Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin

The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

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Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not

Journal of General Virology ◽

10.1099/0022-1317-82-8-1959 ◽

2001 ◽

Vol 82 (8) ◽

pp. 1959-1963 ◽

Cited By ~ 20

Author(s):

P. D. Drew ◽

M. T. Moss ◽

T. J. Pasieka ◽

C. Grose ◽

W. J. Harris ◽

...

Keyword(s):

Varicella Zoster Virus ◽

Cultured Cells ◽

Cross Reactivity ◽

Antibody Fragments ◽

Single Chain ◽

Single Chain Antibody ◽

Neutralizing Activity ◽

Varicella Zoster ◽

Complementarity Determining Region

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the VH to the Vκ domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.

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

Toxins ◽

10.3390/toxins12040256 ◽

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.

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Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3343-3349.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3343-3349 ◽

Cited By ~ 40

Author(s):

William J. J. Finlay ◽

Iain Shaw ◽

Joanna P. Reilly ◽

Marian Kane

Keyword(s):

Domoic Acid ◽

Recombinant Antibody ◽

Variable Region ◽

Antibody Fragments ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Single Chain Antibody ◽

High Affinity ◽

Single Chain Fv ◽

Recombinant Antibody Fragments

ABSTRACT Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single VH and VL genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.

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Generation of recombinant scFv antibody against Ochratoxin A (OTA)

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.31121 ◽

2018 ◽

Vol 22 (2) ◽

pp. 107 ◽

Cited By ~ 1

Author(s):

Ranya Pranomphon ◽

Witsanu Srila ◽

Montarop Yamabhai

Keyword(s):

Ochratoxin A ◽

Recombinant Antibody ◽

Cross Reactivity ◽

Agricultural Product ◽

Binding Property ◽

Single Chain ◽

Single Chain Antibody ◽

Recombinant Scfv ◽

Contaminated Food ◽

Soluble Scfv

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.

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Generation of a Broadly Cross-Neutralizing Antibody Fragment against Several Mexican Scorpion Venoms

Toxins ◽

10.3390/toxins11010032 ◽

2019 ◽

Vol 11 (1) ◽

pp. 32 ◽

Cited By ~ 8

Author(s):

Lidia Riaño-Umbarila ◽

Ilse Gómez-Ramírez ◽

Luis Ledezma-Candanoza ◽

Timoteo Olamendi-Portugal ◽

Everardo Rodríguez-Rodríguez ◽

...

Keyword(s):

Recombinant Antibody ◽

Antibody Fragment ◽

Cross Reactivity ◽

Site Directed Mutagenesis ◽

Significant Advance ◽

Single Chain ◽

Single Chain Antibody ◽

Promising Alternative ◽

Neutralization Capacity ◽

Scorpion Venoms

The recombinant antibody fragments generated against the toxic components of scorpion venoms are considered a promising alternative for obtaining new antivenoms for therapy. Using directed evolution and site-directed mutagenesis, it was possible to generate a human single-chain antibody fragment with a broad cross-reactivity that retained recognition for its original antigen. This variant is the first antibody fragment that neutralizes the effect of an estimated 13 neurotoxins present in the venom of nine species of Mexican scorpions. This single antibody fragment showed the properties of a polyvalent antivenom. These results represent a significant advance in the development of new antivenoms against scorpion stings, since the number of components would be minimized due to their broad cross-neutralization capacity, while at the same time bypassing animal immunization.

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