Luciferase fragment system with universal antibody and protein adapters for homogeneous detection of substances in a sample or molecular interaction assays

Abstract

New protein adapters and their fusions with fragments of the reporter enzyme luciferase (FLuc) are disclosed, as well as methods for determining the presence of a detected substance in a test sample and for studying interactions between two molecules.

Description

[0001] Luciferase Fragment System with Universal Antibody and Protein Adapters for Homogeneous Detection of Substances in a Sample or Molecular Interaction Assays

[0002] The invention relates to novel protein adapters and their fusions with fragments of the reporter enzyme luciferase FLuc, as well as methods (configurations of the proprietary ECHIA assay) for determining the presence of a target substance in a tested sample, and for studying the interaction of two molecules.

[0003] The invention may find application in broadly understood laboratory diagnostics, particularly in medical or research settings, utilizing, for example, the measurement of light produced in an enzymatic bioluminescence reaction to determine the presence and / or concentration of the target substance in the tested sample. This can be implemented either as a standalone reagent kit for diagnostic laboratories or as reagents forming an integral part of cartridges for portable POCT diagnostic devices. The invention may also be applied in biotechnology and pharmaceutical laboratories for the detection of cytokines and other biomarkers in cell cultures, in tested biological samples, or in screening for new drugs.

[0004] State of the Art

[0005] From W02016038750A1 , the luciferase protein FLuc derived from the Japanese firefly (Pyrocoelia matsumurai) is known, as well as its C-terminal and N-terminal fragments, which restore the enzymatic activity of native luciferase upon association.

[0006] From the article by Akiko Saito et al., “High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A,” published in Protein Engineering, Design and Selection, Volume 2, Issue 6, March 1989, Pages 481-487 the B domain, a fragment of Staphylococcus aureus protein A, is known to have high affinity for the Fc domain of antibody heavy chains.

[0007] From the article by Masayasu Mie et aL, published in Analyst, 2012, 137, 1085-1089, the concept of a homogeneous immunoassay system using fragmented Renilla luciferase (Rluc) is known. In this system, a double B domain of protein A was fused to two fragments of Rluc. The idea is that when non-covalent complexes (chemical affinity) between “sandwich” ELISA antibodies and the fragmented Rluc fusion proteins bind to target molecules / antigens, the Rluc fragments come into proximity, and the bioluminescent activity of the fragmented Rluc is restored through complementation. In practice, the researchers did not provide convincing evidence of achieving a specific signal for homogeneous one-step antigen detection in solution. Mie et al. rely on multi-step heterogeneous detection and additional secondary antibodies. They overlook the crucial issue of properly preparing the double B domain of protein A to ensure 1 :1 stoichiometric, non-covalent (chemical affinity) binding of a single luciferase fragment to the double protein A domain per IgG molecule. (Each IgG molecule contains a dimeric Fc domain capable of binding protein As B domains at two independent sites.) This is a necessary condition to prevent two luciferase fragments from binding to the B domains of protein A on a single IgG molecule. In light of the results presented in the present patent application, improper preparation of the double B domain of protein A, or the use of a single protein A domain, prevents the intended one-step homogeneous detection of antigen based on a pair of “sandwich” ELISA antibodies.

[0008] From the article by Yan Ni et al., published in Nature Communications 2021 , 12, 4586- 4598, the homogeneous RAPPID immunoassay system using fragmented NanoLuc® luciferase is known. A single Gx domain of protein G, incorporating the unnatural photosensitive amino acid p-benzoyl-L-phenylalanine, was fused to NanoLuc® fragments, i.e., LgBiT and SmBiT. When covalent complexes (chemical bonds) between “sandwich” ELISA antibodies and the Gx adapter fragments of NanoLuc bind to target molecules / antigens, the LgBiT and SmBiT fragments come into proximity, and the bioluminescent activity of NanoLuc is restored through complementation. The RAPPID immunoassay system requires complex production of NanoLuc fragment fusions with obligatory incorporation of the unnatural amino acid p-benzoyl-L-phenylalanine into the Gx protein adapters, followed by an additional covalent light-induced (UV photochemistry “click”) conjugation of the Gx luciferase fragments to the “sandwich” ELISA antibodies.

[0009] From the articles by Byounghoon Hwang et al., published in Communications Biology, 2020, 3, 8:1-12, and in Methods in Molecular Biology, 2023, 2612: 195-224, the homogeneous LUMIT immunoassay system using fragmented NanoLuc® luciferase, proprietary to Promega, is known. In this system, instead of linking protein A or G domains to luciferase fragments, direct covalent conjugation of LgBiT and SmBiT fragments of NanoLuc® to “sandwich” ELISA antibodies (direct LUMIT assay) or to secondary antibodies (indirect LUMIT assay) is employed using conventional “click” chemistry. When covalent complexes (chemical bonds) between “sandwich” ELISA antibodies or secondary antibodies and NanoLuc® fragments bind to target molecules / antigens, the LgBiT and SmBiT fragments come into proximity, and the bioluminescent activity of NanoLuc is restored through complementation. The LUMIT immunoassay system requires covalent conjugation of antibodies to luciferase fragments for each successive antigen / biomarker (direct LUMIT) or the use of an additional pair of antibodies when using secondary antibodies covalently linked to NanoLuc® fragments (indirect LUMIT).

[0010] Technical Problem

[0011] In light of the solutions known from the prior art concerning homogeneous and universal immunoassays based on biological reagents, significant problems remain unresolved. First, it would be desirable to eliminate the need for covalent conjugation of luciferase fragments to antibodies using chemical or photochemical “click” chemistry, which may negatively affect the activity of the conjugated molecules. Second, it would be desirable to extend the universality and functionality of a homogeneous assay based on luciferase fragment complementation (or other reporter enzymes) beyond the detection of antigens and biomarkers to include the detection of antibodies, protein-protein interactions (PPIs), and the screening of new chemical and biological drugs.

[0012] The objective of the invention is to provide easily manufacturable, universal protein fusions of specialized adapters with fragments of reporter enzymes composed of natural amino acids, without the need for obligatory covalent conjugation of the prepared protein fusions to antibodies or Fc-tagged proteins. These fusions enable straightforward detection of (i) specific antigens / biomarkers or molecular interactions, and (ii) general IgG antibodies with gradation of their activity toward Fc receptors.

[0013] Ease of detection should rely on monitoring the generated signal, i.e., visible light emission (bioluminescence) in the case of luciferase as a reporter enzyme, in a one-step reaction detecting specific molecular interactions in solution due to the enzymatic activity of associating luciferase fragments, without the need for excitation light and filtering as used in conventional fluorescence-based methods.

[0014] The universality of the reagents should allow their use with any antibodies or recombinant Fc-tagged proteins, particularly as a universal component of diagnostic tests detecting antigens / biomarkers using unmodified antibodies, but also as a method for screening new drugs, studying molecular interactions, or assessing the potential of therapeutic antibodies with varying Fc receptor binding strength.

[0015] The technical problem described above has been unexpectedly solved by the present invention.

[0016] Detailed Description of the Invention The subject of the invention is a first fusion protein comprising a polypeptide of the general formula:

[0017] [N-terminal luciferase fragment] - [linker 1] - [protein A domain B], wherein:

[0018] • [N-terminal luciferase fragment] denotes a peptide derived from the luciferase protein shown as Seq. No. 1 , comprising at least 398 consecutive amino acids of this protein and including its N-terminus, preferably shown as Seq. No. 2,

[0019] • [linker 1] denotes a peptide of at least 12 amino acids in length, preferably comprising Seq. No. 5 and / or 61 , or selected from Seq. Nos. 6-10 or 26-27, especially Seq. Nos. 6 or 26-27,

[0020] • [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably shown as Seq. No. 4.

[0021] Preferably, the invention also relates to a second fusion protein, comprising a polypeptide of the general formula:

[0022] [N-terminal luciferase fragment] - [linker 1] - [protein A domain B] - [linker 2] - [protein A domain B], wherein:

[0023] • [N-terminal luciferase fragment] denotes a peptide derived from luciferase protein shown as Seq. No. 1 , comprising at least 398 consecutive amino acids and including its N-terminus, preferably shown as Seq. No. 2,

[0024] • [linker 1] denotes a peptide of at least 12 amino acids, preferably comprising Seq. No. 5 and / or 61 , or selected from Seq. Nos. 10 or 26-27,

[0025] • [linker 2] denotes a peptide of at least 12 amino acids, preferably comprising Seq. No. 5 and / or 61 , or selected from Seq. Nos. 13-14 or 31-33,

[0026] • [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably shown as Seq. No. 4.

[0027] Preferably, the fusion protein according to the invention further comprises a peptide facilitating the isolation of the fusion protein, preferably selected from Seq. Nos. 16-17 or Preferably, it constitutes the fusion protein NLuc-1 B, particularly selected from Seq. Nos. 18-21 , 38, 59, or the fusion protein NLuc-2B, particularly selected from Seq. Nos. 22-23, 40, 48-54.

[0028] Another subject of the invention is a third fusion protein comprising a polypeptide of the general formula:

[0029] [C-terminal luciferase fragment] - [linker 1] - [protein A domain B], wherein:

[0030] • [C-terminal luciferase fragment] denotes a peptide derived from luciferase protein shown as Seq. No. 1 , comprising at least 132 consecutive amino acids of this protein and including its C-terminus, preferably shown as Seq. No. 3,

[0031] • [linker 1] denotes a peptide containing at least one amino acid, especially Gly, preferably comprising Seq. No. 5 and / or 61 , or selected from Seq. Nos. 11 or 26- 27, particularly Seq. Nos. 26-27,

[0032] • [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably shown as Seq. No. 4.

[0033] Preferably, the invention also relates to a fourth fusion protein, comprising a polypeptide of the general formula:

[0034] [C-terminal luciferase fragment] - [linker 1] - [protein A domain B] - [linker 2] - [protein A domain B], wherein:

[0035] • [C-terminal luciferase fragment] denotes a peptide derived from luciferase protein shown as Seq. No. 1 , comprising at least 132 consecutive amino acids and including its C-terminus, preferably shown as Seq. No. 3,

[0036] • [linker 1] denotes a peptide of at least 1 amino acid, especially Gly, preferably comprising Seq. No. 5 and / or 61 , or selected from Seq. Nos. 26-27,

[0037] • [linker 2] denotes a peptide of at least 12 amino acids, preferably comprising Seq. No. 5 and / or 61 , or selected from Seq. Nos. 15 or 28-33,

[0038] • [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably shown as Seq. No. 4. Preferably, it further comprises a peptide facilitating the isolation of the fusion protein, preferably selected from Seq. Nos. 16-17 or 34-37.

[0039] Preferably, it constitutes the fusion protein CLuc-1 B, particularly selected from Seq. Nos. 24, 39, 60, 63.

[0040] Preferably, it constitutes the fusion protein CLuc-2B, particularly selected from Seq. Nos. 25, 41-47, 55-58, or 62.

[0041] Another subject of the invention is a reagent kit comprising the first or second fusion protein according to the invention and the third or fourth fusion protein according to the invention, as defined above.

[0042] Preferably, the reagent kit according to the invention comprises the second and fourth fusion proteins as defined above.

[0043] Preferably, the reagent kit further comprises a reagent serving as a substrate for luciferase.

[0044] Preferably, the reagent kit further comprises an antibody having affinity for the target substance.

[0045] Another subject of the invention is a method for determining the presence of a target substance in a tested sample, characterized by the following steps: a) contacting the tested sample in vitro with an antibody having affinity for the target substance and the reagent kit defined above, comprising the second and fourth fusion proteins according to the invention, b) measuring luciferase activity in the presence of a luciferase substrate reagent, c) the detection of luciferase activity indicating the presence of the target substance in the tested sample.

[0046] Another subject of the invention is a method for determining the presence of an antibody in a tested sample, characterized by the following steps: a) contacting the tested sample in vitro with the reagent kit according to the invention, as defined above, b) measuring luciferase activity in the presence of a luciferase substrate reagent, c) the detection of luciferase activity indicating the presence of the antibody in the tested sample. Preferably, in step c) of the methods according to the invention, the intensity of light produced in the enzymatic bioluminescence reaction is measured, and its value is considered proportional to the concentration of the detected substance (antigen, biomarker) or antibody.

[0047] In a preferred embodiment, the invention enables the development of protein fusions of FLuc luciferase fragments with universal protein adapters specifically and strongly binding Fc antibody heavy chains in a 1 :1 ratio (adapter 2B) or 2:1 ratio (adapter 1 B). Due to their universality, the fusion proteins proposed according to the invention allow various applications of the resulting ECHIA immunoassay (Enzyme Complementation Homogeneous Immuno Assay).

[0048] In an exemplary embodiment, the ECHIA 2B assay enables detection of antigens / biomarkers or protein-protein interactions (PPIs) along with PPI inhibitors using luciferase fragments with the 2B adapter and a commercial pair of sandwich ELISA antibodies or recombinant Fc-tagged proteins. In another exemplary embodiment, the ECHIA 1 B assay enables detection of individual IgG antibodies and grading of their relative Fc receptor binding strength using luciferase fragments with the 1 B adapter and commercial Fc receptors.

[0049] According to the invention, besides the FLuc luciferase used in the examples, other luciferases or reporter enzymes, such as p-galactosidase, alkaline phosphatase, or horseradish peroxidase, may also be used as reporter enzymes whose fragments are employed to obtain fusion proteins according to the invention.

[0050] Definitions

[0051] In the context of the present invention, the term “tested sample” should be understood in the broadest possible sense. In one interpretation, it may include a sample or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and may include fluids, solids, and tissues. Biological samples may include blood components, such as plasma, serum, and the like, as well as saliva and nasopharyngeal swabs. The sample may also refer to cell lysates or purified forms of peptides and / or polypeptides described herein. Cell lysates may include cells lysed with a lysing agent or lysates such as rabbit reticulocyte lysates or wheat embryo lysates. The sample may also include cell-free expression systems. Environmental samples include environmental material such as surface material, soil, water, crystals, and industrial samples. Industrial samples may include food or pharmaceutical products, as well as research samples in the form of potential biological or chemical therapeutics. However, such examples should not be interpreted as limiting the types of samples applicable in the present invention.

[0052] In the context of the present invention, the term “N-terminal luciferase fragment” refers to a peptide derived from the luciferase protein shown as Seq. No. 1 , comprising at least 398 consecutive amino acids of this protein and including its N-terminus (i.e., at least N1-N399; the FLuc luciferase used in the invention from the Japanese firefly Pyrocoelia matsumurai belongs to the same Firefly luciferase class and undergoes similar fragmentation and complementation as the extensively studied FLuc from the American firefly Photinus pyralis-, see patent application W02016038750A1 and Ramasamy Paulmurugan et al., Anal. Chem., 2007, 79(6): 2346-2353), preferably shown as Seq. No. 2. This term also encompasses mutants of such a peptide which, in combination with a C- terminal luciferase fragment, allow detectable luciferase activity.

[0053] Analogously, in the context of the present invention, the term “C-terminal luciferase fragment” refers to a peptide derived from the luciferase protein shown as Seq. No. 1 , comprising at least 132 consecutive amino acids of this protein and including its C-terminus (i.e., at least C416-C548; the FLuc luciferase used in the invention from the Japanese firefly Pyrocoelia matsumurai belongs to the same Firefly luciferase class and undergoes similar fragmentation and complementation as the extensively studied FLuc from the American firefly Photinus pyralis', see W02016038750A1 and Ramasamy Paulmurugan et aL, Anal. Chem., 2007, 79(6): 2346-2353), preferably shown as Seq. No. 3. This term also encompasses mutants of such a peptide which, in combination with an N-terminal luciferase fragment, allow detectable luciferase activity.

[0054] In the context of the present invention, the term “luciferase substrate” refers to any substance that may serve as a substrate in the bioluminescence reaction catalyzed by luciferase or its analogue, particularly in the functional complex formed according to the invention by the N-terminal and C-terminal luciferase fragments. In an exemplary embodiment, D-luciferin, ATP, and atmospheric oxygen were used as substrates for the FLuc enzymatic reaction. However, it may be any component that facilitates the formation of molecular complexes resulting in luciferase fragment complementation and thereby contributes to signal generation in the presence of other enzyme substrates. In the context of the present invention, the term “linker” refers to a flexible peptide containing at least one amino acid. It may also refer to any similar chemical moiety suitable for providing an effective connection between two adjacent functional fragments of a fusion protein such that they perform their functions according to the invention. In exemplary embodiments, the linker may be linker 1 or linker 2. In particularly advantageous embodiments, linker 1 consists of at least one amino acid (see construct 29CLuc-2B #25 and derivatives #62 and #63), for example 5 amino acids (see 6CLuc-1 B #24), or at least 12 amino acids, e.g., 12 or 54 amino acids. Linker 2 in exemplary embodiments comprises at least 12 amino acids, preferably at least 14 amino acids, advantageously at least 27 amino acids. Linker 1 connects the N- or C-terminal luciferase fragment with the 1 B or 2B adapter based on protein A’s B domains, whereas linker 2 connects two protein A’s B domains within the 2B adapter. Numerous examples of such linkers and methods for obtaining them are known in the art (see Chen X, Zaro JL, Shen WC, Fusion protein linkers: property, design and functionality, Adv Drug Deliv Rev, 2013 Oct; 65(10): 1357-69). In the exemplary embodiments described herein, linker 1 may comprise Seq. No. 5 and / or 61 or be selected from Seq. Nos. 10 or 26-27, especially Seq. Nos. 26-27, while linker 2 may comprise Seq. No. 5 and / or 61 , preferably defined by the formula GSAGSAAGSGEF(GGGGS)n, where n is a natural number from 0 to 5, 6, 7, 8, 9, or 10, particularly preferably selected from Seq. Nos. 15 or 28-33.

[0055] In the context of the present invention, the term “protein A domain B” refers to a peptide derived from staphylococcal protein A having affinity for antibody constant chains (Fc dimeric domain) or a derivative retaining such function, in particular a polypeptide forming the domain described by Akiko Saito et al. In the present invention, this peptide may occur as a single B domain in the 1B adapter, with a defined function of binding a single antibody molecule in a 2:1 ratio, or as a double B domain in the 2B adapter, wherein the B domains are connected by a specially selected linker 2 to bind a single antibody molecule in a 1 :1 ratio. In the exemplary embodiments described herein, this peptide is defined as Seq. No. 4.

[0056] In the context of the present invention, the term “reporter enzyme fragments” preferably refers to fragments of Firefly FLuc luciferase disclosed herein, emitting visible light at longer wavelengths (Amax -550 nm) with prolonged “glow” bioluminescence kinetics, compared to other luciferases such as Renilla and Gaussia (Amax -450 nm, fast “flash” kinetics), as well as to artificial luciferase Oplophorus (NanoLuc®, Amax -450 nm, prolonged “glow” kinetics). Longer wavelength bioluminescence is advantageous due to lower interference in measurements of complex biological samples. Prolonged “glow” kinetics are desirable for observing a stable luciferase signal over several minutes in a one-step reaction detecting molecules in solution with enzyme substrates according to the ECHIA method. In contrast, “flash” kinetics result in near-complete signal decay within seconds of reaction initiation in the presence of enzyme substrates (e.g., Renilla RLuc as used by Mie et al., cited above). Other suitable reporter enzyme fragments include complementary fragments of the luciferases mentioned above, 0-galactosidase (bGAL), alkaline phosphatase (ALP) for colorimetric readout, and horseradish peroxidase (HRP) for colorimetric, chemiluminescent, or voltamperometric readouts.

[0057] In the context of the present invention, there is a single, strictly defined, optimal configuration for linking the protein adapters comprising protein As B domains (1 B or 2B) with reporter enzyme fragments. The 1 B or 2B adapters are linked exclusively to the natural N- or C-termini of the FLuc reporter enzyme, in contrast to alternative configurations linking polypeptides / proteins to newly generated N’ and C’ termini created by cleavage, which typically show lower activity (e.g., as used by Mie et aL, cited above).

[0058] In the context of the present invention, the “ECHIA” (Enzyme Complementation Homogeneous Immuno-Assay) system should be understood as a homogeneous immunoassay performed in solution, in a single step, in a single vessel, without washing steps, in contrast to multi-step, time-consuming heterogeneous immunodetection on surfaces with washing, as in conventional ELISA (Enzyme-Linked ImmunoSorbent Assay). The ECHIA assay may be configured in one of three ways:

[0059] 1. For the detection of antigens, biomarkers, allergens, cytokines, or drugs using luciferase fragment fusions and 2B adapters along with commercial sandwich ELISA antibodies;

[0060] 2. For the detection of protein-protein interactions and their inhibition using recombinant Fc-tagged proteins with luciferase fragment fusions and 2B adapters;

[0061] 3. For the detection of general IgG antibodies and assessment of ADCC potential (Antibody-Dependent Cellular Cytotoxicity) in terms of relative IgG binding strength to recombinant Fc receptors using luciferase fragment fusions and 1 B adapters.

[0062] Detailed Description of the Invention In an exemplary embodiment, the fusion protein according to the invention is composed of several functional elements. In this embodiment, peptides obtained by cleavage of the natural Firefly luciferase sequence from the Japanese firefly Pyrocoelia matsumurai are used to generate the fusion proteins. Each luciferase fragment is then linked to a single or double B domain derived from natural protein A using peptide linkers (linker 1) composed of flexible sequences such as (GGGGS)n and GSAGSAAGSGEF, or combinations of flexible and rigid helical sequences (EAAAK)5 or ABD (Albumin Binding Domain). The luciferase sequence is known from W02016038750A1 , and the natural B domain sequence is disclosed in Saito et al., 1989.

[0063] The purpose of such a fusion is to enable rapid and universal detection of either a single type of antibody (adapter 1 B) or an antigen / biomarker using an antibody pair (adapter 2B). The fusion protein according to the invention can be readily combined with commercially available ELISA antibodies specific for a given antigen (the 2B adapter, comprising a double protein A domain B, binds both rabbit and human IgG antibodies), and the resulting conjugates can be placed in a single reaction vessel with luciferase substrates to create a detection kit for the antigen. When a specific antigen is added to such a vessel, a strong bioluminescent signal is produced within approximately 10 minutes due to complementation of the luciferase fragments and restoration of enzymatic activity (see Fig. 1). No signal is generated when a non-specific antigen or water / buffer is added.

[0064] The luciferase fragments do not associate or complement each other in the absence of the antigen, due to their weak micromolar affinity. The presence of a specific antigen brings the antibodies with attached luciferase fragments into close proximity, resulting in high nanomolar / picomolar affinity interactions. Thus, the method according to the invention enables rapid, sensitive (via enzymatic amplification and bioluminescence), and specific (via ELISA antibodies) antigen detection in a one-step reaction, suitable for implementation on a simple light reader. This method is particularly suited for modern, rapid, and point-of- care (POCT) medical diagnostics but may also be used for dynamic measurements in laboratory settings.

[0065] Fig. 2 illustrates an exemplary application of the invention, referred to herein as ECHIA (Enzyme Complementation Homogeneous Immuno-Assay), in a homogeneous, one-step immunoassay configuration for rapid (~10 min) and sensitive antigen detection, in comparison to traditional laboratory methods. These include time-consuming, heterogeneous, multi-step ELISA and the homogeneous AlphaScreen bead-based assay requiring laser excitation.

[0066] Fig. 3 shows an example of applying the method according to the invention for high- throughput screening, e.g., inhibition of protein-protein interactions (PPI), providing an alternative to AlphaScreen technology commonly used in biotech and pharmaceutical drug discovery. Unlike fluorescent protein fragments, luciferase fragments undergo reversible association / dissociation, enabling their use in PPI inhibition assays.

[0067] The examples in Figs. 2 and 3 demonstrate that the ECHIA technology can perform on par with established laboratory methods for antigen or protein-protein interaction detection, such as ELISA and AlphaScreen. The competitive advantage of ECHIA lies in the radical simplification of molecular detection procedures, reduced to a rapid, one-step reaction with a technically simple signal readout.

[0068] The assays shown in Figs. 2 and 3 were conducted using a reagent set comprising preselected exemplary fusion proteins according to the invention, designated 17NLuc-2B (#22) and 29CLuc-2B (#25).

[0069] The development of the invention first required determining the optimal configuration for linking luciferase fragments to the interacting proteins. The intact luciferase possesses natural N- and C-terminal ends. Cleavage of the enzyme generates two additional N’ and C’ termini. To identify the optimal configuration, luciferase fragments NLuc [N1-416C] and CLuc [N'359-548C] were connected via a flexible peptide linker (GGGGS)n to leucine zipper domains on either the original N / C or the cleavage-generated N7C termini (see Indraneel Ghosh et al., Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein, JACS 2000, 122(23): 5658-5659). Model experiments in bacteria demonstrated a markedly higher efficiency of luciferase fragment complementation when the B domains were linked to the natural N / C termini, rather than to N7C’ termini of the cleaved luciferase. Based on these bacterial experiments, various fusion variants of luciferase fragments linked to protein As B domains were prepared for universal and rapid antigen detection using commercial ELISA antibodies.

[0070] Fusion proteins of luciferase fragments with 1B adapter (single protein A domain B fused to luciferase fragments) In an initial exemplary embodiment, fusion proteins according to the invention, termed 1 B fusion proteins, comprise N- or C-terminal luciferase fragments linked to a single protein A domain B. Exemplary 1 B luciferase fragment fusion proteins are shown in Fig. 4.

[0071] Purified N- and C-terminal 1B luciferase fragments expressed in bacteria exhibit nonspecific, spontaneous association (complementation) in buffer or on NiNTA resin only at high fragment concentrations, corresponding to weak micromolar affinity (Table 1 , Fig. 5). This low intrinsic affinity is essential for the method according to the invention, wherein proximity of the luciferase fragments at low concentrations is induced by high nanomolar affinity interactions mediated by the proteins fused to the fragments. Fragments linked via B domains to the original N / C termini exhibit relatively higher spontaneous complementation efficiency than fragments linked to N7C’ termini of cleaved luciferase. Fragment 8NLuc-1 B, linked via N7C’, shows no detectable spontaneous complementation with the corresponding C fragment across the tested concentration range.

[0072] Moreover, no bioluminescent signal is observed for isolated N or C 1 B fragments (Table 1 , Fig. 5), a prerequisite for achieving a specific enzymatic complementation assay, in which individual N or C luciferase fragments are enzymatically inactive.

[0073] Table 1 presents results for spontaneous, nonspecific complementation of N and C 1 B luciferase fragment pairs, as well as bioluminescence of isolated luciferase fragments at final concentrations eight-fold lower (test dilutions) than the original fragment concentrations indicated in the first column. Spontaneous complementation was tested in buffer (mix buffer) or with NiNTA resin (mix beads), which brings His-tagged luciferase fragments into proximity and increases bioluminescence signal.

[0074] Table 1.

[0075] Fig. 5 presents an approximate estimation of the interaction strength, i.e., the chemical affinity, of exemplary 1 B luciferase fragments 9NLuc-1 B (#18) and 6CLuc-1 B (#24), determined by titration of increasing concentrations of fragment 6CLuc-1B (#24) against a fixed, low concentration of fragment 9NLuc-1 B (#18).

[0076] Following confirmation of spontaneous / nonspecific complementation of 1B luciferase fragments at relatively high concentrations, the behavior of these fragments at lower concentrations was tested with respect to antigen-specific detection using an exemplary pair of ELISA antibodies. Fig. 6 illustrates antibody-dependent but antigen-independent complementation of the N-terminal 9NLuc-1 B (#18) and C-terminal 6CLuc-1 B (#24) fragments. The bioluminescent signal is shown for increasing equimolar concentrations of N- and C-terminal 1 B luciferase fragments preincubated with ELISA antibodies or buffer, with either the target antigen or buffer added.

[0077] Results shown in Fig. 7 clarify the origin of the high bioluminescent signal observed for the 9NLuc-1 B (#18) and 6CLuc-1 B (#24) pair, which is dependent solely on the presence of ELISA antibodies. Fig. 7 illustrates the effect of 250 nM protein A (ProtA) on the reaction of 200 nM of the 1B luciferase fusion fragments with either a single 15 nM antibody or a 15 nM pair of ELISA antibodies, as well as the effect of subsequent addition of the specific SARS-CoV-2 N protein antigen. High bioluminescence was observed both in the presence of antibody pairs and for single IgG molecules from the ELISA pair. Similar results were obtained for three different ELISA antibody pairs specific for the SARS-CoV-2 N protein antigen. Moreover, high bioluminescence was observed in the presence of general, nonspecific hlgG. In all cases, this high bioluminescent signal was abolished and returned to baseline spontaneous bioluminescence upon addition of excess natural protein A. Since protein A competes for Fc binding sites on IgG molecules, the observed high bioluminescence is attributed to N- and C-terminal 1 B luciferase fragments binding to general IgG. As previously observed in Figs. 6 and 7, addition of the specific antigen to the system containing ELISA antibodies and 1 B luciferase fragments did not generate the desired ECHIA bioluminescent signal, and only slightly reduced the signal generated by antibody binding.

[0078] The direct effect of protein A on the high bioluminescent signal of complementing 1 B luciferase fragments in the presence of antibodies is shown in Fig. 8. This figure illustrates the effect of protein A and DTT on 1 B luciferase fragments spontaneously complementing in buffer or in the presence of IgG, as well as the effect of DTT on the catalytic activity of the full-length luciferase over time. Addition of excess protein A induced dissociation of 1 B fragments from antibody Fc domains in real time, resulting in a decrease in bioluminescent signal. The simplest explanation for these results is that N- and C-terminal 1 B luciferase fragments complement on a single IgG molecule and subsequent dissociation caused by protein A releases the complemented fragments into solution, where they reversibly dissociate back to free fragments. It is well known that, unlike fluorescent protein fragments, luciferase fragments undergo reversible association and dissociation. Fig. 8 also shows the positive effect of DTT on luciferase fragments and the full-length enzyme. Firefly FLuc contains multiple cysteine residues whose reduced form ensures correct enzymatic catalysis. The figure illustrates the pronounced increase / reset of catalytic activity for cleaved luciferase fragments due to DTT reduction and the prolonged half-life of catalysis for the full-length luciferase, which shows only minor activity reset, as the cysteines in the full-length enzyme are more protected from oxidation than exposed cysteines in cleaved fragments.

[0079] The absence of antigen-dependent specific complementation of the 1 B luciferase fragment pair 9NLuc-1 B (#18) and 6CLuc-1 B (#24) may be attributed to geometric constraints, i.e., insufficient separation of the protein A domain B from the luciferase fragments. To address this, N-terminal fragments with longer linkers separating the antibody-binding B domain from the luciferase fragment, specifically 38NLuc-1 B (#19) and 42NLuc-1 B (#20), were tested. Fig. 9 presents attempts to achieve ECHIA signals, i.e., antigen-dependent specific complementation of successive 1 B luciferase fragment pairs with protein As B domains in the presence of ELISA antibodies. No desired ECHIA signal was observed for any tested 1B fragment pair; as in previous experiments (Figs. 6 and 7), addition of antigen slightly decreased the signal, while antibody presence consistently led to a significant, multi-fold increase in bioluminescence.

[0080] To further investigate the antibody-concentration-dependent high complementation of luciferase fragments, reactions with increasing concentrations of N- and C-terminal 1 B fragments (concentration matrices) were performed in buffer alone or in the presence of varying concentrations of general IgG molecules. Fig. 10 shows bioluminescence signals for these matrices, illustrating spontaneous / nonspecific complementation only at high concentrations in buffer, due to the fragments’ weak micromolar affinity. Addition of a single type of hlgG induced maximal bioluminescence at relatively low fragment concentrations, with the signal diminishing at higher fragment concentrations. Optimal fragment concentrations for maximal bioluminescence shift depending on IgG concentration. This behavior represents a typical solution-phase interaction between two A / B molecules (two 1B luciferase fragments) binding the same site on a third C molecule (single antibody). Specific concentrations of A and B yield maximal ABC complex formation (signal); above these concentrations, signal diminishes due to AAC and BBC complexes lacking signal. Because each natural IgG has a dimeric Fc domain, at certain concentrations, one N and one C fragment may bind a single antibody, whereas at higher concentrations, two N or two C fragments may bind a single antibody (Fig. 11).

[0081] Fig. 11 schematically illustrates 1B luciferase fragment complementation on a single IgG molecule with a dimeric Fc domain, i.e., two identical binding sites for the protein A domain B.

[0082] Since 1 B luciferase fragments (1 B fusion proteins) could not generate an ECHIA immunoassay signal for antigen detection using specific ELISA antibodies, it was tested whether conformational / geometric changes of a single antibody-antigen complex are sufficient to be reported by 1 B luciferase fragments. Fig. 12 demonstrates detection of SARS-CoV-2 N protein antigen binding to its specific antibody R040 or R004, with no detection for nonspecific hlgG. Detection based on a single antibody and 1B fragments results in a very low signal-to-background ratio (S / B), illustrating the limitation for detecting low antigen concentrations. This minor antigen effect on 1B fragment complementation was also observed in previous experiments using ELISA antibody pairs rather than single antibodies (Figs. 6, 7, 9). Fig. 12 further shows the effect of protein A on bioluminescence of 1 B luciferase fragments complementing in the presence of various single antibodies and Fc-containing proteins. This phenomenon prevents use of 1 B fusion proteins for PPI inhibition assays for the same reasons they are unsuitable for ECHIA antigen detection with ELISA antibodies. However, 1 B ECHIA can be employed for general IgG detection or for assessing ADCC potential of individual IgGs through measurements of their interaction with Fc receptors.

[0083] Fusion proteins of luciferase fragments with the 2B adapter, two B domains of protein A linked to Luc fragments

[0084] In the second exemplary embodiment, the fusion proteins according to the invention are referred to as 2B fusion proteins, i.e. fusion proteins comprising N- or C-terminal fragments of luciferase connected to two B domains of protein A. Exemplary 2B luciferase fragment fusion proteins are shown in Fig. 13.

[0085] In order to enable a universal ECHIA antigen detection assay based on pairs of ELISA antibodies, the 1 B luciferase fragments were equipped with an additional, appropriately linked B domain of protein A. In principle, the two B domains of a single 2B luciferase fragment are intended to saturate the dimeric Fc domain of a single antibody molecule. Consequently, the use of 2B fragments should prevent the coexistence of two luciferase fragments on a single antibody molecule (as occurs in the case of 1 B luciferase fragment fusions). The main difference among the above protein fusion schemes of 2B luciferase fragments is the length of linker 2 connecting the two separate B domains of protein A, which were designed to test the appropriate distance required for the primary function of the 2B fusion protein, namely complete blocking of a single IgG antibody.

[0086] To test the utility of 2B luciferase fragments in the ECHIA assay, complementation of combinations of 1 B and 2B luciferase fragments was evaluated in the presence of a single type of IgG antibody. Fig. 14 presents a comparison of the bioluminescence signal resulting from complementation of 1B and 2B luciferase fragments in the presence of a single IgG antibody. Figure 14 demonstrates the repeatedly observed high bioluminescence signal of the 1 B luciferase fragments 9NLuc-1 B (#18) and 6CLuc-1 B (#24) in the presence of IgG, compared with the bioluminescence signal obtained for combinations of newly generated N-terminal and C-terminal 2B luciferase fragments reacted with the complementary N and C fragments 9NLuc-1B (#18) and 6CLuc-1B (#24), respectively. The bioluminescence results show that the 2B fragment with the relatively strongest ability to block luciferase fragment complementation on a single antibody molecule is 29CLuc-2B (#25) (1% complementation of 1B fragments), followed by 17NLuc-2B (#22) (5% complementation of 1B fragments), and finally 25NLuc-2B (30% complementation of 1 B fragments). The unfavorable, insufficiently blocking 2B fragment, i.e. 25NLuc-2B, contains the shortest 14- aa linker 2 between the B domains of protein A in its structure. (Mie et al., in the publication cited above, connected two B domains of protein A in an even more unfavorable manner from the perspective of ECHIA 2B assay feasibility, namely in a tandem arrangement without an additional linker). In contrast, the most favorable pair of 2B fragments, i.e. 17NLuc-2B (#22) and 29CLuc-2B (#25), exhibits the lowest <1 % complementation on a single IgG molecule relative to 1 B fragments.

[0087] To further verify the utility of 2B luciferase fragments in the ECHIA assay, a classical screening was performed using three different pairs of commercial antibodies, R004 / R040, R004 / R001 , and R040 / R001 , detecting the SARS-CoV-2 N protein antigen. Fig. 15 presents the screening of 1B and 2B luciferase fragment pairs using different pairs of ELISA antibodies specific for the SARS-CoV-2 N protein antigen. The signal-to-background ratio (S / B) was determined for all tested ECHIA 2B assay conditions. The data in Fig. 15 show that the 2B fragment pair with the highest S / B ratio (>40) is the pair 17NLuc-2B (#22) and 29CLuc-2B (#25) in configuration with the R040 / R004 or R001 / R004 antibody pairs. In contrast, the S / B signal for luciferase fragment pairs containing the unfavorable 25NLuc- 2B (#23), which only partially blocks the antibody Fc domains (Fig. 14), remained at a much lower, unfavorable level (S / B < 5). Meanwhile, the S / B signal of luciferase fragment pairs containing 9NLuc-1 B (#18) remained at S / B = 1 , indicating a complete lack of antigen detection capability for assays based on this luciferase fragment.

[0088] Because the 2B luciferase fragment pair 17NLuc-2B (#22) and 29CLuc-2B (#25) exhibits the best functional parameters with respect to the assumptions of the ECHIA immunoassay, this pair was used in the experiments described below as well as in the final tests of antigen detection limits and drug-screening assay configurations presented earlier. Figure 16 presents the Hook effect of the ECHIA immunoassay for varying concentrations of 2B luciferase fragments at a fixed ELISA antibody concentration, as well as for varying ELISA antibody concentrations at a fixed excess of 2B luciferase fragments. Figure 16 demonstrates the classical behavior of a homogeneous immunoassay in solution such as ECHIA, in which the saturating antigen concentration resulting in the peak assay signal depends on the concentration of the biological detection elements of the assay, in this case the antigen-binding ELISA antibodies. Upon further increase of antigen concentration, a decrease in immunoassay signal is observed due to decoupling of the detection system, i.e. the Hook effect. The antigen concentration corresponding to the Hook point does not depend on the concentration of the ECHIA signal-generating components, in this case the 2B luciferase fragments, which only affect the intensity of the immunoassay signal. Therefore, the dynamic range of the ECHIA assay (approximately three orders of magnitude of detectable antigen, e.g. 10 pM to 10 nM) can be modulated to some extent by adjusting the concentration of detection antibodies, i.e. shifted toward detection of higher or lower antigen concentrations.

[0089] A further objective of the invention was to address specific problems related to fusion proteins of Luc fragments with 1B and 2B adapters, in particular optimization of the 2B adapter sequence. The essential goal of the invention, as illustrated in Fig. 17, is to propose constructs that enable selective signal generation indicating the presence of free antibodies (1 B constructs) or antibodies forming complexes with antigen (2B constructs).

[0090] In preliminary work leading to the invention, a series of constructs comprising fusion proteins with 1B and 2B adapters was prepared (sequences 6-25 from the attached sequence listing). Some of these constructs were problematic in expression and purification, and most contained different peptide linkers (linkers 1 and 2), which hindered systematic comparison among constructs. Nevertheless, the obtained results confirmed the enormous potential of the ECHIA immunoassay and identified two particularly advantageous pairs of 1 B and 2B fusion proteins that formed the basis for further studies. The first pair of luciferase fusion proteins with 1 B adapters was 9NLuc-1 B (#18) and 6CLuc-1 B (#24), and the second pair of fusion proteins with 2B adapters was 17NLuc-2B (#22) and 29CLuc-2B (#25).

[0091] In subsequent work, new versions of these two fusion protein pairs were generated and analyzed (sequences 26-60 and 62-63 from the attached sequence listing). In these constructs, the same core sequences of luciferase fragments and protein As B domains were used throughout (Seq. 1-5 of the sequence listing). However, in this case, both 1 B and 2B fusion proteins contained identical linker 1 sequences. As a result, the 2B fusion proteins contained within their sequences the corresponding 1B fusion proteins, to which an additional linker 2 sequence and a second protein A domain B were added. Initially, two N- and C-terminal 1B fusions with a long linker 1 (L1+54; SEQ B4 and SEQ B5) were prepared, as well as one N-terminal 2B fusion with a long linker L1+54 and an identical linker 2 L2+54 (SEQ B6). Subsequently, C-terminal 2B fusions with varying linker 2 lengths were prepared (SEQ B7-B13). The aim of these studies was to determine the minimal linker 2 length required for proper functioning of the 2B adapter. The prepared C-terminal 2B fusions were tested (i) alone, (ii) with the above-prepared 1 B fusion pair (SEQ 38 or 59 and SEQ 39 or 60), or (iii) with the complementary N-terminal 2B fusion (SEQ 40). Several linker 2 sequences, or their absence, were tested, starting from the version identical to the prior art (Masayasu Mie et aL, Analyst, 2012, 137, 1085-1089), in which linker 2 is absent (L2-3, three amino acids removed at the head-to-tail junction of two protein As B domains), followed by linker 2 variants of defined amino acid length: L2+12, L2+17, L2+22, L2+27, L2+54, and L2+75 (SEQ 28-33).

[0092] The optimal linker 2 sequence was determined based on a series of tests and the resulting parameters:

[0093] • a direct binding assay of 2B and 1 B fusion proteins to biotinylated hlgG using biolayer interferometry (BLI) on streptavidin SAX sensors (parameter: Kd);

[0094] • an indirect binding assay of 2B and 1 B fusion proteins to biotinylated hlgG using AlphaScreen with donor and acceptor beads modified with streptavidin and protein A, respectively (parameter: IC50 / ON for signal increase);

[0095] • a complementation assay of 2B / 1 B fusions with complementary 1 B fusions in the presence of a pair of specific sandwich antibodies R004 and R040 in samples with zero concentration of SARS-CoV-2 N protein antigen, to determine ECHIA 1 B activity (parameter: ACT-1 B);

[0096] • a complementation assay of 2B / 1B fusions with complementary 2B fusions using the specific antibody pair R004 and R040 in samples with a fixed concentration of SARS-CoV-2 N protein antigen and in zero-antigen samples, to determine ECHIA 2B activity (parameter: ACT-2B).

[0097] The results obtained are summarized in the table below. Notably, head-to-tail linkage of protein As B domains (L2-3, #41 , #62) is insufficient to perform an efficient antigen detection assay (Table 2, ACT-2B, #22, Fig. 20). Figure 20 shows the measured ACT-1 B (antibody detection) and ACT-2B (antigen detection) activities. Antigen detection was performed with ACT-2B defined as the signal-to-background ratio (S / B) during detection of 20 nM SARS-CoV-2 N protein antigen using a system containing 2 nM specific antibodies R004 and R040 (Sino Biological) bound to the appropriate N / C-type 2B / 1 B fusions (20 nM). Background signal was recorded using 0 nM antigen. Antibody detection was also performed, with ACT-1 B defined as the S / B ratio for binding of 2 nM antibodies R004 and R040 (Sino Biological) by the indicated N / C-type 2B / 1 B fusions (20 nM) in a system devoid of specific antigen (SARS-CoV-2 N protein). The background signal was consistently defined as the lowest signal value generated by the 46CLuc-2B (#46) fusion. Table 2 and Fig. 20 show that elongation of linker L2 between the protein As B domains in 2B fusions results in increased antigen detection signal (ACT-2B, #22) accompanied by decreased antibody detection signal (ACT-1 B, #18). Conversely, shortening linker L2 in 2B fusions, up to its complete removal and retention of a single B domain in 1B-type fusions, leads to increased antibody detection signal (ACT-1 B, #18) and a simultaneous decrease in antigen detection signal (ACT-2B, #22) (Table 2 and Fig. 20).

[0098] A similar trend was observed in binding analyses (AlphaScreen, BLI) of 2B- and 1 B-type fusions with the Fc domain of IgG. Stronger binding affinity correlates with elongation of linker L2 between the protein As B domains in 2B fusions and with increased ACT-2B activity (#22), whereas weaker binding correlates with shortening of linker L2 — down to the presence of a single B domain in 1 B-type fusions — which corresponds to higher ACT-1 B activity (#18) (Table 2, Fig. 21). Figure 21 presents representative molecular interaction data showing the relative binding strength (IC50) of CLuc-1 B / 2B fusions to Fc IgG-biot in the AlphaScreen assay, as well as direct binding data obtained by biolayer interferometry (BLI), estimating KD based on kinetic constants kon and koff (KD = kotr / kon).

[0099] 2B fusions containing two protein As B domains connected by a sufficiently long linker (L2 > 27 aa) bind the Fc domain of IgG with affinity (Kd, IC50) comparable to full-length protein A. In contrast, 1 B fusions containing a single protein A domain B exhibit markedly weaker binding to Fc IgG (Fig. 21 , Table 2). According to the results of AR Cruz et aL, PNAS, 2021 , 118(7): 1-11 , full-length protein A interacts with the Fc fragment of IgG with a 1 :1 stoichiometry, whereas individual protein A domains bind Fc IgG with a 2:1 stoichiometry. This suggests analogous 1 :1 stoichiometry for 2B fusions with L2 > 27 aa and 2:1 stoichiometry for the 1B fusions described in the present application. Based on the results in Table 2, linker L2+27 can be considered the minimal required length of linker 2 for an efficient ECHIA 2B assay. Additionally, it was found that 1 B fusion proteins efficiently report the presence of IgG (ACT-1 B, #18) independently of antigen, which is exploited in ECHIA 1B assays for detection of IgG and their relative Fc receptor binding strength.

[0100] Furthermore, Fig. 22 presents antigen detection (ACT-2B) using different antibody pairs, defined based on the signal-to-background ratio (S / B) during detection of 20 nM SARS- CoV-2 N protein antigen relative to antigen-free samples, using a system containing different configurations of specific antibodies R001 , R004, and R040 (2 nM each, Sino Biological) bound to the appropriate N / C-type 2B / 1 B fusions (20 nM).

[0101] Table 2. Summary data on the binding strength of C-terminal 1B and 2B fusions to IgG (ICS0, KD) and their complementation with N-terminal 1B and 2B fusions on IgG molecules (ECHIA 1B, ACT-1 B) or as a result of formation of trimolecular complexes with antigen (ECHIA 2B, ACT-2B).

[0102] *KD estimated based on 1-2 sensorgrams (illustrative data). The KD for full-length wt protein A is approximately 1 nM.

[0103] **ICS0analysis based on three independent experiments performed in duplicate. The IC50for wt protein A is 0.35 nM.

[0104] ***S / B ACT-1 B is the signal-to-background ratio (background is signal #46; 46Luc-2B) obtained in the absence of antigen in a system based on 2 nM antigen-binding specific antibodies preincubated with 20 nM N- and C-terminal Luc fusions.

[0105] ****S / B ACT-2B denotes the signal-to-background ratio obtained at antigen concentrations of 20 nM and 0 nM in a system based on 2 nM antigen-binding antibodies preincubated with 20 nM N- and C-terminal Luc fusions. It is also worth noting that in the ECHIA 2B antigen detection assay (ACT-2B), regardless of the antibody pair used, application of an N-terminal 2B-type fusion in combination with various C-terminal 1B-type fusions consistently results in a partial S / B signal (Table 2, ACT-2B, #22; Figs. 20 and 22). In contrast, this phenomenon is not observed when C-terminal 2B-type fusions are used in combination with various N-terminal 1B-type fusions, for which S / B » 1 (Table 2, ACT-2B, #18; Figs. 20 and 22). This effect most likely arises from partial contamination of the N-terminal 2B fusions with N-terminal 1 B fusions that are capable of detecting antibodies alone in the ECHIA 1 B (ACT-1 B) format, whereas all C-terminal 2B fusion proteins remain pure, as confirmed by SDS-PAGE analysis (Fig. 23). Figure 23 shows reducing SDS-PAGE gels of purified 1B and 2B fusion proteins. The C-terminal 1 B and 2B fusions exhibit high purity, whereas the N-terminal 1 B and 2B fusions — particularly the N-terminal 2B fusion — are purified as two fractions despite the use of two affinity chromatography methods (Co-NTA and FLAG). This is further supported by the partial activity of N-terminal 2B fusions in combination with C-terminal 1 B fusions (Table 2, ACT-1 B; Figs. 20-21).

[0106] For the pair of 2B fusion proteins that exhibited the highest reproducible signal-to- background ratio (S / B) in the ECHIA 2B antigen detection assay — namely 40NLuc-2B (#40) and 46CLuc-2B (#46) — resistance to nonspecific, general IgG antibodies was evaluated. The results are shown in Fig. 24. Figure 24A presents the effect of nonspecific IgG antibodies on the ECHIA 2B signal during detection of 20 nM SARS-CoV-2 N protein using 2 nM of the specific antibodies R004 and R040, preincubated with 20 nM of the 2B fusion proteins 40NLuc-2B (#40) and 46CLuc-2B (#46). Figure 24B shows a non-reducing SDS-PAGE analysis demonstrating bioconjugation of 46CLuc-2B (#46) with a general hlgG antibody using the cross-linking reagent DSS (100-fold molar excess of DSS relative to hlgG and 46CLuc-2B (#46), preincubated at a 1 :1 ratio in 50 mM HEPES buffer, pH 8.2).

[0107] Figure 24A shows that the presence of nonspecific IgG in solution at a concentration of 33 pg / mL (220 nM) causes an approximately 50% decrease in signal in a detection system based on 2 nM R004 and R040 antibodies, preincubated with 20 nM of the fusion proteins 40NLuc-2B (#40) and 46CLuc-2B (#46), reacting with a constant antigen concentration of 20 nM. In this context, the possibility of stabilizing and rendering the detection system resistant to antibody dissociation from the 2B fusion proteins was also confirmed by additional covalent coupling using an excess of the cross-linker DSS (Fig. 24B). Such coupling is required only when the ECHIA 2B system is applied to antigen detection in samples containing high concentrations of nonspecific IgG, for example in 100% serum containing approximately 10 mg / mL total IgG. The conjugation process is preferably performed during purification of the fusion proteins on a column, which enables straightforward separation of the conjugate from unreacted molecules. For this purpose, use of the widely adopted protocol described by James DeCaprio in Cold Spring Harbor Protocols 2019, 2, 159-162, is recommended.

[0108] After establishing the optimal linker 2 sequence for an efficient ECHIA 2B assay, the universality of the ECHIA 2B format was confirmed using an independent second antigen: human C-reactive protein (CRP, 115 kDa). Readouts across the full concentration range and the respective limits of detection (LOD) for ECHIA 2B were compared with laboratory ELISA and AlphaScreen assays for the same antigens and using the same antibodies. Unique example applications of 1 B fusion proteins (SEQ 38 and SEQ 39), which to date had been used only as counter-examples to 2B fusion proteins in antigen detection assays, were also investigated. Methods for detecting general monoclonal and polyclonal IgG in the ECHIA 1B assay were proposed, together with determination of the respective limits of detection (LoD). In addition, a specific application of 1 B fusion proteins in the ECHIA 1 B assay was proposed for readout of the relative binding strength of different IgG molecules, including the human therapeutic monoclonal antibodies lgG1 adalimumab and lgG4 pembrolizumab, to human FcyRllla-V158 / F158 receptors responsible for ADCC responses. The ECHIA 1B assay determining the ADCC cellular response potential of IgG is comparable to the commercial Lumit™ FcyRllla-V158 / F158 Binding Immunoassay.

[0109] Examples

[0110] General method for preparing 1 B and 2B fusion proteins described in the examples below

[0111] Cloning of genetic constructs

[0112] Genetic constructs encoding 1B and 2B luciferase fragment fusions, connected via linkers 1 and 2 to the B domain of protein A, were generated using restriction-free cloning methods with NEBuilder® HiFi DNA Assembly reagents in accordance with the manufacturer’s instructions, or by the SLIC method (Sequence and Ligation Independent Cloning) using T4 DNA polymerase in CutSmart buffer at 21 °C, employing E. coll cloning strains NEB® Turbo Competent E. coliand JM109. For this purpose, appropriate plasmid backbones and DNA insert sequences were linearized and amplified by PCR. The generated DNA fragments were verified by agarose gel electrophoresis, treated with Dpnl to remove the original DNA templates, purified using the Wizard® SV Gel and PCR Clean-Up System, and their concentrations were determined by UV-Vis spectrophotometry.

[0113] Historically, the first genetic constructs (sequences A) were generated based on ordered (GenScript) pUC19 plasmids containing the core sequences of the full-length luciferase gene, the B domain of protein A, linkers incorporating leucine zipper domains, or the ABD domain. Initially, 1 B luciferase fragment fusions were generated, which subsequently served as templates for the generation of 2B luciferase fragment fusions.

[0114] In a subsequent series of genetic constructs (sequences B), the same cloning methods were used to generate luciferase fragment fusions incorporating appropriately designed and custom-ordered (Thermo Fisher Scientific) sequences connecting the 1 B or 2B domains with linkers 1 and 2 in pMX vectors. Correct cloning was verified in each case by DNA sequencing.

[0115] Expression of fusion proteins

[0116] 1B and 2B fusion proteins were overexpressed using the pET28a vector in E. coli expression strains BL21(DE3) or JM109. For this purpose, a single colony of bacteria transformed with the pET28a vector carrying the respective genetic construct was used to inoculate an overnight starter culture in 10-20 mL LB medium supplemented with 30 pg / mL kanamycin, incubated at 37 °C and 250 rpm.

[0117] On the following day, the starter cultures were used to inoculate (1 :500 dilution) smaller (4 x 25 mL) or larger (4 250 mL) expression cultures in LB or 2YT medium supplemented with 30 pg / mL kanamycin and 1 % glucose to prevent premature activation of the lac operon prior to induction in the pET expression system.

[0118] The inoculated cultures were grown at 37 °C and 250 rpm to an OD600of approximately 0.6, then cooled for 15 min to 20 °C and induced with 100 pM IPTG to enable slow protein expression (to reduce aggregation) for 5 h or overnight at 20 °C with shaking at 250 rpm. After the expression period, cultures were centrifuged for 10 min at 4 °C at 10,000 x g.

[0119] Fusion proteins produced by the bacteria were released from the harvested cells by lysis in the presence of the protease inhibitor PMSF, using either sonication on ice or chemical lysis with BugBuster and Benzonase reagents (Novagen) at room temperature for 30 min. Following cell lysis, cellular debris was removed by centrifugation at 20,000 x g for 30 min at 4 °C, and the resulting supernatant containing the soluble fusion proteins was subjected to purification.

[0120] Purification of fusion proteins

[0121] A one-step or two-step purification procedure was performed using either Ni-NTA resin alone (for the initial sequence A fusion proteins containing HIS and AVI tags) or Ni-NTA resin in the first step followed by anti-FLAG M2 resin in the second step (for subsequent sequence B fusion proteins containing HIS and FLAG tags).

[0122] Proteins were eluted from the Ni-NTA column using 250-500 mM imidazole, and from the anti-FLAG M2 column using 100 pg / mL 3xFLAG peptide in PBS buffer, pH 7.4. The eluted fusion proteins were subsequently desalted using Sephadex G25 resin (5 kDa cutoff) or Zeba™ desalting columns (7 kDa cutoff) to remove imidazole and the 3xFLAG peptide and to exchange the buffer to PBS, pH 7.4.

[0123] Protein purity and the correct molecular weight of each fusion protein were confirmed by SDS-PAGE. Protein concentrations were determined using the BOA assay and / or UV-Vis spectrophotometry based on the Beer-Lambert law and the molar extinction coefficient calculated for each fusion protein from its amino acid sequence (ProtParam / ExPASy).

[0124] To the resulting solutions of each fusion protein (approximately 0.5-1 mg / mL in PBS, pH 7.4), glycerol was added to a final concentration of 10%. The protein solutions were then aliquoted into 20 pL and 50 pL portions, flash-frozen in liquid nitrogen, and stored at -80 °C until use in subsequent assays.

[0125] Example 1. Fusion proteins comprising a polypeptide of the general formula: [N-terminal luciferase fragment] - [linker 1] - [B domain of protein A]

[0126] Representative sequences of fusion proteins having this structure are listed in the appended Sequence Listing as Sequence Nos. 18-21 , 38, and 59. These proteins were obtained using the method described above.

[0127] Sequences Nos. 18-21 , 38, and 59 of Example 1 encode fusion proteins consisting of the N-terminal fragment of luciferase linked to a 1 B adapter.

[0128] The N-terminal 1B fusion proteins 18-21 , 38, and 59 bind the Fc domains of human or rabbit IgG antibodies in direct binding assays using Bio-Layer Interferometry (BLI) and the proximity assay AlphaScreen.

[0129] The N-terminal 1B fusion proteins 18-21, 38, and 59, in the absence of any complementary C-terminal luciferase fragment fusions, are not capable of generating even residual bioluminescence characteristic of an active FLuc enzyme.

[0130] The N-terminal 1B fusion proteins 18-21, 38, and 59, when combined with any complementary C-terminal luciferase fragment fusions, generate a relatively low basal bioluminescence signal resulting from spontaneous, nonspecific complementation of FLuc enzyme fragments in buffer.

[0131] The N-terminal 1B fusion proteins 18-21, 38, and 59, when combined with C-terminal luciferase fragments bearing the 1B adapter, i.e. C-terminal 1B fusion proteins 24, 39, 60, and 63 of Example 3, and in the presence of human or rabbit IgG antibodies, generate a relatively high bioluminescence signal corresponding to specific complementation of FLuc enzyme fragments on the Fc domain of a single IgG antibody molecule.

[0132] The N-terminal 1B fusion proteins 18-21, 38, and 59, when combined with any C- terminal luciferase fragment fusions, are not capable of generating a bioluminescence signal for antigen detection using a pair of antigen-specific ELISA antibodies.

[0133] Example 2. Fusion proteins comprising a polypeptide of the general formula: [N-terminal luciferase fragment] - [linker 1] - [B domain of protein A] - [linker 2] - [B domain of protein A]

[0134] Representative sequences of fusion proteins having this structure are listed in the appended Sequence Listing as Sequence Nos. 22-23, 40, and 48-54. These proteins were obtained using the method described above.

[0135] Sequences Nos. 22-23, 40, and 48-54 of Example 2 encode fusion proteins consisting of the N-terminal fragment of luciferase linked to a 2B adapter.

[0136] The N-terminal 2B fusion proteins 22-23, 40, and 48-54 bind the Fc domains of human or rabbit IgG antibodies in direct binding assays using Bio-Layer Interferometry (BLI) and the AlphaScreen proximity assay. The binding stoichiometry of N-terminal 2B fusion proteins to IgG is 1 :1 , or a mixture of 2:1 and 1 :1 for less optimal constructs such as 23. The N-terminal 2B fusion proteins 22-23, 40, and 48-54, in the absence of any complementary C-terminal luciferase fragment fusions, are not capable of generating even residual bioluminescence characteristic of an active FLuc enzyme.

[0137] The N-terminal 2B fusion proteins 22-23, 40, and 48-54, when combined with any complementary C-terminal luciferase fragment fusions, generate a relatively low basal bioluminescence signal resulting from spontaneous, nonspecific complementation of FLuc enzyme fragments in buffer.

[0138] The N-terminal 2B fusion proteins 22-23, 40, and 48-54, when combined with C-terminal luciferase fragments bearing the 2B adapter, primarily C-terminal 2B fusion proteins 25, 45-47, and 55-58 of Example 4, generate a high antigen-detection signal using a pair of antigen-specific ELISA antibodies.

[0139] The N-terminal 2B fusion proteins 22-23, 40, and 48-54, when combined with C-terminal luciferase fragments bearing the 1B adapter, i.e. C-terminal 1B fusion proteins 24, 39, 60, and 63 of Example 3, do not generate a bioluminescence signal corresponding to specific complementation of FLuc enzyme fragments on the Fc domain of a single IgG antibody molecule.

[0140] Example 3. Fusion proteins comprising a polypeptide of the general formula: [C-terminal luciferase fragment] - [linker 1] - [B domain of protein A]

[0141] Representative sequences of fusion proteins having this structure are listed in the appended Sequence Listing as Sequence Nos. 24, 39, 60, and 63. These proteins were obtained using the method described above.

[0142] Sequences Nos. 24, 39, 60, and 63 of Example 3 encode fusion proteins consisting of the C-terminal fragment of luciferase linked to a 1 B adapter.

[0143] The C-terminal 1B fusion proteins 24, 39, 60, and 63 bind the Fc domains of human or rabbit IgG antibodies in direct binding assays using Bio-Layer Interferometry (BLI) and the AlphaScreen proximity assay. The binding stoichiometry of C-terminal 1 B fusion proteins to IgG is 2:1.

[0144] The C-terminal 1B fusion proteins 24, 39, 60, and 63, in the absence of any complementary N-terminal luciferase fragment fusions, are not capable of generating even residual bioluminescence characteristic of an active FLuc enzyme.

[0145] The C-terminal 1B fusion proteins 24, 39, 60, and 63, when combined with any complementary N-terminal luciferase fragment fusions, generate a relatively low basal bioluminescence signal resulting from spontaneous, nonspecific complementation of FLuc enzyme fragments in buffer.

[0146] The C-terminal 1B fusion proteins 24, 39, 60, and 63, when combined with N-terminal luciferase fragments bearing the 1B adapter, i.e. the N-terminal fusion proteins of Example 1 , and in the presence of human or rabbit IgG antibodies, generate a relatively high bioluminescence signal corresponding to specific complementation of FLuc enzyme fragments on the Fc domain of a single IgG antibody molecule.

[0147] The C-terminal 1B fusion proteins 24, 39, 60, and 63, when combined with any N- terminal luciferase fragment fusions, do not generate a bioluminescence signal for antigen detection using a pair of antigen-specific ELISA antibodies.

[0148] Example 4. Fusion proteins comprising a polypeptide of the general formula: [C-terminal luciferase fragment] - [linker 1] - [B domain of protein A] - [linker 2] - [B domain of protein A]

[0149] Representative sequences of fusion proteins having this structure are listed in the appended Sequence Listing as Sequence Nos. 25, 41-47, and 55-58. These proteins were obtained using the method described above.

[0150] Sequences Nos. 25, 41-47, and 55-58 of Example 4 encode fusion proteins consisting of the C-terminal fragment of luciferase linked to a 2B adapter.

[0151] The C-terminal 2B fusion proteins 25, 41-47, and 55-58 bind the Fc domains of human or rabbit IgG antibodies in direct binding assays using bio-layer interferometry (BLI) and the AlphaScreen proximity assay. The binding stoichiometry of C-terminal 2B fusion proteins to IgG is 1 :1 , or a mixture of 2:1 and 1 :1 for less optimal constructs such as 41- 44.

[0152] The C-terminal 2B fusion proteins 25, 41-47, and 55-58, in the absence of any complementary N-terminal luciferase fragment fusions, are not capable of generating even residual bioluminescence characteristic of an active FLuc enzyme.

[0153] The C-terminal 2B fusion proteins 25, 41-47, and 55-58, when combined with any complementary N-terminal luciferase fragment fusions, generate a relatively low basal bioluminescence signal resulting from spontaneous, nonspecific complementation of FLuc enzyme fragments in buffer.

[0154] The C-terminal 2B fusion proteins 25, 41-47, and 55-58, when combined with N-terminal luciferase fragments bearing the 2B adapter, i.e. the N-terminal 2B fusion proteins 22-23, 40, and 48-54 of Example 2, generate an antigen-detection signal using a pair of antigen-specific ELISA antibodies, with the S / B detection ratio depending on the specific selection of N- and C-terminal 2B fusion protein pairs.

[0155] The C-terminal 2B fusion proteins 25, 41-47, and 55-58, when combined with N-terminal luciferase fragments bearing the 1B adapter, i.e. the N-terminal 1B fusion proteins 18-21, 38, and 59 of Example 1 , and in the presence of human or rabbit IgG antibodies, do not generate a bioluminescence signal corresponding to specific complementation of FLuc enzyme fragments on the Fc domain of a single IgG antibody molecule.

[0156] Example 5. Reagent kit comprising fusion proteins of Examples 1 and 3

[0157] In particular, the kit comprises the following N- and C-terminal pairs of fusion proteins bearing the 1B adapter, i.e. 18 + 24, 38 + 39, or 59 + 60, which in the presence of human or rabbit IgG antibodies generate the highest relative signal-to-background (S / B) ratios of bioluminescence corresponding to specific complementation of FLuc enzyme fragment fusions on the Fc domain of a single IgG antibody molecule, relative to the basal signal arising from nonspecific spontaneous complementation of luciferase fragment fusions in the absence of IgG.

[0158] The above N- and C-terminal pairs of fusion proteins bearing the 1B adapter do not generate a bioluminescence signal for antigen detection using a pair of antigen-specific ELISA antibodies.

[0159] Example 6. Reagent kit comprising fusion proteins of Examples 2 and 4

[0160] In particular, the kit comprises the following pairs of N- and C-terminal luciferase fragment fusion proteins bearing the 2B adapter, i.e. 22 + 25, or pairs composed of combinations of 40 and 48-54 with 45-47 and 55-58, which generate the highest relative signal-to-background (S / B) ratios of bioluminescence for specific antigen detection using a pair of antigen-specific ELISA antibodies, relative to the basal signal arising from nonspecific spontaneous complementation of FLuc enzyme fragment fusions in the absence of antigen.

[0161] The above N- and C-terminal pairs of fusion proteins bearing the 2B adapter do not generate a bioluminescence signal corresponding to specific complementation of FLuc enzyme fragment fusions on the Fc domain of a single IgG antibody molecule.

[0162] Example 7. Method for determining the presence of a detectable substance in a test sample using the kit of Example 6, including a direct method for antigen detection and an indirect method for detecting inhibitors of protein-protein interactions using the ECHIA 2B assay. Procedure (conditions, equipment, reagents), exemplary results

[0163] All reactions involved in the detection process using the ECHIA method are carried out in buffer containing 50 mM Tris, pH 8.0, 10 mM MgCI2, 1 mg / mL BSA, 0.05% Tween 20, and 1-5 mM DTT.

[0164] The final working concentration of the FLuc enzyme substrate mixture is 250 pM D- luciferin and 6 mM ATP, with ambient molecular oxygen present.

[0165] Direct detection of specific antigens or biomarkers

[0166] As part of the preparation of a direct antigen detection system, each of the fusion proteins comprising the N- and C-terminal fragments of luciferase with the 2B adapter is reacted with one antibody from a pair of commercial antibodies (1 and 2) binding different epitopes of the same target antigen. The ELISA antibodies are reacted with the 2B fusion proteins at a ratio not exceeding 1 :10 for 15 min at room temperature with shaking at 250 rpm.

[0167] For example, 2 nM antibody 1 is reacted with 20 nM of the N-terminal luciferase fragment with the 2B adapter (bioconjugate A), and 2 nM antibody 2 is reacted with 20 nM of the C- terminal luciferase fragment with the 2B adapter (bioconjugate B). Subsequently, a reaction mixture ready for antigen detection in the sample is prepared.

[0168] In the case of antigen detection using a multiwell plate and a plate reader, the following components are added to each well of a white half-area multiwell plate (Greiner Bio-One, PerkinElmer, or Corning): 1) 10 pL of bioconjugate A at a fixed concentration of 2 nM antibody 1 and 20 nM N-terminal 2B fusion protein, 2) 10 pL of bioconjugate B at a fixed concentration of 2 nM antibody 2 and 20 nM C-terminal 2B fusion protein, 3) 10 pL of an FLuc substrate mixture at fixed concentrations of 1 mM D-luciferin and 24 mM ATP.

[0169] The dispensed plate is centrifuged for 30 s at 500 g to ensure that the added reagent mixtures reach the bottom of the wells. Subsequently, 10 pL of the test sample containing antigen is added to the wells designated for antigen measurement, while 10 pL of the solution in which the antigen is dissolved, but without antigen, is added to the control wells intended for background signal measurement (negative control; bioluminescence background; baseline signal for spontaneous, non-specific complementation of the N- and C-terminal FLuc fusion fragments). The plate is centrifuged again for 30 s at 500 g, and a bioluminescence detection protocol lasting several minutes is initiated using a multiwell plate reader capable of recording bioluminescence in the wavelength range of 500-700 nm.

[0170] Figure 18 shows examples of real-time signal monitoring for the detection of the N protein antigen using a system composed of the protein fusions 17NLuc-2B (#22) and 29CLuc-2B (#25) conjugated with appropriate ELISA antibodies and FLuc substrates.

[0171] A procedure for quantitative determination of the concentration of the analyte using a calibration curve is based on plotting the bioluminescence of the ECHIA 2B detection system as a function of 8-12 known concentrations of the target antigen, tested in duplicate in 2- or 3-fold serial dilutions starting from an antigen concentration of 20 nM, on the same plate as the test samples containing unknown antigen concentrations. Alternatively, the unknown antigen concentration in a sample may be determined based on a previously established model equation of the standard curve for a given antigen. An example is presented below, in which the standard curve equation was determined using a nonlinear regression mathematical model, namely a four-parameter logistic (4PL) curve (GraphPad Prism), for the data shown in Figure 2.

[0172] Figure 19 presents an example of dose-response curve fitting using four-parameter nonlinear regression for data related to detection of the N protein antigen using the protein fusions 17NLuc-2B (#22) and 29CLuc-2B (#25) conjugated with appropriate ELISA antibodies. Similar results of specific detection of the SARS-CoV-2 N protein antigen were obtained for a pair of 2B fusions with unified optimal linkers L1+54 and L2+54, namely 40NLuc-2B (#40) and 46CLuc-2B (#46) (Figure 25).

[0173] Figure 25 shows results of detection of a specific antigen, the SARS-CoV-2 N protein, using 20 nM of the 2B fusions 40NLuc-2B (#40) and 46CLuc-2B (#46) in complex with 2 nM of antibodies R004 and R040 (Sino Biological). No ECHIA-2B signal was observed for other non-specific antigens from different viruses.

[0174] Example 7 encompasses a universal method for direct detection of various antigens and biomarkers, not only pathogenic antigens such as the N protein — the main antigen of the SARS-CoV-2 virus detected in COVID-19 — but also biomarkers such as CRP or IL-6, which are measured in inflammatory diseases, including sepsis requiring rapid and accurate diagnostics. Figure 26 shows an example of specific detection of another antigen, human CRP protein, also obtained using the pair of 2B fusions with unified optimal linkers L1+54 and L2+54, namely 40NLuc-2B (#40) and 46CLuc-2B (#46). Detection of the specific antigen, human CRP protein, was performed using 20 nM of the 2B fusions 40NLuc-2B (#40) and 46CLuc-2B (#46) in complex with 8 nM of antibodies R106 and M07 (Sino Biological). No ECHIA-2B signal was observed for other non-specific antigens. Below, a comparative AlphaScreen assay performed using the same antibodies (R106-biotin on STRP donor beads and M07 on ProtA acceptor beads) and antigens is presented.

[0175] Example 7 includes direct testing of antigens and biomarkers in buffer, throat swabs, cell cultures, and blood samples (serum or whole blood). Testing of antigens or biomarkers in blood samples requires additional stabilization of bioconjugates A and B using the crosslinking reagent DSS applied in PBS pH 7.4, which subsequently rapidly hydrolyzes in the TRIS pH 8.0 reaction buffer (Figure 24). The antigen detection method is not limited to the use of multiwell plates and plate readers. Antigen detection according to the ECHIA 2B method may also be performed using a simple handheld bioluminescence reader with disposable cartridges containing lyophilized reagents of N- and C-terminal luciferase fragment fusions 2B conjugated with antibodies detecting a specific antigen, and FLuc substrates, wherein the detection reaction is initiated upon dissolution of the powdered reagents by addition of the sample solution containing the antigen.

[0176] Indirect detection of protein-protein interaction (PPI) inhibitors

[0177] Another method for determining the presence of a target substance in a test sample using a preferentially selected set of 2B fusion proteins from Example 6 is the indirect detection of a competitive inhibitor of protein-protein interactions (PPI). This includes interactions between two recombinant Fc-tagged proteins, wherein one of the Fc-tagged proteins may be an IgG antibody, the PPI inhibitor represents a potential drug candidate, and the ECHIA 2B method enables screening of potential drugs in multiwell plate formats.

[0178] As part of the preparation of an indirect PPI inhibitor detection system, each of the fusion proteins comprising the N- and C-terminal fragments of luciferase with the 2B adapter is reacted with one of a pair of interacting recombinant Fc-tagged proteins, or with a pair consisting of a recombinant Fc-tagged protein and an IgG antibody. The Fc-tagged proteins are reacted with the 2B fusion proteins at a ratio not exceeding 1 :10 for 15 min at room temperature with shaking at 250 rpm.

[0179] For example, 2 nM of Fc-tagged protein 1 is reacted with 20 nM of the N-terminal luciferase fragment with the 2B adapter (bioconjugate A), and 2 nM of Fc-tagged protein 2 is reacted with 20 nM of the C-terminal luciferase fragment with the 2B adapter (bioconjugate B). Subsequently, a reaction mixture is prepared for detection and testing of PPI inhibitor activity in a biotechnology or pharmaceutical laboratory.

[0180] For this purpose, the following components are added to each well of a white half-area multiwell plate (Greiner Bio-One, PerkinElmer, or Corning): 1) 10 pL of a PPI inhibitor solution at a test concentration depending on the single-point screening procedure or IC50determination, 2) 10 pL of bioconjugate A containing Fc-tagged protein 1 that interacts with the PPI inhibitor, 3) 10 pL of bioconjugate B containing Fc-tagged protein 2 that interacts with Fc-tagged protein 1. The dispensed plate is centrifuged for 30 s at 500 g and incubated for 15 min at room temperature to ensure that the added reagent mixtures reach the bottom of the wells of the multiwell plate and that interactions occur between the Fc-tagged proteins and the PPI inhibitor. Subsequently, in step 4), 10 pL of an FLuc substrate mixture with initial concentrations of 1 mM D-luciferin and 24 mM ATP is added. The plate is centrifuged again for 30 s at 500 g, and a bioluminescence detection protocol lasting several minutes is initiated using a multiwell plate reader capable of recording bioluminescence in the wavelength range of 500-700 nm.

[0181] Each assay plate includes at least 8 positive control wells (addition of buffer instead of the PPI inhibitor in step 1), defining the maximum bioluminescence signal for the PPI interaction, and at least 8 negative control wells (addition of buffer instead of the PPI inhibitor in step 1 and the N-terminal luciferase fragment with the 2B adapter instead of bioconjugate A in step 2), defining the minimum bioluminescence signal for the PPI interaction. These controls allow assessment of the required screening assay quality expressed as a Z-factor > 0.65.

[0182] In a drug screening format, the remaining wells of the plate are filled with duplicate measurements of one or two selected test concentrations (e.g., 100 pM and 1 pM) of each potential PPI inhibitor (one- or two-point screening). In an assay format for determining IC50values of PPI inhibitors, the remaining wells of the plate are allocated to duplicate 10- point concentration series to generate a dose-dependent curve for each PPI inhibitor.

[0183] An example of the use of the ECHIA 2B assay to determine the IC50activity of the inhibitor LCB3, which blocks the PPI between ACE2-Fc and S1-Fc proteins, i.e., the cellular receptor of SARS-CoV-2 and the SARS-CoV-2 spike protein, is shown in Figure 3. Additionally, Figure 27 shows an example of the activity of LCB3 and other inhibitors of the ACE2-Fc and S1-Fc interaction using an optimal pair of 2B fusions with unified optimal linkers L1+54 and L2+54, namely 40NLuc-2B (#40) and 46CLuc-2B (#46).

[0184] Figure 27 illustrates the effect of inhibitors on the activity of the ECHIA 2B assay based on the interaction between the SARS-CoV-2 S1-Fc protein and the human ACE2-Fc receptor. In the assay setup, 8 nM S1-Fc protein was conjugated with 20 nM of the 46CLuc-2B (#46) fusion, while 8 nM ACE2-Fc was conjugated with 20 nM of the 40NLuc-2B (#40) fusion. The S1 proteins tested as inhibitors possessed a His-tag instead of an Fc-tag and therefore were able to compete with S1-Fc for binding to the ACE2 receptor.

[0185] Example 8. Method for determining the presence of antibodies in a test sample using the kit of Example 5

[0186] This includes a direct method for detecting general IgG antibodies and an indirect method for detecting interactions between IgG antibodies and Fc receptors using the ECHIA 1 B assay.

[0187] Procedure (conditions, equipment, reagents), exemplary results

[0188] All reactions of the substance detection process using the ECHIA method are carried out in a buffer comprising 50 mM Tris pH 8.0, 10 mM MgCI2, 1 mg / mL BSA, and 0.05% Tween 20, with 1-5 mM DTT. The FLuc substrate mixture corresponds to working / final concentrations of 250 pM D-luciferin and 6 mM ATP, with ambient oxygen present.

[0189] Direct detection of general IgG antibodies

[0190] As part of the preparation of a direct IgG antibody detection system, each of the fusion proteins comprising the N- and C-terminal fragments of luciferase with the 1 B adapter is mixed at a stoichiometric ratio of 1 :1 . For example, no more than 200 nM of the N-terminal luciferase fragment with the 1 B adapter is mixed with 200 nM of the C-terminal luciferase fragment with the 1 B adapter. During IgG antibody detection using a multiwell plate and a plate reader, the following components are placed into each well of a white half-area multiwell plate (Greiner Bio-One, PerkinElmer, or Corning): 1) 10 pL of the N-terminal luciferase fragment with the 1 B adapter at a constant concentration < 200 nM, 2) 10 pL of the C-terminal luciferase fragment with the 1 B adapter at a constant concentration < 200 nM, 3) 10 pL of an FLuc substrate mixture at constant concentrations of 1 mM D-luciferin and 24 mM ATP. The dispensed plate is centrifuged for 30 s at 500 g to ensure that the added reagent mixtures reach the bottom of the wells of the multiwell plate. Subsequently, in the wells designated for antibody measurements, 10 pL of the test sample containing the antibody at the concentration under investigation is added, whereas in the control wells designated for background measurements, 10 pL of the solution in which the antibody is dissolved, but without the antibody, is added. (The negative background control of bioluminescence corresponds to the baseline signal of spontaneous, nonspecific complementation of FLuc fragment fusions). The plate is centrifuged again for 30 s at 500 g, and a bioluminescence detection protocol lasting several minutes is initiated using a multiwell plate reader capable of recording bioluminescence in the wavelength range of 500-700 nm. The procedure for quantitative determination of the concentration of the analyte is performed using a calibration curve procedure analogous to that described in Example 7 above.

[0191] Example 8 demonstrates a universal direct method for quantitative detection of recombinant rabbit or human antibodies produced in in vitro cell-based expression systems. Detection of general antibodies using the ECHIA 1 B method may be performed using any bioluminescence reader, including a handheld reader, with single-use cartridges containing lyophilized reagent kits composed of N- and C-terminal luciferase fragment fusions with the 1B adapter in the presence of FLuc substrates, wherein the detection reaction is initiated upon dissolution of the lyophilized reagents by the added sample solution containing the antibody. An example of the use of the ECHIA 1 B assay for IgG detection in a sample is confirmed by data demonstrating detection of general monoclonal and polyclonal IgG in buffer (Fig. 28). Figure 28 shows detection of monoclonal and polyclonal IgG antibodies of human and rabbit origin using Firefly luciferase fragment fusions containing a single B domain of protein A, namely the 1 B fusions 38NLuc-1 B (#38) and 39CLuc-1 B (#39). The LoD and S / B values were estimated based on raw data prior to normalization performed for illustrative purposes.

[0192] Indirect detection of IgG antibody ADCC potential

[0193] Another method for determining the presence of the analyte in a test sample using a suitably selected set of 1 B fusion proteins from Example 6 is the indirect detection of interactions between IgG antibodies and human FcyRllla-V158 / F158 receptors, indicative of the ADCC potential of the antibodies. In this context, the IgG antibody constitutes a potential therapeutic agent, and the ECHIA 1 B method enables screening of ADCC properties of candidate IgG drugs in multiwell plate formats.

[0194] During determination of the relative binding strength of an IgG antibody to Fc receptors using a multiwell plate and a plate reader, the following components are placed into each well of a white half-area multiwell plate (Greiner Bio-One, PerkinElmer, or Corning): 1) 8 pL of the N-terminal luciferase fragment with the 1 B adapter at a constant concentration < 200 nM, 2) 8 pL of the C-terminal luciferase fragment with the 1 B adapter at a constant concentration 200 nM, 3) 8 pL of human FcYRIHa-V158 or FcyRllla-F158 receptor at a test concentration dependent on the procedure used for single-point screening or ICS0determination, 4) 8 pL of the test antibody at a constant concentration < 20 nM. The dispensed plate is centrifuged for 30 s at 500 g to ensure that the added reagent mixtures reach the bottom of the wells of the multiwell plate. Subsequently, in step 5), 8 pL of an FLuc substrate mixture at constant concentrations of 1.25 mM D-luciferin and 30 mM ATP is added to the measurement wells. The plate is centrifuged again for 30 s at 500 g, and a bioluminescence detection protocol lasting several minutes is initiated using a multiwell plate reader capable of recording bioluminescence in the wavelength range of 500-700 nm.

[0195] Each test plate includes at least 8 positive control wells (addition of a reference IgG antibody instead of the FcyRllla-V158 / F158 receptor in step 3 and addition of buffer instead of the test IgG in step 4), defining the maximum bioluminescence signal of the assay, and at least 8 negative control wells (addition of buffer instead of the test antibody in step 4), defining the minimum bioluminescence signal of the assay. These controls allow determination of the required screening assay quality in terms of a Z-factor > 0.65.

[0196] In an IgG screening format, the remaining wells of the plate are occupied by duplicate measurements at one or two selected test concentrations (e.g., 10 pM and 100 nM) of each tested IgG antibody (single- or two-point screening). In an assay format for determining ADCC activity expressed as the IC50of IgG binding to Fc receptors, the remaining wells of the plate are allocated to duplicate 10-point concentration series to generate dose-response curves for each tested IgG antibody.

[0197] An example of the use of the ECHIA 1 B assay for determining the ADCC potential of IgG antibodies is confirmed by binding of known therapeutic monoclonal antibodies, lgG1 adalimumab and lgG4 pembrolizumab, to human FcyRllla-V158 / F158 receptors responsible for mediating ADCC responses.

Claims

Patent Claims1. A fusion protein comprising a polypeptide of the general formula:[N-terminal luciferase fragment] - [linker 1] - [protein A domain B], wherein:• [N-terminal luciferase fragment] denotes a peptide derived from a luciferase protein shown as SEQ ID NO: 1 , comprising at least 398 consecutive amino acids of said protein and including its N-terminus, preferably represented by SEQ ID NO: 2,• [linker 1] denotes a peptide having a length of at least 12 amino acids, preferably comprising SEQ ID NO: 5 and / or 61 or selected from SEQ ID NOs: 6-10 or 26-27, particularly from SEQ ID NOs: 6, 26-27,• [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably represented by SEQ ID NO: 4.

2. The fusion protein according to claim 1 , characterized in that it is a polypeptide of the general formula:[N-terminal luciferase fragment] - [linker 1] - [protein A domain B] - [linker 2] - [protein A domain B], wherein:• [N-terminal luciferase fragment] denotes a peptide derived from a luciferase protein shown as SEQ ID NO: 1 , comprising at least 398 consecutive amino acids of said protein and including its N-terminus, preferably represented by SEQ ID NO: 2,• [linker 1] denotes a peptide having a length of at least 12 amino acids, preferably comprising SEQ ID NO: 5 and / or 61 or selected from SEQ ID NO: 10 or 26-27,• [linker 2] denotes a peptide having a length of at least 12 amino acids, preferably comprising SEQ ID NO: 5 and / or 61 or selected from SEQ ID NOs: 13-14 or SI- 33,[protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably represented by SEQ ID NO: 4.

3. The fusion protein according to claim 1 or 2, characterized in that it further comprises a peptide facilitating isolation of the fusion protein, preferably selected from SEQ ID NOs: 16-17 or 34-37.

4. The fusion protein (NLuc-1 B) according to claims 1-3, characterized in that it is selected from SEQ ID NOs: 18-21 , 38, 59.

5. The fusion protein (NLuc-2B) according to claims 2-3, characterized in that it is selected from SEQ ID NOs: 22-23, 40, 48-54.

6. A fusion protein comprising a polypeptide of the general formula:[C-terminal luciferase fragment] - [linker 1] - [protein A domain B], wherein:• [C-terminal luciferase fragment] denotes a peptide derived from a luciferase protein shown as SEQ ID NO: 1 , comprising at least 132 consecutive amino acids of said protein and including its C-terminus, preferably represented by SEQ ID NO: 3,• [linker 1] denotes a peptide comprising at least one amino acid, particularly Gly, preferably comprising SEQ ID NO: 5 and / or 61 or selected from SEQ ID NO: 11 or 26-27, particularly from SEQ ID NOs: 26-27,• [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably represented by SEQ ID NO: 4.

7. The fusion protein according to claim 6, characterized in that it is a polypeptide of the general formula:[C-terminal luciferase fragment] - [linker 1] - [protein A domain B] - [linker 2] - [protein A domain B], wherein:• [C-terminal luciferase fragment] denotes a peptide derived from a luciferase protein shown as SEQ ID NO: 1 , comprising at least 132 consecutive amino acids of said protein and including its C-terminus, preferably represented by SEQ ID NO: 3,• [linker 1] denotes a peptide having a length of at least one amino acid, particularly Gly, preferably comprising SEQ ID NO: 5 and / or 61 or selected from SEQ ID NOs:• [linker 2] denotes a peptide having a length of at least 12 amino acids, preferably comprising SEQ ID NO: 5 and / or 61 or selected from SEQ ID NOs: 15 or 28-33,• [protein A domain B] denotes a peptide derived from staphylococcal protein A having affinity for antibody constant chains, preferably represented by SEQ ID NO: 4.

8. The fusion protein according to claim 6 or 7, characterized in that it further comprises a peptide facilitating isolation of the fusion protein, preferably selected from SEQ ID NOs: 16-17 or 34-37.

9. The fusion protein (CLuc-1 B) according to claims 6-8, characterized in that it is selected from SEQ ID NOs: 24, 39, 60 or 63.

10. The fusion protein (CLuc-2B) according to claims 7-8, characterized in that it is selected from SEQ ID NOs: 25, 41-47, 55-58 or 62.

11. A reagent kit comprising the fusion protein defined in claims 1-5 and the fusion protein defined in claims 6-10, and optionally further comprising a reagent constituting a substrate for luciferase and / or an antibody having affinity for the detected substance.

12. The kit according to claim 11 , characterized in that it comprises the fusion protein defined in claims 2-5 and the fusion protein defined in claims 7-10.

13. A method for determining the presence of a detected substance in a test sample, characterized in that it comprises the following steps: a) contacting in vitro the test sample with an antibody having affinity for the detected substance and with the reagent kit defined in claims 11-12, b) assaying luciferase activity in the presence of a reagent constituting a substrate for luciferase, c) detection of luciferase activity indicates the presence of the detected substance in the test sample.

14. A method for determining the presence of an antibody in a test sample, characterized in that it comprises the following steps: a) contacting in vitro the test sample with the reagent kit defined in claims 11-12, b) assaying luciferase activity in the presence of a reagent constituting a substrate for luciferase, c) detection of luciferase activity indicates the presence of the antibody in the test sample.

15. The method according to claim 13 or 14, characterized in that in step c) the intensity of light produced in the enzymatic bioluminescence reaction is measured and its value is considered proportional to the concentration of the detected substance, in particular an antigen, biomarker, or antibody.

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