CN105646660A - Human HSP90[alpha] antigen epitope peptide, antigen and antibody, application of antibody and kit - Google Patents
Human HSP90[alpha] antigen epitope peptide, antigen and antibody, application of antibody and kit Download PDFInfo
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Abstract
The invention relates to a human HSP90[alpha] antigen epitope peptide, antigen and antibody, an application of the antibody and a kit. The amino acid sequence of the human HSP90[alpha] antigen epitope peptide provided by the invention is a sequence shown in SEQ ID NO.2 of a sequence table. The human HSP90[alpha] antigen provided by the invention is prepared by coupling of the human HSP90[alpha] antigen epitope peptide and a protein carrier. The human HSP90[alpha] monoclonal antibody or polyclonal antibody provided by the invention is prepared from the human HSP90[alpha] antigen provided by the invention. The human HSP90[alpha] monoclonal antibody or polyclonal antibody provided by the invention is used for preparation of the human HSP90[alpha] in-vitro diagnostic kit. The human HSP90[alpha] antigen epitope peptide provided by the invention has good antigenicity, the antigen (immunogen) prepared from the human HSP90[alpha] antigen epitope peptide is used for immunization of animals and the highly specific monoclonal antibody and polyclonal antibody can be produced, therefore, the human HSP90[alpha] antigen epitope peptide can be applied to in-vitro detection of human HSP90[alpha].
Description
Technical field
The application on preparation people's HSP90 �� external diagnosis reagent case of the HSP90 alpha specific antigen the invention belongs to chemiluminescent polypeptide and field of immunology, be specifically related to HUMAN HEAT SHOCK PROTEINS 90 �� (HSP90 ��) epitope peptide, prepared with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, described antibody and people's HSP90 �� external diagnosis reagent case.
Background technology
In recent years, the research of tumor markers has been caused the attention of many investigators. Find a kind of reliably, non-intrusion type, the generation of tumour can be reflected, common aspiration that the tumor markers of proliferation and differentiation is numerous investigator. By the research to blood, tissue juice, find that the generation of tumour, proliferation and differentiation can be predicted by some tumor markerses at present. Wherein, HUMAN HEAT SHOCK PROTEINS 90 �� (HSP90 ��) is exactly such tumor markers.
Heat shock protein72 comprises Heat shock protein72-1 and Heat shock protein72-2, they are the important albumen in heat-shock protein family, it participates in, maintains conformation and the function of multiple protein in cell as molecular chaperones, regulating cell growth, break up and wither die etc. in play an important role. In recent years finding, the pernicious transformation of tumour, growth, propagation and invasion and attack are had important effect as chaperone by Heat shock protein72. Having many reports to confirm, in body tumor tissue, the expression of heat shock protein(HSP) there occurs change. In the tumor tissues of different sites, the change of heat shock protein expression is also inconsistent. Find the grade malignancy of the HSP90 alpha content in affinity antibody to SpA and tumour at present, especially shift positive correlation. Therefore, HSP90 �� is hopeful the mark as diagnosing tumor and prognosis.
The optimal method of the HSP90 alpha levels in detection serum is immunodetection. Therefore, find the suitable HSP90 �� epitope peptide with immunogenicity, the preparation HSP90 �� antigen of specificity and antibody emphasis.
Summary of the invention
For solving problem existing in above-mentioned prior art, the present invention provides a kind of people's HSP90 �� epitope peptide, the HSP90 alpha specific antigen prepared with this epitope peptide and corresponding monoclonal antibody or polyclonal antibody, it is in the application prepared on people's HSP90 �� test kit, and people's HSP90 �� external diagnosis reagent case.
Specifically, the present invention provides:
A kind of people's HSP90 �� epitope peptide, the aminoacid sequence of wherein said HSP90 �� epitope peptide is:
Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Ser-Ala-Ala-Val-Thr-Glu��
Present invention also offers a kind of HSP90 �� antigen, it is by making described people's HSP90 �� epitope peptide and carrier protein couplet be prepared from.
Present invention also offers a kind of people's HSP90 Alpha antibodies, it is the monoclonal antibody or polyclonal antibody that are prepared from by described HSP90 �� antigen, and wherein said HSP90 �� antigen is by making described people's HSP90 �� epitope peptide and carrier protein couplet be prepared from.
Present invention also offers the application of described people's HSP90 Alpha antibodies on preparation people's HSP90 �� external diagnosis reagent case.
Present invention also offers a kind of people's HSP90 �� external diagnosis reagent case, it comprises described people's HSP90 Alpha antibodies as coated antibody or binding antibody.
Preferably, described test kit also comprises another kind of people's HSP90 Alpha antibodies, and the one in above-mentioned people's HSP90 Alpha antibodies and another kind of described people's HSP90 Alpha antibodies is as coated antibody, another one is as binding antibody, and another kind of wherein said people's HSP90 Alpha antibodies is prepared from by another kind of HSP90 �� antigen comprising the epitope as shown in following sequence:
Tyr-Pro-Arg-Asp-Arg-Leu-Asp-Pro-Arg-Pro-Gly-Ser-Pro-Ser-Glu-Ala-Ser��
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 following advantages and positively effect:
1. people's HSP90 �� epitope peptide of the present invention has good antigenicity, and antigen (immunogen) immune animal prepared with it can produce monoclonal antibody and the polyclonal antibody of high degree of specificity.
2. the HSP90 alpha monoclonal antibodies prepared with the present invention and polyclonal antibody can high special HSP90 �� in blood sample be combined.
3. people's HSP90 �� external diagnosis reagent case of the present invention can detect the level of the HSP90 �� in serum effectively, can be used to judge the grade malignancy of tumour, and transfer and prognosis is predicted.
Embodiment
Below by way of the description of embodiment, the invention will be further described, but this is not limitation of the present invention, those skilled in the art are according to the basic thought of the present invention, various modifications may be made or improves, as long as but do not depart from the basic thought of the present invention, all within the scope of the present invention.
One, people HSP90 �� epitope peptide
People's HSP90 �� albumen described herein is known in the art, and its aminoacid sequence is known in the art, it is possible to find in the expert data storehouses such as NCBI.
The present invention provides people HSP90 �� epitope peptide (1) and (2), its aminoacid sequence respectively as shown in sequence table SEQ IDNo.1 and SEQIDNo.2, for:
(1) Y-P-R-D-R-L-D-P-R-P-G-S-P-S-E-A-S; 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 a large amount of theoretical investigationes and experiment, and final screening obtains two kinds and has good antigenic epitope peptide.
One section that HSP90 �� epitope peptide (1) is the 29th to the 45th, people's HSP90 ��-1 albumen (NCBI accession number NP_001017963.2) N end contains 17 amino acid whose peptide sections.
The aminoacid sequence of HSP90 �� epitope peptide (2) belongs to a section in HSP90 ��-1 albumen (NCBI accession number NP_001017963.2) aminoacid sequence. Specifically, HSP90 �� epitope peptide (2) comprises the peptide section of the 819th to the 836th, people's HSP90 ��-1 albumen (NCBI accession number NP_001017963.2) C end, and add Y and R at the N-terminal of this peptide section, thus constitute containing 20 amino acid whose HSP90 �� epitope peptide (2).
The aminoacid sequence of HSP90 �� epitope peptide (2) also belongs to a section in HSP90 ��-2 albumen (NCBI accession number NP_005339.3) aminoacid sequence. Specifically, HSP90 �� epitope peptide (2) comprises the peptide section of the 697th to the 714th, people's HSP90 ��-2 albumen (NCBI accession number NP_005339.3) C end, and add Y and R in the N section of this peptide section, thus constitute this HSP90 �� epitope peptide (2).
It is strong and be easy to the feature of synthesis that above-mentioned peptide Duan Jun has wetting ability, antigenicity.
At present, the present invention studies discovery, and the HSP90 �� epitope peptide of the present invention has following function:
1. there is antigenicity; 2. the antibody of specificity after being connected, is produced with carrier proteins as immunogen stimulating animal; 3. the antibody prepared with epitope peptide can be combined with people HSP90 �� specifically.
The preparation method of the HSP90 �� epitope peptide of the present invention can by chemical synthesis: utilize American AB I431A type polypeptide automatic DNA synthesizer DNA, by Solid phase synthesis epitope peptide. The epitope peptide (1) of the present invention and the molecular weight of (2) are respectively 1898.91,2483.39, and available mass spectrum is determined, and measure the epitope peptide sequence synthesized by qualification by peptide sequence. The purity of peptide section can be evaluated with thin-layer chromatography and high performance liquid chromatography, and measures the concentration of epitope peptide.
Two, HSP90 �� antigen
Present invention also offers HSP90 �� antigen, it is prepared from by the one in people HSP90 �� epitope peptide (1) and (2) making the present invention and carrier protein couplet. Specifically, the present invention provides HSP90 �� antigen (1) and (2), and by making, people HSP90 �� epitope peptide (1) of the present invention and carrier protein couplet are prepared from described HSP90 �� antigen (1); By making, people HSP90 �� epitope peptide (2) of the present invention and carrier protein couplet are prepared from described HSP90 �� antigen (2). The HSP90 �� antigen of the present invention has immunogenicity and specificity, is a kind of immunogen, can be used to immune animal thus prepare the HSP90 Alpha antibodies of specificity. In the present invention, the example of available carrier proteins comprises 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 relatively far away with immune animal sibship, not easily causes cross reaction with it as carrier proteins, is therefore preferred.
Three, HSP90 alpha monoclonal antibodies, HSP90 �� polyclonal antibody and people's HSP90 �� external diagnosis reagent case
Present invention also offers people's HSP90 Alpha antibodies, comprising people's HSP90 alpha monoclonal antibodies and people's HSP90 �� polyclonal antibody, they can utilize HSP90 �� antigen (1) or (2) (immunogen) the immune animal preparation of the present invention separately and obtain. Preparation method can adopt the ordinary skill in the art, specifically can see embodiment 2.
Herein, term " another kind of HSP90 �� antigen " is HSP90 �� antigen (1).When the present invention people's HSP90 Alpha antibodies by HSP90 �� antigen (1) (namely, another kind of HSP90 �� antigen) preparation and time, this antibody can be called " people's HSP90 Alpha antibodies (1) " (or be called for short antibody (1)) or " another kind of people's HSP90 Alpha antibodies ".
The HSP90 alpha monoclonal antibodies of the present invention and polyclonal antibody may be used for preparation people's HSP90 �� external diagnosis reagent case, HSP90 �� in tissue, cell or body fluid can be detected by this test kit based on immunization method, it is preferable that is detected by the HSP90 �� in blood preparation.
Therefore, the present invention provides a kind of people's HSP90 �� external diagnosis reagent case, and it comprises people's HSP90 alpha monoclonal antibodies or the polyclonal antibody of the present invention.
The known immunization experiment method that can be used for Clinical Laboratory mainly comprises following several at present: ELISA method, chemoluminescence method, fluorescent chromatographic method, colloid gold immune assay method etc.
And ELISA method comprises following several types: double antibody sandwich method detectable antigens, dual-antigen sandwich method detection antibody, indirect method survey antibody, competition law surveys antibody, competition law surveys antigen, catch bag is surveyed antibody etc. by method.
People's HSP90 �� external diagnosis reagent case of the present invention preferably adopts ELISA double antibody sandwich method to detect HSP90 �� albumen. The test kit of the present invention 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 �� external diagnosis reagent case adopts people's HSP90 alpha monoclonal antibodies of the present invention as coated antibody. At this, term " coated antibody " refers to the antibody on the enzyme plate being coated in solid phase. In addition, described people's HSP90 �� external diagnosis reagent case is it is also preferred that comprise people's HSP90 �� polyclonal antibody using as binding antibody, wherein, when described binding antibody derives from the one in people HSP90 �� 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 the anti-antibody of antigen to be measured and enzyme labelling. 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, alkaline phosphatase etc.
Such as, in the test kit of the present invention, it is also possible to comprise any reagent needed for detection or instrument, pre-coated plate, washings, developer, stop buffer etc.
The content of the present invention is further explained and described, but these examples should not be understood to the restriction to protection scope of the present invention below by way of the mode of example.
Embodiment
Unless otherwise explanation, the following stated solution is the aqueous solution, and the percentage ratio in solution is percent by volume.
The preparation of embodiment 1:HSP90 �� epitope peptide (1) and (2).
Preparation method's chemical synthesis: utilize American AB I431A type polypeptide automatic DNA synthesizer DNA, synthesizes HSP90 �� epitope peptide (1) and (2) respectively by solid phase method. The purity high performance liquid chromatography of epitope peptide is evaluated, and measures the concentration of peptide section. The epitope peptide (1) of the present invention and the molecular weight of (2) are respectively 1898.91,2483.39, utilize mass spectrum to determine, measure the peptide sequence synthesized by qualification by peptide sequence.
One, the synthesis of HSP90 �� epitope peptide (1) and (2)
Above-mentioned peptide section 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 linkage form by the carboxyl being synthesized the carboxyl-terminus amino acid of peptide chain; then using this amino acid being combined on solid phase carrier as amino group part; through deaminize protecting group and with the reaction of excessive activated carboxyl group part, connect long 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 HSP90 �� 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, can purchased from sigma company)
Fmoc-AA (amino acid of 9-fluorenylmethoxycarbonyl carbonyl acyl group protection, can purchased from Merck company)
NMP (N-methyl pyrrolidone, can purchased from sigma company)
DCM (methylene dichloride, can purchased from Central Plains chemical company)
MeoH (methyl alcohol, can purchased from Central Plains chemical company)
Piperidines (Piperidine, can purchased from sigma company)
DMAP (dimethyl aminopyridine, can purchased from sigma company)
HOBT (hydroxybenzotriazole, can purchased from sigma company)
DCC (dicyclohexylcarbodiimide, can purchased from sigma company)
TFA (trifluoroacetic acid, can purchased from sigma company)
EDT (1,2-dithioglycol, can purchased from sigma company)
Thio phenyl first ether, can purchased from Guangzhou Wei Bai Chemical Co., Ltd.
Crystallization phenol, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Acetonitrile, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
2. use instrument:
Polypeptide automatic DNA synthesizer DNA, model 431A, can purchased from ABI company
Rotary Evaporators, model R-201, can purchased from Shanghai Shen Shun company
High performance liquid chromatograph, Waters600, can purchased from Waters, US
Freeze drier, model VFD-2000, can purchased from Beijing rich doctor Kanggong department
3. synthetic method and process:
Taking HMP resin 100mg, replacing equivalent is 1.0meq, is placed in by 0.1mmol in the reaction chamber of American AB I431A type polypeptide automatic DNA synthesizer DNA, is automatically linked in sequence by different by specific amino acid by synthesizer, and Conjugate ratio reaches 99%. React as follows:
(1) amino acid whose activation (HOBt/DCC method)
The amino acid of Fmoc protection
(2) amino acid is connected on resin
(3) the Fmoc protecting group of deaminize acid
(4) another amino acid whose activation (HOBt/DCC method)
(5) coupling
Peptide-the resin of new coupling
(6) repeating step (3) to (5) is until synthesis terminates.
Obtain the peptide resin 178mg of the peptide resin 214mg and HSP90 �� peptide section (2) of HSP90 �� peptide section (1) respectively.
(7) peptide resin:
Peptide chain is cut with TFA (trifluoroacetic acid), scavenging agent is made with EDT (2.5 volume %), thio phenyl first ether (2.5 volume %), at room temperature react 3.0 hours, remove cutting reagent, again by extracted with diethyl ether, obtain the crude product of HSP90 �� peptide section (1) and (2) respectively.
Two, the purifying of HSP90 �� epitope peptide (1) and (2) crude product:
Adopt high performance liquid chromatography separation and purification:
Condition: chromatographic column: C810 �� 100mm, can purchased from Waters, US
Chromatographic instrument: Waters600, Waters, US
Moving phase: A:0.1%TFA (trifluoroacetic acid) aqueous solution
B:0.1%TFA (trifluoroacetic acid) is in 60% acetonitrile
Determined wavelength: 214nm
Flow velocity: 4ml/ minute
Gradient: 20-60%B, 30 minutes
HPLC (high performance liquid chromatography) analyzes
Chromatographic column: C184.6 �� 150mm, can purchased from Waters, US
Moving phase: A:0.1%TFA (trifluoroacetic acid) aqueous solution
B:0.1%TFA (trifluoroacetic acid) is in acetonitrile
Determined wavelength: 214nm
Flow velocity: 1ml/ minute
Gradient: 0-60%B, 30 minutes
The purity of HSP90 �� epitope peptide (1) and (2) that peptide section analytical results shows the present invention is more than 95%.
Three, the qualification of HSP90 �� epitope peptide (1) and (2)
1. utilize mass spectrum to measure the molecular weight of HSP90 �� epitope peptide (1) and (2) of purifying gained respectively.
(1) reagent raw material
TFA (trifluoroacetic acid, can purchased from sigma company)
HCCA (alpha-cyano-4-hydroxycinnamic acid, can purchased from sigma company)
Acetonitrile (can purchased from Chemical Reagent Co., Ltd., Sinopharm Group)
(2) instrument
Ground substance assistant laser desorption ionization time-of-flight mass spectrometer MALDI-TOF-MS (model: REFLEXIII, Bruker company of Germany);
(3) matrix liquid: ��-CCA is dissolved in the 50%ACN solution containing 0.1%TFA, makes saturated solution, centrifugal, get supernatant;
(4) instrument testing conditions: reflection detection mode; The long 3m of tof tube; Nitrogen laser: wavelength 337nm, acceleration voltage 20KV; Reflected voltage 23KV.
(5) operation steps: the sample getting the above-mentioned purified polypeptide of 1 �� L (1) and (2) respectively, mix with the saturated matrix supernatant liquor mixing equal-volume of 1 �� L separately, get 1 �� L point respectively on sample target, send in ion source and detect.
Result, the molecular weight recording gained HSP90 �� epitope peptide (1) is 1899.1, the molecular weight of HSP90 �� epitope peptide (2) is 2483.5, consistent with theoretical molecular 1898.91,2483.39, proves product for the purpose of improvement on synthesis is namely.
2. the sequence identifying gained HSP90 �� 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 the chemical reaction process of a circulation formula. Comprise three main chemical steps: (1) coupling: the N-end residue reaction of thiocarbanil and proteins and peptides, forms phenylamino sulphur formyl (PTC) derivative, i.e. PTC-peptide. (2) cyclisation cracking: PTC-peptide cyclisation cracking. (3) transform: thiazole purine ketone benzene ammonia (ATZ) is converted into the different sulphur urine amino acid of benzene (PTH-amino acid). Staying the peptide decreasing an amino-acid residue in the solution to repeat above-mentioned reaction process again, whole order-checking process 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
Thiocarbanil PITC, can purchased from sigma company
Normal heptane, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
The Trimethylamine 99 TMA aqueous solution, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
TFA (trifluoroacetic acid, can purchased from sigma company)
Ethyl acetate, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
Chlorobutane, can purchased from sigma company
Acetonitrile, can purchased from Chemical Reagent Co., Ltd., Sinopharm Group
(4) measure
Undertaken by instrument specification sheets.
Result: through qualification, the sequence of gained HSP90 �� epitope peptide (1) and (2) is respectively:
(1) Y-P-R-D-R-L-D-P-R-P-G-S-P-S-E-A-S and
(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 �� antigen (1) and (2) with carrier proteins by HSP90 �� epitope peptide (1) and (2) of embodiment 1 gained respectively, utilize gained antigen (1) and (2) immune animal respectively, thus utilize antigen (1) to prepare monoclonal antibody and the polyclonal antibody of specificity, and antigen (2) is utilized to prepare monoclonal antibody and the polyclonal antibody of specificity.
1. the preparation of antigen: by BDB (Bis-diazotizedbenzidinedichloride) method HSP90 �� peptide section (1) and (2) is connected with carrier proteins KLH (keyhole limpet hemocyanin) (must from sigma company) respectively and is prepared into HSP90 �� antigen (1) and (2).
Get HSP90 �� peptide section (1) or (2) 10.0mg, dissolve with 1ml0.1MPBS damping fluid (pH7.4); KLH10mg, dissolves with 0.2M borate buffer solution (pH9.0) 20ml; Then both are mixed, it is cooled to 0 DEG C, gets BDBCl2110 �� L, react 1.5h under room temperature, packing after dialysed overnight ,-20 DEG C of preservations.
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. after HSP90 �� antigen (1) and (2) (immunogen) getting above-mentioned preparation fully mixes with isopyknic Freund's complete adjuvant (purchased from Shanghai Yuan Ju biotech firm) respectively, immunity Balb/c mouse, 50 �� g antigens/only, subcutaneous multi-point injection. serum titer is surveyed after 4 weeks, select the good mouse booster immunization again of immunoreactivity: get after antigen fully mixes with isopyknic incomplete Freund's adjuvant (purchased from Shanghai Yuan Ju biotech firm), antigen dose 25 �� 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, booster immunization twice continuously in addition before merging, each interval 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 fusion2In incubator, 37 DEG C of cultivations are after 9��11 days, occur bigger cell clone in hole. Within 11 days, start to screen with indirect ELISA. The hole that just sieve is positive utilizes limiting dilution assay carry out 4 time cloningizations cultivate (even if a large amount of fissiparity of cell after screening), afterwards amplifying cells, frozen, preparation ascites.
2.2. Balb/c mouse pristane (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: tiring of monoclonal antibody (1) utilizing HSP90 �� antigen (1) to prepare by indirect ELISA method mensuration, result tiring of monoclonal antibody of display reaches more than 1:32000.
The tiring of monoclonal antibody (2) utilizing HSP90 �� antigen (2) to prepare also utilizes identical method to measure, and it is tired and also reaches more than 1:32000.
3. immune animal prepares polyclonal antibody:
3.1. select New Zealand white rabbit that three monthly ages, body weight are about about 2kg as immune animal. In fundamental immunity, HSP90 �� 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, after booster immunization 6 times, antigen and incomplete Freund's adjuvant (purchased from Shanghai Yuan Ju biotech firm) are fully emulsified, with 100 �� g/ only in back multiple spot subcutaneous injection. Carotid artery bloodletting in 10th day after final boost, separation of serum.
3.2. measuring antibody titer: tiring of polyclonal antibody (1) utilizing HSP90 �� antigen (1) to prepare by indirect elisa method mensuration, result display antibody titer reaches more than 1:32000.
The tiring of polyclonal antibody (2) utilizing HSP90 �� antigen (2) to prepare also utilizes identical method to measure, and it is tired and also reaches more than 1:32000.
3.3. blood and separation of serum is got: carotid artery intubate gets blood, separation of serum.
4. separation and purification antibody: after ammonium sulfate precipitation, then through ProteinG (purchased from sigma company) affinity purification.
5. freeze-drying after antibody packing, cryopreservation.
Embodiment 3: people's HSP90 alpha monoclonal antibodies (1) and (2) is for the specificity identification of HSP90 ��-1 albumen
Detect with ELISA. Respectively taking people's HSP90 ��-1 albumen, S-100B albumen, neuronspecific enolase NSE (all purchased from Shanghai Lian Shuo company) as detectable antigens bag is by elisa plate, prepared HSP90 alpha monoclonal antibodies (1) and the specific reaction of (2) and this people's HSP90 ��-1 albumen is detected respectively by ELISA, making negative control with normal BALB/c mouse serum, PBS liquid makes blank.
Result: HSP90 alpha monoclonal antibodies (1) and (2) are only reacted with HSP90 ��-1 for positive (P/N > 2.1) respectively, and react for negative with S-100B albumen, neuronspecific enolase NSE, illustrate that the HSP90 alpha monoclonal antibodies (1) of the present invention and (2) have specificity respectively.
Embodiment 4: people HSP90 �� polyclonal antibody (1) and (2) is for the specificity identification of HSP90 ��-1 albumen
The method identical with above-mentioned qualification monoclonal antibody specificity is utilized to identify.
Result shows: HSP90 �� polyclonal antibody (1) and (2) reacts with HSP90 ��-1 respectively for positive (P/N > 2.1), and react for negative with S-100B albumen, neuronspecific enolase NSE, illustrate that HSP90 �� polyclonal antibody (1) and (2) of the present invention has specificity respectively.
Embodiment 5: people's HSP90 alpha monoclonal antibodies (2) and people HSP90 �� polyclonal antibody (2) are for the specificity of HSP90 ��-2 albumen
Utilizing the method qualification specificity identical with embodiment 3, difference is to change people's HSP90 ��-1 albumen into people HSP90 ��-2 albumen.
Result shows: people's HSP90 alpha monoclonal antibodies (2) and people HSP90 �� polyclonal antibody (2) are only positive (P/N > 2.1) respectively with people's HSP90 ��-2 albumen test, and react for negative with S-100B albumen, neuronspecific enolase NSE, illustrate that HSP90 ��-2 albumen is also had specificity by the monoclonal antibody (2) of the present invention and polyclonal antibody (2).
Embodiment 6: utilize HSP90 alpha monoclonal antibodies and HSP90 �� polyclonal antibody preparation HSP90 �� external diagnosis reagent case.
In the present embodiment, the coated antibody of monoclonal antibody (1) in this test kit that HSP90 �� epitope peptide (1) will be utilized in embodiment 2 to prepare; Using embodiment 2 utilizes HSP90 �� epitope peptide (2) prepare polyclonal antibody (2) as binding antibody, for detection sample in HSP90 ��-1.
Preparation and the operation of HSP90 �� external diagnosis reagent case are as follows:
1. the preparation of various damping fluid and reagent:
A, bag are buffered liquid: the CB (carbonate buffer solution) of 0.050M, pH9.6
Na2CO3: 16.0 grams
NaHCO3: 29.0 grams
Distilled water is molten to 1000ml
10 �� the PBS-Tween20 of B, sample/lavation buffer solution: pH7.2
Na2HPO4��12H2O:58 gram
KH2PO4: 4 grams
NaCl:100 gram
KCl:4 gram
Distilled water is molten to 1000ml
Add Tween20:20ml
C, enzyme marker diluent
10 �� PBS-Tween20:10ml
FCS (calf serum): 20ml
Distilled water is molten to 1000ml
Enzyme stabilizers (can purchased from Shanghai Xi Bao company): 1 gram
Biological preservative (can purchased from Shanghai Xi Bao company): 1ml
D, developer A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
Distilled water is molten to 1000ml
Tween20:10ml
E, developer B:
Citric acid: 120 grams
EDTA-2Na:1 gram
TMB 2HCl:2 gram
Distilled water is molten to 1000ml
F, stop buffer: 2MH2SO4
The vitriol oil (95-98%): 22.2ml
Distilled water: 177.3ml
The vitriol oil is slowly dripped in distilled water by timing, and Bian Jiabian shakes even.
2. the preparation of pre-coated plate:
HSP90 alpha monoclonal antibodies (1) is dissolved in the carbonate buffer solution of 0.05M of pH=9.6, make pre-coated liquid, 100 �� l are added by 0.1 �� g/ hole in the upper every hole of enzyme plate (can purchased from Shenzhen Jin Canhua company), put 4 DEG C to place 18-24 hour, take out, get rid of coating buffer, with sample/lavation buffer solution washing, load after closed 16 hours of 1 (w/v) %BSA-0.05M thanomin, dried overnight and aluminide-coating bag vacuumizes sealing, and be placed in 4 DEG C of preservations.
3. the Dilution ratio of binding antibody (HSP90 �� 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 ��-1 calibration object (raw material is purchased from Shanghai Lian Shuo company): 7: 7 �� 1.0ml (concentration is respectively 25ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1ng/ml, 0.5ng/ml, 0.25ng/ml)
HSP90 �� binding antibody: 1 �� 10ml (through 1:5000 dilution)
Enzyme connection thing: 1 �� 10ml (through 1:5000 dilution)
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 operation steps of test kit:
Each hole of pre-coated plate adds blood sample to be checked and standard substance 100 �� l/ hole respectively, is diplopore, hatches 60 minutes for 37 DEG C, wash 5 times with 1 �� lavation buffer solution, pat dry. In each hole, add HSP90 �� binding antibody 100 �� 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 �� 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 �� l in every hole, mixed even, hatch 15 minutes for 37 DEG C. Add stop buffer 50 �� l/ hole termination reaction, join detector (model RT-6000, can purchased from Lei Du company) with enzyme and detect absorbancy with dual wavelength (450nm, 620nm).
6. result judges:
Table 1: standard substance concentration and corresponding mean light absorbency (OD) value
| Concentration ng/ml | 0.25 | 0.5 | 1 | 2 | 5 | 10 | 25 |
| Mean OD value | 0.067 | 0.124 | 0.215 | 0.384 | 0.713 | 1.236 | 2.155 |
With the logarithmic value drawing standard curve of standard substance concentration and corresponding absorbancy, the R of typical curve2=0.978.
HSP90 ��-1 concentration results in the sample detected is calculated according to typical curve.
30 example tumour patients and 81 example healthy persons being carried out serum HSP90 ��-1 in a manner described detect, HSP90 ��-1 content in affinity antibody to SpA is obviously higher than normal healthy controls group, and difference has statistical significance (P < 0.01), in table 2.
Table 2: two groups of sample HSP90 ��-1 concentration compare
By above data it will be seen that the test kit of the present invention can effectively and specifically detect HSP90 ��-1 content in serum, thus detect out HSP90 ��-1 content difference between tumour patient and normal people, thus can judge the generation of tumour.
Embodiment 7: for the preparation of the HSP90 �� external diagnosis reagent case of the HSP90 ��-2 in detection sample.
In the present embodiment, composition, the preparation of HSP90 �� external diagnosis reagent case are identical with embodiment 6 with operation, difference is: by HSP90 ��-2 monoclonal antibody as the coated antibody in this test kit, replaces HSP90 ��-1 calibration object with HSP90 ��-2 calibration object (raw material is purchased from Shanghai Lian Shuo company).
Described HSP90 ��-2 monoclonal antibody is that the epitope peptide being following by sequence is prepared from: Y-E-K-E-S-E-D-K-P-E-I-E-D-V-G-S-D-E (shown in SEQIDNo.3). The preparation of this epitope peptide and prepare described HSP90 ��-2 monoclonal antibody with this epitope peptide and all carry out according to the method described in the embodiment of the present application 1 and 2. Further, this HSP90 ��-2 monoclonal antibody is identified to the specificity of people's HSP90 ��-2 albumen by the method identical with embodiment 3.
As follows by the result of the HSP90 ��-2 in the HSP90 �� external diagnosis reagent case of the present embodiment detection sample:
Table 3: standard substance 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 logarithmic value drawing standard curve of standard substance concentration and corresponding absorbancy, the R of typical curve2=0.976.
HSP90 ��-2 concentration results in the sample detected is calculated according to typical curve.
30 example tumour patients and 81 example healthy persons being carried out serum HSP90 ��-2 in a manner described detect, HSP90 ��-2 content in affinity antibody to SpA is obviously higher than normal healthy controls group, and difference has statistical significance (P < 0.01), in table 4.
Table 4: two groups of sample HSP90 ��-2 concentration compare
By above data it will be seen that the test kit of the present invention can effectively and specifically detect HSP90 ��-2 content in serum, thus detect out HSP90 ��-2 content difference between tumour patient and normal people, thus can judge the generation of tumour.
Claims (9)
1. a people HSP90 �� epitope peptide, the aminoacid sequence of wherein said HSP90 �� epitope peptide is:
Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Ser-Ala-Ala-Val-Thr-Glu��
2. a HSP90 �� antigen, by making, people's HSP90 �� epitope peptide according to claim 1 and carrier protein couplet are prepared from for it.
3. a people HSP90 Alpha antibodies, it is the monoclonal antibody or polyclonal antibody that are prepared from by HSP90 �� antigen according to claim 2.
4. people's HSP90 Alpha antibodies according to claim 3 is in the application prepared on people's HSP90 �� external diagnosis reagent case.
5. a people HSP90 �� external diagnosis reagent case, it comprises people's HSP90 Alpha antibodies according to claim 3 as coated antibody or binding antibody.
6. people's HSP90 �� external diagnosis reagent case according to claim 5, wherein said test kit also comprises another kind of people's HSP90 Alpha antibodies, and the one in described people's HSP90 Alpha antibodies and another kind of described people's HSP90 Alpha antibodies is as coated antibody, another one is as binding antibody, and another kind of wherein said people's HSP90 Alpha antibodies is prepared from by another kind of HSP90 �� antigen comprising the epitope as shown in following sequence:
Tyr-Pro-Arg-Asp-Arg-Leu-Asp-Pro-Arg-Pro-Gly-Ser-Pro-Ser-Glu-Ala-Ser��
7. HSP90 �� external diagnosis reagent case according to claim 5 or 6, wherein said coated antibody is monoclonal antibody.
8. HSP90 �� external diagnosis reagent case according to claim 5 or 6, wherein said binding antibody is polyclonal antibody.
9. HSP90 �� external diagnosis reagent case according to claim 5 or 6, wherein said test kit also comprises the anti-antibody of enzyme labelling.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111116743A (en) * | 2018-10-30 | 2020-05-08 | 迈威(上海)生物科技有限公司 | Hsp90 antibodies and their use against fungal infections |
| CN111197040A (en) * | 2020-01-21 | 2020-05-26 | 福建亿彤生物科技有限公司 | Chitinase 3-like protein 1(CHI3L1) epitope peptide, antigen, antibody, application and kit |
| CN117388494A (en) * | 2023-10-08 | 2024-01-12 | 烟台普罗吉生物科技发展有限公司 | Application of tumor marker in tumor diagnosis and tumor treatment monitoring products |
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| CN101215323A (en) * | 2007-12-28 | 2008-07-09 | 首都医科大学宣武医院 | Human AD7C-NTP antigen determinant polypeptide, antibody and application thereof in diagnosis kit |
| CN101942017A (en) * | 2009-07-07 | 2011-01-12 | 清华大学 | Novel tumor marker |
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| CN101215323A (en) * | 2007-12-28 | 2008-07-09 | 首都医科大学宣武医院 | Human AD7C-NTP antigen determinant polypeptide, antibody and application thereof in diagnosis kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111116743A (en) * | 2018-10-30 | 2020-05-08 | 迈威(上海)生物科技有限公司 | Hsp90 antibodies and their use against fungal infections |
| CN111116743B (en) * | 2018-10-30 | 2022-01-28 | 迈威(上海)生物科技股份有限公司 | Hsp90 antibodies and their use against fungal infections |
| CN111197040A (en) * | 2020-01-21 | 2020-05-26 | 福建亿彤生物科技有限公司 | Chitinase 3-like protein 1(CHI3L1) epitope peptide, antigen, antibody, application and kit |
| CN111197040B (en) * | 2020-01-21 | 2023-08-08 | 福建亿彤生物科技有限公司 | Chitinase 3-like protein 1 (CHI 3L 1) epitope peptide, antigen, antibody, application and kit |
| CN117388494A (en) * | 2023-10-08 | 2024-01-12 | 烟台普罗吉生物科技发展有限公司 | Application of tumor marker in tumor diagnosis and tumor treatment monitoring products |
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