Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

Rosa, Sofía Tabares-da; Rossotti, Martín A.; Carleiza, Carmen; Carrión, Federico; Pritsch, Otto; Ahn, Ki Chang; Hammock, Bruce D.; González‐Sapienza, Gualberto

doi:10.1021/ac201824z

Cited by 70 publications

(86 citation statements)

References 22 publications

(38 reference statements)

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“…A VHH phage display library was built as previously described [26]. Briefly, about 10 8 lymphocytes were obtained from 150 mL of blood using Histopaque 1077 (Sigma) gradients.…”

Section: Methodsmentioning

confidence: 99%

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Cui

Morisseau

et al. 2015

Anal Bioanal Chem

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The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer and other diseases. However, there is not a simple, inexpensive and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal-variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the ELISA and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney and lung). The VHH based ELISA appears to be a new reliable method for monitoring the sEH, and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

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“…A VHH phage display library was built as previously described [26]. Briefly, about 10 8 lymphocytes were obtained from 150 mL of blood using Histopaque 1077 (Sigma) gradients.…”

Section: Methodsmentioning

confidence: 99%

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Cui

Morisseau

et al. 2015

Anal Bioanal Chem

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“…29 The expression level of the bivalent antibodies was lower than that of the monomeric domains, but still about 5 mg/L of purified end product were obtained in shake flasks. The neutralization activity of these constructs was tested under challenging conditions, using a 10 £ LD 100 of the toxin preparation.…”

Section: Selection Of Tetc Specific Vh/vhhsmentioning

confidence: 99%

“…A VHH phage display library was built as previously described. 29 Briefly, about 10 8 lymphocytes were obtained from 150 mL of blood using Histopaque 1077 (Sigma) gradients. The RNA was extracted and reverse transcribed using the primer JH.…”

Section: Library Constructionmentioning

confidence: 99%

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Rossotti

González-Techera

Guarnaschelli

et al. 2015

mAbs

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Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (»30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti-Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.

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“…TCC may cause adverse biological and toxicological effects on humans and the environment. In this work [29], TCC was used as a model hapten to study the practical aspects of producing VHH against small molecules. The titer and overall affinity of the IgG1 (conventional) subclass was relatively higher (IC 50 = 51 ng/mL) than that of IgG2 and IgG3 (monodomain).…”

Section: Summary Of Vhh Assays To Environmental Chemicalsmentioning

confidence: 99%

“…However, another VHH selective for caffeine generated by Franco et al [20] showed weaker temperature-tolerance, because the mid-point of the unfolding transition (T m ) was only 56 °C. Tabares-da Rosa et al [29] obtained five different anti-TCC VHHs. All of these VHHs were more stable than rabbit pAbs at 85 °C and 100°C.…”

Section: Advantages Of Vhhsmentioning

confidence: 99%

VHH antibodies: emerging reagents for the analysis of environmental chemicals

et al. 2016

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A VHH antibody (or nanobody) is the antigen binding fragment of heavy chain only antibodies. Discovered nearly 25 years ago, they have been investigated for their use in clinical therapeutics and immunodiagnostics, and more recently for environmental monitoring applications. A new and valuable immunoreagent for the analysis of small molecular weight environmental chemicals, VHH will overcome many pitfalls encountered with conventional reagents. In the work so far, VHH antibodies often perform comparably to conventional antibodies for small molecule analysis, are amenable to numerous genetic engineering techniques, and show ease of adaption to other immunodiagnostic platforms for use in environmental monitoring. Recent reviews cover the structure and production of VHH antibodies as well as their use in clinical settings. However, no report focuses on the use of these VHH antibodies to small environmental chemicals (MW <1,500 Da). This review article summarizes the efforts made to produce VHHs to various environmental targets, compares the VHH-based assays with conventional antibody assays, and discusses the advantages and limitations in developing these new antibody reagents particularly to small molecule targets.

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Competitive Selection from Single Domain Antibody Libraries Allows Isolation of High-Affinity Antihapten Antibodies That Are Not Favored in the llama Immune Response

Cited by 70 publications

References 22 publications

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

VHH antibodies: emerging reagents for the analysis of environmental chemicals

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