CN105669836A - Human HSP90 alpha-2 antigen epitope peptide, HSP90 alpha-2 antigen, HSP90 alpha-2 antibody, HSP90 alpha-2 kit and application - Google Patents
Human HSP90 alpha-2 antigen epitope peptide, HSP90 alpha-2 antigen, HSP90 alpha-2 antibody, HSP90 alpha-2 kit and application Download PDFInfo
- Publication number
- CN105669836A CN105669836A CN201410670399.4A CN201410670399A CN105669836A CN 105669836 A CN105669836 A CN 105669836A CN 201410670399 A CN201410670399 A CN 201410670399A CN 105669836 A CN105669836 A CN 105669836A
- Authority
- CN
- China
- Prior art keywords
- hsp90
- antibody
- antigen
- people
- hsp90 alpha
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a human HSP90 alpha-2 antigen epitope peptide, a human HSP90 alpha-2 antigen, a human HSP90 alpha-2 antibody, a human HSP90 alpha-2 kit and application. An amino acid sequence of the human HSP90 alpha-2 antigen epitope peptide is as shown in sequence table SEQ ID NO. 1. The human HSP90 alpha-2 antigen is prepared from the human HSP90 alpha-2 antigen epitope peptide and a protein carrier by coupling. A human HSP90 alpha-2 monoclonal or polyclonal antigen is prepared from the human HSP90 alpha-2 antigen. The human HSP90 alpha-2 monoclonal or polyclonal antigen is used for the preparation of a human HSP90 alpha-2 in-vitro diagnostic kit. The human HSP90 alpha-2 antigen epitope peptide has good antigenicity, highly specific monoclonal and polyclonal antibodies can be produced by use of an antigen (immunogen) produced from the human HSP90 alpha-2 antigen epitope peptide for immunizing an animal, and the human HSP90 alpha-2 antigen epitope peptide can be applied to human HSP90 alpha-2 in vitro testing.
Description
Technical field
The invention belongs to chemiluminescent polypeptide and field of immunology, HSP90 α-2 specific antigen be specifically related to HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 90 α-2 (HSP90 α-2) epitope peptide, preparing with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, the application on preparation people's HSP90 α-2 external diagnosis reagent case of the described antibody, people's HSP90 α-2 external diagnosis reagent case.
Background technology
In recent years, the research of tumor markers has been caused the attention of many researcheres. Find a kind of reliably, non-intrusion type, generation that tumor can be reflected, increment differentiation tumor markers be the common aspiration of numerous researcher. By the research to blood, tissue fluid, have now been found that the generation of tumor, proliferation and differentiation can be predicted by some tumor markerses. Wherein, HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 90 α-2 (HSP90 α-2) a kind of tumor markers thus.
Heat shock protein72-2 is one of important albumen in heat-shock protein family, and it participates in regulation and control, the conformation maintaining multiple protein in cell and function as molecular chaperones, plays an important role in regulating Growth of Cells, differentiation and apoptosis etc. In recent years finding, the malignant transformation of tumor, growth, propagation and invasion and attack are had important effect as chaperone by Heat shock protein72-2. Having many reports to confirm, in body tumor tissue, the expression of heat shock protein there occurs change. In the tumor tissues of different parts, the change of heat shock protein expression is also inconsistent. Have now been found that the grade malignancy of HSP90 α-2 content in affinity antibody to SpA and tumor, especially shift positive correlation. Therefore, HSP90 α-2 holds promise as the mark of diagnosing tumor and prognosis.
The optimal method of HSP90 α-2 level in detection serum is immune detection. Therefore, find suitable have immunogenic HSP90 α-2 epitope peptide, prepare specific HSP90 α-2 antigen and antibody emphasis.
Summary of the invention
For solving problem existing in above-mentioned prior art, the invention provides a kind of people's HSP90 α-2 epitope peptide, HSP90 α-2 specific antigen prepared with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, its application on preparation people's HSP90 α-2 test kit, and people's HSP90 α-2 external diagnosis reagent case.
Specifically, the invention provides:
A kind of people's HSP90 α-2 epitope peptide, the aminoacid sequence of wherein said HSP90 α-2 epitope peptide is:
Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-Gly-Ser-Asp-Glu。
Present invention also offers a kind of HSP90 α-2 antigen, it is by making described people's HSP90 α-2 epitope peptide and carrier protein couplet be prepared from.
Present invention also offers a kind of people's HSP90 α-2 antibody, it is the monoclonal antibody or polyclonal antibody that are prepared from by described HSP90 α-2 antigen, and wherein said HSP90 α-2 antigen is by making described people's HSP90 α-2 epitope peptide and carrier protein couplet be prepared from.
Present invention also offers the application on preparation people's HSP90 α-2 external diagnosis reagent case of described people's HSP90 α-2 antibody.
Present invention also offers a kind of people's HSP90 α-2 external diagnosis reagent case, it comprises described people's HSP90 α-2 antibody as coated antibody or binding antibody.
Preferably, described test kit also comprises another kind of people's HSP90 α-2 antibody, and the one in above-mentioned people's HSP90 α-2 antibody and described another kind of people's HSP90 α-2 antibody is as coated antibody, another one is as binding antibody, and wherein said another kind of people's HSP90 α-2 antibody is prepared from by another kind of HSP90 α-2 antigen of the epitope comprised as shown in following sequence:
Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Ser-Ala-Ala-Val-Thr-Glu。
Preferably, described coated antibody is monoclonal antibody. Preferably, described binding antibody is polyclonal antibody.
Preferably, described test kit also comprises the anti antibody of enzyme labelling.
The present invention compared with prior art has the advantages that:
1. people's HSP90 α-2 epitope peptide of the present invention has good antigenicity, can produce monoclonal antibody and the polyclonal antibody of high degree of specificity with its antigen prepared (immunogen) immune animal.
2. HSP90 α-2 monoclonal antibody prepared by the present invention and polyclonal antibody can high special HSP90 α-2 in blood sample be combined.
3. people's HSP90 α-2 external diagnosis reagent case of the present invention can detect the level of the HSP90 α-2 in serum effectively, can be used to judge the grade malignancy of tumor, and transfer and prognosis are predicted.
Detailed description of the invention
Below by way of the description of detailed description of the invention, the invention will be further described, but this is not limitation of the present invention, those skilled in the art's basic thought according to the present invention, various modifications may be made or improves, but without departing from the basic thought of the present invention, all within the scope of the present invention.
One, people HSP90 α-2 epitope peptide
People's HSP90 α-2 albumen specifically described herein is that to it known in the art, its aminoacid sequence be it known in the art, to find in the specialized databases such as NCBI.
The invention provides people HSP90 α-2 epitope peptide (1) and (2), its aminoacid sequence respectively as shown in sequence table SEQ IDNo.1 and SEQIDNo.2, for:
(1) Y-E-K-E-S-E-D-K-P-E-I-E-D-V-G-S-D-E; With
(2)Y-R-G-I-D-E-D-D-P-T-A-D-D-T-S-A-A-V-T-E。
The present inventor gropes through substantial amounts of theoretical research and experiment, and final screening obtains two kinds and has good antigenic epitope peptide.
HSP90 α-2 epitope peptide (1) comprises the peptide fragment of the 249th to the 265th, people's HSP90 α-2 albumen (NCBI accession number NP_005339.3) N end, and the N section at this peptide fragment adds Y, comprises 18 amino acid whose HSP90 α-2 epitope peptide (1) thus constituting.
HSP90 α-2 epitope peptide (2) comprises the 697th to the 714th peptide fragment of people's HSP90 α-2 albumen (NCBI accession number NP_005339.3) C end, and the N section at this peptide fragment adds Y and R, comprises 20 amino acid whose HSP90 α-2 epitope peptide (2) thus constituting.
The two peptide fragment is respectively provided with the feature that hydrophilic, antigenicity are strong and are readily synthesized.
At present, the present invention studies discovery, and HSP90 α-2 epitope peptide of the present invention has following function:
1. there is antigenicity; 2. after being connected with carrier protein, produce specific antibody as immunogen stimulating animal; 3. the antibody prepared with epitope peptide can be combined with people HSP90 α-2 specifically.
The preparation method useful chemical synthetic method of HSP90 α-2 epitope peptide of the present invention: utilize the many automatic peptide synthesizers of American AB I431A type, by Solid phase synthesis epitope peptide. The epitope peptide (1) of the present invention and the molecular weight of (2) respectively 2404.35,2483.39, available mass spectrum is determined, and is measured by peptide sequence and identify synthesized epitope peptide sequence. The purity of peptide fragment can be evaluated by thin layer chromatography and high performance liquid chromatography, and measures the concentration of epitope peptide.
Two, HSP90 α-2 antigen
Present invention also offers HSP90 α-2 antigen, it passes through to make the one in people HSP90 α-2 epitope peptide (1) and (2) of the present invention be prepared from carrier protein couplet. Specifically, the invention provides HSP90 α-2 antigen (1) and (2), described HSP90 α-2 antigen (1) is prepared from by making people HSP90 α-2 epitope peptide (1) of the present invention and carrier protein couplet; Described HSP90 α-2 antigen (2) is prepared from by making people HSP90 α-2 epitope peptide (2) of the present invention and carrier protein couplet. HSP90 α-2 antigen of the present invention has immunogenicity and specificity, being a kind of immunogen, can be used to immune animal thus preparing specific HSP90 α-2 antibody. In the present invention, the example of available carrier protein includes KLH (keyhole limpet hemocyanin), bovine serum albumin (BSA), ovalbumin OVA etc. Owing to KLH (keyhole limpet hemocyanin) immunogenicity is strong, binding site is many, and immune effect is better, and with immune animal sibship farther out, not easily causes cross reaction with it as carrier protein, is therefore preferred.
Three, HSP90 α-2 monoclonal antibody, HSP90 α-2 polyclonal antibody and people's HSP90 α-2 external diagnosis reagent case
Present invention also offers people's HSP90 α-2 antibody, including people's HSP90 α-2 monoclonal antibody and people's HSP90 α-2 polyclonal antibody, described antibody can be prepared each with HSP90 α-2 antigen (1) or (2) (immunogen) immune animal of the present invention and obtain. Preparation method can adopt the ordinary skill in the art, specifically can referring to embodiment 2.
In this article, term " another kind of HSP90 α-2 antigen " is HSP90 α-2 antigen (2). When the present invention people's HSP90 α-2 antibody by HSP90 α-2 antigen (2) (namely, another kind of HSP90 α-2 antigen) preparation and time, this antibody is properly termed as " people HSP90 α-2 antibody (2) " (or being called for short antibody (2)) or " another kind of people's HSP90 α-2 antibody ".
HSP90 α-2 monoclonal antibody of the present invention and polyclonal antibody may be used for preparation people's HSP90 α-2 external diagnosis reagent case, HSP90 α-2 in tissue, cell or body fluid can be detected by this test kit based on immunization method, it is preferable that the HSP90 α-2 in blood preparation is detected.
Therefore, the invention provides a kind of people's HSP90 α-2 external diagnosis reagent case, its people's HSP90 α-2 monoclonal antibody comprising the present invention or polyclonal antibody.
It is currently known the immunization experiment method that can be used for Clinical Laboratory and mainly includes 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-antigen sandwich method detect antibody, indirect method surveys antibody, competition law surveys antibody, competition law surveys antigen, catch the method that is coated surveys antibody etc.
People's HSP90 α-2 external diagnosis reagent case of the present invention preferably employs ELISA double antibody sandwich method to detect HSP90 α-2 albumen. This test kit can comprise coated antibody, binding antibody, the anti antibody of enzyme labelling and/or the instrument of necessity and reagent etc.
Preferably, described people's HSP90 α-2 external diagnosis reagent case adopts people's HSP90 α-2 monoclonal antibody of the present invention as coated antibody. At this, term " coated antibody " refers to the antibody in the ELISA Plate being coated in solid phase. In addition, described people's HSP90 α-2 external diagnosis reagent case is it is also preferred that comprise people's HSP90 α-2 polyclonal antibody using as binding antibody, wherein, when described binding antibody derives from the one in people HSP90 α-2 epitope peptide (1) and (2) of the present invention, described coated antibody derives from the another one in described epitope peptide (1) and (2). At this, term " binding antibody " refers to the specific antibody can being combined in test kit with determined antigen and enzyme labelling anti antibody. Described test kit can also comprise the anti antibody of enzyme labelling, and this anti antibody can be goat anti-rabbit igg antibody, and described enzyme labelling can be horseradish peroxidase, alkali phosphatase etc.
In the test kit of the present invention, it is also possible to comprise the required any reagent of detection or instrument, for instance pre-coated plate, cleaning mixture, developer, stop buffer etc.
Mode below by way of example further explains and describes present disclosure, but these examples are understood not to the restriction to protection scope of the present invention.
Embodiment
Except as otherwise noted, the following stated solution is aqueous solution, and the percent in solution is percentage by volume.
The preparation of embodiment 1:HSP90 α-2 epitope peptide (1) and (2).
Preparation method chemical synthesis: utilize the many automatic peptide synthesizers of American AB I431A type, is respectively synthesized HSP90 α-2 epitope peptide (1) and (2) by solid phase method. The purity high performance liquid chromatography of epitope peptide is evaluated, and measures the concentration of peptide fragment. The epitope peptide (1) of the present invention and the molecular weight of (2) respectively 2404.35,2483.39, utilizes mass spectrum to be determined, and is measured by peptide sequence and identifies synthesized peptide sequence.
One, the synthesis of HSP90 α-2 epitope peptide (1) and (2)
Above-mentioned peptide fragment adopts Solid phase synthesis. The main thought of Solid phase peptide synthesis is: be first connected with the same insoluble macromolecular compound (resin) of covalent bond form by the carboxyl synthesizing the carboxyl-terminus amino acid of peptide chain; then it is combined in aminoacid on solid phase carrier as moiety using this; through sloughing amino protecting group and with excessive activated carboxyl component reaction, spreading peptide chain.Such step can repeatedly go on repeatedly, finally reaches the length of the peptide chain of required synthesis. This building-up process is as follows.
The respective concrete preparation process of the HSP90 α-2 epitope peptide (1) and (2) of the present invention is as follows:
1. raw materials used:
HMP resin (the many polyvinyl resins of P-hydroxymethyl phenoxy methyl, be purchased from sigma company)
Fmoc-AA (aminoacid of 9-fluorenylmethoxycarbonyl carbonyl acyl group protection, be purchased from Merck company)
NMP (N-methyl ketopyrrolidine is purchased from sigma company)
DCM (dichloromethane is purchased from Central Plains chemical company)
MeoH (methanol is purchased from Central Plains chemical company)
Piperidines (Piperidine is purchased from sigma company)
DMAP (dimethyl aminopyridine is purchased from sigma company)
HOBT (hydroxybenzotriazole is purchased from sigma company)
DCC (dicyclohexylcarbodiimide is purchased from sigma company)
TFA (trifluoroacetic acid is purchased from sigma company)
EDT (1，2-ethandithiol is purchased from sigma company)
Thioanisole, is purchased from Guangzhou Wei Bai Chemical Co., Ltd.
Crystalline phenol, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Acetonitrile, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
2. use instrument:
Many automatic peptide synthesizers, model 431A, it is purchased from ABI company
Rotary Evaporators, model R-201, it is purchased from Shanghai Shen Shun company
High performance liquid chromatograph, Waters600, it is purchased from Waters, US
Freezer dryer, model VFD-2000, it is purchased from Beijing rich doctor Kanggong department
3. synthetic method and process:
Weighing HMP resin 100mg, replacing equivalent is 1.0meq, is placed in the reaction chamber of the many automatic peptide synthesizers of American AB I431A type by 0.1mmol, synthesizer is automatically coupled together in a different order by specific aminoacid, and Conjugate ratio reaches 99%. React as follows:
(1) amino acid whose activation (HOBt/DCC method)
The aminoacid of Fmoc protection
(2) aminoacid is connected to resin
(3) slough amino acid whose Fmoc and protect base
(4) another amino acid whose activation (HOBt/DCC method)
(5) coupling
Peptide-the resin of new coupling
(6) step (3) to (5) is repeated until end of synthesis.
Respectively obtain the peptide resin 178mg of the peptide resin 214mg and HSP90 Α-2 peptide fragment (2) of HSP90 α-2 peptide fragment (1).
(7) peptide resin:
Peptide chain is cut with TFA (trifluoroacetic acid), scavenger is made with EDT (2.5 volume %), thioanisole (2.5 volume %), at room temperature reaction 3.0 hours, remove cutting reagent, extract with ether again, respectively obtain the crude product of HSP90 α-2 peptide fragment (1) and (2).
Two, the purification of HSP90 α-2 epitope peptide (1) and (2) crude product:
Adopt high performance liquid chromatography separation purification:
Condition: chromatographic column: C810 × 100mm, is purchased from Waters, US
Chromatograph: Waters600, 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/ minute
Gradient: 20-60%B, 30 minutes
HPLC (high performance liquid chromatography) analyzes
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/ minute
Gradient: 0-60%B, 30 minutes
The purity of the HSP90 α-2 epitope peptide (1) and (2) of the peptide piecewise analysis result display present invention is more than 95%.
Three, the qualification of HSP90 α-2 epitope peptide (1) and (2)
1. utilize mass spectrum to measure the molecular weight of HSP90 α-2 epitope peptide (1) and (2) of purification gained respectively.
(1) reagent raw material
TFA (trifluoroacetic acid is purchased from sigma company)
HCCA (alpha-cyano-4-hydroxycinnamic acid, be purchased from sigma company)
Acetonitrile (is purchased from Chemical Reagent Co., Ltd., Sinopharm Group)
(2) instrument
Matrix Assisted Laser Desorption ionization time-of-flight mass spectrometer MALDI-TOF-MS (model: REFLEXIII, Bruker company of Germany);
(3) matrix liquid: α-CCA is dissolved in the 50%ACN solution containing 0.1%TFA, makes saturated solution, centrifugal, take 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: take the sample of the above-mentioned purified polypeptide of 1 μ L (1) and (2) respectively, each mix with the saturated stromal supernatant mixing equal-volume of 1 μ L, take 1 μ L point respectively on sample target, send in ion source and detect.
Result, the molecular weight recording gained HSP90 α-2 epitope peptide (1) is 2404.5, the molecular weight of HSP90 α-2 epitope peptide (2) is 2483.5, consistent with theoretical molecular 2404.35,2483.39, it was demonstrated that synthesis polypeptide namely for the purpose of product.
2. the sequence identifying gained HSP90 α-2 epitope peptide (1) and (2) respectively is measured by peptide sequence.
(1) principle: the ultimate principle of polypeptid acid sequence analysis is Edman degraded, is a circulating chemical reaction process. Including three main chemical steps: (1) coupling: the N-end residue of phenyl isothiocyanate and proteins and peptides reacts, form phenylamino formyl sulfide (PTC) derivant, i.e. PTC-peptide. (2) cyclisation cracking: PTC-peptide cyclisation cracks. (3) convert: thiazole purine ketone phenylamino (ATZ) is converted into the different sulfur urine amino acid of benzene (PTH-aminoacid). Staying the peptide decreasing an amino acid residue in the solution to repeat and carry out above-mentioned course of reaction, whole sequencing procedure is all automatically carried out by sequenator now.
(2) instrument: American AB I company 491 type protein/polypeptide-terminal amino acid sequenator
(3) reagent raw material
Phenyl isothiocyanate PITC, is purchased from sigma company
Normal heptane, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Trimethylamine TMA aqueous solution, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
TFA (trifluoroacetic acid is purchased from sigma company)
Ethyl acetate, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Chlorobutane, is purchased from sigma company
Acetonitrile, is purchased from Chemical Reagent Co., Ltd., Sinopharm Group
(4) measure
Undertaken by instrument description.
Result: identified, the sequence of gained HSP90 α-2 epitope peptide (1) and (2) is respectively as follows:
(1) Y-E-K-E-S-E-D-K-P-E-I-E-D-V-G-S-D-E; With
(2)Y-R-G-I-D-E-D-D-P-T-A-D-D-T-S-A-A-V-T-E。
This result is consistent with target section of synthesized peptide.
Embodiment 2: be connected to prepare HSP90 α-2 antigen (1) and (2) with carrier protein by the HSP90 α-2 epitope peptide (1) and (2) of embodiment 1 gained respectively, utilize gained antigen (1) and (2) immune animal respectively, thus utilizing antigen (1) to prepare specific monoclonal antibody and polyclonal antibody, and antigen (2) is utilized to prepare specific monoclonal antibody and polyclonal antibody.
1. the preparation of antigen: by BDB (Bis-diazotizedbenzidinedichloride) method HSP90 α-2 peptide fragment (1) is connected with carrier protein KLH (keyhole limpet hemocyanin) (deriving from sigma company) respectively with (2) and prepares into HSP90 α-2 antigen (1) and (2).
Take HSP90 α-2 peptide fragment (1) or (2) 10.0mg, dissolve with 1ml0.1MPBS buffer (pH7.4); KLH10mg, dissolves with 0.2M borate buffer solution (pH9.0) 20ml; Then both are mixed, be cooled to 0 DEG C, take BDBCl2110 μ L, react 1.5h, subpackage after dialysed overnight ,-20 DEG C of preservations under room temperature.
In the present embodiment, the formula of PBS is: the Na of 0.2mol/L2HPO481ml adds the NaH of 0.2mol/L2PO419ml mixes.
The formula of borate buffer solution is: 0.05mol/L Borax 80ml, adds 0.2mol/L boric acid 20ml and mixes.
2. immune animal prepares monoclonal antibody:
2.1. take after the HSP90 α-2 antigen (1) and (2) (immunogen) of above-mentioned preparation is sufficiently mixed with isopyknic Freund's complete adjuvant (purchased from Shanghai Yuan Ju biotech firm) respectively, immunity Balb/c mice, 50 μ g antigens/only, subcutaneous multi-point injection. serum titer is surveyed after 4 weeks, select the good mice booster immunization again of immunoreactivity: take after antigen is sufficiently mixed with isopyknic incomplete Freund's adjuvant (purchased from Shanghai Yuan Ju biotech firm), antigen dose 25 μ g/ is only, subcutaneous multi-point injection, the number of times of booster immunization is 6 times, each At intervals of two to three weeks, additionally booster immunization twice continuously before merging, every minor tick 1-2 week, extracting spleen cell and Sp2/0 myeloma cell are merged with 50%PEG (MW4000) (purchased from Central Plains chemical company) mediation according to a conventional method afterwards, and select to cultivate with HAT conditioned medium (purchased from sigma company). CO is put into after fusion2Incubator is cultivated after 9～11 days for 37 DEG C, the cell clone that appearance is bigger in the hole in. Within 11 days, start to screen with indirect ELISA. The hole that primary dcreening operation is positive utilizes limiting dilution assay carry out 4 time cloningizations and cultivates (even if a large amount of schizogamy of cell after screening), afterwards amplifying cells, frozen, preparation ascites.
2.2. Balb/c mice norphytane (purchased from sigma company) 0.5ml/ is only processed, one week pneumoretroperitoneum inoculation hybridoma 2 × 106Individual/only, collect ascites after 10 days.
2.3. measuring antibody titer: measure the titer of the monoclonal antibody (1) utilizing HSP90 α-2 antigen (1) to prepare with indirect ELISA method, the titer of result display monoclonal antibody reaches more than 1:32000.
The titer utilizing HSP90 α-2 antigen (2) monoclonal antibody (2) prepared also utilizes identical method to be measured, and its titer also reaches more than 1:32000.
3. immune animal prepares polyclonal antibody:
3.1. three monthly ages, body weight are selected to be about the New Zealand white rabbit of about 2kg as immune animal. In fundamental immunity, the HSP90 α-2 antigen (1) and (2) (immunogen) of above-mentioned for 1-2mg preparation is mixed with isopyknic complete Freund's adjuvant (purchased from Shanghai Yuan Ju biotech firm) respectively-fully emulsified after carry out multiple spot subcutaneous injection at rabbit back. Every 4 weeks booster immunizations once, booster immunization 6 times, after antigen is fully emulsified with incomplete Freund's adjuvant (purchased from Shanghai Yuan Ju biotech firm), with 100 μ g/ only in back multiple spot subcutaneous injection. Carotid artery blood-letting in 10th day after final boost, separates serum.
3.2. measuring antibody titer: measure the titer of the polyclonal antibody (1) utilizing HSP90 α-2 antigen (1) to prepare with indirect elisa method, result display antibody titer reaches more than 1:32000.
The titer utilizing HSP90 α-2 antigen (2) polyclonal antibody (2) prepared also utilizes identical method to be measured, and its titer also reaches more than 1:32000.
3.3. take blood and separate serum: carotid artery intubates and takes blood, separating serum.
4. separate antibody purification: after ammonium sulfate precipitation, then through ProteinG (purchased from sigma company) affinity purification.
5. lyophilizing after antibody subpackage, cryopreservation.
Embodiment 3: the specificity identification of people HSP90 α-2 monoclonal antibody (1) and (2)
Detect with ELISA. Respectively with people's HSP90 α-2 albumen, S-100B albumen, neuronspecific enolase NSE (all purchased from Shanghai Lian Shuo company) for detecting antigen coated elisa plate, the specific reaction of prepared HSP90 α-2 monoclonal antibody (1) and (2) and this people's HSP90 α-2 albumen is detected respectively by ELISA, making negative control with normal BALB/c mouse serum, PBS liquid makes blank.
Result: HSP90 α-2 monoclonal antibody (1) and (2) only reacts with HSP90 α-2 for positive (P/N > 2.1) respectively, and react for negative with S-100B albumen, neuronspecific enolase NSE, illustrate that the HSP90 α-2 monoclonal antibody (1) and (2) of the present invention is respectively provided with specificity.
Embodiment 4: the specificity identification of people HSP90 α-2 polyclonal antibody (1) and (2)
The method identical with above-mentioned qualification monoclonal antibody specificity is utilized to identify.
Result shows: HSP90 α-2 polyclonal antibody (1) and (2) reacts with HSP90 α-2 respectively for positive (P/N > 2.1), and react for negative with S-100B albumen, neuronspecific enolase NSE, illustrate that the HSP90 α-2 polyclonal antibody (1) and (2) of the present invention is respectively provided with specificity.
Embodiment 5: utilize HSP90 α-2 monoclonal antibody and HSP90 α-2 polyclonal antibody preparation HSP90 α-2 external diagnosis reagent case.
In the present embodiment, using the monoclonal antibody (1) that utilizes HSP90 α-2 epitope peptide (1) to prepare in embodiment 2 as the coated antibody in this test kit; Using the polyclonal antibody (2) that utilizes HSP90 α-2 epitope peptide (2) to prepare in embodiment 2 as binding antibody.
Preparation and the operation of HSP90 α-2 external diagnosis reagent case are as follows:
1. the preparation of various buffer and reagent:
A, it is coated buffer: the CB (carbonate buffer solution) of 0.050M, pH9.6
Na2CO3: 16.0 grams
NaHCO3: 29.0 grams
Distill water-soluble to 1000ml
B, sample/lavation buffer solution: pH7.2 10 × PBS-Tween20
Na2HPO4·12H2O:58 gram
KH2PO4: 4 grams
NaCl:100 gram
KCl:4 gram
Distill water-soluble to 1000ml
Add Tween20:20ml
C, enzyme marker diluent:
10 × PBS-Tween20:10ml
FCS (calf serum): 20ml
Distill water-soluble to 1000ml
Enzyme stabilizers (is purchased from Shanghai Xi Bao company): 1 gram
Biological preservative (is purchased from Shanghai Xi Bao company): 1ml
D, developer A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
Distill water-soluble to 1000ml
Tween20:10ml
E, developer B:
Citric acid: 120 grams
EDTA-2Na:1 gram
TMB 2HCl:2 gram
Distill water-soluble to 1000ml
F, stop buffer: 2MH2SO4
Concentrated sulphuric acid (95-98%): 22.2ml
Distilled water: 177.3ml
Concentrated sulphuric acid is slowly dropped in distilled water by timing, and limit edged shakes up.
2. the preparation of pre-coated plate:
HSP90 α-2 monoclonal antibody (1) is dissolved in the carbonate buffer solution of 0.05M of pH=9.6, make pre-coated liquid, 100 μ l are added by 0.1 μ g/ hole in the upper every hole of ELISA Plate (being purchased from Shenzhen Jin Canhua company), put 4 DEG C to place 18-24 hour, take out, get rid of and be coated liquid, with sample/lavation buffer solution washing, after 1 (w/v) %BSA-0.05M ethanolamine closing 16 hours, dried overnight, load evacuation in aluminide-coating bag seal, be placed in 4 DEG C of preservations.
3. the dilution ratio of binding antibody (HSP90 α-2 polyclonal antibody (2)) and enzyme connection thing (goat anti-rabbit igg antibody of horseradish peroxidase-labeled) (purchased from Beijing company of Zhong Shan Golden Bridge) is determined by square formation titration experiments, and the goat anti-rabbit igg antibody of horseradish peroxidase-labeled uses enzyme marker diluted.
4. the composition of test kit:
Pre-coated plate: 48/96 hole
HSP90 α-2 calibration object (raw material is purchased from Shanghai Lian Shuo company): 7: 7 × 1.0ml (concentration is 25ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml, 0.5ng/ml, 0.25ng/ml respectively)
HSP90 α-2 binding antibody: 1 × 10ml (dilutes through 1:5000)
Enzyme connection thing: 1 × 10ml (diluting through 1:5000)
Concentrated cleaning solution (25 × PBS-Tween20): 1 × 20ml
Developer A:1 × 6.0ml
Developer B:1 × 6.0ml
Stop buffer: 1 × 6.0ml
5. the operating procedure of test kit:
Each hole of pre-coated plate is separately added into blood sample to be checked and standard substance 100 μ l/ hole, is diplopore, hatch 60 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry. In each hole, add HSP90 α-2 binding antibody 100 μ l/ hole, hatch 30 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry. In each hole, add enzyme connection thing 100 μ l/ hole again, hatch 30 minutes for 37 DEG C, wash 5 times with 1 × lavation buffer solution, pat dry. Add developer A, B liquid, each 50 μ l in every hole, mixing, hatch 15 minutes for 37 DEG C. Add stop buffer 50 μ l/ hole and terminate reaction, join detector (model RT-6000 is purchased from Lei Du company) with enzyme and detect absorbance with dual wavelength (450nm, 620nm).
6. result judges:
Table 1: standard concentration and corresponding mean light absorbency (OD) value
| Concentration ng/ml | 0.25 | 0.5 | 1 | 2 | 5 | 10 | 25 |
| Mean OD value | 0.066 | 0.123 | 0.214 | 0.385 | 0.714 | 1.236 | 2.155 |
With the logarithm value drawing standard curve of standard concentration and corresponding absorbance, the R of standard curve2=0.976.
HSP90 α-2 concentration results in the specimen detected is calculated according to standard curve.
30 example tumour patients and 81 example healthy persons carry out serum HSP90 α-2 detection in a manner described, and HSP90 α-2 content in affinity antibody to SpA is apparently higher than normal healthy controls group, and difference statistically significant (P < 0.01), in Table 2.
2: two groups of sample HSP90 α-2 concentration of table compare
By data above it can be seen that the test kit of the present invention can effectively and specifically detect HSP90 α-2 content in serum, thus HSP90 α-2 content difference detected between tumour patient and normal person, thus can determine whether the generation of tumor.
Claims (9)
1. people HSP90 α-2 epitope peptide, the aminoacid sequence of wherein said HSP90 α-2 epitope peptide is:
Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-Gly-Ser-Asp-Glu。
2. HSP90 α-2 antigen, it is by making people's HSP90 α-2 epitope peptide described in claim 1 be prepared from carrier protein couplet.
3. people HSP90 α-2 antibody, it is the monoclonal antibody that is prepared from of HSP90 α-2 antigen described in claim 2 or polyclonal antibody.
4. people's HSP90 α-2 antibody according to claim 3 application on preparation people's HSP90 α-2 external diagnosis reagent case.
5. people HSP90 α-2 external diagnosis reagent case, it comprises people's HSP90 α-2 antibody described in claim 3 as coated antibody or binding antibody.
6. people's HSP90 α-2 external diagnosis reagent case according to claim 5, wherein said test kit also comprises another kind of people's HSP90 α-2 antibody, and the one in described people's HSP90 α-2 antibody and described another kind of people's HSP90 α-2 antibody is as coated antibody, another one is as binding antibody, and wherein said another kind of people's HSP90 α-2 antibody is prepared from by another kind of HSP90 α-2 antigen of the epitope comprised as shown in following sequence:
Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Ser-Ala-Ala-Val-Thr-Glu。
7. HSP90 α-2 external diagnosis reagent case according to claim 5 or 6, wherein said coated antibody is monoclonal antibody.
8. HSP90 α-2 external diagnosis reagent case according to claim 5 or 6, wherein said binding antibody is polyclonal antibody.
9. HSP90 α-2 external diagnosis reagent case according to claim 5 or 6, wherein said test kit also comprises the anti antibody of enzyme labelling.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410670399.4A CN105669836B (en) | 2014-11-20 | 2014-11-20 | People HSP90 α -2 epitope peptide, antigen, antibody, application and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410670399.4A CN105669836B (en) | 2014-11-20 | 2014-11-20 | People HSP90 α -2 epitope peptide, antigen, antibody, application and kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105669836A true CN105669836A (en) | 2016-06-15 |
| CN105669836B CN105669836B (en) | 2019-05-21 |
Family
ID=56958015
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410670399.4A Active CN105669836B (en) | 2014-11-20 | 2014-11-20 | People HSP90 α -2 epitope peptide, antigen, antibody, application and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105669836B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215323A (en) * | 2007-12-28 | 2008-07-09 | 首都医科大学宣武医院 | Human AD 7C-NTP antigenic determinant polypeptide, antibody and application thereof in diagnostic kit |
| CN101942017A (en) * | 2009-07-07 | 2011-01-12 | 清华大学 | Novel tumor marker |
-
2014
- 2014-11-20 CN CN201410670399.4A patent/CN105669836B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215323A (en) * | 2007-12-28 | 2008-07-09 | 首都医科大学宣武医院 | Human AD 7C-NTP antigenic determinant polypeptide, antibody and application thereof in diagnostic kit |
| CN101942017A (en) * | 2009-07-07 | 2011-01-12 | 清华大学 | Novel tumor marker |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105669836B (en) | 2019-05-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111197040B (en) | Chitinase 3-like protein 1 (CHI 3L 1) epitope peptide, antigen, antibody, application and kit | |
| CN106556592B (en) | The chemical luminescence reagent kit and preparation method thereof of quantitative detection people TK1 | |
| CN108084257B (en) | Human atresia protein epitope peptide, antigen, antibody, kit and application | |
| CN104142404B (en) | Fluorescence immune chromatography test paper of detection people's GFAP albumen and preparation method thereof | |
| CN106554950A (en) | People's TK1 epitope peptides, antigen, antibody, application and test kit | |
| EP2105447A1 (en) | Avian-derived antibody capable of binding specifically to human hmgb1, immunological determination method for human hmgb1, and immunological determination reagent for human hmgb1 | |
| CN112457392B (en) | Soluble ST2 protein antigenic determinant polypeptide and application thereof | |
| CN100554278C (en) | Human tumor M 2-type pyruvate kinase antigen determinant polypeptide, antibody and the application on diagnostic kit thereof | |
| CN105606814B (en) | Detect fluorescence immune chromatography test paper of the albumen of people ApoE ε 4 and preparation method thereof | |
| CN108929374B (en) | Human HBP epitope peptide, antigen, antibody, application and kit | |
| CN105646660B (en) | People HSP90 α epitope peptide, antigen, antibody, application and kit | |
| CN104231052B (en) | People Lp PLA2 epitope peptides, antigen, antibody, purposes and kit | |
| CN104418937B (en) | People PGII epitope peptides, antigen, antibody, purposes and kit | |
| CN104140464B (en) | People's GFAP epitope peptide, antigen, antibody, purposes and test kit | |
| CN105652010B (en) | Detect fluorescence immune chromatography test paper of the albumen of people HSP90 α 1 and preparation method thereof | |
| CN107446040B (en) | Human ST2 epitope peptide, antigen, antibody, kit and application | |
| CN104422773B (en) | Fluorescence immune chromatography test paper of detection people's PGI albumen and preparation method thereof | |
| CN105669834B (en) | People HSP90 α -1 epitope peptide, antigen, antibody, application and kit | |
| CN110317246B (en) | Human MOG epitope peptide, antigen, antibody, application and chemiluminescence kit | |
| CN110470847B (en) | ELISA kit for quantitatively detecting glutathione reductase and detection method thereof | |
| CN104418939B (en) | People PGI epitope peptides, antigen, antibody, purposes and kit | |
| CN105669836B (en) | People HSP90 α -2 epitope peptide, antigen, antibody, application and kit | |
| CN105652011B (en) | Detect fluorescence immune chromatography test paper of the albumen of people HSP90 α 2 and preparation method thereof | |
| CN105669835B (en) | 4 epitope peptide of people ApoE- ε, antigen, antibody, application and kit | |
| CN110317254A (en) | People MBP epitope peptide, antigen, antibody, application and chemical luminescence reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |