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Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens. Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized. Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues. Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications. [ABSTRACT FROM AUTHOR]

Objective: To screen mimic epitope of Norovirus (NoV) by Ph.D.-12 phage display peptide library. Methods: Single-chain variable fragment antibody (scFv) with high specificity and neutralizing ability against G II.4/GII.6/G II. 17 NoV were coated and screened for 3 rounds with Ph.D.-12 phage library. ELISA was used to identify binding activity of phage with scFv and its competition with NoV P protein. Positive clones were sequenced and bioinformatic analysis was performed, antigenicity was identified by synthetic peptides. Results: A sequence was found to be with highly homologous to GII.6 VP1 region: MG-D-W, suggesting that it was a specific epitope of GII.6 NoV. Peptide containing "MG-D-W" was further synthesized, which could competitively inhibit binding of P protein to HBGA receptor. Conclusion: MG-D-W is a peptide with high affinity for NoV scFv and likely mimics antigenic epitope of GII.6 NoV bound to scFv. [ABSTRACT FROM AUTHOR]

Abstract Background Oxidation-specific epitopes (OSEs) are rich in atherosclerotic plaques. Innate and adaptive immune responses to OSEs play an important role in atherosclerosis. The purpose of this study was to develop novel human single-chain variable fragment (scFv) antibody specific to OSEs to image and inhibit atherosclerosis. Results Here, we screened a novel scFv antibody, named as ASA6, from phage-displayed human scFv library. ASA6 can bind to oxidized LDL (Ox-LDL) and atherosclerotic plaques. Meanwhile, ASA6 can also inhibit the uptake of Ox-LDL into macrophage to reduce macrophage apoptosis. The atherosclerotic lesion area of ApoE −/− mice administrated with ASA6 antibody was significantly reduced. Transcriptome analysis reveals the anti-atherosclerosis effect of ASA6 is related to the regulation of fatty acid metabolism and inhibition of M1 macrophage polarization. Moreover, we conjugated ASA6 antibody to NaNdF4@NaGdF4 nanoparticles for noninvasive imaging of atherosclerotic plaques by magnetic resonance (MR) and near-infrared window II (NIR-II) imaging. Conclusions Together, these data demonstrate the potential of ASA6 antibody in targeted therapy and noninvasive imaging for atherosclerosis. Graphic abstract

To determine furaltadone metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) residual levels in ctenopharyngodon idellus, direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. Firstly, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive VH and VL genes were assembled to a scFv gene and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step dcELISA against AMOZ derivative was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 10.62 and 4.83 ng/mL, respectively. The recovery values of AMOZ from spiked fish samples determined by dcELISA were in 71.31%∼82.88%. The results suggested that the prepared scFv-AP fusion protein in this work can be used for rapid and convenient immunoassays to detect AMOZ residues in fish samples.

To determine furaltadone metabolites 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) residual levels in ctenopharyngodon idellus, direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed based on a single-chain variable fragment (scFv)-alkaline phosphatase (AP) fusion protein. Firstly, VH and VL gene sequences were cloned from hybridoma cell lines secreting monoclonal antibody against AMOZ derivative, and then the productive VH and VL genes were assembled to a scFv gene and engineered to produce scFv-AP fusion protein. The fusion protein was further identified as a bifunctional reagent for immunoassay, then a sensitive one-step dcELISA against AMOZ derivative was developed with a half-maximal inhibitory concentration (IC50) and limit of detection (LOD) of 10.62 and 4.83 ng/mL, respectively. The recovery values of AMOZ from spiked fish samples determined by dcELISA were in 71.31%∼82.88%. The results suggested that the prepared scFv-AP fusion protein in this work can be used for rapid and convenient immunoassays to detect AMOZ residues in fish samples. [ABSTRACT FROM AUTHOR]

Background: Oxidation-specific epitopes (OSEs) are rich in atherosclerotic plaques. Innate and adaptive immune responses to OSEs play an important role in atherosclerosis. The purpose of this study was to develop novel human single-chain variable fragment (scFv) antibody specific to OSEs to image and inhibit atherosclerosis. Results: Here, we screened a novel scFv antibody, named as ASA6, from phage-displayed human scFv library. ASA6 can bind to oxidized LDL (Ox-LDL) and atherosclerotic plaques. Meanwhile, ASA6 can also inhibit the uptake of Ox-LDL into macrophage to reduce macrophage apoptosis. The atherosclerotic lesion area of ApoE−/− mice administrated with ASA6 antibody was significantly reduced. Transcriptome analysis reveals the anti-atherosclerosis effect of ASA6 is related to the regulation of fatty acid metabolism and inhibition of M1 macrophage polarization. Moreover, we conjugated ASA6 antibody to NaNdF4@NaGdF4 nanoparticles for noninvasive imaging of atherosclerotic plaques by magnetic resonance (MR) and near-infrared window II (NIR-II) imaging. Conclusions: Together, these data demonstrate the potential of ASA6 antibody in targeted therapy and noninvasive imaging for atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a common cause of dementia among elderly people. Hyperphosphorylation and aggregation of tau correlates with the clinical progression of AD; therefore, therapies targeting the aggregation of tau may have potential applications for anti-AD drug development. Several inhibitors of tau aggregation, including small molecules and antibodies, have been found to decrease the aggregation of tau and the corresponding pathology.Objective: To screen one kind of single-chain variable fragment (scFv) antibody which could inhibit the aggregation of tau and ameliorate its cytotoxicity.Methods/results: Using phosphorylated tau (pTau) as an antigen, we obtained a scFv antibody via the screening of a high-capacity phage antibody library. Biochemical analysis revealed that this scFv antibody (scFv T1) had a strong ability to inhibit pTau aggregation both in dilute solutions and under conditions of macromolecular crowding. ScFv T1 could also depolymerize preformed pTau aggregates in vitro. Furthermore, scFv T1 was found to be able to inhibit the cytotoxicity of extracellular pTau aggregates and ameliorate tau-mediated toxicity when coexpressed with a hTauR406W mutant in the eye of transgenic Drosophila flies.Conclusion: This scFv T1 antibody may be a potential new therapeutic agent against AD. Our methods can be used to develop novel strategies against protein aggregation for the treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

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Heat shock protein 70.1 (Hsp70.1), also known as Hsp70, is a highly conserved member of the heat shock protein family that exists in all living organisms and determines the protein fate as molecular chaperones. Hsp70 basal expression is undetectable or low in most unstressed normal cells, however, its abundant presence in several types of human cancer cells is reported. Several studies support upregulated Hsp70 involved in tumor progression and drug resistance through modulation of cell death pathways and suppresses anticancer immune responses. However, numerous studies have confirmed that Hsp70 can also induce anticancer immune responses through the activation of immune cells in particular antigen-presenting cells (APCs). Regarding the significant and the promising role of vaccines in cancer immunotherapy, identification and characterization of the overexpressed Hsp70 as a potential immune stimulatory factor can pave the path for development of highly effective anticancer vaccines. In this review, we will discuss the interactions of Hsp70 with components of the immune system in cancers as well as possible strategies to harness Hsp70 for eliciting anticancer immune responses.

Heat shock protein 70.1 (Hsp70.1), also known as Hsp70, is a highly conserved member of the heat shock protein family that exists in all living organisms and determines the protein fate as molecular chaperones. Hsp70 basal expression is undetectable or low in most unstressed normal cells, however, its abundant presence in several types of human cancer cells is reported. Several studies support upregulated Hsp70 involved in tumor progression and drug resistance through modulation of cell death pathways and suppresses anticancer immune responses. However, numerous studies have confirmed that Hsp70 can also induce anticancer immune responses through the activation of immune cells in particular antigen-presenting cells (APCs). Regarding the significant and the promising role of vaccines in cancer immunotherapy, identification and characterization of the overexpressed Hsp70 as a potential immune stimulatory factor can pave the path for development of highly effective anticancer vaccines. In this review, we will discuss the interactions of Hsp70 with components of the immune system in cancers as well as possible strategies to harness Hsp70 for eliciting anticancer immune responses. [ABSTRACT FROM AUTHOR]

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens., Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized., Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues., Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications., Competing Interests: B-YT is the chairman of Navi Bio-Therapeutics Inc. Taipei, Taiwan. The company has no role in the study’s experimental design, data collection, or interpretation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Leu, Lee, Lin, Wang, Tsai, Lee, Chen, Yang and Lin.)

Mu-2-related death-inducing (MuD) gene is involved in apoptosis in tumor cells. Although we have previously produced mouse monoclonal antibodies (MAbs) that specifically recognize human MuD, the application scope of MuD MAbs was restricted due to their mouse origin. Therefore, we attempted the generation of single-chain variable fragment (scFv) against MuD. The heavy- and light-chain variable region genes from two MuD hybridomas were isolated by PCR and joined by DNA encoding a (Gly4Ser1)3 linker. These scFv fragments were cloned into a phagemid vector and expressed as E-tagged fusion proteins in Escherichia coli HB2151. The reactivity of selected Abs was evaluated using ELISA. Selected MuDscFv Abs specifically recognized human MuD, retaining ~ 50% potency of the parent MAbs. MuDscFv-M3H9 recognized the middle region of MuD, while MuDscFv-C22B3 recognized a broad region. Intracellular expression of MuDscFvs-C22B3 protected cells from TRAIL-induced apoptosis. These MuDscFv Abs may help in the study of intracellular signaling pathway centered on MuD and of drug use target and points. [ABSTRACT FROM AUTHOR]

Abstract Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease characterized by motor neuron loss in the brain and spinal cord. Mutations in Cu-Zn superoxide dismutase (SOD1) are the first identified genetic mutations that are causative for familial ALS. Soluble SOD1 oligomers are considered the most toxic species and play a key role in the pathologic process of ALS. Here we present a therapeutic strategy for ALS with an oligomer-specific antibody (W20) targeting toxic SOD1 oligomers. Our study showed that W20 significantly improved motor neuron survival and motor performance in SOD1-G93A mouse model of ALS when administrated even at low dose within short time. Further investigation demonstrated that the beneficial effects of W20 resulted from the reduction of SOD1 oligomer levels and the inhibition of gliosis and neuroinflammation in the spinal cords and brain stems of ALS model mice. These findings for the first time suggest that an oligomer-specific antibody has promising therapeutic potential for ALS and open a new way for ALS treatment. Highlights • Antibody W20 improved motor neuron survival and motor performance in SOD1-G93A mice. • Antibody W20 reduced SOD1 aggregates and oligomer levels in SOD1-G93A mice. • Antibody W20 inhibited gliosis and neuroinflammation in SOD1-G93A mice. [ABSTRACT FROM AUTHOR]

To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction. Finally, the library for anti-VP3 scFv was screened by four rounds of adsorption–elution–enrichment with the purified VP3 protein. The characters of binding ability, specificity and neutralization of soluble antibodies expressed were evaluated by ELISA. The results showed 7 VP3-specific scFvs have been screened and identified with high both sensitivity and specificity for binding DHAV-1. To our knowledge, this is the first report for VP3-specific scFv of DHAV-1 and potentially promising application used in prevention and treatment of duck viral hepatitis. [ABSTRACT FROM AUTHOR]

Background and aims Early detection and evaluation of vulnerable atherosclerotic plaque are important for risk stratification and timely intervention, and vascular cell adhesion molecule 1 (VCAM1) assists in adhesion and recruitment of inflammatory cells to vulnerable lesions. We labeled a single-chain variable fragment (scFv) of VCAM1 with 99m technetium ( 99m Tc) and fluorescent markers to investigate its potential utility in detecting vulnerable plaques in animal models of atherosclerosis. Methods We labeled VCAM1 scFv with 99m Tc and cyanine5 (CY5) and evaluated the probes on apolipoprotein E gene-deficient mice and New Zealand White rabbits with induced atherosclerosis. Histopathology and Western blot examinations confirmed atherosclerotic plaque and VCAM1 expression in the aortas. In vivo biodistribution of 99m Tc-scFv-VCAM1 was studied. Abdominal organs of mice were removed after CY5-scFv-VCAM1 administration for aortic fluorescence imaging. Rabbits SPECT imaging of 99m Tc-scFv-VCAM1 was performed and autoradiography (ARG) of the aortas was checked to confirm the tracer uptake. Results The radiochemical purity of 99m Tc-scFv-VCAM1 was 98.72± 1.04% ( n = 5) and its specific activity was 7.8 MBq/μg. Biodistribution study indicated predominant probe clearance by kidneys. In fluorescence imaging, stronger signal from CY5-scFv-VCAM1 in the aorta was observed in atherosclerotic mice than that in controls. SPECT imaging with 99m Tc-scFv-VCAM1 showed tracer uptake in the abdominal aorta and the aortic arch of atherosclerotic animals. ARG confirmed tracer uptake in the aortas of atherosclerotic rabbits, with higher uptake ratios of aortic arch/descending aorta in experimental animals (4.45 ± 0.63, n = 5) than controls (1.12 ± 0.15, n = 5; p < 0.05). Conclusions SPECT and fluorescence imaging results showed the feasibility and effectiveness of detecting vulnerable plaque with scFv of VCAM1, indicating its potential for early diagnosis and evaluation of atherosclerosis. [ABSTRACT FROM AUTHOR]

A novel monoclonal antibody against human epidermal growth factor receptor 2 (HER2), i.e., pertuzumab (Perjeta®) developed by Genentech, has been verified to be effective in treating metastatic HER2-overexpressing breast cancer. The fact that the presence of the Fc region of the anti-HER2 is uncritical for growth inhibition of tumor cells suggests the potential biological activity of the associated antibody fragments. In the present study, we report functional expression of anti-HER2his-scFv, a single-chain variable fragment (scFv) derived from pertuzumab, in the periplasm of Escherichia coli and its purification. Biological activity of the soluble scFv produced in this manner was characterized using immunofluorescent staining, immunocytochemistry, flow cytometry and cytotoxicity assay. The effect of anti-HER2his-scFv on HER2 dimerization was also assessed by tyrosine kinase assay. It was observed that the purified scFv had a high specificity and affinity to HER2 receptors expressed on the surface of tumor cells with a selective cytotoxic effect on HER2-overexpressing SK-OV-3 cells. In addition, anti-HER2his-scFv was able to suppress phosphorylation of HER2 in the presence of heregulin. The results suggest that anti-HER2his-scFv can be a potential candidate for various therapeutic and diagnosis applications. [ABSTRACT FROM AUTHOR]

The human epidermal growth factor receptor (HER) family plays an important role in cell growth and signaling and alteration of its function has been demonstrated in many different kinds of cancer. Receptor dimerization is necessary for the HER signal transduction pathway and tyrosine kinase activity. Recently, several monoclonal antibodies have been developed to directly interfere with ligand-HER receptor binding and receptor dimerization. A single chain variable fragment (ScFv) is a valuable alternative to an intact antibody. This report describes the production and purification of an ScFv specific for domain II of the HER2 receptor in Escherichia coli BL21 (DE3) cytoplasm. The majority of expressed of anti-her2his-ScFv protein was produced as inclusion bodies. A Ni-NTA affinity column was used to purify the anti-her2his-ScFv protein. The molecular weight of anti-her2his-ScFv protein was estimated to be approximately 27 kDa, as confirmed by SDS-PAGE and Western blotting assay. The anti-her2his-ScFv showed near 95 % purity and reached a yield of approximately 29 mg/l in flask fermentation. The purified anti-her2his-ScFv showed its biological activity by binding to HER2 receptor on the surface of BT-474 cells. This ScFv may be a potential pharmaceutical candidate for targeting tumour cells overexpressing HER2 receptor. [ABSTRACT FROM AUTHOR]

Nervous necrosis virus (NNV), causing almost 100% mortality among infected larvae and juvenile fish, is severely affecting the aquaculture industry. Although various vaccines against NNV were studied, they can hardly provoke strong enough immunity in fry with immature immune systems. To eliminate the infection possibility during fertilization, we applied an anti-NNV strategy by neutralization based on a single-chain variable fragment (ScFv) antibody, named Sc3F9, against orange-spotted grouper nervous necrosis virus (OGNNV). Sc3F9 binds to the shell domain of viral capsid protein (CP) with a dissociation constant (K D) of 48.6 nM by interacting with two discontinuous target sites on CP fragments of G 122 -L 128 and L 141 -A 148. The binding capacity was enhanced by prokaryotic expressing double copies of Sc3F9 (DSc3F9) in tandem, and the augmented avidity (K D = 29.5 nM) with irreversible binding to OGNNV was stable even in high salinity, indicating the wide application potential in seawater. DSc3F9 exhibited great recognition breadth within the Betanodavirus genus to bind all NNV serotypes in working concentration (40 μg/mL) and distinguished these three serotypes by different levels of binding capacity in low concentration (10 μg/mL). DSc3F9 blocked virus-like particle entry by destructing its virion as well as strongly neutralized OGNNV with an average half-maximal inhibitory concentration (IC50) value of 0.64 μM. Moreover, DSc3F9 efficiently eliminated the possible NNV infection during fertilization by washing grouper eggs before artificial insemination without reducing the fertility rate and hatchability. Thus, our results not only obtained a strong anti-NNV candidate but also provided an approach to NNV decontamination, especially in obtaining NNV-free grouper eggs. • The OGNNV-specific ScFv antibody (Sc3F9) binds two discontinuous target sites of shell domain on one capsid protein. • Double Sc3F9 (DSc3F9) strongly neutralizes NNV by destructing virion structure via irreversible binding. • DSc3F9 can recognize all NNV serotypes in high concentration and distinguish them in low concentration. • Before artificial insemination, DSc3F9 has practical value for eliminating NNV infection via grouper egg wash. [ABSTRACT FROM AUTHOR]

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica. [ABSTRACT FROM AUTHOR]

Abstract: Increasing evidence indicates that amyloid aggregates, including oligomers, protofibrils or fibrils, are pivotal toxins in the pathogenesis of many amyloidoses such as Alzheimer''s disease (AD), Parkinson''s disease, Huntington''s disease, prion-related diseases, type 2 diabetes and hereditary renal amyloidosis. Various oligomers assembled from different amyloid proteins share common structures and epitopes. Here we present data indicating that two oligomer-specific single chain variable fragment (scFv) antibodies isolated from a naïve human scFv library could conformation-dependently recognize oligomers assembled from α-synuclein, amylin, insulin, Aβ1–40, prion peptide 106–126 and lysozyme, and fibrils from lysozyme. Further investigation showed that both scFvs inhibited the fibrillization of α-synuclein, amylin, insulin, Aβ1–40 and prion peptide 106–126, and disaggregated their preformed fibrils. However, they both promoted the aggregation of lysozyme. Nevertheless, the two scFv antibodies could attenuate the cytotoxicity of all amyloids tested. Moreover, the scFvs recognized the amyloid oligomers in all types of plaques, Lewy bodies and amylin deposits in the brain tissues of AD and PD patients and the pancreas of type 2 diabetes patients respectively, and showed that most amyloid fibril deposits were colocalized with oligomers in the tissues. Such conformation-dependent scFv antibodies may have potential application in the investigation of aggregate structures, the mechanisms of aggregation and cytotoxicity of various amyloids, and in the development of diagnostic and therapeutic reagents for many amyloidoses. [Copyright &y& Elsevier]

The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 30% of human breast cancers. One of the mechanisms by which tumor cell proliferation can be inhibited consists in hampering HER2 dimerization by targeting its extracellular domain with specific antibodies. In recent clinical practice, a valuable alternative to entire IgGs resides in the use of smaller molecules, such as single-chain variable fragments (scFv), developed for selective molecular targeting. In this paper, we report on the production and purification of a soluble anti-HER2 scFv antibody secreted by Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at the 3′-end was inserted into the expression vector pPICZα and transformed in P. pastoris. The highest expression level was obtained in presence of 0.5% methanol and 0.8% glycerol in the culture medium after 48 h of induction. The use of P. pastoris proved very valuable as an expression system, allowing the isolation of 10 mg/L of highly purified antibody, remarkably higher than previously reported data. The functionality of purified anti-HER2 scFv was assessed by cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer cells, showing good affinity and high selectivity for the target membrane receptor. These findings confirm that P. pastoris is a suitable host for high level expression of antibody fragments and highlight the potential role of scFv800E6 in diagnostic and therapeutic application. [ABSTRACT FROM AUTHOR]

single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (GlySer) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding. [ABSTRACT FROM AUTHOR]

Connective tissue growth factor (CTGF) has been identified as playing critical roles in fibrosis and is a promising therapeutic target. In a previous study, we used a phage display library to develop a humanized single-chain variable fragment antibody (scFv) against CTGF. In the present study, the protective effect of anti-CTGF scFv against bleomycin (BL)-induced pulmonary fibrosis was investigated in mice. The expression of α-smooth muscle actin in human embryonic lung fibroblast (HELF) cells was analysed by western blotting. A mouse model of pulmonary fibrosis was established by tracheal injection of BL (5 mg/kg). Mice received anti-CTGF scFv (4 mg/kg, three times a week) by i.v. injection. The effects of anti-CTGF scFv were evaluated by leukocyte counts in BAL fluid, hydroxyproline measurements in lung tissue and pathological examination. α-Smooth muscle actin expression was decreased in HELF cells treated with anti-CTGF scFv. Anti-CTGF scFv significantly reduced the numbers of inflammatory leukocytes (total and differential count) in BAL fluid, as well as the hydroxyproline content of lung tissue. The severity of alveolitis and fibrosis in the mouse model was markedly attenuated by treatment with anti-CTGF scFv. Anti-CTGF scFv may potentially be developed as a useful inhibitor of pulmonary fibrosis. Connective tissue growth factor (CTGF) plays a critical role in fibrosis. A humanized single-chain variable fragment antibody (scFv) against CTGF inhibited collagen deposition and the severity of alveolitis and fibrosis in a mouse model. Anti-CTGF scFv may potentially be developed as a useful inhibitor of pulmonary fibrosis. [ABSTRACT FROM AUTHOR]

single-chain variable fragment antibody (scFv) against ginsenoside Re (G-Re) was constructed and applied to an enzyme-linked immunosorbent assay (ELISA) for determining the total concentration of ginsenosides in various ginsengs. The variable heavy and light chain genes were cloned directly from the cDNA of the 4G10 hybridoma cell line and assembled by means of splicing by overlapping extension PCR (SOE-PCR) using specific primers designed to have flexible peptide (GlySer) between the variable heavy chain and light chain domains. The constructed scFv gene was ligated into the pET28a expression vector and transformed into E. coli BL21 (DE3). The recombinant scFv against G-Re (GRe-scFv) was expressed as a chimera protein containing the His6-tag at its N-termini, purified by immobilized metal ion affinity chromatography (IMAC), and refolded by a stepwise dialysis method. The yield of GRe-scFv after purification was 1.7 mg per liter of culture medium. Characterization of GRe-scFv revealed that it retained the characteristics of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10) which has wide cross-reactivity with 20( S)-protopanaxadiol- and 20( S)-protopanaxatriol-type ginsenosides. The detectable range for G-Re in ELISA using scFv antibody was 0.02-10 µg/ml. Based on validation analysis, the use of GRe-scFv in ELISA is a precise, accurate, and sensitive method. In light of the time-consuming and labor-intensive procedures for the preparation of MAb, speedy bacterial expression of GRe-scFv is a powerful alternative tool for producing MAb to use in ELISA for quantitative analysis of total ginsenoside concentrations. [ABSTRACT FROM AUTHOR]

A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05–10 μg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. [ABSTRACT FROM PUBLISHER]

The administration of antibodies against the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a promising approach in the upregulation of immune responses in many cancers and infectious diseases. The single-chain variable fragment of antibody against CTLA4 is also useful in developing immunotoxins that might be used in the treatment of cancer, transplant rejection, and autoimmune diseases. Here, we report the production of a soluble and functional scFv antibody against CTLA4 by using Pichia pastoris as the expression system. The gene encoding scFv hS83 with an additional 6His-tag at the 5’-end was inserted into the expression vector pPIC9K. Then, the transformants were double-screened on plates containing 0.25 mg/mL and 1.5 mg/mL of neomycin G418 and many clones with different levels of G418-resistance were selected for further studies on expression. After induction by the addition of methanol, various levels of hS83 were detected in the supernatant of P. pastoris containing pPIC9K-hS83. Clones with low G418-resistance produced more hS83 than those with higher G418-resistance. Under the optimized conditions (initial inoculum, 40 A600nm AU/mL; pH 6.0; methanol concentration, 3.0%; induction time, 72 h), approximately 16–20 mg protein could be recovered from 1 L of the culture. The purified hS83 had a stronger binding ability towards CTLA4-positive Raji cells than CTLA4-negative ECV304 cells. This finding indicates that the antibody produced by P. pastoris is functional and may be used in immunotherapy for cancer, infection, transplant rejection, and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Abstract: A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the β′ subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a “linker” sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60mg purified His-tagged scFv per liter of culture (3.3g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73. [Copyright &y& Elsevier]

• Single-chain variable fragment antibody-based rapid test strip has been developed. • The immunochromatographic strip (ICS) showed good specificity for FB 1. • The ICS prepared with scFv was 8 times more sensitive than with monoclonal antibody. A rapid and sensitive immunochromatographic strip (ICS) based on a single-chain variable fragment (scFv) was developed for detecting fumonisin B 1 (FB 1). The ICS was based on a competitive reaction for colloidal gold-labeled scFv between FB 1 and FB 1 -BSA, which was used along with sheep anti-mouse IgG as capture reagents immobilized at test and control lines, respectively, on a nitrocellulose membrane of the strip. The limit of detection of the ICS was 2.5 ng/mL (25 μg/kg) FB 1 in buffer, and the sensitivity was eight times higher than that of monoclonal antibodies for the preparation of the scFv. The cross-reactivity of the scFv with common mycotoxins was determined by ICS, the results showed that the scFv were not against other mycotoxins. Eight naturally contaminated maize samples were analyzed with the scFv-based ICS and by LC-MS/MS. The results of analysis obtained with the strip assay showed good agreement with those obtained by LC-MS/MS. [ABSTRACT FROM AUTHOR]

Background: The venom of bamboo vipers ( Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites., Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins., Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 10 7 and 6.8 × 10 7 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2 nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins., Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future., Competing Interests: Competing interests: The authors declare that they have no competing interests.