CN106432492A - Antibody combination established aiming at tumor marker and ELISA method thereof - Google Patents
Antibody combination established aiming at tumor marker and ELISA method thereof Download PDFInfo
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- CN106432492A CN106432492A CN201510480951.8A CN201510480951A CN106432492A CN 106432492 A CN106432492 A CN 106432492A CN 201510480951 A CN201510480951 A CN 201510480951A CN 106432492 A CN106432492 A CN 106432492A
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Abstract
The invention belongs to the technical field of biology, and particularly relates to an antibody combination established aiming at a tumor marker and an ELISA method thereof. Compared with an antibody, an antibody analogue has the advantages of having good solubleness, good tissue penetration, good heat stability, good enzyme stability, low production cost and the like. Therefore, the invention discloses the antibody combination established aiming at the tumor marker, wherein the combination comprises stable and specific a pairing antibody analogue, and is expected to replace conventional antibody and be applied in a high-throughout technology (antibody chip) in screening antibody analogue libraries of various kinds to conduct development of antibody analogue bonded reagents.
Description
Technical field
The present invention relates to biological technical field is and in particular to be directed to Antibody Combination and its ELISA method that tumor markerses are set up.
Background technology
The most important feature of malignant tumor is local infiltration and metastasis, and is lethal main cause.It is formed by cell transformation continuous hypertrophy procreation, can develop infiltration and arrive local organization, and shift distant sites, this tumor being diffused into other positions already belongs to high malignancy level, the probability of its healing has been very little.At present, the Clinics and Practices of developed country's tumor many in early stage, some tumor markerses are as essential items for inspection accordingly, so tomour specific label is the key of early diagnosis of tumor, Personalized medicine and tumor prediction and prevention.But seldom there is special label in the world at present it is impossible to simple make a definite diagnosis tumor according to tumor markerses.Therefore, make full use of the clinical resources locally enriching and carry out biomarker（Especially serious to Guangdong harm malignant tumor）Research and development and application, both complied with the actual demand in market, also comply with country strategic demand.In the commitment of cancer, only very micro specific tumor mark is secreted and is discharged in blood, and direct detecting method is difficult to detect the change of these biomarkers.Even however, when these micro biomarkers are as autoantigen, can be with stimulating immune system, output substantial amounts of specific aim antibody, it is more beneficial for the detection of tumor using the result that this signal amplifies.
Antibody is so far by the most deep the one group bioconjugation molecule of human research.They are widely used in biological and medical field, serve very important effect.But, affected by volume, low stability etc., they are restricted in the application of diagnostic field.The exploitation of antibody analog solves this difficult problem, its small volume but there is antigenic determinant, can be bundled on target protein, therefore possess the potential as biology tool.Additionally, by being combined with new medical and health-care products, they can be used as therapeutic agent and the protein for detecting in patient's sample and be allowed to the instrument being imaged.They may be utilized for the neoplasm mark on identification of cell system and tissue samples.
The difference being subject between volume, low stability, high production cost and batch due to the critical material detection antibody in antibody chip and self-molecules present amount big and lead to sterically hindered affect, antibody is often limited in the application of the diagnostic field including antibody chip.In order to solve these problems, scientists develop small volume and the strong recombinant antibodies analog of activity, for molecular recognition.Antibody analog has the advantages that good dissolubility, tissue permeability, heat stability, enzyme stability, low production cost compared with antibody, therefore, filters out stable antibody analog, can extensively replace antibody for high-throughput techniques（Antibody chip）Sieve the exploitation that multiple different antibodies analog storehouses carry out antibody analog binding reagents.
Content of the invention
5 reasonable antibody analogs of the open tumor markerses S100B of the present invention are S100B-A10, S100B-A12, S100B-B11, S100B-D10 and S100B-D12；The antibody analog of tumor markerses GP73 is GP73-A5, GP73-B4, GP73-F6 and GP73-G4；The antibody analog of tumor markerses CK8 is CK8-F5 and CK8-12.But the application of these antibody analogs is not also studied.Especially by these antibody analogs after active and antibody is to detection, best target antibody to the preparation being applied to antibody chip, lay the foundation for exploitation stable performance, high and high specificity the novel tumor markers immunity detection reagent of diagnostic sensitivity.This will provide a kind of reliable approach for the infantile tumour early diagnosiss of tumor, Personalized medicine and tumor prediction.
To achieve these goals, the present invention is achieved by below scheme.
The ELISA Antibody Combination set up for tumor markerses, is matched with its conventional antibody with the antibody analog of tumor markerses.
Preferably, described tumor markerses are S100B, GP73 or CK8.
The ELISA Antibody Combination set up for tumor markerses S100B, the Antibody Combination of S100B mark is with the conventional antibody of anti-S100B as coated antibody, with antibody analog S100B-A10, S100B-A12, S100B-B11, S100B-D10 or S100B-D12 of S100B as detection antibody.
The ELISA Antibody Combination set up for tumor markerses GP73, the Antibody Combination of GP73 mark is with the conventional antibody of anti-GP73 as coated antibody, with antibody analog GP73-A5, GP73-B4, GP73-F6 or GP73-G4 of GP73 as detection antibody.
The ELISA Antibody Combination set up for tumor markerses CK8, the antibody of CK8 mark is made up of the conventional antibody of the antibody analog F5 and anti-CK8 of anti-CK8.
Preferably, the Antibody Combination of S100B mark is with the conventional antibody of anti-S100B as coated antibody, with the antibody analog S100B-D10 of S100B as detection antibody.
Preferably, the Antibody Combination of GP73 mark is with the conventional antibody of anti-GP73 as coated antibody, with the antibody analog GP73-G4 of GP73 as detection antibody.
Preferably, the Antibody Combination of CK8 mark is using CK8 antibody analog F5 as capture antibody, and corresponding conventional antibody 237 or 260 does detection antibody；
Or using CK8 antibody analog F5 as detection antibody, corresponding conventional antibody 260 or 251 does capture antibody.
The present invention has filtered out the antibody analog for 3 kinds of tumor markerses respectively, including S100B, GP73 and CK8, for identifying made binding proteins.
The present invention is using a kind of affine technology selecting to combine with gene expression product, with the phage of transformation as carrier, the exogenous dna fragment of encoding exogenous polypeptide or protein is cloned into the appropriate location of the structural gene of bacteriophage coat protein, in the case of not affecting other coat protein normal functions, allogenic polypeptide or protein and bacteriophage coat protein amalgamation and expression, re-assemblying with progeny phage, fusion protein will be illustrated in the surface of virion, and the DNA encoding this fusant is then located in this phagocytosis body, the DNA fragmentation of expression allogenic polypeptide or albumen can be learnt by sequencing.
The present invention adopts an antibody sequence library, makes to establish between a large amount of rondom polypeptides and its DNA encoding sequence and directly contacts so that the polypeptide ligand of various target molecule is able to Rapid identification by a kind of external option program being referred to as elutriation.
Phage elutriation and amplification, using the specific affinity of Ag-Ab, first, the target protein of biotin labeling are coated in the magnetic bead ELISA Plate of marked by streptavidin, add phage（Storage capacity 3 × 1010）, it is impossible to the phage combining still in the solution, can be removed by washing, then the bacteriophage elution of specific bond is got off after reaction 1h, through breeding amplification after the phage-infect host cell of eluting, carry out next round eluting, so
" absorption-eluting-amplification " through 3 wheel～5 wheel, you can useful gene is separated from up to more than million phage clone.
This experiment is taken turns through repeating elutriation 3, and application Phage-ELISA carries out positive verification.First by antigen coat in ELISA Plate, add the culture supernatant containing phage antibody after closing, be subsequently adding the antiphagin antibody of HRP labelling；Finally by TMB colour developing, the phage that such as elutriation goes out is target phage, then positive reaction shows blueness, and compares and do not develop the color.
Preferably, after reaction being sequenced for positive clone, import carrier, proceed to escherichia coli and carry out protein expression.
The gene of positive stronger phage is entered after performing PCR amplification extraction, is connected to pET11A carrier with double digestion method, is transformed into BL21 and carries out abduction delivering target protein, then used column chromatography is extracted to target protein and purification.The corresponding albumen obtaining, as antibody analog.
Preferably, after obtaining antibody analog, to carrying out filler test, the exploitation further being intended to be antibody analog chip immunoreagent provides optimal antibody pair for the activity of research team's antagonist analog and corresponding antibody.
The method of generally screening antibodies pair is sandwich ELISA, and the sandwich ELISA scheme that the present invention adopts is：
（1）Antibody analog is coated in NUNC microwell plate, after incubation, closing, adds target antigen incubation 1h；Subsequently operation such as Phage-ELISA, add detached antibody analog in phage, be subsequently adding the antiphagin antibody of HRP labelling, finally by TMB colour developing, can be made into the aobvious blueness of antibody analog reaction, and fail the analog that matches and matched group does not develop the color.
（2）As coated antibody, it is coated in ELISA Plate using for respective target target monoclonal antibody, 4 DEG C are incubated overnight；After room temperature with block solution closing 2h, add the corresponding target of gradient dilution（Antigen）, incubation at room temperature 2h；The antibody analog of above-mentioned biotin labeling, incubation at room temperature 1.5～2h is added after the completion of washing；The horseradish peroxidase of marked by streptavidin, incubation at room temperature 45 is added after the completion of washing
min；It is eventually adding nitrite ion colour developing, colour developing is added completely into terminate liquid and is terminated to displaing yellow and carries out OD with microplate reader450Mensure reading；According to the data obtained（OD value）Whether observation is proportional with antigen concentration, if representing：Antibody pair being become, if it is not, not becoming antibody pair, can not measure its target, gradient is more obvious more mensure that be conducive to target.
Compared with prior art, the invention has the advantages that：
For the antibody analog of pairing, there is small volume and the strong characteristic of activity, there is compared with antibody good dissolubility, tissue permeability, heat stability, enzyme stability, low production cost.
Brief description
Fig. 1 is the phage-ELISA specific reaction result of GP73.
Fig. 2 is the phage-ELISA specific reaction result of S100B.
Fig. 3 is the phage-ELISA specific reaction result of CK8.
Fig. 4 is S100B antibody analog determination of activity result.
Fig. 5 is GP73 antibody analog determination of activity result.
Fig. 6 is S100B antibody analog antibody to the selection result.
Fig. 7 is GP73 antibody analog antibody to the selection result.
Specific embodiment
Below by Figure of description and specific embodiment, the present invention is specifically described further, if following used experimental technique no specified otherwise, it is the existing conventional method of the art, the dispensing being used or material, if no special instructions, it is by the available dispensing of commercial sources or material.The above be only the preferred embodiment of the present invention it is noted that for those skilled in the art, under the premise without departing from the principles of the invention, some improvement can also be made, these improvement also should be regarded as protection scope of the present invention.
Embodiment
1
5 reasonable antibody analogs of the open tumor markerses S100B of the present invention are S100B-A10, S100B-A12, S100B-B11, S100B-D10 and S100B-D12；The antibody analog of tumor markerses GP73 is GP73-A5, GP73-B4, GP73-F6 and GP73-G4；The antibody analog of tumor markerses CK8 is CK8-F5 and CK8-12.
The preparation of antibody analog, flow process is as follows：
First, the target protein of biotin labeling is coated in the magnetic bead ELISA Plate of marked by streptavidin, adds phage（Storage capacity 3 × 1010）；
It is impossible to the phage combining still in the solution, can be removed by washing, then the bacteriophage elution of specific bond is got off after reaction 1h；
Through breeding amplification after the phage-infect host cell of eluting, carry out next round eluting；
So through " absorption-eluting-amplification " of 3～5 wheels, you can separate useful gene from up to more than million phage clone.
This experiment is taken turns through repeating elutriation 3, and application Phage-ELISA carries out positive verification.
First by antigen coat in ELISA Plate, after closing, add the culture supernatant containing phage antibody；
It is subsequently adding the antiphagin antibody of HRP labelling；
Finally by TMB colour developing, the phage that such as elutriation goes out is target phage, then positive reaction shows blueness, and compares and do not develop the color；For target CK8, then the optical density value of test experience group and matched group at a particular wavelength（OD value）, positive colony is screened, as shown in figure 3, wherein experimental group OD value significantly increases than matched group（>100）, only individual other clone do not specifically bind with protein target.
Complete the screening of antibody analog, after reaction is sequenced for positive clone, imports carrier, proceed to escherichia coli and carry out protein expression.
First, the gene of positive stronger phage is entered after performing PCR amplification extraction, be connected to pET11A carrier with double digestion method, be transformed into BL21 and carry out abduction delivering target protein；
Then used column chromatography is extracted to target protein and purification；
The corresponding albumen obtaining, as antibody analog.
Embodiment
2
For the antibody analog of report, the present invention have studied its activity first, preferably combines originality to identify whether it has for corresponding antigen.
Using Salmonella method, and before carrying out Salmonella mensure, antagonist analog is needed to carry out biotin labeling.Then corresponding antigen gradient is coated in elisa plate, 4 DEG C be incubated overnight after room temperature carry out closing 1.5 h；The good antibody analog of biotin labeling, incubation at room temperature 2h is added after to be closed；Add the horseradish peroxidase of marked by streptavidin afterwards（streptavidin-HRP）, incubation at room temperature 45 min；It is eventually adding TMB colour developing, add terminate liquid to be terminated to displaing yellow after the completion of colour developing, and carry out OD with microplate reader450Mensure reading；According to the data obtained observation whether reduction with antigen concentration, assuming corresponding gradient and reduce, combining originality if it is, indicating, be otherwise then not bound with originality or bad with reference to originality.
At present, for corresponding target, research team of the present invention has completed to S100B（Containing 5 analog）、GP73（Containing 4 analog）Combination originality mensure, result such as Fig. 4（S100B）, Fig. 5（GP73）Shown.Can be seen that each antibody analog its corresponding target is all shown from two in figures and combine originality, also stronger in conjunction with originality.
Embodiment
3
Sandwich ELISA method screens respective target target antibody pair, and the method for screening antibodies pair is sandwich ELISA：
Scheme 1：Antibody analog is coated in NUNC microwell plate, after incubation, closing, adds target antigen incubation 1h；
Subsequently operation such as Phage-ELISA, add detached antibody analog in phage, be subsequently adding the antiphagin antibody of HRP labelling, finally by TMB colour developing, can be made into the aobvious blueness of antibody analog reaction, and fail the analog that matches and matched group does not develop the color.
Scheme 2：Using own for respective target target monoclonal antibody as coated antibody, be coated in ELISA Plate, 4 DEG C are incubated overnight；
After room temperature with block solution closing 2h, add the corresponding target of gradient dilution（Antigen）, incubation at room temperature 2h；The antibody analog of above-mentioned biotin labeling, incubation at room temperature 1.5～2h is added after the completion of washing；The horseradish peroxidase of marked by streptavidin, incubation at room temperature 45 is added after the completion of washing
min；It is eventually adding nitrite ion colour developing, colour developing is added completely into terminate liquid and is terminated to displaing yellow and carries out OD with microplate reader450Mensure reading；
According to the data obtained（OD value）Whether observation is proportional with antigen concentration, if represented：Antibody pair being become, if it is not, not becoming antibody pair, can not measure its target, gradient is more obvious more mensure that be conducive to target.
For corresponding target, the present invention has completed the mensure of the antibody pair to S100B and GP73 antibody analog, is carried out using sandwich ELISA scheme 2, result such as Fig. 6（S100B）And Fig. 7（GP73）Shown, and successfully obtain corresponding antibodies pair.
The antibody centering of S100B, most preferably conventional antibody（Anti- S100B antibody）And antibody analog（S100B-D10）Antibody to best；Can draw in Fig. 7, its conventional antibody（Anti- GP73 antibody）As coated antibody, corresponding antibody analog（GP73-A5, GP73-B4, GP73-F6 and GP73-G4）When doing detection antibody, all can become preferable antibody pair, the antibody that wherein GP73-G4 becomes therewith is to being best.
Embodiment
4
According to the method for scheme 2 in embodiment 2, the pairing situation of CK8 antibody analog is identified, such as table 1.
Table 1
Can be drawn by table 1, with CK8 antibody analog F5 as capture antibody, corresponding conventional antibody（237,260）When doing detection antibody, all can become preferable antibody pair, and not advantageous when matching with another CK8 antibody analog 12, corresponding conventional antibody（251,259）Pairing result is poor.
In addition, with CK8 antibody analog F5 as detection antibody, corresponding conventional antibody（260,251）When doing detection antibody, all can become preferable antibody pair, and and conventional antibody（237,259）Pairing result is poor（It is shown in Table 2）.
Table 2
.
Claims (8)
1. it is directed to the ELISA Antibody Combination of tumor markerses foundation it is characterised in that matching with its conventional antibody with the antibody analog of tumor markerses.
2. it is directed to the ELISA Antibody Combination of tumor markerses foundation according to claim 1 it is characterised in that described tumor markerses are S100B, GP73 or CK8.
3. it is directed to the ELISA Antibody Combination that tumor markerses S100B sets up, it is characterized in that, the Antibody Combination of S100B mark is with the conventional antibody of anti-S100B as coated antibody, with antibody analog S100B-A10, S100B-A12, S100B-B11, S100B-D10 or S100B-D12 of S100B as detection antibody.
4. it is directed to the ELISA Antibody Combination that tumor markerses GP73 sets up, it is characterized in that, the Antibody Combination of GP73 mark is with the conventional antibody of anti-GP73 as coated antibody, with antibody analog GP73-A5, GP73-B4, GP73-F6 or GP73-G4 of GP73 as detection antibody.
5. the ELISA Antibody Combination being directed to tumor markerses CK8 foundation is it is characterised in that the antibody of CK8 mark is made up of the conventional antibody of the antibody analog F5 and anti-CK8 of anti-CK8.
6. according to claim 3 combination it is characterised in that the Antibody Combination of S100B mark is with the conventional antibody of anti-S100B as coated antibody, with the antibody analog S100B-D10 of S100B as detection antibody.
7. according to claim 4 combination it is characterised in that the Antibody Combination of GP73 mark is with the conventional antibody of anti-GP73 as coated antibody, with the antibody analog GP73-G4 of GP73 as detection antibody.
8. it is characterised in that the Antibody Combination of CK8 mark is as capture antibody with CK8 antibody analog F5, corresponding conventional antibody 237 or 260 does detection antibody for combination according to claim 5;Or with CK8 antibody analog F5 as detection antibody, corresponding conventional antibody 260 or 251 does capture antibody.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009103542A1 (en) * | 2008-02-22 | 2009-08-27 | Mubio Products Bv | Small cell lung carcinoma biomarker panel |
| CN101988926A (en) * | 2009-08-03 | 2011-03-23 | 中国科学院广州生物医药与健康研究院 | Sandwich ELISA quantitative detection method of Golgi protein GP73 and detection kit thereof |
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