CN109627332A - A kind of immune antiboidy and preparation method thereof - Google Patents
A kind of immune antiboidy and preparation method thereof Download PDFInfo
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- CN109627332A CN109627332A CN201910036677.3A CN201910036677A CN109627332A CN 109627332 A CN109627332 A CN 109627332A CN 201910036677 A CN201910036677 A CN 201910036677A CN 109627332 A CN109627332 A CN 109627332A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Mycobacteriaceae (F)
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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
- C07K16/065—Purification, fragmentation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to Fluorescent antib fields, and disclose a kind of immune antiboidy and preparation method thereof, pass through the multiple immune generated antibody of sample drawn blood progress out of rabbit body, specific steps: step 1: the selection of animal, step 2: the selection of antigen, step 3: the preparation of adjuvant and antigen emulsion, step 4: immune, step 5: sero-fast preparation.The immune antiboidy and preparation method thereof, it is immunized by carrying out three phases to rabbit, the immune antiboidy of high-purity can be prepared, high specificity, potency are high and affinity is strong, sample blood is taken in vivo from rabbit by specification, so that the immune antiboidy taken out improves the purity of immune antiboidy not by germ contamination.
Description
Technical field
The present invention relates to Fluorescent antib field, specially a kind of immune antiboidy and preparation method thereof.
Background technique
Blood group antibody is generally divided into two classes: natural antibody and immune antiboidy, and general natural antibody is IgM, and immune antiboidy is
IgG.1. IgM class antibody can be aggregated the red blood cell with corresponding antigens in brine media, this kind of antibody be mainly ABO and
P, MN, Lewis antibody is often natural antibody.Energy activating complement, occurs intravascular hemolysis.Such antibody cannot penetrate placenta, and
Its optimal reaction temperature is 4 DEG C so being called cold antibody.2. IgG class antibody is corresponding to having in brine media in most cases
The red blood cell of antigen is not aggregated, and is only just aggregated in colloidal medium, enzyme or antiglobulin, cohesion amine medium, this
Class antibody is mainly the blood group antibodies such as Rh, Kidd, Duffy.Usual situation is generated by immune (blood transfusion, gestation, organ transplant)
Antibody, also known as immune antiboidy.Mostly occur extravascular hemolysis.37 DEG C of such antibody optimal reaction temperature is so be called warm antibodies.
Current immune antiboidy is not ideal enough to the immune stage level of animal during preparation, and to dynamic
Object not enough standardizes when taking sample blood, is easy so that obtained immune antiboidy is not pure and mild contaminated, specificity is low, potency is low and affine
Power is low.For this purpose, the invention proposes a kind of immune antiboidies and preparation method thereof.
Summary of the invention
(1) the technical issues of solving
In view of the deficiencies of the prior art, the present invention provides a kind of immune antiboidy and preparation method thereof, have by rabbit
Son carries out the immune of three phases, can prepare the immune antiboidy of high-purity, takes sample in vivo from rabbit by specification
Blood, so that the advantages that immune antiboidy taken out not by germ contamination, improves the purity of immune antiboidy, solves immune antiboidy
It is not ideal enough to the immune stage level of animal during preparation, and not enough standardized when taking sample blood to animal, hold
It is easier that obtained immune antiboidy is not pure and mild contaminated, specific low, the low problem low with affinity of potency.
(2) technical solution
In order to achieve the above object, the invention provides the following technical scheme: a kind of immune antiboidy, by being taken out out of rabbit body
Sample blood is taken to carry out repeatedly immune generated antibody.
A kind of preparation method of immune antiboidy, the specific steps are as follows:
Step 1: the selection of animal selects 2-3 months or more rabbits, immune to perform label, then carries out a point cage again and raises
It supports；
Step 2: antigen (cellulase) is diluted to 4mg/ml, can be mixed and made into emulsion with adjuvant by the selection of antigen
It is immune for rabbit；
Step 3: the preparation of adjuvant and antigen emulsion, adjuvant Split completely: light mineral oil (Valelinum Liquidum) 8.5ml, sheep
Hair rouge 1.5ul inactivates tubercle bacillus 10mg, the preparation of antigen emulsion；
(1) finish: mineral oil and lanolin mix in proportion, finish are used as after 8 pounds of sterilizings in 20 minutes of high pressure, using preceding micro-
Fire melts；
(2) aqua: inactivation tubercle bacillus 1mg/ul+ antigen 4mg/ml；
(3) finish and aqua 1:1 mixing；
Step 4: it is immune, then the subcutaneous multi-point injection of arteria auricularis of rabbit, every 0.1m1, immune for the first time: experimental group exists
Add the antigen of adjuvant in rabbit body,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphoric acid buffer
Liquid；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain south liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis, total injection 0.4ml add the anti-of adjuvant
Former emulsion；
Second is immune: adjuvant antigen is not added in experimental group rabbit within the 3rd week,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphoric acid buffer
Liquid；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain alcohol liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain enzyme solution；
Third time is immune: second is immune 1 week latter, and adjuvant antigen is not added in experimental group rabbit,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphoric acid buffer
Liquid；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain enzyme solution；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain alcohol liquid；
Step 5: sero-fast preparation, third time is immune, after a week, from the arteria auricularis of rabbit is subcutaneous or cardiac sample product
Blood, the blood of collection is placed in 1h or so at room temperature, after solidification, sets at 4 DEG C, and serum is precipitated in (being sure not to freeze) overnight, is centrifuged,
Serum is sucked out in aseptic condition in 4000rpa, 10min, is then carried out with the antigen (having epitope 2) with same epitope anti-
It answers, to remove non-specific antibody, the specific immunity for being directed to (epitope 2) is anti-.
Preferably, the hybrid mode of the finish and aqua is grinding or syringe mixing.
Preferably, the serum being sucked out in the step 5 is dispensed (0.05~0.2ul), stores in -40 DEG C or less refrigerators,
Or it is stored in 4 DEG C of refrigerators after freeze-drying and saves.
Preferably, the inactivation tubercle bacillus and antigen are final concentration.
Preferably, described the step of taking sample blood are as follows: the ear of rabbit is exposed to outside boxes and baskets, can also be caught and be exempted from by another people
Body cuts off the hair of ear edge, smears auricle with a little dimethylbenzene, after 30s, ear blood vessel dilatation, hyperemia gently draw have sharp ears with hand, with list
The knife blade of face shaver or point quickly cuts ear artery, and blood flows out, and collects 30~40ml every time, then uses
Cotton balls hemostasis by compression washes away dimethylbenzene after blood coagulation, after two weeks, can in another ear bloodletting, this method can repeated multiple times bloodletting, put
It strictly to be carried out by sterility requirements during blood.
(3) beneficial effect
Compared with prior art, the present invention provides a kind of immune antiboidy and preparation method thereof, have it is following the utility model has the advantages that
1, immune antiboidy and preparation method thereof is immunized by carrying out three phases to rabbit, can be prepared high-purity
The immune antiboidy of degree, high specificity, potency are high and affinity is strong, sample blood are taken in vivo from rabbit by specification, so that taking
Immune antiboidy out by germ contamination, does not improve the purity of immune antiboidy.
Specific embodiment
The invention discloses a kind of immune antiboidy and preparation method thereof, those skilled in the art can use for reference present disclosure,
It is suitably modified realization of process parameters.It is important to note that all similar substitutions and modifications carry out those skilled in the art
Say it is it will be apparent that they are considered as being included in the present invention.Preparation method of the invention passed through preferred embodiment into
Gone description, related personnel obviously can not depart from the content of present invention, in spirit and scope to preparation method as described herein into
Row change or appropriate changes and combinations, carry out implementation and application the technology of the present invention.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.Below with reference to embodiment, the present invention is further explained.
A kind of immune antiboidy passes through the multiple immune generated antibody of sample drawn blood progress out of rabbit body.
A kind of preparation method of immune antiboidy, the specific steps are as follows:
Step 1: the selection of animal selects 2-3 months or more rabbits, immune to perform label, then carries out a point cage again and raises
It supports；
Step 2: antigen (cellulase) is diluted to 4mg/ml, can be mixed and made into emulsion with adjuvant by the selection of antigen
It is immune for rabbit；
Step 3: the preparation of adjuvant and antigen emulsion, adjuvant Split completely: light mineral oil (Valelinum Liquidum) 8.5ml, sheep
Hair rouge 1.5ul inactivates tubercle bacillus 10mg, the preparation of antigen emulsion；
(1) finish: mineral oil and lanolin mix in proportion, finish are used as after 8 pounds of sterilizings in 20 minutes of high pressure, using preceding micro-
Fire melts；
(2) aqua: inactivation tubercle bacillus 1mg/ul+ antigen 4mg/ml；
(3) finish and aqua 1:1 mixing；
Step 4: it is immune, then the subcutaneous multi-point injection of arteria auricularis of rabbit, every 0.1m1, immune for the first time: experimental group exists
Add the antigen of adjuvant in rabbit body,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphoric acid buffer
Liquid；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain south liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis, total injection 0.4ml add the anti-of adjuvant
Former emulsion；
Second is immune: adjuvant antigen is not added in experimental group rabbit within the 3rd week,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphoric acid buffer
Liquid；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain alcohol liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain enzyme solution；
Third time is immune: second is immune 1 week latter, and adjuvant antigen is not added in experimental group rabbit,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphoric acid buffer
Liquid；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain enzyme solution；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml fiber
Plain alcohol liquid；
Step 5: sero-fast preparation, third time is immune, after a week, from the arteria auricularis of rabbit is subcutaneous or cardiac sample product
Blood, the blood of collection is placed in 1h or so at room temperature, after solidification, sets at 4 DEG C, and serum is precipitated in (being sure not to freeze) overnight, is centrifuged,
Serum is sucked out in aseptic condition in 4000rpa, 10min, is then carried out with the antigen (having epitope 2) with same epitope anti-
It answers, to remove non-specific antibody, the specific immunity for being directed to (epitope 2) is anti-.
The hybrid mode of finish and aqua is grinding or syringe mixing.
The serum being sucked out in step 5 is dispensed (0.05~0.2ul), is store after storing in -40 DEG C or less refrigerators, or freeze-drying
4 DEG C of refrigerators are stored in save.
It inactivates tubercle bacillus and antigen is final concentration.
The step of taking sample blood are as follows: the ear of rabbit is exposed to outside boxes and baskets, can also be caught by another people and exempt from body, cut off ear edge
Hair, smear auricle with a little dimethylbenzene, after 30s, ear blood vessel dilatation, hyperemia gently draw have sharp ears with hand, with single side shaver or point
Knife blade, quickly cut ear artery, blood i.e. flow out, every time collect 30~40ml, then use cotton balls hemostasis by compression,
Dimethylbenzene is washed away after blood coagulation, after two weeks, can in another ear bloodletting, this method can repeated multiple times bloodletting, it is tight during bloodletting
Lattice are carried out by sterility requirements.
In conclusion the preparation method of the immune antiboidy, is immunized by carrying out three phases to rabbit, can be prepared
The immune antiboidy of high-purity, high specificity, potency are high and affinity is strong, take sample blood in vivo from rabbit by specification, make
The immune antiboidy that must be taken out by germ contamination, does not improve the purity of immune antiboidy.
It should be noted that term " includes " or any other variant thereof is intended to cover non-exclusive inclusion, thus
So that the process, method, article or equipment for including a series of elements not only includes those elements, but also including not clear
The other element listed, or further include for elements inherent to such a process, method, article, or device.Do not having more
In the case where more limitations, the element that is limited by sentence "including a ...", it is not excluded that including process, the side of the element
There is also other identical elements in method, article or equipment.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of immune antiboidy and preparation method thereof, it is characterised in that: by out of rabbit body sample drawn blood carry out it is multiple
Antibody caused by immune.
2. a kind of preparation method of immune antiboidy, the specific steps are as follows:
Step 1: the selection of animal selects 2-3 months or more rabbits, immune to perform label, then carries out sub-cage rearing again；
Step 2: antigen (cellulase) is diluted to 4mg/ml, can be mixed and made into emulsion with adjuvant and be used for by the selection of antigen
Rabbit is immune；
Step 3: the preparation of adjuvant and antigen emulsion, adjuvant Split completely: light mineral oil (Valelinum Liquidum) 8.5ml, lanolin
1.5ul inactivates tubercle bacillus 10mg, the preparation of antigen emulsion；
(1) finish: mineral oil and lanolin mix in proportion, are used as finish after 8 pounds of sterilizings in 20 minutes of high pressure, are melted using preceding small fire
Change；
(2) aqua: inactivation tubercle bacillus 1mg/ul+ antigen 4mg/ml；
(3) finish and aqua 1:1 mixing；
Step 4: it is immune, then the subcutaneous multi-point injection of arteria auricularis of rabbit, every 0.1m1, immune for the first time: experimental group is in rabbit
Add the antigen of adjuvant in vivo,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphate buffer；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml cellulose south
Liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis, total injection 0.4ml add the antigen cream of adjuvant
Agent；
Second is immune: adjuvant antigen is not added in experimental group rabbit within the 3rd week,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphate buffer；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml cellulose alcohol
Liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml cellulase
Liquid；
Third time is immune: second is immune 1 week latter, and adjuvant antigen is not added in experimental group rabbit,
A group 5 (there is yellow on head): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml phosphate buffer；
B group 5 (there is yellow in tail portion): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml cellulase
Liquid；
C group 8 (there is yellow at middle part): the every subcutaneous multi-point injections of rabbit arteria auricularis amount to injection 0.4ml2mg/ml cellulose alcohol
Liquid；
Step 5: sero-fast preparation, third time is immune, after a week, from the arteria auricularis of rabbit is subcutaneous or cardiac sample product blood, receives
The blood of collection is placed in 1h or so at room temperature, after solidification, sets at 4 DEG C, and serum is precipitated in (being sure not to freeze) overnight, is centrifuged, 4000rpa,
Serum is sucked out in aseptic condition in 10min, is then reacted with the antigen (having epitope 2) with same epitope, to remove
Non-specific antibody, the specific immunity for being directed to (epitope 2) are anti-.
3. a kind of preparation method of immune antiboidy according to claim 2, it is characterised in that: the finish and aqua it is mixed
Conjunction mode is grinding or syringe mixing.
4. a kind of preparation method of immune antiboidy according to claim 2, it is characterised in that: be sucked out in the step 5
Serum is dispensed (0.05~0.2ul), is stored in 4 DEG C of refrigerators preservations after storing in -40 DEG C or less refrigerators, or freeze-drying.
5. a kind of immune antiboidy according to claim 2 and preparation method thereof, it is characterised in that: the inactivation tubercle bacillus
It is final concentration with antigen.
6. a kind of immune antiboidy according to claim 2 and preparation method thereof, it is characterised in that: the step for taking sample blood
Suddenly are as follows: the ear of rabbit is exposed to outside boxes and baskets, can also be caught by another people and exempt from body, cut off the hair of ear edge, is applied with a little dimethylbenzene
Smear auricle, after 30s, ear blood vessel dilatation, hyperemia gently draw have sharp ears with hand, with single side shaver or the knife blade of point, quickly cut
Ear artery, blood flow out, and collect 30~40ml every time, then use cotton balls hemostasis by compression, wash away dimethylbenzene after blood coagulation, and two
After week, can in another ear bloodletting, this method can repeated multiple times bloodletting, strictly to be carried out by sterility requirements during bloodletting.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5962258A (en) * | 1995-08-23 | 1999-10-05 | Diversa Corporation | Carboxymethyl cellulase fromthermotoga maritima |
| CN1544630A (en) * | 2003-11-12 | 2004-11-10 | 浙江大学 | Method for preparing recombinant duck interleukin-2 protein and its application |
| CN108948185A (en) * | 2018-07-17 | 2018-12-07 | 遵义医学院 | The rabbit alliin antibody that alliin antigen and its immune response obtain |
-
2019
- 2019-01-15 CN CN201910036677.3A patent/CN109627332A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5962258A (en) * | 1995-08-23 | 1999-10-05 | Diversa Corporation | Carboxymethyl cellulase fromthermotoga maritima |
| CN1544630A (en) * | 2003-11-12 | 2004-11-10 | 浙江大学 | Method for preparing recombinant duck interleukin-2 protein and its application |
| CN108948185A (en) * | 2018-07-17 | 2018-12-07 | 遵义医学院 | The rabbit alliin antibody that alliin antigen and its immune response obtain |
Non-Patent Citations (5)
| Title |
|---|
| ROY SEXTON等: ""Use of cellulase antibodies to study leaf abscission"", 《NATURE》 * |
| 依兹拉依里斯基: "《植物细菌病害 上》", 31 July 1955, 财政经济出版社 * |
| 安天琛: ""海南东寨港红树林大型底栖动物纤维素酶的鉴定及纯化"", 《中国优秀博硕士学位论文全文数据库（硕士）基础科学辑》 * |
| 王佩铭等: "《相关医学大辞典》", 30 April 1994, 天津科技翻译出版公司 * |
| 邹义洲等: "《医学免疫学》", 31 December 2018, 中国医药科技出版社 * |
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Application publication date: 20190416 |