Cloning and Expression inEscherichia coliof an Anti-Cyclohexanedione Single-Chain Variable Antibody Fragment and Comparison to the Parent Monoclonal Antibody

1997 ◽  
Vol 45 (2) ◽  
pp. 535-541 ◽  
Author(s):  
Steven R. Webb ◽  
Hung Lee ◽  
J. Christopher Hall
Keyword(s):  
Monoclonal Antibody ◽  
Antibody Fragment ◽  
Cloning And Expression ◽  
Single Chain

Inhibitory Effect of an Anti-Factor VIII Antibody Fragment On Factor VIII Activity in Different Functional Assays

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4387-4387
Author(s):  
Mikhail V Ovanesov ◽  
James H Kurasawa ◽  
Naveen Jha ◽  
Svetlana A Shestopal ◽  
Timothy K Lee ◽  
...  
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Antibody Fragment ◽  
Inhibitory Effect ◽  
Host Cells ◽  
Cloning And Expression ◽  
Single Chain ◽  
High Affinity ◽  
Functional Assays ◽  
Fviii Activity

Abstract Abstract 4387 Background. A single-chain variable antibody fragment (scFv) is an engineered protein corresponding to the antibody's antigen-recognizing (variable) domains (VH and VL), connected with an artificial linker. Due to the inherited specificity to the respective antigen, scFv are useful in a broad range of applications. A relatively small size (∼30 kDa) of scFv makes them easy to produce using the recombinant DNA technology. A particular scFv, known as KM33, was previously derived from a patient with anti-factor VIII (FVIII) auto-antibodies manifested in a functional deficiency of FVIII (inhibitory Hemophilia A). KM33 was described as a high affinity ligand of FVIII inhibiting its major functions: procoagulant activity and interactions with von Willebrand factor, phospholipids and catabolic receptors (van den Brink E. et al, 2001 and Limburg V. et al, 2005). Objective. Our goal was to develop a methodology for fast and easy production of KM33 and test its applicability for selective inhibition of FVIII in functional assays. Experimental Approach. As an alternative to a previously described expression of KM33 in bacteria, we used insect cells capable to perform majority of posttranslational modifications and produce correctly folded proteins. Previously published KM33 coding sequence (Mertens K. et al, 2008) was codon-optimized to the insect host cells (Spodoptera frugiperda), followed by the gene's chemical synthesis, cloning and expression in a baculovirus system. A secretable form of KM33 was purified from the culture media using nickel-affinity and size-exclusion chromatography. The structure and properties of the expressed protein were tested by binding to FVIII in a surface plasmon resonance assay and by measuring its effect on FVIII activity in a chromogenic substrate (CS) assay, clotting assay (APTT), thrombin-generation test (TG) and video microscopy of clot growth (CG). Results. The insect-cell derived KM33 demonstrated high affinity to FVIII (KD 0.1–0.2 nM) and strong inhibitory effect on the FVIII activity in all assays. At the same time, the inhibition efficiency, assessed by KM33/FVIII molar ratio corresponding to 50% of the inhibitory magnitude, was different between assays. The strongest inhibition was observed in the TG assay, and the weakest in the APTT assay (Fig. 1 and 2). Furthermore, the effect reached an apparent saturation: about 5%, 28% and 42% of FVIII activity remained uninhibited in the TG, CS and APTT assays, respectively. Control experiments performed at highest concentrations of KM33 in the absence of FVIII showed absence of non-specific effects of KM33. Conclusions. Altogether, our data shows highly specific interaction of KM33 and FVIII including the complex milieu of plasma, and applicability of KM33 for the inhibition of FVIII in functional assays. Strong inhibition of the FVIII activity in the TG test is consistent with the expected physiological relevance of this “global hemostasis” assay. In turn, our data demonstrate limitations of the classical APTT-based Bethesda assay typically used to analyze FVIII inhibitors in comparison with the CS and TG methods. The discrepant inhibitory effect on the FVIII activity between the assays may indicate a need for optimization or even revising the current methodology of analysis of FVIII inhibitors in Hemophilia A patients. Future studies are needed to evaluate relative clinical relevance of in vitro tests that quantitate activity of the FVIII inhibitory antibodies. Disclosures: No relevant conflicts of interest to declare.

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.

Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency

2001 ◽  
Vol 359 (2) ◽  
pp. 369-374 ◽  
Author(s):  
Xiaojun REN ◽  
Shujuan GAO ◽  
Delin YOU ◽  
Hualiang HUANG ◽  
Zi LIU ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Glutathione Peroxidase ◽  
Binding Site ◽  
Active Form ◽  
Catalytic Efficiency ◽  
Catalytic Antibodies ◽  
Rabbit Liver ◽  
Cloning And Expression ◽  
Single Chain ◽  
Variable Regions

Glutathione peroxidase (GPX) has a powerful role in scavenging reactive oxygen species. In previous papers we have developed a new strategy for generating abzymes: the monoclonal antibody with a substrate-binding site is first prepared, then a catalytic group is incorporated into the monoclonal antibody's binding site by using chemical mutation [Luo, Zhu, Ding, Gao, Sun, Liu, Yang and Shen (1994) Biochem. Biophys. Res. Commun. 198, 1240–1247; Ding, Liu, Zhu, Luo, Zhao and Ni (1998) Biochem. J. 332, 251–255]. Since then we have established a series of catalytic antibodies capable of catalysing the decomposition of hydroperoxides by GSH. The monoclonal antibody 2F3 was raised against GSH-S-2,4-dinitrophenyl t-butyl ester and exhibited high catalytic efficiency, exceeding that of rabbit liver GPX, after chemical mutation. To produce pharmaceutical proteins and to study the reason why it exhibits high catalytic efficiency, we sequenced, cloned and expressed the variable regions of 2F3 antibody as a single-chain Fv fragment (2F3-scFv) in different bacterial strains. The amounts of 2F3-scFv proteins expressed from JM109 (DE3), BL21 (DE3), and BL21 (coden plus) were 5–10%, 15–20% and 25–30% of total bacterial proteins respectively. The 2F3-scFv was expressed as inclusion bodies, purified in the presence of 8M urea by Co2+-immobilized metal-affinity chromatography (IMAC) and renatured to the active form in vitro by gel filtration. The binding constants of the active 2F3-scFv for GSH and GSSG were 2.46×105M−1 and 1.03×105M−1 respectively, which were less by one order of magnitude than that of the intact 2F3 antibody. The active 2F3-scFv was converted into selenium-containing 2F3-scFv (Se-2F3-scFv) by chemical modification of the reactive serine; the GPX activity of the Se-2F3-scFv was 3394units/μmol, which approaches the activity of rabbit liver GPX.

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.

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 Functional Production and Characterization of a Fibrin-Specific Single-Chain Antibody Fragment from Bacillus subtilis: Effects of Molecular Chaperones and a Wall-Bound Protease on Antibody Fragment Production

2002 ◽  
Vol 68 (7) ◽  
pp. 3261-3269 ◽  
Author(s):  
Sau-Ching Wu ◽  
Jonathan C. Yeung ◽  
Yanjun Duan ◽  
Ruiqiong Ye ◽  
Steven J. Szarka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bacillus Subtilis ◽  
Molecular Chaperones ◽  
Blood Clot ◽  
Expression System ◽  
Antibody Fragment ◽  
High Sensitivity ◽  
Single Chain ◽  
Single Chain Antibody ◽  
Major Factors

ABSTRACT To develop an ideal blood clot imaging and targeting agent, a single-chain antibody (SCA) fragment based on a fibrin-specific monoclonal antibody, MH-1, was constructed and produced via secretion from Bacillus subtilis. Through a systematic study involving a series of B. subtilis strains, insufficient intracellular and extracytoplasmic molecular chaperones and high sensitivity to wall-bound protease (WprA) were believed to be the major factors that lead to poor production of MH-1 SCA. Intracellular and extracytoplasmic molecular chaperones apparently act in a sequential manner. The combination of enhanced coproduction of both molecular chaperones and wprA inactivation leads to the development of an engineered B. subtilis strain, WB800HM[pEPP]. This strain allows secretory production of MH-1 SCA at a level of 10 to 15 mg/liter. In contrast, with WB700N (a seven-extracellular-protease-deficient strain) as the host, no MH-1 SCA could be detected in both secreted and cellular fractions. Secreted MH-1 SCA from WB800HM[pMH1, pEPP] could be affinity purified using a protein L matrix. It retains comparable affinity and specificity as the parental MH-1 monoclonal antibody. This expression system can potentially be applied to produce other single-chain antibody fragments, especially those with folding and protease sensitivity problems.

Sign in / Sign up

Export Citation Format

Share Document