Knowledge-based Design of Reagentless Fluorescent Biosensors from Recombinant Antibodies

Renard, Martial; Belkadi, Laurent; Hugo, Nicolas; England, Patrick; Altschuh, Danièle; Bedouelle, Hugues

doi:10.1016/s0022-2836(02)00023-2

Cited by 65 publications

(49 citation statements)

References 38 publications

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“…The binding of lysozyme could reorientate the two fluorescent groups toward the solvent and thus increase the mean distance between them. This mechanism is consistent with the peripheral location of the fluorescent groups, relative to the paratope, in our conjugates, and their lack of effect on the affinity of the scFv fragment for lysozyme (3).…”

Section: Discussionsupporting

confidence: 89%

Improving the Sensitivity and Dynamic Range of Reagentless Fluorescent Immunosensors by Knowledge-Based Design

Renard

Bedouelle

2004

Biochemistry

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The variable fragment (Fv) of an antibody can be transformed into a reagentless fluorescent biosensor by mutating a residue into a cysteine in the neighborhood of the paratope (antigen-binding site) and then coupling an environment-sensitive fluorophore, e.g., N- ((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester), to the mutant cysteine. For some residues, named operational, the formation of the conjugate does not affect the affinity of the Fv fragment for the antigen, and the binding of the antigen generates a measurable variation in the fluorescence intensity of the conjugate. We tested if this signal variation could be increased by coupling several molecules of fluorophores to the same molecule of Fv. Seven operational residues have been previously identified in the single-chain Fv (scFv) of monoclonal antibody D1.3 (mAbD1.3), directed against lysozyme. Ten double mutants of scFvD1.3, involving these residues, were constructed and coupled to the IANBD ester. The fluorescence of the double conjugates revealed a transfer of resonance energy between the two identical fluorescent groups. This homotranfer could be more important in the free state of the conjugate than in its antigenbound state and increase its sensitivity for the detection of the antigen by up to 2.9-fold. A poorly sensitive conjugate could be improved by coupling a second molecule of fluorophore to residues located far from the paratope. Mutations altering the affinity of scFvD1.3 for lysozyme were introduced into one of its fluorescent conjugates. Using a mixture of three mutant derivatives of this unique conjugate, we could titrate lysozyme with precision in a concentration range encompassing 3 orders of magnitude.A signal-transducing receptor generally comprises an extracellular domain that recognizes a specific molecular signal, a transmembrane domain through which the signal is transmitted to the interior of the cell and an intracellular domain through which the signal is transformed and amplified, eventually in the form of second messenger molecules. These three domains are assembled in a single-receptor macromolecule (1). Similarly, a biosensor transforms a specific molecular signal into an electrical signal and comprises several modules: a recognition module, which can be biological or biomimetic; a transduction module, which tranforms the recognition event into a measurable signal; and a module of data evaluation. The recognition and transduction modules are integrated into a compact device. A biosensor can function without additional reagent, provide quantitative analytical information, and follow the concentration of an analyte continuously. Important characteristics of a biosensor are its specificity and selectivity, the sensitivity, linearity and speed of the response, the dynamic range of the measurements, the possibility of calibration and accuracy (2).In a previous work, we have shown that it is possible to transform any antibody into a reagentless fluorescent immunosensor. The antibody is...

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Section: Discussionsupporting

confidence: 89%

Improving the Sensitivity and Dynamic Range of Reagentless Fluorescent Immunosensors by Knowledge-Based Design

Renard

Bedouelle

2004

Biochemistry

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“…A purified preparation was dialysed extensively against 50 mM ammonium carbonate (pH 9.5) and then lyophilized. The sample was dissolved in a mixture of water, methanol and formic acid, and the measurement of its mass was performed exactly as described previously (Renard et al, 2002).…”

Section: Methodsmentioning

confidence: 99%

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

Lisova

Hardy

Petit

et al. 2007

Journal of General Virology

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Dengue is caused by a taxonomic group of four viruses, dengue virus types 1-4 (DENV1-DENV4). A molecular understanding of the antibody-mediated protection against this disease is critical to design safe vaccines and therapeutics. Here, the energetic epitope of antibody mAb4E11, which neutralizes the four serotypes of DENV but no other flavivirus, and binds domain 3 (ED3) of their envelope glycoprotein, was characterized. Alanine-scanning mutagenesis of the ED3 domain from serotype DENV1 was performed and the affinities between the mutant domains and the Fab fragment of mAb4E11 were measured. The epitope residues (307-312, 387, 389 and 391) were at the edges of two distinct b-sheets. Four residues constituted hot spots of binding energy. They were aliphatic and contributed to form a hydrophobic pocket (Leu308, Leu389), or were positively charged (Lys307, Lys310). They may bind the diversity residues of mAb4E11, H-Trp96-Glu97. Remarkably, cyclic residues occupy and block the hydrophobic pocket in all unrelated flaviviruses. Transplanting the epitope from the ED3 domain of DENV into those of other flaviviruses restored affinity. The epitope straddles residues of ED3 that are involved in virulence, e.g. Asn/Asp390. These results define the epitope of mAb4E11 as an antigenic signature of the DENV group and suggest mechanisms for its neutralization potency. INTRODUCTIONDengue is a re-emerging viral disease. It is caused by four types of virus, dengue virus types 1-4 (DENV1-DENV4), which belong to the species Dengue virus in the genus Flavivirus (Heinz et al., 2000). They are transmitted by Aedes mosquitoes and infect between 50 million and 100 million persons each year. The disease generally takes a mild form, dengue fever, but severe forms, dengue haemorragic fever and dengue shock syndrome, have recently become epidemic (Gubler, 2002).The immune response against an infection by DENV involves both a humoral component, in the form of neutralizing antibodies, and a cell-mediated component (Guzman & Kouri, 2002). The preferential reactivation of the memory B cells that correspond to a primary infection, and an antibody-dependent enhancement of infection, might constitute triggering mechanisms of the severe forms during a secondary infection by a different viral serotype (Halstead, 2003; Mongkolsapaya et al., 2003). A molecular understanding of the events that lead to antibody neutralization, enhancement or escape is critical to the development of efficient and secure vaccines and therapeutics. DENV1-DENV4 are enveloped RNA viruses, like all flaviviruses. The structures of the whole virus and of the envelope glycoprotein E (gpE) have been solved by electron cryomicroscopy and X-ray crystallography (Modis et al., 2003(Modis et al., , 2005 Zhang et al., 2003). Ninety dimers of gpE cover the surface of the virus. Each monomer comprises three ectodomains, ED1-ED3, and a transmembrane segment. ED2 includes the dimerization interface, a glycosylation site and the peptide of fusion with the cellular membrane. ED3 is continuou...

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“…The cells were harvested by centrifugation at 2,500 ϫ g for 15 min at 4°C. scFv's were extracted with polymyxin B sulfate (47) and purified on a nickel-nitrilotriacetic acid spin column (QIAGEN, Valencia, CA) according to the manufacturer's instructions. An anti-His tag antibody (QIAGEN, Courtaboeuf, France) was used in ELISA to detect scFv's.…”

Section: Animal Immunizationmentioning

confidence: 99%

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

Pelat

Hust

Laffly

et al. 2007

Antimicrob Agents Chemother

101

112

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The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 ؋ 10 8 clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 ؎ 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

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Knowledge-based Design of Reagentless Fluorescent Biosensors from Recombinant Antibodies

Cited by 65 publications

References 38 publications

Improving the Sensitivity and Dynamic Range of Reagentless Fluorescent Immunosensors by Knowledge-Based Design

Improving the Sensitivity and Dynamic Range of Reagentless Fluorescent Immunosensors by Knowledge-Based Design

Mapping to completeness and transplantation of a group-specific, discontinuous, neutralizing epitope in the envelope protein of dengue virus

High-Affinity, Human Antibody-Like Antibody Fragment (Single-Chain Variable Fragment) Neutralizing the Lethal Factor (LF) of Bacillus anthracis by Inhibiting Protective Antigen-LF Complex Formation

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