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Activation of a Refolded, Berberine-specific, Single-chain Fv Fragment by Addition of Free Berberine
A single-chain variable fragment (scFv) specific for berberine was produced in Escherichia coli. The anti-berberine scFv gene was cloned from hybridoma 1D5-3B-7 producing the monoclonal antibody. The variable regions of the heavy (VH) and light chain (VL) genes were connected with a flexible linker using an assembly PCR. The VH-linker-VL gene was inserted into a plasmid, pET28a (+), then overexpressed in E. coli BL21 (DE3). The active of the scFv by refolding based on stepwise dialysis methods and an artificial chaperone was determined by direct and competitive enzyme-linked immunosorbent assay (ELISA). The results of direct ELISA showed that the anti-berberine scFv retained specific binding activity to berberine. In competitive ELISA, however, activity was increased depending on the concentration of berberine. 相似文献
3.
Wongi Min Jin-Kyoo Kim Hyun Soon Lillehoj Eun Jung Sohn Jae Yong Han Ki Duk Song Erik Peter Lillehoj 《Biotechnology letters》2001,23(12):949-955
The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding V
H and V
L genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l–1 and exhibited virtually identical antigen reactivity as the original mAb. 相似文献
4.
Hong Wang Jinyi Yang Xixia Liu Yan Liang Hongtao Lei Yudong Shen Xiaoyan Xu Yuanming Sun Zhenlin Xu Yongsheng He 《Biotechnology progress》2009,25(4):1018-1024
Phage display method was used to clone anti‐carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF‐specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly4Ser)3. The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti‐CBF antibody libraries were then constructed. After one round of panning with CBF‐ovalbumin (CBF‐OVA) as a conjugate, antigen‐binding positive recombinant phage clones were successfully selected by enzyme‐linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS‐PAGE and showed HRP‐anti‐E‐tag antibody‐recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC50 value of 1.07 ng/mL. The cross reactivity studies showed that the anti‐CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
5.
Sergey M. Kipriyanov Stefan Dübel Frank Breitling Roland E. Kontermann Stefan Heymann Melvyn Little 《Cell biochemistry and biophysics》1995,26(3):187-204
Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed inEscherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase
II ofDrosophila melanogaster and rat MAb Yol1/34, specific for pig brain α-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus.
After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by
size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv′)2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly
of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation
procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by
enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays
showed that covalently linked (scFv′)2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv′ monomers. ScFv derivatives,
specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot
analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection. 相似文献
6.
Tatsuro Shibui Teruaki Kobayashi Keiichiro Kanatani Hirohisa Koga Satoru Misawa Tetsu Isomura Tooru Sasaki 《Applied microbiology and biotechnology》2009,84(4):725-732
Synthetic DNA libraries encoding human antibody VL and VH fragments were designed, constructed, and enriched using mRNA display. The enriched libraries were then combined to construct
a scFv library for mRNA display. Sequencing revealed that 46% of the library coded for full-length scFvs. Considering the
number of molecules used in mRNA display, the size of the library displayed was calculated to be >1010. To verify this, we tried to isolate a scFv against human RANK. A scFv was successfully isolated in the sixth round of panning
and was synthesized in wheat embryo cell-free (WE) and Escherichia coli cell systems. In the WE system, even though the production level was high, the product was almost soluble. However, in the
E. coli system, it was over-produced as inclusion bodies. The inclusion bodies were successfully refolded and showed approximately
the same binding affinity as the WE product. These results demonstrate that using mRNA display with synthetic libraries and
WE and E. coli cell production systems, a system for in vitro selection and small- to large-scale production of scFvs has been established. 相似文献
7.
《MABS-AUSTIN》2013,5(6):1058-1071
Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability. 相似文献
8.
Yinting Chen Kaihong Huang Xuexian Li Xiangan Lin Zhaohua Zhu Ying Wu 《Cancer immunology, immunotherapy : CII》2010,59(6):933-942
CD44v6 is a cancer-associated antigen that mainly expresses in a subset of adenocarcinomas. Therefore, in this study, anti-human
CD44v6 single-chain variable fragment (scFv) has been selected and characterized because it is the first step of primary importance
towards the construction of a novel cancer-targeted agent for cancer diagnosis and therapy. In our study, anti-human CD44v6
scFv was selected from a human phage-displayed scFv library based on its ability to bind in vitro to CD44v6 antigen. Subsequently,
immunofluorescent staining and Western blot analyses were performed to measure the binding characteristics of this scFv. In
addition, flow cytometric analysis was done to verify its cancer-targeting ability in vitro. And a flow cytometry-based assay
was used to determine its equilibrium dissociation constant (K
D). Finally, one functional anti-CD44v6 scFv was selected and characterized. Nucleotide sequencing verified that it was an
incomplete scFv gene but had a variable heavy chain (VH) alone. However, anti-CD44v6 scFv demonstrated cell-binding and antigen-binding activities by immunofluorescent staining
and Western blot analyses. Furthermore, flow cytometric analysis proved that this scFv specifically targeted CD44v6-expressing
cancer cells other than CD44v6 non-expressing normal cells or tumor cells in vitro. The K
D of this scFv was calculated to be 7.85 ± 0.93 × 10−8 M. In summary, the selected human scFv against CD44v6 has specific binding activity and favorable binding affinity despite
lacking a variable light chain (VL). Moreover, it can effectively and specifically target CD44v6-expressing cancer cells. All these characteristics make anti-CD44v6
scFv a promising agent for cancer detection and anti-cancer therapy. 相似文献
9.
Robyn L. Malby J. Bruce Caldwell L. Clem Gruen Vincent R. Harley Neva Ivancic Alexander A. Kortt Glenn G. Lilley Barbara E. Power Robert G. Webster Peter M. Colman Peter J. Hudson 《Proteins》1993,16(1):57-63
The variable heavy (VH) and variable light (VL) genes of NC10, a monoclonal antibody with specificity toward N9 neuraminidase (NA), were cloned and sequenced. A single chain Fv (scFv) fragment of NC10, consisting of VH and VL domains joined by a peptide linker, was designed, constructed and expressed in the E. coli expression vector pPOW. The N-terminal secretion signal PelB directed the synthesized protein into the periplasm where it was associated with the insoluble membrane fraction. An octapeptide (FLAG) tail was fused to the C-terminus of the single chain Fv to aid in its detection and remained intact throughout the protein purification process. NC10 scFv was purified by solubilization of the E. coli membrane fraction with guanidinium hydrochloride followed by column chromatography. The purified NC10 scFv showed binding affinity for its antigen, NA, 2-fold lower than that of the parent Fab. The complex between NA and the scFv has been crystallized by the vapor diffusion method. The crystals are tetragonal, space group P4212, with unit cell dimensions a = b = 141 Å, c = 218 Å. © 1993 Wiley-Liss, Inc. 相似文献
10.
Sean A. Gray John R. Barr Suzanne R. Kalb James D. Marks Cheryl L. Baird Gerard A. Cangelosi Keith D. Miller Michael J. Feldhaus 《Biotechnology and bioengineering》2011,108(10):2456-2467
A non‐immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody‐mediated labeling strategy was used in which antigen‐binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, 3 also bound to full‐length BoNT/A toxin complex with affinities ranging from 5 to 48 nM. Epitope binning showed that the three unique clones recognized at least two epitopes distinct from one another as well as from the detection MAbs. After production in E. coli, scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep‐MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A‐specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep‐MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep‐MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigens. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support. Biotechnol. Bioeng. 2011;108: 2456–2467. © 2011 Wiley Periodicals, Inc. 相似文献
11.
Power BE Caine JM Burns JE Shapira DR Hattarki MK Tahtis K Lee FT Smyth FE Scott AM Kortt AA Hudson PJ 《Cancer immunology, immunotherapy : CII》2001,50(5):241-250
A single-chain antibody fragment (scFv) of the humanised monoclonal antibody, hu3S193, that reacts specifically with Ley antigen expressed in numerous human epithelial carcinomas was constructed. A five-residue linker joined the C-terminus of
the VH and the N-terminus of the VL, which prevented V-domain association into a monomeric scFv and instead directed non-covalent association of two scFvs into
a dimer or diabody. The diabody was secreted into the E. coli periplasm using a heat-inducible vector, pPOW3, and recovered as a soluble, correctly processed protein, following osmotic
shock or solubilised with 4M urea from the insoluble fraction. The diabody from both fractions was isolated by a rapid batch
affinity chromatography procedure, using the FLAG affinity tag to minimise degradation and aggregation. The purified diabody
has an Mr of ˜54 kDa, was stable and demonstrated similar binding activity as the parent monoclonal antibody, as measured by FACS and
BIAcore analyses. The radiolabelled diabody showed a rapid tumour uptake, with fast blood clearance, proving it to be an excellent
potential candidate as a tumour-imaging agent.
Received: 16 November 2000 / Accepted: 8 March 2001 相似文献
12.
Andreas Lehmann Josephine H F Wixted Maxim V Shapovalov Heinrich Roder Roland L Dunbrack Jr. Matthew K Robinson 《MABS-AUSTIN》2015,7(6):1058-1071
Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability. 相似文献
13.
Duanpen Sandee Sumalee Tungpradabkul Manae Tsukio Tadayuki Imanaka Masahiro Takagi 《BMC biotechnology》2002,2(1):16-8
Background
Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques.Results
The sequences encoding the variable regions of heavy (VH) and light (VL) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab).Conclusion
This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy. 相似文献14.
Sun-Ae Jun Dong Hyun Nam Young-Seob Lo Ju-Jin Kim Hyunju Chang Jeong-O Lee Yong Hwan Kim Byoung-In Sang 《Biotechnology and Bioprocess Engineering》2010,15(5):810-816
The preS2 antigens of hepatitis B virus (HBV), which causes a serious health problem in the world, have been implicated in
hepatocyte cell binding and viral penetration. Therefore, the importance of antibody production against preS2 antigen for
early diagnosis of HBV has been well established. In this study, the recombinant HBV preS2 single chain variable fragment
(scFv) antibody was successfully expressed in E. coli with the novel cold shock vector (pCold) under the cspA promoter, and its expression level was compared with the pET vector under the T7 promoter. Additionally, a host with an oxidizing
cytoplasm, E. coli trxB/gor double mutant, was used to improve the soluble expression. The anti-HBV preS2 scFv using pCold vector was successfully expressed
in a soluble and functional form in both wild type and double mutant E. coli, while the scFv using the pET vector was expressed in an insoluble form in spite of using a double mutant providing an oxidizing
environment. The induction with 0.05 mM IPTG showed a 2-fold higher functional expression compared to induction with 1 mM
IPTG, and the functional expression at the induction temperature (15°C), which is optimal temperature for pCold vector, was
improved 2-fold and 3- fold at 4 and 25°C, respectively. The efficacy of anti-HBV preS2 scFv for detecting HBV preS2 antigen
was tested and verified by using Ni-decorated single-walled carbon nanotube (SWNT) field effect transistors. 相似文献
15.
Michael Hust Thomas Jostock Christian Menzel Bernd Voedisch Anja Mohr Mariam Brenneis Martina I Kirsch Doris Meier Stefan Dübel 《BMC biotechnology》2007,7(1):14
Background
The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. 相似文献16.
Park KJ Park DW Kim CH Han BK Park TS Han JY Lillehoj HS Kim JK 《Biotechnology letters》2005,27(5):289-295
Chicken monoclonal antibody (mAb), 8C3, which is reactive with a sporozoite antigen of Eimeria acervulina, is a potential therapeutic agent against avian coccidiosis caused by Eimeria spp. However, production of large amounts of 8C3 mAb in cell culture system is labor intensive and not cost-effective. Accordingly, recombinant single chain variable fragment (ScFv) antibody was constructed by amplification of the VH and VL genes from chicken hybridoma, 8C3 and when expressed in E. coli gave 5 mg l–1. The expressed protein showed antigen binding activity equivalent to that of the parental mAb. In addition, nucleotide sequence comparison of 8C3 gene to the germline chicken VL genes suggested that the gene conversion with V pseudogenes might contribute to the diversification of VL genes in chickens. 相似文献
17.
Bühler P Wolf P Gierschner D Schaber I Katzenwadel A Schultze-Seemann W Wetterauer U Tacke M Swamy M Schamel WW Elsässer-Beile U 《Cancer immunology, immunotherapy : CII》2008,57(1):43-52
Background Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists
after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking
of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific
cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy
with bispecific diabodies could be a promising novel treatment option for prostate cancer.
Methods A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA
single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv VHCD3-VLPSMA and VHPSMA-VLCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography
(IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as
on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation
the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model.
Results By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both
to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to
be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor
xenografts with the diabody and PBL efficiently inhibited tumor growth.
Conclusions The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal
residual disease.
P. Bühler and P. Wolf equally contributed to the work. 相似文献
18.
Peter Iliades David A. Dougan Geoffrey W. Oddie Dennis W. Metzger Peter J. Hudson Alexander A. Kortt 《Journal of Protein Chemistry》1998,17(3):245-254
A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E. coli (10 mg/L). The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 g/ml) was stable for 7 days at 21°C and 30 days at 4°C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomer–dimer equilibrium mixture after 30 days at 4°C. The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of M
r 156,000. Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-1G10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair. This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab. 相似文献
19.
Yonghua Qi Congming Wu Suxia Zhang Zhanhui Wang Siyang Huang Lei Dai Shaochen Wang Lining Xia Kai Wen Xingyuan Cao Yongning Wu Jianzhong Shen 《PloS one》2009,4(7)
Background
Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes.Methodology/Principal Findings
In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM2) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA–ribosome–antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM2-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM2 by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis.Conclusions/Significance
The selection of anti-SM2 specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs. 相似文献20.
Gorawit Yusakul Seiichi Sakamoto Benyakan Pongkitwitoon Satoshi Morimoto 《Bioscience, biotechnology, and biochemistry》2016,80(7):1306-1312
The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs. 相似文献