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We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L- assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L- assay were compared. PERT and S+/L- assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.
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Vetores Genéticos , Vírus da Leucemia do Macaco Gibão , DNA Polimerase Dirigida por RNA , Humanos , Vírus da Leucemia do Macaco Gibão/genética , Vetores Genéticos/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas do Envelope Viral/genética , Linhagem Celular , Replicação Viral , Virologia/métodos , Retroviridae/genética , Células HEK293 , Glicoproteínas de MembranaRESUMO
The lack of intrinsic active sites for photocatalytic CO2 reduction reaction (CO2RR) and fast recombination rate of charge carriers are the main obstacles to achieving high photocatalytic activity. In this work, a novel phosphorus and boron binary-doped graphitic carbon nitride, highly porous material that exhibits powerful photocatalytic CO2 reduction activity, specifically toward selective CO generation, is disclosed. The coexistence of Lewis-acidic and Lewis-basic sites plays a key role in tuning the electronic structure, promoting charge distribution, extending light-harvesting ability, and promoting dissociation of excitons into active carriers. Porosity and dual dopants create local chemical environments that activate the pyridinic nitrogen atom between the phosphorus and boron atoms on the exposed surface, enabling it to function as an active site for CO2RR. The P-N-B triad is found to lower the activation barrier for reduction of CO2 by stabilizing the COOH reaction intermediate and altering the rate-determining step. As a result, CO yield increased to 22.45 µmol g-1 h-1 under visible light irradiation, which is ≈12 times larger than that of pristine graphitic carbon nitride. This study provides insights into the mechanism of charge carrier dynamics and active site determination, contributing to the understanding of the photocatalytic CO2RR mechanism.
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Pancreatic adenocarcinoma, a highly aggressive form of cancer with a poor prognosis, necessitates the development of innovative treatment strategies. Our prior research showcased the growth-inhibiting effects of the anti-EphA2 antibody drug hSD5 on pancreatic cancer tumors. This antibody targets and induces the degradation of the EphA2 receptor while also prompting the antibody's internalization. A deeper dive into the hSD5 Fab crystallographic structure and docking studies revealed that hSD5's CDRH3 drives the primary interaction between hSD5 and the EphA2 active site. In this study, we developed a novel antibody-drug conjugate (ADC)-the auristatin-based hSD5-vedotin specifically targeting EphA2 in pancreatic cancer cells. This ADC aims at the tumor-specific antigen EphA2, triggering endocytosis and releasing the conjugated payload molecule Monomethyl auristatin E (MMAE), amplifying the tumor-killing effect. Upon cellular entry, hSD5-vedotin demonstrated an impressive tumor-killing response, inhibiting tumor cell growth and promoting apoptosis even at lower antibody concentrations. In a pancreatic cancer xenograft animal model, hSD5-vedotin showcased the potential to suppress tumor growth entirely. Notably, potential immune resistance responses were also observed in recurrent pancreatic cancer tumors. Our empirical results underscore the possibility of developing hSD5-vedotin further, which we anticipate will have a broader and more potent therapeutic impact on pancreatic cancer and other EphA2-related cancers.
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Adenocarcinoma , Imunoconjugados , Neoplasias Pancreáticas , Animais , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Neoplasias Pancreáticas/patologia , Adenocarcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Erythropoietin-producing hepatocyte receptor type A2 (EphA2) is a tyrosine kinase that binds to ephrins (e.g., ephrin-A1) to initiate bidirectional signaling between cells. The binding of EphA2 and ephrin-A1 leads to the inhibition of Ras-MAPK activity and tumor growth. During tumorigenesis, the normal interaction between EphA2 and ephrin-A1 is hindered, which leads to the overexpression of EphA2 and induces cancer. The overexpression of EphA2 has been identified as a notable tumor marker in diagnosing and treating pancreatic cancer. In this study, we used phage display to isolate specific antibodies against the active site of EphA2 by using a discontinuous recombinant epitope for immunization. The therapeutic efficacy and inhibition mechanism of the generated antibody against pancreatic cancer was validated and clarified. The generated antibodies were bound to the conformational epitope of endogenous EphA2 on cancer cells, thus inducing cellular endocytosis and causing EphA2 degradation. Molecule signals pAKT, pERK, pFAK, and pSTAT3 were weakened, inhibiting the proliferation and migration of pancreatic cancer cells. The humanized antibody hSD5 could effectively inhibit the growth of the xenograft pancreatic cancer tumor cells BxPc-3 and Mia PaCa-2 in mice, respectively. When antibody hSD5 was administered with gemcitabine, significantly improved effects on tumor growth inhibition were observed. Based on the efficacy of the IgG hSD5 antibodies, clinical administration of the hSD5 antibodies is likely to suppress tumors in patients with pancreatic cancer and abnormal activation or overexpression of EphA2 signaling.
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Studies have shown that the high expression of EphA4 in gastric cancer tissues may correlate with unfavorable clinical pathological characteristics. Therefore, EphA4 may be an effective target for treating gastric cancer in addition to HER-2/neu. In this study, generated scFv S3 can bind endogenous EphA4 of gastric cancer cells and has significant membrane staining. Additionally, scFv S3 binding to EphA4 inhibits the growth and migration of cancer cells and the growth induction that ephrinA1 generates in gastric cancer cells. We found that EphA4 molecules may degrade through antibody treatment of cells, and the increase in LAMP1 and LAMP2 indicates that lysosome is involved in the degradation. The scFv S3 administration leads to the signals pAKT, pERK, and pSTAT3 decrease in cancer cells. The xenograft model of HER-2/neu low expressing gastric cancer cell SNU-16 exhibits better therapeutic effects by scFv S3 than trastuzumab scFv. The scFv S3 administration in vivo can degrade EphA4 molecules in tumor tissues, decreasing Ki67 and increasing cleaved C3 molecule expression. Furthermore, we identified and validated that scFv S3 generates essential ionic bonding with R162 on EphA4. The antibody may provide effective treatment for patients with gastric cancer and abnormal activation or overexpression of EphA4 signaling.
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Anticorpos de Cadeia Única , Neoplasias Gástricas , Humanos , Transdução de Sinais , Neoplasias Gástricas/tratamento farmacológico , Anticorpos de Cadeia Única/farmacologia , AnimaisRESUMO
Structural flexibility is a critical issue that limits the application of metal-organic framework (MOF) membranes for gas separation. Herein we propose a mixed-linker approach to suppress the structural flexibility of the CAU-10-based (CAU = Christian-Albrechts-University) membranes. Specifically, pure CAU-10-PDC membranes display high separation performance but at the same time are highly unstable for the separation of CO2/CH4. A partial substitution (30 mol.%) of the linker PDC with BDC significantly improves its stability. Such an approach also allows for decreasing the aperture size of MOFs. The optimized CAU-10-PDC-H (70/30) membrane possesses a high separation performance for CO2/CH4 (separation factor of 74.2 and CO2 permeability of 1,111.1 Barrer under 2 bar of feed pressure at 35°C). A combination of in situ characterization with X-ray diffraction (XRD) and diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, as well as periodic density functional theory (DFT) calculations, unveils the origin of the mixed-linker approach to enhancing the structural stability of the mixed-linker CAU-10-based membranes during the gas permeation tests.
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BACKGROUND: The main commercially available methods for detecting small molecules of mycotoxins in traditional Chinese medicine (TCM) and functional foods are enzyme-linked immunosorbent assay and mass spectrometry. Regarding the development of diagnostic antibody reagents, effective methods for the rapid preparation of specific monoclonal antibodies are inadequate. METHODS: In this study, a novel synthetic phage-displayed nanobody Golden Glove (SynaGG) library with a glove-like cavity configuration was established using phage display technology in synthetic biology. We applied this unique SynaGG library on the small molecule aflatoxin B1 (AFB1), which has strong hepatotoxicity, to isolate specific nanobodies with high affinity for AFB1. RESULT: These nanobodies exhibit no cross-reactivity with the hapten methotrexate, which is recognized by the original antibody template. By binding to AFB1, two nanobodies can neutralize AFB1-induced hepatocyte growth inhibition. Using molecular docking, we found that the unique non-hypervariable complementarity-determining region 4 (CDR4) loop region of the nanobody was involved in the interaction with AFB1. Specifically, the CDR4's positively charged amino acid arginine directed the binding interaction between the nanobody and AFB1. We then rationally optimized the interaction between AFB1 and the nanobody by mutating serine at position 2 into valine. The binding affinity of the nanobody to AFB1 was effectively improved, and this result supported the use of molecular structure simulation for antibody optimization. CONCLUSION: In summary, this study revealed that the novel SynaGG library, which was constructed through computer-aided design, can be used to isolate nanobodies that specifically bind to small molecules. The results of this study could facilitate the development of nanobody materials to detect small molecules for the rapid screening of TCM materials and foods in the future.
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Integrating vectors are associated with alterations in cellular function related to disruption of normal gene function. This has been associated with clonal expansion of cells and, in some instances, cancer. These events have been associated with replication-defective vectors and suggest that the inadvertent exposure to a replication-competent virus arising during vector manufacture would significantly increase the risk of treatment-related adverse events. These risks have led regulatory agencies to require specific monitoring for replication-competent viruses, both prior to and after treatment of patients with gene therapy products. Monitoring the risk of cell expansion and malignancy is also required. In this review, we discuss the rational potential approaches and challenges to meeting the US FDA expectations listed in current guidance documents.
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The clinical impact of any therapy requires the product be safe and effective. Gammaretroviral vectors pose several unique risks, including inadvertent exposure to replication competent retrovirus (RCR) that can arise during vector manufacture. The US FDA has required patient monitoring for RCR, and the National Gene Vector Biorepository is an NIH resource that has assisted eligible investigators in meeting this requirement. To date, we have found no evidence of RCR in 338 pre-treatment and 1,595 post-treatment blood samples from 737 patients associated with 60 clinical trials. Most samples (75%) were obtained within 1 year of treatment, and samples as far out as 9 years after treatment were analyzed. The majority of trials (93%) were cancer immunotherapy, and 90% of the trials used vector products produced with the PG13 packaging cell line. The data presented here provide further evidence that current manufacturing methods generate RCR-free products and support the overall safety profile of retroviral gene therapy.
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Retroviridae , Replicação Viral , Humanos , Retroviridae/genética , Vetores Genéticos/genética , Linhagem Celular , Terapia Genética/efeitos adversosRESUMO
For highly conserved mammalian protein, chicken is a suitable immune host to generate antibodies. Monoclonal antibodies have been successfully targeted with immunity checkpoint proteins as a means of cancer treatment; this treatment enhances tumor-specific immunity responses through immunoregulation. Studies have identified the importance of B7-H4 in immunoregulation and its use as a potential target for cancer treatment. High levels of B7-H4 expression are found in tumor tissues and are associated with adverse clinical and pathological characteristics. Using the phage display technique, this study isolated specific single-chain antibody fragments (scFvs) against B7-H4 from chickens. Our experiment proved that B7-H4 clearly induced the inhibition of T-cell activation. Therefore, use of anti-B7-H4 scFvs can effectively block the exhaustion of immunity cells and also stimulate and activate T-cells in peripheral blood mononuclear cells. Sequence analysis revealed that two isolated scFv S2 and S4 have the same VH complementarity-determining regions (CDRs) sequence. Molecule docking was employed to simulate the complex structures of scFv with B7-H4 to analyze the interaction. Our findings revealed that both scFvs employed CDR-H1 and CDR-H3 as main driving forces and had strong binding effects with the B7-H4. The affinity of scFv S2 was better because the CDR-L2 loop of the scFv S2 had three more hydrogen bond interactions with B7-H4. The results of this experiment suggest the usefulness of B7-H4 as a target for immunity checkpoints; the isolated B7-H4-specific chicken antibodies have the potential for use in future cancer immunotherapy applications.
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Galinhas/imunologia , Leucócitos Mononucleares/imunologia , Anticorpos de Cadeia Única/imunologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/imunologia , Animais , Linfócitos T/imunologiaRESUMO
The Astragalus membranaceus polysaccharides (APS) can improve immunity and enhance treatment reactions. This study analyzed the effects of effective antivascular endothelial growth factor (anti-VEGF) antibody production in mice treated with APS. After APS treatment, the serum of mice produced the antibody reactions that can cross-validate VEGF. The isolated single-chain fragment variable (scFv) antibodies could neutralize VEGF and inhibit in vivo tumor growth. Of the scFvs, scFv 4E can significantly compete the interaction of bevacizumab with VEGF. In cell experiments, scFv 4E effectively inhibited human umbilical vein endothelial cells induced by VEGF in vitro. In a matrix gel-assisted angiogenesis model, scFv 4E significantly inhibited angiogenesis reactions. In addition, in a xenograft model established in the colorectal cancer cell strain HCT116, scFv 4E treatment inhibited tumor growth by up to 52.7%. Finally, molecule docking was performed to simulate the complex interactions of scFv 4E and VEGF, the main driving forces of which involve the hydrophobic interactions and hydrogen bonds of Tyr108 and Tyr 109 of the complementarity-determining region H3 loop with VEGF. The results help in establishing antibody library with high diversity for selecting antibodies with specificity. In addition, this study indirectly expounded the correlations of APS enhancing immunity regulation in vivo.
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Astrágalo/química , Polissacarídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Indutores da Angiogênese , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Bevacizumab , Biomarcadores Tumorais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Células HCT116 , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Neoplasias Experimentais , Neovascularização Patológica , Biblioteca de Peptídeos , Conformação Proteica , Anticorpos de Cadeia ÚnicaRESUMO
BACKGROUND: ZNF322A is an oncogenic transcription factor that belongs to the Cys2His2-type zinc-finger protein family. Accumulating evidence suggests that ZNF322A may contribute to the tumorigenesis of lung cancer, however, the ZNF322A-mediated downstream signaling pathways remain unknown. METHODS: To uncover ZNF322A-mediated functional network, we applied phosphopeptide enrichment and isobaric labeling strategies with mass spectrometry-based proteomics using A549 lung cancer cells, and analyzed the differentially expressed proteins of phosphoproteomic and proteomic profiles to determine ZNF322A-modulated pathways. RESULTS: ZNF322A highlighted a previously unidentified insulin signaling, heat stress, and signal attenuation at the post-translational level. Consistently, protein-phosphoprotein-kinase interaction network analysis revealed phosphorylation of IRS1 and HSP27 were altered upon ZNF322A-silenced lung cancer cells. Thus, we further investigated the molecular regulation of ZNF322A, and found the inhibitory transcriptional regulation of ZNF322A on PIM3, which was able to phosphorylate IRS1 at serine1101 in order to manipulate glucose uptake via the PI3K/AKT/mTOR signaling pathway. Moreover, ZNF322A also affects the unfolded protein response by phosphorylation of HSP27S82 and eIF2aS51, and triggers autophagosome formation in lung cancer cells. CONCLUSIONS: These findings not only give new information about the molecular regulation of the cellular proteins through ZNF322A at the post-translational level, but also provides a resource for the study of lung cancer therapy.
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Autofagossomos/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Neoplasias Pulmonares/genética , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética , Resposta a Proteínas não Dobradas , Células A549 , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Oncogênicas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Astragalus membranaceus polysaccharide (APS) components are main ingredients of TCM and have proven efficacy to activate T cells and B cells, enhancing immunity in humans. In this study, elevated cytokine and anti-PD-1 antibody titers were found in mice after immunization with APS. Therefore, phage-display technology was utilized to isolate specific anti-programmed death-1 (PD-1) antibodies from mice stimulated by APS and to confirm whether the isolated anti-PD-1 antibody could inhibit the interaction of PD-1 with the programmed death-ligand 1 (PD-L1), resulting in tumor growth inhibition. The isolated single-chain fragment variable (scFv) S12 exhibited the highest binding affinity of 20 nM to PD-1, completed the interaction between PD-1 and PD-L1, and blocked the effect of PD-L1-induced T cell exhaustion in peripheral blood mononuclear cells in vitro. In the animal model, the tumor growth inhibition effect after scFv S12 treatment was approximately 48%. However, meaningful synergistic effects were not observed when scFv S12 was used as a cotreatment with ixabepilone. Moreover, this treatment caused a reduction in the number of tumor-associated macrophages in the tumor tissue. These experimental results indirectly indicate the ability of APS to induce specific antibodies associated with the immune checkpoint system and the potential benefits for improving immunity in humans.
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Anticorpos Monoclonais/imunologia , Anticorpos/uso terapêutico , Astragalus propinquus/química , Antígeno B7-H1/imunologia , Fatores Imunológicos/uso terapêutico , Neoplasias/tratamento farmacológico , Extratos Vegetais/farmacologia , Aloenxertos , Animais , Modelos Animais de Doenças , Feminino , Humanos , Imunidade , Antígeno Ki-67 , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos BALB C , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Anticorpos de Cadeia Única/farmacologia , Anticorpos de Cadeia Única/uso terapêutico , Linfócitos T/imunologia , Microambiente TumoralRESUMO
In clinical trials, the efficacy of treatment might be dependent on the value of a covariate variable. Therefore, it might be possible to detect the region over the covariate variable where the two treatments under investigation do not have significantly different efficacy or the region of superiority of one treatment. The non-significant region can be verified to be a confidence interval for the abscissa of the intersection point of two regression lines, and each of the complementary regions of the confidence interval corresponds to a region of superiority. In this study, we develop a method of constructing the confidence interval based on the concept of a generalized pivotal quantity, so as to perform the task of detecting the possible three regions for a clinical trial. Two real-world examples are given to illustrate the application of our proposed method, and a simulation study is conducted to evaluate its performance.
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Ensaios Clínicos Controlados Aleatórios como Assunto/estatística & dados numéricos , Projetos de Pesquisa/estatística & dados numéricos , Anti-Hipertensivos/uso terapêutico , Cálcio/uso terapêutico , Simulação por Computador , Interpretação Estatística de Dados , Feminino , Humanos , Hipertensão/diagnóstico , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Recém-Nascido de Baixo Peso , Recém-Nascido Pequeno para a Idade Gestacional , Comportamento Materno , Modelos Estatísticos , Gravidez , Nascimento Prematuro/etiologia , Efeitos Tardios da Exposição Pré-Natal , Medição de Risco , Fatores de Risco , Fumar/efeitos adversos , Resultado do TratamentoRESUMO
Air pollution is one of the most important global environmental issues. Taiwan Environmental Protection Agency (EPA) is currently using an Air Quality Index (AQI) to measure and monitor its national air quality. The main objective of this study is to assess the air quality per hour every month in Taichung City of Taiwan from 2014 to 2016 based on the nonconformance probability of the AQI index. The nonconformance probability is defined as the probability that a characteristic of interest falls outside of an acceptance region. A lower confidence bound for the nonconformance probability is applied to test whether the AQI index value exceeds a warning threshold, and then the government could issue warnings according to the decision made by such statistical inference. An unbalanced two-way random effects model is presented for fitting the AQI index values. We evaluate three different lower confidence bound construction methods, including a t-based, an adjusted t-based and a generalized pivotal quantity (GPQ) based methods, through a detailed simulation study. Finally, a hybrid method of the t-based and the adjusted t-based estimators is recommended for practical use.
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Tuning the electronic band structure of black titania to improve photocatalytic performance through conventional band engineering methods has been challenging because of the defect-induced charge carrier and trapping sites. In this study, KSCN-modified hydrogenated nickel nanocluster-modified black TiO2 (SCN-H-Ni-TiO2) exhibits enhanced photocatalytic CO2 reduction due to the interfacial dipole effect. Upon combining the experimental and theoretical simulation approach, the presence of an electrostatic interfacial dipole associated with chemisorption of SCN has dramatic effects on the photocatalyst band structure in SCN-H-Ni-TiO2. An interfacial dipole possesses a more negative zeta potential shift of the isoelectric point from 5.20 to 3.20, which will accelerate the charge carrier separation and electron transfer process. Thiocyanate ion passivation on black TiO2 demonstrated an increased work function around 0.60 eV, which was induced by the interracial dipole effect. Overall, the SCN-H-Ni-TiO2 photocatalyst showed an enhanced CO2 reduction to solar fuel yield by 2.80 times higher than H-Ni-TiO2 and retained around 88% product formation yield after 40 h.
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The human cluster of differentiation 19 (CD19) is highly expressed in most leukemia, rendering is a promising therapeutic target. In this study, we generated anti-CD19 single-chain variable fragments (scFv) from immunized chickens by phage display technology. After constructing a scFv antibody library with 2.5 × 108 compositional diversity for panning, one representative scFv clone S2 which can specifically recognize to the CD19 protein was isolated and characterized. The binding reactivity of the scFv S2 to the endogenous CD19 protein of the ARH-77 leukemia cancer cell was verified through flow cytometry and the binding affinity of scFv S2 is 6.9 × 10-8 M determined by the surface plasmon resonance system. Compared with the chicken germline, hyper mutation in the complementarity-determining regions (CDRs) suggested that scFv S2 could be generated through an antigen-driven humoral response. By molecular modeling, the possible CDR configurations of scFv S2 were constructed rationally. Furthermore, the characteristics of chicken antibodies of a protein database were investigated. The findings in this study contribute to antibody development and engineering because they reveal the geometric structures and properties of the CDRs in chicken antibodies.
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Antígenos CD19/imunologia , Anticorpos de Cadeia Única/química , Animais , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Galinhas/imunologia , Regiões Determinantes de Complementaridade/imunologia , Humanos , Modelos Moleculares , Anticorpos de Cadeia Única/sangue , Anticorpos de Cadeia Única/genética , Ressonância de Plasmônio de SuperfícieRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0186784.].
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To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10-8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.