Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis

2014 ◽  
Vol 90 (1) ◽  
pp. 74-80 ◽  
Author(s):  
R. Kamatchi ◽  
J. Charumathi ◽  
R. Ravishankaran ◽  
P. Kaliraj ◽  
S. Meenakshisundaram
Keyword(s):  
Monoclonal Antibody ◽  
Lymphatic Filariasis ◽  
Antibody Fragment ◽  
Wuchereria Bancrofti ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Recombinant Scfv ◽  
Overlap Extension ◽  
Field Samples ◽  
Filarial Antigen

AbstractGlobal programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ–Linker–VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples.

scholarly journals A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1794-1800 ◽  
Author(s):  
PJ Declerck ◽  
HR Lijnen ◽  
M Verstreken ◽  
H Moreau ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

scholarly journals Endothelial targeting of a recombinant construct fusing a PECAM-1 single-chain variable antibody fragment (scFv) with prourokinase facilitates prophylactic thrombolysis in the pulmonary vasculature

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4191-4198 ◽  
Author(s):  
Bi-Sen Ding ◽  
Claudia Gottstein ◽  
Andrea Grunow ◽  
Alice Kuo ◽  
Kumkum Ganguly ◽  
...  
Keyword(s):  
Antibody Fragment ◽  
Pulmonary Vasculature ◽  
Single Chain Variable Fragment ◽  
Wild Type ◽  
Single Chain ◽  
Recombinant Scfv ◽  
Platelet Endothelial Cell Adhesion ◽  
Limited Effectiveness ◽  
Recombinant Molecule ◽  
Cell Adhesion Molecule 1

Means to prevent thrombus extension and local recurrence remain suboptimal, in part because of the limited effectiveness of existing thrombolytics. In theory, plasminogen activators could be used for this purpose if they could be anchored to the vascular lumen by targeting stably expressed, noninternalized determinants such as platelet-endothelial-cell adhesion molecule 1 (PECAM-1). We designed a recombinant molecule fusing low-molecular-weight single-chain prourokinase plasminogen activator (lmw-scuPA) with a single-chain variable fragment (scFv) of a PECAM-1 antibody to generate the prodrug scFv/lmw-scuPA. Cleavage by plasmin generated fibrinolytically active 2-chain lmw-uPA. This fusion protein (1) bound specifically to PECAM-1-expressing cells; (2) was rapidly cleared from blood after intravenous injection; (3) accumulated in the lungs of wild-type C57BL6/J, but not PECAM-1 null mice; and (4) lysed pulmonary emboli formed subsequently more effectively than lmw-scuPA, thereby providing support for the concept of thromboprophylaxis using recombinant scFv-fibrinolytic fusion proteins that target endothelium.

scholarly journals Soluble Expression of a Neo2/15-Conjugated Single Chain Fv against PD-L1 in Escherichia coli

2022 ◽  
Vol 44 (1) ◽  
pp. 301-308
Author(s):  
Sun-Hee Kim ◽  
Hee-Jin Jeong
Keyword(s):  
Fusion Protein ◽  
Expression System ◽  
Antibody Fragment ◽  
Therapeutic Index ◽  
High Yield ◽  
Soluble Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Single Chain Fv ◽  
Programmed Death

Immunocytokines, antibody-cytokine fusion proteins, have the potential to improve the therapeutic index of cytokines by delivering the cytokine to the site of localized tumor cells using antibodies. In this study, we produced a recombinant anti-programmed death-ligand 1 (PD-L1) scFv, an antibody fragment against PD-L1 combined with a Neo2/15, which is an engineered interleukin with superior function using an E. coli expression system. We expressed the fusion protein in a soluble form and purified it, resulting in high yield and purity. The high PD-L1-binding efficiency of the fusion protein was confirmed via enzyme-linked immunosorbent assay, suggesting the application of this immunocytokine as a cancer-related therapeutic agent.

scholarly journals Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Marcelo Arantes Levenhagen ◽  
Fabiana de Almeida Araújo Santos ◽  
Patrícia Tiemi Fujimura ◽  
Ana Paula Carneiro ◽  
Julia Maria Costa-Cruz ◽  
...  
Keyword(s):  
Phage Display ◽  
Functional Characterization ◽  
Positive Likelihood Ratio ◽  
Antibody Fragment ◽  
The Body ◽  
Enzyme Linked Immunosorbent Assay ◽  
Heat Shock Protein 60 ◽  
Single Chain ◽  
Serum Samples ◽  
Antigenic Fraction

Abstract Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis.

scholarly journals Assessment of the usefulness of anti-Wb123 antibody for post-elimination surveillance of lymphatic filariasis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Ameyo Monique Dorkenoo ◽  
Adjaho Koba ◽  
Wemboo A. Halatoko ◽  
Minongblon Teko ◽  
Komlan Kossi ◽  
...  
Keyword(s):  
Lymphatic Filariasis ◽  
Specific Antigen ◽  
Public Health Problem ◽  
Wuchereria Bancrofti ◽  
World Health ◽  
School Age Children ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cross Sectional ◽  
Apparent Lack ◽  
Endemic District

Abstract Background The World Health Organization has targeted lymphatic filariasis (LF) for elimination as a public health problem and recommends, among other measures, post-elimination surveillance of LF. The identification of sensitive and specific surveillance tools is therefore a research priority. The Wuchereria bancrofti-specific antigen Wb123-based enzyme-linked immunosorbent assay (Wb123 ELISA) detects antibodies to the recombinant Wb123 antigen of W. bancrofti and may be useful as a surveillance tool for LF. Six years after stopping mass drug administration to eliminate LF and recording successful results on two post-treatment transmission assessment surveys, a study was conducted in Togo aimed at helping to identify the role of the Wb123 ELISA in post-validation surveillance of LF. Methods This was a cross-sectional study in eight previously LF-endemic districts and one non-endemic district in Togo. In each sub-district of these nine districts, two schools were selected and 15 children aged 6 to 9 years old at each school provided finger-stick blood for testing for antibodies to Wb123 using the Filaria Detect™ IgG4 ELISA kit® (InBios, International, Inc., Seattle, WA, USA). Results A total of 2654 children aged 6 to 9 years old were tested in 134 schools in the nine districts. Overall, 4.7% (126/2654) children tested positive for antibodies to the Wb123 antigen of W. bancrofti. The prevalence of Wb123 antibodies varied across the eight previously endemic LF districts, from 1.56 to 6.62%. The highest prevalence, 6.99%, was found in the non-endemic district, but this was not significantly different from the average of all the LF districts (4.49%, P = 0.062). Conclusions The Wb123 ELISA was positive in 4.7% of Togolese school-age children who were almost certainly unexposed to LF. This apparent lack of specificity in the Togo context makes it difficult to establish a seroprevalence threshold that could serve to signal LF resurgence in the country, precluding the use of this test for post-validation surveillance in Togo. There remains a need to develop a useful and reliable test for post-elimination surveillance for LF in humans.

scholarly journals Characterization of a Monoclonal Antibody and Its Single-Chain Antibody Fragment Recognizing the Nucleoside Triphosphatase/Helicase Domain of the Hepatitis C Virus Nonstructural 3 Protein

2000 ◽  
Vol 7 (1) ◽  
pp. 58-63 ◽  
Author(s):  
Zhu-Xu Zhang ◽  
Una Lazdina ◽  
Margaret Chen ◽  
Darrell L. Peterson ◽  
Matti Sällberg
Keyword(s):  
Monoclonal Antibody ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antibody Fragment ◽  
Expression Vectors ◽  
Helicase Domain ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Variable Regions

ABSTRACT We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors.Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371–1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.

scholarly journals Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 256
Author(s):  
Sabrina Karim-Silva ◽  
Alessandra Becker-Finco ◽  
Isabella Gizzi Jiacomini ◽  
Fanny Boursin ◽  
Arnaud Leroy ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Therapeutic Potential ◽  
Antibody Fragment ◽  
Binding Activity ◽  
Antibody Fragments ◽  
Antigen Binding ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Humanized Antibody ◽  
Neutralizing Capacity

Envenoming due to Loxosceles spider bites still remains a neglected disease of particular medical concern in the Americas. To date, there is no consensus for the treatment of envenomed patients, yet horse polyclonal antivenoms are usually infused to patients with identified severe medical conditions. It is widely known that venom proteins in the 30–35 kDa range with sphingomyelinase D (SMasesD) activity, reproduce most of the toxic effects observed in loxoscelism. Hence, we believe that monoclonal antibody fragments targeting such toxins might pose an alternative safe and effective treatment. In the present study, starting from the monoclonal antibody LimAb7, previously shown to target SMasesD from the venom of L. intermedia and neutralize its dermonecrotic activity, we designed humanized antibody V-domains, then produced and purified as recombinant single-chain antibody fragments (scFvs). These molecules were characterized in terms of humanness, structural stability, antigen-binding activity, and venom-neutralizing potential. Throughout this process, we identified some blocking points that can impact the Abs antigen-binding activity and neutralizing capacity. In silico analysis of the antigen/antibody amino acid interactions also contributed to a better understanding of the antibody’s neutralization mechanism and led to reformatting the humanized antibody fragment which, ultimately, recovered the functional characteristics for efficient in vitro venom neutralization.

scholarly journals Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization

2015 ◽  
Vol 71 (4) ◽  
pp. 896-906 ◽  
Author(s):  
Jennifer L. Johnson ◽  
Kevin C. Entzminger ◽  
Jeongmin Hyun ◽  
Sibel Kalyoncu ◽  
David P. Heaner ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Antibody Fragment ◽  
Dynamics Simulation ◽  
Peptide Sequence ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Fab Fragment ◽  
Biophysical Characterization ◽  
Peptide Epitope

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

scholarly journals Rapid Wuchereria bancrofti-Specific Antigen Wb123-Based IgG4 Immunoassays as Tools for Surveillance following Mass Drug Administration Programs on Lymphatic Filariasis

2013 ◽  
Vol 20 (8) ◽  
pp. 1155-1161 ◽  
Author(s):  
Cathy Steel ◽  
Allison Golden ◽  
Joseph Kubofcik ◽  
Nicole LaRue ◽  
Tala de los Santos ◽  
...  
Keyword(s):  
Lymphatic Filariasis ◽  
Drug Administration ◽  
Mass Drug Administration ◽  
Wuchereria Bancrofti ◽  
Lateral Flow ◽  
Enzyme Linked Immunosorbent Assay ◽  
Predictive Values ◽  
Content Type ◽  
Helminth Infections ◽  
Highly Sensitive

ABSTRACTThe Global Programme to Eliminate Lymphatic Filariasis has an urgent need for rapid assays to detect ongoing transmission of lymphatic filariasis (LF) following multiple rounds of mass drug administration (MDA). Current WHO guidelines support using the antigen card immunochromatographic test (ICT), which detects active filarial infection but does not detect early exposure to LF. Recent studies found that antibody-based assays better serve this function. In the present study, two tests, a rapid IgG4 enzyme-linked immunosorbent assay (ELISA) and a lateral-flow strip immunoassay, were developed based on the highly sensitive and specificWuchereria bancroftiantigen Wb123. A comparison ofW. bancrofti-infected and -uninfected patients (with or without other helminth infections) demonstrated that both tests had high sensitivities and specificities (93 and 97% [ELISA] and 92 and 96% [strips], respectively). When theW. bancrofti-uninfected group was separated into those with other filarial/helminth infections (i.e., onchocerciasis, loiasis, and strongyloidiasis) and those who were parasite uninfected, the specificities of the assays varied between 91 and 100%. In addition, the geometric mean response by ELISA ofW. bancrofti-infected patients was significantly higher than the response of those withoutW. bancroftiinfection (P< 0.0001). Furthermore, the Wb123 ELISA and the lateral-flow strips had high positive and negative predictive values, giving valuable information on the size of survey population needed to be reasonably certain whether or not transmission is ongoing. These highly sensitive and specific IgG4 tests to theW. bancroftiWb123 protein give every indication that they will serve as useful tools for post-MDA monitoring.

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