CN106674347A - Novel ear chalone antibody - Google Patents
Novel ear chalone antibody Download PDFInfo
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- CN106674347A CN106674347A CN201610680614.8A CN201610680614A CN106674347A CN 106674347 A CN106674347 A CN 106674347A CN 201610680614 A CN201610680614 A CN 201610680614A CN 106674347 A CN106674347 A CN 106674347A
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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
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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Abstract
The invention provides a novel ear chalone antibody or a functional fragment of the novel ear chalone antibody. The novel ear chalone antibody has relatively good affinity and high sensitivity. The invention further provides a method for detecting ear chalone and a coupling medicine of the ear chalone by using the novel ear chalone antibody or the functional fragment of the novel ear chalone antibody.
Description
Technical field
The present invention relates to a kind of Novel ear chalone antibody or its functional fragment, it includes heavy chain and light chain, and the present invention is also
It is related to the kit with the Antibody preparation, and the use during the antibody is used for quantitative determination ear chalone and its coupling drug
On the way.
Technical background
Ear chalone (dolastatin) is also referred to as aplysiatoxin, is without being extracted one in shell mollusk truncation sea hare from ocean
The linear depsipeptides toxin protein of class, there is various derivatives, it is common as：Dolastatin-10、Dolastatin-15、TZT-
1027th, MMAE (Monomethyl Auristatin E), MMAF (Monomethyl Auristatin F), Cemadotin, he
(Bai RL, Pettit GR, Hamel E.Dolastatin 10, a powerful cytostaticpeptide such as Xi Duoding
derived from a marine animal.Inhibition of tubulinpolymerization mediated
through the vinca alkaloid bindingdomain[J].Biochem harmacol,1990,39(12)：
1941-1949.).Ear chalone plays powerful cytotoxic activity by acting on tubulin, and various aplysiatoxin derivatives are
It is exploited for drug therapy (Bai RL, Pettit GR, Hamel E.Binding of dolastatin 10
totubulin at a distinct site for peptide anti-mitotic agents near
theexchangeable nucleotide and vinca alkaloid sites[J].J BiolChem,1990,265
(28)：17141-17149.).
Because ear chalone has very strong cytotoxicity, direct drug injection security is relatively low, and " therapeutic window " is narrower, therefore main
One of strategy be ear chalone targeting medication treatment.There is targeting work(by being prepared into the preparation with target function or connection
The molecule of energy, by the privileged site of ear chalone carrier band to organism drug effect is played.Modal targeting administration form is antibody idol
Connection medicine (Antibody Drug Conjugations, ADC), the ADC medicines development clinic that multiple ear chalones are had at present is ground
Study carefully, wherein having 1 medicine (Adcetris, SGN-35) approved listing (Deng CC, Pan BQ, Owen A.Brentuximab
Vedotin [J] .Clin Cancer Res, 2013,19 (1)：22-27.).
In drug effect, medicine generation, the poison generation research for carrying out ear chalone correlation targeted drug, quantitative pharmacological research is important grinding
Study carefully one of aspect.The quantitative detecting method for setting up ear chalone coupling drug is to support the studies above
Key, these quantitative detecting methods include liquid phase-mass spectrography (LC-MS, LC-MS/MS), ELISA
(ELISA) etc..Wherein ELISA (ELISA) is first known antigen or antibody to be fixed on into surface of solid phase carriers, to be measured
Thing generally corresponds to antibody or antigen, specifically binds with the antigen or antibody combined in surface of solid phase carriers, determinand
Enzyme is connected with, (depth of color and the concentration of test substance are in certain proportion to the color reaction occurred by enzyme-to-substrate after reaction
Relation) carry out quantitative analysis.
For one of detection ear chalone and its coupling drug, main policies of ELISA quantitative determinations are exactly to utilize antiradiation drug
Antibody is captured or detected.During examinations, the acquisition of the high anti-drug antibodies of high specificity, affinity is to close
Key, and most small-molecule drug only has reactionogenicity and non-immunogenicity, this just for ELISA quantitative determination ear chalones and its
Coupling drug brings insoluble puzzlement.
The invention provides the technical scheme for solving the above problems, is prepared for specifically binding the antibody of ear chalone, specifically
Property it is strong, affinity is high, and sensitivity is high, can be used for the quantitative determination of ELISA.
The content of the invention
In order to solve the above problems, the invention provides a kind of new ear chalone antibody or its functional fragment, it is
By the way that ear chalone is coupled from different carrier proteins, artificial antigen is formed, carry out animal immune, then Jing screens what is obtained
With the antibody that ear chalone drug molecule has high-affinity.Its coupling strategies is even mainly by after the group activation of drug molecule
The different carrier protein of connection prepares artificial antigen, and one kind is used as immunizing antigen, and another kind is used to screen antigen, to ensure to produce
Antibody be for ear chalone molecule rather than carrier protein.Present invention also offers using the new ear chalone antibody or its work(
The method of energy property fragment quantitative determination ear chalone or its conjugate, and using the new ear chalone antibody or its functional fragment
For preparing the kit of the free ear chalone of detection or ear chalone coupling drug.
Specifically technical scheme is the present invention：
On the one hand, the invention provides a kind of new ear chalone antibody or its functional fragment, it includes heavy chain and light
Chain, particularly：I () described heavy chain includes at least three CDR regions, the CDR region has such as SEQ ID NO:1、2、3、7、8、9、
13rd, the amino acid sequence shown in 14,15；And/or (ii) described light chain includes at least three CDR regions, the CDR region has such as
SEQ ID NO:4th, the amino acid sequence shown in 5,6,10,11,12,16,17.
Specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, (i) described
Heavy chain includes three CDR regions, and the CDR region has such as SEQ ID NO:1st, the amino acid sequence shown in 2,3；And/or (ii) institute
Light chain is stated comprising three CDR regions, the CDR region has such as SEQ ID NO:4th, the amino acid sequence shown in 5,6.
Specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, (i) described
Heavy chain includes three CDR regions, and the CDR region has such as SEQ ID NO:7th, the amino acid sequence shown in 8,9；And/or (ii) institute
Light chain is stated comprising three CDR regions, the CDR region has such as SEQ ID NO:10th, the amino acid sequence shown in 11,12.
Specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, particularly：
I () described heavy chain includes three CDR regions, the CDR region has such as SEQ ID NO:13rd, the amino acid sequence shown in 14,15；
And/or (ii) described light chain includes three CDR regions, the CDR region has such as SEQ ID NO:16th, the amino acid sequence shown in 5,17
Row.
More specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, (i) institute
Heavy chain is stated comprising three CDR regions, CDR1 has such as SEQ ID NO:Amino acid sequence shown in 1, CDR2 has such as SEQ ID
NO:Amino acid sequence shown in 2, CDR3 has such as SEQ ID NO:Amino acid sequence shown in 3；(ii) light chain includes three
Individual CDR region, CDR1 has such as SEQ ID NO:Amino acid sequence shown in 4, CDR2 has such as SEQ ID NO:Amino shown in 5
Acid sequence, CDR3 has such as SEQ ID NO:Amino acid sequence shown in 6.
More specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, (i) institute
Heavy chain is stated comprising three CDR regions, CDR1 has such as SEQ ID NO:Amino acid sequence shown in 7, CDR2 has such as SEQ ID
NO:Amino acid sequence shown in 8, CDR3 has such as SEQ ID NO:Amino acid sequence shown in 9；(ii) light chain includes three
Individual CDR region, CDR1 has such as SEQ ID NO:Amino acid sequence shown in 10, CDR2 has such as SEQ ID NO:Ammonia shown in 11
Base acid sequence, CDR3 has such as SEQ ID NO:Amino acid sequence shown in 12.
More specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, (i) institute
Heavy chain is stated comprising three CDR regions, CDR1 has such as SEQ ID NO:Amino acid sequence shown in 13, CDR2 has such as SEQ ID
NO:Amino acid sequence shown in 14, CDR3 has such as SEQ ID NO:Amino acid sequence shown in 15；(ii) light chain is included
Three CDR regions, CDR1 has such as SEQ ID NO:Amino acid sequence shown in 16, CDR2 has such as SEQ ID NO:Shown in 5
Amino acid sequence, CDR3 has such as SEQ ID NO:Amino acid sequence shown in 17.
Further, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, its feature
It is：I () is comprising such as SEQ ID NO:18th, the weight chain variable district of the amino acid sequence shown in 20,22；(ii) comprising such as SEQ ID
NO:19th, the light chain variable district of the amino acid sequence shown in 21,23.
Specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, and its feature exists
In：I () is comprising such as SEQ ID NO:The weight chain variable district of the amino acid sequence shown in 18；(ii) comprising such as SEQ ID NO:19 institutes
The light chain variable district of the amino acid sequence for showing.
Specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, and its feature exists
In：I () is comprising such as SEQ ID NO:The weight chain variable district of the amino acid sequence shown in 20；(ii) comprising such as SEQ ID NO:21 institutes
The light chain variable district of the amino acid sequence for showing.
Specifically, described new ear chalone antibody or its functional fragment, it includes heavy chain and light chain, particularly：
I () is comprising such as SEQ ID NO:The weight chain variable district of the amino acid sequence shown in 22；(ii) comprising such as SEQ ID NO:Shown in 23
Amino acid sequence light chain variable district.
Further, described new ear chalone antibody or its functional fragment, wherein described antibody is monoclonal
Antibody.
Further, described new ear chalone antibody or its functional fragment, wherein described functional fragment is
Refer to Fv, scFv, Fab, F (ab ') 2, Fab ', scFv-Fc fragments or Fv-PEG, scFv-PEG, Fab-PEG.
Further, described new ear chalone antibody or its functional fragment, wherein described ear chalone is
Dolastatin-10、Dolastatin-15、TZT-1027、MMAE(Monomethyl Auristatin E)、MMAF
(Monomethyl Auristatin F), Cemadotin, his western many fourths etc., it is therefore preferable to MMAE, MMAF.
Further, the antibody of affiliated Novel ear chalone and MMAE affinity are 10-8~10-10Between.
On the other hand, the invention provides arbitrary ear chalone antibody described above or its functional fragment can be encoded
Detached polynucleotides.
On the other hand, the invention provides expression vector, comprising described detached polynucleotides.
On the other hand, the invention provides host cell, comprising described expression vector.
On the other hand, present invention also offers the antibody containing described new ear chalone antibody or its functional fragment
Coupling drug.
On the other hand, present invention also offers the medicine containing described new ear chalone antibody or its functional fragment
Thing, detection reagent or kit.
On the other hand, present invention also offers the new ear chalone antibody or its functional fragment are in detection ear chalone
Or the application in its coupling drug, described application refers to application of the ear chalone antibody in ELISA method.
Description of the drawings：
Fig. 1 shows the specificity of ear chalone monoclonal antibody and sensitivity, and anti-ear chalone monoclonal antibody is added while conjugated antigen
Ear chalone evaluates its specificity and sensitivity as competition binding agent.Wherein A is control antibodies (control), its with antigen without
Specific bond, adds after variable concentrations ear chalone without obvious Competition, and B, C, D are respectively the competition of 1G1,2F10,4C3 monoclonal antibody
With reference to effect.
Fig. 2 shows that the Inhibition ELISA that ear chalone monoclonal antibody is set up determines the calibration curve of rat blood serum middle ear chalone.
Fig. 3 shows that the ELISA method that ear chalone monoclonal antibody is set up determines monkey serum middle ear chalone conjugate quantitative measurement standard
Curve.
Specific embodiment：
Definition：
Unless otherwise defined, the phase that there are all scientific and technical terminologies used herein those of ordinary skill in the art to be understood
Same implication.Definition and term with regard to this area, professional specifically refers to Current Protocols in
Molecular Biology(Ausubel).The abbreviation of amino acid residue is used in this area to refer to 20 conventional L- amino
The letter of standard 3 of one of acid and/or 1 alphanumeric codes.
Although the digital scope and parameter approximation shown in the broad scope of the present invention, shown in specific embodiment
Numerical value is accurately recorded as far as possible.However, any numerical value natively necessarily contains certain error, it is by each of which
Measurement present in caused by standard deviation.In addition, all ranges disclosed herein is interpreted as covering wherein include any
With all subranges.The scope of " 1 to 10 " of such as record is considered as including between minimum of a value 1 and maximum 10 (comprising end points)
Any and all subrange；That is, it is all with minimum of a value 1 or it is bigger starting subrange, such as 1 to 6.1, and with
Maximum 10 or the subrange of less termination, such as 5.5 to 10.In addition, any bibliography referred to as " being expressly incorporated herein " should be managed
Solution is to be integrally incorporated with it.
It is further noted that as used in this description, singulative includes the plural form of its referent, unless
Understand and be clearly limited to a referent.Term "or" can with term "and/or" used interchangeably, unless the context otherwise clearly
Chu Zhiming.
" CDR region " used herein or " CDR " refer to the heavy chain of immunoglobulin (Ig) and the hypervariable region of light chain, such as Kabat et
Al. (Kabat et al., Sequences of proteins of immunological interest, 5th are defined
Ed., U.S.Department of Health and Human Services, NIH, 1991, and version later).Have three
Individual heavy chain CDR and three light chain CDR.According to circumstances, terms used herein CDR or CDRs be in order to indicate one of these regions,
Either these regions several or or even all, the region comprising by antibody to antigen or the affinity of its identification epi-position
And it is responsible for the Most amino-acids residue for combining.
For purposes of the invention, two kinds of " uniformity ", " homogeneity " or " similitude " between nucleic acid or amino acid sequence are
Refer to, identical nucleotides or same amino acid are residual between two sequences obtained afterwards in optimal comparison (optimum is compared), to be compared
The percentage of base, the percentage is the difference random distribution between pure statistical and two kinds of sequences and covers its total length.Two
Plant the sequence between nucleic acid or amino acid sequence to compare typically after in an optimal manner matching them, by comparing this
A little sequences and carry out, it is described to compare and implement by section or by " comparing window ".Except can be used in addition to manual implementation
The optimum comparison of comparative sequences, additionally it is possible to by Smith and Waterman (1981) [Ad.App.Math.2:482] local is same
Source property algorithm, by (1970) [J.MoI.Biol.48 of Neddleman and Wunsch:443] local homology algorithm, pass through
(1988) [Proc.Natl.Acad.Sci.USA85 of Pearson and Lipman:2444) search for similarity method, by using
The computer software of these algorithms implements (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics
Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or pass through
BLAST N or BLAST P comparison softwares).
Term " antibody " means complete antibody and its any Fab (" antigen-binding portion thereof ") or single-stranded.It is " complete
Long antibody " means the albumen comprising at least two weight (H) chains and two light (L) chains interconnected by disulfide bond.Every heavy chain
Comprising a weight chain variable district (being abbreviated as VH) and a CH.The CH include three domains (domain), CH1,
CH2 and CH3.Every light chain includes a light chain variable district (being abbreviated as VL) and a constant region of light chain.The constant region of light chain includes one
Individual domain, CL.VH and VL regions can also be sub-divided into having high variable multiple areas, be referred to as complementary determining region (CDR), its
Between be scattered with the more conservative multiple regions for being referred to as framework region (FR).Each VH and VL is by three CDR and four FR structures
Into the arrangement from aminoterminal to c-terminus in the following order：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4.Heavy chain and light chain
These variable regions comprising binding domain with AI.The constant region of antibody can mediated immunity globulin and host group
Knit or the factor is combined, including the first composition (Clq) of immune various cells (such as effector cell) and classical complement system.
Chimeric or humanized antibody is also covered by antibody of the invention.
Term " monoclonal antibody " means the prepared product with single molecular antibody molecule.Monoclonal antibody cocktail
Thing shows the single binding specificity and compatibility for defined epitope.
Term as used herein " functional fragment " refers in particular to antibody fragment such as Fv, scFv (sc refers to single-stranded), Fab, F
(ab ') 2, Fab ', scFv-Fc fragments or double antibody (diabody) or by chemical modification or by mix liposome in
Any fragment of half life should be able to be increased, poly- (alkylidene) glycol such as polyethylene glycol (" poly- second is for example added in the chemical modification
It is diolation, PEGization ") (it is referred to as the polyethylene glycol of Fv-PEG, scFv-PEG, Fab-PEG, F (ab') 2-PEG or Fab'-PEG
Change fragment) (" PEG " is polyethylene glycol), the fragment has ear chalone binding activity.Preferably, the function fragment will be by it
The partial sequence of the weight of derived antibodies or light variable chains is constituted or comprising them, and the partial sequence be enough to retain and originate with it
Antibody identical binding specificity and sufficient affinity, for ear chalone E, preferably at least equal to its derived antibodies affinity
1/100, at least equal to 1/10 in more preferably mode.This function fragment will be comprising minimum 5 amino acid, and preferably it is originated
10,15,25,50 and 100 continuous amino acids of antibody sequence.
Generally, in order to prepare monoclonal antibody or its function fragment, the especially monoclonal antibody in mouse source or its functional sheet
Section, may be referred to technology (Harlow and Lane, the Antibodies being especially described in handbook " Antibodies ":A
Laboratory Manual,Cold Spring Harbor Laboratory,Cold Spring Harbor NY,pp.726,
1988) or with reference to preparing from hybridoma of describing of Kohler and Milstein technology (Nature, 256:495-497,
1975)。
Terms used herein " ear chalone ", is also called " aplysiatoxin peptide " and refers to and be isolated from a kind of marine organisms truncation sea
The polypeptide and its derivative of rabbit (Dollabella auricularia), it includes but is not limited to aplysiatoxin peptide 10
(Dolastatin-10, ear chalone E), aplysiatoxin peptide 15 (Dolastatin-15, ear chalone F).Ear chalone (aplysiatoxin
Peptide) it is mitotic inhibitor, it shows strong active anticancer, therefore by the candidate as cancer therapy drug.Researcher enters
One step finds and has synthesized the derivative of many aplysiatoxin peptides, such as ear chalone E (MMAE) and ear chalone F (MMAF), MMAE
(Monomethyl Auristatin E), MMAF (Monomethyl Auristatin F), TZT-1027, Cemadotin, he
Xi Duoding etc..
" coupling drug " in terms used herein " ear chalone and its coupling drug " refers to all of and ear chalone idol
The medicine that connection is generated, the most common are antibody coupling medicine (Antibody Drug Conjugations, ADC) form.
Following examples are used for proving and being explained further some of the invention preferred embodiment and aspect, but this is simultaneously
Do not limit the scope of the invention.
The preparation and sequence analysis of the anti-ear chalone mouse resource monoclonal antibody of embodiment 1
1) preparation of ear chalone artificial antigen
The activation of drug molecule is carried out using azo method or Maleimide method.Wherein azo method can refer to document
(Erlanger BF.The preparation of antigenic hapten-carrier conjugate:a survey
method in enzymology[M].New York:Academic Press.1980:85-103；) carry out：Take ear chalone
(MMAE/MMAF) 0.5% storing solution 1.5ml adjusts pH to pH2.5 in test tube with the hydrochloric acid of 1mol/L, separately takes natrium nitrosum
Solution 0.5ml is added dropwise over, and side edged shake, 4 DEG C of lucifuges are incubated 1 hour, adds 2~3 to drip sulfamic acid ammonia solution, removes trip
From nitrite ion.Finally measure stereometer calculation ear chalone concentration to be coupled with BSA.By 20:1 (azo anakmetomeres：
BSA ratio) adds the 2% BSA aqueous solution, adjusts pH to 7.5 with NaOH and is incubated 24 hours at 4 DEG C, subsequently uses
The PBS of 0.01mol/L, pH7.4 carry out 106 times of dialysis, and 4 DEG C of preservations, KLH, immunoglobulin (Ig) (IgG) is coupled with reference to above-mentioned side
Method；It is coupled using Maleimide and refers to document (Greg T.Hermanson.Bioconjugate Techniques (2nd
edition),Chapter19.Preparation of Hapten-carrier Immunogen
Conjugates.Elsevier Inc.Academic Press.2008:The connexon synthesis of ear chalone 746-782) is carried out, and
Reference literature (Hamblett, K.J., et al., Effects of drug loading on the antitumor
activity of a monoclonal antibody drug conjugate.Clin Cancer Res,2004.10(20):
P.7063-70. the coupling of carrier protein) is carried out, 1% BSA protein solutions are cooled to 4 DEG C, plus final concentration 25%DMSO, according to
Drug molecule and protein mole ratios example 20：1 adds MC-linker-MMAE/MMAF (0.01mol/L, DMSO dissolve), and room temperature is stirred
Reaction 1h is mixed, 106 times of process of dialysing.4 DEG C of preservations, KLH, IgG is coupled with reference to said method.
2) immunity and the preparation of hybridoma
Monoclonal antibody is prepared using the ear chalone-carrier protein of above-mentioned preparation as mice immunized with antigen.Immune response,
Hybridoma cell fusion and primary dcreening operation are all according to standard step (bibliography：WHO Technical Report Series,
No.822,1992 Annex 3) carry out.6 Balb/c mouse (purchased from Shanghai Slac Experimental Animal Co., Ltd.) are taken,
It is randomly divided into 2 groups, 3 per group.One group with ear chalone-BSA inoculations, another group carries out immunity with ear chalone-KLH.First
The immune μ L of immunogene 50 for taking 1mg/ml respectively, mouse neck hypodermic injection fully emulsified with the Freund's complete adjuvant of equivalent,
The 2nd injection was carried out after 2 weeks, with incomplete Freund's adjuvant (Difco Lab), amount of antigen is that 25-50 μ g/0.05ml/ are only little
Mouse, interval carries out the 3rd injection after 3 weeks, injection dosage takes blood in 10 days with the 2nd time after the 3rd injection.Use Enzyme-linked Immunosorbent Assay
Test (ELISA) detects the serum of mouse, detects that antigen is coupled different from the artificial of immunogenic carriers albumen using ear chalone
Antigen.The spleen of the maximum mouse of anti-ear chalone antibody titer in serum is taken out, then with myeloma cell P3X63Ag8
(ATCC CRL-1580) merges.Fused cell is diluted on 10 piece of 96 orifice plate, according to the knot with ear chalone antibody-ear chalone
Conjunction ability carries out primary dcreening operation with ELISA method.In typical ELISA experiments, with ear chalone-BSA/KLH, (0.2-1 (g/ml) is wrapped
By the orifice plates of Nunc Maxisorb 96, then it is incubated with the mice serum or doma supernatant (100 μ L) of gradient dilution.Mouse source
Anti- ear chalone antibody horseradish peroxidase goat F (ab')2Anti- mouse IgG Fc it is special two resist
(Invitrogen companies) is detected.
The supernatant of 400 hybridoma cell strains is screened with ELISA method, wherein 36 show strong ear suppression
Element-ECD adhesions.Ten strain of hybridoma for selecting ear chalone binding ability most strong, by sub- gram of limited dilution method screening
Grand hybridoma cell strain.By the subclone hybridoma cell strain culture that suspends, protein purification determines the knot of ear chalone with ELISA
Affinity is closed, with the Inhibition ELISA of ear chalone molecule its binding ability to ear chalone is further tested.Eventually through sequence
Analysis determines 3 strain of hybridoma systems：1G1,2F10,4C3, they have strong ear chalone binding ability, subsequently through
ELISA and test cell line are further analyzed to it.
3) sequence analysis of anti-ear chalone hybridoma cell clone
The sequence of the variable region of above-mentioned hybridoma cell clone heavy chain and light chain utilizes commercial reagents box SMARTTM RACE
CDNA Amplification Kit (Clontech companies) end of rapid amplifying 5 ' is sequenced and is obtained.Use RNApure
Tissue Kit (Beijing Kang Wei centuries bio tech ltd) extract total serum IgE from hybridoma, use
SMARTTMRACE cDNA Amplification Kit carry out the reverse transcription of total serum IgE, with total serum IgE as template, using in kit
Primer, add reverse transcriptase SMARTScribeTM Reverse Transcriptase, enter the step of provide according to kit
Row reverse transcription obtains RACE-Ready the first chain cDNA, then carries out two-wheeled PCR, first round PCR with the cDNA that obtains as template,
The UPM provided in kit is 5 ' end primers, and 3 ' hold primers.PCR reaction conditions are：94 DEG C of denaturations 5min；25 amplifications are followed
Ring (94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C of extension 2min)；Last 72 DEG C of extensions 10min.Second wheel PCR, with the first round
The product of PCR is template, and the NUP provided with kit is 5 ' end primers, 3 ' end primers, and PCR reaction conditions are：94 DEG C of pre- changes
Property 5min；25 amplification cycles (94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C of extension 2min)；72 DEG C of extension 10min.Obtain
The variable region of above-mentioned hybridoma cell clone heavy chain and light chain.Three cell line expression monoclonal antibody sequences are as shown in table 1：
The variable region amino acid sequence of the anti-ear chalone monoclonal antibody of table 1
PCR primer is purified by agarose gel electrophoresis, and is subcloned on pCR2.1TOPO cloning vectors
(Invitrogen companies).By PCR, the DNA of independent cloning is obtained, and then be sequenced with M13 forward and reverses primer.Mutually
Mend and determine that the amino acid sequence of area (CDR) encodes table definition by Kabat, and list in table 2.
The amino acid sequence of anti-ear chalone monoclonal antibody CDR of table 2
The specificity of the ear chalone monoclonal antibody of embodiment 2. and sensitivity
To detect specificity and the sensitivity of ear chalone monoclonal antibody, the IgG albumen that coupling has ear chalone is coated in into 96 orifice plates
In (300ng/well), by monoclonal antibody (1G1,2F10,4C3) gradient dilution (1:500,1:1000,1:2000,1:5000,
1:10000,1:50000) add hole in, every kind of gradient add antibody while add 3 kinds of concentration ear chalone (10ng/ml,
50ng/ml, 500ng/ml) be at war with incubation, adds the sheep anti-mouse igg that detection HRP is coupled to be detected, while adding not phase
Closing antibody carries out control test.As a result as shown in figure 1, as a result showing：2F10,4C3,1G1 can affect after pleasant chalone is added
It is coupled the affine of IgG with ear chalone and the increase competition with ear chalone concentration increases, in antibody 1:Under 2000 dilution factor
The ear chalone of 500ng almost can perfect competition antibody and be coupled ear chalone combination.Thus can be shown that the spy of anti-ear chalone antibody
Different in nature strong, sensitivity is high.
The ear chalone monoclonal antibody of embodiment 3. detection ear chalone (being coupled ear chalone and ear chalone)
1) the competitive ELISA method of ear chalone molecular method quantification detection is set up
Competitive ELISA is that antibody with high specificity is adsorbed on solid carrier, makes free ear chalone and chemical synthesis ear
The binding site of chalone conjugate (rabbit igg be coupled ear chalone) sequentially competition binding antibody, when the amount of antibody and ear chalone of absorption
When conjugate amount is fixed, the more, the ear chalone conjugate being incorporated on antibody is fewer for the amount of the ear chalone that dissociates in testing sample, that
With lacking that goat-anti rabbit ELIAS secondary antibody is combined so that substrate colour developing is more shallow, make the amount of ear chalone to be measured and absorbance into anti-
The relation of ratio.
Through coated antibody and ear chalone coupling-rabbit igg proportioning, the optimal screening of two anti-concentration of detection, it is determined that competition
ELISA method operating process and for rat blood serum middle ear chalone concentration detection.Operation sequence and result are as follows：
Operation sequence：
1) monoclonal antibody coating：By purified monoclonal antibody 2F10 and 4C3 carbonate buffer solutions (pH9.4~9.8) in molar ratio 1:1 mixes
It is diluted to 5 μ g/ml after conjunction to be coated with, 100 μ L/ holes, 2~8 DEG C overnight (16~18 hours).
2) board-washing：Enzyme mark version is taken out, with board-washing liquid (PBS+0.05% polysorbas20s, 350 μ L/ holes board-washing 3 of pH7.2~7.4)
It is secondary.
3) close：Addition confining liquid (1%BSA+1% gelatin PBS solutions), 200 μ L/ holes, 25 ± 3 DEG C are closed 2 hours.
4) board-washing：With reference to step (2)
5) sample preparation：A calibration curves：By ear chalone gradient dilution it is 0.25ng/mL, 0.5ng/mL with rat blood serum,
The ear chalone standard liquid of 1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL, 16ng/mL, 32ng/mL, as detection mark song sample.
B standard curve：Ear chalone is diluted into 0.8ng/mL with rat blood serum, basic, normal, high three concentration of 5ng/mL, 25ng/mL,
As detection Quality Control sample.
6) sample treatment：By calibration curve sample, Quality Control sample and sample to be detected, with dilution, (1%BSA-PBS is molten
Liquid) press 1:10 (such as：The μ L dilution → 300 μ L of 30 μ L samples+270) pre-treatment.
7) it is loaded：The all samples for having pre-processed are entered into hole, 100 μ L/ holes, 37 DEG C are incubated 1 hour.
8) add and be coupled ear chalone：After step (2) board-washing, the μ g/mL of addition rabbit igg coupling ear chalone 8,100 μ L/ holes,
37 DEG C are incubated 1 hour.
9) detection two is added to resist：After board-washing, 1 is added:The HRP coupling goat-anti rabbit enzyme labelled antibodies of 5000 dilutions, 100 μ L/ holes, 37
DEG C incubation 1 hour.
10) substrate is added：After step (2) board-washing, the new tmb substrate working solution prepared, 100 μ L/ holes, room temperature lucifuge are added
Incubation 10-15 minutes.
11) terminating reaction：Add the sulfuric acid solution of 2M, 50 μ l/ holes.
12) detect：ELIASA OD450Reading.
13) mark song is fitted using four parameter fitting methods, calculates the concentration for obtaining each sample, and to calibration curve and
Quality Control sample carries out rate of recovery calculating.
As a result show foundation rat blood serum ear chalone quantitation curves scope be 0.25-32ng/mL, each concentration point
It is good (calibration curve legend is shown in Fig. 2) with the goodness of fit of curve.The multiple batches of analyses of Jing, reappearance is good between calibration curve batch, accurate
Exactness RE% scope is：- 2.4%≤RE%≤5.2%；Favorable reproducibility between each concentration point multiple holes, precision CV%≤
5.5%.Degree of accuracy RE% scopes between the quality-control sample plate of basic, normal, high concentration are：- 13.5%≤RE%≤- 13.1%, plate
Between precision CV%≤10.2%.As shown by data, the method can be used for the quantitative determination of rat blood serum middle ear chalone.
Using identical competition principle, the monkey set up and human serum ear chalone calibration curve quantification range are respectively 0.39-
50ng/mL and 0.625-40ng/mL, method validation result shows, can be applicable to the quantitative of corresponding serum matrix middle ear chalone
Detection.
2) the double crush syndrome method for being coupled ear chalone quantitative determination is set up
Double crush syndrome is that antibody with high specificity is adsorbed on solid carrier, makes ear chalone conjugate (human IgG idol
Connection ear chalone) ear chalone be incorporated into the binding site of antibody, the amount of testing sample middle ear chalone conjugate is the more incorporated into anti-
Ear chalone conjugate on body is the more, then many with what goat-anti people's ELIAS secondary antibody was combined, so that substrate colour developing is deeper, makes to be measured
The amount of ear chalone conjugate is proportional with absorbance.
Through coated antibody and the optimal screening for detecting two anti-concentration, it is determined that double crush syndrome method operating process
And for the detection of monkey serum middle ear chalone conjugate concentration.Operation sequence and result are as follows：
Operation sequence：
1) monoclonal antibody coating：By purified monoclonal antibody 2F10 and 4C3 carbonate buffer solutions (pH9.4~9.8) in molar ratio 1:1 mixes
It is diluted to 4 μ g/mL after conjunction to be coated with, 100 μ L/ holes, 2~8 DEG C overnight (16~18 hours).
2) board-washing：Enzyme mark version is taken out, with board-washing liquid (PBS+0.05% polysorbas20s, 350 μ L/ holes board-washing 3 of pH7.2~7.4)
It is secondary.
3) close：Addition confining liquid (3%BSA-PBS solution), 200 μ L/ holes, 25 ± 3 DEG C are closed 2 hours.
4) board-washing：With reference to step (2)
5) sample preparation：A calibration curves：By ear chalone conjugate gradient dilution it is 6.25ng/mL (grapplings with monkey serum
Point), the ear of 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 800ng/mL (anchor point)
Chalone standard liquid, as detection mark song sample.B standard curve：Ear chalone conjugate is diluted into 32ng/mL with monkey serum,
Basic, normal, high three concentration of 80ng/mL, 320ng/mL, as detection Quality Control sample.
6) sample treatment：Calibration curve sample, Quality Control sample and sample dilution to be detected (1%BSA-PBS) are pressed
1:10 (such as：The μ L dilution → 300 μ L of 30 μ L samples+270) pre-treatment.
7) it is loaded：The all samples for having pre-processed are entered into hole, 100 μ L/ holes, 37 DEG C are incubated 1 hour.
8) detection two is added to resist：After board-washing, 1 is added:The HRP coupling goat-anti rabbit enzyme labelled antibodies of 10000 dilutions, 100 μ L/ holes,
37 DEG C are incubated 1 hour.
9) substrate is added：After step (2) board-washing, the new tmb substrate working solution prepared, 100 μ L/ holes, room temperature lucifuge are added
Incubation 10-15 minutes.
10) terminating reaction：Add the sulfuric acid solution of 2M, 50 μ L/ holes.
11) detect：ELIASA OD450Reading.
12) mark song is fitted using four parameter fitting methods, calculates the concentration for obtaining each sample, and to calibration curve and
Quality Control sample carries out rate of recovery calculating.
As a result show foundation monkey serum ear chalone quantitation curves scope be 12.5-400ng/mL, each concentration point
It is good (calibration curve legend is shown in Fig. 3) with the goodness of fit of curve.The multiple batches of analyses of Jing, reappearance is good between calibration curve batch, accurate
Exactness RE% scope is：- 0.9%≤RE%≤3.8%；Favorable reproducibility between each concentration point multiple holes, precision CV%≤
2.1%.Degree of accuracy RE% scopes between the quality-control sample plate of basic, normal, high concentration are：- 10.8%≤RE%≤- 4.3%, between plate
Precision CV%≤10.5%.As shown by data, the method degree of accuracy set up by ear chalone antibody is very high, and the method can be used for monkey
The quantitative determination of serum middle ear chalone conjugate.
Using double crush syndrome method, the rat set up and human serum ear chalone conjugate calibration curve quantification range
Respectively 6.25-800ng/mL and 39.06-500ng/mL, method validation shows, can be applicable to corresponding serum matrix middle ear and press down
The quantitative determination of plain conjugate.
Claims (10)
1. a kind of new ear chalone antibody or its functional fragment, wherein the antibody includes heavy chain and light chain, its feature exists
In：
I the heavy chain described in () includes at least three CDR regions, the CDR region has such as SEQ ID NO:1、2、3、7、8、9、13、
14th, the amino acid sequence shown in 15；
(ii) light chain described in includes at least three CDR regions, and the CDR region has such as SEQ ID NO:4、5、6、10、11、12、
16th, the amino acid sequence shown in 17.
2. new ear chalone antibody according to claim 1 or its functional fragment, it is characterised in that：
A () described heavy chain includes three CDR regions, the CDR region has such as SEQ ID NO:1st, the amino acid sequence shown in 2,3；
The light chain includes three CDR regions, and the CDR region has such as SEQ ID NO:4th, the amino acid sequence shown in 5,6；Or
B () described heavy chain includes three CDR regions, the CDR region has such as SEQ ID NO:7th, the amino acid sequence shown in 8,9；
The light chain includes three CDR regions, and the CDR region has such as SEQ ID NO:10th, the amino acid sequence shown in 11,12；Or
C () described heavy chain includes three CDR regions, the CDR region has such as SEQ ID NO:13rd, the amino acid sequence shown in 14,15
Row；The light chain includes three CDR regions, and the CDR region has such as SEQ ID NO:16th, the amino acid sequence shown in 5,17.
3. new ear chalone antibody according to claim 1 or its functional fragment, it includes heavy chain and light chain, and it is special
Levy and be：
A () is comprising such as SEQ ID NO:The weight chain variable district of the amino acid sequence shown in 18；Comprising such as SEQ ID NO:Shown in 19
Amino acid sequence light chain variable district；Or
B () is comprising such as SEQ ID NO:The weight chain variable district of the amino acid sequence shown in 20；Comprising such as SEQ ID NO:Shown in 21
Amino acid sequence light chain variable district；Or
C () is comprising such as SEQ ID NO:The weight chain variable district of the amino acid sequence shown in 22；Comprising such as SEQ ID NO:Shown in 23
Amino acid sequence light chain variable district.
4. the new ear chalone antibody or its functional fragment according to claim 1-3, wherein described antibody is single
Clonal antibody.
5. the new ear chalone antibody or its functional fragment according to right 1-3, wherein described functional fragment is
Refer to antibody fragment such as Fv, scFv, Fab, Fab ', the fragment of scFv-Fc, F (ab ') 2 or Fv-PEG, scFv-PEG, Fab-PEG.
6. the new ear chalone antibody or its functional fragment according to right 1-3, wherein described ear chalone is
Dolastatin-10, Dolastatin-15, TZT-1027, MMAE, MMAF, Cemadotin, his western many fourths.
7. new ear chalone antibody according to claim 6 or its functional fragment, wherein described ear chalone is preferred
Ground is MMAE (Monomethyl Auristatin E).
8. the medicine comprising the new ear chalone antibody described in claim 1-3 or its functional fragment, detection reagent or examination
Agent box.
9. it is a kind of detection ear chalone and its coupling drug method, it is characterised in that usage right require 1-3 described in ear chalone
Antibody or its functional fragment.
10. it is according to claim 12 detection ear chalone and its coupling drug method, it is characterised in that claim 1-
Ear chalone antibody or functional fragment described in 3 is used for ELISA method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171152A (en) * | 2020-01-15 | 2020-05-19 | 吉林医药学院 | PCSK9 antibody, and preparation method and application thereof |
| WO2023108102A1 (en) * | 2021-12-10 | 2023-06-15 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Anti-lcn-2 antibodies as a treatment for eye disorders |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1482972A4 (en) * | 2001-11-20 | 2005-11-23 | Seattle Genetics Inc | Treatment of immunological disorders using anti-cd30 antibodies |
| CN1938046A (en) * | 2003-11-06 | 2007-03-28 | 西雅图基因公司 | Monomethylvaline compounds capable of conjugation to ligands |
| US20080213289A1 (en) * | 2002-07-31 | 2008-09-04 | Seattle Genetics, Inc. | Recombinant anti-cd30 antibodies and uses thereof |
| US20120039915A1 (en) * | 2010-06-30 | 2012-02-16 | The Regents Of The University Of California | Glioblastoma multiforme-reactive antibodies and methods of use thereof |
| CN103145847A (en) * | 2013-02-05 | 2013-06-12 | 浙江大学 | Anti-CD20 antibody-monomethyl auristatin E conjugate, its preparation method and application |
| CN103702996A (en) * | 2011-05-27 | 2014-04-02 | Ambrx公司 | Compositions containing, methods involving, and uses of non-natural amino acid linked dolastatin derivatives |
| CN104640842A (en) * | 2012-09-20 | 2015-05-20 | 赛特瑞恩股份有限公司 | Dolastatin-10 derivative, method of producing same and anticancer drug composition containing same |
| US20180339061A1 (en) * | 2016-03-29 | 2018-11-29 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
-
2016
- 2016-08-16 CN CN201610680614.8A patent/CN106674347B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1482972A4 (en) * | 2001-11-20 | 2005-11-23 | Seattle Genetics Inc | Treatment of immunological disorders using anti-cd30 antibodies |
| US20080213289A1 (en) * | 2002-07-31 | 2008-09-04 | Seattle Genetics, Inc. | Recombinant anti-cd30 antibodies and uses thereof |
| CN1938046A (en) * | 2003-11-06 | 2007-03-28 | 西雅图基因公司 | Monomethylvaline compounds capable of conjugation to ligands |
| US20120039915A1 (en) * | 2010-06-30 | 2012-02-16 | The Regents Of The University Of California | Glioblastoma multiforme-reactive antibodies and methods of use thereof |
| CN103702996A (en) * | 2011-05-27 | 2014-04-02 | Ambrx公司 | Compositions containing, methods involving, and uses of non-natural amino acid linked dolastatin derivatives |
| CN104640842A (en) * | 2012-09-20 | 2015-05-20 | 赛特瑞恩股份有限公司 | Dolastatin-10 derivative, method of producing same and anticancer drug composition containing same |
| CN103145847A (en) * | 2013-02-05 | 2013-06-12 | 浙江大学 | Anti-CD20 antibody-monomethyl auristatin E conjugate, its preparation method and application |
| US20180339061A1 (en) * | 2016-03-29 | 2018-11-29 | Obi Pharma, Inc. | Antibodies, pharmaceutical compositions and methods |
Non-Patent Citations (1)
| Title |
|---|
| BAI RL等: "Binding of dolastatin 10 totubulin at a distinct site for peptide anti-mitotic agents near thexchangeable nucleotide and vinca alkaloid sites", 《J. BIOLCHEM》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171152A (en) * | 2020-01-15 | 2020-05-19 | 吉林医药学院 | PCSK9 antibody, and preparation method and application thereof |
| CN111171152B (en) * | 2020-01-15 | 2023-04-18 | 吉林医药学院 | PCSK9 antibody, and preparation method and application thereof |
| WO2023108102A1 (en) * | 2021-12-10 | 2023-06-15 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Anti-lcn-2 antibodies as a treatment for eye disorders |
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