CN1866022A - Construction method for sandwiched antibody chip detection system based on single chain antibody fusion protein - Google Patents
Construction method for sandwiched antibody chip detection system based on single chain antibody fusion protein Download PDFInfo
- Publication number
- CN1866022A CN1866022A CN 200610019362 CN200610019362A CN1866022A CN 1866022 A CN1866022 A CN 1866022A CN 200610019362 CN200610019362 CN 200610019362 CN 200610019362 A CN200610019362 A CN 200610019362A CN 1866022 A CN1866022 A CN 1866022A
- Authority
- CN
- China
- Prior art keywords
- sbp
- antibody
- fusion protein
- sandwich
- chip
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 69
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 69
- 238000001514 detection method Methods 0.000 title claims abstract description 30
- 238000010276 construction Methods 0.000 title claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 102000029797 Prion Human genes 0.000 claims abstract description 16
- 108091000054 Prion Proteins 0.000 claims abstract description 16
- 230000035945 sensitivity Effects 0.000 claims abstract description 12
- 239000013604 expression vector Substances 0.000 claims abstract description 10
- 238000003118 sandwich ELISA Methods 0.000 claims abstract description 9
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 4
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 4
- 239000013642 negative control Substances 0.000 claims description 26
- 230000004927 fusion Effects 0.000 claims description 23
- 230000029087 digestion Effects 0.000 claims description 18
- 238000011534 incubation Methods 0.000 claims description 17
- 108010090804 Streptavidin Proteins 0.000 claims description 13
- 102000012410 DNA Ligases Human genes 0.000 claims description 12
- 108010061982 DNA Ligases Proteins 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 11
- 241000283690 Bos taurus Species 0.000 claims description 10
- 210000004556 brain Anatomy 0.000 claims description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 10
- 239000002096 quantum dot Substances 0.000 claims description 8
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 6
- 241001529936 Murinae Species 0.000 claims description 6
- 210000004958 brain cell Anatomy 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 239000012528 membrane Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 102000002933 Thioredoxin Human genes 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 4
- 238000010168 coupling process Methods 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 108060008226 thioredoxin Proteins 0.000 claims description 4
- 229940094937 thioredoxin Drugs 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 3
- 229940098773 bovine serum albumin Drugs 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 239000013612 plasmid Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 230000008521 reorganization Effects 0.000 claims description 2
- 101000692225 Homo sapiens Selenocysteine insertion sequence-binding protein 2 Proteins 0.000 claims 1
- 102100026077 Selenocysteine insertion sequence-binding protein 2 Human genes 0.000 claims 1
- 102100028298 Vacuolar protein sorting-associated protein VTA1 homolog Human genes 0.000 claims 1
- 101710111280 Vacuolar protein sorting-associated protein VTA1 homolog Proteins 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 22
- 201000010099 disease Diseases 0.000 abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 10
- 238000000034 method Methods 0.000 abstract description 8
- 230000003141 anti-fusion Effects 0.000 abstract 1
- 244000052769 pathogen Species 0.000 abstract 1
- 230000001717 pathogenic effect Effects 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000011160 research Methods 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 5
- 210000003050 axon Anatomy 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000000427 antigen Substances 0.000 description 3
- 108091007433 antigens Proteins 0.000 description 3
- 102000036639 antigens Human genes 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000001360 synchronised effect Effects 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 238000012270 DNA recombination Methods 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- NVKAWKQGWWIWPM-ABEVXSGRSA-N 17-β-hydroxy-5-α-Androstan-3-one Chemical compound C1C(=O)CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CC[C@H]21 NVKAWKQGWWIWPM-ABEVXSGRSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 101000582398 Staphylococcus aureus Replication initiation protein Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000003498 protein array Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种基于单链抗体融合蛋白的夹心抗体芯片检测系统的构建方法。本发明首先是单链抗融合蛋白Z186－L－SBP和Z163－L－SBP表达载体的构建，设计上下游引物，双链两端分别为NotI和BamHI限制性酶切位点，连接产物；其次是单链抗体融合蛋白Z163－Fc表达载体的构建。通过夹心ELISA方法筛选用于夹心抗体芯片配对的单链抗体融合蛋白，并在此基础上建立了特异性和灵敏度更高的夹心抗体芯片检测系统，该系统的模式不仅适合朊病毒的检测，还适用于其他疾病的病原检测。The invention discloses a construction method of a sandwich antibody chip detection system based on a single-chain antibody fusion protein. The present invention firstly constructs the expression vectors of single-chain anti-fusion proteins Z186-L-SBP and Z163-L-SBP, designs upstream and downstream primers, and the two ends of the double chains are respectively NotI and BamHI restriction enzyme sites, and connects the products; secondly It is the construction of single-chain antibody fusion protein Z163-Fc expression vector. The single-chain antibody fusion protein used for sandwich antibody chip pairing was screened by sandwich ELISA method, and on this basis, a sandwich antibody chip detection system with higher specificity and sensitivity was established. The mode of this system is not only suitable for the detection of prions, but also Pathogen detection applicable to other diseases.
Description
Technical field
The present invention relates to gene fusion and protein chip technology field, relate more specifically to a kind of construction method of the sandwich antibody chip detection system based on scfv fusion protein, adopt gene fusion technique construction scfv fusion protein to be used for sandwich antibody chip; Set up two kinds of sandwich antibody chip detection systems based on scfv fusion protein.The pattern of this system not only can be used for the detection of prion, is applicable to that also the cause of disease of other diseases detects, and this pattern has good using value at the cause of disease detection range.
Background technology
Antibody molecule fragment and other albumen are merged, can obtain having the antibody fusion protein of antibody activity and other biologically active or function, antibody fusion protein can improve immunoassay, immunization therapy and antibody purification.The structure form of antibody fusion protein mainly contains two kinds, promptly utilizes respectively according to the variable fragment of its antibody and two big functions of constant fragment, makes antibody fusion protein have a certain characteristic of antibody, forms two big antibody-like fusions.One class is that the variable fragment with functional protein and antibody merges, and another kind of is that constant fragment with functional protein and antibody merges.The structure of antibody fusion protein in the past is the method that adopts chemical crosslinking, but so antibody linked albumen is formed heterogeneity, unstable properties, and molecule is big, and penetration capacity is low, and immunogenicity is big.Along with genetic engineering and genetic engineering antibody technology rapid development, the particularly development of phage antibody technology since the nineties, not only make the screening and the preparation of antibody fragment more and more convenient, and because the molecule of genetic engineering antibody fragment is little, function strong, stability is high, be easy to gene fusion, the structure of antibody fusion protein is also more and more convenient.Adopt the method for gene fusion to comprise with one section polypeptied chain and connect antibody molecule and functional protein and the antibody fusion protein structural stability height that makes up, performance is also more reliable.Being applied to the more antibody molecule of antibody fusion protein at present is Fab and scFv.The small peptide mark of they and the various features of tool merges, as with fusions such as E-tag, C-myc, His-tag, can be used for the analyzing and testing of the fast purifying and the antigen of antagonist; Merge with the albumen of other signatures, it is mainly used in the research in conjunction with back character of the analyzing and testing of target antigen and antibody antigen, the antibody fusion protein that constructs as Fab, scFv and alkaline phosphatase, streptavidin, staphylococcus aureus protein A (spA) etc. can improve immunoassay, simplifies analytic process.
At present, detection and the protein chip in the proteome analysis for disease marker mainly are divided into forward chip (forward phase array) (or antibody chip, antibody array), reverse protein chip (reverse-phase protein array, RPA) and sandwich protein chip, the forward chip is a sessile antibody on chip, add sample mixture to be checked again, carry out input, be mainly used in the research of proteinogram by mark; Anti-phase protein chip is that sample mixture to be checked directly is fixed on the specific solid support of chip, and tagged then albumen or antibody detect, and are mainly used in the functional analysis of albumen.Sandwich protein chip is at first capture antibody to be fixed on the chip substrate, catch the analyte that will detect, add the detection antibody that has mark then, capture antibody requires to be mainly used in the specific detection of target analytes at the different epitope of target analytes with detection antibody.Present double-antibody sandwich protein chip is used for the detection of analyte and mainly adopts two monoclonal antibodies.Though monoclonal antibody has higher affinity, the screening of monoclonal antibody and preparation are more loaded down with trivial details does not still satisfy the actual detected requirement with costliness and prion detection sensitivity.
Summary of the invention
The object of the present invention is to provide a kind of construction method of the sandwich antibody chip detection system based on scfv fusion protein, by the DNA recombination to construct have a Streptavidin affinity peptide (SBP) the scfv fusion protein orientation that is used for albumen fix, the scfv fusion protein scFv-Fc that makes up, affinity is significantly improved.Sandwich antibody chip based on the double single chain antibody fusion is the detection system of forming by DNA recombination to construct scfv fusion protein fully, scfv fusion protein molecule and sandwich antibody test system, not only provide technological approaches, but also detected the new pattern that provides for the cause of disease of other diseases for solving quick, the sensitive detection of rabid ox disease.
In order to achieve the above object, the present invention adopts following technical measures:
A, three scfv fusion protein: Z186-L-SBP, Z163-L-SBP, Z163-Fc (Z1030-Fc is eliminated) have been made up in the sandwich ELISA process.
The structure of scfv fusion protein Z163-L-SBP and Z186-L-SBP expression vector
Utilize SBP fragment (L-SBP) (the KEEFEAD.et al.2001 of upstream primer SBP15 ' ATATAGCGGCCGCTTCGAGCTCAGGAG3 ' and downstream primer SBP25 ' TGACCGGATCCTGGTTCACGTTGACCTT3 ' amplification N end band Linker (SSSGGSGSG), Protein Expression and Purification, 23 (3): 440-446), double-stranded two ends are respectively NotI and BamHI restriction enzyme site.Double-stranded then sequence with mix (Wang T.et al.2003 through the linearization plasmid pOPE101-215 of NotI and BamHI double digestion (Yol) fragment, ZhonghuaShi Yan He Lin Chuang Bing Du Xue Za Zhi.17 (2): 116-20), spend the night through the connection under 16 ℃ of T4DNA ligase, connecting product is carrier pOPE-L-SBP.The positive colony that screens is carried out linearization with NcoI and NotI double digestion, with the pCANTAB5E-Z163 (pCANTAB 5E:Pharmacia company product) of the same double digestion of warp and the product Z163 and the Z186 of pCANTAB5E-Z186 glue recovery, spend the night with the connection under 16 ℃ of T4DNA ligase with 1: 3 mole, connecting product is pOPE-Z163-L-SBP and pOPE-Z186-L-SBP.
The structure of scfv fusion protein Z163-Fc expression vector
Downcut the Fc gene segment with BamHI and NotI from carrier pPICZaFc (Eeckhout D.et al.2004.J ImmunolMethods 294:181-187), be connected with 3: 1 mol ratio with the carrier pOPE of same double digestion then, connect under 16 ℃ of conditions with the T4DNA ligase and to spend the night, connect product has Fc for coding pOPE-Fc.
With NcoI and NotI double digestion pCANTAB5E-Z163, be connected with 3: 1 mol ratio with the carrier pOPE-Fc of same double digestion, connect under 16 ℃ of conditions with the T4DNA ligase and spend the night, connect product has scFv and Fc for coding pOPE-Z163-Fc.
With sandwich ELISA (enzyme-linked immunosorbent assay) scfv fusion protein and anti-bovine prion protein monoclonal antibody KG9 are matched experiment, Z186-L-SBP/Z163-Fc and KG9/Z163-L-SBP pairing show tangible positive findings.
B, pass through sandwich ELISA, screen pairing antibody Z186-L-SBP/Z163-Fc and KG9/Z163-L-SBP, developed the sandwich antibody chip of two kinds of high specifics and sensitivity on this basis: based on the sandwich antibody chip of monoclonal antibody and scfv fusion protein with based on the sandwich antibody chip of double single chain antibody fusion, 1pg/mL can be reached to reorganization bovine prion protein detectability, and natural PrPC in the mouse brain can be detected.Two kinds of sandwich antibody chips can develop into the prion detection system of new high sensitivity and high specific.
The objective of the invention is to make up sandwich antibody chip, develop the new high sensitivity and the prion detection system of high specific, and detect the new pattern that provides for the cause of disease of other disease based on scfv fusion protein.
The present invention compared with prior art has following advantage and effect: SBP to be blended in the C end of scFv, can be used for purifying, and fluorescent nano particle or the quantum dot specific bond that can modify with Streptavidin again is used for target protein and detects as detecting antibody.ScFv-L-SBP fusion another outstanding characteristics in this research are to combine with the chip of Streptavidin bag quilt, realize that the orientation of scFv-L-SBP fusion is fixed, and as catching property antibody, are used for catching and detecting of target protein.Fusion Z163-Fc had both kept the selectivity of scFv, had kept the affinity of complete antibody again.In actual applications, carry out purifying and directed fixing, can be used as detection property antibody again by proteinA, protein G.
At present, the antibody that uses all is monoclonal antibody, and monoclonal antibody has very high selectivity, but the reaction of reporting to the leadship after accomplishing a task usually occurs, and the very strong recombinant antibodies of selectivity, particularly single-chain antibody show tangible advantage.This research adopts monoclonal antibody and scfv fusion protein pairing to make up sandwich antibody chip, and it combines the selectivity of the high affinity and the single-chain antibody of monoclonal antibody.
The sandwich antibody chip of another kind of the present invention is based on the sandwich antibody chip of the double single chain antibody fusion of the directed fixing and scFv-Fc of scfv fusion protein (scFv-L-SBP), has very strong selectivity and sensitivity, this protein chip detectability can reach 1pg/mL, and the quantitative sandwich ELISA method (50pg/mL) that adds hyperfluorescence than highly sensitive time resolution at present is sensitiveer.
Description of drawings
Fig. 1 is the structure synoptic diagram of a kind of scfv fusion protein Z163-L-SBP and Z186-L-SBP expression vector
Fig. 2. be the structure synoptic diagram of a kind of scfv fusion protein Z163-Fc and Z1030-Fc expression vector
Fig. 3. sandwich ELISA screening monoclonal antibody and scfv fusion protein pairing
1.KG9/Z163-L-SBP pairing, rb-PrP
c(grey rectangle);
(2.TrxA negative control, white rectangle);
3.KG9/Z1030-Fc pairing, rb-PrP
c(grey rectangle);
(4.TrxA negative control, white rectangle);
5.KG9/Z186-L-SBP pairing, rb-PrP
c(grey rectangle);
(6.TrxA negative control, white rectangle).
Fig. 4. be a kind of mode chart of the sandwich antibody chip based on monoclonal antibody and scfv fusion protein
(I) chip of poly-D-lysine bag quilt; (II) monoclonal antibody KG9 is coated on the poly-D-lysine chip; (III) add rb-PrP
c(IV) add scFv-L-SBP; (V) add QD635nm-streptavidin conjugate.
Fig. 5. detect the selectivity of bovine prion protein based on the sandwich antibody chip of monoclonal antibody and scfv fusion protein
A：a1-3：rb-PrP
c(10ng/mL)；b1-3：TrxA(10ng/mL)；c1-3：BSA(10ng/mL)；d1-3：OVA(10ng/mL)。
The B:635nm fluorescence intensity.The corresponding rb-PrP of black rectangle
c, the corresponding TrxA of striped rectangle, the corresponding BSA of grey rectangle, the corresponding OVA of space rectangles.Each rectangle is represented three repetitions.
Fig. 6. the sandwich antibody chip based on monoclonal antibody and scfv fusion protein detects bovine prion protein sensitivity.
A: detection by quantitative rb-PrP
c: (a1-a3), 100pg/mL rb-PrP
c(b1-b3), 10pg/mLrb-PrP
c(c1-c3), 1pg/mL rb-PrP
cNegative control, (d4-d6), 100pg/mL TrxA; (e4-e6), 10pg/mLTrxA; (f4-f6): 1pg/mL TrxA.
The B:635nm fluorescence intensity, the corresponding rb-PrP of grey rectangle
c, the corresponding TrxA of striped rectangle, each rectangle is represented three repetitions.
Fig. 7. the sandwich protein chip based on monoclonal antibody and scfv fusion protein detects the mouse PrPC
A:a1-a3: mouse brain PrP
cB1-b3:TrxA, 10ng/mL.
The B:635nm fluorescence intensity, the corresponding mouse brain of grey rectangle PrP
cThe corresponding negative control of striped rectangle, each rectangle is represented three repetitions.
Fig. 8. the pairing of sandwich ELISA screening scfv fusion protein
1.Z186-L-SBP/Z163-Fc pairing, rb-PrP
c(grey rectangle);
(2.TrxA negative control, white rectangle);
3.Z186-L-SBP/Z1030-Fc pairing, rb-PrP
c(grey rectangle);
(4.TrxA negative control, white rectangle);
5.Z163-L-SBP/Z1030-Fc pairing, rb-PrP
c(grey rectangle);
(6.TrxA negative control, white rectangle).
Fig. 9. based on the mode chart of the sandwich antibody chip of double single chain antibody fusion.
I) chip of streptavidin modification; II) by interact the fixing of antibody chip of fixing Z186-L-SBP of SBP-streptavidin; III) by the fixing rb-PrP of antibody chip
cIV) detect antibody Z163-Fc-Cy3 and the rb-PrP that catches
cIn conjunction with, detect fluorescence signal.
Figure 10. detect the selectivity of bovine prion protein based on the sandwich antibody chip of double single chain antibody fusion
A：a1-3：rb-PrPc(10ng/mL)；b1-3：TrxA(10ng/mL)；c1-3：BSA(10ng/mL)；d1-3：OVA(10ng/mL)。
The B:532nm fluorescence intensity.The corresponding rb-PrP of black rectangle
c, the corresponding TrxA of striped rectangle, the corresponding BSA of grey rectangle, the corresponding OVA of space rectangles.Each rectangle is represented three repetitions.
Figure 11. detect the sensitivity of bovine prion protein based on the sandwich protein chip of double single chain antibody fusion
A: detection by quantitative rb-PrP
c: (a1-a3), 100pg/mL rb-PrP
c(b1-b3), 10pg/mLrb-PrP
c(c1-c3), 1pg/mL rb-PrP
c(d1-d3), negative control, 100pg/mL TrxA.
The B:532nm fluorescence intensity, the corresponding rb-PrP of grey rectangle
c, the corresponding TrxA of striped rectangle, each rectangle is represented three repetitions.
Figure 12. the sandwich antibody chip based on scfv fusion protein detects the mouse PrPC
A:a1-a3: mouse brain PrP
cB1-b3:TrxA, 10ng/mL.
The B:532nm fluorescence intensity, the corresponding mouse brain of grey rectangle PrP
cThe corresponding negative control of striped rectangle, each rectangle is represented three repetitions.
Embodiment
The structure of embodiment 1 scfv fusion protein Z163-L-SBP and Z186-L-SBP expression vector
According to Fig. 1 as can be known: Streptavidin binding peptide (SBP) fragment (L-SBP) (the KEEFE A D.et al.2001 that utilizes upstream primer SBP15 ' ATATAGCGGCCGCTTCGAGCTCAGGAG3 ' and downstream primer SBP25 ' TGACCGGATCCTGGTTCACGTTGACCTT3 ' amplification N end band Linker (SSSGGSGSG), Protein Expression and Purification, 23 (3): 440-446), double-stranded two ends are respectively NotI and BamHI restriction enzyme site.Double-stranded then sequence with mix (Wang T.et al.2003 through the linearization plasmid pOPE101-215 of NotI and BamHI double digestion fragment, Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi.17 (2): 116-20), spend the night through the connection under 16 ℃ of T4DNA ligase, connecting product is carrier pOPE-L-SBP.The positive colony that screens is carried out linearization with NcoI and NotI double digestion, with the pCANTAB5E-Z163 (pCANTAB 5E:Pharmacia company product) of the same double digestion of warp and the product Z163 and the Z186 of pCANTAB5E-Z186 glue recovery, spend the night with the connection under 16 ℃ of T4DNA ligase with 1: 3 mole, connecting product is pOPE-Z163-L-SBP and pOPE-Z186-L-SBP.
The structure of embodiment 2 scfv fusion protein Z163-Fc and Z1030-Fc expression vector
Downcut the Fc gene segment with BamHI and NotI from carrier pPICZaFc (Eeckhout D.et al.2004.JImmunol Methods 294:181-187), be connected with 3: 1 mol ratio with the carrier pOPE-215 of same double digestion then, connect under 16 ℃ of conditions with the T4DNA ligase and to spend the night, connect product has Fc for coding pOPE-Fc.
According to Fig. 2 as can be known, with NcoI and NotI double digestion pCANTAB5E-Z163 and pCANTAB5E-Z1030, be connected with 3: 1 mol ratio with the carrier pOPE-Fc of same double digestion, connect under 16 ℃ of conditions with the T4DNA ligase and to spend the night, connect product has scFv and Fc for coding pOPE-Z163-Fc and pOPE-Z1030-Fc.
1. monoclonal antibody and scfv fusion protein pairing
According to Fig. 3 as can be known, by 96 hole ELISA Plate, the PBSM with 4% seals 2h in 37 ℃ with 1 μ g/mL monoclonal antibody KG9 bag, and PBS gives a baby a bath on the third day after its birth time, adds 10 μ g/mL rb-PrP
c, simultaneously, with the negative contrast of 10 μ g/mL TrxA, 37 ℃ of incubation 2h; The scFv-L-SBP and the scFv-Fc that add 25 μ g/mL respectively, 37 ℃ of incubation 2h; PBS gives a baby a bath on the third day after its birth inferior, adds the streptavidin-AP coupling matter in the scFv-L-SBP group, and 37 ℃ of incubation 1h add 100 μ L/ hole alkaline phosphatase substrate pNPP colour developing, measure light absorption value in the 405nm place; In the scFv-Fc group, add the anti-people Fc of the horse of being with HRP antibody, 37 ℃ of incubation 1h, add 100 μ L/ hole substrate TMB colour developing, measure light absorption value in the 450nm place, more than the OD value (S/N)＞2 of the OD value/negative control of reaction compound o'clock is judged to the positive, and every assembly is to establishing 3 repetitions.
In the pairing experiment that monoclonal antibody and scfv fusion protein carry out, with monoclonal antibody KG9 as capture antibody, serve as to detect antibody with scfv fusion protein Z163-L-SBP, Z1030-Fc and Z186-L-SBP respectively, be combined as KG9/Z163-L-SBP, KG9/Z1030-Fc and KG9/Z186-L-SBP, the sandwich ELISA result shows the rb-PrP that 3 assembly are right
c/ TrxA OD
450nmRatio is respectively 3.4,2.0 and 1.1, the KG9/Z163-L-SBP assembly is to showing tangible positive reaction, KG9/Z1030-Fc shows positive reaction, but ratio is lower, therefore selects for use the KG9/Z163-L-SBP assembly to being used for the research that sandwich protein chip detects bovine prion protein.
2. based on the structure of the sandwich antibody chip of monoclonal antibody and scfv fusion protein
Fig. 4 is the mode chart based on the sandwich antibody chip generation of monoclonal antibody and scfv fusion protein.Put the rb-PrP that 0.1 μ L/ is ordered by array being coated with on the poly-D-lysine chip of monoclonal antibody KG9
cOr the extract of murine brain cell membrane, room temperature (20-25 ℃) is incubation 1h down, it is inferior respectively to give a baby a bath on the third day after its birth with PBST and PBS, simultaneously, be made as negative control with TrxA, thioredoxin (TrxA) is used in sandwich antibody chip specificity experiment respectively, bovine serum albumin(BSA) (BSA) and oralbumin (OVA) are as negative control, the Z163-L-SBP that on each point, adds 10 μ g/mL then, room temperature (20-25 ℃) is incubation 1h down, respectively gives a baby a bath on the third day after its birth time with PBST and PBS, adds the quantum dot that the streptavidin coupling joins after air-dry, 37 ℃ of incubation 30min, with respectively the give a baby a bath on the third day after its birth time unconjugated quantum dot of flush away of PBST and PBS, quantum dot is at 635nm (Geho D, et al.2005, Bioconjug Chem, 16 (3): fluorescence signal 559-66) detects and uses GenePix 4000B (Axon Instrument) fluorescent scanning instrument.GenePix Pro 4.0 analysis software (Axon Instruments) are used in all signal analysis, and detectability is defined as concentration (Cretich M.et al.2004, Anal Biochem, 332 (1): 67-74) of signal to noise ratio (S/N ratio) (S/N) 〉=3 o'clock tested PrPC.
3. based on the selectivity of the sandwich antibody chip of monoclonal antibody and scfv fusion protein
In order to study the selectivity that detects bovine prion protein based on the sandwich antibody chip of monoclonal antibody and scfv fusion protein, put the sample that 0.1 μ L/ orders by array and add 10ng/mL rb-PrP being coated with on the poly-D-lysine chip of monoclonal antibody KG9
c, simultaneously, with TrxA, BSA and the negative contrast of OVA, it adds the Z163-L-SBP of 10 μ g/mL then on each point, and the quantum dot that joins with the streptavidin coupling shows as the detection signal result: rb-PrP
cBe respectively 5.6,4.2 and 3.5 with negative control in the signal to noise ratio (S/N ratio) (S/N) of the fluorescence intensity of 635nm, show that this sandwich protein chip adopts different albumen all can show tangible positive findings as negative control, have very strong selectivity (Fig. 5 A, B).
4. based on the sensitivity of the sandwich antibody chip of monoclonal antibody and scfv fusion protein
In order to study the sensitivity that detects bovine prion protein based on the sandwich antibody chip of monoclonal antibody and scfv fusion protein, add different dilution rb-PrP
c(available from German Roboscreen company) compares rb-PrP
cDilutability is respectively 100pg/mL, 10pg/mL and 1pg/mL, and signal to noise ratio (S/N ratio) (S/N) is respectively 4.0,3.4 and 3.2, the result show detectability can reach 1pg/mL (Fig. 6 A, B).
5. the sandwich antibody chip based on monoclonal antibody and scfv fusion protein detects the mouse PrPC
Be coated with on the poly-D-lysine chip of monoclonal antibody KG9, put the extract of 0.1 μ L/ point murine brain cell membrane by array, with TrxA synchronous detection survey in contrast, testing result shows: the extract of murine brain cell membrane and negative control are 3.1 in the signal to noise ratio (S/N ratio) (S/N) of the fluorescence intensity of 635nm, show tangible positive findings, show this sandwich antibody chip can detect PrPC natural in the mouse brain (Fig. 7 A, B).
Embodiment 4 is based on the structure of the sandwich antibody chip of double single chain antibody fusion
1. scfv fusion protein pairing
Wrap respectively by 96 hole ELISA Plate with Z186-L-SBP and Z163-L-SBP (10 μ g/mL in PBS), the PBSM with 4% is in 37 ℃ of sealing 2h, and PBS gives a baby a bath on the third day after its birth time, adds 10 μ g/mL rb-PrP
c, simultaneously, with the negative contrast of 10 μ g/mL TrxA, 37 ℃ of incubation 2h; Z163-Fc or 37 ℃ of incubation 2h of Z1030-Fc of adding 1 μ g/mL; PBS gives a baby a bath on the third day after its birth inferior, adds the anti-people Fc-HRP of horse antibody, and 37 ℃ of incubation 1h add substrate TMB colour developing, 1M H
2SO
4Cessation reaction is measured light absorption value in the 450nm place, more than the OD value (S/N)＞2 of the OD value/negative control of reaction compound o'clock is judged to the positive, and every assembly is to establishing 3 repetitions.
Match by scfv fusion protein, four scfv fusion proteins have been carried out pairing in twos, two Z186-L-SBP and Z163-L-SBP are as capture antibody, two Z163-Fc and Z1030-Fc are as detecting antibody, scfv fusion protein pairing be combined as Z186-L-SBP/Z163-Fc, Z186-L-SBP/Z1030-Fc and Z163-L-SBP/Z1030-Fc, the result shows the rb-PrP that three assembly are right
c/ TrxA OD
450nmRatio is respectively 4.2,1.3 and 1.1, and the Z186-L-SBP/Z163-Fc assembly is to showing tangible positive reaction, and this pairing is used for the research (Fig. 8) that sandwich protein chip detects bovine prion protein.
2. based on the structure of the sandwich antibody chip of double single chain antibody fusion
Fig. 9 is the mode chart based on the sandwich antibody chip generation of double single chain antibody fusion.Be fixed with in orientation on the chip of Streptavidin bag quilt of scfv fusion protein Z186-L-SBP and put the rb-PrP that 0.1 μ L/ is ordered by array
cOr the homogenate of mouse brain, room temperature (20-25 ℃) is incubation 1h down, it is inferior respectively to give a baby a bath on the third day after its birth with PBST and PBS, simultaneously, be made as negative control with TrxA, TrxA is used in sandwich antibody chip specificity experiment respectively, BSA and OVA are as negative control, on each point, add the fluorescently-labeled albumen Z163-Fc of 1ug/mL Cy3 then, room temperature (20-25 ℃) is incubation 1h down, respectively give a baby a bath on the third day after its birth time with PBST and PBS, air-dry after, the fluorescently-labeled albumen Z163-Fc of the Cy3 of flush away detects at the fluorescence signal of 532nm and uses GenePix 4000B (Axon Instrument) fluorescent scanning instrument.GenePix Pro 4.0 analysis software (Axon Instruments) are used in all signal analysis, and detectability is defined as signal to noise ratio (S/N ratio) (S/N) 〉=3 o'clock, the concentration of tested PrPC.
3. based on the selectivity of the sandwich antibody chip of double single chain antibody fusion
According to Figure 10 A, shown in the B, be fixed with chip (Nallur G, the et al.2001.Nucleic Acids Res.29 (23): 118) upward put 0.1 μ l/ point rb-PrP of the Streptavidin bag quilt of scfv fusion protein Z186-L-SBP in orientation by array
c, simultaneously, with 10ng/mL TrxA, BSA and the negative contrast of OVA, the fluorescently-labeled albumen Z163-Fc of Cy3 of 1 μ g/mL on each point then is at the rb-PrP of 532nm
cGroup is respectively 6.8,8.5 and 13.3 with the signal to noise ratio (S/N ratio) (S/N) of the fluorescence intensity of negative control group, shows that this sandwich protein chip adopts different albumen all can show tangible positive findings as negative control, also has very strong selectivity.
4. based on the sensitivity of the sandwich antibody chip of double single chain antibody fusion
According to Figure 11 A, shown in the B, be fixed with the rb-PrP that is respectively 100pg/mL, 10pg/mL and 1pg/mL on the chip of Streptavidin bag quilt of scfv fusion protein Z186-L-SBP by array point concentration in orientation
c, with TrxA synchronous detection in contrast, testing result shows: the signal to noise ratio (S/N ratio) (S/N) in the 532nm fluorescence intensity is respectively 6.1,3.8 and 3.1, and the result shows that detectability also can reach 1pg/mL.
5. the sandwich antibody chip based on the double single chain antibody fusion detects the mouse PrPC
According to Figure 12 A, shown in the B, the Streptavidin that is fixed with scfv fusion protein Z186-L-SBP in orientation wraps on the chip of quilt, put the extract of 0.1 μ L/ point murine brain cell membrane by array, with TrxA synchronous detection in contrast, testing result shows: the extract group of murine brain cell membrane and negative control group are 13 in the signal to noise ratio (S/N ratio) (S/N) of the fluorescence intensity of 532nm, show tangible positive findings, show that this sandwich antibody chip also can detect PrPC natural in the mouse brain.
Claims (3)
1, a kind of construction method of the sandwich antibody chip detection system based on scfv fusion protein, this system comprises the following steps:
A, make up three kinds of scfv fusion protein Z163-L-SBP, Z186-L-SBP and Z163-Fc: the structure that at first is scfv fusion protein Z163-L-SBP and Z186-L-SBP expression vector, utilize the SBP fragment of upstream primer SBP1 5 ' ATATAGCGGCCGCTTCGAGCTCAGGAG3 ' and downstream primer SBP2 5 ' TGACCGGATCCTGGTTCACGTTGACCTT3 ' amplification N end band Linker, double-stranded two ends are respectively NotI and BamHI restriction enzyme site, double-stranded then sequence with mix through the linearization plasmid pOPE101-215 of NotI and BamHI double digestion fragment, spend the night through the connection under 16 ℃ of T4 dna ligase, connecting product is carrier pOPE-L-SBP, the positive colony that screens is carried out linearization with NcoI and NotI double digestion, with the pCANTAB5E-Z163 of the same double digestion of warp and the product Z163 and the Z186 of pCANTAB5E-Z186 glue recovery, spend the night with the connection under 16 ℃ of T4 dna ligase with 1: 3 mole, connecting product is pOPE-Z163-L-SBP and pOPE-Z186-L-SBP; Next is the structure of scfv fusion protein Z163-Fc expression vector, downcut the Fc gene segment with BamHI and NotI from carrier pPICZaFc, be connected with 3: 1 mol ratio with the carrier pOPE-215 of same double digestion then, connect under 16 ℃ of conditions with the T4 dna ligase and to spend the night, connecting product has the pOPE-Fc of Fc for coding, use NcoI and NotI double digestion pCANTAB5E-Z163 then, be connected with 3: 1 mol ratio with the carrier pOPE-Fc of same double digestion, connect under 16 ℃ of conditions with the T4 dna ligase and to spend the night, connect product has scFv and Fc for coding pOPE-Z163-Fc;
B, pass through sandwich ELISA, screening pairing antibody Z186-L-SBP/Z163-Fc and KG9/Z163-L-SBP, the sandwich antibody chip of two kinds of high specifics and sensitivity: based on the sandwich antibody chip of monoclonal antibody and scfv fusion protein with based on the sandwich antibody chip of double single chain antibody fusion, detection reaches 1pg/mL to the reorganization bovine prion protein, and detects natural PrPC in the mouse brain.
2, the construction method of a kind of sandwich antibody chip detection system based on scfv fusion protein according to claim 1, it is characterized in that:, at first put the rb-PrP that 0.1 μ L/ is ordered by array being coated with on the poly-D-lysine chip of monoclonal antibody KG9 based on the structure of the sandwich antibody chip of monoclonal antibody and scfv fusion protein
cOr the extract of murine brain cell membrane, incubation 1h under the room temperature, it is inferior respectively to give a baby a bath on the third day after its birth with PBST and PBS, is made as negative control with thioredoxin, thioredoxin is used in sandwich antibody chip specificity experiment respectively, and bovine serum albumin(BSA) and oralbumin are as negative control; Next is the Z163-L-SBP that adds 10 μ g/mL on point, incubation 1h under the room temperature, it is inferior respectively to give a baby a bath on the third day after its birth with PBST and PBS, the quantum dot that adds streptavidin coupling connection after air-dry, 37 ℃ of incubation 30min, with PBST and the unconjugated quantum dot of PBS flush away, quantum dot detects use GenePix 4000B fluorescent scanning instrument at the fluorescence signal of 635nm.
3, the construction method of a kind of sandwich antibody chip detection system based on scfv fusion protein according to claim 1, it is characterized in that:, at first on orientation is fixed with the chip of Streptavidin bag quilt of scfv fusion protein scFv-L-SBP, put the rb-PrP that 0.1 μ L/ is ordered by array based on the structure of the sandwich antibody chip of double single chain antibody fusion
cOr the homogenate of mouse brain, incubation 1h under the room temperature, it is inferior respectively to give a baby a bath on the third day after its birth with PBST and PBS, simultaneously, be made as negative control with TrxA, thioredoxin is used in sandwich antibody chip specificity experiment respectively, bovine serum albumin(BSA) and oralbumin are as negative control, on point, add the fluorescently-labeled albumen Z163-Fc of 1 μ g/mLCy3 then, incubation 1h under the room temperature, respectively give a baby a bath on the third day after its birth time with PBST and PBS, air-dry after, the fluorescently-labeled albumen Z163-Fc of the Cy3 of flush away detects at the fluorescence signal of 532nm and uses GenePix 4000B fluorescent scanning instrument.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610019362 CN1866022A (en) | 2006-06-15 | 2006-06-15 | Construction method for sandwiched antibody chip detection system based on single chain antibody fusion protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610019362 CN1866022A (en) | 2006-06-15 | 2006-06-15 | Construction method for sandwiched antibody chip detection system based on single chain antibody fusion protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1866022A true CN1866022A (en) | 2006-11-22 |
Family
ID=37425096
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610019362 Pending CN1866022A (en) | 2006-06-15 | 2006-06-15 | Construction method for sandwiched antibody chip detection system based on single chain antibody fusion protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1866022A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267841A (en) * | 2013-05-15 | 2013-08-28 | 湖南农业大学 | Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle |
| CN103487589A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | Protein chip kit marked by quantum dot and preparation method thereof |
| CN108700576A (en) * | 2017-01-20 | 2018-10-23 | 深圳市新产业生物医学工程股份有限公司 | The system for preparing the application of the method, kit and kit of antibody pair and preparing antibody pair |
| CN109709331A (en) * | 2019-01-29 | 2019-05-03 | 广州瑞博奥生物科技有限公司 | Purposes of the GDF15 in the kit that preparation is used for quantitative detection liver cancer marker |
-
2006
- 2006-06-15 CN CN 200610019362 patent/CN1866022A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103267841A (en) * | 2013-05-15 | 2013-08-28 | 湖南农业大学 | Method for preparing ELISA (enzyme-linked immuno sorbent assay) enzyme-labelled antigen through utilizing avidin binding peptide and avidin combining principle |
| CN103487589A (en) * | 2013-10-16 | 2014-01-01 | 深圳市大爱医疗科技有限公司 | Protein chip kit marked by quantum dot and preparation method thereof |
| CN108700576A (en) * | 2017-01-20 | 2018-10-23 | 深圳市新产业生物医学工程股份有限公司 | The system for preparing the application of the method, kit and kit of antibody pair and preparing antibody pair |
| CN109709331A (en) * | 2019-01-29 | 2019-05-03 | 广州瑞博奥生物科技有限公司 | Purposes of the GDF15 in the kit that preparation is used for quantitative detection liver cancer marker |
| CN109709331B (en) * | 2019-01-29 | 2022-02-25 | 瑞博奥(广州)生物科技股份有限公司 | Application of GDF15 in preparation of kit for quantitatively detecting liver cancer marker |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8969255B2 (en) | Compound arrays for sample profiling | |
| US9366668B2 (en) | Device, array, and methods for disease detection and analysis | |
| US9970932B2 (en) | Non-covalent patterned chemical features and use thereof in MALDI-based quality control | |
| US20240077492A1 (en) | Array-based peptide libraries for therapeutic antibody characterization | |
| Gupta et al. | An overview of innovations and industrial solutions in Protein Microarray Technology | |
| Ofir et al. | Versatile protein microarray based on carbohydrate‐binding modules | |
| Thaler et al. | Highly sensitive immunodiagnostics at the point of care employing alternative recognition elements and smartphones: hype, trend, or revolution? | |
| AU2013344322A1 (en) | Diagnostic, prognostic, therapeutic and screening protocols | |
| Yang et al. | Cancer biomarkers detection using 3D microstructured protein chip: Implementation of customized multiplex immunoassay | |
| CN1866022A (en) | Construction method for sandwiched antibody chip detection system based on single chain antibody fusion protein | |
| US20050048545A1 (en) | Universal detection of binding | |
| CN1589404A (en) | Calibrating microarrays | |
| CN1584592A (en) | Immune microarray protein chip | |
| CN114755424A (en) | Supports, kits and methods for fluorescence polarization immunoassays | |
| CN1766621A (en) | Immune complex specific antibody for decreasing blank value in array testing form when detecting specific immunoglobulin type antibody which has been combined by antigen-antibody form | |
| CN116120440B (en) | Monoclonal antibodies against SARS-CoV-2 spike protein S1 and their applications | |
| US20120316078A1 (en) | Methods and materials for producing polypeptides in vitro | |
| CN105683755B (en) | Use of trap molecules and detection molecules and related kits and compositions | |
| Nishimura et al. | The development of a highly sensitive and quantitative SARS-CoV-2 rapid antigen test applying newly developed monoclonal antibodies to an automated chemiluminescent flow-through membrane immunoassay device | |
| Liu et al. | A simplified direct on‐chip forward or reverse immunoassay for evaluating protein–protein interactions in the serum | |
| Kaew-Amdee et al. | Identification of novel human IgE-binding peptides from a phage display library for total IgE detection | |
| Du | A simplified direct on-chip forward or reverse immune assay for understanding protein-protein interactions in the serum | |
| Anaya et al. | A multiplex extracellular interactome screening method employing high-avidity nanoparticles | |
| Park | Development of Recombinant Secondary Antibody Mimics (rSAMs) for Immunoassays using a Monomeric Alkaline Phosphatase | |
| CN1428606A (en) | Antigen detection method and detection device made up by using said method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |