CN106243222A - A kind of doxycycline broad-spectrum monoclonal antibody and preparation technology - Google Patents
A kind of doxycycline broad-spectrum monoclonal antibody and preparation technology Download PDFInfo
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- CN106243222A CN106243222A CN201610707258.4A CN201610707258A CN106243222A CN 106243222 A CN106243222 A CN 106243222A CN 201610707258 A CN201610707258 A CN 201610707258A CN 106243222 A CN106243222 A CN 106243222A
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
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Abstract
nullThe invention discloses a kind of doxycycline broad-spectrum monoclonal antibody and preparation technology，It is made up of following raw material according to parts by weight: doxycycline 40 52 parts、Tetracycline 4 20 parts、Minocycline 26 parts、Metacycline 24 parts、Bovine serum albumin 35 parts、Freund's complete adjuvant 58 parts、Incomplete Freund's adjuvant 12 parts、HRP sheep anti-mouse igg 23 parts、36 parts of ammonium sulfate、Disodium hydrogen phosphate 9 10 parts、Potassium dihydrogen phosphate 16 20 parts、Citric acid 12 16 parts、Ammonium dihydrogen phosphate 8 12 parts、Dehydrated alcohol 9 15 parts、Isobutyl chlorocarbonate 13 parts、It is coated buffer 24 parts、Confining liquid 57 parts、Lavation buffer solution 34 parts、Substrate 6 10 parts、Cell culture fluid 20 25 parts，With doxycycline and equal amido phenenyl acid as primary raw material，Use heavy nitrogen to prepare and there is antiserum specific doxycycline broad-spectrum monoclonal antibody，There is excellent biological property，And the antiserum titre of this antibody、Inhibition is preferable.
Description
Technical field
The present invention relates to doxycycline antibody preparation technology field, be specifically related to a kind of doxycycline broad-spectrum monoclonal antibody
And preparation technology.
Background technology
Doxycycline is one of important member of tetracycline antibiotics, very extensive in animal and veterinary clinical practice, along with
The fast development of China's animal husbandry, plant (community) is under the ordering about of interests, and unreasonable abuse veterinary drug causes medicine animal
Accumulation residual in edible tissue, this behavior not only causes fastbacteria to produce, and has had a strong impact on China's livestock products
Outlet, causes heavy economic losses, brings harm greatly, additionally, raiser there is also also to consumers in general's physical and mental health
Do not meet the medication regulations such as dosage, medicine-feeding part and medication animal species, and it is different to reuse several trade name
But the phenomenon of composition same medicine, therefore, strengthens the monitoring of animal-derived food veterinary drug residue the most necessary.
Summary of the invention
For problem above, the invention provides a kind of doxycycline broad-spectrum monoclonal antibody and preparation technology, with strength
Mycin and equal amido phenenyl acid are primary raw material, add a certain amount of sodium nitrite solution, sodium carbonate liquor, hydrochloric acid, use weight
Nitrogen method is prepared and to have antiserum specific doxycycline broad-spectrum monoclonal antibody, has excellent biological property, Ke Yiyou
Effect solves the problem in background technology.
To achieve these goals, the technical solution used in the present invention is as follows: a kind of doxycycline broad-spectrum monoclonal antibody,
It is made up of following raw material according to parts by weight:
Doxycycline 40-52 part, tetracycline 4-20 part, minocycline 2-6 part, metacycline 2-4 part, bovine serum albumin 3-5
Part, Freund's complete adjuvant 5-8 part, incomplete Freund's adjuvant 1-2 part, HRP-sheep anti-mouse igg 2-3 part, ammonium sulfate 3-6 part, phosphoric acid
Disodium hydrogen 9-10 part, potassium dihydrogen phosphate 16-20 part, citric acid 12-16 part, ammonium dihydrogen phosphate 8-12 part, dehydrated alcohol 9-15 part,
Isobutyl chlorocarbonate 1-3 part, it is coated buffer 2-4 part, confining liquid 5-7 part, lavation buffer solution 3-4 part, substrate 6-10 part, cell
Culture fluid 20-25 part.
According to technique scheme, described in be coated buffer select concentration be 0.05mol/L, pH value is the carbonate of 9.6
Buffer.
According to technique scheme, described confining liquid uses 2g import gelatin, adds to be coated in buffer 100ml to boil and takes advantage of
Heat filtering gained.
According to technique scheme, the preparation of described substrate is to be dissolved in 5ml dehydrated alcohol by the tetramethyl benzidine of 10mg,
Itself 0.5mL with 10mL substrate buffer solution is mixed, add 10ul 30% H2O2。
According to technique scheme, the Pidolidone solution that described cell culture fluid selects concentration to be 0.2mol/L.
Additionally the present invention have also been devised the preparation technology of a kind of doxycycline broad-spectrum monoclonal antibody, comprises the steps:
(1) 0.4g doxycycline is dissolved in 5mL sodium carbonate liquor；
(2) 151mg equal amido phenenyl acid is dissolved in 2mL HCL (0.3mol/L), is cooled to 4 DEG C；
Under the conditions of (3) 4 DEG C of lucifuges, in (2) liquid, it is slowly added dropwise sodium nitrite solution while stirring；Course of reaction is all the time with forming sediment
Powder-potassium iodide starch paper monitoring, when reagent paper becomes indigo plant, stops dropping；
(4) liquid in (1) is slowly dropped in (3) liquid.
(5) with HCL(0.1mol/L) adjust PH to 3.6, continue 4 DEG C of lucifuge stirring 30min；
(6) vacuum filtration, obtains red precipitate, 50mL ultrapure water, dries；
(7) it is loaded on ampere bottle and seals 4 DEG C of preservations.
According to technique scheme, in described step (1), the concentration of sodium carbonate liquor is 0.1mol/L.
According to technique scheme, described step (3), the concentration of the sodium nitrite solution of instillation is 0.1mol/L；Described
In step (4), need under the conditions of 4 DEG C of lucifuges, stir 30min, until red precipitate occurs.
Beneficial effects of the present invention:
The present invention, with doxycycline and equal amido phenenyl acid as primary raw material, adds a certain amount of sodium nitrite solution, sodium carbonate
Solution, hydrochloric acid, use heavy nitrogen to prepare and have antiserum specific doxycycline broad-spectrum monoclonal antibody, have excellent
Biological property.
Accompanying drawing explanation
Fig. 1 is the graph of relation of foamed heat insulating time of the present invention and bubble diameter.
Fig. 2 is the schematic diagram of different level of PbO addition of the present invention and blowing temperature.
Fig. 3 is the schematic diagram of PbO of the present invention and density.
Fig. 4 is the schematic diagram of PbO addition of the present invention and high-density foam glass mechanical property.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, to the present invention
It is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to
Limit the present invention.
Embodiment 1:
A kind of doxycycline broad-spectrum monoclonal antibody, is made up of following raw material according to parts by weight:
Doxycycline 40 parts, tetracycline 4 parts, minocycline 2 parts, metacycline 2 parts, bovine serum albumin 3 parts, Freund are helped completely
Agent 5 parts, incomplete Freund's adjuvant 1 part, HRP-sheep anti-mouse igg 2 parts, 3 parts of ammonium sulfate, disodium hydrogen phosphate 9 parts, potassium dihydrogen phosphate
16 parts, citric acid 12 parts, ammonium dihydrogen phosphate 8 parts, dehydrated alcohol 9 parts, isobutyl chlorocarbonate 1 part, be coated buffer 2 parts, close
Liquid 5 parts, lavation buffer solution 3 parts, substrate 6 parts, cell culture fluid 20 parts.
The described buffer that is coated selects concentration to be 0.05mol/L, and pH value is the carbonate buffer solution of 9.6；Described confining liquid
Use 2g import gelatin, add to be coated in buffer 100ml and boil filtered while hot gained；The preparation of described substrate is by the four of 10mg
Methyl biphenyl amine is dissolved in 5ml dehydrated alcohol, mixed by itself 0.5mL with 10mL substrate buffer solution, adds the 30% of 10ul
H2O2；The Pidolidone solution that described cell culture fluid selects concentration to be 0.2mol/L.
Its preparation technology, comprises the steps:
(1) 0.4g doxycycline being dissolved in 5mL sodium carbonate liquor, the concentration of described sodium carbonate liquor is 0.1mol/L；
(2) 151mg equal amido phenenyl acid is dissolved in 2mL HCL (0.3mol/L), is cooled to 4 DEG C；
Under the conditions of (3) 4 DEG C of lucifuges, in (2) liquid, it is slowly added dropwise sodium nitrite solution while stirring, the sodium nitrite solution of instillation
Concentration be 0.1mol/L；Course of reaction monitors with starch-kalium iodide reagent paper all the time, when reagent paper becomes indigo plant, stops dropping；
(4) liquid in (1) is slowly dropped in (3) liquid, under the conditions of 4 DEG C of lucifuges, stirs 30min, until occurring that redness is heavy
Till shallow lake.
(5) with HCL(0.1mol/L) adjust PH to 3.6, continue 4 DEG C of lucifuge stirring 30min；
(6) vacuum filtration, obtains red precipitate, 50mL ultrapure water, dries；
(7) it is loaded on ampere bottle and seals 4 DEG C of preservations.
Embodiment 2:
A kind of doxycycline broad-spectrum monoclonal antibody, is made up of following raw material according to parts by weight:
Doxycycline 46 parts, tetracycline 12 parts, minocycline 4 parts, metacycline 3 parts, bovine serum albumin 4 parts, Freund are complete
Adjuvant 6.5 parts, incomplete Freund's adjuvant 1.5 parts, HRP-sheep anti-mouse igg 2.5 parts, 4.5 parts of ammonium sulfate, disodium hydrogen phosphate 9.5
Part, potassium dihydrogen phosphate 18 parts, citric acid 14 parts, ammonium dihydrogen phosphate 10 parts, dehydrated alcohol 12 parts, isobutyl chlorocarbonate 2 parts, it is coated
Buffer 3 parts, confining liquid 6 parts, lavation buffer solution 3.5 parts, substrate 8 parts, cell culture fluid 22.5 parts.
The described buffer that is coated selects concentration to be 0.05mol/L, and pH value is the carbonate buffer solution of 9.6；Described confining liquid
Use 2g import gelatin, add to be coated in buffer 100ml and boil filtered while hot gained；The preparation of described substrate is by the four of 10mg
Methyl biphenyl amine is dissolved in 5ml dehydrated alcohol, mixed by itself 0.5mL with 10mL substrate buffer solution, adds the 30% of 10ul
H2O2；The Pidolidone solution that described cell culture fluid selects concentration to be 0.2mol/L.
Its preparation technology, comprises the steps:
(1) 0.4g doxycycline being dissolved in 5mL sodium carbonate liquor, the concentration of described sodium carbonate liquor is 0.1mol/L；
(2) 151mg equal amido phenenyl acid is dissolved in 2mL HCL (0.3mol/L), is cooled to 4 DEG C；
Under the conditions of (3) 4 DEG C of lucifuges, in (2) liquid, it is slowly added dropwise sodium nitrite solution while stirring, the sodium nitrite solution of instillation
Concentration be 0.1mol/L；Course of reaction monitors with starch-kalium iodide reagent paper all the time, when reagent paper becomes indigo plant, stops dropping；
(4) liquid in (1) is slowly dropped in (3) liquid, under the conditions of 4 DEG C of lucifuges, stirs 30min, until occurring that redness is heavy
Till shallow lake.
(5) with HCL(0.1mol/L) adjust PH to 3.6, continue 4 DEG C of lucifuge stirring 30min；
(6) vacuum filtration, obtains red precipitate, 50mL ultrapure water, dries；
(7) it is loaded on ampere bottle and seals 4 DEG C of preservations.
Embodiment 3:
A kind of doxycycline broad-spectrum monoclonal antibody, is made up of following raw material according to parts by weight:
Doxycycline 52 parts, tetracycline 20 parts, minocycline 6 parts, metacycline 4 parts, bovine serum albumin 5 parts, Freund are complete
Adjuvant 8 parts, incomplete Freund's adjuvant 2 parts, HRP-sheep anti-mouse igg 3 parts, 6 parts of ammonium sulfate, disodium hydrogen phosphate 10 parts, biphosphate
20 parts of potassium, citric acid 16 parts, ammonium dihydrogen phosphate 12 parts, dehydrated alcohol 15 parts, isobutyl chlorocarbonate 3 parts, be coated buffer 4 parts,
Confining liquid 7 parts, lavation buffer solution 4 parts, substrate 10 parts, cell culture fluid 25 parts.
The described buffer that is coated selects concentration to be 0.05mol/L, and pH value is the carbonate buffer solution of 9.6；Described confining liquid
Use 2g import gelatin, add to be coated in buffer 100ml and boil filtered while hot gained；The preparation of described substrate is by the four of 10mg
Methyl biphenyl amine is dissolved in 5ml dehydrated alcohol, mixed by itself 0.5mL with 10mL substrate buffer solution, adds the 30% of 10ul
H2O2；The Pidolidone solution that described cell culture fluid selects concentration to be 0.2mol/L.
Its preparation technology, comprises the steps:
(1) 0.4g doxycycline being dissolved in 5mL sodium carbonate liquor, the concentration of described sodium carbonate liquor is 0.1mol/L；
(2) 151mg equal amido phenenyl acid is dissolved in 2mL HCL (0.3mol/L), is cooled to 4 DEG C；
Under the conditions of (3) 4 DEG C of lucifuges, in (2) liquid, it is slowly added dropwise sodium nitrite solution while stirring, the sodium nitrite solution of instillation
Concentration be 0.1mol/L；Course of reaction monitors with starch-kalium iodide reagent paper all the time, when reagent paper becomes indigo plant, stops dropping；
(4) liquid in (1) is slowly dropped in (3) liquid, under the conditions of 4 DEG C of lucifuges, stirs 30min, until occurring that redness is heavy
Till shallow lake.
(5) with HCL(0.1mol/L) adjust PH to 3.6, continue 4 DEG C of lucifuge stirring 30min；
(6) vacuum filtration, obtains red precipitate, 50mL ultrapure water, dries；
(7) it is loaded on ampere bottle and seals 4 DEG C of preservations.
Embodiment 4:
Reflected by UV scanning method, envelope antigen and antiserum working concentration identification method, the method for testing such as mensuration of serum titer
The biological property of order clonal antibody.
(1) UV scanning method identifies the effect of monoclonal antibody
Hapten and BSA(OA) it is coupled together as " spacerarm " by Isosorbide-5-Nitrae-tincture diether, as it is shown in figure 1, purple at conjugate
In outer scanning curve, containing hapten and BSA(OA) uv absorption feature, may determine that coupling according to the additivity of absworption peak
Success.The coupling ratio calculating doxycycline and BSA according to formula is 16:1, and doxycycline is 11:1 with the coupling ratio of OA.Exempt from
The concentration of epidemic focus DC-BSA is 21.87mg/mL.The concentration of coating antigen DC-OA is 6.81mg/mL.
If immunogenic carrier protein with between hapten be by longer spacerarm coupled together with, and containing relatively
The homogenous immunogen of short spacerarm is compared, and it is easier to stimulate body immune system to produce hapten and mechanism's analog tool thereof
Have a universal antibody of higher discernment and adhesion, two kinds of immunogenic core textures of this research synthesis all pass through longer between
Every arm away from carrier protein, therefore, it should body immune system can be stimulated to produce preferable universal antibody.
(2) envelope antigen and antiserum working concentration identify the effect of monoclonal antibody
The optimal envelope antigen CTC-C-OA concentration determining ELISA method according to square formation method is 2.1 μ g/mL, and positive serum is optimal
Dilution factor is 1:20000, and result is shown in Fig. 2.
Mice identifies the blood sampling of one week after eye socket, surveys titer with indirect ELISA method, it is determined that standard is with 3.1, and mice serum is imitated
Valency is all up to 1:4 × 104, result is as shown in Figure 3.
By synthesis coating antigen (concentration is 0.17mg/mL) be diluted to concentration be 4.2 μ g/mL, 2.1 μ g/mL, 1.05 μ g/
Coated elisa plate after mL, be separately added into variable concentrations standard substance (0,25,50,100ng/mL) and 1:20000 dilution Mus blood
Clearly, after same dilution coating antigen adds standard substance and antibody, OD value increases with standard concentration and is gradually lowered, and illustrates to resist
Serum is for DC, and result is as shown in Figure 4.
Therefore, from Fig. 2,3,4 it can be seen that the antiserum titre of this antibody, inhibition are preferable, antiserum titre reaches
The requirement of preparation monoclonal antibody.
Based on above-mentioned, it is an advantage of the current invention that the present invention, with doxycycline and equal amido phenenyl acid as primary raw material, adds
Enter a certain amount of sodium nitrite solution, sodium carbonate liquor, hydrochloric acid, use heavy nitrogen to prepare and there is the specific strength of antiserum
Mycin broad-spectrum monoclonal antibody, has an excellent biological property, and the antiserum titre of this antibody, inhibition are preferable so that work
Skill production efficiency is greatly improved, convenient production process operation, and cost is relatively low.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.
Claims (9)
1. a doxycycline broad-spectrum monoclonal antibody, it is characterised in that be made up of following raw material according to parts by weight:
Doxycycline 40-52 part, tetracycline 4-20 part, minocycline 2-6 part, metacycline 2-4 part, bovine serum albumin 3-5
Part, Freund's complete adjuvant 5-8 part, incomplete Freund's adjuvant 1-2 part, HRP-sheep anti-mouse igg 2-3 part, ammonium sulfate 3-6 part, phosphoric acid
Disodium hydrogen 9-10 part, potassium dihydrogen phosphate 16-20 part, citric acid 12-16 part, ammonium dihydrogen phosphate 8-12 part, dehydrated alcohol 9-15 part,
Isobutyl chlorocarbonate 1-3 part, it is coated buffer 2-4 part, confining liquid 5-7 part, lavation buffer solution 3-4 part, substrate 6-10 part, cell
Culture fluid 20-25 part.
A kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 1, it is characterised in that described in be coated buffer
Selection concentration is 0.05mol/L, and pH value is the carbonate buffer solution of 9.6.
A kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 1, it is characterised in that described confining liquid uses
2g import gelatin, adds to be coated in buffer 100ml and boils filtered while hot gained.
A kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 1, it is characterised in that the preparation of described substrate is
The tetramethyl benzidine of 10mg is dissolved in 5ml dehydrated alcohol, itself 0.5mL with 10mL substrate buffer solution is mixed, adds
The H of the 30% of 10ul2O2。
A kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 1, it is characterised in that described cell culture fluid
The Pidolidone solution selecting concentration to be 0.2mol/L.
6. the preparation technology of a doxycycline broad-spectrum monoclonal antibody, it is characterised in that comprise the steps:
(1) 0.4g doxycycline is dissolved in 5mL sodium carbonate liquor；
(2) 151mg equal amido phenenyl acid is dissolved in 2mL HCL (0.3mol/L), is cooled to 4 DEG C；
Under the conditions of (3) 4 DEG C of lucifuges, in (2) liquid, it is slowly added dropwise sodium nitrite solution while stirring；Course of reaction is all the time with forming sediment
Powder-potassium iodide starch paper monitoring, when reagent paper becomes indigo plant, stops dropping；
(4) liquid in (1) is slowly dropped in (3) liquid；
(5) with HCL(0.1mol/L) adjust PH to 3.6, continue 4 DEG C of lucifuge stirring 30min；
(6) vacuum filtration, obtains red precipitate, 50mL ultrapure water, dries；
(7) it is loaded on ampere bottle and seals 4 DEG C of preservations.
The preparation technology of a kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 6, it is characterised in that described
In step (1), the concentration of sodium carbonate liquor is 0.1mol/L.
The preparation technology of a kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 6, it is characterised in that described
In step (4), need under the conditions of 4 DEG C of lucifuges, stir 30min, until red precipitate occurs.
The preparation technology of a kind of doxycycline broad-spectrum monoclonal antibody the most according to claim 6, it is characterised in that described
Step (3), the concentration of the sodium nitrite solution of instillation is 0.1mol/L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610707258.4A CN106243222A (en) | 2016-08-23 | 2016-08-23 | A kind of doxycycline broad-spectrum monoclonal antibody and preparation technology |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610707258.4A CN106243222A (en) | 2016-08-23 | 2016-08-23 | A kind of doxycycline broad-spectrum monoclonal antibody and preparation technology |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1798241A1 (en) * | 2005-12-13 | 2007-06-20 | Etat-Francais représenté par le Délégué Général pour L'Armement | Method of manufacturing a hapten between doxycycline and bovine serum albumin and application for detecting doxycycline in humans or animals |
-
2016
- 2016-08-23 CN CN201610707258.4A patent/CN106243222A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1798241A1 (en) * | 2005-12-13 | 2007-06-20 | Etat-Francais représenté par le Délégué Général pour L'Armement | Method of manufacturing a hapten between doxycycline and bovine serum albumin and application for detecting doxycycline in humans or animals |
Non-Patent Citations (1)
| Title |
|---|
| 高峰: "强力霉素广谱单克隆抗体的制备及应用", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
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