CN109679924A - The anti-human monoclonal antibody and the preparation method and application thereof with peptide element of high-affinity - Google Patents
The anti-human monoclonal antibody and the preparation method and application thereof with peptide element of high-affinity Download PDFInfo
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- CN109679924A CN109679924A CN201811587898.1A CN201811587898A CN109679924A CN 109679924 A CN109679924 A CN 109679924A CN 201811587898 A CN201811587898 A CN 201811587898A CN 109679924 A CN109679924 A CN 109679924A
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Classifications
-
- 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
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a kind of high-affinity the anti-human monoclonal antibody and the preparation method and application thereof with peptide element, by the way that common be immunized is combined with immune modulatory molecules, assisted delivery cell factor Flt3l, mGM-CSF and CCL20 while conventional common immune, to increase the immune response of immune animal, improve effective submission of body endoantigen molecule, promote antibody effectively mature, and then obtain the antibody with high-affinity, obtained affinity of antibody is 10~100 times high compared with the antibody that conventional method is prepared, and can be very good to be applied to and peptide element immune detection.
Description
Technical field
The invention belongs to field of biomedicine, in particular to a kind of high-affinity is anti-human and peptide element monoclonal antibody and its system
Preparation Method and application.
Background technique
It is a kind of homologous with arginine vasopressin (AVP) more comprising 39 amino acid residues with peptide plain (copeptin)
Peptide is the C-terminal part peptide fragment of arginine vasopressin former (pro-VAP).Recent studies have found that and the peptide element biology new as one
Marker can replace AVP, early stage the diseases such as cardiovascular and cerebrovascular disease, diabetic nephropathy, septicopyemia and urinary tract infections
In terms of diagnosis and prognostic evaluation, there is certain clinical value, is a kind of important clinical disease predictive factors.Currently, needle
To and the detection of peptide element mainly include chemo-immunity luminescence method and enzyme linked immunosorbent assay.Two methods are all based on immune detection
The detection technique of principle detects sensitivity and specificity that antibody mass used is directly related to detection, therefore, two methods
Detection time be all larger than 3 hours, seriously limit the clinical value with peptide element.Exploitation and peptide are induced for polypeptide antigen
For plain monoclonal antibody, because its own immunogenicity is lower, it is difficult to cause the immune response of immune animal, it is generally the case that
For this kind of immunogene, generally enhance immune response by way of coupling carrier large protein.But it is nonetheless, common to exempt from
Epidemic disease mode can not improve clone's positive rate well, be difficult to develop high-affinity and peptide element monoclonal antibody.It answers at present
For still being limited to resist with the antibody of peptide element immune detection more, more anti-the shortcomings that there is poor specificities in itself.Although market is gushed
Reveal some monoclonal antibodies about with peptide element, but its affinity is general.If while wanting to greatly shorten detection time and not influencing
Sensitivity, then exploitation is high affine and peptide element monoclonal antibody is imperative.
Under normal circumstances, the effect of adjuvant is only in that the immune system of stimulation body, and exempts to immune animal body
Epidemic disease power is without humidification.The immunity power of body is directly concerning the power to immune response, and the production of high-quality monoclonal antibody
The raw power for directly depending on immune animal body immunological effect.Cell factor prepares monoclonal antibody in a large amount of DNA immunization methods at present
It is applied in experiment, and achieves remarkable result.But there are certain technical difficulty for DNA immunization, and operation difficulty degree is high, exempts from
Epidemic disease dosage is uncontrollable, and it is also high that cost is immunized.Therefore, it is necessary to provide a kind of preparation method, it is anti-that high-affinity can be prepared
People and peptide element monoclonal antibody.
Summary of the invention
Based on this, the purpose of the present invention is to provide a kind of high-affinity is anti-human and peptide element monoclonal antibody and its preparation side
Method.
To achieve the above object, the present invention provides the following technical scheme that
A kind of preparation method for secreting anti-human and peptide element monoclonal antibody hybridoma, comprising the following steps:
With people and peptide element mice immunized with antigen；
To the plasmid of the mouse delivering expression cell factor after being immunized；
Collect the splenocyte or lymph node cells of the mouse through plasmid delivery, and by the splenocyte or lymph node cells with
Murine myeloma cell merge to obtain hybridoma to get.
The people and peptide element antigen are to contain amino acid sequence SEQ ID NO:1, SEQ ID in one of the embodiments,
The polypeptide of NO:2 or SEQ ID NO:3.
The people and peptide element antigen are people and the peptide element antigen that coupling has high molecular weight protein in one of the embodiments,
The high molecular weight protein is at least one of KLH, OVA, BSA.Preferably, the people and peptide element antigen are that C-terminal coupling has KLH
People and peptide element antigen.
The cell factor includes mFlt3l, mGM-CSF and mCCL20 in one of the embodiments,.
The carrier of the plasmid is pCAGGS in one of the embodiments,.
Immune mouse described in one of the embodiments, is repeatedly to be immunized, and the 6th~8 day after being immunized every time carries out
Once to the plasmid of the mouse delivering expression cell factor, each immunization interval time is 13~15 days；And/or
The immune mouse includes:
Initial immunity: antigen is mixed in equal volume with Freund's complete adjuvant, after fully emulsified, according to every 90~110 μ g
Carry out back of mice subcutaneously more site injections；
Second immune: antigen being mixed in equal volume with freund 's incomplete adjuvant, after fully emulsified, according to every 45~55
μ g carries out back of mice subcutaneously more site injections；
To the plasmid of the mouse delivering expression cell factor after being wherein immunized every time.
Preferably, the immune mouse further includes that third time is immune: antigen is mixed in equal volume with freund 's incomplete adjuvant,
After fully emulsified, back of mice subcutaneously more site injections are carried out according to every 45~55 μ g.
Preferably, first 3 days progress abdominal cavity booster immunizations are merged.It is highly preferred that abdominal cavity booster immunization specifically includes: with 45~
55 μ g antigen diluents are in physiological saline, intraperitoneal injection.
Anti-human and peptide element monoclonal antibody hybridoma is secreted the present invention also provides a kind of, specific technical solution is such as
Under:
It is a kind of to secrete anti-human and peptide element monoclonal antibody hybridoma, it is prepared by preparation method as described above
It arrives.
The present invention also provides a kind of anti-human and peptide element monoclonal antibody preparation method, specific technical solution is as follows:
A kind of anti-human and peptide element monoclonal antibody preparation method, the hybridization for taking preparation method as described above to be prepared
Oncocyte, culture, secrete monoclonal antibody to get.
The present invention also provides a kind of anti-human and peptide element monoclonal antibody, specific technical solution is as follows:
A kind of anti-human and peptide element monoclonal antibody, is secreted to obtain, or by as described above by hybridoma as described above
Anti-human and peptide element monoclonal antibody preparation method be prepared.
Based on the above-mentioned technical proposal, the invention has the following advantages:
The present inventor stumbles on, relative to common immune, will it is common it is immune combined with DNA immunization for
Immune response has significant adjustment effect, while the present invention expresses on the basis of conventional common immune in conjunction with assisted delivery
The plasmid of cell factor improves effective submission of body endoantigen molecule to increase the immune response of immune animal, promotes anti-
Body is effectively mature, and then obtains the antibody with high-affinity, what obtained affinity of antibody was prepared compared with conventional method
Antibody is 10~100 times high, can be very good to be applied to in peptide element immune detection, on the basis of guaranteeing detection sensitivity significantly
Detection time is shortened, is applied so as to effectively realize with the POCT of peptide element, the clinical value with peptide element is increased, is severe
Monitoring and prognosis treatment provide more effective reference index.
Detailed description of the invention
Fig. 1 is the anti-human technology of preparing route with peptide element monoclonal antibody of high-affinity；
Fig. 2 is the corresponding monoclonal antibody gel electrophoresis figure being prepared of 8 plants of stable hybridoma positive cells；
Fig. 3 is the titration result figure for the high-affinity antibody that embodiment 1 is prepared；
Fig. 4~7 are the subtype identification result for the high-affinity antibody that embodiment 1 is prepared；
Fig. 8 is the result of the high-affinity antibody that embodiment 1 is prepared and the measurement of common affinity of antibody；
Fig. 9 is the result that high-affinity antibody and common antibody are used for the test of cellular immunity groupization；
Figure 10 is the chemiluminescence standard for detecting CPP based on the Full-automatic chemiluminescence method of MPs using high-affinity antibody
Curve graph.
Specific embodiment
To facilitate the understanding of the present invention, it below with reference to embodiment to invention is more fully described, is given below
Presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to described herein
Embodiment.Purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.It should be understood that
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, for example (,) Sambrook et al., molecule gram
It is grand: condition described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989),
Or according to the normal condition proposed by manufacturer.Used various common agents, are commercial product in embodiment.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Used term is intended merely to describe specific reality in the description of the invention
Apply the purpose of example, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more relevant institutes
Any and all combinations of list of items.
A kind of preparation method for secreting anti-human and peptide element monoclonal antibody hybridoma of the invention, including following step
It is rapid: with people and peptide element mice immunized with antigen；To the plasmid of the mouse delivering expression cell factor after being immunized；It collects through plasmid delivery
Mouse splenocyte or lymph node cells, and the splenocyte or lymph node cells are hybridized with mouse tumor cell fusion
Oncocyte to get.
Optionally, the people and peptide element antigen are to contain amino acid sequence SEQ ID NO:1, SEQ ID NO:2 or SEQ
The polypeptide of ID NO:3.Preferably, people and peptide element antigen are people and the peptide element antigen that coupling has high molecular weight protein, the macromolecular
Albumen is at least one of KLH, OVA, BSA.It is highly preferred that the people and peptide element antigen are people and the peptide that C-terminal coupling has KLH
Plain antigen.
In wherein some embodiments, the cell factor includes mFlt3l, mGM-CSF and mCCL20.Preferably, table
Plasmid up to cell factor is pCAGGS.
Preferably, the plasmid to the mouse delivering expression cell factor after being immunized are as follows: building can be thin in eukaryon lactation
The plasmid of cellular expression cell factor passes through the plasmid of the mouse tail vein injection expression cell factor.Specifically, it designs and constructs
To the plasmid of mFlt3l, mGM-CSF and mCCL20, including pCAGGS-mFlt3L, pCAGGS- can be expressed in eukaryon mammalian cell
MGM-CSF and pCAGGS-mCCL20, three kinds of plasmids, being capable of the expression cell factors after by tail vein injection to Mice Body
MFlt3l, mGM-CSF and mCCL20 play immunoregulation effect.Preferably, the expression cell factor is delivered to the mouse after being immunized
Plasmid are as follows: pCAGGS-mFlt3L, pCAGGS-mGM-CSF and pCAGGS-mCCL20, every mouse each 9~11 μ g are more excellent
The mass ratio of selection of land, pCAGGS-mFlt3L, pCAGGS-mGM-CSF and pCAGGS-CCL20 is (0.5~1.5): (0.5~
1.5):1.In some other embodiment, to the plasmid of the mouse delivering expression cell factor after being immunized, base can also be passed through
Because the modes such as rifle, electroporation, subcutaneous injection, intracutaneous injection, intraperitoneal injection are realized, and best Delivery time, dosage and delivery side
Formula is related.
Specifically, immune mouse is repeatedly immune, and the 6th~8 day after being immunized every time carries out a cell factor delivering, often
The secondary immunization interval time is 13~15 days.It is when being immunized, antigen is isometric with Freund's complete adjuvant in some of embodiments
Mixing, after fully emulsified, initial immunity carries out back of mice subcutaneously more site injections according to the amount of every 90~110 μ g, is immunized
Carrying out within the 6th~8 day afterwards can be in the tail vein injection of the plasmid of the expression of eukaryon mammalian cell mFlt3l, mGM-CSF and mCCL20
(pCAGGS-mCCL20, pCAGGS-mFlt3L, pCAGGS-mGM-CSF: each 9~11 μ g/ are only)；It is carried out after another week second
Immune, antigen dosage is 45~55 μ g, cannots be used up full adjuvant substitution Freund's complete adjuvant.Hereafter, the latter Zhou Jinhang of each subcutaneous inoculation
Cell factor delivering.Preferably, it is immunized and the process of cell factor delivering carries out altogether three times.Optionally, exempt from for the second time
After epidemic disease, third time are immune antibody titer measurement can be carried out to mice serum.Preferably, go mouse liver or lymphocyte with it is small
When rat bone marrow tumour cell fusion is prepared before hybridoma 3 days, abdominal cavity booster immunization can be carried out.
In some other embodiment, to the plasmid of the mouse delivering expression cell factor after being immunized, it can also use
3 ligand of mouse tyrosine kinase receptor (the Mouse fms-like tyrosinekinase of direct tail vein injection recombination
3ligand, mFlt3l), mouse granulocyte-macrophage colony stimutaing factor (Mouse granulocyte-macrophage
Colony-stimulating factor, mGM-CSF) and 20 (Mouse CC subtype of mouse CC hypotype chemotactic factor (CF)
Chemokine ligand 20, mCCL20).
On the basis of mouse is immunized in above-mentioned immunization strategy, immune mouse spleen and/or lymph node are taken, with mouse
Myeloma cell SP2/0 fusion, obtains hybridoma.Specifically, fused cell is trained in the Selective agar medium containing HAT
It supports, cell culture supernatant antibody titer is detected after 7~10d, screen positive hybridoma cell.Further, using limited dilute
Interpretation of the law is subcloned through 3~4 times, until the cloned culture supernatant antibody positive that individual cells are formed, measures supernatant potency, obtain
Obtain the monoclonal cell strain of stably excreting antibody.The monoclonal cell strain of the stably excreting antibody have can stably excreting it is anti-human
With peptide element monoclonal antibody, and antibody affinity with higher has good antigen rich when for detecting people and peptide element
Collection ability can effectively improve detection sensitivity.
The present invention also provides a kind of anti-human and peptide element monoclonal antibody preparation methods, are resisted by stably excreting as described above
The monoclonal cell strain of body is secreted to obtain.Wherein, further comprise expanding the hybridoma to cultivate, induce legal system in vivo
The step of standby ascites.Preferably, by the ascites being prepared centrifugation, the list for taking supernatant, purifying plain to get the anti-human and peptide of purifying
Clonal antibody.Specifically, described to induce method preparation ascites in vivo are as follows: with every 4.5 × 105A~5.5 × 105A cell abdominal cavity
Mouse is injected, collects ascites after 7~10d；The centrifugation are as follows: 9000r/min~11000r/min is centrifuged 8~12min.Further
Ground, monoclonal antibody purification are the continuous double dilution mouse ascites supernatant of PBS (pH 7.4) using 0.01mol/L, use grape
Pneumococcal proteins A affinity chromatography (is operated) monoclonal antibody purification by reagent specification.After the monoclonal antibody purified, also
Spectrophotometry instrument method can be selected and measure protein concentration after purification, be convenient for subsequent storage and application.
The anti-human preparation method with peptide element monoclonal antibody of 1 high-affinity of embodiment
1.1 Peptide systhesis
Synthesis and peptide element antigen polypeptide (cppF, N-cpp, C-cpp) are simultaneously coupled KLH and prepare comlete antigen, obtain KLH-cppF,
KLH-N-cpp,KLH-C-cpp.Amino acid sequence therein are as follows:
CppF:ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY (SEQ ID NO:1),
N-cpp:CATQLDGPAGALLLRLV (SEQ ID NO:2),
C-cpp:CLAGAPEPFEPAQPDAY (SEQ ID NO:3),
That is the comlete antigen that coupling KLH is obtained are as follows:
KLH-cppF (KLH-C-ATQLDGPAGALLLRLVQLAGAPEPFEPAQPDAY),
KLH-N-cpp (KLH-C-ATQLDGPAGALLLRLV),
KLH-C-cpp(KLH-C-LAGAPEPFEPAQPDAY)；
It Peptide systhesis and couples using conventional method, gill biochemistry Shanghai Co., Ltd is entrusted to complete.High performance liquid chromatography
(HPLC) method measures purity, equal > 95%.
1.2 plasmid construction
Using pCAGGS carrier, building can be in the matter of eukaryon mammalian cell expression people and peptide element and immunity regulatin remedy molecule respectively
Grain.Immune modulatory molecules are respectively as follows: 3 ligand of mouse tyrosine kinase receptor (mouse Fms-like tyrosinekinase
3ligand, mFlt3l), mouse granulocyte-macrophage colony stimutaing factor (mouse granulocyte-macrophage
Colony-stimulating factor, mGM-CSF), 20 (mouse CC subtype of mouse CC hypotype chemotactic factor (CF)
Chemokine ligand 20, mCCL20).It can divide in the plasmid that eukaryon mammalian cell expresses Flt3l, mGM-CSF and CCL20
Not are as follows: 1. pCAGGS-mFlt3L；②pCAGGS-mGM-CSF；③pCAGGS-CCL20.Plasmid commission general strong biological (Wuhan) is raw
The preparation of object Science and Technology Ltd..
1.3 experimental animals and cell line
8~12 week old female BAl BIcs/c mouse is purchased from Nanjing Medical University's Experimental Animal Center.SP2/0 cell line is Guangzhou
Ten thousand, which inspire confidence in biotechnology joint-stock company, freezes, and Human cervical cancer cell lines are purchased from China typical culture collection center.
1.4 reagent
Fu Shi is helped completely, freund 's incomplete adjuvant, (H- hypoxanthine, A- aminopterin-induced syndrome, T- thymus gland are phonetic by selection system HAT
Pyridine), source of people and peptide element, horseradish peroxidase (HRP) label sheep anti-mouse igg, tetramethyl benzidine (TMB) and IgG subclass
It identifies Isotype specific kit (Sigma company)；Staphylococcal protein A (Amersham company)；Immunohistochemistry is used
SABC-AP immunohistochemical kit (Bioengineering Research Institute is built up in Nanjing)；Positive control people and peptide element monoclonal antibody 4081/
4082/4084/4086 (Medix company).
1.5 experimental method
1.5.1 immunization strategy
Antigen KLH-cppF is mixed into (in other embodiments, antigen with Freund's complete adjuvant in equal volume in the present embodiment
It includes the people of KLH-C-cpp sequence and the sequence of peptide element C-terminal that KLH-C-cpp or other, which can also be selected), after fully emulsified,
Back of mice subcutaneously more site injections are carried out according to the amount of every 100 μ g, are initial immunity；Cell factor is carried out after a week to pass
It send: by plasmid (pCAGGS-mCCL20, pCAGGS-mFlt3L, pCAGGS-mGM- of the tail vein injection expression cell factor
CSF: each 10 μ g/ only)；It carries out second after another week to be immunized, antigen dosage is 50 μ g, cannots be used up full adjuvant substitution assistant completely
Agent.Hereafter, cell factor delivering of the latter Zhou Jinhang of each subcutaneous inoculation.It carries out 3 times altogether.It is imitated after two exempt from, three exempt from
Valence measurement.Merge first 3 days progress abdominal cavity booster immunizations.Control group 1 is delivered without cell factor, and other conditions are consistent.It is right
According to group 2 after the initial immunity of subcutaneous loci injection, pass through the plasmid of the tail vein injection expression cell factor after a week:
PCAGGS-mFlt3L and pCAGGS-mGM-CSF does not inject pCAGGS-mCCL20 that is, compared with experimental group.
1.5.2 cell fusion, screening and clonal cell line
It is sterile to take immune mouse spleen, lymph node, progress is merged with murine myeloma cell SP2/0 using cell fusion apparatus
Fusion.Fused cell is cultivated in the Selective agar medium containing HAT, with indirect elisa method detection cell training after 7~10d
Object supernatant antibody titer is supported, positive hybridoma cell is screened.It is subcloned using limiting dilution assay through 3~4 times, until individual cells
The cloned culture supernatant antibody positive of formation measures supernatant potency with indirect elisa method, obtains the list of stably excreting antibody
Clonal cell line.Expand culture, dispenses postposition liquid nitrogen cryopreservation.
1.5.3 it merges positive rate and stablizes strain number and calculate
Hybridoma hole count × 100% after positive rate=positive hole count/selection culture
Wherein, stablizing strain number is the cell strain number that finally subclone culture obtains in Single cell fusion.
1.5.4 the preparation of anti-human and peptide element monoclonal antibody
The hybridoma cell strain of acquisition is expanded into culture, induces method preparation ascites in vivo, i.e., with every 5 × 105A cell
Intraperitoneal injection of mice, ascites is collected after 7~10d, and 12000r/min is centrifuged 10min, stays supernatant.Utilize the PBS of 0.01mol/L
(pH 7.4) continuous double dilution mouse ascites supernatant (is operated) with staphylococcal protein A affinity chromatography by reagent specification
Monoclonal antibody purification, Nanodrop2000 spectrophotometry instrument method measure protein concentration after purification, dispense, -70
DEG C save.The technical solution of above-mentioned preparation method and subsequent characterizations is as shown in Figure 1.
2 immune effect of embodiment and antibody titer measurement
Cell factor group: immunization strategy described in embodiment 1: i.e. cell of the latter Zhou Jinhang of each subcutaneous inoculation because
Sub- tail vein injection.
Control group 1: compared with Example 1, without cell factor tail vein injection, remaining step side after being immunized every time
Method is same as Example 1.
Control group 2: compared with Example 1, after the initial immunity of subcutaneous loci injection, pass through tail vein injection after a week
The plasmid of the expression cell factor: pCAGGS-mFlt3L and pCAGGS-mGM-CSF does not inject pCAGGS- that is, compared with experimental group
mCCL20。
Above-mentioned two groups of immune effect comparing result is as shown in table 1.
The comparison of 1 immune effect of table
As shown in Table 1, cell factor group, that is, method described in embodiment 1 is immunized, and immune effect is obviously preferable.Its
In, in cell factor group, 8 plants of stable hybridoma positive cells have been prepared, after it is expanded culture respectively, have induced in vivo
Method prepares ascites, i.e., with every 5 × 105A cell intraperitoneal injection of mice collects ascites, 12000r/min centrifugation after 7~10d
10min stays supernatant.Monoclonal antibody purification (is operated) by reagent specification using staphylococcal protein A affinity chromatography,
Nanodrop2000 spectrophotometry instrument method measures protein concentration after purification.Gel electrophoresis therapy determining antibody purification
Purity.As a result as shown in Figure 2.Wherein, Lane1 Marker, Lane2~Lane9 (9#~16#) are respectively cell factor group institute
Obtain the corresponding monoclonal antibody being prepared of 8 plants of stable hybridoma positive cells.As it can be seen that method preparation described in embodiment 1
The purity of obtained antibody is 90% or more.And the positive hybrid rate of control group 1 is very low, only obtains 1 plant of stable cell strain, and
Control group 2 does not screen the cell strain for stablizing expression.
Coating is added in (1:1000,1:10000,1:100000,1:1000000) after 10 times of gradient dilutions of antibody after purification
In the ELISA reaction plate micropore of people and peptide element, after 37 DEG C of water-bath 30min, secondary antibody (1: 20000 dilution), 37 DEG C of water-baths are added
After 30min, 50 μ L 2mol/L H are added in the colour developing of TMB developing solution2SO4Terminate reaction, microplate reader (Thermo fisher company)
Measure absorbance value at 450nm.As a result as shown in Figure 3.Wherein, OD 450 >=0.3 is considered as the positive.As it can be seen that 9#~16# antibody is pressed
After 1:1000,1:10000,1:100000,1:1000000 dilution, OD 450 be all larger than 0.3,2# antibody (control group 1 gained),
The potency of 9#~16# (obtained by cell factor group) antibody is up to 10-6, with positive control people and peptide element monoclonal antibody
The potency of Medix4084 and Medix4086 is suitable.
3 monoclonal antibody subgroup identification of embodiment
Packet is added in (1:1000,1:10000,1:100000,1:1000000) after 10 times of gradient dilutions of antibody after purification
By in the ELISA reaction plate micropore of people and peptide element, after 37 DEG C of water-bath 30min, it is separately added into IgG subgroup identification Isotype
Subgroup identification secondary antibody (including IgA, IgG1, IgG2a, IgG2b, IgG3, IgM) in specific kit (Sigma company),
After 1: 1000 dilution, after 37 DEG C of water-bath 30min, 50 μ L 2mol/L H are added in the colour developing of TMB developing solution2SO4Terminate reaction, enzyme
Absorbance value at instrument (Thermo fisher company) measurement 450nm is marked, and Subclass of antibody is judged according to result.As a result as Fig. 4~
Shown in 7, the subtype identification result that Fig. 4 is the subtype identification result of 9# antibody, Fig. 5 is 13# antibody, the hypotype that Fig. 6 is 14# antibody
Qualification result, the subtype identification result that Fig. 7 is 15# antibody.Complex chart 4~7 can determine whether the anti-human and peptide that the present invention is prepared
The hypotype of plain monoclonal antibody is IgG1 type.
The measurement of 4 affinity of antibody of embodiment
It is measured using biomembrane interference technique.Avidin sensor solidifies antigen molecule, and antibody gradient dilution passes through
Association and dissociation rate characterizes affinity of antibody size.As a result as shown in Figure 8.Wherein Fig. 8 A is positive control monoclonal antibody
The affinity measurement result of MEDIX 4084, obtains affinity 1.693nM, and Fig. 8 B is the height that the embodiment of the present invention 1 is prepared
The anti-human affinity measurement result with peptide element antibody 9# of affinity, affinity 0.14nM, Fig. 8 C are common in control group 1
The affinity determination of antibody (2#) (being delivered in immunization method without cell factor, remaining step method is same as Example 1)
As a result, its affinity is 19.3nM.As it can be seen that the anti-human and peptide element affinity of antibody that the embodiment of the present invention 1 is prepared is compared with market
Upper common 4084 affinity of control antibodies MEDIX improves 10 times or more, relative to not carrying out the immune of cell factor delivering
The common affinity of antibody that mouse is prepared has more obvious raising.
The test of 5 cellular immunity groupization of embodiment
It is (immune using human cervical carcinoma cell (hela) and high-affinity antibody (embodiment 1 is prepared), common antibody
Delivered in method without cell factor, remaining step method is same as Example 1) immunohistochemical assay is carried out, as a result such as Fig. 9
It is shown.Wherein, Fig. 9 A, which is the result of high affinity antibody (9#), 9B is negative control, 9C is that control group 1 is prepared common resists
Body (2#) ImmunohistochemistryResults Results, the ImmunohistochemistryResults Results that 9D is control antibodies (MEDIX 4084).As it can be seen that the present invention is prepared
Antibody can be in conjunction with natural and peptide element.For high affinity antibody due to its affinity outstanding, the more common antibody of background colour developing is obvious
It reduces.
The anti-human application with peptide element monoclonal antibody of 6 high-affinity of embodiment
1.CPP antibody is coated with MPs
High-affinity CPP monoclonal antibody obtained by cell factor skeptophylaxis is total by N-terminal amino and magnetic particle (MPs)
Valence coupling, as capture antibody.Firstly, the MPs of 20mg/mL is placed in 2.0mL Ep pipe.MPs five is washed with combination buffer
It is secondary.In washing process, pipe is placed on magnetic force inspissator and removes supernatant.Then, MPs is resuspended in 2mL and combines buffering
In liquid, antibody-solutions are added to above-mentioned suspension, shaken cultivation is stayed overnight at 37 DEG C.After incubation, it is dense that Ep pipe is placed in magnetic force
With by they and supernatant separation in contracting device.The residual binding sites on MPs are closed with 3% bovine serum albumin(BSA) (BSA), 37
DEG C incubate and gentle agitation 2 hours.After washing 5 times, coated immunomagnetic beads (mAb-MPs) are dispersed in 2mL buffer simultaneously
It is saved at 4 DEG C, it is spare.
The preparation of 2.AP label CPP antibody
The antibody in another site alkaline phosphatase (AP) marker recognition CPP.Firstly, AP and anti-CPP antibody are hanged respectively
It floats in ultrapure water and is diluted to 4 and 8mg/mL.The 4mg/mL AP solution of 250 μ L is transferred in 1.5mL Ep pipe and with 250
The anti-CPP antibody-solutions mixing of μ L 8mg/mL.Secondly, the 0.1mol L that 0.5mL contains 1% glutaraldehyde is added into solution-1Phosphate
Buffer (pH7.4).By gained mixture, slight oscillatory is incubated 4 hours in the dark at 37 DEG C.Third step, into mixture
0.1mL 1molL is added-1Monoethanolamine solution, then incubated under agitation 2 hours at room temperature.Mixture is used into PBS at 4 DEG C
Solution dialysed overnight.After dialysis, enzyme mark CPP antibody is transferred in Ep pipe, and is mixed with isometric glycerol and 1%BSA.Most
Afterwards, enzyme mark CPP antibody (AP-mAbs) is stored in -20 DEG C, it is spare.
3. the Full-automatic chemiluminescence method based on MPs detects CPP
The dilution ratio of 1:100 and 1:50 is selected to be used for AP-mAbs and mAb-MPs, incubation time is the condition of 30min,
Measure a series of standard CPP solution (S0~S9,0~1250pmol/L) of concentration.The calibration curve of CPP standard items is constructed, such as
Shown in Figure 10.By adding twice of standard deviation to determine minimum detection limit (LOD) average value in 10 holes S0.The inspection of standard curve
Survey range is 1.2~1250pmol/L, and detection limit (LOD) is 6.25pmol/L, related coefficient 0.9993.As it can be seen that using this
The high-affinity antibody that inventive embodiments 1 are prepared detects CPP, inspection applied to the Full-automatic chemiluminescence method based on MPs
Limit is surveyed down to 6.25pmol/L, when detection a length of 30min.Significantly shorten detection time, while ensure that the spirit of detection
Sensitivity.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to the above reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Guangzhou Wondfo Biotech. Co., Ltd.
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1 5 10 15
Gln Leu Ala Gly Ala Pro Glu Pro Phe Glu Pro Ala Gln Pro Asp Ala
20 25 30
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Cys Ala Thr Gln Leu Asp Gly Pro Ala Gly Ala Leu Leu Leu Arg Leu
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Cys Leu Ala Gly Ala Pro Glu Pro Phe Glu Pro Ala Gln Pro Asp Ala
1 5 10 15
Tyr
Claims (10)
1. a kind of preparation method for secreting anti-human and peptide element monoclonal antibody hybridoma, which is characterized in that including following
Step:
With people and peptide element mice immunized with antigen；
To the plasmid of the mouse delivering expression cell factor after being immunized；
Collect the splenocyte or lymph node cells of the mouse being immunized through plasmid delivery, and by the splenocyte or lymph node cells with
Murine myeloma cell merge to obtain hybridoma to get.
2. preparation method according to claim 1, which is characterized in that the people and peptide element antigen are to contain amino acid sequence
The polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.
3. preparation method according to claim 1, which is characterized in that the people and peptide element antigen are that coupling has macromolecular egg
White people and peptide element antigen, the high molecular weight protein are at least one of KLH, OVA, BSA.
4. preparation method according to claim 1, which is characterized in that the cell factor include mFlt3l, mGM-CSF and
mCCL20。
5. preparation method according to claim 1, which is characterized in that the carrier of the plasmid is pCAGGS.
6. described in any item preparation methods according to claim 1~5, which is characterized in that the immune mouse is multi-time no
Epidemic disease carried out once to the plasmid of the mouse delivering expression cell factor, each immunization interval time for the 6th~8 day after being immunized every time
It is 13~15 days；And/or
The immune mouse includes:
Initial immunity: antigen is mixed in equal volume with Freund's complete adjuvant, after fully emulsified, is carried out according to every 90~110 μ g
The subcutaneous more site injections of back of mice；
Second immune: antigen is mixed in equal volume with freund 's incomplete adjuvant, after fully emulsified, according to every 45~55 μ g into
The subcutaneous more site injections of row back of mice；
To the plasmid of the mouse delivering expression cell factor after being wherein immunized every time.
7. a kind of secrete anti-human and peptide element monoclonal antibody hybridoma, which is characterized in that any by Claims 1 to 5
Preparation method described in is prepared.
8. a kind of anti-human and peptide element monoclonal antibody preparation method, which comprises the following steps:
Take the described in any item secretions of claim 1~6 anti-human and the preparation method of the hybridoma of peptide element monoclonal antibody
The hybridoma being prepared, culture, secrete monoclonal antibody to get.
9. a kind of anti-human and peptide element monoclonal antibody, which is characterized in that secreted by hybridoma as claimed in claim 7
It arrives；Or it is prepared by according to any one of claims 8 anti-human and peptide element monoclonal antibody preparation method.
10. anti-human and peptide element monoclonal antibody as claimed in claim 9 answering in the kit of preparation detection people and peptide element
With.
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| CN110195079A (en) * | 2019-05-27 | 2019-09-03 | 广州万孚生物技术股份有限公司 | Prepare plasmid, antibody, preparation method and the screening technique of AMH antibody, kit and detection method |
| CN114184603A (en) * | 2021-11-11 | 2022-03-15 | 宁波海壹生物科技有限公司 | Kit for determining copeptin by magnetic particle chemiluminescence method |
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