CN106554950A - People's TK1 epitope peptides, antigen, antibody, application and test kit - Google Patents
People's TK1 epitope peptides, antigen, antibody, application and test kit Download PDFInfo
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Abstract
The present invention relates to people's TK1 epitope peptides, antigen, antibody, application and test kit.The aminoacid sequence of people's TK1 epitope peptides of the present invention is the sequence shown in sequence table SEQ ID NO.1.The people TK1 antigens of the present invention are coupled with protein carrier by people TK1 epitope peptides and are obtained.The people TK1 monoclonal antibodies or polyclonal antibody of the present invention is obtained by people's TK1 antigens of the present invention.The people TK1 monoclonal antibodies or polyclonal antibody of the present invention is used to prepare people's TK1 external diagnosis reagent cases.People's TK1 epitope peptides of the present invention have good antigenicity, and antigen (immunogen) immune animal prepared with which can produce the monoclonal antibody and polyclonal antibody of high degree of specificity, so as to can be applicable to the vitro detection of people TK1.
Description
Technical field
The invention belongs to chemiluminescent polypeptide and field of immunology, and in particular to people's thymidine kinase 1
(TK1) epitope peptide, the TK1 specific antigens prepared with the epitope peptide and phase
The monoclonal antibody answered or polyclonal antibody, the antibody are preparing the examination of people TK1 in-vitro diagnosis
Application and people's TK1 external diagnosis reagent cases on agent box.
Background technology
TK1 is a kind of kinases closely related with cell DNA synthesis, is included in normal human
Amount is extremely low or can not be detected.When there is abnormality proliferation such as tumor in vivo, TK1 in serum
Concentration is substantially increased.Therefore the generation of tumor can be indicated in advance by detecting the change of TK1,
Can be used for tumor early stage Risk Screening, prognosis and Treatment monitoring.
Thymidine kinase (TK) is thymidine (abbreviation thymidine) synthetic DNA
One of key enzyme.TK occurs in the form of two kinds of isozymes：Cytoplasmic thymidine kinase (TK1)
With thymidine kinase,mitochondrial (TK2), but only TK1 be raised and lowered be assessment proliferative cell
Propagation degree important symbol, the synthesis of TK1 and DNA raises into positive correlation.Malignant tumor
In cell, the synthesis of DNA increases severely causes the drastically propagation of tumor cell, the rising of TK1 with it is swollen
Tumor cell growth state is closely related.TK1 in the akinete and serum of normal adult
Enzymatic activity and concentration are atomic, once cell occurs canceration, the activity of TK1 enzymes and content can be big
Exceed greatly normal level.In malignant cell, the synthesis of DNA increases severely causes tumor cell
Drastically breed, it is demonstrated experimentally that having been found that TK1 has difference in 95% malignant cell
The rising of degree, rising and the growth of tumour cell state of TK1 are closely related.Therefore TK1
Rising is closely related with cell cycle, proliferative activity, is the cell propagation mark of a broad spectrum activity
Will thing.Can be clinically used for tumor early screening, tumor patient treatment curative effect monitoring and prognosis
With recurring risk assessment.
Lesion detection means at present using iconography are almost helpless for infantile tumour,
The optimal method of the TK1 levels in detection serum is immune detection.Additionally, applying TK1
Antibody and advanced enzyme immunity, chemiluminescence, can be checked various by conventional blood drawing
Cell proliferation in vivo before and after therapeutic scheme (such as operation, chemotherapy, radiotherapy and Biotherapeutics etc.)
Mobility, so as to play the effect of monitoring treatment and prognosis evaluation.Therefore, find suitable
With immunogenic TK1 epitope peptides, the TK1 antigens and antibody that prepare specificity i.e.
Into emphasis.
The content of the invention
In the presence of solving the problems, such as above-mentioned prior art, the invention provides a kind of people
TK1 epitope peptides, TK1 specific antigens prepared with the epitope peptide and corresponding
Monoclonal antibody or polyclonal antibody, its application on people's TK1 test kits are prepared, and
People's TK1 external diagnosis reagent cases.
Specifically, the invention provides：
A kind of people TK1 epitope peptides, wherein the aminoacid sequence of the TK1 epitope peptides
It is classified as：
Tyr-Lys-Ser-Thr-Pro-Gly-Ser-Pro-Ser-Lys-Thr-Arg-Gly-Gln。
Present invention also offers a kind of TK1 antigens, which is by making described people's TK1 antigens
Epitope peptide is prepared from carrier protein couplet.
Present invention also offers a kind of people TK1 antibody, which is prepared by described TK1 antigens
Monoclonal antibody or polyclonal antibody, wherein the TK1 antigens are described by making
People TK1 epitope peptides and carrier protein couplet be prepared from.
Present invention also offers described people TK1 antibody is preparing people's TK1 external diagnosis reagents
Application on box.
Present invention also offers a kind of people TK1 external diagnosis reagent cases, which includes described people
TK1 antibody is used as coated antibody or binding antibody.
Preferably, the test kit also includes another kind of people's TK1 antibody, and above-mentioned people
One of TK1 antibody and described another kind of people TK1 antibody are as coated antibody, another
Person is used as binding antibody, wherein described another kind of people TK1 antibody is by comprising such as following sequence
Another kind of TK1 antigens of shown epitope are prepared from：
Tyr-Arg-Gly-Thr-Glu-Lys-Glu-Val-Glu-Val-Ile-Gly-Gly-Ala-A
sp-Lys
Preferably, the coated antibody is monoclonal antibody.Preferably, the binding antibody
For polyclonal antibody.
Preferably, the test kit also anti antibody comprising enzyme labelling.
Preferably, the test kit is used to detect the people's TK1 albumen in serum.
The present invention is had the advantages that compared with prior art：
1. people's TK1 epitope peptides of the present invention have good antigenicity, are prepared with which
Antigen (immunogen) immune animal can produce the monoclonal antibody and polyclone of high degree of specificity
Antibody.
2. the TK1 monoclonal antibodies and polyclonal antibody for being prepared with the present invention being capable of high special
Ground is combined with the TK1 in blood (particularly serum) sample.
3. the people TK1 external diagnosis reagent cases of the present invention can effectively detect blood sample
The level of the TK1 in (particularly serum sample), can be used for the early screening of tumor, swell
Tumor patient treatment curative effect monitoring and prognosis and recurring risk assessment.
Specific embodiment
Below by way of the description of specific embodiment, the invention will be further described, but this is simultaneously
Non- is limitation of the present invention, those skilled in the art's basic thought of the invention, can be with
Various modifications or improvement are made, but without departing from the basic thought of the present invention, at this
Within the scope of bright.
First, people TK1 epitope peptides
People's TK1 albumen specifically described herein is known in the art, and its aminoacid sequence is this
Known to field, can find in the specialized databases such as NCBI.
The invention provides people's TK1 epitope peptides (1) and (2), its aminoacid sequence is respectively such as
Shown in sequence table SEQ ID No.1 and SEQ ID No.2, it is：
(1) Y-K-S-T-P-G-S-P-S-K-T-R-G-Q and
(2)Y-R-G-T-E-K-E-V-E-V-I-G-G-A-D-K。
The present inventor gropes through substantial amounts of theoretical research and experiment, finally screens
There is good antigenic epitope peptide to two kinds.
TK1 epitope peptides (1) include people's TK1 albumen (NCBI accession number AAH07986.1)
The peptide fragment that N-terminal is the 11st to the 20th, and the N-terminal in the peptide fragment adds Y, K, S
And T, so as to constitute the TK1 epitope peptides (1) containing 14 aminoacid.
TK1 epitope peptides (2) include people's TK1 albumen (NCBI accession number AAH07986.1)
The peptide fragment that C-terminal is the 167th to the 180th, and the peptide fragment N-terminal plus Y and
R, so as to constitute the TK1 epitope peptides (2) containing 16 aminoacid.
The characteristics of the two peptide fragments are respectively provided with hydrophilic, antigenicity by force and are readily synthesized.
At present, present invention research finds that the TK1 epitope peptides of the present invention have following work(
Energy：
1. there is antigenicity；2. produce as immunogen stimulating animal after being connected with carrier protein
The antibody of specificity；3. specifically can be tied with people TK1 with antibody prepared by epitope peptide
Close.
The preparation method of the TK1 epitope peptides of the present invention can use chemical synthesiss：Using U.S.
The many automatic peptide synthesizers of state's ABI431A types, by Solid phase synthesis epitope peptide.This
The molecular weight of bright epitope peptide (1) and (2) is respectively 1727.83,2021.15, can use matter
Spectrum is determined, and determines the synthesized epitope peptide sequence of identification by peptide sequence.Peptide
The purity of section can be evaluated with thin layer chromatography and high performance liquid chromatography, and determines epitope peptide
Concentration.
2nd, TK1 antigens
Present invention also offers TK1 antigens, its people's TK1 epitope by making the present invention
One of peptide (1) and (2) are prepared from carrier protein couplet.Specifically, the present invention is carried
TK1 antigens (1) and (2), the TK1 antigens (1) have been supplied to resist by making the people TK1 of the present invention
Former epitope peptide (1) is prepared from carrier protein couplet；The TK1 antigens (2) are by making this
Bright people's TK1 epitope peptides (2) is prepared from carrier protein couplet.The TK1 of the present invention
Antigen has immunogenicity and specificity, is a kind of immunogen, can be used to immune animal so as to make
The TK1 antibody of standby specificity.In the present invention, the example of available carrier protein includes KLH
(keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin OVA etc..By
Strong in KLH (keyhole limpet hemocyanin) immunogenicity, binding site is more, and immune effect is preferable,
And with immune animal sibship farther out, it is difficult to cause intersection anti-as carrier protein with which
Should, therefore be preferred.
3rd, TK1 monoclonal antibodies, TK1 polyclonal antibodies and people's TK1 external diagnosis reagents
Box
Present invention also offers people's TK1 antibody, including people TK1 monoclonal antibodies and people TK1
Polyclonal antibody, they can be each with the TK1 antigens (1) of the present invention or (2) (immunogen)
Immune animal is prepared.Preparation method can adopt the ordinary skill in the art, specifically can be found in
Embodiment 2.
Herein, term " another kind of TK1 antigens " is TK1 antigens (2).When this
Bright people TK1 antibody is prepared by TK1 antigens (2) (that is, another kind of TK1 antigens)
When, the antibody is properly termed as " people's TK1 antibody (2) " (or abbreviation antibody (2)) or " another
Plant people's TK1 antibody ".
The TK1 monoclonal antibodies and polyclonal antibody of the present invention can be used for preparing people's TK1 bodies
Outer diagnostic kit, the test kit can be based on immunization methods to tissue, cell, body fluid
In TK1 detected that the TK1 in preferred pair blood preparation, particularly serum is examined
Survey.
Therefore, the invention provides a kind of people TK1 external diagnosis reagent cases, which includes this
Bright people TK1 monoclonal antibodies or polyclonal antibody.
The immunization experiment method that can be used for Clinical Laboratory is currently known mainly including following several：
ELISA method, chemoluminescence method, fluorescent chromatographic method, colloid gold immune algoscopy etc..
And ELISA method includes following several types：Double antibody sandwich method detection antigen, dual anti-
Former sandwich assay detection antibody, indirect method survey antibody, competition law survey antibody, competition law survey antigen,
Capture coating method surveys antibody etc..
The people TK1 external diagnosis reagent cases of the present invention preferably adopt ELISA double antibody sandwich methods
To detect TK1 albumen.The test kit can include coated antibody, binding antibody, enzyme labelling
Anti antibody and/or necessary instrument and reagent etc..
Preferably, the people TK1 external diagnosis reagent cases are mono- using the people TK1 of the present invention
Clonal antibody is used as coated antibody.Here, term " coated antibody " is referred to and is coated in solid phase
Antibody in ELISA Plate.Additionally, the people TK1 external diagnosis reagent cases further preferably include people
TK1 polyclonal antibodies using as binding antibody, wherein, when the binding antibody is from this
During one of people's TK1 epitope peptides (1) and (2) of invention, the coated antibody is derived from
The other of the epitope peptide (1) and (2).Here, term " binding antibody " is referred to
The specific antibody that can be combined with the anti antibody of determined antigen and enzyme labelling in test kit.The examination
Agent box can also include the anti antibody of enzyme labelling, and the anti antibody can be goat anti-rabbit igg antibody,
The enzyme labelling can be horseradish peroxidase, alkali phosphatase etc..
TK1 in people's TK1 external diagnosis reagent case preferred pair serum of the present invention is examined
Survey.
In the test kit of the present invention, any reagent or instrument needed for detection can also be included,
Such as pre-coated plate, cleaning mixture, developer, terminate liquid etc..
Present disclosure, but these are further explained and described below by way of the mode of example
Example is understood not to the restriction to protection scope of the present invention.
Embodiment
Unless otherwise stated, described below solution is aqueous solution, and the percent in solution is equal
For percentage by volume.
Embodiment 1：TK1 epitope peptides (1) and the preparation of (2).
Preparation method chemical synthesiss：Using many automatic peptide synthesizers of American AB I431A type,
TK1 epitope peptides (1) and (2) are respectively synthesized by solid phase method.The purity of epitope peptide is used
High performance liquid chromatography is evaluated, and determines the concentration of peptide fragment.The epitope peptide (1) of the present invention
(2) molecular weight is respectively 1727.83,2021.15, is determined using mass spectrum, by many
The synthesized peptide sequence of peptide sequence determination identification.
First, the synthesis of TK1 epitope peptides (1) and (2)
Above-mentioned peptide fragment adopts Solid phase synthesis.The main thought of Solid phase peptide synthesis is：First by institute
The carboxyl of the carboxyl-terminus amino acid synthesized by peptide chain is same insoluble with covalent bond form
Macromolecular compound (resin) is connected, and then combines the aminoacid on solid phase carrier with this and makees
For moiety, through sloughing amino protecting group and reacting with excessive activated carboxyl component, connect
Long peptide chain.Such step repeatedly repeatedly can go on, and finally reach required synthesis
Peptide chain length.This building-up process is as follows.
The TK1 epitope peptides (1) of the present invention and the respective concrete preparation process of (2) are as follows：
1. raw materials used：
HMP resins (many polyvinyl resins of P- hydroxymethyl phenoxy methyl, be purchased from sigma companies)
Fmoc-AA (aminoacid of 9- fluorenylmethoxycarbonyl carbonyls acyl group protection, be purchased from Merck companies)
NMP (N-methyl ketopyrrolidine is purchased from sigma companies)
DCM (dichloromethane, commercially available eleutheromorph garden chemical company)
MeoH (methanol, commercially available eleutheromorph garden chemical company)
Piperidines (Piperidine is purchased from sigma companies)
DMAP (dimethyl aminopyridine is purchased from sigma companies)
HOBT (hydroxybenzotriazole is purchased from sigma companies)
DCC (dicyclohexylcarbodiimide is purchased from sigma companies)
TFA (trifluoroacetic acid is purchased from sigma companies)
EDT (1，2-ethandithiol is purchased from sigma companies)
Thioanisole, is purchased from Guangzhou Wei Bai Chemical Co., Ltd.s
Crystalline phenol, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Acetonitrile, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
2. instrument is used：
Many automatic peptide synthesizers, model 431A are purchased from ABI companies
Rotary Evaporators, model R-201 are purchased from Shanghai Shen Shun companies
High performance liquid chromatograph, Waters 600, is purchased from Waters, US
Freezer dryer, model VFD-2000 are purchased from the rich doctor Kanggong department in Beijing
3. synthetic method and process：
Weigh HMP resin 100mg, replace equivalent to be 1.0meq, will 0.1mmol put
In the reaction intracavity of many automatic peptide synthesizers of American AB I431A type, by synthesizer automatically by spy
Fixed aminoacid is coupled together in a different order, and Conjugate ratio is up to 99%.Reaction is as follows：
(1) activation (HOBt/DCC methods) of aminoacid
(2) connection aminoacid is on resin
(3) the Fmoc protection groups of aminoacid are sloughed
(4) activation (HOBt/DCC methods) of another aminoacid
(5) it is coupled
(6) repeat step (3) to (5) is until end of synthesis.
Respectively obtain the peptide resin of peptide resin 136mg and TK1 peptide fragment (2) of TK1 peptide fragments (1)
107mg。
(7) peptide resin：
Peptide chain is cut with TFA (trifluoroacetic acid), with EDT (2.5 volume %), thio phenyl
Methyl ether (2.5 volume %) makees scavenger, reacts 3.0 hours at room temperature, removes cutting examination
Agent, then extracted with ether, respectively obtain the crude product of TK1 peptide fragments (1) and (2).
2nd, the purification of TK1 epitope peptides (1) and (2) crude product：
Using high performance liquid chromatography separation purification：
Condition：Chromatographic column：C810 × 100mm, is purchased from Waters, US
Chromatograph：Waters 600, Waters, US
Mobile phase：A：0.1%TFA (trifluoroacetic acid) aqueous solution
B：0.1%TFA (trifluoroacetic acid) is in 60% acetonitrile
Detection wavelength：214nm
Flow velocity：4ml/ minutes
Gradient：20-60%B, 30 minutes
HPLC (high performance liquid chromatography) is analyzed
Chromatographic column：C184.6 × 150mm, is purchased from Waters, US
Mobile phase：A：0.1%TFA (trifluoroacetic acid) aqueous solution
B：0.1%TFA (trifluoroacetic acid) is in acetonitrile
Detection wavelength：214nm
Flow velocity：1ml/ minutes
Gradient：0-60%B, 30 minutes
The TK1 epitope peptides (1) of the peptide fragment analysis result display present invention and the purity of (2) are equal
For more than 95%.
3rd, the identification of TK1 epitope peptides (1) and (2)
1. is determined using mass spectrum dividing for TK1 epitope peptides (1) obtained by purification and (2) respectively
Son amount.
(1) reagent raw material
TFA (trifluoroacetic acid is purchased from sigma companies)
HCCA (alpha-cyano -4- hydroxycinnamic acids, be purchased from sigma companies)
Acetonitrile (is purchased from Chemical Reagent Co., Ltd., Sinopharm Group)
(2) instrument
MALDI-TOF-MS instrument MALDI-TOF-MS (models：
REFLEX III, German Bruker companies)；
(3) matrix liquid：α-CCA are dissolved in the 50%ACN solution containing 0.1%TFA,
Saturated solution is made, is centrifuged, is taken supernatant；
(4) instrument testing conditions：Reflection detection mode；Flight pipe range 3m；Nitrogen laser：
Wavelength 337nm, accelerating potential 20KV；Reflected voltage 23KV.
(5) operating procedure：The sample of the above-mentioned purified polypeptides (1) of 1 μ L and (2) is taken respectively, respectively
Mix from the saturation stromal supernatant mixing equal-volume with 1 μ L, 1 μ L points are taken respectively in sample
On target, detected in sending into ion source.
As a result, the molecular weight for measuring gained TK1 epitope peptides (1) is anti-for 1727.6, TK1
The molecular weight of former epitope peptide (2) is 2020.9, with theoretical molecular 1727.83,2021.15 1
Cause, it was demonstrated that synthesis polypeptide is purpose product.
2. TK1 epitope peptides (1) and (2) as obtained by peptide sequence determines identification respectively
Sequence.
(1) principle：The ultimate principle of polypeptid acid sequence analysis is Edman degradeds,
It is a circulating chemical reaction process.Including three main chemical steps：(1) it is even
Connection：Phenyl isothiocyanate and the N- ends residue reaction of proteins and peptides, form phenylamino sulfur
Formyl (PTC) derivant, i.e. PTC- peptides.(2) cyclisation cracking：The cyclisation cracking of PTC- peptides.
(3) convert：Thiazole purine ketone phenylamino (ATZ) is converted into different sulphur urine aminoacid (the PTH- ammonia of benzene
Base acid).The peptide for reducing an amino acid residue for staying in the solution repeat carry out it is above-mentioned anti-
Process is answered, whole sequencing procedure is all to be carried out by sequenator automatically now.
(2) instrument：491 type protein/polypeptide -terminal amino acid sequence of American AB I company
Row analyser
(3) reagent raw material
Phenyl isothiocyanate PITC, is purchased from sigma companies
Normal heptane, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Trimethylamine TMA aqueous solutions, are purchased from Chemical Reagent Co., Ltd., Sinopharm Group
TFA (trifluoroacetic acid is purchased from sigma companies)
Ethyl acetate, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Chlorobutane, is purchased from sigma companies
Acetonitrile, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
(4) determine
Carry out by instrument description.
As a result：Identified, the sequence of gained TK1 epitope peptides (1) and (2) is respectively：
(1) Y-K-S-T-P-G-S-P-S-K-T-R-G-Q and
(2)Y-R-G-T-E-K-E-V-E-V-I-G-G-A-D-K。
The result is consistent with target section of synthesized peptide.
Embodiment 2：Respectively by the TK1 epitope peptides (1) obtained by embodiment 1 and (2) and load
Body protein connects to prepare TK1 antigens (1) and (2), is exempted from using gained antigen (1) and (2) respectively
Epidemic disease animal, so as to the monoclonal antibody and polyclonal antibody of specificity are prepared using antigen (1),
And the monoclonal antibody and polyclonal antibody of specificity are prepared using antigen (2).
1. the preparation of antigen：With BDB (Bis-diazotizedbenzidine dichloride) method
TK1 peptide fragments (1) and (2) (are derived from carrier protein KLH (keyhole limpet hemocyanin) respectively
Sigma companies) connection be prepared into TK1 antigens (1) and (2).
TK1 peptide fragments (1) or (2) 10.0mg are taken, with 1ml 0.1M PBSs (pH 7.4)
Dissolving；KLH 10mg, are dissolved with 0.2M borate buffer solutions (pH 8.6) 20ml；So
Afterwards both are mixed, 0 DEG C is cooled to, is taken BDBCl2110 μ L, react 1.5h under room temperature,
Subpackage after dialysed overnight, -20 DEG C of preservations.
In embodiments, the formula of PBS (if use) is：0.2mol/L
Na2HPO481ml adds the NaH of 0.2mol/L2PO419ml is mixed.
The formula of borate buffer solution is：0.05mol/L Borax 80ml, plus 0.2mol/L boron
Sour 20ml is mixed.
2. immune animal prepares monoclonal antibody：
2.1. take the TK1 antigens (1) and (2) (immunogen) of above-mentioned preparation respectively with it is isopyknic
After Freund's complete adjuvant (being purchased from sigma biotech firms) is sufficiently mixed, immune Balb/c mices,
50 μ g antigens/only, subcutaneous multi-point injection.Serum titer is surveyed after 4 weeks, immunoreactivity is selected
Good mice booster immunization again：Antigen is taken with isopyknic incomplete Freund's adjuvant (purchased from sigma
Biotech firm) be sufficiently mixed after, 25 μ g/ of antigen dose only, strengthen exempting from by subcutaneous multi-point injection
The number of times of epidemic disease is 6 times, each At intervals of two to three weeks, merges front booster immunization continuous in addition twice,
All per minor tick 1-2, extracting spleen cell is used according to a conventional method with Sp2/0 myeloma cell afterwards
50%PEG (MW4000) (being purchased from Jing Yuan chemical companies) is merged by mediation, and uses HAT
Conditioned medium (being purchased from sigma companies) selects culture.CO is put into after fusion2In incubator
After 37 DEG C are cultivated 9～11 days, in hole, there is larger cell clone.Start within 11 days with indirectly
ELISA is screened.To primary dcreening operation, positive hole carries out 4 time clonings using limiting dilution assay
(even if a large amount of schizogamies of cell after screening) is cultivated, afterwards amplifying cells, frozen, system
Standby ascites.
2.2. Balb/c mices are only processed with norphytane (being purchased from sigma companies) 0.5ml/,
Pneumoretroperitoneum is inoculated with hybridoma 2 × 10 within one week6Individual/only, ascites is collected after 10 days.
2.3. determine antibody titer：Determined using TK1 antigens (1) with indirect ELISA method
The potency of the monoclonal antibody (1) of preparation, as a result shows that the potency of monoclonal antibody reaches 1:32000 with
On.
The potency of the monoclonal antibody (2) prepared using TK1 antigens (2) also utilizes identical side
Method is measured, and its potency also reaches 1:More than 32000.
3. immune animal prepares polyclonal antibody：
3.1. it is about the New Zealand white rabbit of 2kg or so as immunity from three monthly ages, body weight
Animal.In fundamental immunity, by the TK1 antigens (1) and (2) (immunogen) of 1-2mg above-mentioned preparation
Mix with isopyknic complete Freund's adjuvant (being purchased from sigma biotech firms) respectively-fully newborn
Multiple spot subcutaneous injection is carried out at rabbit back after change.Every 4 weeks booster immunizations once, strengthen exempting from
Epidemic disease 6 times, after antigen is fully emulsified with incomplete Freund's adjuvant (being purchased from sigma biotech firms),
With 100 μ g/ only in back multiple spot subcutaneous injection.After final boost, the 10th day carotid artery is put
Blood, separates serum.
3.2. determine antibody titer：Determined with indirect elisa method and made using TK1 antigens (1)
The potency of standby polyclonal antibody (1), as a result shows that antibody titer reaches 1:More than 32000.
The potency of the polyclonal antibody (2) prepared using TK1 antigens (2) also utilizes identical side
Method is measured, and its potency also reaches 1:More than 32000.
3.3. take blood and separate serum：Carotid artery intubation takes blood, separates serum.
4. antibody is isolated and purified：After ammonium sulfate precipitation, then Jing Protein G (are purchased from sigma
Company) affinity purification.
5. lyophilizing after antibody subpackage, cryopreservation.
Embodiment 3：The specificity identification of people's TK1 monoclonal antibodies (1) and (2)
Detected with ELISA.Respectively with people's TK1 albumen, TK2 albumen, S-100B eggs
In vain, neuronspecific enolase NSE (being purchased from Shanghai Lian Shuo companies) is anti-for detection
Primordial covering elisa plate, detects prepared TK1 monoclonal antibodies (1) respectively by ELISA
(2) specific reaction with people's TK1 albumen, makees cloudy with normal BALB/c mouse serum
Property control, PBS liquid makees blank.
As a result：TK1 monoclonal antibodies (1) and (2) are only reacted for the positive (P/N with TK1 respectively>
2.1) it is, anti-with S-100B albumen, neuronspecific enolase NSE, TK2 albumen
Should be negative, the TK1 monoclonal antibodies (1) and (2) for illustrating the present invention has specificity respectively.
Embodiment 4：The specificity identification of people's TK1 polyclonal antibodies (1) and (2)
Using being identified with above-mentioned identification monoclonal antibody specificity identical method.
As a result show：TK1 polyclonal antibodies (1) and (2) are reacted for the positive (P/N with TK1 respectively
>2.1), and with S-100B albumen, neuronspecific enolase NSE, TK2 albumen
React for feminine gender, illustrate the present invention TK1 polyclonal antibodies (1) and (2) respectively with specificity.
Embodiment 5：TK1 bodies are prepared using TK1 monoclonal antibodies and TK1 polyclonal antibodies
Outer diagnostic kit.
In the present embodiment, the list that will be prepared using TK1 epitope peptides (1) in embodiment 2
Clonal antibody (1) is used as the coated antibody in this test kit；To be resisted using TK1 in embodiment 2
Polyclonal antibody (2) prepared by former epitope peptide (2) is used as binding antibody.
The preparation and operation of TK1 external diagnosis reagent cases is as follows：
1. the preparation of various buffer and reagent：
A, coating buffer：The CB (carbonate buffer solution) of 0.050M, pH9.6
Na2CO3：16.0 grams
NaHCO3：29.0 grams
Distillation is water-soluble to 1000ml
B, sample/lavation buffer solution：10 × the PBS-Tween 20 of pH7.2
Na2HPO4·12H2O：58 grams
KH2PO4：4 grams
NaCl：100 grams
KCl：4 grams
Distillation is water-soluble to 1000ml
Plus Tween 20：20ml
C, enzyme marker diluent
10×PBS-Tween 20：10ml
FCS (calf serum)：20ml
Distillation is water-soluble to 1000ml
Enzyme stabilizers (are purchased from Shanghai Xi Bao companies)：1 gram
Biological preservative (is purchased from sigma companies)：1ml
D, developer A：
Citric acid：35.5 grams
Urea peroxide：10 grams
Distillation is water-soluble to 1000ml
Tween 20：10ml
E, developer B：
Citric acid：120 grams
EDTA-2Na：1 gram
TMB·2HCl：2 grams
Distillation is water-soluble to 1000ml
F, terminate liquid：2M H2SO4
Concentrated sulphuric acid (95-98%)：22.2ml
Distilled water：177.3ml
Timing is slowly dropped into concentrated sulphuric acid in distilled water, and side edged shakes up.
2. the preparation of pre-coated plate：
In the carbonate buffer solution of the 0.05M that TK1 monoclonal antibodies (1) are dissolved in pH=9.6,
Pre-coated liquid is made, per hole by 0.1 μ g/ in ELISA Plate (being purchased from Shenzhen Jin Canhua companies)
Hole add 100 μ l, put 4 DEG C placement 18-24 hours, take out, get rid of coating buffer, with sample/
Lavation buffer solution wash, 1 (w/v) %BSA-0.05M ethanolamine of Jing close 16 hours, overnight
Load evacuation sealing in aluminide-coating bag after drying, be placed in 4 DEG C of preservations.
3. binding antibody (TK1 polyclonal antibodies (2)) and enzyme-linked thing (horseradish peroxidase
The goat anti-rabbit igg antibody of labelling) (be purchased from Beijing company of Zhong Shan Golden Bridge) dilution ratio it is equal
Determined by square formation titration experiments, the goat anti-rabbit igg antibody of horseradish peroxidase-labeled is used
Enzyme marker diluted.
4. the composition of test kit：
Pre-coated plate：48/96 hole
TK1 calibration objects (raw material is purchased from Abcan companies)：6：6 × 1.0ml (concentration point
Wei 20pM, 10pM, 5pM, 2pM, 1pM, 0pM)
TK1 binding antibodies：1 × 10ml (Jing 1:5000 dilutions)
Enzyme-linked thing：1 × 10ml (Jing 1:5000 dilutions)
Concentrated cleaning solution (25 × PBS-Tween 20)：1×20ml
Developer A：1×6.0ml
Developer B：1×6.0ml
Terminate liquid：1×6.0ml
5. the operating procedure of test kit：
100 μ l/ holes of serum to be checked and calibration object are separately added in each hole of pre-coated plate,
Diplopore is, 37 DEG C are incubated 60 minutes, are washed with 1 × lavation buffer solution 5 times, are patted dry.
100 μ l/ holes of TK1 binding antibodies are added in each hole, 37 DEG C are incubated 30 minutes, slow with 1 × washing
Rush liquid to wash 5 times, pat dry.100 μ l/ holes of enzyme-linked thing are added in each hole again, 37 DEG C are incubated
30 minutes, washed with 1 × lavation buffer solution 5 times, patted dry.Developer A, B liquid is added,
Per each 50 μ l in hole, mix, 37 DEG C are incubated 15 minutes.Plus 50 μ l/ holes terminating reaction of terminate liquid,
With enzyme-linked detector (model RT-6000 is purchased from Lei Du companies) dual wavelength (450nm,
Absorbance is detected 620nm).
6. result judgement：
Table 1：Calibration object concentration and corresponding mean light absorbency (OD) value
| Concentration pM | 0 | 1 | 2 | 5 | 10 | 20 |
| Mean OD value | 0.066 | 0.127 | 0.215 | 0.374 | 0.714 | 1.236 |
Standard curve, standard curve are drawn with the logarithm value of calibration object concentration and correspondence absorbance
R2=0.977.
TK1 concentration results in detected specimen are calculated according to standard curve.
The inspection of serum T K1 is carried out in a manner described to 54 tumour patients and 103 healthy persons
Survey, apparently higher than healthy control group, difference has statistics to the TK1 contents in affinity antibody to SpA
Learn meaning (P<0.01) 2, are shown in Table.
Table 2：Two groups of sample TK1 concentration compare
From data above, the test kit of the present invention effectively and specifically can detect serum
TK1 contents, so as to detect the TK1 content differences between tumour patient and normal person,
Thus can determine whether the generation of tumor.
Claims (10)
1. a kind of people TK1 epitope peptides, wherein the aminoacid sequence of the TK1 epitope peptides is：
Tyr-Lys-Ser-Thr-Pro-Gly-Ser-Pro-Ser-Lys-Thr-Arg-Gly-Gln。
2. a kind of TK1 antigens, which is prepared from by making the people TK1 epitope peptides described in claim 1 and carrier protein couplet.
3. a kind of people TK1 antibody, which is the monoclonal antibody or polyclonal antibody being prepared from by the TK1 antigens described in claim 2.
4. application of the people TK1 antibody according to claim 3 on people's TK1 external diagnosis reagent cases are prepared.
5. a kind of people TK1 external diagnosis reagent cases, which includes people TK1 antibody described in claim 3 as coated antibody or binding antibody.
6. people TK1 external diagnosis reagent cases according to claim 5, wherein described test kit is also comprising another kind of people's TK1 antibody, and one of described people TK1 antibody and described another kind of people TK1 antibody are used as coated antibody, another one is used as binding antibody, wherein described another kind of people TK1 antibody is prepared from by another kind of TK1 antigens comprising the epitope as shown in following sequence：
Tyr-Arg-Gly-Thr-Glu-Lys–Glu-Val-Glu-Val-Ile-Gly-Gly-Ala-Asp-Lys 。
7. TK1 external diagnosis reagent cases according to claim 5 or 6, wherein the coated antibody is monoclonal antibody.
8. TK1 external diagnosis reagent cases according to claim 5 or 6, wherein the binding antibody is polyclonal antibody.
9. TK1 external diagnosis reagent cases according to claim 5 or 6, wherein the test kit also anti antibody comprising enzyme labelling.
10. TK1 external diagnosis reagent cases according to claim 5 or 6, wherein the TK1 external diagnosis reagent cases are used to detecting the people's TK1 albumen in serum.
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| CN112940131A (en) * | 2021-02-04 | 2021-06-11 | 福建亿彤生物科技有限公司 | Rabbit monoclonal antibody aiming at TK1 in human serum and application |
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