CN106810608A - Anti- Cobratoxin serum of high-titer and preparation method and application - Google Patents
Anti- Cobratoxin serum of high-titer and preparation method and application Download PDFInfo
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- CN106810608A CN106810608A CN201710122170.0A CN201710122170A CN106810608A CN 106810608 A CN106810608 A CN 106810608A CN 201710122170 A CN201710122170 A CN 201710122170A CN 106810608 A CN106810608 A CN 106810608A
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- neurotoxin
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- antiserum
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/35—Valency
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
Anti- Cobratoxin serum the invention discloses a kind of high-titer and preparation method thereof, comprises the following steps：First weaken the neurotoxicity of cobra venom, be then embedded in polyacrylamide gel, finally by the levigate rear inoculation rabbit of gel containing neurotoxin, obtain antiserum, freezen protective, you can.The cobra venom of present invention various dose, neurotoxicity is weakened using heating, the treatment of SDS and β mercaptoethanols, polyacrylamide gel embedding replaces emulsification, it is ensured that the survival of immunized animal health, the antiserum for reducing sero-fast production cost, having prepared high-titer.
Description
Technical field
A kind of preparation method of the anti-Cobratoxin serum of high-titer, belongs to immunological technique field.
Background technology
Cobra (Naja naja atraCantor Elapidae, Elaps) are belonged to, it is widely distributed in China, be
Nervous system type poisonous snake, its primary toxins are neurotoxin.Neurotoxin is divided into two classes：Presynaptic neurotoxin and postsynaptic neuronal
Toxin, the former suppresses the release of acetylcholine in nerve conduction, and the latter can be combined with motor end plate acetylcholinergic receptor, the two
Result in nerve conduction blocking.Two class neurotoxins are all basic protein, and without non-protein composition, molecular weight is small, containing 4-5 two
Sulphur is good for, and heat resistance is very strong.
Because the stability of Cobra neurotoxin is very strong, general method of attenuating is difficult to change its toxicity, is preparing
During neurotoxin antiserum, the little animal of amount of antigen of injection could survive, and amount of antigen is small to cause antiserum titre low, controls
Therapeutic effect is not good.Prepare High-titre antiserum significant for effectively treatment bite by Naja naja atra, the reduction death rate.
The content of the invention
Anti- Cobratoxin serum the invention provides a kind of high-titer and preparation method thereof, the present invention utilizes eye
The resistant to elevated temperatures feature of mirror snake venom neurotoxin, is centrifuged after heating and obtains neurotoxin, by neurotoxin SDS and beta -mercaptoethanol
Treatment reduces neurotoxicity, and is embedded in polyacrylamide gel and reduces rate of release, and the gel containing neurotoxin is levigate
Inoculation rabbit, obtains the anti-Cobratoxin serum of high-titer afterwards.
Concrete technical scheme of the invention is as follows：
The preparation method of the anti-Cobratoxin serum of a kind of high-titer, it is characterised in that comprise the following steps：
A, according to 0.08-0.12g cobra venom 0.8-1.2 mL physiological saline solutions, in boiling water bath heat 8-12 minutes, from
The heart goes unless neurotoxin precipitation, then adds 48-52 mg SDS and 1.8-2.2 uL beta -mercaptoethanols to be sufficiently mixed and weaken it
Neurotoxicity；
The embedding of b, neurotoxin：To be embedded in polyacrylamide gel by the neurotoxin of attenuation treatment；
C, will contain neurotoxin the levigate rear inoculation rabbit of gel, through inoculation several times after, to cut off arteria carotis mode
Kill rabbit and take blood, centrifugation obtains antiserum.
The preparation method of the anti-Cobratoxin serum of described high-titer, it is characterised in that exempting from described in step c
Epidemic disease injects the concrete operations side of rabbit：By the levigate rear inoculation rabbit of gel containing neurotoxin, 4 inoculations are carried out,
Per injection is spaced 7 days, for the first time the injection mL of hypodermic injection 1.25, the mL of intraperitoneal injection 2.25, and second, third is noted with the 4th time
Equal intraperitoneal injection 3.5 mL is penetrated, after the 4th injection, rabbit is killed cutting off arteria carotis mode within the 3rd day and is taken blood, be centrifuged after 6 hours
Obtain antiserum.
Antiserum obtained in the preparation method of the anti-Cobratoxin serum of described high-titer.
Application of the described antiserum on treatment bite by Naja naja atra patient.
The technology of the present invention effect is embodied in two aspects：
1st, ensure to prepare sero-fast high-titer
It is suppressed because neurotoxin activity is most of, injection volume increases, and being injected animal will not be because of the effect of neurotoxin
And it is dead, it is ensured that the antiserum titre of preparation is high.By comparing, the present invention is higher by than antiserum titre prepared by conventional method
About 7 times, therapeutic effect is greatly improved；
2nd, the freund adjuvant of costliness is not used, reduces sero-fast production cost.
Be embedded in neurotoxin in netted polyacrylamide gel by the present invention, and neurotoxin is from netted polyacrylamide
It is released slowly into animal body in amine gel, the residence time is long in animal body for antigen, can abundant stimulating immune system, Er Qieyong
Polyacrylamide gel instead of the freund adjuvant of costliness.
Specific embodiment
Embodiment one：
The first step：The acquisition of Cobratoxin
0.1g cobra venom is dissolved in 1.0 mL physiological saline, is heated 10 minutes in boiling water bath, centrifugation（13000 revs/min）
Go unless neurotoxin precipitation, then adds 50 mg SDS and 2 uL beta -mercaptoethanols to be sufficiently mixed and weaken its neurotoxicity；
Second step：The embedding of neurotoxin
Above-mentioned neurotoxin solution is added into the acrylamides of 1.4mL 30%, the % ammonium persulfates of 50uL 10,2uL tetramethyls second two
Amine (TEMED), is fully mixed, and 0.5mL isopropanols are slowly added to along test tube wall, is solidified after 1 hour, removes isopropanol, is taken out
Colloid scalpel is divided into 4 parts, every part of about 1 g, freezen protective；
3rd step：Injecting immune rabbit, obtains antiserum
1 part of above-mentioned colloid, plus 2.0 mL physiological saline are taken, in fully levigate in mortar, the disposable injection of 5mL specifications is drawn into
In device, the rabbit of cumulative volume about 3.5mL, inoculation 2.5kg or so, co-injection 4 times, secondary immunity is spaced 7 days, notes for the first time
Hypodermic injection 1.25mL, intraperitoneal injection 2.25mL are penetrated, second, third injects equal intraperitoneal injection 3.5mL, the 4th note with the 4th time
Rabbit is killed cutting off arteria carotis mode and takes blood within the 3rd day after penetrating, be centrifuged after 6 hours and obtain antiserum, freezen protective.
4th step：The measure of antiserum titre
The measure of antiserum titre is similar to ELISA method.
By cobra venom coating buffer（The carbonate buffer solution of 0.1mol/L pH 9.5）10 μ g/mL are configured to as anti-
Raw sample, doubling dilution is carried out by the antiserum of acquisition, replaces antigen samples as blank using the coating buffer without snake venom, with just
Normal rabbit anteserum（Non-immune rabbit anteserum）Antiserum to be measured is substituted as negative control, with the antiserum for preparing as primary antibody,
Goat anti-rabbit igg-alkaline phosphatase is secondary antibody, and the p-NPP of 1 mg/mL is substrate, and colour developing is determined after 45 minutes on ELIASA
OD405Value, is returned to zero with blank, and the potency of 2 times of negative control light absorption values of sero-fast light absorption value to be measured is set to 1, antiserum
Extension rate both be sero-fast potency.
（1）Antigen coat：ELISA Plate adds 100 μ L envelope antigens, ELISA Plate to put 4 per holeoC refrigerator overnights.
（2）Closing：Plus confining liquid, per the μ L of hole 120,37oC is incubated 1 h.
（3）Plus primary antibody：Add the antiserum of doubling dilution（ELISA buffer solutions dilute）, feminine gender is non-immune rabbit blood
Clearly, 37oC is incubated 1 h.
（4）Plus ELIAS secondary antibody：Add 1/10000 goat anti-rabbit igg-alkaline phosphatase（Sigma, ELISA buffer solution are dilute
Release）, 37oC is incubated 1 h.
（5）Plus substrate：The p-NPP of 1 mg/mL（Sigma, is dissolved in substrate buffer solution, pH 9.8）, 37oIt is incubated in C dark
45 minutes, survey OD405Value.
Note：Washed 3 times, every time more than 5 minutes with PBST after the completion of each step.
By calculating, antiserum titre prepared by embodiment one reaches 560, and adds prepared by adjuvant emulsion with conventional snake venom
Antiserum titre only has 80, it is seen that antiserum titre prepared by the present invention improves about 7 times.
Embodiment two：
The first step：The acquisition of Cobratoxin
Except identical with " embodiment one " first step with other steps in addition to 0.09g cobra venom.
Second step：The embedding of neurotoxin
The neurotoxin solution that the first step is obtained adds the acrylamides of 1.3mL 30%, 50uL10 % ammonium persulfates, 2uL
TEMED, fully mixes, and other steps are identical with " embodiment one " second step；
3rd step：Injecting immune rabbit, obtains antiserum
It is identical with " embodiment one " the 3rd step.
4th step：The measure of antiserum titre
Continuous mode is identical with " embodiment one " the 4th step.
By calculating, antiserum titre prepared by " embodiment two " reaches 544, and adds adjuvant emulsion to prepare with conventional snake venom
Antiserum titre there was only 80, it is seen that the present invention prepare antiserum titre improve about 6.8 times.
Embodiment three：
The first step：The acquisition of Cobratoxin
Except with addition to 0.11g cobra venom, other steps are identical with " embodiment one " first step.
Second step：The embedding of echidnotoxin
The neurotoxin solution that the first step is obtained adds the acrylamides of 1.5mL 30%, 50uL10 % ammonium persulfates, 2uL
TEMED, fully mixes, and other steps are identical with " embodiment one " second step；
3rd step：Injecting immune rabbit, obtains antiserum
It is identical with " embodiment one " the 3rd step.
4th step：The measure of antiserum titre
Detection process is identical with " embodiment one " the 4th step.
By calculating, antiserum titre prepared by " embodiment three " reaches 584, and adds adjuvant emulsion to prepare with conventional snake venom
Antiserum titre there was only 80, it is seen that the present invention prepare antiserum titre improve 7.3 times.
The cobra venom of present invention various dose, Nervous toxicity is weakened using heating, three kinds for the treatment of of SDS and beta -mercaptoethanol
Property, polyacrylamide gel embedding replacement emulsification, the result stablized and be satisfied with illustrates the technical application, reliability.
Claims (4)
1. the preparation method of the anti-Cobratoxin serum of a kind of high-titer, it is characterised in that comprise the following steps：
A, according to 0.08-0.12g cobra venom 0.8-1.2 mL physiological saline solutions, in boiling water bath heat 8-12 minutes, from
The heart goes unless neurotoxin precipitation, then adds 48-52 mg SDS and 1.8-2.2 uL beta -mercaptoethanols to be sufficiently mixed and weaken it
Neurotoxicity；
The embedding of b, neurotoxin：To be embedded in polyacrylamide gel by the neurotoxin of attenuation treatment；
C, will contain neurotoxin the levigate rear inoculation rabbit of gel, through inoculation several times after, to cut off arteria carotis mode
Kill rabbit and take blood, centrifugation obtains antiserum.
2. the preparation method of the anti-Cobratoxin serum of high-titer according to claim 1, it is characterised in that step
The concrete operations side of the inoculation rabbit described in rapid c：By the levigate rear inoculation rabbit of gel containing neurotoxin, 4 are carried out
Secondary inoculation, per injection is spaced 7 days, for the first time the injection mL of hypodermic injection 1.25, intraperitoneal injection 2.25 mL, second, the
Inject equal intraperitoneal injection 3.5 mL for three and the 4th times, after the 4th injection, kill rabbit cutting off arteria carotis mode within the 3rd day and take
Blood, is centrifuged after 6 hours and obtains antiserum.
3. resist obtained in the preparation method of the anti-Cobratoxin serum of the high-titer described in any one of claim 1 or 2
Serum.
4. application of the antiserum described in claim 3 on treatment bite by Naja naja atra patient.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710122170.0A CN106810608A (en) | 2017-03-02 | 2017-03-02 | Anti- Cobratoxin serum of high-titer and preparation method and application |
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| CN201710122170.0A CN106810608A (en) | 2017-03-02 | 2017-03-02 | Anti- Cobratoxin serum of high-titer and preparation method and application |
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Citations (8)
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|---|---|---|---|---|
| CN1858064A (en) * | 2006-06-02 | 2006-11-08 | 广州蛇毒研究所 | Chicken yoke antibody resisting Bengalese cobra venom and its preparation |
| CN101012279A (en) * | 2007-01-12 | 2007-08-08 | 广州医学院 | Vitelline antibody for resisting king cobra poison and its preparing method and use |
| CN101816789A (en) * | 2010-04-08 | 2010-09-01 | 成都军区疾病预防控制中心军事医学研究所 | Lyophilized viper antivenin and preparation method thereof |
| CN102133232A (en) * | 2011-03-18 | 2011-07-27 | 苏州大学 | Cobra venom physical modification method and application in preparation of analgesia or immunosuppressive drugs |
| CN102241760A (en) * | 2011-05-20 | 2011-11-16 | 上海赛伦生物技术有限公司 | Inactivation method for snake venom and inactivated snake venom prepared thereby |
| CN103910798A (en) * | 2014-04-24 | 2014-07-09 | 上海赛伦生物技术有限公司 | Antibody for resisting vipera berus toxin and long-noded pit viper toxin and preparation method and application thereof |
| WO2016061611A1 (en) * | 2014-10-23 | 2016-04-28 | Q-Sera Pty Ltd | Improved clotting composition |
| CN106008713A (en) * | 2016-08-05 | 2016-10-12 | 安徽威尔试剂盒科技有限责任公司 | Preparation method and application of Agkistrodon acutus venom high-titer antiserum |
-
2017
- 2017-03-02 CN CN201710122170.0A patent/CN106810608A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1858064A (en) * | 2006-06-02 | 2006-11-08 | 广州蛇毒研究所 | Chicken yoke antibody resisting Bengalese cobra venom and its preparation |
| CN101012279A (en) * | 2007-01-12 | 2007-08-08 | 广州医学院 | Vitelline antibody for resisting king cobra poison and its preparing method and use |
| CN101816789A (en) * | 2010-04-08 | 2010-09-01 | 成都军区疾病预防控制中心军事医学研究所 | Lyophilized viper antivenin and preparation method thereof |
| CN102133232A (en) * | 2011-03-18 | 2011-07-27 | 苏州大学 | Cobra venom physical modification method and application in preparation of analgesia or immunosuppressive drugs |
| CN102241760A (en) * | 2011-05-20 | 2011-11-16 | 上海赛伦生物技术有限公司 | Inactivation method for snake venom and inactivated snake venom prepared thereby |
| CN103910798A (en) * | 2014-04-24 | 2014-07-09 | 上海赛伦生物技术有限公司 | Antibody for resisting vipera berus toxin and long-noded pit viper toxin and preparation method and application thereof |
| WO2016061611A1 (en) * | 2014-10-23 | 2016-04-28 | Q-Sera Pty Ltd | Improved clotting composition |
| CN106008713A (en) * | 2016-08-05 | 2016-10-12 | 安徽威尔试剂盒科技有限责任公司 | Preparation method and application of Agkistrodon acutus venom high-titer antiserum |
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Effective date of registration: 20180322 Address after: 236000 Anhui Province, Lu'an City Yun Lu Street West of West Anhui University Applicant after: West Anhui University Applicant after: Wei Chuanbao Address before: 237000 Lu'an centralized demonstration park in Lu'an, Anhui province (Lu'an University Science Park) Applicant before: Anhui Weier reagent box science and Technology Co Ltd |
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Application publication date: 20170609 |