CN105669836B - People HSP90 α -2 epitope peptide, antigen, antibody, application and kit - Google Patents
People HSP90 α -2 epitope peptide, antigen, antibody, application and kit Download PDFInfo
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Abstract
The present invention relates to people HSP90 α -2 epitope peptide, antigen, antibody, application and kits.The amino acid sequence of people's HSP90 α -2 epitope peptide of the invention is sequence shown in sequence table SEQ ID NO.1.People's HSP90 α -2 antigen of the invention is coupled by people's HSP90 α -2 epitope peptide and protein carrier and is made.People's HSP90 α -2 monoclonal antibody or polyclonal antibody of the invention is made by people's HSP90 α -2 antigen of the invention.People's HSP90 α -2 monoclonal antibody of the invention or polyclonal antibody are used to prepare people's HSP90 α -2 external diagnosis reagent case.People's HSP90 α -2 epitope peptide of the invention has good antigenicity, animal, which is immunized, with the antigen (immunogene) of its preparation can generate the monoclonal antibody and polyclonal antibody of high degree of specificity, so as to be applied to the vitro detection of people HSP90 α -2.
Description
Technical field
The invention belongs to chemiluminescent polypeptides and field of immunology, and in particular to 90 α -2 of human heat shock protein (HSP90 α -2) is anti-
Former epitope peptide, HSP90 α -2 specific antigen and corresponding monoclonal antibody or Anti-TNF-α prepared with the epitope peptide
The application of body, the antibody on preparation people HSP90 α -2 external diagnosis reagent case, people's HSP90 α -2 external diagnosis reagent case.
Background technique
In recent years, the attention of many researchers has been caused to the research of tumor markers.Find a kind of reliable, non-invade
Tumor markers entering formula, can reflecting the generation of tumour, rise in value differentiation are the common aspirations of numerous researchers.By to blood
The research of liquid, tissue fluid, have now been found that some tumor markers can generation to tumour, Proliferation, Differentiation predict.Wherein,
90 α -2 of human heat shock protein (HSP90 α -2) is exactly a kind of such tumor markers.
Heat shock protein72-2 is one of the important albumen in heat-shock protein family, is joined as molecular chaperones
With the conformation and function for regulating and controlling, maintaining intracellular multiple protein, adjust cell growth, differentiation and in terms of play it is important
Effect.In recent years it finds, Heat shock protein72-2 has the malignant transformation of tumour, growth, proliferation and invasion as chaperone
Important role.There are many reports to confirm, in body tumor tissue, the expression of heat shock protein is changed.In difference
In the tumor tissues at position, the change of heat shock protein expression is not consistent.Have now been found that the HSP90 α-in affinity antibody to SpA
The grade malignancy of 2 contents and tumour, especially transfer are positively correlated.Therefore, HSP90 α -2 holds promise as diagnosing tumor and prognosis
Marker.
The optimal method for detecting HSP90 α -2 level in serum is immune detection.Therefore, finding suitably has
HSP90 α -2 epitope peptide of immunogenicity, HSP90 α -2 antigen of preparation specificity and antibody emphasis.
Summary of the invention
For solve it is above-mentioned the problems of in the prior art, the present invention provides a kind of people HSP90 α -2 epitopes
Peptide, HSP90 α -2 specific antigen and corresponding monoclonal antibody or polyclonal antibody prepared with the epitope peptide,
Prepare the application on people HSP90 α -2 kit and people's HSP90 α -2 external diagnosis reagent case.
Specifically, the present invention provides:
A kind of people HSP90 α -2 epitope peptide, wherein the amino acid sequence of HSP90 α -2 epitope peptide are as follows:
Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-Gly-Ser-Asp-
Glu。
The present invention also provides a kind of HSP90 α -2 antigen, be by make people's HSP90 α -2 epitope peptide with
What carrier protein couplet was prepared.
It is the list being prepared by HSP90 α -2 antigen the present invention also provides a kind of people HSP90 α -2 antibody
Clonal antibody or polyclonal antibody, wherein HSP90 α -2 antigen be by make people's HSP90 α -2 epitope peptide with
What carrier protein couplet was prepared.
The present invention also provides people's HSP90 α -2 antibody on preparation people HSP90 α -2 external diagnosis reagent case
Using.
The present invention also provides a kind of people HSP90 α -2 external diagnosis reagent cases, and it includes people's HSP90 α -2 antibody
As coated antibody or binding antibody.
Preferably, the kit also includes another people HSP90 α -2 antibody, and above-mentioned people's HSP90 α -2 antibody
Be used as coated antibody with one of another people HSP90 α -2 antibody, another one as binding antibody, wherein described in
Another people HSP90 α -2 antibody as comprising the epitope as shown in following sequence another HSP90 α -2 antigen preparation
It forms:
Tyr-Arg-Gly-Ile-Asp-Glu-Asp-Asp-Pro-Thr-Ala-Asp-Asp-Thr-Ser-Ala-Ala-
Val-Thr-Glu。
Preferably, the coated antibody is monoclonal antibody.Preferably, the binding antibody is polyclonal antibody.
Preferably, the kit also includes the antiantibody of enzyme label.
Compared with the prior art, the present invention has the following advantages and good effect:
1. people's HSP90 α -2 epitope peptide of the invention has good antigenicity, the antigen (immunogene) prepared with it
Immune animal can generate the monoclonal antibody and polyclonal antibody of high degree of specificity.
2. with HSP90 α -2 monoclonal antibody prepared by the present invention and polyclonal antibody can high special with blood sample
HSP90 α -2 in this is combined.
3. the water that the HSP90 α -2 in serum can be effectively detected in people's HSP90 α -2 external diagnosis reagent case of the invention
It is flat, it can be used to the grade malignancy for judging tumour, and predict transfer and prognosis.
Specific embodiment
The following describes the present invention further through the description of specific embodiments, but it is to limit of the invention that this, which is not,
System, those skilled in the art's basic thought according to the present invention can make various modifications or improvements, but without departing from this
The basic thought of invention, is all within the scope of the present invention.
One, people HSP90 α -2 epitope peptide
People's HSP90 α -2 albumen described herein be it is known in the art, amino acid sequence be it is known in the art,
It can be found in the specialized databases such as NCBI.
The present invention provides people's HSP90 α -2 epitope peptides (1) and (2), and amino acid sequence is respectively such as sequence table SEQ
Shown in ID No.1 and SEQ ID No.2, are 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。
The present inventor gropes by a large amount of theoretical research and experiment, finally screens to obtain two kinds with good
Antigenic epitope peptide.
HSP90 α -2 epitope peptide (1) includes people HSP90 α -2 albumen (NCBI accession number NP_005339.3) N-terminal the
249 to the 265th peptide fragments, and Y is added in the N of peptide fragment section, to constitute the HSP90 α -2 comprising 18 amino acid
Epitope peptide (1).
HSP90 α -2 epitope peptide (2) includes people HSP90 α -2 albumen (NCBI accession number NP_005339.3) C-terminal the
697 to the 714th peptide fragments, and Y and R is added in the N of peptide fragment section, to constitute the HSP90 α -2 comprising 20 amino acid
Epitope peptide (2).
The characteristics of the two peptide fragments all have hydrophily, antigenicity by force and are readily synthesized.
Currently, the research of the invention finds that, HSP90 α -2 epitope peptide of the invention has following function:
1. having antigenicity；2. the antibody of specificity is generated after connecting with carrier protein as immune primary stimuli animal；3.
It can specifically be combined with people HSP90 α -2 with antibody prepared by epitope peptide.
The preparation method of HSP90 α -2 epitope peptide of the invention can use chemical synthesis: utilize American AB I431A type
Polypeptide automatic synthesizer passes through Solid phase synthesis epitope peptide.The molecular weight of epitope peptide (1) and (2) of the invention point
Not Wei 2404.35,2483.39, can be determined with mass spectrum, and pass through the synthesized epitope peptide of polypeptide sequence measurement identification
Sequence.The purity of peptide fragment can be evaluated with thin-layer chromatography and high performance liquid chromatography, and measure the concentration of epitope peptide.
Two, HSP90 α -2 antigen
The present invention also provides HSP90 α -2 antigen, by make people's HSP90 α -2 epitope peptide (1) of the invention and
One of (2) it is prepared with carrier protein couplet.Specifically, the present invention provides HSP90 α -2 antigen (1) and (2),
HSP90 α -2 antigen (1) is and preparing people's HSP90 α -2 epitope peptide (1) and carrier protein couplet of the invention
At；HSP90 α -2 antigen (2) is by preparing people's HSP90 α -2 epitope peptide (2) and carrier protein couplet of the invention
It forms.HSP90 α -2 antigen of the invention has immunogenicity and specificity, is a kind of immunogene, can be used to immune animal to
Prepare HSP90 α -2 antibody of specificity.In the present invention, the example of available carrier protein includes KLH (keyhole blood indigo plant egg
It is white), bovine serum albumin(BSA) (BSA), ovalbumin OVA etc..Since KLH (keyhole limpet hemocyanin) immunogenicity is strong, binding site
More, immune effect is preferable, and farther out with immune animal affiliation, it is used to be not easy to cause cross reaction as carrier protein,
It is therefore preferred.
Three, HSP90 α -2 monoclonal antibody, HSP90 α -2 polyclonal antibody and people's HSP90 α -2 external diagnosis reagent case
The present invention also provides people's HSP90 α -2 antibody, including people's HSP90 α -2 monoclonal antibody and more grams of people HSP90 α -2
Grand antibody, the antibody can each with HSP90 α -2 antigen (1) or (2) (immunogene) of the invention be immunized animal preparation and
?.The ordinary skill in the art can be used in preparation method, and for details, reference can be made to embodiments 2.
Herein, term " another HSP90 α -2 antigen " is HSP90 α -2 antigen (2).As people of the invention
When HSP90 α -2 antibody is prepared by HSP90 α -2 antigen (2) (that is, another kind HSP90 α -2 antigen), which is properly termed as
" people's HSP90 α -2 antibody (2) " (or abbreviation antibody (2)) or " another people HSP90 α -2 antibody ".
HSP90 α -2 monoclonal antibody of the invention and polyclonal antibody can be used for preparing people's HSP90 α -2 in-vitro diagnosis
Kit, the kit can detect the HSP90 α -2 in tissue, cell or body fluid based on immunization method, preferably
HSP90 α -2 in blood preparation is detected.
Therefore, the present invention provides a kind of people HSP90 α -2 external diagnosis reagent cases, and it includes people HSP90 α-of the invention
2 monoclonal antibodies or polyclonal antibody.
It is currently known that can be used for the immunization experiment method of clinical examination mainly include following several: ELISA method, chemiluminescence
Method, fluorescent chromatographic method, colloid gold immune measuring method etc..
And ELISA method includes following several types: double antibody sandwich method detects antigen, dual-antigen sandwich method detects antibody,
Indirect method surveys antibody, competition law surveys antibody, competition law surveys antigen, capture coating method surveys antibody etc..
People's HSP90 α -2 external diagnosis reagent case of the invention preferably uses ELISA double antibody sandwich method to detect HSP90
α -2 albumen.The kit may include coated antibody, binding antibody, the antiantibody of enzyme label and/or necessary tool and reagent
Deng.
Preferably, people HSP90 α -2 external diagnosis reagent case uses people's HSP90 α -2 monoclonal antibody of the invention
As coated antibody.Here, term " coated antibody " refers to the antibody being coated on the ELISA Plate of solid phase.In addition, the people
HSP90 α -2 external diagnosis reagent case further preferably includes people's HSP90 α -2 polyclonal antibody using as binding antibody, wherein works as institute
When stating binding antibody from one of people's HSP90 α -2 epitope peptide (1) and (2) of the invention, the coated antibody comes
Derived from the other of the epitope peptide (1) and (2).Here, term " binding antibody " refer in kit can with it is to be measured
The specific antibody that antigen and enzyme label antiantibody combine.The kit can also be comprising the antiantibody of enzyme label, this is anti-
Body can be goat anti-rabbit igg antibody, and the enzyme label can be horseradish peroxidase, alkaline phosphatase etc..
, can also be comprising any reagent or tool needed for detection in kit of the invention, such as pre-coated plate, wash
Wash liquid, color developing agent, terminate liquid etc..
The contents of the present invention are further explained and described below by way of the mode of example, but these examples are understood not to
Limitation to protection scope of the present invention.
Embodiment
Unless otherwise indicated, solution as described below is aqueous solution, and the percentage in solution is percentage by volume.
The preparation of embodiment 1:HSP90 α -2 epitope peptide (1) and (2).
Preparation method chemical synthesis: American AB I431A type polypeptide automatic synthesizer is utilized, is closed respectively by solid phase method
At HSP90 α -2 epitope peptide (1) and (2).The purity of epitope peptide is evaluated with high performance liquid chromatography, and measures peptide
The concentration of section.The molecular weight of epitope peptide (1) and (2) of the invention is respectively 2404.35,2483.39, is carried out using mass spectrum
It determines, passes through the synthesized polypeptide sequence of polypeptide sequence measurement identification.
One, the synthesis of HSP90 α -2 epitope peptide (1) and (2)
Above-mentioned peptide fragment uses Solid phase synthesis.The main thought of Solid phase peptide synthesis is: first by the carboxyl for the peptide chain of being synthesized
The carboxyl of end amino acid same insoluble high-molecular compound (resin) in the form of covalent bond is connected, and is then tied with this
Amino acid on solid phase carrier is closed as moiety, through sloughing amino protecting group and with excessive activated carboxyl component it is anti-
It answers, spreading peptide chain.Such step repeatedly can repeatedly go on, the length of the peptide chain synthesized required for finally reaching.
This synthesis process is as follows.
Specific preparation process is as follows for HSP90 α -2 epitope peptide (1) and (2) of the invention respective:
1. raw materials used:
HMP resin (P- hydroxymethyl phenoxy methyl poly vinyl, be purchased from sigma company)
Fmoc-AA (amino acid of 9- fluorenylmethoxycarbonyl carbonyl acyl group protection, be purchased from Merck company)
NMP (N-methyl pyrrolidones is purchased from sigma company)
DCM (methylene chloride 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- dithioglycol is purchased from sigma company)
Thioanisole is purchased from Guangzhou Wei Bai Chemical Co., Ltd.
Crystalline phenol is purchased from Sinopharm Chemical Reagent Co., Ltd.
Acetonitrile is purchased from Sinopharm Chemical Reagent Co., Ltd.
2. using instrument:
Polypeptide automatic synthesizer, model 431A are purchased from ABI company
Rotary Evaporators, model R-201 are purchased from Shanghai Shen Shun company
High performance liquid chromatograph, Waters 600, is purchased from Waters, US
Freeze drier, model VFD-2000 are purchased from the rich doctor Kanggong department in Beijing
3. synthetic method and process:
HMP resin 100mg is weighed, replacing equivalent is 1.0meq, i.e., 0.1mmol is placed in American AB I431A type polypeptide certainly
In the reaction chamber of dynamic synthesizer, specific amino acid is connected in a different order automatically by synthesizer, Conjugate ratio reaches
99%.It reacts as follows:
(1) activation (HOBt/DCC method) of amino acid
Fmoc-protected amino acid
(2) it connects on amino acid to resin
(3) the Fmoc protecting group of amino acid is sloughed
(4) activation (HOBt/DCC method) of another amino acid
(5) it is coupled
Peptide-resin of new coupling
(6) step (3) to (5) are repeated until synthesis terminates.
Respectively obtain the peptide resin 178mg of 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), is removed with EDT (2.5 volume %), thioanisole (2.5 volume %)
Agent is reacted 3.0 hours at room temperature, is removed cutting reagent, then extracted with ether, is respectively obtained HSP90 α -2 peptide fragment (1) and (2)
Crude product.
Two, the purifying of HSP90 α -2 epitope peptide (1) and (2) crude product:
It is purified using high performance liquid chromatography separation:
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) analysis
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 purity of peptide piecewise analysis HSP90 α -2 epitope peptide (1) and (2) of the invention as the result is shown is 95% or more.
Three, the identification of HSP90 α -2 epitope peptide (1) and (2)
1. measuring the molecular weight of purifying resulting HSP90 α -2 epitope peptide (1) and (2) respectively using mass spectrum.
(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 Sinopharm Chemical Reagent Co., Ltd.)
(2) instrument
Matrix-Assisted Laser Desorption Ionization Time of Flight instrument MALDI-TOF-MS (model: REFLEX III, Germany
Bruker company)；
(3) matrix liquid: α-CCA being dissolved in the 50%ACN solution containing 0.1%TFA, saturated solution is made, and centrifugation takes
Clearly；
(4) instrument testing conditions: reflection detection mode；Flight pipe range 3m；Nitrogen laser: wavelength 337nm, acceleration voltage
20KV；Reflected voltage 23KV.
(5) operating procedure: taking the sample of the above-mentioned purified polypeptide of 1 μ L (1) and (2) respectively, respectively and in the saturation matrix of 1 μ L
The isometric mixing of clear liquid mixing, takes 1 μ L point on sample target respectively, is sent into ion source and is detected.
As a result, the molecular weight for measuring gained HSP90 α -2 epitope peptide (1) is 2404.5, HSP90 α -2 epitope peptide
(2) molecular weight is 2483.5, consistent with theoretical molecular weight 2404.35,2483.39, it was demonstrated that synthesis polypeptide is purpose product.
2. the sequence of HSP90 α -2 epitope peptide (1) and (2) as obtained by polypeptide sequence measurement identification respectively.
(1) principle: the basic principle of polypeptid acid sequence analysis is Edman degradation, is that a circulating chemistry is anti-
Answer process.Including three main chemical steps: (1) be coupled: the end the N- residue of phenyl isothiocyanate and proteins and peptides is anti-
It answers, forms phenylamino formyl sulfide (PTC) derivative, i.e. PTC- peptide.(2) cyclisation cracking: PTC- peptide cyclisation cracking.(3) it converts: thiophene
Azoles purine ketone phenylamino (ATZ) is converted into the different sulphur urine amino acid of benzene (PTH- amino acid).Stay in the solution reduce an amino acid
The peptide of residue, which repeats, carries out above-mentioned reaction process, and entire sequencing procedure is carried out automatically by sequenator now.
(2) instrument: 491 type protein/polypeptide -terminal amino acid sequenator of American AB I company
(3) reagent raw material
Phenyl isothiocyanate PITC is purchased from sigma company
Normal heptane is purchased from Sinopharm Chemical Reagent Co., Ltd.
Trimethylamine TMA aqueous solution, is purchased from Sinopharm Chemical Reagent Co., Ltd.
TFA (trifluoroacetic acid is purchased from sigma company)
Ethyl acetate is purchased from Sinopharm Chemical Reagent Co., Ltd.
Chlorobutane is purchased from sigma company
Acetonitrile is purchased from Sinopharm Chemical Reagent Co., Ltd.
(4) it measures
It is carried out by instrument specification.
As a 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。
The result is consistent with target section of synthesized peptide.
Embodiment 2: resulting HSP90 α -2 epitope peptide (1) of embodiment 1 and (2) are connect with carrier protein respectively with
It prepares HSP90 α -2 antigen (1) and (2), animal is immunized respectively using gained antigen (1) and (2), to be prepared using antigen (1)
The monoclonal antibody and polyclonal antibody of specificity, and prepare using antigen (2) monoclonal antibody and polyclonal of specificity
Antibody.
1. the preparation of antigen: using BDB (Bis-diazotizedbenzidine dichloride) method by HSP90 α -2 peptide
Section (1) and (2) is connect with carrier protein KLH (keyhole limpet hemocyanin) (derive from sigma company) respectively to be prepared into HSP90 α -2 and resists
Former (1) and (2).
HSP90 α -2 peptide fragment (1) or (2) 10.0mg are taken, is dissolved with 1ml 0.1M PBS buffer solution (pH7.4)；KLH
10mg is dissolved with 0.2M borate buffer solution (pH 9.0) 20ml；Then the two is mixed, is cooled to 0 DEG C, takes BDBCl2110μ
L reacts 1.5h at room temperature, dispenses after dialysed overnight, -20 DEG C of preservations.
In the present embodiment, the formula of PBS buffer solution are as follows: the Na of 0.2mol/L2HPO481ml adds 0.2mol/L's
NaH2PO419ml is mixed.
The formula of borate buffer solution are as follows: 0.05mol/L borax 80ml adds 0.2mol/L boric acid 20ml to mix.
2. immune animal prepares monoclonal antibody:
2.1. HSP90 α -2 antigen (1) and (2) (immunogene) for taking above-mentioned preparation are helped with isometric Freund completely respectively
After agent (being purchased from Shanghai Yuan Ju biotech firm) is sufficiently mixed, immune Balb/c mouse, 50 μ g antigens/only, subcutaneous multi-point injection.4
It surveys serum titer after week, the mouse for selecting immunoreactivity good booster immunization again: takes antigen and isometric incomplete Freund
After agent (being purchased from Shanghai Yuan Ju biotech firm) is sufficiently mixed, 25 μ of antigen dose g/, subcutaneous multi-point injection, time of booster immunization
Number is 6 times, each At intervals of two to three weeks, before merging in addition continuous booster immunization twice, every minor tick 1-2 week, later extracting spleen cell and
Sp2/0 myeloma cell is mediated with 50%PEG (MW4000) (being purchased from Central Plains chemical company) merge according to a conventional method, is used in combination
HAT conditioned medium (being purchased from sigma company) selection culture.CO is put into after fusion2In incubator 37 DEG C culture 9~11 days after,
Occurs biggish cell clone in hole.Start to be screened with indirect ELISA within 11 days.Limiting dilution is utilized to the hole of the primary dcreening operation positive
Method carries out 4 time cloning cultures (even if screening after a large amount of schizogamies of cell), later amplifying cells, freeze, prepare ascites.
2.2. Balb/c mouse is only handled with norphytane (being purchased from sigma company) 0.5ml/, intraperitoneal inoculation is miscellaneous after a week
Hand over oncocyte 2 × 106A/only, ascites is collected after 10 days.
2.3. antibody titer is measured: anti-using the monoclonal of HSP90 α -2 antigen (1) preparation with indirect ELISA method measurement
The potency of body (1), the potency of monoclonal antibody reaches 1:32000 or more as the result is shown.
It is also measured using identical method using the potency of the monoclonal antibody (2) of HSP90 α -2 antigen (2) preparation,
Its potency also reaches 1:32000 or more.
3. immune animal prepares polyclonal antibody:
3.1. select the New Zealand White Rabbit that three monthly ages, weight are about 2kg or so as immune animal.In fundamental immunity,
HSP90 α -2 antigen (1) of the above-mentioned preparation of 1-2mg and (2) (immunogene) (are purchased from isometric complete Freund's adjuvant respectively
Shanghai Yuan Ju biotech firm) mixing-it is fully emulsified after rabbit back carry out multiple spot subcutaneous injection.Every 4 weeks booster immunizations one
It is secondary, booster immunization 6 times, after antigen and incomplete Freund's adjuvant (being purchased from Shanghai Yuan Ju biotech firm) are fully emulsified, with 100 μ g/
Only it is subcutaneously injected in back multiple spot.Arteria carotis bloodletting in 10th day after final boost separates serum.
3.2. antibody titer is measured: the polyclonal antibody with indirect elisa method measurement using HSP90 α -2 antigen (1) preparation
(1) potency, antibody titer reaches 1:32000 or more as the result is shown.
It is also measured using identical method using the potency of the polyclonal antibody (2) of HSP90 α -2 antigen (2) preparation,
Its potency also reaches 1:32000 or more.
3.3. take blood and separation serum: arteria carotis intubation takes blood, separates serum.
4. isolating and purifying antibody: after ammonium sulfate precipitation, then through Protein G (being purchased from sigma company) affinity purification.
5. being lyophilized after antibody packing, cryo-conservation.
Embodiment 3: the specificity identification of people's HSP90 α -2 monoclonal antibody (1) and (2)
It is detected with ELISA.Respectively with people HSP90 α -2 albumen, S-100B albumen, neuronspecific enolase
NSE (being purchased from Shanghai Lian Shuo company) is detection antigen coat elisa plate, detects prepared HSP90 α-respectively by ELISA
The specific reaction of 2 monoclonal antibodies (1) and (2) and the people HSP90 α -2 albumen is made negative with normal BALB/c mouse serum
Control, PBS liquid make blank control.
As a result: HSP90 α -2 monoclonal antibody (1) and (2) only reacts respectively with HSP90 α -2 for the positive (P/N > 2.1), and
It is negative for reacting with S-100B albumen, neuronspecific enolase NSE, illustrates HSP90 α -2 monoclonal antibody of the invention
(1) and (2) are respectively provided with specificity.
Embodiment 4: the specificity identification of people's HSP90 α -2 polyclonal antibody (1) and (2)
It is identified using method identical with above-mentioned identification monoclonal antibody specificity.
As the result is shown: HSP90 α -2 polyclonal antibody (1) and (2) are reacted respectively with HSP90 α -2 for positive (P/N > 2.1),
And reacting with S-100B albumen, neuronspecific enolase NSE is feminine gender, illustrates HSP90 α -2 Anti-TNF-α of the invention
Body (1) and (2) are respectively provided with specificity.
Embodiment 5: it is examined in vitro using HSP90 α -2 monoclonal antibody and HSP90 α -2 polyclonal antibody preparation HSP90 α -2
Disconnected kit.
In the present embodiment, the monoclonal antibody (1) of HSP90 α -2 epitope peptide (1) preparation will be utilized in embodiment 2
As the coated antibody in this kit；The polyclonal antibody of HSP90 α -2 epitope peptide (2) preparation will be utilized in embodiment 2
(2) it is used as binding antibody.
The preparation and operation of HSP90 α -2 external diagnosis reagent case are as follows:
1. the preparation of various buffers and reagent:
A, it is coated with buffer: the CB (carbonate buffer solution) of 0.050M, pH9.6
Na2CO3: 16.0 grams
NaHCO3: 29.0 grams
It distills water-soluble to 1000ml
B, sample/washing buffer: 10 × PBS-Tween 20 of pH7.2
Na2HPO4·12H2O:58 grams
KH2PO4: 4 grams
NaCl:100 grams
KCl:4 grams
It distills water-soluble to 1000ml
Add Tween 20:20ml
C, enzyme marker dilution:
10 × PBS-Tween 20:10ml
FCS (calf serum): 20ml
It distills water-soluble to 1000ml
Enzyme stabilizers (are purchased from Shanghai Xi Bao company): 1 gram
Biological preservative (is purchased from Shanghai Xi Bao company): 1ml
D, color developing agent A:
Citric acid: 35.5 grams
Urea peroxide: 10 grams
It distills water-soluble to 1000ml
Tween 20:10ml
E, color developing agent B:
Citric acid: 120 grams
EDTA-2Na:1 grams
TMB2HCl:2 grams
It distills water-soluble to 1000ml
F, terminate liquid: 2M H2SO4
The concentrated sulfuric acid (95-98%): 22.2ml
Distilled water: 177.3ml
The concentrated sulfuric acid is slowly dropped into distilled water by timing, is shaken well while adding.
2. the preparation of pre-coated plate:
In the carbonate buffer solution for the 0.05M that HSP90 α -2 monoclonal antibody (1) is dissolved in pH=9.6, it is made pre-coated
Liquid, 100 μ l are added by 0.1 hole μ g/ in every hole on ELISA Plate (being purchased from Shenzhen Jin Canhua company), and it is small to set 4 DEG C of placement 18-24
When, take out, get rid of coating buffer, wash with sample/washing buffer, through 1 (w/v) %BSA-0.05M ethanol amine closing 16 hours,
It is fitted into after being dried overnight in aluminide-coating bag and vacuumizes sealing, be placed in 4 DEG C of preservations.
3. binding antibody (HSP90 α -2 polyclonal antibody (2)) and enzyme-linked object (the goat-anti rabbit of horseradish peroxidase-labeled
IgG antibody) dilution ratio of (be purchased from company, Beijing Zhong Shan Golden Bridge) determines by square matrix titration experiments, horseradish peroxidase mark
The goat anti-rabbit igg antibody of note uses enzyme marker diluted.
4. the composition of kit:
Pre-coated plate: 48/96 hole
HSP90 α -2 calibration object (raw material be purchased from Shanghai Lian Shuo company): 7: 7 × 1.0ml (concentration be respectively 25ng/ml,
10ng/ml、5ng/ml、2.5ng/ml、1ng/ml、0.5ng/ml、0.25ng/ml)
HSP90 α -2 binding antibody: 1 × 10ml (dilutes) through 1:5000
Enzyme-linked object: 1 × 10ml (dilutes) through 1:5000
Concentrated cleaning solution (25 × PBS-Tween 20): 1 × 20ml
Color developing agent A:1 × 6.0ml
Color developing agent B:1 × 6.0ml
Terminate liquid: 1 × 6.0ml
5. the operating procedure of kit:
It is separately added into 100 hole μ l/ of blood sample and standard items to be checked in each hole of pre-coated plate, is diplopore, 37 DEG C incubate
It educates 60 minutes, is washed 5 times, patted dry with 1 × washing buffer.Addition 100 hole μ l/ of HSP90 α -2 binding antibody in each hole, 37
DEG C be incubated for 30 minutes, washed 5 times, patted dry with 1 × washing buffer.Enzyme-linked 100 hole μ l/ of object is added in each hole again, 37 DEG C incubate
It educates 30 minutes, is washed 5 times, patted dry with 1 × washing buffer.Color developing agent A, B liquid is added, every each 50 μ l in hole is mixed, 37 DEG C of incubations
15 minutes.Add 50 hole μ l/ of terminate liquid to terminate reaction, uses double wave with enzyme detector (model RT-6000 is purchased from Lei Du company)
Long (450nm, 620nm) detects absorbance.
6. result judgement:
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 |
Standard curve, the R of standard curve are drawn with the logarithm of standard concentration and corresponding absorbance2=0.976.
HSP90 α -2 concentration results in sample detected are calculated according to standard curve.
Serum HSP90 α -2 detection, affinity antibody to SpA are carried out in a manner described to 30 tumour patients and 81 healthy persons
In HSP90 α -2 content be apparently higher than healthy control group, difference is statistically significant (P < 0.01), is shown in Table 2.
2: two groups of sample HSP90 α -2 concentration of table compare
From the above data, the HSP90 α -2 that kit of the invention effectively and can be detected specifically in serum contains
Amount, to detect HSP90 α -2 content difference between tumour patient and normal person, thus can determine whether the generation of tumour.
Claims (2)
1. a kind of people HSP90 α -2 epitope peptide, wherein the amino acid sequence of HSP90 α -2 epitope peptide are as follows:
Tyr-Glu-Lys-Glu-Ser-Glu-Asp-Lys-Pro-Glu-Ile-Glu-Asp-Val-Gly-Ser-Asp-Glu。
2. a kind of HSP90 α -2 antigen is by making people HSP90 α -2 epitope peptide described in claim 1 and carrier egg
What white coupling was prepared.
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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 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 |
| CN101942017A (en) * | 2009-07-07 | 2011-01-12 | 清华大学 | Novel tumor marker |
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