Journal of Molecular Biology
Volume 326, Issue 1, 7 February 2003, Pages 167-175
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Deriving Topological Constraints from Functional Data for the Design of Reagentless Fluorescent Immunosensors

https://doi.org/10.1016/S0022-2836(02)01334-7Get rights and content

Abstract

The possibility of obtaining, from any antibody, a fluorescent conjugate which responds to the binding of the antigen by a variation of fluorescence, would be of great interest in the micro- and nano-analytical sciences. This possibility was explored with antibody mAb4E11, which is directed against the dengue virus and for which no structural data is available. Three rules of design were developed to identify residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. (i) The target residue belonged to the hypervariable loops of the antibody. (ii) It was adjacent, along the amino acid sequence of the antibody, to a residue which was functionally important for the interaction with the antigen. (iii) It was not important in itself for the interaction with the antigen. Eight conjugates between a single chain variable fragment of mAb4E11 and an environment-sensitive fluorophore were constructed. Three of them showed an increase in their fluorescence intensity by 1.5–2.8-fold on antigen binding, without loss of affinity. This increase allowed the titration of the antigen in serum above a threshold concentration of 10 nM. Experiments of quenching with potassium iodide suggested that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the binding of the antigen, and that therefore its mechanism is general.

Introduction

A biosensor comprises two major components: a biological receptor, which specifically recognizes a ligand, and a transducer, which detects the recognition event and transforms it into a measurable signal. A biosensor functions reagentless if the different steps going from the ligand recognition to the signal measurement do not imply any change in its composition. This characteristic is necessary for a continuous measurement. Monoclonal antibodies seem ideally suited to provide the biological receptor of biosensors, since they can be directed against most haptens or macromolecules. However, they are not adapted to the simple mechanisms of signal transduction.1

Several solutions have been developed to solve this problem of signal transduction by antibodies. Some consist in labelling in vitro all the proteins of the sample under analysis, as in the present use of antibody micro-arrays.2., 3. Others measure the mass increase on the binding of the antigen to the immobilized antibody, by means of apparatuses based on surface plasmon resonance or piezoelectric quartz microcrystals.4., 5. However, most of these solutions do not allow either continuous or high throughput measurements.

About 15 years ago, a general solution to the problem of signal transduction by antibodies was suggested. North has proposed the insertion of a reporter group close to the paratope (binding-site) of the antibody, for example a fluorophore that is sensitive to changes in its electronic environment.6 Schultz and co-workers have proposed to use oligonucleotide-directed mutagenesis to introduce a cysteine in a specific site of the antibody paratope, chosen on the basis of the crystal structure, then to chemically couple a reactive fluorophore to this mutant Cys.7 To be operational, the resulting conjugate must obviously show a variation of fluorescence on antigen binding that is measurable, and have an affinity for the antigen that is comparable to the one of the parental antibody. Recently and for the first time, we demonstrated the feasibility of this general approach.8 We developed quantitative rules of design, based mainly on the crystal structure of the complex between antibody and antigen, to choose the site of fluorophore coupling. We applied these rules to a single chain variable fragment (scFv) of antibody mAbD1.3, which is directed against hen egg white lysozyme and for which numerous structural and functional data are available. In all, 60% of the tested residues gave operational biosensors. Two observations were instrumental in our demonstration. We found that a mild reducing treatment was necessary to reactivate the mutant cysteine residue before its coupling with a reactive fluorophore, and that the reactions of reduction and coupling could be adjusted so as to preserve the essential disulphide bonds of the antibody variable domains.

To be useful, the above approach must imperatively be generalized to antibodies of unknown structure, because structural data are rarely available. Here, we used the following rationale for this generalization. First, we considered that an antibody residue that contributes to the energy of interaction with an antigen, is generally located in the neighbourhood of the antigen in the corresponding complex. The apparent contribution of a residue to an interaction energy can be measured easily, by mutagenesis. Second, we considered that residues that are adjacent in the sequence of a protein, are necessarily adjacent in its spatial structure. These two considerations imply that an antibody residue that is immediately adjacent, along the sequence, to a residue that is energetically important for the interaction with an antigen, will often be located in the three-dimensional neighbourhood of the antigen in the corresponding complex without contributing to its binding. Such a residue constitutes a target of choice for the coupling of an environment-sensitive fluorophore. Thus, sequence and mutagenesis data might be sufficient to deduce the topological data that are necessary for the design of a fluorescent biosensor.

We used mAb4E11, a murine monoclonal antibody (mAb), which is directed against the dengue virus (serotype DEN1) and whose three-dimensional structure is unknown, to test this new approach. The dengue virus infects 2% of the world population every year, i.e. 100 millions of individuals. It can induce a haemorragic fever or shock and be deadly. It is an expanding re-emerging disease. Up until now, no vaccine or specific treatment is available.9., 10. The epitope of mAb4E11 is included within domain 3 (residues 296–400) of the viral envelope protein Env.11 A recombinant Fab fragment, derived from mAb4E11, and domain 3 of Env can be produced in Escherichia coli and purified in quantity.12 We showed that mutagenesis data on the CDR3 hypervariable loops of Fab4E11 were sufficient to choose sites of coupling for a fluorophore and obtain operational fluorescent conjugates with a high success rate. We used experiments of quenching with potassium iodide (KI) to explore the physico-chemical mechanism by which the fluorescence of the conjugates varied on antigen binding, and showed that this mechanism is general. Our results pave the way to the fast generation of reagentless fluorescent biosensors from any antibody, for fundamental or applied purposes.

Section snippets

Search for coupling sites

We chose the sites of fluorophore coupling in mAb4E11, according to the following rules of design. (1) The target residue was in the hyper-variable loops of the antibody, as defined by Chothia.13 (2) It was adjacent, along the amino acid sequence of the antibody, to a residue that is functionally important for the interaction with the antigen. (3) It was not important itself for the interaction with the antigen. We considered that this last condition was fulfilled if the change of the target

Efficiency of the design rules

Here, we describe new rules of design to choose coupling sites for fluorophores on an antibody and transform it into a reagentless fluorescent biosensor. These rules were based: (1) on the target residue belonging to the neighbourhood of functionally important residues along the sequence of the antibody, and (2) on its absence of functional importance for the interaction with the antigen. We validated these rules with antibody mAb4E11, whose three-dimensional structure is unknown. Among the

Recombinant plasmids and proteins

Plasmid pLB1, coding for the scFv of mAb4E11, had the same structure as pMR1, coding for scFvD1.3,8 except that the sequences coding for VH and VL were specific for mAb4E11. They were retrieved from plasmid pMad4E11, coding for Fab4E11, by PCR12 (L.B. et al.,unpublished results). scFv4E11 carried the same His6 extension at its C-terminal end as scFvD1.3. Selected residues of scFv4E11 were changed into cysteine by oligonucleotide site-directed mutagenesis as described.8 The antigen of mAb4E11

Acknowledgements

We thank Shamila Naı̈r for critical reading of the manuscript and Philippe Thullier for the gift of plasmid pMad4E11. This research was funded by grants from the French Ministry of Defense to H.B. (DGA No. 20218/DSP/SREA/F) and from the Fondation pour la Recherche Médicale to M.R. (FRM No. FDT20020920140/1).

References (37)

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