CN110452296A - A kind of rabbit monoclonal antibodies preparation method - Google Patents
A kind of rabbit monoclonal antibodies preparation method Download PDFInfo
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- CN110452296A CN110452296A CN201910640933.XA CN201910640933A CN110452296A CN 110452296 A CN110452296 A CN 110452296A CN 201910640933 A CN201910640933 A CN 201910640933A CN 110452296 A CN110452296 A CN 110452296A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- 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
- 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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Abstract
The invention discloses a kind of rabbit monoclonal antibodies preparation methods, include the following steps: step 1, immune serum preparation；Step 2, the affinity purification of antibody；Step 3, mass spectrum；Step 4, antibody variable gene spectrum obtain；Step 5, monoclonal antibody obtain；Wherein in above-mentioned step one, the special-shaped rabbit with bas mutation and wild type b9 is chosen, the subcutaneous multiple spot of Freund's complete adjuvant is inoculated with rabbit, it is immune primary every three days, it is immune altogether to be immunized after two weeks that same injection is primary again three times, rabbit tail vein injection NG-XMT after being immunized one monthTMAlbumen collects immunizing rabbit blood with sterile cuvette, send preparation room after label, it is compared with non-immune mouse serum, measure antibody titer with ELISA, by using microplate reader measurement OD490 absorbance value qualitatively and quantitatively judge immune serum whether be it is positive, choose positive immune serum；The present invention more efficiently quickly has high-affinity.
Description
Technical field
The present invention relates to rabbit monoclonal antibodies preparation technical field, specially a kind of rabbit monoclonal antibodies preparation method.
Background technique
Monoclonal antibody be the height that is generated by single B cell clone it is uniform, only for the anti-of a certain specific antigen epitope
Body, referred to as monoclonal antibody, rabbit antibody library are the very good sources of rabbit source polyclonal antibody and monoclonal antibody, and generation resists
Body possesses the advantages of high specific, high activity and high-affinity, and rabbit monoclonal antibodies preparation in the prior art is all by miscellaneous
Tumor technology is handed over, slowly, not enough efficiently, affinity of antibody is not high for preparation, and therefore, designing a kind of rabbit monoclonal antibodies preparation method is
It is necessary to.
Summary of the invention
The purpose of the present invention is to provide a kind of rabbit monoclonal antibodies preparation methods, to solve to propose in above-mentioned background technique
The problem of.
To achieve the above object, the invention provides the following technical scheme:
A kind of rabbit monoclonal antibodies preparation method includes the following steps: step 1, immune serum preparation；Step 2, the parent of antibody
And purifying；Step 3, mass spectrum；Step 4, antibody variable gene spectrum obtain；Step 5, monoclonal antibody obtain；
Wherein in above-mentioned step one, the special-shaped rabbit with bas mutation and wild type b9 is chosen, Freund is helped completely
The subcutaneous multiple spot of agent is inoculated with rabbit, is immunized every three days once, total to be immunized three times, is immunized and equally injects once again after two weeks, immune
Rabbit tail vein injection NG-XMT after one monthTMAlbumen collects immunizing rabbit blood with sterile cuvette, send preparation room after label,
It is compared with non-immune mouse serum, antibody titer is measured with ELISA, it is quantitative by using microplate reader measurement OD490 absorbance value
Whether qualitatively judge immune serum is the positive, when the ratio of test hole and negative hole is greater than 2.1, regards as the positive,
Choose positive immune serum；
Wherein in above-mentioned step two, albumin A sepharose CL-4B filler is filled into column, washes 3 ~ 5 columns with elution buffer
Bed volume washes off ethyl alcohol and impurity, washes 5 ~ 10 bed volume balance columns with equalizing and buffering liquid stream, blood serum sample upper prop, uniformly on
Efflux loading repeatedly can be washed again 5 ~ 10 bed volumes with equilibration buffer by sample, until baseline tends towards stability, respectively with
Relatively rapid and elution buffer is added at a slow speed, pH is down to about 3.0, and antibody is eluted from filler, immediately among the above and
In buffer and eluent to pH be neutrality, prevent the acidic environment of elution buffer from antibody being caused to inactivate, obtain sample；
Wherein in above-mentioned step three, LC-ESI-MS/MS mass spectrograph is opened, using nanometer Flowing liquid chromatography to be measured
Sample is isolated and purified, and the sample after separation enters mass spectrograph, the antibody component being digested with tandem mass spectrum combination analysis；
Wherein in above-mentioned step four, the next-generation DNA sequence dna by searching for immunizing rabbit b cell immunoglobulin library is established
Reference database, obtain antibody variable region high confidence level peptide spectrum matching；
Wherein in above-mentioned step five, CDR3 sequence and corresponding heavy chain are chosen from antibody variable gene spectrum and light chain is anti-
Body nucleic acid sequence constructs heavy chain and light chain expression vector as candidate matched sequence respectively, using suitable thin after mixing pairing
Dysuria with lower abdominal colic dyeing technique and expression system carry out antibody expression, carry out Activity determination to expression product, positive combination are obtained, by positive group
Heavy chain and light chain in conjunction are split, and are matched according to the principle of 1 heavy chain and 1 light chain, are utilized suitable cell transfecting skill
Art and expression system carry out antibody expression, carry out Activity determination to expression product, and obtained positive combination is and purpose antigen
Monoclonal antibody with high-affinity.
According to the above technical scheme, in the step 1, pay attention to sterile working, and acquisition serum as much as possible.
According to the above technical scheme, in the step 2, equilibration buffer is pH 7.4,20mmol/L phosphate-buffered
Liquid, elution buffer are pH 2.8, and 0.1mol/L glycine buffer, neutralization buffer is pH 9.0,1mol/L Tris-HCl
Buffer.
According to the above technical scheme, in the step 1,30min, 2000rpm centrifugation 10 are stored at room temperature to immunizing rabbit blood
Minute, it draws serum and sets 1.5ml plastic tube, mark.
According to the above technical scheme, in the step 2, buffer is using being preceding both needed to through 0.45 μm of membrane filtration.
According to the above technical scheme, in the step 1, inoculation dosage is 50 ~ 100 μ g/.
Compared with prior art, the beneficial effects of the present invention are: using proteomics, by antigentic specificity, directly
Direct Identification and antigentic specificity monoclonal antibody is cloned from the blood circulation of immune animal, more compared to conventional hybridoma technology
Efficiently, more rapidly, moreover, the monoclonal antibody of preparation has high-affinity, it can be applied to the relevant people of exploitation treatment disease
Monoclonal antibody, and help deeply to understand amynologic basis problem.
Detailed description of the invention
Fig. 1 is preparation method flow chart of the invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Referring to Fig. 1, the present invention provides a kind of technical solution:
A kind of rabbit monoclonal antibodies preparation method includes the following steps: step 1, immune serum preparation；Step 2, the parent of antibody
And purifying；Step 3, mass spectrum；Step 4, antibody variable gene spectrum obtain；Step 5, monoclonal antibody obtain；
Wherein in above-mentioned step one, the special-shaped rabbit with bas mutation and wild type b9 is chosen, Freund is helped completely
The subcutaneous multiple spot of agent is inoculated with rabbit, is immunized every three days once, total to be immunized three times, is immunized and equally injects once again after two weeks, immune
Rabbit tail vein injection NG-XMT albumen after one month collects immunizing rabbit blood with sterile cuvette, send preparation room after label,
It is compared with non-immune mouse serum, antibody titer is measured with ELISA, it is quantitative by using microplate reader measurement OD490 absorbance value
Whether qualitatively judge immune serum is the positive, when the ratio of test hole and negative hole is greater than 2.1, regards as the positive,
Choose positive immune serum；
Wherein in above-mentioned step two, albumin A sepharose CL-4B filler is filled into column, washes 3 ~ 5 columns with elution buffer
Bed volume washes off ethyl alcohol and impurity, washes 5 ~ 10 bed volume balance columns with equalizing and buffering liquid stream, blood serum sample upper prop, uniformly on
Efflux loading repeatedly can be washed again 5 ~ 10 bed volumes with equilibration buffer by sample, until baseline tends towards stability, respectively with
Relatively rapid and elution buffer is added at a slow speed, pH is down to about 3.0, and antibody is eluted from filler, immediately among the above and
In buffer and eluent to pH be neutrality, prevent the acidic environment of elution buffer from antibody being caused to inactivate, obtain sample；
Wherein in above-mentioned step three, LC-ESI-MS/MS mass spectrograph is opened, using nanometer Flowing liquid chromatography to be measured
Sample is isolated and purified, and the sample after separation enters mass spectrograph, the antibody component being digested with tandem mass spectrum combination analysis；
Wherein in above-mentioned step four, the next-generation DNA sequence dna by searching for immunizing rabbit b cell immunoglobulin library is established
Reference database, obtain antibody variable region high confidence level peptide spectrum matching；
Wherein in above-mentioned step five, CDR3 sequence and corresponding heavy chain are chosen from antibody variable gene spectrum and light chain is anti-
Body nucleic acid sequence constructs heavy chain and light chain expression vector as candidate matched sequence respectively, using suitable thin after mixing pairing
Dysuria with lower abdominal colic dyeing technique and expression system carry out antibody expression, carry out Activity determination to expression product, positive combination are obtained, by positive group
Heavy chain and light chain in conjunction are split, and are matched according to the principle of 1 heavy chain and 1 light chain, are utilized suitable cell transfecting skill
Art and expression system carry out antibody expression, carry out Activity determination to expression product, and obtained positive combination is and purpose antigen
Monoclonal antibody with high-affinity.
According to the above technical scheme, in step 1, pay attention to sterile working, and acquisition serum as much as possible.
According to the above technical scheme, in step 2, equilibration buffer is pH 7.4, and 20mmol/L phosphate buffer is washed
De- buffer is pH 2.8, and 0.1mol/L glycine buffer, neutralization buffer is pH 9.0,1mol/L Tris-HCl buffering
Liquid.
According to the above technical scheme, in step 1,30min is stored at room temperature to immunizing rabbit blood, 2000rpm is centrifuged 10 points
Clock draws serum and sets 1.5ml plastic tube, marks.
According to the above technical scheme, in step 2, buffer is using being preceding both needed to through 0.45 μm of membrane filtration.
According to the above technical scheme, in step 1, inoculation dosage is 50 ~ 100 μ g/.
Based on above-mentioned, it is an advantage of the current invention that using proteomics, by antigentic specificity, directly from immune dynamic
Direct Identification and antigentic specificity monoclonal antibody is cloned in the blood circulation of object, it is more efficient compared to conventional hybridoma technology, more
Quickly, moreover, the monoclonal antibody of preparation has high-affinity, it is anti-to can be applied to the relevant human monoclonal of exploitation treatment disease
Body, and help deeply to understand amynologic basis problem.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a reality
Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation
In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to
Non-exclusive inclusion, so that the process, method, article or equipment including a series of elements is not only wanted including those
Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment
Intrinsic element.
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 rabbit monoclonal antibodies preparation method includes the following steps: step 1, immune serum preparation；Step 2, antibody
Affinity purification；Step 3, mass spectrum；Step 4, antibody variable gene spectrum obtain；Step 5, monoclonal antibody obtain；Its feature
It is:
Wherein in above-mentioned step one, the special-shaped rabbit with bas mutation and wild type b9 is chosen, Freund is helped completely
The subcutaneous multiple spot of agent is inoculated with rabbit, is immunized every three days once, total to be immunized three times, is immunized and equally injects once again after two weeks, immune
Rabbit tail vein injection NG-XMT after one monthTMAlbumen collects immunizing rabbit blood with sterile cuvette, send preparation room after label,
It is compared with non-immune mouse serum, antibody titer is measured with ELISA, it is quantitative by using microplate reader measurement OD490 absorbance value
Whether qualitatively judge immune serum is the positive, when the ratio of test hole and negative hole is greater than 2.1, regards as the positive,
Choose positive immune serum；
Wherein in above-mentioned step two, albumin A sepharose CL-4B filler is filled into column, washes 3 ~ 5 columns with elution buffer
Bed volume washes off ethyl alcohol and impurity, washes 5 ~ 10 bed volume balance columns with equalizing and buffering liquid stream, blood serum sample upper prop, uniformly on
Efflux loading repeatedly can be washed again 5 ~ 10 bed volumes with equilibration buffer by sample, until baseline tends towards stability, respectively with
Relatively rapid and elution buffer is added at a slow speed, pH is down to about 3.0, and antibody is eluted from filler, immediately among the above and
In buffer and eluent to pH be neutrality, prevent the acidic environment of elution buffer from antibody being caused to inactivate, obtain sample；
Wherein in above-mentioned step three, LC-ESI-MS/MS mass spectrograph is opened, using nanometer Flowing liquid chromatography to be measured
Sample is isolated and purified, and the sample after separation enters mass spectrograph, the antibody component being digested with tandem mass spectrum combination analysis；
Wherein in above-mentioned step four, the next-generation DNA sequence dna by searching for immunizing rabbit b cell immunoglobulin library is established
Reference database, obtain antibody variable region high confidence level peptide spectrum matching；
Wherein in above-mentioned step five, CDR3 sequence and corresponding heavy chain are chosen from antibody variable gene spectrum and light chain is anti-
Body nucleic acid sequence constructs heavy chain and light chain expression vector as candidate matched sequence respectively, using suitable thin after mixing pairing
Dysuria with lower abdominal colic dyeing technique and expression system carry out antibody expression, carry out Activity determination to expression product, positive combination are obtained, by positive group
Heavy chain and light chain in conjunction are split, and are matched according to the principle of 1 heavy chain and 1 light chain, are utilized suitable cell transfecting skill
Art and expression system carry out antibody expression, carry out Activity determination to expression product, and obtained positive combination is and purpose antigen
Monoclonal antibody with high-affinity.
2. a kind of rabbit monoclonal antibodies preparation method according to claim 1, it is characterised in that: in the step 1, note
Meaning sterile working, and acquisition serum as much as possible.
3. a kind of rabbit monoclonal antibodies preparation method according to claim 1, it is characterised in that: in the step 2, put down
Weighing apparatus buffer is pH 7.4, and 20mmol/L phosphate buffer, elution buffer is pH 2.8,0.1mol/L glycine buffer
Liquid, neutralization buffer are pH 9.0,1mol/L Tris-HCl buffer.
4. a kind of rabbit monoclonal antibodies preparation method according to claim 1, it is characterised in that: right in the step 1
Immunizing rabbit blood is stored at room temperature 30min, and 2000rpm is centrifuged 10 minutes, draws serum and sets 1.5ml plastic tube, marks.
5. a kind of rabbit monoclonal antibodies preparation method according to claim 1, it is characterised in that: in the step 2, delay
Fliud flushing is using being preceding both needed to through 0.45 μm of membrane filtration.
6. a kind of rabbit monoclonal antibodies preparation method according to claim 1, it is characterised in that: in the step 1, exempt from
Epidemic disease injection dosage is 50 ~ 100 μ g/.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111217905A (en) * | 2019-11-18 | 2020-06-02 | 安徽环球基因科技有限公司 | Preparation method of recombinant rabbit monoclonal antibody |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103003697A (en) * | 2010-05-17 | 2013-03-27 | 得克萨斯系统大学董事会 | Rapid isolation of monoclonal antibodies from animals |
| CN103842379A (en) * | 2011-03-09 | 2014-06-04 | 细胞信号科技公司 | Methods and reagents for creating monoclonal antibodies |
| CN108456682A (en) * | 2017-02-17 | 2018-08-28 | 苏州金唯智生物科技有限公司 | A kind of screening technique of monoclonal antibody and its application |
-
2019
- 2019-07-16 CN CN201910640933.XA patent/CN110452296A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103003697A (en) * | 2010-05-17 | 2013-03-27 | 得克萨斯系统大学董事会 | Rapid isolation of monoclonal antibodies from animals |
| CN103842379A (en) * | 2011-03-09 | 2014-06-04 | 细胞信号科技公司 | Methods and reagents for creating monoclonal antibodies |
| CN108456682A (en) * | 2017-02-17 | 2018-08-28 | 苏州金唯智生物科技有限公司 | A kind of screening technique of monoclonal antibody and its application |
Non-Patent Citations (2)
| Title |
|---|
| DANIEL R. BOUTZ ET AL.: ""Proteomic Identification of Monoclonal Antibodies from Serum"", 《ANAL. CHEM.》 * |
| WAN CHEUNG CHEUNG ET AL.: ""A proteomics approach for the identification and cloning of monoclonal antibodies from serum"", 《NATURE BIOTECHNOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111217905A (en) * | 2019-11-18 | 2020-06-02 | 安徽环球基因科技有限公司 | Preparation method of recombinant rabbit monoclonal antibody |
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Application publication date: 20191115 |