15:1), generated bromadiolone haptens BSA binding substances.
Embodiment 8
Adopt the bromadiolone haptens of embodiment 2 to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:2 in the methyl-sulphoxide, the mass ratio of bromadiolone haptens and BSA is 4:1, per 100 milligrams of bromadiolone haptens need 1～3 milliliter of methyl-sulphoxide, need pH is 2.5 milliliters of 7 phosphate buffer solutions], and then to add pH be 7 phosphate buffer solution, stirs under 30 ℃ condition; The bromadiolone complete antigen is made in dialysis after the lyophilize, calculate the productive rate of bromadiolone complete antigen as 88% take the bromadiolone haptens.
Bromadiolone complete antigen to the present embodiment preparation carries out ultra-violet analysis, infrared analysis and flight mass spectrum analysis, the result shows: have the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also there is the haptenic absorption peak of bromadiolone in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows and bromadiolone haptens and BSA linked reaction has occured (ratio of amount of substance is the bromadiolone haptens: BSA=15:1), generated bromadiolone haptens BSA binding substances.
Embodiment 9
Adopt the bromadiolone haptens of embodiment 3 to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.3 in the methyl-sulphoxide, the mass ratio of bromadiolone haptens and BSA is 2:1, per 100 milligrams of bromadiolone haptens need 2 milliliters of methyl-sulphoxides, need pH is 2.2 milliliters of 7 phosphate buffer solutions], and then to add pH be 7 phosphate buffer solution, stirs under 20 ℃ condition; The bromadiolone complete antigen is made in dialysis after the lyophilize, calculate the productive rate of bromadiolone complete antigen as 92% take the bromadiolone haptens.
Bromadiolone complete antigen to the present embodiment preparation carries out ultra-violet analysis, infrared analysis and flight mass spectrum analysis, the result shows: have the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also there is the haptenic absorption peak of bromadiolone in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows and bromadiolone haptens and BSA linked reaction has occured (ratio of amount of substance is the bromadiolone haptens: BSA=15:1), generated bromadiolone haptens BSA binding substances.
Embodiment 10
Adopt the bromadiolone haptens of embodiment 4 to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.8 in the methyl-sulphoxide, the mass ratio of bromadiolone haptens and BSA is 2.6:1, per 100 milligrams of bromadiolone haptens need 1.5 milliliters of methyl-sulphoxides, need pH is 2.5 milliliters of 6 phosphate buffer solutions], and then to add pH be 6 phosphate buffer solution, stirs under 26 ℃ condition; The bromadiolone complete antigen is made in dialysis after the lyophilize, calculate the productive rate of bromadiolone complete antigen as 93% take the bromadiolone haptens.
Bromadiolone complete antigen to the present embodiment preparation carries out ultra-violet analysis, infrared analysis and flight mass spectrum analysis, the result shows: have the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also there is the haptenic absorption peak of bromadiolone in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows and bromadiolone haptens and BSA linked reaction has occured (ratio of amount of substance is the bromadiolone haptens: BSA=15:1), generated bromadiolone haptens BSA binding substances.
Embodiment 11
Adopt the bromadiolone haptens of embodiment 5 to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.7 in the methyl-sulphoxide, the mass ratio of bromadiolone haptens and BSA is 3.5:1, per 100 milligrams of bromadiolone haptens need 2 milliliters of methyl-sulphoxides, need pH is 2.5 milliliters of 7 phosphate buffer solutions], and then to add pH be 7 phosphate buffer solution, stirs under 19 ℃ condition; The bromadiolone complete antigen is made in dialysis after the lyophilize, calculate the productive rate of bromadiolone complete antigen as 94% take the bromadiolone haptens.
Bromadiolone complete antigen to the present embodiment preparation carries out ultra-violet analysis, infrared analysis and flight mass spectrum analysis, the result shows: have the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also there is the haptenic absorption peak of bromadiolone in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows and bromadiolone haptens and BSA linked reaction has occured (ratio of amount of substance is the bromadiolone haptens: BSA=15:1), generated bromadiolone haptens BSA binding substances.
Embodiment 12
Adopt the bromadiolone haptens of embodiment 6 to prepare anticoagulation raticide bromadiolone complete antigen: respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA to be joined that [mass ratio of bromadiolone haptens and dicyclohexylcarbodiimide is 1:1.6 in the methyl-sulphoxide, the mass ratio of bromadiolone haptens and BSA is 3.8:1, per 100 milligrams of bromadiolone haptens need 2.6 milliliters of methyl-sulphoxides, need pH is 2.5 milliliters of 6.5 phosphate buffer solutions], and then to add pH be 6.5 phosphate buffer solution, stirs under 30 ℃ condition; The bromadiolone complete antigen is made in dialysis after the lyophilize, calculate the productive rate of bromadiolone complete antigen as 90% take the bromadiolone haptens.
Bromadiolone complete antigen to the present embodiment preparation carries out ultra-violet analysis, infrared analysis and flight mass spectrum analysis, the result shows: have the haptenic absorption peak of bromadiolone in the UV spectrum of bromadiolone complete antigen, also there is the haptenic absorption peak of bromadiolone in the infrared spectra of bromadiolone complete antigen, flight mass spectrum shows and bromadiolone haptens and BSA linked reaction has occured (ratio of amount of substance is the bromadiolone haptens: BSA=15:1), generated bromadiolone haptens BSA binding substances.
The following institute of bromadiolone and the Succinic anhydried reaction preparation haptenic reaction formula of bromadiolone among above-described embodiment 1-6
Above-described embodiment only is for the invention example clearly is described, and is not the restriction 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 need not also can't give all embodiments exhaustive.And the apparent variation of being amplified out thus or change still are among the protection domain of the invention claim.
Claims (10)
1. the haptenic preparation method of anticoagulation raticide bromadiolone is characterized in that, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined respectively in Isosorbide-5-Nitrae-dioxane, under agitation react; Add again Succinic anhydried, reflux; React as follows:
(2) mixture that reaction in the step (1) is obtained is poured into water, and has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the bromadiolone haptens.
2. the haptenic preparation method of anticoagulation raticide bromadiolone according to claim 1 is characterized in that, in step (1), is to stir 5-20h under 15-40 ℃ the condition in temperature; Reflux 1-6h.
3. the haptenic preparation method of anticoagulation raticide bromadiolone according to claim 1 is characterized in that, in step (1), is to stir 10h under 25-27 ℃ the condition in temperature; Reflux 4h.
4. the haptenic preparation method of anticoagulation raticide bromadiolone according to claim 1 is characterized in that, in step (2), the temperature of water is 0 ℃.
5. the haptenic preparation method of arbitrary described anticoagulation raticide bromadiolone according to claim 1-4, it is characterized in that, 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.
6. the haptenic preparation method of anticoagulation raticide bromadiolone according to claim 5 is characterized in that, bromadiolone is 1:1.5～6 with the ratio of the amount of substance of Succinic anhydried.
7. the haptenic preparation method of anticoagulation raticide bromadiolone according to claim 5 is characterized in that, the concentration of bromadiolone in Isosorbide-5-Nitrae-dioxane is 0.3mol/L.
8. the preparation method of anticoagulation raticide bromadiolone complete antigen is characterized in that, comprises the steps:
(1) bromadiolone and Anhydrous potassium carbonate are joined respectively in Isosorbide-5-Nitrae-dioxane, under agitation react; Add again Succinic anhydried, reflux;
(2) mixture that reaction in the step (1) is obtained is poured into water, and has pale solid to separate out;
(3) filter, ethyl alcohol recrystallization obtains the bromadiolone haptens;
(4) respectively bromadiolone haptens, dicyclohexylcarbodiimide and BSA are joined in the methyl-sulphoxide, and then to add pH be 6～7 phosphate buffer solution, stir;
(5) the bromadiolone complete antigen is made in dialysis after the lyophilize.
9. the preparation method of described anticoagulation raticide bromadiolone complete antigen according to claim 8, it is characterized in that, 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, and per 100 milligrams of bromadiolone haptens need 1～3 milliliter of methyl-sulphoxide, need pH is 2～2.5 milliliters of 6～7 phosphate buffer solutions.
10. the preparation method of described anticoagulation raticide bromadiolone complete antigen according to claim 8 is characterized in that, in the step (4): stir under 15～30 ℃ condition.
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| CN110950961A (en) * | 2019-12-11 | 2020-04-03 | 中国农业大学 | Bromadiolone nano antibody and application thereof |
| CN111289753A (en) * | 2020-04-13 | 2020-06-16 | 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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