HK1095881B - Biosensors, process for obtaining them and their applications - Google Patents

Description

The present invention relates to a process for the preparation of biosensors which allows the production of biosensors usable for the detection, dosing or localization, by direct immunofluorescence, of a ligand such as an antigen or a hapten in a heterogeneous population.

A biosensor is a bifunctional biological macromolecule: on the one hand, it is able to specifically fix its ligand in a mixture, and on the other hand, it translates the fixation event into a measurable signal directly and instantly: by means of a simple diode system, a spectrofluorometer, a fluorescence microscope, a confocal microscope or any other apparatus, resulting in the immediacy of the detection thus carried out.

While DNA chips for detection of nucleic acid sequences already exist, a general solution for the construction of biosensors and protein-based chips for detection of other ligands does not exist and therefore remains to be developed.

Several approaches have been tried: In the case of proteins that do not have cysteine residues (Cys) or disulfide bridges, a cysteine has been introduced into the protein by directed mutagenesis. The cysteine is introduced either in the immediate vicinity of the ligand binding site (GB1 protein; Sloan et al., 1998) or at a distance from that site (MBP protein; Gilardi et al., 1994 Marvin et al., 1997; GBP protein; Marvin et al., 1998; Tolosa et al., 1999). The added thiol function allows coupling to a fluorophore sensitive to its electronic environment.The binding of the ligand then changes this environment and the change in the properties of the biosensor is thus directly detectable by spectrofluorimetry. For example, Sloan et al. (1998) have shown that the fluorophore must be positioned in a region at the protein-protein interface and they have investigated the coupling of a fluorophore in such a region, for the GB1-Fc complex domain: in this context, the formation of the complex is analyzed; the best results are obtained when the fluorophore is coupled at the level of an amino acid involved in the formation of the complex. - What? However, this approach only applies to very specific proteins without free cysteine residues and without cysteine residues involved in disulfide bridges.In addition, the use of the above biocaptors is limited to the detection of a few particular ligands (maltose, glucose and Fc fragment of antibody). Indeed, in this approach, the main difficulty for creating biocaptors from a protein containing disulfide bridges comes from the fact that the steps of coupling the fluorophore to a Cys residue can result in an attack of disulfide bridges essential for the structure and stability of the molecule and inactivation. Among these proteins, antibodies represent the class of proteins naturally dedicated to the specific binding of protein, peptide, polysaccharide or haptacids (antigen) ligands with high affinity.However, antibody molecules have Cys residues forming intra- and inter-chain disulfide bridges. In particular, Fv fragments of antibodies have two disulfide bridges, one in each of the VH and VL domains, which are essential for the stability of the Fv fragment or single-chain scFv fragments (Glockshuber et al., 1992). a biosensor was derived from an antibody that binds 2,4-dinitrophenol by the following approach: a thiol group was introduced into the vicinity of the ligand binding site by means of an affinity marker that combined in one molecule a 2,4-dinitrophenol group specifically recognised by the antibody, a thiophenyl bond or disulfide,The affinity marker was able to bind to the antibody binding site via the 2,4-dinitrophenol group and then to bind to the antibody by randomly reacting its aldehyde or alpha-bromo ketone group with the side chains of nearby Lys, His or Tyr residues. Treatment of the coupled antibody with dithiothreate released an SH group which could in turn be coupled to a fluorophore (Pollack et al. 1988).

This method has the disadvantage of being randomized and not generalizable and only applicable to antibodies directed against haptens as it depends on the presence of Lys, His or Tyr residues at a sufficient distance from the antibody binding site and the possibility of synthesizing a tripartite affinity marker as described above.

The Inventors set out to make it possible to obtain protein-based biosensors by a rational, general and generalizable method capable of identifying amino acid residues from a protein receptor which can be substituted for cysteine residues. Such biosensors are better suited to practical needs, in particular by allowing the coupling of a fluorophore to a position inducing a change in the structural environment of the fluorophore at ligand attachment, whether or not the crystal structure of the complex between the receptor and the ligand is known.

Such biosensors can be constructed from any type of protein, including proteins with cysteine residues important for their structure or stability, and are new tools for detecting, dosing and locating a wide variety of ligands in a heterogeneous population of molecules.

The present invention is a process for preparing a biosensor, consisting of (i) at least one protein receptor fragment capable of binding to a suitable ligand via a binding site, in which at least one of its amino acid residues, located in the vicinity of the binding site, is selected from the group consisting of residues that are in direct contact with the ligand, those that are in contact via a water molecule, and those whose surface is modified to the solvent by ligand binding and is naturally present as Cys residue or is substituted as Cys residue,and (ii) a fluorophore coupled to the so-called residues Cys, the process being characterised by the following steps: (a) identification of amino acid residues at the receptor binding site by mutagenesis of all or a subset of the receptor amino acid residues, and determination of changes in ligand interaction parameters (KD, kon, koff) due to each mutation or limited groups of mutations;in a Cys residue where that residue is not naturally a Cys residue; (d) a conserved reduction of the receptor obtained by (b) or (c), and (e) coupling of the Sγ atom of at least one Cys residue obtained by (b) or (c) with a fluorophore.

Non-essential Cys residues, e.g. those located far from the active site, may be converted into other residues by directed mutagenesis, including Ser or Ala residues, to avoid parasitic coupling with fluorophores.

The savings (step (d)) can significantly improve the coupling performance (step (e)).

A receptor, as defined in the present invention, is a protein macromolecule with an active site capable of binding to a ligand, including an antibody, hormone or bacterial receptor, an affinity protein, a transport protein or a viral receptor, or any polypeptide with a specific affinity for a ligand.

For the purposes of this invention, a ligand is any molecule capable of binding to this receptor via the active site, including: a protein, peptide or haptenic antigen, such as a bacterial antigen, or a hormone, cytokine, interleukin, tumor necrosis factor (TNF), growth factor, viral protein, or peptide or nucleotide sequence.

For the purposes of the present invention, the active site of the receptor or receptor fragment is the set of residues involved in ligand binding.

For the purposes of the present invention, the term neighbourhood is understood as defined by the mathematical theory of topological spaces; the receptor residues in the vicinity of the active site are those residues which are in direct contact with the ligand, those which are in contact through a water molecule, and those whose surface accessible to the solvent (ASA; Creighton, 1993) is altered by the ligand's attachment. The use of increasing radius spheres, at a maximum of 30 Å, preferably from 1.4 to 2.9 Å, i.e. larger than that of a high volume water molecule (1,4 Å), e.g. 1,4 Å; 1.7 to 2.7 Å; 2.3 to 2.6 and 2.9 Å; if the solvent is to be calculated, a good fluorofluorophore marker must be defined in order to allow a greater affinity of the receptor surface and thus to increase the binding potential of the receptor, taking into account the large volume of the fluorofluorophore molecule; if the solvent is to be conserved, a greater affinity of the receptor site must be defined in order to increase the binding potential of the receptor, and thus to increase the affinity of the binding site, which is not accessible by the whole fluorofluorophore molecule.

For the purposes of this invention, fluorophore means any molecule whose fluorescence is sensitive to its microenvironment and which may be coupled to a Cys residue.

The biosensor preferably comprises a fragment of a receptor which has one or more disulfide bridges essential for its activity or maintenance of its structure; it includes in particular an antibody or an antibody fragment, such as an Fv, scFv, Fab or mini-antibody fragment; the antibody is preferably a natural or artificial monoclonal antibody.

In accordance with the invention, the fluorophore is selected from the group consisting of: N-((2-iodoacetoxy) ethyl) -N-methyl) amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD), 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (CNBD), acrylodone, 5-iodoacetamidofluorescein (5-IAF), or a fluorophore with an aliphatic chain of 1 to 6 carbon atoms.

The biocensor is preferably in soluble form or fixed on a suitable solid support of plastic or glass; preferably such solid support is a microplate or optical fiber.

The methods of immobilization include those described in Piervincenzi et al., 1998; Yoshioka et al., 1991; Turkova, 1999; Saleemuddin, 1999; Weetall, 1993; Sara et al., 1996.

Where the medium is an optical fiber, such biosensors may be implanted in situ, for example in a patient's vein, an animal's vein or in an individual cell, to continuously dose the ligand in vivo and thus monitor its kinetics of onset and disappearance.

The biosensor obtained by the process of the invention may be used for the preparation of a protein chip, consisting of a solid support on which at least one biosensor obtained by the process of the invention is fixed.

Such protein-based chips include a solid support, e.g. a microplate, on which the molecules of different biosensors are advantageously immobilized in a matrix and included in micro-fluid circuits.

The biocenters obtained by the process of the invention have a number of advantages: the receptor residue on which the fluorophore coupling is performed is predetermined and is a Cys residue in the vicinity of the receptor active site.The fluorophore coupling on predetermined cysteine residues is performed under conditions where it does not attack any disulfide bridges present on the receptor.

For example, the sequences of the hypervariable loops of the mAbD1.3 antibody, called CDRs (Complementary Determining Regions), can be defined by sequence comparisons (England et al., 1999). The active site of mAbD1.3 for its interaction with the lysozyme has been characterized by changes in CDR residues, mainly in Ala (Dall'Acqua et al., 1996, 1998; England et al., 1997, 1999; Ito et al., 1993, 1995; Hawkins et al., 1993; Ysern et al., 1994; Goldbaum et al., 1996). The residual dissociation chains (L-H30A32A, L-YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY

Therefore, residues of L-Thr51, L-Thr52, L-Thr53, L-Ser93, L-Thr94, H-Thr30, H-Asn56, H-Asp58 and H-Arg99 which are polar and therefore likely to be exposed to the surface of the mAbD1.3 antibody and which are close to residues of the active site in the sequence would constitute potential coupling sites according to purely functional criteria.

In an advantageous implementation of this method, prior to step (a), non-essential Cys residues from the receptor are replaced by Ser or Ala residues by directed mutagenesis.

In another advantageous implementation of this process, in step (d), the fluorophore is selected from the group consisting of N-((2-iodoacetoxy) ethyl) -N-methyl) amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD), 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (CNBD), acrylodone, 5-iodoacetamidofluorescein or a fluorophore with an aliphatic chain of 1 to 6 carbon atoms.

Another advantageous implementation of this process is that after step (e) it includes an additional step (f) of purification of the biosensor, e.g. on an exclusion or affinity column, in particular a column of immobilized nickel ions, if the receptor or antibody fragment includes an extension of His residues.

Another advantageous implementation of this method is that after step (f) it includes an additional step of measuring the equilibrium constant (KD or K'D), or rate of dissociation (koff) and association (kon) constants between the receptor and the ligand.

The equilibrium constant is called KD when the complex is in solution and K'D when one of the complex's partners is immobilized.

The data obtained (KD, K'D, koff, kon, amplitude of fluorescence change during ligand fixation, displacement of maximum emission) allow the selection of the most suitable receptor residues for fluorophore coupling to be refined.

Another advantageous implementation of this method, after step (e) or step (f), is an additional step of immobilization of the biosensor on a suitable solid support as mentioned above.

The biosensors obtained by the process of the invention can be used alone or in combination for ligand detection, dosing and localization applications.

For example, they allow the detection, dosing or localization of an antigen or hapten in a heterogeneous population of molecules, instantaneously.

In the health sector, these biosensors have applications: to monitor the progression or regression of a disease in response to treatment; to dose an infectious agent (bacteria, virus), a pathogen (tumor cell), a macromolecule (hormone, cytokine) or a hapten in a body fluid (e.g. blood, semen, etc.); to determine the serotype of an infectious agent; to detect a cell marker and determine its location; to sort cells based on the presence of a surface marker; to sort molecules; to quantify an intracellular or extracellular component, to monitor its kinetics of formation and disappearance, and to track its location; to determine the half-life of a molecule (dependent on its composition, instability, or elimination of metabolism); to describe its concentration and its effect on tissue, particularly its chemical structure (e.g. its effect on the cell wall).

In the fields of environment, industry and fraud prevention, these biosensors also have applications: to detect and dose a pollutant and monitor its elimination by natural or bioremediation processes; to dose an active ingredient in a preparation in the process of manufacture or marketing; to monitor the kinetics of a chemical synthesis reaction.

These biosensors are also used in the manufacture of protein chips.

They may also be used for the preparation of reagents for detection, dosing and/or localization of ligands.

These reagents may be implemented in a process for detecting, assessing or locating a ligand in a heterogeneous sample, characterised by the inclusion of contact between the heterogeneous sample and at least one such reagent.

They may also be used in the preparation of a ligand detection, dosing and/or localization kit.

The bio-sensors can also be used to prepare a screening kit for ligand/receptor inhibitors.

The pMR1 plasmid of sequence SEQ ID NO10, registered with the National Collection of Microorganisms Culture (CNCM), under number I-2386, dated 29 February 2000, may be used for the implementation of the process according to the invention.

The use of the plasmid pMR1 ((VL-S93C) sequence SEQ ID NO:11, registered with the National Collection of Microorganism Culture (CNCM), under number I-2387, dated 29 February 2000, for the implementation of the process according to the invention is also possible.

In addition to the foregoing, the invention contains further provisions, which will be shown in the following description, which refers to examples of the process described in the present invention and the accompanying drawings, in which: Figure 1 shows the criteria for determining residues of the Fv fragment of the antibody mAbD1.3 in the vicinity of the active site of lysozyme binding, which can be used for fluorophore coupling.Columns 3 and 4 indicate the ASA values in free Fv (using R = 1,4 Å) of atoms at γ and δ, possibly of each of the selected Xi residues, at position i of the Fv sequence, as percentages of the corresponding ASAs in a Gly-Xi-Gly tripeptide that would adopt the same conformation as the Xi-Xi1+ tripeptide F-1-v in the structure.Columns 5, 6 and 7 indicate the ASA in the native complex structure, groupings at γ, δ and ε positions in the initial side chain. Columns 8 and 9 indicate the mutations of these residues known in mAbD1.3 and their effects, as a variation ΔΔG of interaction energy with the lysozyme. Column 10 summarizes the classification of residues in descending order of priority (+) corresponding to residues whose atoms at γ position are accessible to the solvent in the free Fv structure, and which are in contact with the lysozyme either directly or via a water molecule in the complex structure,(+/-) corresponds to residues where the γ-atom is accessible and not in direct contact with the lysozyme and (?) corresponds to residues where the δ-atom is accessible but not the γ-atom.Figure 2 illustrates the optimisation of the production of Fv from recombinant mAbD1.3 (scFv-His6) by prokaryotic expression vectors (pMR1 and pMR5).The periplasmic content of HB2151 and BL21 (DE3) strains of E. coli transformed by the pMR5 and pMR1 plasmids, respectively, induced by IPTG (BL21DE (3)) or anhydrocyline (HB2151) and cultured to vary temperature, inducer time and concentration,The results are expressed in arbitrary units.Figure 3 shows the analysis by electrophoresis in polyacrylamide-SDS gel and Coomassie blue staining of the purification fractions of the scFv-His6 ((VL-S93C) mutant, on a nickel ion column. EP: unpurified periplasmic extract, NR: unconserved fractions, M: molecular mass marker, L20 and L40: 20 and 40 mM imida wash fractions, EL scovalence fractions. Figures 4 and 5 show the non-covalent oligomerization of wild-type scFv-His6L and VC-S93 mutant, excluding chromatography.Purified scFv-His6 was injected at a concentration of 50 μg/ml for the wild type and for the mutant, in a volume of 200 μL, at the top of a SuperdexR 75 HR 10/30 (Pharmacia) column. The chromatogram was developed at 25°C, pH=7.5 for the wild type and pH=7.0 for the mutant.Figure 6 represents the chemical structures of CNBD, IANBD, 5-iodoacetamidofluorescein (5-IAF) and acrylodone.Figure 7 illustrates the proportionality between fluorescence intensity (excitation at 469 nm, concentration at 535 nm) and protein in the elevation fractions resulting from the separation between conjugate and free fluorophore.Figure 8 shows the fluorescence emission by derivatives of scFv-His6 for excitation at 469 nm, in the absence of lysozyme.The following species were analysed: wild-type scFv-His6 and scFv-His6 ((VL-S93C) after couplings with IANBD and separations; scFv-His6 ((VL-S93C) before coupling.Figure 9 shows the evolution of coupling efficiency (proportion of mutant scFv-His6 bound to a fluorophore, here IANBD) and material efficiency during biocaptor preparation, as a function of the concentration of 2-mercaptools used for reduction.Figure 10 shows the long-term fluorescent spectrum for the 250-VL-HIS6S, recorded at 250 nm.The peak height at 278 nm, corresponding to the protein, and the peak height at 500 nm, corresponding to the coupled IANBD, allowed the coupling efficiency to be calculated (around 83%).Figure 11 illustrates the fluorescence emission by the derivative scFv-His6 ((VL-S93ANBD) for an excitation at 469 nm expressed in abritractory units (v.a.). The spectra were recorded in the absence of lysozyme, or 1 minute after addition of lysozyme at 100 nM and 1 μM (final concentrations) and brief homogenization.The same maximum fluorescence exaltation (+27%) is observed for three preparations of the same device for which different coupling methods (with or without reducing pre-treatment) were used,The fluorescence intensities were recorded in the absence or presence of varying concentrations of lysozyme (10 nM to 2 μM) in a 50 mM Tris-HCl, 150 mM NaCl, pH=7.5 buffer. In this buffer, the fluorescence intensity of the conjugate scFv-His6 ((VL-S93ANBD) is proportional to the lysozyme concentration up to 400 MM and increased by 91% at saturation. The directly obtained lysozyme titer is between 10 and 400 M.Figure 13 shows the coupling yields and fluorescence properties of the biosensors. Column 1 shows the residues of the Fv fragment of the antibody called mAbD1.3 which were mutated into Cys residues; these residues were selected according to the structural data shown in Figure 1. Column 2 shows the percentage of mutant scFv-His6 molecules coupled to the IANB, compared to wild-type scFv-His6 (wt). Column 3 shows the fluorescence variation at 535 nm between the free and lysozyme complex forms of the FV-CysB-AND conjugates, measured in a 20 mM T-HrisCl buffer,500 mM NaCl, 160 mM imidazole, pH=7.9 Significant fluorescence excitations are obtained for more than half of the coupling positions. - What? It must be held, however, that these examples are given merely for the purpose of illustrating the subject-matter of the invention, and in no way constitute a limitation of the subject-matter.

EXAMPLE 1: Materials and methods. 1-Parent bacterial strains, plasmids and phages

Souche	Principaux caractères	Référence
HB2151	Carter et al., 1985
RZ1032	Kunkel et al., 1987
BL21(DE3)	Studier et Moffatt, 1986

The pASK98-D1.3 vector codes for a hybrid between a scFv of the monoclonal mAbD1.3 antibody and a streptavidin-like label. The corresponding gene is controlled by the promoter and tetracycline operator (Skerra, 1994). pSPI1.0 is a derivative of pET26b (Novagen) and allows the subcloning of scFvs under the control of the T7 phage promoter.

2-Culturing media and buffers

The LB and 2xYT media have been described (Sambrook et al., 1989). The SB medium contains 16 g/l bacto-tryptone, 10 g/l bacto-yeast, 10 g/l NaCl, 50 mM K2HPO4, 5 mM MgSO4 and 1% glucose (Plückthun et al., 1996). Ampicillin is used at 200 μg/ml and kanamycin at 30 μg/ml in the growth media. PBS and TBE buffers have been described (Sambrook et al., above). Tampon: P 20 mM Tris-HCl pH7,9 ; 500 mM NaCl. Tampon Q: 50 mM Tris-HCl pH7,5 150 mM NaCl. Tampon: 50 mM CCl; sodium phosphate pH7,0 150 mM NaCl.

Other, of a kind used for the manufacture of motor vehicles

The sequences of oligonucleotides used for mutagenesis or genetic constructs are given in the following table.

Nom	Séquence
pMR1-VL-H30C	SEQ ID NO :20
pMR1-VL-N31C	SEQ ID NO :17
pMR1-VL-Y49C	SEQ ID NO :21
pMR1-VL-Y50C	SEQ ID NO :22
pMR1-VL-T52C	SEQ ID NO :13
pMR1-VL-T53C	SEQ ID NO :1
pMR1-VL-D56C	SEQ ID NO :16
pMR1-VL-W52C	SEQ ID NO :12
pMR1-VL-S93C	SEQ ID NO :2
pMR1-VL-T94C	SEQ ID NO :3
pMR1-VH-S28C	SEQ ID NO :18
pMR1-VH-G31C	SEQ ID NO :24
pMR1-VH-T30C	SEQ ID NO :15
pMR1-VH-Y32C	SEQ ID NO :14
pMR1-VH-G53C	SEQ ID NO :25
pMRI-VH-N56C	5'-ttatagtctgtgcacccatcaccccaaatcat-3'	SEQ ID NO :19
pMR1-VH-R99C	SEQ ID NO :23
Séquençage:
VL-CDR1-Rev	5'-agaatattgtgttcctga-3'	SEQ ID NO :4
VH-CDR1-Rev	5'-tgctgatgctcagtctgg-3'	SEQ ID NO :5
VH-Seq-CDR3	5'-ggtgatggaaacacagac-3'	SEQ ID NO :6
pASK98-seq-His	5'-cgccgcgcttaatgc-3'	SEQ ID NO :7
Construction de pMR1:
pASK98-His6-For:	5'-tcgagatcaagcggccgctggaacaccatcaccatcaccatta-3'SEQ	ID NO :8
pASK98-His6-Back :	5'-agcttaatggtgatggtgatggtggtccagcggccgcttgatc-3'	SEQ ID NO :9

4- Recombinant DNA techniques

Plasmid DNA preparation: Plasmid DNA mini-preparations are made from 5 ml of bacterial culture by the alkaline lysis method (Sambrook et al., above).

Processing: the preparation of competent bacteria by the simple CaCl2 method and the heat shock process are carried out as described in Sambrook et al., above.

Purification of DNA fragments by electrophoresis: the restriction mixture is loaded onto a 1-2% agarose gel (Easy Bag Agarose, Quantum Bioprobe). The electrophoresis is conducted for 1-2 hours at 8 V/cm in TBE buffer. The gel is then stained with ethidium bromide at 1 μg/ml in water. The agarose strip containing the DNA fragment is cut under a U.V lamp and then the DNA is extracted and purified using the QIAquick Gel Extraction Kit (QIAgen).

Binding of a DNA fragment to a plasmid vector: for binding of restriction fragments, their mixture is precipitated with Precipitator (Appligene), resuspended in water and then applied for 5 minutes at 50°C. 400 IU of T4 (New England Biolabs) DNA ligase and its buffer are added and the binding is continued overnight at 16°C. The HB2151 strains are then processed with the binding products.

Mutagenesis: directed mutagenesis is performed as described in Kunkel et al., (1987) using T4 polynucleotide kinase, T4 DNA ligase and T4 DNA polymerase (New England Biolabs).

Sequencing: the presence of mutation or control of genetic constructs is carried out by sequencing using the T7-Sequencing Kit (Pharmacia), with the mixtures of nucleotides for short reading, dATP-35S and a primer. The matrix consists of a mini-preparation of plasmidic DNA, denatured by soda and then neutralized by passing on a Microspin S-400-HR column. The sequence gels (6% acrylamide 1:19 bis, 42% urate in TBE, 30 x 40 cm) are subjected to a 45 min pre-electrophoresis at 40 W, then loaded with the previously scrubbed samples (Sokak et al., above). The migration is conducted at 40 W for 2 to 3 h. gels are dried and exposed to an MRI film (Max Kodak).

5-Constructions of recombinant plasmids

pMR1 : the pASK98-D1.3 vector is cut by the XhoI and HindIII enzymes. The restriction mixture is passed to the Microspin S-400-HR column to remove the smallest restriction fragment (43 pb). The pASK98-his6-for and pASK98-his6-back oligonucleotides are phosphorylated and hybridized at 60°C to form a cohesive end adapter which is inserted into the large XhoI-HindIII fragment of pASK98-D1.3. The resulting phagemide, pMR1 is controlled by restriction analysis with the NotI and PvuI enzymes and by sequencing by the pASK98-Hq-H6 oligonucleotide, corresponding to the IDQ of SE10.

PMR 5: the pFBX and pSPI1.0 plasmids are cut by SfiI and NotI. The small fragment of pFBX (750 pb) containing the scFv gene from D1.3 and the large fragment of pSPI1.0 (4650 pb) are purified on agarose gel and then assembled by ligation. The resulting plasmid, pMR5 is controlled by restriction analysis with the enzyme AflIII.

6- Production of antibody fragments

Production in small quantities from pMR1 or pMR5: 200 ml of ampicillin 2xYT medium is inoculated with a colony isolated from the recombinant strains HB2151 ((pMR1) or BL21 ((DE3, pMR5). The culture is agitated at 22, 30 or 37°C until A600nm = 0.5 where it is induced with 0.2 to 1.0 μg/ml anhydrotetracycline for HB2151 or with 0.2 to 1.0 mM IPTG for BL21 ((3) and then agitation is continued for 2 h, 5 or 16 h. A periplasmic extract is prepared by osmotic peristaltic (see below) from 50 ml of culture mined at 20 500 g 10 500 g, at 4°C.

Production of large quantities: to produce scFv-His6, SB (100 ml) medium with ampicillin is inoculated with a colony isolated from the recombinant strain HB2151 ((pMR1). The culture is agitated overnight at 37°C and then 25 ml of the contents are transferred to 750 ml of the same pre-balanced medium at 22°C. The culture is agitated at 22°C to A600nm = 2.0 and then induced by 0.22 μg/ml anhydrotetracycline. Growth is continued for approximately 2 h 30 min, to A600 = 4.

7- Preparation of periplasmic extracts

The bacterial cultures are centrifuged and periplasmic extracts are prepared from the bacterial collets by one of the following four methods.

The bacterial coating is resuspended in 20 per cent sucrose in 100 mM Tris-HCl pH 7.5 (1/20th volume). The suspension is held on ice for 10 minutes and then centrifuged for 15 minutes at 10,000 g at 4°C. The coating is resuspended in 0.5 mM MgCl2 (1/20th volume). The suspension is again held on ice for 10 minutes and centrifuged in the same way. This second supernatant constitutes the actual pelasmic extract.

Tris-EDTA method: the bacterial coating is resuspended in 100 mM Tris-HCl pH7.5 ; 1 mM EDTA (1/20th volume). The suspension is homogenised for 30 minutes by magnetic agitation at 4°C and then centrifuged for 30 minutes at 35000 g. The supernatant is the periplasmic extract. The coating is resuspended in 1/20th volume of SDS at 1% in water. It contains the insoluble, membrane or cytoplasmic proteins.

Tris-EDTA-NaCl method: this method is identical to the Tris-EDTA method except that the bacterial collet is resuspended in 100 mM Tris-HCl pH7,5; 1 mM EDTA and 1 M NaCl.

Polymyxin method: this method is identical to the Tris-EDTA-NaCl method with the only difference that the bacterial collet is resuspended in a P-pad containing 1 mg/ml of polymyxin B sulphate (ICN).

8-Purification of antibody fragments

The column is successively washed with 10 ml of 5 mM imidazole, 20 mM imidazole, 40 mM imidazole in buffer P. The scFv-His6 is electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically electrolytically elec

9-Quantification of scFv-His6 by indirect ELISA

A microtitration plate (Nunc) is coated with chicken egg white lysozyme (HEL, 10 μg/ml) in a 50 mM NaHCO3 pH9.6 buffer and incubated overnight at room temperature. The plate is saturated for 2 hours at 37°C with BSA (3% by weight/volume) in PBS buffer. The samples are diluted at least 2 times in PBS at 3% BSA, deposited on the plate and left for 1 hour at room temperature. The plate is then washed 5 times with PBS at 0.05 % Tween 20, incubated for 1 hour in the presence of a primary antibody (mouse monoclonal antibodies against H5,The plaque is washed as before and then incubated for 1 hour in the presence of a secondary antibody (mouse monoclonal anti-Fc antibody coupled with alkaline phosphatase) diluted to 1/10,000. The plaque is washed as before and the immune complexes are revealed with 2 mg/ml of p-nitrophenylphosphate (Sigma) in 1MClthanolamine-HCl pH9,8, 10 mMSO Mg4. The A405 signal is measured on a multiscan MS microplate scale. In the experimental conditions used, the linearity zone is obtained for concentrations of F-H6v less than 0.8 μg/ml.

10- Electrophoresis of proteins and Western-blot or immuno-fingerprinting techniques.

The concentration gel is a 4 per cent acrylamide gel:bisacrylamide (1:29) in 125 mM Tris-HCl pH6.8 ; 0.1 per cent SDS and the separation gel is a 15 per cent acrylamide gel in 375 mM Tris-HCl pH8.8 ; 0.1 per cent SDS. The electrophoresis pad contains 190 mM Glycine, 25 mM Tris-base and 0.1 per cent SDS. The Bio-gels (8 x 7 cm) are poured and then electrophoresis is performed using a Hoefer Mighty Small II device. The samples are added with a 2x loaded heat pad (125 mM Tris-HCl pH 21.8 ; 0.1 per cent SDS; 20 per cent Glycerol 2 mg/ day; 2 per cent Marginal blue; 10 per cent Marginal blue; and 10 per cent Marginal blue; in the case of masses of S-R, the low-molecular acids are reduced to 1 per cent L-methanol and 10 per cent Marginal blue; and in the case of R-R, the low-molecular acids are reduced to 1 per cent L-methanol, 1 per cent Marginal blue; and 10 per cent Marginal blue; the following tests are performed at a temperature of 30°C.

Western-blot technique: The proteins are separated by SDS-PAGE and then transferred to a Hybond-C (Amersham) nitrocellulose membrane overnight at 10V in a TE22 Series Transphor Electrophoresis Unit HSI (Hoefer) tank containing 25 mM Tris-base, 190 mM glycine, 20% methanol. The membrane is saturated by 3% BSA (weight:volume) in TBS buffer (100 mM Tris-HCl pH 7.5 ; 150 mM NaCl), then incubated for 1 ml at room temperature with primary and secondary antibodies, diluted in TBS at 3% BSA, followed by 0.2 ml in TBS at 0.2 mg NaCl, diluted in TBS at 3% BSA, at concentrations appropriate for the TISA (Example 1-9).

11- Analysis of the interaction between scFv of mAbD1.3 and the chicken lysozyme by BIAcore

The measurements are made on a CM5 chip (BIACORE AB) with a carboxymethyldextran surface, as described in England et al., (1999). Briefly, the lysozyme is immobilized in a channel by amide bonding up to a level of 500 RU (1 RU = 1 ng/mm2). The rate of association (kon) and rate of dissociation (koff) constants at the interface, and their ratio K'D (dissociation constant at the interface), are measured at 20°C with a flow of 10 μl/min of scFv-His6 in a PBS buffer added 0.005 % Tween 20. The buffer also contains 1 μM of lysozyme during the dissociation phase to prevent the re-excitation of scFv-His6 without specifically immobilizing the lysozyme channel.

12- Chromatography of exclusion

The scFv-His6 is dialyzed against Q buffer and then concentrated by ultrafiltration on CentriconR 10 (Amicon). The chromatography is performed with a SuperdexR 75 HR10/30 (Pharmacia) column in Q buffer at a flow of 0.5 ml/min. The column is pre-balanced under the same conditions. The proteins are detected in the column effluent by A280nm. The samples prepared in Q buffer are injected by means of a 200 μl loop. They have the following compositions: scFv-His6, 200 μL at 50 μg/ml; BSA, 40 μg chymotrypsinogen, 14 μg acetone, 0.1 μg acetone. The ratio of concentrations to dimethane is determined after the peak surface.

13-Coupled fluorophores on free cysteine of a mutant scFv-His6

Alternatively, the purified mutant scFv-His6 is adjusted to a concentration of at least 100 μg/ml by ultrafiltration on CentriconR 10 (Amicon) filter and then reduced by addition of 2-mercaptoethanol to a final concentration of 0.1 mM, 1 mM, 10 mM or 50 mM. The mixture is incubated for 1 hour at 22°C. The reduced protein is separated from the excess reducer by desalting, using a highly balanced HiTrap Desalting (Pharmacia) column in pre-de-gased C-buffer.

The coupling is performed by adding NBD chloride, IANBD ester, 5-IAF or acrylodone (BioProbe) in a molar ratio (protein:fluorophore) of at least 1:20. The mixture is incubated for 24 hours at 22°C and then centrifuged for at least 15 minutes at 10,000 g at 4°C. The marked scFv-His6 is then purified. The reaction mixture is then diluted 7 times in 5 mM imidazole in P-buffer and charged successively 3 times on a column containing 500 μl of Ni-NTA. The excess fluorophore is removed by washing 6 times in 5 ml of 5 mM imidazole in P-buffer. The marked scFv-His6 is excreted 1 time in 40 mL of P-buffer in 500 mM imidazole, 4 times in 100 mL of P-buffer.It is used as a control for protein dosing and fluorescence measurements. The coupling efficiency can be estimated by the ratio of fluorophore to protein absorbances: ε280nm(scFv-His6) = 51.13 mM-1.cm-1, ε435nm (CNBD) = 9.6 mM-1.cm-1, ε500nm (IANBD) = 31.1 mM-1.cm-1, ε492nm (5-IAF) = 75 mM-1.cm-1, ε391nm (acryl) = 20M-1.cm-1, as described in Haugland et al., (1996).

EXAMPLE 2: Example of the implementation of the process according to the invention. 1-Search for residues of the free Fv fragment of mAbD1.3 which can be used for fluorophore coupling (Figure 1).

The structure of the free Fv fragment of mAbD1.3 and that of its complex with lysozyme are known (Bhat et al., 1994). Nevertheless, to simplify the search for Fv residues that are located in the vicinity of the active site of lysozyme binding and usable for fluorophore coupling, and to make this search applicable to other macromolecule pairs for which only the structure of the complex exists, experimental data regarding the free Fv of mAbD1.3 were not used.The structures were analysed with the WHAT IF software suite (Vriends, 1990; http://www.sander.embl-heidelberg.de/whatif/). Three types of residues were investigated in mAbD1.3: those in direct contact with the lysozyme, those in contact via a water molecule, and those whose solvent-accessible surface (SAS) is altered by the fixation of the lysozyme when spheres of radius 1.4· 1.7· 2.0· 2.3· 2.6 or 2.9 Å are used for the solvent molecule.The residues of mAbD1.3 which satisfy one of these criteria, and the nature of the criterion used, are shown in columns 1 and 2 of Figure 1. Columns 8 and 9 of Figure 1 give the nature of the known mutations in mAbD1.3 and their effects, in the form of a ΔΔG variation in the energy of interaction with the lysozyme 2; the residues of the receptor selected in (1) must be changed into Cys mutagenesis,The Cys mutant residue Sγ must therefore be exposed to the solvent in the free Fv, in order to be attacked by the fluorophore molecule. It can be assumed, as a first approximation, that the Sγ of the Cys mutant residue overlaps with the γ-position of the wild type residue. This γ-position atom must therefore be exposed to the solvent in the structure of the free wild Fv. When an atom is present at δ-position in the wild type residue, it can mask the γ-position atom from the solvent, but this masking will no longer exist after its change to Cys.The ASA values of the atoms in the γ position and possibly δ, for each residue selected in point 1), are given in columns 3 and 4 of Figure 1 (using R = 1.4 Å) ;3- The fluorophore can be advantageously coupled to the Sγ of the Cys residue by means of an aliphatic chain ranging in length from 1 to 6 carbon atoms for the most common fluorophores, but may be longer.After coupling, the polar and aromatic groups of the fluorophore should preferably be located at the periphery of the contact interface between the antibody and the antigen, and not at this interface where they would conflict with the antigen. This requires that the aliphatic arm of the fluorophore has access to the solvent in the complex structure between the marked antibody and the antigen.In columns 5, 6 and 7 of Figure 1 ;4- The mutations in the receptor residues must not be too deleterious for interaction with the antigen if the antibody to be marked is to maintain sufficient affinity. Columns 8 and 9 of Figure 1 give the nature of the mutations known in mAbD1.3 and their effects, as a ΔΔG variation in the interaction energy with the lysozyme (England et al., 1997 and 1999; Hawkins et al., 1993; Dall'Acqua et al. 1996; Dall'Acqua and Carter, 1998).

The residue classification is divided into three different priority classes: the first class contains those residues whose γ-atom is accessible to the solvent in the free Fv structure, and which are in contact with the lysozyme either directly or via a water molecule: VL-Thr53, VL-Ser93, VL-Thr94 and VH-Thr30. The second class contains those residues whose γ-atom is accessible but which are not in direct contact with the lysozyme: VL-Asn31, VL-Thr52, VL-Asp56, VH-Ser28, VH-Asn56.

2-Construction of expression vectors for scFv-His6 and optimization of production of scFv-His6 (Figure 2) (a) Construction of vectors for expression of scFv-His6

The study of the effect of fluorophore coupling to mAbD1.3 scFv residues as defined in 1, on lysozyme binding, was performed using recombinant mAbD1.3 scFvs with a C-terminal extension of 6 histidine residues (scFv-His6), expressed by prokaryotic expression vectors.

Two vectors encoding for scFv-His6, pMR1 and pMR5, were constructed according to the protocol described in example 1-5. They carry the kanamycin or ampicillin resistance gene, the origin of M13 phage replication (f1IG), and periplasmic addressing sequences, derived from pelB or ompA genes, upstream of the scFv gene. The pMR5 vector carries the scFv-His6 gene under the control of a T7 phage promoter and lactose operator, and the promoter of the lactose recycler. Expression occurs in the tDE21 gene that carries the polymerase RNA receptor T7 (tP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/TP/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/T/

(b) Optimization of the production of scFv-His6

The production of scHis6, estimated by the amount of scFv-His6 present in a raw periplasmic extract, is measured by indirect ELISA using an anti-His5 antibody, using the techniques described in Example 1-9.

Production of small amounts of antibody fragments from pMR1 or pMR5 under the conditions described in Example 1-6 showed that the pMR1 vector yielded best at 22°C, inducing 2 hours with 0.2 μg/ml anhydrotetracycline.

Production of large amounts of antibody fragments from either pMR1 or pMR5 under the conditions described in example 1-6 showed that production yields were better when induction occurred in an exponential growth phase. At 22°C, the 2xYT medium only allowed a short exponential growth phase for the HB2151 strain, between A600nm=0.3 and A600nm=1.1 and bacteria were observed to live in this medium. The latter was therefore supplemented with a low base volume (K2HPO4), to prevent its acidification during culture, and with compounds that stabilized the outer bacterial membrane (MgSO4, NaCl, NaCl).

Two types of methods exist to extract periplasmic fluid from a bacterium: (1) the periplasmic contents are then balanced in a medium of high osmotic pressure and then transferred to a hypoosmotic buffer. The osmotic shock ruptures the outer membrane and the periplasmic contents are recovered in the second buffer; (2) the bacteria are resuspended in a buffer that lixiviates the outer membrane (EDTA, amphiphilic peptides such as polymyxin B). The periplasmic contents are then recovered in one step. The methods for preparing the periplasmic extract of HB2151MR1p1 described in Examples 1-7 were therefore saved, and the amount of TNF-H6 was described in the samples for each of the samples. The best measurements for the TNF-H6 and TNF-H6 were given by the following method: the maximum volume of the extract was approximately 6 ± 10 ± 10 ± 10 ± 10 mm, and the maximum value was given by the following method: the maximum value was given by the following method: TNF-H6 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10 ± 10

3- Purification of scFv-His6

The polyhistidine tail of scFv-His6 allows for purification on a column of immobilized Ni-NTA nickel, according to the protocol described in example 1-8. After loading the column with the periplasmic extract, washing and elution are done with different concentrations of imidazole, which competes with the histidines for coordination of nickel. The concentration of imidazole for washing, which allows for the removal of impurities without moving the scFv-His6 itself, and the concentration for elution, which allows for the removal of scFv-His6 into a minimum of fractions without denaturing it, are specified in example 1-8.The product of the measurements of A405nm by the volume of each fraction yielded the following relative values: of the 100% contained in the raw extract, 6.7% of the scFv-His6 does not attach to the column, 7.6% is detached during washing and 85.6% is found in the elution fractions.Under optimal culture, extraction and purification conditions, 650 μg of purified wild scFv-His6 was obtained per litre of culture.

4-Oligomerization and lysozyme binding properties of wild type scFv-His6 (Figure 4)

The BIAcore device allows rapidly obtaining the kon-association rate and koff-dissociation constants between antibody and antigen, as well as the K'D=koff/kon equilibrium constant, at the interface between the buffer and the surface on which the antigen has been immobilized. The measurements are performed in continuous flow on the surface, they can be distorted by recapture of recently dissociated scFv at another site of immobilized antigen. This recapture has little influence with a fragment of monovalent antibodies, and is limited by the addition of antigen in the solution of the dissociation buffer.

The proportions of monomer and dimer at pH used for BIAcore (pH7.5) and at a concentration of 50 μg/ml were estimated by the height of the corresponding peaks in exclusion chromatography, according to the protocol of example 1-12, and the results are presented in Figure 4. The ratio of the surfaces of the peaks of the dimer and the monomer gives a dimerization rate of 9.8%. $k_{\mathrm{on}}=1,15(\pm 0,15)\mathrm{.}{10}^5{M}^{-1}\mathrm{.}{s}^{-1}$ $k_{\mathrm{off}}=1,20(\pm 0,22)\mathrm{.}{10}^{-3}{s}^{-1}$ ${\mathrm{Kʹ}}_D=10,5(\pm 3,3)\mathrm{nM}$

5-Construction, production and purification of mutant scFv-His6 (Figure 3)

The cysteine mutations of the mAbD1.3 antibody residues, placed in the first class (VH-T30C, VL-T53C, VL-S93C and VL-T94C, see also paragraph 1 of this example and column 10 of Figure 1) were introduced into the pMR1 expression vector by the Kunkel method described in example 1-4. The oligonucleotides used, described in example 1-3, were designed to introduce or remove sites of deletion. The mutation is then detectable by analysis of fragments obtained after the mutation. The restriction clones were further sequenced in the region of the mutation, using oligonucleotides described in example 1-3,The ability of several mutant pMR1 derivatives to express scFv-His6 was tested under the conditions described for the production of wild scFv-His6 from pMR1 (example 2-2). The results show that these mutants are efficiently produced in comparison to wild: 100 ± 12 for the wild type, 100 ± 29 for VL-S93C, 97 ± 5 for VL-T94C. The VL-S93C mutant was purified with yields of 575 μg per litre of culture,The results of the analysis were comparable to those obtained with wild scFv-His6.

The purity of the elution fractions was analysed by polyacrylamide-SDS gel electrophoresis and Coomassie blue staining, according to the protocol described in Example 1-10. The results obtained are shown in Figure 3. These fractions contain a protein which has an apparent molecular weight equal to the expected mass for scFv-His6, and are 95% pure.

6-Absent of covalent dimerization of the mutant VL-S93C (Figure 5)

The presence of free cysteine on mutant scFv-His6 could lead to the formation of parasitic intermolecular disulfide bridges during production, purification, or during fluorophore coupling. scFv-His6 ((VL-S93C), dialysed against the coupling buffer, was analyzed by exclusion chromatography under conditions used for wild-type scFv-His6 (example 1-12 and example 2-4). The chromatogram shows only one peak, suggesting that scFv-His6 ((VL-S93C) exists in only one state of oligerization in the buffer used (Figure 5). The position of this almost corresponds to that of the wild-type scFv-His6 monomer. The difference between the two positions of the pH peaks could be due to the fact that the two chromatograms have been developed in different low pH and slightly different positions.

7-Coupled fluorophores with the VL-S93C mutant (Figures 6 to 10)

The reaction is done at pH=7.0 to avoid parasitic coupling on deprotonated lysines at basic pH (Houk et al., 1983 ; Del Boccio et al., 1991). After coupling, the excess fluorophore and the derivative of the scFv-His6 ((VL-S93C) are separated by a new Nickel column purification. Several arguments converge to confirm the specific intensity of the coupling on the fluorescent protein V-LysCv93F. In the mutant coupling, the amount of fluorophore is effectively re-saturated and the amount of fluorescent is proportional to the time of the coupling (Figure 7).

In the absence of reduction treatment, wild-type scFv-His6 does not react with IANBD, whereas scFv-His6 ((VL-S93C) is marked at 8% by fluorophore (Figure 8). This latter performance is greatly improved when scFv-His6 ((VL-S93C) is reduced by 2-mercaptoethanol, rapidly de-capped and placed in the presence of IANBD. In contrast, this treatment results in a loss of material as much as the reduction is pushed. It is likely that scFv-His6 is made partially insoluble by reduction of disulfides.

8- Changes in fluorescence of the VL-S93C mutant coupled with a fluorophore according to antigen concentration (Figure 11 and Figure 12)

A spectrum of emission of the conjugate scFv-His6(VL-S93ANBD), taken immediately after the addition of the antigen (lysozyme), shows an exaltation of fluorescence which depends on the final antigen concentration. The fluorescence intensity saturates to 1.26 ± 0.05 times that of the free biosensor when the antigen concentration is sufficient, and reproducibly on several biosensor preparations. No significant wavelength shift of the maximum emission is observed (Figure 11).

Fluorescence intensities were recorded in the absence or presence of varying lysozyme concentrations (10 nM to 2 μM) in a 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 buffer.

The results shown in Figure 12 show that in this buffer the fluorescence intensity of the conjugate scFv-His6 (VL-S93ANBD) is proportional to the lysozyme concentration up to 400 nM and increased by 91% at saturation.

Therefore, the results obtained in Figure 12 show that the biosensor of the invention is advantageous for directly titrating an antigen or hapten, for example in body fluid.

9- Coupling yield and fluorescence variation according to the antigen concentration of a series of scFv-His6 mutants (Figure 13).

ScFv-His6 mutants, selected by the process described in Example 2-1, were produced in a similar manner to the VL-S93C mutant (Example 2-5), using the methods described in Example 1 (1-1 to 1-12). Thus, pMR1-derived plasmids (SEQ ID NO:10) carrying the mutations described in Figure 12 were constructed by directed mutagenesis using the oligonucleotides shown in Table II. The scFv-His6 mutants produced from these plasmids were coupled to the IANBD, similarly to the VL-S93C mutant (Example 2-7), using a concentration of mercaptofluoroethane of almost 10 mM, according to the results described in Example 1-13. The results for these samples show that the most important positions for the selection of the most important positions of the coupling and the exaltations of the coupling are obtained for the satisfaction of the results of the samples.

The Commission shall be assisted by the European Parliament.

The following is a list of the most commonly used chemical names in the field of immunology: Arndt K.M. et al., Biochemistry, 1998, 37, 12918-12926.Bhat T.N. et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 1089-1096.Braden B.C. et al., Immunological Review, 1998, 163, 45-57.Carter P. et al., Nucleic Acids Research, 1985, 13, 4431-4443.Creighton T.E., Proteins: Structure and molecular properties (Second edition).W.H. Freeman and Cie, 1996, 227-229.Dall'Ac W. et al., Biochemistry, 1996, 35, 9667-9676 Dall'Acqua W. et al., Opinion in Structural Biology, 1998, 8, 443-450 Del Boquaccio G.et al., The Journal of Biochemistry, 1996, 137-1376, 137-1372.England and Chemistry, P. and al., 1997, 162-1722, and The Journal of Immunology, 1997, 162-1372, and G. and al., P. and G., 162-1372, 162-1722, and 163-172.The following is a list of the most commonly used methods of determining the concentration of a substance in a sample:The following is a list of the most commonly used methods of determining the concentration of a substance in a food: the most commonly used methods are the use of a chemical, the most commonly used methods are the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, the use of a chemical, a chemical, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, a, aThe following is a list of the most commonly used methods of determining the concentration of a protein in a cell, and the number of cells that are required for the determination of the concentration of a protein:

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The Commission has also adopted a proposal for a directive on the protection of workers from the risks related to exposure to ionising radiation. - I'm not going to do it. I'm going to have to go. I'm not sure. BEDOUELLE, Hugues<120> BIOCAPTURES, their production process and their applications<130> 226 cases 82<140> The Commission has decided to initiate the procedure provided for in Article 93 (1) of the Treaty. The number of employees The following is a list of the main types of DNA sequences: The following information is provided for the purpose of the analysis: The following shall be added: The number of employees The following are the types of products: The following table shows the number of samples of the test chemical: The following information is provided for the purpose of the analysis: The following are the types of aid: The number of employees The following are the types of products: The following table shows the number of samples of the test chemical: The following information is provided for the purpose of the analysis: The following are the types of aid: The number of employees The following are the types of products: The following table shows the number of samples of the test chemical: The following are the main characteristics of the product: The number of employees The following are the types of products: The following table shows the number of samples of the test chemical: The following information is provided for the purpose of the analysis: The number of employees is calculated as follows: The number of employees The following are the types of products: The following table shows the number of samples of the test chemical: The following information is provided for the purpose of the analysis: The following are the main categories of products: The number of employees The following are the types of products: The following table shows the number of samples of the test chemical: The following are the main characteristics of the product: Other The following are the types of products: The following table shows the number of samples of the test chemical: The following shall be considered as a single measure: Other The following are the types of products: The following table shows the number of samples of the test chemical: The following information is provided for the purpose of the analysis: The following shall be reported in the table: Other, including: The following are the types of products: The number of samples is calculated as follows: The number of employees Other, including: The following are the types of products: The following table shows the number of samples of the test chemical: - What?

Claims (6)

1. Process of preparing a biosensor consisting of (i) at least a protein-type receptor fragment capable of binding to an appropriate ligand via a binding site and in which fragment at least one of its amino acid residues, located in the proximity of said binding site, is selected from the group formed by the residues which are in direct contact with the ligand, those which are in contact via a molecule of water, and those whose surface accessible to the solvent is modified by binding the ligand and is naturally present in the form of a Cys residue or is substituted for a Cys residue, and (ii) a fluorophore coupled with said Cys residue(s), said process being characterised in that it comprises the following steps:

   a) identification of receptor binding site amino acid residues by mutagenesis of all or a sub-group of the receptor amino acid residues and determination of variations in ligand interaction parameters (K_D, k_on, k_off) which are due to each mutation or limited groups of mutations;

   b) selection of Cys residues or residues to be mutated into cysteine among receptor amino acid residues which are located in the proximity of binding site residues along the receptor sequence;

   c) directed mutation of at least one of the amino acid residues selected in (b) into a Cys residue, in the event that said residue is not naturally a Cys residue;

   d) controlled reduction of the receptor obtained in (b) or in (c), and

   e) coupling of the Sγ atom of at least one Cys residue obtained in (b) or in (c) with a fluorophore.
2. Process of preparing according to claim 1, characterised in that, prior to step (a), the non-essential Cys residues of the receptor are substituted with Ser or Ala residues by directed mutagenesis.
3. Process of preparing according to claim 1 or claim 2, characterised in that, at step (e), said fluorophore is selected from the group formed by N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3diazole, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, acrylodan, 5-iodoacetamidofluoresceine or a fluorophore with an aliphatic chain of 1 to 6 carbon atoms.
4. Process of preparing according to any of claims 1 to 3, characterised in that it comprises an additional step (f) of purification of the biosensor obtained in (e).
5. Process of preparing according to claim 4, characterised in that it comprises an additional step of measuring the equilibrium constant (K_D or K'_D) or the dissociation (k_off) and association rate constants (k_on) between the purified biosensor obtained in (f) and the ligand.
6. Process of preparing according to any of claims 1 to 5, characterised in that after step (e) or step (f) it comprises an additional step of immobilisation of the biosensor on a solid support.