Anti-g protein alpha antibody

Abstract

The present invention relates to an antibody or antibody fragment capable of binding to G protein alpha, a nucleic acid sequence encoding said antibody, a vector comprising said nucleic acid sequence, a cell comprising said vector or said nucleic acid sequence and a kit comprising: i) said antibody or antibody fragment or said composition and ii) a GTP source labeled with a member of a RET partner pair.

Description

TECHNICAL FIELD

[0001] The present invention relates to a new antibody or antibody fragment capable of binding to the G protein alpha. BACKGROUND

[0002] G protein-coupled receptors (RCPG or GPCR in English) are a family of membrane receptors in mammals and throughout the animal kingdom. The G protein is a heterotrimeric protein (3 subunits: alpha, beta and gamma) activated by the RCPG. Through the RCPG, the G protein has a signal transduction effect from the outside of the cell to the inside of the cell (i.e. the cell reacts to an external stimulus). The generally described mechanism of action is summarized as follows:

[0003] - in the unactivated resting state of the RCPG, the alpha subunit of the G protein is bound to the nucleotide GDP (holo-G protein bound to GDP;

[0004] - upon activation of the RCPG, the RCPG binds to the alpha subunit of the G protein and triggers the activation process of the G protein, comprising two steps: 1) ejection of GDP from the G protein to produce an empty G protein, and formation of the unactivated RCPG / empty G protein complex, and 2) fixation of GTP, which leads to the formation of the activated G protein in the form of GTP (holo-G protein bound to GTP). In the first step, the receptor-bound G protein is in the form called "empty". This state is described in the literature as transient, since this state describes the rapid binding of the nucleotide GTP to the alpha subunit of the G protein. In addition, the beta / gamma subunits of the activated G protein are separated from the alpha subunit;

[0005] - the alpha subunit of the holo-G protein bound to GTP subsequently binds to effectors to activate them. In turn, the effectors activate signaling pathways, thus generating a cellular response;

[0006] - then, GTP is hydrolyzed to GDP by the alpha subunit of the G protein, and the alpha subunit reassociates with the beta / gamma subunits to form again the holo-G protein bound to GDP (unactivated state).

[0007] Given that RCPGs are involved in many signal transduction pathways, tools for studying RCPG activity have been developed in the prior art, with the aim of identifying new ligands for these receptors with potential therapeutic activity. As examples of these tools, we can mention the use of non-hydrolyzable or slowly hydrolyzable radioactive derivatives of GTP, particularly GTP-γ-S (which binds to G protein α when the receptor is activated). There are also recombinant systems based on enzyme activity measurements, such as luciferase, whose expression is controlled by a second messenger generated by receptor activation. Antibodies for detecting RCPG activation at the cell surface level have also been synthesized [1]. Also refer to patent EP2723764 B1, which proposes nanobodies that bind to the interface between G protein α and G protein β / γ, thereby stabilizing the RCPG / G protein complex.

[0008] However, the prior art does not describe the ability to generate a RET signal when an antibody or antibody fragment labeled with a member of a RET pair is used in conjunction with GTP labeled with a member of a RET pair.

[0009] The inventors have developed antibodies or antibody fragments that perform the RET method for detecting RCPG activation using member-labeled GTPs of RET couples. The inventors have also demonstrated that these antibodies or antibody fragments possess these properties because they are able to bind to G protein α in very specific regions. Summary of the Invention

[0010] The first object of the present invention relates to an antibody or antibody fragment capable of binding to G protein α, comprising:

[0011] - Heavy chain variable domain containing CDR1 of amino acid sequence SEQ ID NO: 1, CDR2 of amino acid sequence SEQ ID NO: 2, and CDR3 of amino acid sequence SEQ ID NO: 3; and

[0012] - Light chain variable structural domain containing CDR1 of amino acid sequence SEQ ID NO: 4, CDR2 of amino acid sequence DTS (i.e., three amino acids Asp Thr Ser), and CDR3 of amino acid sequence SEQ ID NO: 5.

[0013] The second object of the present invention relates to an antibody or antibody fragment that competitively binds to G protein α in accordance with the antibody or antibody fragment of the first object of the present invention.

[0014] A third object of the present invention relates to compositions comprising antibodies or antibody fragments according to the present invention.

[0015] A fourth object of the present invention relates to kits (French trousse de réactifs) which comprise (i) an antibody or antibody fragment or a composition according to the invention and (ii) a GTP source of a member marker of a RET couple.

[0016] A fifth object of the present invention relates to a nucleic acid sequence encoding an antibody or antibody fragment according to the present invention.

[0017] A sixth object of the present invention relates to a vector comprising a nucleic acid sequence according to the present invention.

[0018] A seventh object of the present invention relates to cells comprising a vector according to the invention, or a nucleic acid sequence according to the invention.

[0019] Detailed description

[0020] Definitions

[0021] The terms “antibody against G protein α”, “anti-Gα antibody”, or “antibody (or antibody fragment) capable of binding to G protein α” are interchangeable and refer to an antibody (or antibody fragment) that binds to G protein α with sufficient affinity, and is a reagent for detection (e.g., RET), diagnosis, and / or treatment by targeting G protein α.

[0022] The term "antibody," also known as "immunoglobulin," refers to a heterotetramer composed of two heavy chains (H chains) each approximately 50-70 kDa and two light chains (L chains) each approximately 25 kDa, linked together by intra- and inter-chain disulfide bonds. Each chain consists of a variable region or domain at the N-terminus (VL for the light chain and VH for the heavy chain) and a constant region at the C-terminus (consisting of a single domain called CL for the light chain and three or four domains called CH1, CH2, CH3, and CH4 for the heavy chain). Each variable domain typically contains four "hinge regions" (FR1, FR2, FR3, and FR4) and three regions directly responsible for binding to the antigen, called "CDRs" (CDR1, CDR2, and CDR3). Antibodies can be, for example, mammalian antibodies (such as mouse antibodies), chimeric antibodies, humanized antibodies, or human antibodies.

[0023] "Chimeric antibody" refers to an antibody in which the variable region sequences of the light and heavy chains belong to different species than the constant region sequences of the light and heavy chains. For the purposes of this invention, the variable region sequences of the heavy and light chains are preferably murine, while the constant region sequences of the heavy and light chains belong to non-mouse species. In this respect, for the constant regions, all non-mouse mammal species are available, particularly humans, monkeys, Old World pigs (Suidae), bovids, equines, felines, canids, or birds; this list is not exhaustive. Preferably, the chimeric antibody according to the invention comprises human-derived constant region sequences of the heavy and light chains and murine-derived variable region sequences of the heavy and light chains.

[0024] "Humanized antibody" refers to an antibody in which all or part of the region involved in antigen recognition (hypervariate region or CDR: complementarity-determining region) and sometimes certain amino acids in the FR region (framework region) are non-human, but the sequences of the constant and variable regions that do not participate in antigen recognition are human.

[0025] "Human antibody" refers to an antibody that contains only human sequences for both the variable and constant regions of the light chain and the variable and constant regions of the heavy chain.

[0026] "Antibody fragment" refers to any part of an immunoglobulin obtained through enzymatic digestion or bioproduction that contains at least one disulfide bond and is capable of binding to an antigen recognized by the intact antibody, such as Fab, Fab', F(ab')2, and Fab'-SH. Papain digestion of immunoglobulins produces two identical fragments called Fab (antigen-binding fragments) and fragment Fc (crystallizable fragment). Pepsin digestion of immunoglobulins produces fragment F(ab')2 and fragment Fc, which is divided into several peptides. F(ab')2 is formed by two Fab' fragments linked by interchain disulfide bonds. The Fab moiety consists of a variable region and CH1 and CL domains. The Fab' fragment consists of a Fab region and a hinge region. Fab'-SH refers to a Fab' fragment in which the cysteine ​​residues in the hinge region carry free thiol groups.

[0027] The term "affinity" refers to the force of all non-covalent interactions between a molecule (e.g., an antibody or antibody fragment) and a recognized antigen (e.g., an antigen such as G protein α). Affinity is typically represented by the dissociation constant (Kd). The dissociation constant (Kd) can be measured by known methods, such as FRET or SPR.

[0028] In the context of this invention, "identity" or "homology" is calculated by comparing two sequences aligned within a comparison window. Sequence alignment determines the number of common positions (nucleotides or amino acids) between the two sequences within the comparison window. Therefore, the number of common positions is divided by the total number of positions in the comparison window and multiplied by 100 to obtain the percentage identity. The percentage identity of sequences can be determined manually or using known software.

[0029] In specific embodiments of the invention, identity or homology corresponds to at least one substitution of an amino acid residue, such as substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably conservatively. "Conservatively substituting an amino acid residue" includes replacing an amino acid residue with another amino acid residue having a side chain with similar properties. Families of amino acids with side chains having similar properties are well known; we may mention, for example, basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0030] Therefore, homologous antibodies or antibody fragments, or "variants of antibodies or antibody fragments" (i.e., antibodies or antibody fragments with the same function), have certain amino acids that can be substituted by other amino acids at the level of constant and / or variable regions without losing their ability to bind to antigens. Such substitutions preferably occur within the DNA sequence encoding the antibody or antibody fragment, i.e., the substitutions are inherently conserved. Those skilled in the art apply their general knowledge to determine the number and location of substitutions that can be made to maintain the function of the antibody or antibody fragment. To determine the ability of one or more variants of an antibody or antibody fragment to specifically bind to an antigen, several suitable methods well known to those skilled in the art and described in the prior art can be used. Thus, antibodies or antibody fragments can be determined by binding techniques such as ELISA, affinity chromatography, etc. Variants of antibodies or antibody fragments can be generated, for example, by "phage display" methods (which allow the generation of phage libraries). Numerous methods are known for generating "phage display" libraries and targeting variants of antibodies or antibody fragments with desired functional characteristics.

[0031] "Purified" and "isolated" refer to the presence of antibodies or antibody fragments according to the invention in the absence of other biomacromolecules of the same type. As used herein, the term "purified" preferably means that at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight, of the antibody relative to all macromolecules.

[0032] The term "G protein" refers to a heterotrimeric protein composed of three subunits called G protein α, G protein β, and G protein γ.

[0033] The term "G protein α" or "Gα" refers to the α subunit of a G protein. G protein α has two domains: a GTPase domain and an α-helical domain. There are at least 20 different G protein αs, which can be classified into the following major protein families: Gαs (known to activate adenylate cyclase to increase cAMP synthesis), Gαi (known to inhibit adenylate cyclase), Gαolf (associated with olfactory receptors), Gαt (known to be involved in visual signal transduction in the retina and associated with rhodopsin), Gαq (known to stimulate phospholipase C), or the Gα12 / 13 family (known to regulate the cytoskeleton, cell junctions, and other processes related to cell movement). G protein α can be selected from G protein αi, G protein αo, and / or G protein αz. G protein αi can be selected from G protein αi1, G protein αi2, and / or G protein αi3. G protein αi can be of human or animal origin.

[0034] Advantageously, the antibody or antibody fragment according to the invention binds to G protein αi, G protein αo, and / or G protein αz, for example, the antibody or antibody fragment according to the invention binds to G protein αi1, G protein αi2, and / or G protein αi3. For isoform 1, the human-derived G protein αi1 carries the identifier UniProt P63096-1, and for isoform 2, it carries the identifier UniProt P63096-2. The gene encoding the human-derived G protein αi1 is called “GNAI1” (gene ID: 2770, NCBI).

[0035] The term "GTP" refers to guanosine triphosphate.

[0036] The term "non-hydrolyzable or slowly hydrolyzable GTP" refers to GTP analogues that do not hydrolyze or hydrolyze very little to GDP. Examples of such analogues include, for instance, GTPγS (CAS No. 37589-80-3), GppNHp (CAS No. 148892-91-5), or GppCp (CAS No. 10470-57-2).

[0037] The terms “non-hydrolyzable or slowly hydrolyzable GTP labeled with a member of a RET mating body pair” or “labeled non-hydrolyzable or slowly hydrolyzable GTP” are interchangeable and mean non-hydrolyzable or slowly hydrolyzable GTP labeled with a donor member of a RET mating body pair (“GTP-donor”) or non-hydrolyzable or slowly hydrolyzable GTP labeled with a member of an acceptor member of a RET mating body pair (“GTP-acceptor”).

[0038] The term "RET" (an abbreviation for Resonant Energy Transfer) refers to energy transfer techniques, including FRET and BRET.

[0039] The term "FRET" (an abbreviation for fluorescence resonance energy transfer) refers to energy transfer between two fluorescent molecules. FRET is defined as a nonradiative energy transfer resulting from dipole-dipole interactions between an energy donor and an energy acceptor. This physical phenomenon requires energy compatibility between these molecules. This means that the emission spectrum of the donor must at least partially overlap with the absorption spectrum of the acceptor. In theory, FRET is a method that depends on the distance between two molecules (donor and acceptor): when these molecules are close to each other, they will emit a FRET signal. For example, the dissociation constant (Kd) between an antibody and its target is measured by FRET, as shown in the example.

[0040] The term "BRET" (an abbreviation for bioluminescent resonance energy transfer) refers to the energy transfer between bioluminescent and fluorescent molecules.

[0041] Antibodies or antibody fragments according to the invention

[0042] The first object of the present invention relates to an antibody or antibody fragment capable of binding to G protein α, comprising:

[0043] - Heavy chain variable domain, comprising CDR1 of amino acid sequence SEQ ID NO: 1, CDR2 of amino acid sequence SEQ ID NO: 2, and CDR3 of amino acid sequence SEQ ID NO: 3; and

[0044] - Light chain variable structural domain, which contains CDR1 of amino acid sequence SEQ ID NO:4, CDR2 of amino acid sequence DTS (i.e., three amino acids "Asp Thr Ser", namely aspartic acid, threonine and serine), and CDR3 of amino acid sequence SEQ ID NO:5.

[0045] Heavy-chain variable structural domains can contain:

[0046] - FR1, which has at least 80% homology to the amino acid sequence SEQ ID NO: 6, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0047] - FR2, which has at least 80% homology to the amino acid sequence SEQ ID NO: 7, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0048] - FR3 and / or amino acid sequence SEQ ID NO: 8 having at least 80% homology, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0049] - FR4, which has at least 80% homology with the amino acid sequence SEQ ID NO: 9, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0050] The variable structural domain of a light chain can contain:

[0051] - FR1, which has at least 80% homology to the amino acid sequence SEQ ID NO: 10, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0052] - FR2 has at least 80% homology with the amino acid sequence SEQ ID NO: 11, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0053] - FR3 and / or amino acid sequence SEQ ID NO: 12 having at least 80% homology, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0054] - FR4, which has at least 80% homology with the amino acid sequence SEQ ID NO: 13, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% homology.

[0055] In a specific embodiment of the antibody according to the present invention:

[0056] • Heavy-chain variable structural domains contain:

[0057] -FR1 of the amino acid sequence SEQ ID NO: 6 (i.e., 100% homologous to the amino acid sequence SEQ ID NO: 6),

[0058] -Amino acid sequence FR2 of SEQ ID NO: 7

[0059] -Amino acid sequence FR3 and SEQ ID NO: 8

[0060] -Amino acid sequence FR4 of SEQ ID NO: 9; and

[0061] • The variable structural domain of the light chain contains:

[0062] -Amino acid sequence FR1 of SEQ ID NO: 10

[0063] -Amino acid sequence FR2 of SEQ ID NO: 11

[0064] -Amino acid sequence FR3 and SEQ ID NO: 12

[0065] - FR4 of the amino acid sequence SEQ ID NO: 13.

[0066] In one specific embodiment of the antibody of the present invention, the heavy chain variable domain may have at least 80% homology with the amino acid sequence SEQ ID NO: 14, preferably at least 90% homology, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology, and the light chain variable domain may have at least 80% homology with the amino acid sequence SEQ ID NO: 15, preferably at least 90% homology, for example at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology.

[0067] Therefore, the present invention relates to an antibody or antibody fragment, wherein:

[0068] - The heavy chain variable domain has at least 80% homology with the amino acid sequence SEQ ID NO: 14, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology;

[0069] - The light chain variable domain has at least 80% homology with the amino acid sequence SEQ ID NO: 15, preferably at least 90% homology, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology; and

[0070] - The heavy chain variable domain CDR1 is composed of the amino acid sequence SEQ ID NO: 1, the heavy chain variable domain CDR2 is composed of the amino acid sequence SEQ ID NO: 2, the heavy chain variable domain CDR3 is composed of the amino acid sequence SEQ ID NO: 3, the light chain variable domain CDR1 is composed of the amino acid sequence SEQ ID NO: 4, the light chain variable domain CDR2 is composed of the amino acid sequence DTS, and the light chain variable domain CDR3 is composed of the amino acid sequence SEQ ID NO: 5.

[0071] Advantageously, the heavy chain variable domain is composed of the amino acid sequence SEQ ID NO: 14 (i.e., the heavy chain variable domain is 100% homologous to the amino acid sequence SEQ ID NO: 14) and the light chain variable domain is composed of the amino acid sequence SEQ ID NO: 15.

[0072] The antibody described in the example of reference DSV36S comprises a heavy chain variable domain consisting of the amino acid sequence SEQ ID NO: 14 and a light chain variable domain consisting of the amino acid sequence SEQ ID NO: 15.

[0073] The antibody or antibody fragment for the first purpose described above is referred to below as the "reference antibody or antibody fragment".

[0074] A second object of the present invention relates to an antibody or antibody fragment that competitively binds to G protein α in relation to a reference antibody or antibody fragment, hereinafter referred to as a "competitive antibody or antibody fragment".

[0075] The ability of an antibody or antibody fragment to competitively bind to G protein α against a reference antibody or antibody fragment can be determined by a competitive method. A “competitive method” includes testing the ability of an antibody (or antibody fragment) to block the binding of a reference antibody or antibody fragment to an antigen, or to competitively bind to an antigen against a reference antibody or antibody fragment. In other words, the antibody competing with the reference antibody or antibody fragment binds to the same epitope as the reference antibody or antibody fragment, or binds to an epitope sufficiently close to the epitope recognized by the reference antibody or antibody fragment, thereby preventing the reference antibody or antibody fragment from binding to the epitope due to steric hindrance.

[0076] Many types of competitive methods can be used to determine whether an antibody or antibody fragment competes with a reference antibody or antibody fragment, such as: by competitive ELISA assays, by immunofluorescence assays (e.g., by FRET or HTRF (“homogeneous time-resolved fluorescence”), by immunoluminescence assays, by direct or indirect sandwich assays, by direct or indirect solid-phase radioimmunoassays (RIA), by direct or indirect solid-phase enzyme immunoassays (EIA), by surface plasmon resonance techniques (e.g., BIACORE), by flow cytometry, by fluorescence polarization (e.g., between a fluorescent peptide and the antibody to be tested), etc. For example, competitive ELISA methods involve using a purified antigen bound to a solid surface or cells, a test antibody bound to an unlabeled antigen, and a labeled reference antibody or antibody fragment. Typically, the reference antibody or antibody fragment is present at an unsaturated concentration (relative to its dissociation constant Kd for G protein α), and the signal is measured as the concentration of the test antibody or antibody fragment gradually increases. When an antibody is present in excess, it can block or inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55%-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of a reference antibody or antibody fragment to an antigen. In some cases, binding is inhibited by at least 80 to 85%, 85 to 90%, 90 to 95%, 95 to 97%, or 97% or more.

[0077] Specifically, antibodies or antibody fragments that competitively bind to G protein α against a reference antibody or antibody fragment are identified by HTRF assay or by fluorescence polarization assay, preferably by HTRF assay. When an antibody or antibody fragment competitively binds to G protein α against a reference antibody or antibody fragment, the antibody or antibody fragment can inhibit the HTRF signal generated by the reference antibody or antibody fragment. Conversely, antibodies or antibody fragments that do not competitively bind to G protein α against a reference antibody or antibody fragment cannot inhibit the HTRF signal generated by the reference antibody or antibody fragment.

[0078] The competitive antibody or antibody fragment according to the invention can be obtained, for example, by performing the procedure described in Example 1 using a reference antibody or antibody fragment. Antibody DSV38S is a competitive antibody according to the invention.

[0079] In one specific embodiment, antibody DSV38S and / or antibody DSV36S are excluded from the competitive antibodies or antibody fragments according to the invention.

[0080] The reference antibody or antibody fragment and the competitive antibody or antibody fragment as defined above are collectively referred to below as "antibody or antibody fragment according to the invention".

[0081] The antibodies or antibody fragments according to the invention can bind to isolated G protein α and / or G protein α present in a membrane environment. For example, the antibodies or antibody fragments according to the invention can bind to G protein α present in a membrane preparation carrying one or more RCPGs and one or more G protein αs. For antibodies according to the invention capable of binding G protein α, G protein α complexation with RCPG is not necessary.

[0082] The antibody or antibody fragment according to the invention can bind to G protein α with a dissociation constant (Kd) of less than or equal to 20 nM as measured by FRET. A dissociation constant below 20 nM is preferred for suitable RET performance. Advantageously, the antibody or antibody fragment according to the invention can bind to G protein α with a dissociation constant (Kd) of less than or equal to 20 nM as measured by FRET, for example, with an affinity constant of less than or equal to 10 nM, or less than or equal to 5 nM, for example, from 0 to 20 nM (excluding 0), from 0 to 10 nM (excluding 0), from 0 to 5 nM (excluding 0). The method for measuring the Kd of the antibody or antibody fragment according to the invention by FRET is described in Example 2.

[0083] The antibody or antibody fragment according to the present invention can be used in the RET method of labeling GTP using a member of the RET couple.

[0084] The antibodies or antibody fragments according to the invention are particularly advantageous for performing RET, especially FRET, and especially for detecting G protein α activation. Therefore, the antibodies or antibody fragments according to the invention can be conjugated to molecules that allow for their detection. Advantageously, the antibodies or antibody fragments according to the invention can be labeled with members of RET couples.

[0085] For example, the antibody or antibody fragment according to the present invention can be obtained by performing the operating procedure described in Example 1.

[0086] The inventors also demonstrate that the antibody or antibody fragment according to the invention binds to the SwitchII domain of G protein α, and more specifically to peptides 215-294 of G protein α.

[0087] Labeling of antibodies or antibody fragments according to the invention

[0088] The antibodies or antibody fragments according to the invention can be labeled directly or indirectly by methods familiar to those skilled in the art, such as those described below, but preferably the antibodies are labeled directly with members of the RET couple via covalent bonds.

[0089] The term "RET pair" refers to a pair consisting of an energy donor compound (hereinafter referred to as the "donor compound") and an energy acceptor compound (hereinafter referred to as the "acceptor compound"); these compounds emit RET signals when they are brought close together and excited at the excitation wavelength of the donor compound. It is well known that for two compounds to be RET pairs, the emission spectrum of the donor compound must partially overlap with the excitation spectrum of the acceptor compound. For example, when using a fluorescent donor compound and an acceptor compound, it is called a "FRET pair," or when using a bioluminescent donor compound and an acceptor compound, it is called a "BRET pair."

[0090] Antibodies or antibody fragments can be directly labeled using members of a RET pair (e.g., fluorescent compounds when FRET is used) based on reactive groups present on the antibody or antibody fragment, using conventional methods known to those skilled in the art. For example, the following reactive groups can be used: terminal amino groups, carboxylic acid groups of aspartic acid and glutamic acid, amine groups of lysine, guanidine groups of arginine, thiol groups of cysteine, phenolic groups of tyrosine, indole rings of tryptophan, thioether groups of methionine, and imidazole groups of histidine.

[0091] Reactive groups can form covalent bonds with reactive groups carried by antibodies or antibody fragments. Suitable reactive groups carried by antibodies or antibody fragments are well known to those skilled in the art; for example, donor or acceptor compounds functionalized with maleimide groups can, for instance, covalently bind to thiol groups carried by cysteine ​​residues carried by antibodies or antibody fragments. Similarly, donor / acceptor compounds carrying N-hydroxysuccinimide esters can covalently bind to amines present in antibodies or antibody fragments.

[0092] The antibodies or antibody fragments according to the invention can also be indirectly labeled with fluorescent or bioluminescent compounds, for example, by introducing an antibody or antibody fragment that is covalently bound to an acceptor / donor compound into a measurement medium, the second antibody or antibody fragment specifically recognizing the antibody or antibody fragment according to the invention.

[0093] Another common indirect labeling method involves immobilizing biotin onto the antibody or antibody fragment to be labeled, and then incubating the biotinylated antibody or antibody fragment in the presence of streptavidin labeled with an acceptor / donor compound. Suitable biotinylated antibodies or antibody fragments can be prepared using techniques familiar to those skilled in the art; fluorophores labeled streptavidin, for example, is sold by Cisbio Bioassay under the trade name "d2" (reference number 610SADLA).

[0094] In the context of this invention, the antibody or antibody fragment is labeled with (i) a fluorescent donor compound or a light-emitting donor compound, or (ii) a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher). Preferably, the antibody or antibody fragment is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher).

[0095] According to one embodiment of the invention, the antibody or antibody fragment is labeled with a fluorescent acceptor compound, for example, selected from allophycocyanin, rhodamine, anthocyanins, squaranes, coumarin, proflavin, acridine, fluorescein, boron-dipyrrole methylene derivatives, nitrobenzoxadiazole and quantum dots, GFP, GFP variants selected from GFP10, GFP2 and eGFP, YFP, YFP variants selected from eYFP, YFP topaz, YFP citrine, YFP venus and YPet, mOrange and DsRed.

[0096] According to another embodiment of the invention, the antibody or antibody fragment is labeled with a fluorescent donor compound, for example selected from: europium cavitary compounds, europium chelates, terbium chelates, terbium cavitary compounds, ruthenium chelates, quantum dots, allophycocyanin, rhodamine, anthocyanins, squaric acid cyanide, coumarin, proflavin, acridine, fluorescein, boron-dipyrrole methylene derivatives and nitrobenzidine, preferably selected from: europium cavitary compounds; europium chelates; terbium chelates; terbium cavitary compounds; ruthenium chelates; and quantum dots; particularly preferred are europium chelates and cavitary compounds and terbium chelates and cavitary compounds.

[0097] According to another embodiment of the invention, the antibody or antibody fragment is labeled with a luminescent donor compound, such as selected from luciferase (luc), Renilla luciférase (Rluc), a variant of Renilla luciférase (Rluc8), and firefly luciferase.

[0098] The notation methods and notation descriptions that can be used in the context of this invention are found in French patent application No. FR 1900880, filed on January 30, 2019, which is incorporated herein by reference.

[0099] Compositions of antibodies or antibody fragments

[0100] A third object of the present invention relates to compositions comprising the aforementioned antibodies or antibody fragments. The compositions according to the invention may further comprise a GTP source labeled with a member of the RET mating pair.

[0101] GTP labeled with members of RET couples is described in the “Reagent Kit” section below.

[0102] Kit

[0103] A fourth object of the present invention relates to a reagent kit (kit) comprising (i) an antibody or antibody fragment (as defined above, reference or competitive) according to the invention and (ii) a GTP source of a member marker (hereinafter referred to as “marked GTP”) of a RET couple.

[0104] GTP can be non-hydrolyzable or slowly hydrolyzable, such as GTPgammaS (GTPγS or GTPgS), GppNHp, and GppCp.

[0105] GTP can be labeled directly or indirectly. Preferably, GTP is directly labeled. Based on the reactive groups present on GTP, GTP can be directly labeled using a member of the RET pair (e.g., a fluorescent compound when FRET is used).

[0106] The reactive group can form a covalent bond with the reactive group carried by a member of the RET pair. Suitable reactive groups carried by members of the RET pair are well known to those skilled in the art; for example, donor or acceptor compounds functionalized with maleimide groups can covalently bind thiol groups. Similarly, donor / acceptor compounds carrying N-hydroxysuccinimide esters can covalently bind amines.

[0107] In the context of this invention, GTP is labeled with (i) a fluorescent donor compound, or with (ii) a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher). Preferably, GTP is labeled with a fluorescent donor compound.

[0108] In one specific embodiment, the labeled GTP is a non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent donor compound, said fluorescent donor compound being selected from GTPgN-C2 (GTP-γ-N-C2), GTPgN-C3 (GTP-γ-N-C3), GTPgN-octyl-C2 (GTP-γ-N-octyl-C2), and GTPgN-octyl-C11 (GTP-γ-N-octyl-C11). GTPgN-octyl-C3 (GTP-γ-N-octyl-C3), GTPgO-hexyl-C2 (GTP-γ-O-hexyl-C2), GTPgO-hexyl-C3 (GTP-γ-O-hexyl-C3) or GTP-gN-octyl-thiosuccinimide-C2 (GTP-γ-N-octyl-thiosuccinimide-C2), preferably GTP-gN-octyl-thiosuccinimide-C2.

[0109] According to the present invention, the labeled GTP is a non-hydrolyzable or slowly hydrolyzable GTP labeled with a fluorescent acceptor compound selected from GTPgN-octyl-Cy5, GTPgN-octyl-AF488, GTPgN-L15-fluorescein, GTPgO-connector-Cy5(P), or GTPgS-connector-Cy5(R). GTP can also be labeled with a non-fluorescent acceptor compound (quencher).

[0110] GTP-gN-octyl-thiosuccinimide-C2 is shown below.

[0111]

[0112] In one embodiment of the invention, GTP is labeled with a fluorescent donor compound, and the antibody or antibody fragment is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher). In another embodiment, GTP is labeled with a fluorescent acceptor compound or a non-fluorescent acceptor compound (quencher), and the antibody or antibody fragment is labeled with a fluorescent donor compound or a luminescent donor compound.

[0113] The other GTPs with the above-mentioned markings and their preparation methods are described in French patent application No. FR1900856, filed on January 30, 2019, which is incorporated herein by reference.

[0114] Advantageously, (i) is an antibody comprising a heavy chain variable domain consisting of the amino acid sequence SEQ ID NO: 14 and a light chain variable domain consisting of the amino acid sequence SEQ ID NO: 15 and (ii) is GTP-gN-octyl-thiosuccinimide-C2.

[0115] Other objects

[0116] A fifth object of the present invention relates to a nucleic acid sequence encoding an antibody or antibody fragment according to the present invention (reference or competing).

[0117] A sixth aspect of the invention relates to a vector comprising a nucleic acid sequence according to the invention. Any type of vector suitable for generating antibodies may be used in the context of this invention. Specifically, the vector is a recombinant vector. The vector will contain the nucleic acid sequence necessary for generating antibodies according to the invention, such as a promoter sequence, regulatory sequence, etc. Methods for preparing suitable vectors are widely described in the literature.

[0118] A seventh object of the present invention relates to cells comprising a vector according to the invention or a nucleic acid sequence according to the invention. Cells according to the invention can be obtained by methods widely described in the literature, such as by transfecting cell clones with a vector or nucleic acid sequence according to the invention. The invention is not limited to specific cell types. Any cell capable of producing antibodies can be used in the context of the invention. These cells can be eukaryotic cells (such as mammalian cells, such as human or mouse cells) or prokaryotic cells, such as bacteria or yeast.

[0119] The present invention will be further illustrated by the accompanying drawings and embodiments given below. However, these embodiments and drawings should not in any way be construed as limiting the scope of the invention. Attached Figure Description

[0120] Figure 1 This shows the FRET reaction protocol used to measure Kd of the anti-G protein αi1 antibody.

[0121] Figure 2 The graphs show the affinity of the d2-labeled antibodies SC13533, DSV36S, and DSV38S for human G protein αi1 (or protein Gai1). All antibodies exhibited highly significant HTRF signals in the presence and absence of nucleotides (GTPgS or GDP) in both the presence and absence of G protein αi1. These results demonstrate that antibodies DSV36S, DSV38S, and SC13533 can bind to G protein αi1. These graphs allow for the calculation of the affinity (Kd) of these different antibodies for protein Gαi1 (Table 1).

[0122] Figure 3 This curve illustrates the ability of unlabeled antibodies DSV36S, DSV38S, and SC13533 to inhibit the binding of antibody SC13533-d2 to human G protein α1. Logically, the unlabeled antibody SC13533 completely inhibited the HTRF signal obtained with antibody SC13533-d2. Conversely, antibodies DSV36S and DV38S failed to inhibit the signal generated by SC13533-d2. This indicates that these two antibodies do not bind to the same region of G protein α1 as antibody SC13533. In conclusion, antibodies DSV36S and DSV38S recognize different epitopes than antibody SC13533.

[0123] Figure 4The commercial antibodies sc-13533, sc-56536, and AM05302PU-N were shown to inhibit the binding of antibody DSV SC13533-d2 to human G protein i1. Logically, the unlabeled antibody DSV SC13533 completely inhibited the HTRF signal obtained with antibody SC13533-d2. Furthermore, antibodies sc-56536 and AM05302PU-N completely inhibited the HTRF signal obtained with antibody SC13533-d2. In conclusion, the commercial antibodies sc-13533, sc-56536, and AM05302PU-N are all non-competitive antibodies against antibody DSV 36S. Therefore, all of these antibodies recognize a different epitope than antibody DSV 36S.

[0124] Figure 5 The commercial antibodies sc-13533, sc-56536, and AM05302PU-N were shown to inhibit the binding of antibody DSV 3S-d2 (laboratory-produced) to human G protein i1. Logically, the unlabeled antibody DSV 3S completely inhibited the HTRF signal obtained with antibody DSV 3S-d2. Furthermore, antibodies sc-13533, sc-56536, and AM05302PU-N completely inhibited the HTRF signal obtained with antibody DSV 3S-d2. In summary, these results indicate that the commercial antibodies sc-13533, sc-56536, and AM05302PU-N are non-competitive antibodies against antibody DSV 3S. Therefore, the same applies to laboratory-produced antibody DSV 3S. Consequently, all of these antibodies recognize a different epitope from antibody DSV 36S.

[0125] Figure 6 This curve illustrates the ability of unlabeled antibodies DSV36S and DSV38S to inhibit the binding of antibody DSV36S-d2 to G protein αi1. Logically, the unlabeled antibody DSV36S completely inhibited the HTRF signal acquired by antibody DSV36S-d2. Antibody DSV38S also completely inhibited the signal generated by DSV36S-d2. This indicates that both antibodies bind to the same region of G protein αi1. In conclusion, antibodies DSV38S and DSV36S competitively bind to G protein αi.

[0126] Figure 7The study demonstrated the ability of commercial antibodies DSV 36S, DSV 26S, DSV 3S, and DSV 39S to inhibit the binding of antibody DSV36S-d2 to human G protein α1. Logically, the unlabeled antibody DSV36S completely inhibited the HTRF signal acquired by antibody DSV36S-d2. Conversely, antibodies DSV 26S, DSV 3S, and DSV 39S failed to inhibit the signal generated by DSV 36S-d2. This indicates that these three antibodies do not bind to the same region of human G protein α1 as antibody DSV 36S. In conclusion, all these antibodies recognize different epitopes from antibody DSV 36S.

[0127] Figure 8A and Figure 8B The FRET reaction protocol for measuring the selectivity of antibodies DSV36S, DSV38S, and SC13533 for different types of G protein α is shown.

[0128] Figure 9 This is a graph showing the HTRF signals of different types of G protein α obtained using a pair of antibodies, anti-Twin-Strep-tag-Lumi4 Tb and anti-FLAG-d2, which verifies that all G protein α encoded by the plasmid are indeed overexpressed in HEK293. In fact, the HTRF signals obtained with each G protein α are significantly greater than those obtained using negative conditions (“MOCK”), which correspond to HEK293 cells transfected with a control plasmid that does not encode any protein tag, FLAG+Twin-Strep-tag.

[0129] Figure 10 This chart illustrates the selectivity of antibodies DSV36S, DSV38S, and SC13533 for different types of G protein α. When proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz are overexpressed, antibodies DSV36S and DSV38S produce positive HTRF signals, but when proteins Gαs, Gαq, Gα12, and Gα13 are overexpressed, antibodies DSV36S and DSV38S do not produce HTRF signals. When proteins Gαi1 and Gαi3 are overexpressed, antibody SC13533 produces positive HTRF signals, but when proteins Gαi2, Gαo, Gαz, Gαs, Gαq, Gα12, and Gα13 are overexpressed, antibody SC13533 does not produce HTRF signals. These results confirm that antibodies DSV36S and DSV38S recognize the same epitopes on G protein α (antibodies DSV36S and DSV38S have the same selection profile and are therefore competitive). This epitope is different from the epitope recognized by antibody SC13533, which recognizes a smaller number of G protein αs.

[0130] Figure 11This graph illustrates the ability of antibodies DSV36S, DSV38S, and SC13533 to generate TR-FRET signals when combined with fluorescent GTP analogs (GTPgN-octyl-C2-europium cavitary compounds) in membrane preparations overexpressing RCPG. The condition "GTPgS" represents the nonspecific signal (NS) measured, where excess unlabeled GTPgS (100 μM) inhibits the binding of GTPgN-octyl-C2-europium cavitary compounds to G protein α, thus preventing the appearance of the FRET signal. The condition "Buffer" indicates that a moderate but significant FRET signal can be obtained between GTPgN-octyl-C2-europium cavitary compounds and d2-labeled antibodies DSV36S and DSV 38S. An increase in FRET signal was observed by adding SNC162 (which leads to the activation of the DOR receptor overexpressed in the membrane), which was associated with increased binding of the fluorescent GTP analog to some or all of the proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz recognized by the antibodies DSV36S and DSV38S. Conversely, although antibody SC13533 has the ability to bind proteins Gαi1 and Gαi3 (which are endogenously expressed in HEK293 cells [2]), it failed to give any FRET signal when combined with the GTPgN-octyl-C2-europium cavitary compound, regardless of the presence or absence of SNC162. These results demonstrate that only the antibody according to the invention can generate a FRET signal.

[0131] Figure 12The ability of antibodies DSV 36S, DSV 26S, DSV 3S, and DSV 39S to generate TR-FRET signals in membrane preparations overexpressing unactivated RCPG when combined with a fluorescent GTP analog (GTPgN-octyl-C2-europium cavitary compound). The condition "Non-specific signal" indicates a weak baseline FRET signal detected between the fluorescent GTP analog and the d2-labeled antibody. The condition "Negative control" represents the measured non-specific signal, where excess unlabeled GTPgS (100 μM) inhibited the binding of the fluorescent GTP analog to G protein α, thus preventing the appearance of the FRET signal. The measured inhibition was complete, as the HTRF signal levels measured under the "Non-specific signal" and "Negative control" conditions were nearly identical. An increase in FRET signal was measured after the addition of fluorescent GTP analogs and antibodies. This increase was associated with increased binding of the fluorescent GTP analogs to some or all of the proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz recognized by antibody DSV36S. Conversely, although antibodies DSV 26S, DSV 3S, and DSV 39S have the ability to bind to G protein αi in HEK293 cells, they do not produce any FRET signal when combined with the GTPgN-octyl-C2-europium cavitary compound. In summary, these results demonstrate that only the antibodies described according to the present invention can generate FRET signals.

[0132] Figure 13The results demonstrate the ability of antibodies DSV 36S and sc-13533 to generate TR-FRET signals when combined with a fluorescent GTP analog (GTPgO-linker-Cy5(P)) in membrane preparations overexpressing unactivated RCPG. The condition "Non-specific signal" indicates a weak baseline FRET signal detected between the fluorescent GTP analog and the terbium cavitation compound-labeled antibody. The condition "Negative control" represents the measured non-specific signal, where excess unlabeled GTPgS (100 μM) inhibited the binding of the fluorescent GTP analog to G protein α, thus preventing the appearance of the FRET signal. The measured inhibition was complete, as the HTRF signal levels measured under the "Non-specific signal" and "Negative control" conditions were nearly identical. An increase in FRET signal was measured by adding the fluorescent GTP analog and antibody, which was associated with increased binding of the fluorescent GTP analog to some or all of the proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz recognized by the antibody DSV36S. Conversely, although antibody sc-13533 has the ability to bind proteins Gαi1 and Gαi3 (which are endogenously expressed in HEK293 cells), it does not produce any FRET signal when combined with GTPgO-linker-Cy5(P). In summary, only the antibody described according to the present invention is capable of generating a FRET signal.

[0133] Figure 14A and Figure 14B The diagrams (Symbols 2A and 2B) show two FRETs that can be used with the antibodies according to the present invention.

[0134] Figure 15A and Figure 15B The detection pair used is: GTPgN-octyl-C2+DSV36S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A.

[0135] Figure 16A and Figure 16B The detection pair used is: GTPgN-octyl-C2+DSV36S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A.

[0136] Figure 17A and Figure 17B The detection pair used is: GTPgN-octyl-C2+DSV38S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A.

[0137] Figure 18A and Figure 18B The detection pair used is: GTPgN-octyl-C11+DSV36S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A.

[0138] Figure 19A and Figure 19B The detection pair used is: GTPgO-hexyl-C2+DSV36S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A.

[0139] Figure 20A and Figure 20B The detection pair used is GTPgN-C2+DSV36S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A.

[0140] Figure 21A and Figure 21B The detection pair used is: GTPgN-octyl-C2+DSV36S-d2, and the activation determination of RCPG dopamine D2S is performed according to Scheme 2A.

[0141] Figure 22A and Figure 22B The detection pair used is: GTPgN-octyl-C2+DSV36S-d2, and the activation determination of RCPG dopamine D2S is performed according to Scheme 2A.

[0142] Figure 23A and Figure 23B The assay shows the activation determination of RCPG dopamine D2S using the detection method for GTPgN-octyl-C2+DSV36S-d2 according to Scheme 2A.

[0143] Figure 24A and Figure 24B The detection pair used is: GTPgN-octyl-Cy5+DSV36S-Lumi4Tb, and the activation determination of Δopioid RCPG is performed according to Scheme 2B.

[0144] Figure 25A and Figure 25B The detection pair used is: GTPgN-octyl-AF488+DSV36S-Lumi4Tb. The activation of Δopioid RCPG was determined according to Scheme 2B.

[0145] Figure 26A and Figure 26B The detection pair used is: GTP-gN-octyl-thiosuccinimide-C2+DSV36S-d2, and the activation determination of Δopioid RCPG is performed according to Scheme 2A. Detailed Implementation

[0146] Example

[0147] Materials

[0148] Cell membrane preparations expressing Δ opioid receptors (DOR) were obtained from Euroscreen through service supply.

[0149] Antibodies DSV36S, DSV38S, DSV26S, DSV3S, and DSV39S were produced by Cisbio Bioassays and are available from Cisbio Bioassays as needed (under reference numbers DSV36S, DSV38S, DSV26S, DSV3S, and DSV39S, respectively). Antibody DSV36S comprises a heavy chain variable domain consisting of the amino acid sequence SEQ ID NO: 14 and a light chain variable domain consisting of the amino acid sequence SEQ ID NO: 15. The antibody was labeled with a compatible fluorescent probe for TR-FRET detection (targeting acceptor: Red-d2 or donor: Lumi4Tb).

[0150] Antibodies SC13533 and SC56536 were purchased from Santa Cruz Biotechnology (reference numbers SC13533 and SC56536). Antibody AM05302PU-N was purchased from Acris Antibodies GmbH (reference number AM05302PU-N).

[0151] Anti-Twin-Strep-tag antibodies were purchased from IBA Lifesciences (reference number 2-1517-001) and labeled with fluorescent probes compatible with TR-FRET detection (with Lumi4Tb as the donor).

[0152] Anti-FLAG-d2 antibodies are available from Cisbio Bioassays (reference number 61FG2DLF).

[0153] Nucleotides GDP and GTPγS were purchased from Sigma Aldrich (the respective catalog reference numbers are G7127 and G8634).

[0154] The agonist of opioid RCPG (SNC162) was purchased from Tocris Biosciences (reference number 1529).

[0155] The 384-well low-volume plates (white background) and black 96-well plates (black background) suitable for cell culture were purchased from GreinerBio One (catalog reference numbers 784075 and 665086, respectively).

[0156] The non-hydrolyzable / slowly hydrolyzable GTP analog labeled with the donor fluorophore (europium cavitary compound) (GTPgN-octyl-C2) was synthesized by Cisbio Bioassays. The non-hydrolyzable / slowly hydrolyzable GTP analog labeled with the acceptor fluorophore (Cy5) (GTPgO-linker-Cy5(P)) was purchased from Jena Bioscience (reference number NU-834-Cy5).

[0157] Human recombinant G protein αi1, fused to the N-terminus with a Twin-Strep-tag, was produced and purified by Cisbio Bioassays.

[0158] Plasmids encoding various human G proteins Gαi1, Gαi2, Gαi3, Gαo, Gαz, Gαs, Gαq, Gα12, and Gα13, whose N-terminal portions are fused with the tags Twin-Strep-tag and FLAG, were synthesized and amplified by Genecust (which provides the service).

[0159] HEK293 cells were purchased from ATCC.

[0160] The reagents and media used in the cell experiments, Opti-MEM media, Lipofectamine 2000 and polyornithine were purchased from Thermo Fisher Scientific (reference numbers 51985-026 and 11668-019) and Sigma-Aldrich (reference number P4957), respectively.

[0161] Methods

[0162] Reading of the FRET signal (HTRF technology)

[0163] The HTRF signal was measured on a Pherastar reader (BMG Labtech) with the following configuration:

[0164] • Module: HTRF (excitation 337nm, emission 665nm and 620nm)

[0165] • Excitation: Laser, flashing 40 times or lamp, flashing 100 times

[0166] • Read window: Latency: 60μs – Integration: 400μs.

[0167] Treatment of the HTRF signal

[0168] The HTRF ratio is calculated from the raw signals at 665nm and 620nm using the following formula:

[0169] HTRF ratio = signal at 665nm / signal at 620nm * 10000.

[0170] Example 1 : Operating protocol for obtaining anti-protein Gaii antibodies according to the invention

[0171] Immunization of mice

[0172] Recombinant TST-G protein αi1 (G protein αi1 of sequence UniProt P63096-1, tagged with TwinStreptag(TST)(IBA) at the N-terminus via a TEV linker) was generated in Sf9 insect cells (infected with a baculovirus encoding the protein), and then purified on an affinity column (Strep-Tactin Superflow high-capacity resin (IBA, catalog number: 2-1208-002)) tagged with TwinStreptag(TST).

[0173] BALB / c mice were immunized by injecting TST-G protein αi1, which had been pre-diluted in a buffer containing GTPgS (HEPES 20mM pH8, NaCl 100mM, MgCl2 3mM, CHAPS 11mM, GTPgS 100μM). Three booster injections were given at one-month intervals after the initial injection.

[0174] Fifteen days after each injection, blood samples collected from mice can verify the presence of an immune response.

[0175] Therefore, an ELISA-type assay was established. TST-G protein αi1, pre-diluted to 20 μg / mL in a buffer containing GTPgS (Tris HCl 20 mM pH 8.5, NaCl 140 mM, EDTA 2 mM, MgCl2 10 mM, BSA 0.1%, GTPgS 1 μM), was adsorbed onto a Strep- ELISA-type buffer using the TwinStreptag tag. XT (IBA, catalog number: 2-4101-001) 96-well plates. For this purpose, 100 μl of protein was added to each well, and the plates were incubated at 37°C for 2 hours, followed by washing three times in 1×PBS buffer containing 0.05% Tween 20.

[0176] Then, blood samples serially diluted in factors from 10 to 100 million were added at a rate of 100 μL / well and incubated at 37°C for 2 hours. Unfixed antibodies were removed by three washes in 1× PBS buffer containing 0.05% Tween 20. Fixed antibodies were then detected using a secondary anti-mouse Fc antibody bound to HRP (horseradish peroxidase) (Sigma#A0168, diluted 1 / 10,000 in PBS, 0.1% BSA). After incubation at 37°C for 1 hour, followed by three washes in 1× PBS buffer containing 0.05% Tween 20, and then incubation at room temperature with stirring for 20 minutes with substrate TMB (3,3',5,5'-tetramethylbenzidine, Sigma#T0440), HRP was detected colorimetrically at 450 nm.

[0177] To ensure that the antibodies detected by the ELISA assay were indeed targeting G protein αi1 and not the TwinStrepTag tag, the same blood sample was pre-incubated with an excess of another orthogonal protein tagged with TwinStrepTag (SNAPTag-TwinStrepTag) before being tested in the ELISA assay. Therefore, the anti-tag antibody immobilized on the tagged orthogonal protein and thus not on G protein αi1 attached to the bottom of the well; in this case, no HRP signal was detected or the HRP signal was weakened.

[0178] Mice with the best antibody titers and least signal attenuation in the anti-tag control case were selected for the next step of lymphocyte hybridization, also known as fusion. Mouse spleens were recovered, and a mixture of lymphocytes and plasma cells obtained from the spleen was fused in vitro with a myeloma cell line in the presence of a polyethylene glycol-based cell fusion catalyst. A mutant myeloma cell line lacking the enzyme HGPRT (hypoxanthine-guanosine phosphoribosyltransferase) was used to select hybrid cells (called hybridomas). These cells were cultured in a medium containing hypoxanthine, aminopterin (methotrexate), and thymine (HAT medium) to remove unfused myeloma cells, thus selecting the target hybridoma. Unfused spleen cells died because they could not proliferate in vitro. Therefore, only the hybridomas survived.

[0179] These hybridomas were then cultured in petri dishes. The supernatant from these hybridomas was then tested to assess their ability to produce antibodies against G protein αi1. For this purpose, an ELISA assay as described above was performed.

[0180] To evaluate the selectivity of the antibody with different forms of G protein αi1 (full form bound to GDP vs. full form bound to GTPgS vs. empty form), parallel assays were performed under conditions of pre-incubation of TST-G protein αi1 in buffer containing 1 μM GDP, or 1 μM GTPgS, or a nucleotide-free buffer. Then, the optimal hybridoma was cloned using a finite dilution procedure to obtain the hybridoma clone.

[0181] Then, the clone of the target hybridoma was injected into mice (intraperitoneal injection) to allow for the production of large amounts of antibodies in the ascites fluid.

[0182] The antibody was then purified by affinity chromatography on a column containing protein A.

[0183] Ability of the purified antibodies described above to compete with the antibody DSV 36S for binding to the G protein alpha

[0184] All reagents were diluted in TrisHCl 50 mM pH 7.4 buffer, MgCl2 10 mM, BSA 0.1%, and NaCl 10 mM. G protein αi1 was prepared at 2× to obtain a final concentration of 2.5 nM in wells. GTPgS nucleotides were prepared at 2× to obtain a final concentration of 10 μM in wells. Both reagents were prepared in the same solution, pre-incubated at room temperature for 30 minutes, and then dispensed into wells. The purified antibody was prepared at 4× to obtain a final concentration between 0.01 and 1 μM in wells. The DSV36S-d2 antibody was prepared at 4× to obtain a final concentration of 10 nM. The anti-Twin-Strep-tag-Lumi4 Tb antibody was prepared at 4× to obtain a final concentration of 0.5 nM in wells.

[0185] Dispense the reagents into the 384-well plate as follows:

[0186] 1. Place 10 μl of the pre-incubated mixture of G protein αi1 + GTPgS into each well.

[0187] 2. Add 5 μl of purified antibody to each well.

[0188] 3. Incubate the plate at room temperature for 30 minutes.

[0189] 4. Add 5 μl of a mixture of anti-Twin-Strep-tag-Lumi4 Tb antibody and antibody DSV 36S-d2 to each well.

[0190] Before reading the HTRF signal, incubate the board at room temperature for 1 hour.

[0191] The antibody according to the invention is able to inhibit the HTRF signal obtained by antibody DSV 36S-d2. Conversely, antibodies not according to the invention cannot inhibit the signal generated by DSV36S-d2.

[0192] Example 2: Determination of the affinity of the d2-labeled antibodies SC13533, DSV 36S, DSV38S for the G protein alpha i1

[0193] Experimental operating protocol

[0194] All reagents were diluted in buffer TrisHCl 50 mM pH 7.4, MgCl2 10 mM, BSA 0.1%, and NaCl 10 mM. G protein αi1 was prepared at 2× to obtain a final concentration of 2.5 nM in wells. GTPgS nucleotides were prepared at 2× to obtain a final concentration of 100 μM in wells. Both reagents were prepared in the same solution, pre-incubated at room temperature for 30 minutes, and then dispensed into wells. Antibodies DSV36S-d2, DSV38S-d2, and SC13533-d2 were prepared at 4× to obtain a final concentration between 0.01 and 10 nM in wells (depending on the antibody). Anti-Twin-Strep-tag-Lumi4 Tb antibody was prepared at 4× to obtain a final concentration of 0.25 nM in wells.

[0195] Dispense the reagents into the 384-well plate as follows:

[0196] 1) Place 10 μl of pre-incubated G protein αi1+ buffer alone, or a mixture of GTPgS or GDP into each well.

[0197] 2) Add 5 μl of d2-labeled antibody DSV36S, DSV38S, or SC13533 to each well.

[0198] 3) Add 5 μl of anti-Twin-Strep-tag-Lumi4 Tb antibody to each well.

[0199] Before reading the HTRF signal, incubate the board at room temperature for 24 hours.

[0200] Reaction scheme as follows Figure 1 As shown.

[0201] Results

[0202] Figure 2The results obtained with d2-labeled antibodies DSV36S, DSV38S, and SC13533 are shown. All these antibodies obtained very significant HTRF signals in the presence of G protein αi1, and in both the presence and absence of nucleotides (GTPgS or GDP). These results indicate that antibodies DSV36S, DSV38S, and SC13533 are capable of binding to G protein αi1.

[0203] Furthermore, the obtained antibody titration curves allowed for the calculation of the affinity (Kd) of these different antibodies for G protein αi1. This was performed using GraphPad Prism software by applying a "one-site specific binding" model to the HTRF data. The obtained Kd values ​​are shown in Table 1.

[0204] Table 1:

[0205]

[0206]

[0207] The Kd value indicates that all three antibodies have excellent affinity for G protein αi1, and the Kd value is between 0.1 and 1 nM, regardless of the protein state.

[0208] However, it can be noted that antibodies DSV36S and DSV38S exhibit higher signal strength and higher affinity when G protein αi1 binds to nucleotides (especially GTPgS). Conversely, antibody SC13533 shows similar signal strength and affinity regardless of the state of G protein αi1. This suggests that antibody SC13533 binds to G protein αi1 differently than antibodies DSV36S and DSV38S.

[0209] Example 3: Ability of the unlabeled antibodies DSV36S, DSV38S, SC 13533, SC56536 and AM05302PU-N to inhibit the binding of the antibody SC13533-d2 to the G protein i1 Experimental operating protocol

[0210] Results

[0211] All reagents were diluted in TrisHCl 50 mM pH 7.4 buffer, MgCl2 10 mM, BSA 0.1%, and NaCl 10 mM. G protein αi1 was prepared at 2× to obtain a final concentration of 2.5 nM in wells. GTPgS nucleotides were prepared at 2× to obtain a final concentration of 10 μM in wells. Both reagents were prepared in the same solution, pre-incubated at room temperature for 30 min, and then dispensed into wells. Cold antibodies DSV36S, DSV38S, and SC13533 were prepared at 4× to obtain final concentrations between 0.01 and 100 nM in wells. Antibody SC 13533-d2 was prepared at 4× to a final concentration of 10 nM. Anti-Twin-Strep-tag-Lumi4 Tb antibody was prepared at 4× to obtain a final concentration of 0.5 nM in wells.

[0212] Dispense the reagents into the 384-well plate as follows:

[0213] 1) Place 10 μl of the pre-incubated mixture of G protein αi1 + GTPgS into each well.

[0214] 2) Add 5 μl of unlabeled antibody DSV36S, DSV38S, SC56536, AM05302PU-N, or SC13533 to each well.

[0215] 3) Incubate the plate at room temperature for 30 minutes.

[0216] 4) Add 5 μl of anti-Twin-Strep-tag-Lumi4 Tb antibody and 5 μl of antibody SC13533-d2 to each well.

[0217] Before reading the HTRF signal, incubate the board at room temperature for 1 hour.

[0218] Figure 3

[0219] Figure 4 The results obtained using antibodies DSV 36S, DSV 38S, and SC 13533 are shown. Logically, the unlabeled antibody SC 13533 completely inhibited the HTRF signal obtained using antibody SC 13533-d2. Conversely, antibodies DSV36S and DSV38S failed to inhibit the signal generated by SC 13533-d2. This indicates that these two antibodies do not bind to the same region of G protein αi1 as antibody SC 13533. In conclusion, antibodies DSV36S and DSV38S recognize different epitopes than antibody SC 13533.

[0220] Figure 3The results obtained using antibodies SC13533, SC56536, and AM05302PU-N are shown. Logically, the unlabeled antibody DSV SC13533 completely inhibited the HTRF signal obtained using antibody SC13533-d2. Furthermore, antibodies SC56536 and AM05302PU-N also completely inhibited the HTRF signal obtained using antibody SC13533-d2. Since antibody SC13533 does not compete with antibodies DSV36S and DSV38S, and antibodies SC56536 and AM05302PU-N are able to inhibit the HTRF signal obtained using antibody SC13533-d2, antibodies SC56536 and AM05302PU-N are also non-competitive antibodies against antibody DSV 36S.

[0221] In short, Figure 4 and Example 4: Ability of the unlabeled antibodies DSV3S, SC56536, AM05302PU-N and SC 13533 to inhibit the binding of the antibody DSV3S-d2 to the G protein i1 The results shown indicate that the commercial antibodies sc-13533, sc-56536, and AM05302PU-N are all non-competitive antibodies against antibody DSV 36S. Therefore, all of these antibodies recognize a different epitope from antibody DSV 36S.

[0222] Experimental operating protocol Results

[0223] Figure 5

[0224] All reagents were diluted in buffer TrisHCl 50 mM pH 7.4, MgCl2 10 mM, BSA 0.1%, and NaCl 10 mM. G protein αi1 was prepared at 2× to obtain a final concentration of 2.5 nM in wells. GTPgS nucleotides were prepared at 2× to obtain a final concentration of 10 μM in wells. Both reagents were prepared in the same solution, pre-incubated at room temperature for 30 min, and then dispensed into wells. Cold antibodies DSV3S, SC56536, AM05302PU-N, and SC13533 were prepared at 4× to obtain final concentrations between 0.03 and 300 nM in wells. Antibody DSV36S-d2 was prepared at 4× to a final concentration of 10 nM. Anti-Twin-Strep-tag-Lumi4 Tb antibody was prepared at 4× to obtain a final concentration of 0.25 nM in wells.

[0225] Dispense the reagents into the 384-well plate as follows:

[0226] 1) Place 10 μl of the pre-incubated mixture of G protein αi1 + GTPgS into each well.

[0227] 2) Add 5 μl of unlabeled antibody DSV 3S, SC56536, SC13533, or AM05302PU-N to each well.

[0228] 3) Incubate the plate at room temperature for 30 minutes.

[0229] 4) Add 5 μl of anti-Twin-Strep-tag-Lumi4 Tb antibody and 5 μl of antibody DSV 3S-d2 to each well.

[0230] Before reading the HTRF signal, incubate the board at room temperature for 1 hour.

[0231] Example 5: Ability of the unlabeled antibodies DSV36S, DSV38S, DSV3S, DSV26S and DSV39S to inhibit the binding of the antibody DSV36S-d2 to the G protein alpha i1

[0232] Experimental operating protocol The results are shown. Logically, the unlabeled antibody DSV 3S completely inhibited the HTRF signal acquired by antibody DSV 3S-d2. Furthermore, antibodies sc-13533, sc-56536, and AM05302PU-N completely inhibited the HTRF signal acquired by antibody DSV3S-d2. In summary, these results, combined with those shown in Example 3, indicate that the commercial antibodies sc-13533, sc-56536, and AM05302PU-N are all non-competitive antibodies against antibody DSV 36S. Therefore, this also applies to laboratory-manufactured antibody DSV 3S. Thus, all these antibodies recognize a different epitope from antibody DSV 36S.

[0233] Results Figure 6

[0234] Figure 7

[0235] All reagents were diluted in buffer TrisHCl 50 mM pH 7.4, MgCl2 10 mM, BSA 0.1%, and NaCl 10 mM. G protein αi1 was prepared at 2× to obtain a final concentration of 2.5 nM in wells. GTPgS nucleotides were prepared at 2× to obtain a final concentration of 100 μM in wells. Both reagents were prepared in the same solution, pre-incubated at room temperature for 30 minutes, and then dispensed into wells. Cold antibodies DSV36S, DSV38S, DSV26S, DSV3S, and DSV39S were prepared at 4× to obtain a final concentration between 0.001 and 1 μM in wells. Antibody DSV36S-d2 was prepared at 4× to a final concentration of 10 nM. Anti-Twin-Strep-tag-Lumi4 Tb antibody was prepared at 4× to obtain a final concentration of 0.25 nM in wells.

[0236] Dispense the reagents into the 384-well plate as follows:

[0237] 1) Place 10 μl of the pre-incubated mixture of G protein αi1 + GTPgS into each well.

[0238] 2) Add 5 μl of unlabeled antibodies DSV36S, DSV38S, DSV26S, DSV3S, and DSV39S to each well.

[0239] 3) Incubate the plate at room temperature for 30 minutes.

[0240] 4) Add 5 μl of anti-Twin-Strep-tag-Lumi4 Tb antibody and 5 μl of antibody DSV36S-d2 to each well.

[0241] Before reading the HTRF signal, incubate the board at room temperature for 1 hour.

[0242] Example 6: Selectivity of the antibodies DSV36S, DSV38S and SC13533 for different types of G protein alpha when the G protein alpha is overexpressed in HEK293 cells

[0243] Day 1 The results obtained using antibodies DSV36S and DSV38S are shown. Logically, the unlabeled antibody DSV36S completely suppressed the HTRF signal obtained by antibody DSV36S-d2. Antibody DSV38S also completely suppressed the signal generated by DSV36S-d2. This indicates that both antibodies bind to the same region of G protein αi1. Day 2 The results obtained using antibodies DSV36S, DSV26S, DSV3S, and DSV39S are shown. Logically, the unlabeled antibody DSV36S completely inhibited the HTRF signal obtained by antibody DSV36S-d2. Conversely, antibodies DSV26S, DSV3S, and DSV39S failed to inhibit the signal generated by DSV36S-d2. This indicates that these three antibodies do not bind to the same region of human G protein αi1 as antibody DSV36S.

[0244] In summary, only antibody DSV38S and antibody DSV36S competitively bind to G protein αi, thus sharing the same epitope.

[0245] Figure 8A Figure 8B

[0246] Results HEK293 cells were transfected with plasmids encoding different proteins Gαi1, Gαi2, Gαi3, Gαo, Gαz, Gαs, Gαq, Gα12 and human Gα13.

[0247] A solution containing 1 million HEK cells / ml was prepared in OptiMEM medium. A plasmid / liposome mixture containing 150 ng / well of plasmid and 0.375 μl of liposomes was prepared in OptiMEM medium, and after 30 minutes, the plasmid / liposome mixture was added to a 96-well plate with black-bottomed wells.

[0248] Dispense the reagent into each well of a black microplate suitable for cell culture:

[0249] 1) Incubate 50 μl / well of polyornithine at room temperature for 30 minutes, and then extract the solution from each well by suction.

[0250] 2) Add 50 μl of HEK293 preparation (cell density of 50,000 cells per well) to each well.

[0251] 3) Add 50 μl of a mixture of plasmid and liposome encoding the G protein to each well.

[0252] The microplate was incubated at 37°C and 5% CO2 (in a regulated stove) for 24 hours.

[0253] Figure 9 HTRF assay for selective measurement of antibodies DSV36S, DSV38S, and SC 13533

[0254] All reagents were diluted in the following buffers: TrisHCl 50 mM pH 7.4, MgCl2 10 mM, BSA 0.1%, and Triton × 100 0.02%. Antibodies DSV36S-d2, DSV38S-d2, and SC13533-d2 were prepared at 4× to achieve a final concentration of 10 nM in the wells. Anti-Twin-Strep-tag-Lumi4 Tb antibody was prepared at 4× to achieve a final concentration of 0.5 nM in the wells. Nucleotides GDP and GTPgS were prepared at 4× to achieve a final concentration of 10 μM in the wells.

[0255] Dispense the reagents into the 384-well plate as follows:

[0256] 1) Remove the OptiMEM culture medium.

[0257] 2) Add 25 μl of buffer, or GDP or GTP to each well.

[0258] 3) Add 25 μl of buffer to each well.

[0259] 4) Add 25 μl of antibody DSV36S-d2, DSV38S-d2, SC13533-d2, or anti-FLAG-d2 to each well.

[0260] 5) Add 5 μl of anti-Twin-Strep-tag-Lumi4 Tb antibody to each well.

[0261] Before reading the HTRF signal, the microplate was incubated at room temperature for 20 hours.

[0262] Reaction scheme as follows Figure 10 and Example 7: Ability of the antibodies DSV36S, DSV38S, DSV 26S, DSV 39S, DSV 3S and SC13533 to generate a TR-FRET signal when bound to a fluorescent GTP analogue in membrane preparations with overexpressed RCPG As shown.

[0263] Figure 8A

[0264] Results and Figure 11 The results obtained in this experiment are shown.

[0265] Using a pair of antibodies, anti-Twin-Strep-tag-Lumi4 Tb and anti-FLAG-d2, it was verified that all G protein αs encoded by the plasmid were indeed overexpressed in HEK293 cells. In fact, the HTRF signal obtained with each of the G protein αs was significantly greater than the HTRF signal obtained using a negative condition (“MOCK”), corresponding to HEK293 cells transfected with a control plasmid that does not encode any protein tag, FLAG+Twin-Strep-tag.

[0266] Overexpression of all G protein α was validated, and the selectivity profiles of antibodies DSV36S, DSV38S, and SC 13533 could then be determined by measuring the possible FRET between these d2-labeled antibodies DSV36S, DSV38S, and SC 13533 and the anti-Twin-Strep-tag-Lumi4 Tb antibody. Antibodies DSV36S and DSV38S gave positive HTRF signals when proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz were overexpressed, but did not produce HTRF signals when proteins Gαs, Gαq, Gα12, and Gα13 were overexpressed. When proteins Gαi1 and Gαi3 are overexpressed, antibody SC13533 produces a positive HTRF signal, but when proteins Gαi2, Gαo, Gαz, Gαs, Gαq, Gα12, and Gα13 are overexpressed, antibody SC13533 does not produce an HTRF signal. These results confirm that antibodies DSV36S and DSV38S recognize the same epitope on G protein α (antibodies DSV36S and DSV38S have the same selection profile and are competitive). This epitope is different from the epitope recognized by antibody SC13533, which recognizes a smaller number of G protein αs.

[0267] Figure 12Figure 13

[0268] All reagents were diluted in the following buffers: TrisHCl 50 mM pH 7.4, MgCl2 10 mM, BSA 0.1%, NaCl 300 mM, and GDP 0.5 μM. HEK293 membranes expressing the DOR receptor were prepared at 4× to a final volume of 10 μg in the wells. Antibodies DSV36S-d2, DSV38S-d2, SC13533-d2, and anti-FLAG-d2 were prepared at 4× to a final concentration of 10 nM in the wells. GTPgN-octyl-C2 labeled with a europium cavitation compound was prepared at 4× to obtain a final concentration of 6 nM in the wells. GTPgO-linker-Cy5 was prepared at 4× to obtain a final concentration of 50 nM in the wells. Nucleotides GTPgS and GDP were prepared at 4× to obtain a final concentration of 100 μM in the wells. SNC 162 was prepared at 4× to obtain a final concentration of 10 μM in the wells.

[0269] Dispense the reagents into the 384-well plate as follows:

[0270] 1) Add 5 μl of DOR membrane to each well.

[0271] 2) Add 5 μl of GTPgN-octyl-C2 labeled with europium cavitation compound, or 5 μl of GTPgO-connector-Cy5, to each well.

[0272] 3) Add 5 μl of d2-labeled antibody DSV36S, or DSV38S, or SC13533, or DSV 26S, or DSV3S, or DSV 39S, or DSV 36S or SC13533 labeled with a terbium cavitation compound to each well.

[0273] 4) Add 5 μl of buffer or SNC 162 or GTPgS to each well.

[0274] Before reading the HTRF signal, incubate the board at room temperature for 20 hours.

[0275] Reaction scheme as follows Example 8: Measurement of the activation of RCPG by FRET using labeled antibodies according to the invention As shown.

[0276] Materials

[0277] Cell linesThe results are shown when the analogue GTPgN-octyl-C2-europium cavitary compound is combined with antibodies DSV36S-d2, DSV38S-d2, and SC13533-d2. The condition "GTPgS" represents the nonspecific signal (NS) measured, where excess unlabeled GTPgS (100 μM) inhibits the binding of GTPgN-octyl-C2-europium cavitary compound to G protein α, thus preventing the appearance of the FRET signal. The condition "Buffer" indicates that a moderate but significant FRET signal can be obtained between GTPgN-octyl-C2-europium cavitary compound and antibodies DSV36S and DSV 38S labeled with d2. An increase in FRET signal was observed by adding SNC162 (which leads to activation of the overexpressed DOR receptor in the membrane), which was associated with increased binding of the fluorescent GTP analog to some or all of the proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz recognized by the antibodies DSV36S and DSV38S. Conversely, although antibody SC13533 has the ability to bind proteins Gαi1 and Gαi3 (which are endogenously expressed in HEK293 cells [2]), no FRET signal was generated when antibody SC13533 was combined with the GTPgN-octyl-C2-europium cavitary compound, regardless of the presence or absence of SNC162.

[0278] same, Supplier The results are shown for membrane preparations overexpressing unactivated RCPG, obtained by combining the analog GTPgN-octyl-C2-europium cavitary compound with antibodies DSV36S-d2, DSV26S-d2, DSV39S-d2, and DSV3S-d2. The condition "Non-specific signal" indicates a weak baseline FRET signal detected between the fluorescent GTP analog and the antibody labeled with d2. The condition "Negative control" represents the measured non-specific signal, where excess unlabeled GTPgS (100 μM) inhibited the binding of the fluorescent GTP analog to G protein α, thus preventing the appearance of the FRET signal. The measured inhibition was complete, as the HTRF signal levels measured under the "Non-specific signal" and "Negative control" conditions were almost identical. After the addition of fluorescent GTP analogs and antibodies, an increase in FRET signal was measured, which was associated with an increase in the binding of the fluorescent GTP analogs to some or all of the proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz recognized by antibody DSV36S. Conversely, although antibodies DSV26S, DSV3S, and DSV39S have the ability to bind to G protein αi in HEK293 cells[2], antibodies DSV26S, DSV3S, and DSV39S do not produce any FRET signal when combined with GTPgN-octyl-C2-europium cavitary compound.

[0279] Reference number The results are shown for membrane preparations overexpressing unactivated RCPG, obtained by combining the analog GTPgO-linker-Cy5 with antibodies DSV36S-Tb and SC13533-Tb. The condition "Non-specific signal" indicates a weak baseline FRET signal detected between the fluorescent GTP analog and the antibody labeled with a terbium cavitation compound. The condition "Negative control" represents the measured non-specific signal, where excess unlabeled GTPgS (100 μM) inhibited the binding of the fluorescent GTP analog to G protein α, thus preventing the appearance of the FRET signal. The measured inhibition was complete, as the HTRF signal levels measured under "Non-specific signal" and "Negative control" conditions were nearly identical. An increase in FRET signal was measured by adding the fluorescent GTP analog and antibody, which was associated with increased binding of the fluorescent GTP analog to some or all of the proteins Gαi1, Gαi2, Gαi3, Gαo, and Gαz recognized by the antibody DSV36S. Conversely, although the antibody sc-13533 has the ability to bind proteins Gαi1 and Gαi3 (which are endogenously expressed in HEK293 cells[2]), the antibody sc-13533 does not produce any FRET signal when combined with GTPgO-linker-Cy5(P).

[0280] In summary, these results demonstrate that only the antibody according to the present invention can generate the FRET signal.

[0281] Delta opioid

[0282] HEK293

[0283] Cell membrane preparations for expressing the receptor and G protein αi in the study were purchased from Perkin Elmer or Euroscreen. The table below lists the base cells and reference numbers for the different samples used:

[0284] Table 2:

[0285] Perkin Elmer 6110549400UA Delta opioid CHO-K1 Euroscreen Services Dopamine D2S CHO-K1 Euroscreen Services Methods Assay scheme Figure 14A Figure 14B Activation assay on the delta opioid RCPG (DOR) according to scheme 2A: increase in the TR-FRET signal between the donor GTP and the acceptor anti-G protein alpha i antibody under the stimulation of an agonist

[0286] The antibody DSV36S was labeled using a fluorescent probe compatible with TR-FRET detection (d2 for the red acceptor or Lumi4 Tb for the donor).

[0287] Nucleotides GTP, GDP and GTPγS were purchased from Sigma Aldrich (their respective catalog reference numbers are G8877, G7127 and G8634).

[0288] The agonist of opioid RCPG (SNC162), the agonist of dopamine D2S (PPHT), and the antagonist of opioid RCPG (Naltrindole) were purchased from Tocris (respective catalog reference numbers 1529 and 0740, respectively).

[0289] 384-well low-capacity plate, white background, purchased from Greiner Bio One (catalog reference number 784075).

[0290] Non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with donor or acceptor fluorophores (GTPgN-C2; GTPgN-C3; GTPgN-octyl-C2; GTPgN-octyl-C11; GTPgN-octyl-C3; GTPgO-hexyl-C2; GTPgO-hexyl-C3; GTP-gN-octyl-thiosuccinimide-C2; GTPgN-octyl-Cy5; GTPgN-octyl-AF488) were synthesized by CisbioBioassays.

[0291] Non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with the acceptor fluorophores GTPgO-connector-Cy5(P) and GTPgS-connector-Cy5(R) were purchased from Jena Bioscience, reference numbers NU-834-CY5 and NU-1610-CY5, respectively.

[0292] Figure 15A

[0293] Reagent preparation:

[0294] All reagents were diluted in the following buffers: TrisHCl 50 mM pH 7.4, MgCl2 10 mM, BSA 0.1%, NaCl 10 mM, 100 mM, 300 mM, or 500 mM (concentrations specified in the legend of each figure), and 0, 0.5, or 1 μM GDP (concentrations specified in the legend of each figure). Membranes were prepared at 4× to dispense 1 μg or 10 μg / well (amounts specified in the legend of each figure). GTPgS nucleotides were prepared at 6.67× (condition: nonspecific signal) to obtain a final concentration of 100 μM in the wells. Test compounds (agonists or antagonists) were prepared at 10× to obtain the final concentrations in the wells shown in the graphs. Anti-Gαi antibodies used for detection were prepared in 4× form for the following final concentrations in the wells: antibody DSV36S-d2 (10 nM); antibody DSV36S-Lumi4 Tb (0.5 nM or 1 nM); antibody DSV38S-d2 (10 nM). Non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with donor or acceptor fluorescent probes were prepared in 4× form to obtain the final concentrations in the wells as shown in the legend of each figure.

[0295] Reagent distribution in a 384-well plate:

[0296] • Membranes expressing RCPG and G protein αi: 5 μL

[0297] • Buffer or nucleotide GTPgS (for non-specific signal conditions): 3 μL

[0298] • Non-hydrolyzable / slowly hydrolyzable GTP analogs – donor or acceptor: 5 μL

[0299] • Anti-Gαi antibody - donor or acceptor: 5 μL

[0300] • Buffer solution or test compound (agonist and / or antagonist): 2 μL.

[0301] Non-specific signals (fluorescent background noise) were measured using wells containing excess GTPgS (100 μM).

[0302] Read HTRF signal

[0303] The plate was incubated at 21°C for 20 hours (unless otherwise indicated in the figure), and then the HTRF signal was measured on a PHERAstar reader (BMG Labtech) configured as follows:

[0304] • Module: HTRF (excitation 337nm, emission 665nm and 620nm)

[0305] • Excitation: Laser, flashing 40 times or lamp, flashing 100 times

[0306] • Read window: Latency: 60μs – Integration: 400μs.

[0307] Signal processing

[0308] The HTRF ratio is calculated from the raw signal at 665 nm (for red receptors - Cy5) or 520 nm (for green receptors - AF488 or fluorescein) and 620 nm according to the following formula:

[0309] HTRF ratio = (signal at 665nm or signal at 520nm) / (signal at 620nm) * 10000.

[0310] Figure 16A

[0311] Figure 17AThe diagram illustrates the assay principle using non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with donor RET mates and anti-G protein α antibodies labeled with acceptor RET mates, wherein RCPG is activated with an agonist compound, which induces increased binding of the donor GTP analog to the G protein, thus increasing the RET signal (Symbol 2A).

[0312] ​ The diagram illustrates the assay principle using non-hydrolyzable / slowly hydrolyzable GTP analogs labeled with the acceptor RET mate and anti-G protein α antibodies labeled with the donor RET mate, wherein RCPG is activated with an agonist compound, which induces an increase in the binding of the acceptor GTP analog to the G protein, thus increasing the RET signal (Symbol 2B).

[0313] ​ ​

[0314] First, using CHO-K1 cell membrane preparations expressing Δ opioid RCPG and G protein αi, the ability of donor GTP / receptor anti-Gαi antibodies to generate specific TR-FRET signaling via G protein binding was demonstrated. The following experimental conditions were used:

[0315] - ​ : GTPgN-octyl-C2 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 500 mM; BSA 0.1%.

[0316] - ​ : GTPgN-octyl-C2 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%.

[0317] - ​ : GTPgN-octyl-C2 (final concentration in wells: 6 nM); DSV38S-d2 (final concentration in wells: 10 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%.

[0318] - Figure 18A: GTPgN-octyl-C11 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%.

[0319] - Figure 19A : GTPgN-hexyl-C2 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%.

[0320] - Figure 20A : GTPgN-C2 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO membrane-DOR / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%.

[0321] The membrane was incubated with or without a large amount of excess GTPgS (100 μM). The difference in TR-FRET signal observed under these two conditions (HTRF ratio) indicates that, together with the receptor anti-Gαi antibody, the analogues GTPgN-octyl-C2, GTPgN-octyl-C11, GTPgO-hexyl-C2, and GTPgN-C2 can bind to G protein αi and generate a TR-FRET signal. Figures 16A-20A ).

[0322] Secondly, using the same membrane and experimental conditions as described above, the ability of RCPG agonists to regulate the ratio of G protein α bound to donor GTP was tested. Increased TR-FRET signaling (HTRF ratio) generated by agonist stimulation indicated an increase in the proportion of G protein α bound to donor GTP (i.e., a decrease in the empty form of G protein α). Therefore, RCPG receptors activated by RCPG agonists lead to donor GTP binding to G proteins, followed by G protein conversion to the donor GTP form, resulting in increased TR-FRET signaling. These results show… Figure 15B , Figure 16B , Figure 17B , Figure 18B , Figure 19B and Figure 20B In addition,Figure 16B The second condition was shown, in which activation by a fixed concentration of the RCPG agonist SNC162 (200 nM) was inhibited with increasing concentration of the RCPG antagonist (naltrexedole). This inhibition of activation was observed in the decrease of the TR-FRET signal (HTRF ratio).

[0323] Activation assay on RCPG Dopamine D2S (D2S) according to Figure 2A: increase in TR-FRET signal between donor GTP and acceptor anti-G protein alpha i antibody upon stimulation with agonist Figure 21A

[0324] First, using CHO-K1 cell membrane preparations expressing RCPG dopamine D2S and G protein αi, the ability of donor GTP / receptor anti-Gαi antibodies to generate specific TR-FRET signaling via G protein binding was demonstrated. The following experimental conditions were used:

[0325] - Figure 22A : GTPgN-octyl-C2 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-D2S membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 10 mM; GDP 1 μM; BSA 0.1%.

[0326] - Figure 23A : GTPgN-octyl-C2 (final concentration in wells: 6 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-D2S membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 100 mM; BSA 0.1%.

[0327] - Figures 21A-23A : GTPgN-octyl-C2 (finally 6 nM in wells); DSV36S-d2 (finally 10 nM in wells); 10 μg CHO-D2S membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 100 mM; GDP 1 μM; BSA 0.1%.

[0328] The membrane was incubated with or without a large excess of GTPgS (100 μM). The difference in TR-FRET signal (HTRF ratio) observed under these two conditions indicates that the analog GTPgN-octyl-C2, together with the receptor anti-Gαi antibody, can bind to G protein αi and generate a TR-FRET signal. Figure 21B ).

[0329] Secondly, using the same membrane and experimental conditions as described above, the ability of RCPG agonists to regulate the ratio of G protein α bound to donor GTP was tested. Increased TR-FRET signaling (HTRF ratio) generated by agonist stimulation indicated an increase in the proportion of G protein α bound to donor GTP (i.e., a decrease in the empty form of G protein α). Therefore, RCPG receptor activation by RCPG agonists leads to donor GTP binding to G protein, followed by G protein conversion to the donor GTP form, resulting in increased TR-FRET signaling. These results show… Figure 22B , Figure 23B and Activation assay on Delta Opioid RCPG (DOR) according to Figure 2B: increase in TR-FRET signal between GTP acceptor and donor anti-G protein alpha i antibody upon stimulation with agonist middle.

[0330] Figure 24A Figure 25A

[0331] First, using CHO-K1 cell membrane preparations expressing Δopioid RCPG and G protein αi, the ability of acceptor GTP / donor anti-Gαi antibody to generate specific TR-FRET signaling via G protein binding was demonstrated. The following experimental conditions were used:

[0332] - Figures 24A-25A : GTPgN-octyl-Cy5 (final concentration in wells: 50 nM); DSV36S-Lumi4Tb (final concentration in wells: 1 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%. Read the values ​​after incubation at 21 °C for 3 hours.

[0333] - Figure 24B : GTPgN-octyl-AF488 (final concentration in wells: 50 nM); DSV36S-Lumi4Tb (final concentration in wells: 1 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 10 mM; NaCl 300 mM; GDP 0.5 μM; BSA 0.1%. Read the values ​​after incubation at 21 °C for 3 hours.

[0334] The membrane was incubated with or without a large excess of GTPgS (100 μM). The difference in TR-FRET signal (HTRF ratio) observed under these two conditions indicates that the analogues GTPgN-octyl-Cy5 and GTPgN-octyl-AF488, together with the donor anti-Gαi antibody, can bind to G protein αi and generate a TR-FRET signal. Figure 25B ).

[0335] Secondly, using the same membrane and experimental conditions as described above, the ability of RCPG agonists to regulate the ratio of G protein α bound to receptor GTP was tested. Increased TR-FRET signaling (HTRF ratio) generated by agonist stimulation indicated an increase in the proportion of G protein α bound to receptor GTP (i.e., a decrease in the empty form of G protein α). Therefore, RCPG receptor activation by RCPG agonists leads to receptor GTP binding to G protein, followed by G protein conversion to receptor GTP form, resulting in increased TR-FRET signaling. These results show… Activation assay on Delta Opioid RCPG (DOR) according to Figure 2A: increase in TR-FRET signal between donor GTP and acceptor anti-G protein alpha i antibody upon stimulation with agonist and Figure 26A .

[0336] Figure 26A Figure 26B

[0337] First, using CHO-K1 cell membrane preparations expressing Δ opioid RCPG and G protein αi, the ability of donor GTP / receptor anti-Gαi antibodies to generate specific TR-FRET signaling via G protein binding was demonstrated. The following experimental conditions were used:

[0338] - ​ : GTP-gN-octyl-thiosuccinimide-C2 (final concentration in wells: 7.5 nM); DSV36S-d2 (final concentration in wells: 10 nM); 10 μg CHO-DOR membrane / well; buffer: TrisHCl 50 mM pH 7.4; MgCl2 60 mM; NaCl 150 mM; BSA 0.1%.

[0339] The membrane was incubated with or without a large excess of GTPgS (100 μM). The difference in TR-FRET signal observed under these two conditions (HTRF ratio) indicates that the analog GTP-gN-octyl-thiosuccinimide-C2, together with the receptor anti-Gαi antibody, can bind to G protein αi and generate a TR-FRET signal. ​ ).

[0340] Secondly, using the same membrane and experimental conditions as described above, the ability of RCPG agonists to regulate the ratio of G protein α bound to donor GTP was tested. An increase in the TR-FRET signal (HTRF ratio) generated by agonist stimulation indicated an increase in the proportion of G protein α bound to donor GTP (i.e., a decrease in the empty form of G protein α). Therefore, RCPG receptors activated by RCPG agonists lead to donor GTP binding to G proteins, followed by G protein conversion to the donor GTP form, resulting in increased TR-FRET signaling. These results are as follows: ​ As shown.

[0341] sequence list

[0342] Table 3

[0343]

[0344] References

[0345] [1] Damien Maurel. APPLICATIONS OF TECHNOLOGIES IN FRET AND TECHNOLOGY GABAB.Biologie Cellulaire.Université Montpellier I,2006.

[0346] [2]Atwood et al., BMC Genomics, 2011, 12:14. sequence list <110> CISBIO Biotesting Company <120> Anti-G protein α antibody <130> 1H305110 0037 <150> FR2000923 <151> 2020-01-30 <160> 15 <170> BiSSAP 1.3.6 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH-CDR1 <400> 1 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial sequence <220> <223> VH-CDR2 <400> 2 Ile Asp Pro Glu Asn Gly Asn Thr 1 5 <210> 3 <211> 14 <212> PRT <213> Artificial sequence <220> <223> VH-CDR3 <400> 3 Thr Arg Gly Gly Gly Tyr Tyr Ser Asp Trp Tyr Phe Asp Val 1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VL-CDR1 <400> 4 Ser Ser Val Ser Tyr 1 5 <210> 5 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VL-CDR3 <400> 5 Gln Gln Trp Ser Ser Asn Pro Pro Ile Thr 1 5 10 <210> 6 <211> 25 <212> PRT <213> Artificial sequence <220> <223> VH-FR1 <400> 6 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Leu Val Lys Leu Ser Cys Lys Ala Ser 20 25 <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH-FR2 <400> 7 Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Trp <210> 8 <211> 38 <212> PRT <213> Artificial sequence <220> <223> VH-FR3 <400> 8 Ile Tyr Asp Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr 1 5 10 15 Ser Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 9 <211> 11 <212> PRT <213> Artificial sequence <220> <223> VH-FR4 <400> 9 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 10 <211> 26 <212> PRT <213> Artificial sequence <220> <223> VL-FR1 <400> 10 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser 20 25 <210> 11 <211> 17 <212> PRT <213> artificial sequence <220> <223> VL‑FR2 <400> 11 Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile 1 5 10 15 Taurus <210> 12 <211> 36 <212> PRT <213> artificial sequence <220> <223> VL‑FR3 <400> 12 Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 13 <211> 10 <212> PRT <213> artificial sequence <220> <223> VL‑FR4 <400> 13 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 1 5 10 <210> 14 <211> 121 <212> PRT <213> Artificial sequence <220> <223> Heavy chain variable domain (VH) <400> 14 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Ile Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Gly Gly Tyr Tyr Ser Asp Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 15 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Light chain variable domain (VL) <400> 15 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Pro Ile 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105

Claims

1. An antibody or antibody fragment capable of binding to G protein α, comprising: - Heavy chain variable domain containing CDR1 of amino acid sequence SEQ ID NO: 1, CDR2 of amino acid sequence SEQ ID NO: 2, and CDR3 of amino acid sequence SEQ ID NO: 3; and - Light chain variable domain containing CDR1 of amino acid sequence SEQ ID NO: 4, CDR2 of amino acid sequence DTS and CDR3 of amino acid sequence SEQ ID NO:

5.

2. The antibody or antibody fragment according to claim 1, wherein: -The heavy chain variable domain comprises: FR1, which has at least 80% homology with the amino acid sequence SEQ ID NO: 6; FR2, which has at least 80% homology with the amino acid sequence SEQ ID NO: 7; FR3, which has at least 80% homology with the amino acid sequence SEQ ID NO: 8; and FR4, which has at least 80% homology with the amino acid sequence SEQ ID NO: 9; and - The light chain variable domain includes: FR1, which has at least 80% homology with the amino acid sequence SEQ ID NO: 10; FR2, which has at least 80% homology with the amino acid sequence SEQ ID NO: 11; FR3, which has at least 80% homology with the amino acid sequence SEQ ID NO: 12; and FR4, which has at least 80% homology with the amino acid sequence SEQ ID NO:

13.

3. The antibody or antibody fragment according to claim 1 or claim 2, wherein: -The heavy chain variable domain has at least 80% homology with the amino acid sequence SEQ ID NO: 14; - The light chain variable domain has at least 80% homology with the amino acid sequence SEQ ID NO: 15; and - The heavy chain variable domain CDR1 is composed of the amino acid sequence SEQ ID NO: 1, the heavy chain variable domain CDR2 is composed of the amino acid sequence SEQ ID NO: 2, the heavy chain variable domain CDR3 is composed of the amino acid sequence SEQ ID NO: 3, the light chain variable domain CDR1 is composed of the amino acid sequence SEQ ID NO: 4, the light chain variable domain CDR2 is composed of the amino acid sequence DTS, and the light chain variable domain CDR3 is composed of the amino acid sequence SEQ ID NO:

5.

4. The antibody or antibody fragment according to claim 1, wherein the heavy chain variable domain is composed of the amino acid sequence SEQ ID NO: 14 and the light chain variable domain is composed of the amino acid sequence SEQ ID NO:

15.

5. The antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment is labeled with a member of a RET pair.

6. The antibody or antibody fragment of claim 5, wherein the member of the RET mating pair is: i) Fluorescent donor compounds or light-emitting donor compounds, Or ii) fluorescent acceptor compounds or non-fluorescent acceptor compounds.

7. The antibody or antibody fragment according to claim 5 or claim 6, wherein: - The RET pair members are fluorescent acceptor compounds selected from allophycocyanin, rhodamine, anthocyanins, squaringin, coumarin, proflavin, acridine, fluorescein, boron-dipyrrole methylene derivatives, nitrobenzidine and quantum dots, GFP and YFP; or - The RET pair members are fluorescent donor compounds selected from: europium cavitary compounds, europium chelates, terbium chelates, terbium cavitary compounds, ruthenium chelates, quantum dots, allophycocyanin, rhodamine, anthocyanins, squaric acid cyanide, coumarin, proflavin, acridine, fluorescein, boron-dipyrrole methylene derivatives, and nitrobenzidine; or - The RET couple members are luminescent donor compounds selected from: luciferase (luc), renal luciferase (Rluc), a variant of renal luciferase (Rluc8), and firefly luciferase.

8. A composition comprising the antibody or antibody fragment of any one of claims 1 to 7.

9. A nucleic acid encoding an antibody or antibody fragment according to any one of claims 1 to 4.

10. A vector comprising the nucleic acid of claim 9.

11. A cell comprising the vector of claim 10 or the nucleic acid of claim 9.

12. A kit comprising (i) an antibody or antibody fragment of any one of claims 1 to 7, or a composition of claim 8, and (ii) a source of GTP, wherein the GTP is a non-hydrolyzable or slowly hydrolyzable GTP selected from member-marked GTP gammaS, GppNHp, and GppCp of RET couples.

13. The kit of claim 12, wherein the labeled GTP is a non-hydrolyzable or slowly hydrolyzable GTP labeled with the following: -Fluorescent donor compounds; or - Fluorescent acceptor compounds or non-fluorescent acceptor compounds.

14. The kit according to claim 13, wherein the fluorescent donor compound is selected from GTPgN-C2, GTPgN-C3, GTPgN-octyl-C2, GTPgN-octyl-C11, GTPgN-octyl-C3, GTPgO-hexyl-C2, GTPgO-hexyl-C3 or GTP-gN-octyl-thiosuccinimide-C2.

15. The kit according to claim 13, wherein the fluorescent donor compound is GTP-gN-octyl-thiosuccinimide-C2.

16. The kit according to claim 13, wherein the fluorescent acceptor compound is selected from GTPgN-octyl-Cy5, GTPgN-octyl-AF488, GTPgN-L15-fluorescein, GTPgO-connector-Cy5(P), or GTPgS-connector-Cy5(R), wherein the GTPgO-connector-Cy5(P) is purchased from Jena Bioscience, reference number NU-834-Cy5, and wherein the GTPgS-connector-Cy5(R) is purchased from Jena Bioscience, reference number NU-1610-CY5.

17. The kit of claim 13, wherein the non-fluorescent acceptor compound is a quencher.

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