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WO2009082781A1 - Méthode de dosage - Google Patents

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Publication number
WO2009082781A1
WO2009082781A1 PCT/AU2008/001919 AU2008001919W WO2009082781A1 WO 2009082781 A1 WO2009082781 A1 WO 2009082781A1 AU 2008001919 W AU2008001919 W AU 2008001919W WO 2009082781 A1 WO2009082781 A1 WO 2009082781A1
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WIPO (PCT)
Prior art keywords
reporter
analyte
interest
assay
mab
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PCT/AU2008/001919
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English (en)
Inventor
Chang-Yi Huang
Rachel De Las Heras
Scott Robert Fry
Jun Li
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Alere Switzerland GmbH
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Inverness Medical Switzerland GmbH
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Priority claimed from AU2008900006A external-priority patent/AU2008900006A0/en
Application filed by Inverness Medical Switzerland GmbH filed Critical Inverness Medical Switzerland GmbH
Priority to JP2010540989A priority Critical patent/JP2011508596A/ja
Priority to EP08866208A priority patent/EP2231868A4/fr
Priority to AU2008344987A priority patent/AU2008344987A1/en
Publication of WO2009082781A1 publication Critical patent/WO2009082781A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

Definitions

  • the present invention relates generally to the field of molecular biology and diagnostics. More specifically, the present invention relates to in vitro forced enzyme complementation, assays based on glow luminescence.
  • EIAs enzyme-linked immunosorbent, assays
  • ELISA enzyme-linked immunosorbent, assay
  • the principle drawback associated with heterogeneous EIAs is their lengthy processing time and requirement of multiple wash and incubation steps, necessitating specialized instrumentation for the purpose of automation.
  • Luminescence is broadly used herein to describe chemiluminescence and/or bioluminescence. ' Luminescent detection methodologies typically provide sensitivities that are up to 100-1000 fold higher than may be possible with fluorescent or eolorimetric enzyme substrates, respectively (Olesen et al., 2000, Methods Enzymol. 326: 175-202). As a result, luminescence provides a greater dynamic range (as illustrated in FIG. 1) with broad linearity and low inherent background (Fan and Wood, 2007, Assay Drug Dev. Technol. 5(1): 127-136).
  • homogeneous assays permit the formation and detection of an enzyme-labelled analyte complex (e.g. antibody-antigen complex) within a single solution, obviating the need for a solid support, allowing for more simple and rapid assays. Therefore, luminescent homogeneous EIAs present an alternative platform for diagnosis of disease in. a single reaction vessel with limited processing requirements and 17. The elimination of solid supports and washing to remove unbound label provides significant advantages to the application of homogeneous EIAs to chemistry analysers for high throughput, automated immunoassays.
  • enzyme-labelled analyte complex e.g. antibody-antigen complex
  • FEC forced enzyme complementation
  • PCA protein fragment complementation assays
  • Interactors are separate moieties or domains contained within each member of the fragment pair constructs, and include, but are not limited to, disease-specific antigen, antibodies or protein binding partners. Interactor domains may be joined directly to the reporter fragment domains or may be joined by a linker domain.
  • the protein fragment pair members are typically constructed so that, in the absence of the interactor domain target, the reporter fragments do not spontaneously functionally reassemble. Thus, the interactor domains of each member of the pair assist the reconstitution of a functional reporter protein from its fragments through binding of target to the interactor domains.
  • glow luminescence allows for kinetic determination of reaction rates and is intrinsically more sensitive than endpoint readings provided with flash luminescence. This preference for use of glow luminescence is evidenced by the current multi-mode microplate reader trends, which indicate that glow luminescence detection accounts for 13% of total detection modalities while flash luminescence is still in its infancy (HTStec 2007 Market Report). Flash luminescence is transient in nature and reaches maximal light intensity within seconds or milliseconds compared with glow luminescence, which is a steady-state kinetic approach to signal generation and is long lasting (minutes to hours). Other benefits of glow luminescence mostly relate to instrumentation, as flash luminescence requires reagent dispensing, mixing and measurement onboard the instrument.
  • Flash luminescence is performed on a well-by- well (well mode) basis due to the rapidity of the light emission; therefore limiting its utility for diagnostic purposes, as fluid injectors and detection throughput compromise assay flexibility.
  • glow luminescence reagents can be added outside of the reader and luminescent deterrninations can be made on the entire plate (plate mode). Injectors are not required, there is greater flexibility in plate processing and a single reagent addition and incubation can be performed. For this reason glow luminescence has been the detection method of choice for the ELISA market as glow luminescent detection is compatible with current EIA liquid handling instruments.
  • Glow luminescence also provides the added advantage of dual analyte detection by multiplexing response signals using multirnode detection; by combining color and luminescence; fluorescence and luminescence; flash and glow luminescence into a single reaction for in vitro purposes.
  • Luminescent reporter enzymes known as luciferases produce detectable photon emission as a result of the oxidation of luciferin to oxyluciferin in a process known generally as bioluminescence (Wilson et al., 1998, Annu. Rev. Cell Dev. Biol. 14:197- 230).
  • luciferases have been split and successfully used to determine protein- protein interactions via PCA; including, firefly Photinus pyralis luciferase in Paulmurugan et al., 2002, Proc. Natl. Acad. Sci. USA 99(24): 15608-15613 and Paulmuruga ⁇ et al., 2007, Anal. Chem.
  • Homogeneous assays are typically constituted of isolated, purified components in order to ensure specificity, reliability, manufacturing ease and robust characteristics in use.
  • the design of appropriate protein fragment pairs (including appropriate reporter fragments, interactor domains, Unking domains, and whole fusion constructs or reporter fragment pairs incorporating each) as- well as robust assay conditions that also produce the necessary solubility, stability, and amenability to manufacture for in vitro diagnostic use, is crucial to creating a broadly applicable homogeneous assay platform.
  • in vitro diagnostic assay conditions are extra-cellular and relatively harsh and may not be conducive to appropriate protein folding and protein-protein interactions mat may readily occur in vivo.
  • sample fractions containing analytes may be processed without washing, likely leaving homogeneous platforms, especially those based upon luminescent FEC, susceptible to cross-reactivity, interference and inhibitory effects due to serum components or other contaminants.
  • the present invention is based on the successful development of a sensitive homogeneous FEC assay using a split enzyme reporter system that produces a glow luminescent signal. We have demonstrated the use of such a system in detecting analytes with high sensitivity . (100%) and specificity (100%). Thus, we have demonstrated the successful application of an in vitro homogeneous FEC assay based on extended glow luminescence for the detection of a diagnostically relevant target. Further the timeframe required (approximately 1 hour) is suitable for uptake of this methodology into pathology laboratories. The present invention therefore provides significant advantages to the current immunodiagnostic field.
  • the present invention provides a method of dete ⁇ nining the presence of an analyte of interest in a sample which method comprises contacting the sample with:
  • a purified second reporter fragment pair member comprising an interactor domain with affinity for the analyte of interest and operable in reconstituting an active enzyme upon association with the first reporter fragment pair member through the affinities of the interactor domains of the first and second reporter fragment pair members with the analyte of interest; under in vitro assay conditions which allow the first and second reporter fragment pair members to associate in the presence of analyte of interest in the sample through the affinity of the interactor domains with the analyte to produce a glow luminescent signal through the action of the reconstituted active enzyme on a substrate; and detecting the presence or absence, or extent, of a glow luminescent signal.
  • the present invention provides a method of assaying for the presence of an analyte in a sample, the method comprising the steps of:
  • the active enzyme is a luciferase, preferably a Renilla luciferase or a derivative thereof, such as a luciferase comprising a C124A mutation.
  • the first and second reporter fragment pair members are alpha and omega fragments of a luciferase polypeptide formed by a breakpoint between amino acid residues serine 91 and tyrosine 92 or glycine 229 and lysine 230 of the Renilla luciferase amino acid sequence shown in SEQ ID NO: 10, or the equivalent position in another luciferase sequence.
  • one or more reducing agents such as DTT 5 preferably at a total concentration at from about 1 to about 2% w/v;
  • one or more denaturants such as thiourea, preferably at a total concentration of from about 1 to about 2% w/v;
  • one or more detergents such as CHAPS, preferably at a total concentration of from about 0.1 to about 0.2% w/v;
  • luciferase substrates namely a luciferase substrate selected from the group consisting of coelenterazine analogues f, h and n (see Figure 3).
  • tibe sample is a biological sample, such as a blood or serum sample.
  • the present invention provides a kit for performing a homogeneous in vitro forced enzyme fragment complementation assay to identify the presence of an analyte of interest in a sample, which kit comprises :
  • reagents for providing in vitro assay conditions which allow the first and second reporter fragment pair members to associate in the presence of analyte of interest in the sample, through the affinity of the interactor domains with the analyte, to produce a glow luminescent signal through the action of the active enzyme on a substrate, wherein a glow luminescent signal indicates the presence of the analyte of interest in the sample.
  • the present invention provides the use of a kit for determining the presence of an analyte of interest in a sample, for example an antibody to a pathogen such as Herpes Simplex Virus or a Hepatitis B virus.
  • a kit for determining the presence of an analyte of interest in a sample for example an antibody to a pathogen such as Herpes Simplex Virus or a Hepatitis B virus.
  • the present invention provides an assay reagent storage composition comprising (i) one or more reporter polypeptides at a total concentration of greater than about 10 nM and (ii) one or more sugars and/or one or more inert proteins.
  • the one or more reporter polypeptides are luciferase reporter polypeptides for an in vitro forced enzyme complementation assay.
  • the one or more sugars are selected from the group consisting of sucrose, trehalose and mixtures thereof.
  • the one or more inert proteins are selected from the group consisting of serum albumin, gelatine and mixtures thereof.
  • the present invention provides the use of a composition comprising one or more sugars and/or one or more inert proteins to stabilise one or reporter polypeptides in an assay reagent storage composition comprising the one or more reporter polypeptides at a total concentration of greater than about 10 nM.
  • FIG. 1 Comparison between the colorimetri , fiuorometric and luminescent analyte signal detection range.
  • FIG. 2. Bioluminescent reaction catalysed by Renilla luciferase.
  • FIG. 3. Native coelenterazine synthetic derivatives (A-H) that differ from each other in terms of luminescence intensity and response time.
  • FIG. 4 Renilla luciferase model structure illustrating the position of two functional split points, S91/Y92 (A) and G229/K230 (B).
  • FIG. 5 Schematic illustration of Renilla luciferase FEC assay and its basic components. Enzyme fragments, ⁇ and co, are linked to analyte binding moieties such as a disease-specific antigenic peptide (P) and one domain of protein G. In the presence of analyte (disease-specific antibody), the fragments are forced into close proximity (right), thereby initiating catalysis of a bioluminescent reaction.
  • P disease-specific antigenic peptide
  • analyte disease-specific antibody
  • FIG. 6 Schematic representation of FEC fragments (A and B) used in this study.
  • RLuc refers to Renilla Luciferase alpha ( ⁇ blue) and omega ( ⁇ green) fragments.
  • Analyte binding moieties red
  • ProG refers to the protein G domain
  • P P (Pl and P2) refers to the HSV type-specific antigenic peptides for HSV-I and HSV-2 respectively. Histidine tags are shown in grey. (Gly 4 Ser) linkers were used to join the enzyme fragments to the analyte binding moieties.
  • FIG. 7 Purification of ⁇ protein fragments (RLuC a 229 Pl and RLuc_ ⁇ 229 P2) of the invention.
  • FIG. 8 Purification of ⁇ protein fragment (RLuc_ 23 o ⁇ ProG) of the invention.
  • FIG. 10. Comparison of the use of (A) a commercial Renilla Luciferase 5 x Promega lysis buffer (proprietary composition) with (B) optimised 5 x PB enhancer buffer C (150 niM HEPES pH 8, 1% CHAPS, 5% thiourea and 25% glycerol) of the invention.
  • FIG. 11 Luciferase FEC assay for HSV-I specific IgG.
  • Sensitivity 100 %.
  • Specificity 100 %.
  • the normalized enzyme activity is represented as a box and whiskers plot for each set of 5 patient samples.
  • SEQ ID NO: 7 Nucleotide sequence of Protein G subunit.
  • SEQ ID NO: 8 Amino acid sequence of Protein G subunit.
  • SEQ ID NO: 9 Nucleotide sequence of synthetic/codon optimised Renilla luciferase (RLuc) reporter enzyme incorporating a C124A mutation.
  • SEQ ID NO: 10 Amino acid sequence of synthetic/codon optimised Renilla luciferase (RLuc) reporter enzyme incorporating a C124A mutation.
  • SEQ ID NO: 20 RIUC-(X 91 PI 5 Renilla luciferase alpha fragment (split at S91/Y92) Pl trunc construct amino acid sequence.
  • SEQ ID NO: 22 RLucjirfl, Renilla luciferase alpha fragment (split at G229/K230) Pl trunc construct amino acid sequence.
  • SEQ ID NO: 23 Rluc_ ⁇ 91 P2, Renilla luciferase alpha fragment (split at S91/Y92) P2 trunc construct nucleotide sequence.
  • SEQ ID NO: 24 Rluc_ ⁇ 91 P2, Renilla luciferase alpha fragment (split at S91/Y92) P2 trunc construct amino acid sequence.
  • SEQ lD NO: 28 RLuc ⁇ ProG, Renilla luciferase omega fragment (split at S91/Y92) Protein G s ⁇ bunit construct amino acid sequence.
  • HBeAg Hepatitis B surface antigen
  • scFv single chain construct nucleotide sequence
  • HsAg Hepatitis B surface antigen
  • scFv single chain construct.amino acid sequence.
  • HBeAg Hepatitis B surface antigen
  • scFv Ell single chain construct nucleotide sequence
  • HBeAg Hepatitis B surface antigen
  • scFv Ell single chain construct amino acid sequence
  • SEQ ED NO: 35 RLuc_ ⁇ 9 iA5, Renilla luciferase omega fragment (split at S91/Y92) Hepatitis B surface antigen (HBsAg) A5 single chain (scFv) construct nucleotide sequence.
  • SEQ ID NO: 36 RLucjX9iA5, Renilla luciferase omega fragment (split at S91/Y92) Hepatitis B surface antigen (HBsAg) A5 single chain (scFv) construct amino acid sequence.
  • SEQ ID NO: 37 RLuc_ 92 ⁇ El 1, Renffla luciferase omega fragment (split at S91/Y92) Hepatitis B surface antigen (EIBsAg) Ell single chain (scFv) construct nucleotide sequence.
  • SEQ ID NO: 38 RLuc_jj 2 coEll. Renilla luciferase omega fragment (split at S91/Y92) Hepatitis B surface antigen (HBsAg) Ell single chain (scFv) construct amino acid sequence.
  • the assay methods of the present invention use reporter components that comprise or consist of polypeptides that are adapted for use as isolated and purified components of homogeneous in vitro FEC assays.
  • the reporter components form part of a reporter system.
  • the reporter system in FEC assays includes two or more polypeptide fragments that when they associate, form a reporter protein complex that can give rise to a detectable signal.
  • each reporter component includes at least one such polypeptide fragment or subunit (herein termed a "reporter fragment").
  • a first reporter component may comprise an alpha fragment of an enzyme, such as a luciferase and a second reporter component may comprise an omega fragment of an enzyme, such as a luciferase such that when the two reporter components associate under assay conditions, the resulting complex has luciferase activity.
  • a combination of a first reporter component and a second reporter component which together can give rise to detectable enzyme activity when their respective reporter polypeptide fragments (also termed "subunits") are brought into association, is termed herein a "reporter component pair", the respective reporter polypeptide fragments being collectively termed a "reporter fragment pair”.
  • luciferases have been split and successfully used to determine protein- protein interactions via FEC; including, firefly Photinus pyralis luciferase in Pauhnurugan et al., 2002, Proc. Natl. Acad. Sci. USA 99(24): 15608-15613 and Pauhnurugan et al., 2007, Anal. Chem. 79(6): 2346-2353, sea pansy Renilla remiformis luciferase in Pauhnurugan et al., 2003, Anal. Chem. 75(7): 1584-1589 and Kaihara et al., 2003, Anal. Chem.
  • luciferases have been modified to alter the wavelength of light emitted (see for example US Patent No. 6,495,355 which describes a red-shifted luciferase) and/or to improve in. vitro characterstics.
  • the luciferase variants are within the scope of the present invention.
  • Renilla remiformis luciferase (EC 1.13.12.5) which is responsible for the production of light via oxidation of coelenterazine (FIG. 2) is particularly suitable for application to the FEC format as it is small (36 fcDa), monomeric and contains only three cysteines, none of which form disulphide bonds (Matthews et al., 1977, Biochemistry 16(1): 85-91).
  • split bioluminescent reporter enzymes An alternative strategy to the use of split bioluminescent reporter enzymes as previously described is the use of split reporter enzymes that do not traditionally catalyse chemiluminescent reactions, by combining these with synthetically produced luminescent substrates or pro-luminescent substrates, for example, pro-luciferin (Fan and Wood, 2007, Assay Drug Dev. Technol. 5(1): 127-136) glow luminescence can be produced.
  • Several patents disclose the synthesis and use of luminescent ⁇ -lactamase substrates, for example, those disclosed in; US Patents 5,955,604, 7,018,802 B2 and published US Patent Application 20060292656 Al.
  • Bluco is a pro-luciferin in which the 6-hydroxy group of D-luciferin is coupled to a cephalosporin through an ether bond, ⁇ -lactamase acts to open the ⁇ -lactam ring resulting in spontaneous fragmentation allowing firefly luciferase to subsequently act on the luciferin resulting in light emission.
  • the assay methods of the present invention are not limited to reporter polypeptides based on bioluminescent proteins such as luciferases, but also include the use of any reporter polypeptides that can generate a luminescent signal in the presence of an appropriate substrate, herein termed a luminescence reporter polypeptide.
  • reporter polypeptides that can be used as the basis for reporter fragments and systems include bioluminescent proteins such as luciferases (e.g. firefly and Renilla luciferases) and beta-lactamase (e.g. TEM-I beta- lactamase: EC: 3.5.2.6).
  • the reporter polypeptides are typically split into two fragments which when they associate can reconstitute the activity of the original full length polypeptide.
  • luciferase and beta-lactamase are typically split into two fragments, an alpha and an omega fragment. Suitable breakpoints in the amino acid sequences of these various proteins to generate the two fragments have been described previously.
  • Reporter polypeptides/fragments are selected so that they are suitable for in vitro use (for the avoidance of doubt, in the present context the term in vitro means that the assays take place outside of living cells).
  • the reporter polypeptides are typically variants of wild type sequences that have amino acid changes that improve their suitability for in vitro use, for example to enhance stability, improve solubility and/or reduce aggregation.
  • the reporter polypeptides/fragments have reduced sensitivity, compared to wild type polypeptides, to inhibitors, such as enzyme inhibitors, of the activity of the polypeptide required for reporter function, e.g. beta lactamase activity.
  • inhibitors include compounds found in samples, e.g.
  • inhibitors are inhibitors of beta-lactamase activity found in blood as a result of administering antibiotics to individuals from whom the blood samples are taken.
  • the association of the reporter fragments to form an active complex having reporter activity is typically mediated by the interaction between other regions of the reporter components and a target analyte.
  • the reporter components typically comprise an interactor moiety or domain.
  • Interactor domains have binding specificity for a target analyte of interest.
  • Interactor domains include, for example, peptides, glycoproteins, polysaccharides, antigens, antibodies and antigen-binding fragments of antibodies such as complementarity determining regions (CDRs).
  • Antigens include antigens derived from pathogens, such, as viral or bacterial antigens.
  • Antibodies/CDRs include sequences that bind to antigens derived from pathogens, such as viruses or bacteria.
  • interactor domains include the IgG-binding domain of Protein G, and Herpes Simple Virus antigens (particularly preferred versions of which are truncated glycoprotein Gl envelope proteins from HSVl or truncated glycoprotein G2 envelope proteins from HSV2, such as amino acids 92 to 148 of gGl or 551 to 641 of gG2).
  • Interactor domains may be joined directly to the reporter fragments or via a linker.
  • Suitable linker domains include peptides, such as glycine rich repeat sequences (e.g. G 4 S repeat sequences - i.e. GGGGS sequence repeats).
  • the number of linker domains for example, G 4 S domains may be 1, 2, 3, 4, 5, 6, or more.
  • the number of glycine rich repeat sequences is preferably 2 or 3, particularly where the interactor domain is a polypeptide having fewer than 150 or 100 amino acids. Where larger interactor domains are used, it may be desirable to increase linker length.
  • the linker domain is flexible. In another embodiment the linker domain is rigid.
  • linker domain sequence and/or number of linker domain repeats for any particular interactor and reporter fragment construct and fragment pairing is within the routine level of experimentation by the skilled artisan.
  • Reporter fragment polypeptides, linker domains and interactor domains may be joined by covalent or non-covalent means to form a reporter component.
  • the linker domains and interactor domains are polypeptides.
  • the reporter component may be a single polypeptide.
  • the reporter fragments are united to interactor domains by conjugation e.g. covalent coupling via, for example, thiol-thiol, arnine-carboxyl or amine-aldehyde functional groups.
  • conjugation e.g. covalent coupling via, for example, thiol-thiol, arnine-carboxyl or amine-aldehyde functional groups.
  • Particular examples include cross-linking of polypeptides to glycoproteins via the carbohydrate groups; cross-linking via primary amines(found at the N-terminus and on lysine residues) e.g.
  • heterobifunctional cross linkers with an amine reactive group and a sulfhydryl reactive group; cross- linking via carboxyl groups (found at C-terminus and as side groups on glutamic acid and aspartic acid residues); cross-linking via free sulfhydryl groups; and disulphide exchange.
  • Non-covalent methods include avidin-biotin systems and hybridization of oligonucleotide-protein conjugates.
  • a peptide/polypeptide/protein is said to be "isolated” or “purified” when it is substantially free of cellular material or free of chemical precursors or other chemicals.
  • the peptides may be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use.
  • the intended use in the present invention is as operable components of homogenous in vitro FEC assays for specific analytes.
  • substantially free of cellular material includes preparations of the peptide having less than about 30% (by dry weight) of other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.
  • other proteins i.e., contaminating protein
  • the peptide When the peptide is recombinantly produced, it may also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis, hi one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of the enzyme peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
  • the isolated reporter fragment and polypeptide reporter components may be purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. For example, a nucleic acid molecule encoding the enzyme peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. Suitable host cells are described in more detail below.
  • the protein may then be isolated from the cells by an appropriate purification scheme using appropriate protein purification techniques. Exemplary techniques of the invention are described in detail in the Examples set out below.
  • the reporter polypeptides are proteins that consist of the amino acid sequences provided herein.
  • a protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.
  • the present invention further provides proteins that consist essentially of the amino acid sequences provided.
  • a protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues that do not alter the functional characteristics of the proteins of the invention.
  • the reporter polypeptides are proteins that comprise the amino acid sequences provided.
  • a protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein.
  • the protein may be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are associated with it or heterologous amino acid residues or peptide sequences.
  • additional amino acid molecules such as amino acid residues (contiguous encoded sequence) that are associated with it or heterologous amino acid residues or peptide sequences.
  • Such a protein may have a few additional amino acid residues or may comprise several hundred or more additional amino acids.
  • the reporter polypeptides used in the present invention may be attached to heterologous sequences to form chimeric or fusion proteins.
  • Such chimeric and fusion proteins may comprise an enzyme peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the enzyme peptide. "Operatively linked" indicates that the enzyme peptide and the heterologous protein are fused such that the operability of each is not destroyed.
  • the heterologous protein may be fused to the N-terminus or C4erminus of the enzyme peptide.
  • a chimeric or fusion protein may be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques.
  • the fusion gene may be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification or ligation of gene fragments may be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which may subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al, Current Protocols in Molecular Biology, 1998).
  • many expression vectors are commercially available that already encode a fusion moiety.
  • An enzyme peptide- encoding nucleic acid may be cloned into such an expression vector such that the fusion moiety is linked in-frame to the enzyme peptide, which is one means by which the fusion protein is made without destroying the operability of each component. .
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and a second amino acid or nucleotide sequence for optimal alignment and non-homologous sequences may be disregarded for comparison purposes).
  • at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the molecules are identical at that position and the position is homologous in the two.
  • the percent identity, between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • pairwise alignments and levels of sequence identity and homology may be determined using the BestFit program in the GCG software package.
  • the percent identity between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch (J. MoI. Biol. 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package.
  • the algorithm is typically employed using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences may be determined using the GAP program (Devereux, J., et al, Nucleic Acids Res. 12(1):387 (1984)) with a NSWga ⁇ dna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a substitution at a position in an amino acid sequence or peptide is indicated by the one letter designation for the amino acid, followed by the position number of the relevant non-substituted sequence or peptide, followed by one or more one letter designations for replacement amino acids.
  • substitution of Threonine for Valine at position 74 of the ⁇ fragment of TEM-I Beta-lactamase would be designated as V74T. Similar designations will be clear from the context and further details provided herein.
  • Reporter polypeptide fragments for use in the assays of the invention may comprise amino acid sequence changes or modifications that improve the suitability of the reporter polypeptide for use in such assays, hi particular, in a preferred embodiment, at least one of the reporter polypeptide fragments comprises a variation in or modification to its amino acid sequence which renders a reconstituted active reporter polypeptide complex less susceptible to inhibition by substances that are inhibitors of the unmodified (e.g. wild type) amino acid sequence, especially substances that are found in samples to be tested, such as biological samples including serum.
  • unmodified amino acid sequence especially substances that are found in samples to be tested, such as biological samples including serum.
  • variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
  • variants of altered function may also contain substitution of similar amino acids that result in no change or an insignificant change in function. In one embodiment, variants contain 1, 2, 3, 4 or 5 amino acid changes.
  • Beta-lactamase peptide sequences comprising single amino acid substitutions or multiple substitutions in combination that are especially adapted for use in homogeneous in vitro FEC assays as further exemplified herein.
  • Specific examples of such substitutions are substitutions at amino acid position 69 (preferably M69L or M69I) in the alpha fragment, which reduces inhibition by beta-lactamase inhibitors and substitutions at amino acid position 276 (preferably N276D) in the omega fragment, which also reduces inhibition by beta- lactamase inhibitors.
  • Other examples are selected from substitutions at one or more of amino acid positions 74, 182, 208, 211 and 230 (preferably one or more of V74T, M182T, I208T, M211Q and F230Y).
  • Renilla luciferase stability and light output by mutation of cysteine at position 124 to alanine (Liu et al., 1999, Gene 237: 153-159). Additional improvements to Renilla luciferase stability and light output can be achieved with the use of several supplementary mutations (Loening et al., 2006, Protein Eng. Des. SeI. 19(9): 391-400 and Loening et al. 5 2007, Nat. Methods 4(8): 641-643) when used in combination with analogs of coelenterazine that vary in their light intensity and response times (FIG. 3).
  • Modified reporter fragments having improved properties for use in in vitro assays can be obtained using various techniques. For example, sequence changes may be introduced by site-directed mutagenesis. The selection of suitable sites may, for example, be guided by the primary amino acid sequence and/or secondary/tertiary structural information included structural information determined by techniques such as x-ray crystallography or NMR. For example, the 3D structure of the active site of an enzyme can be used to assist in designing variants which have reduced susceptibility to inhibition (see for example, the crystal structure of TEMl as described in Jelsch, C 5 F. Lieri, et ah, 1992, FEBS Lett 299(2): 135-42)
  • Modified reporter fragments having improved properties can also be obtained by techniques such as random mutagenesis or directed molecular evolution followed by selection of variants having the desired properties (e.g. by testing variants for enzyme activity in the presence of an inhibitor of the unmodified protein).
  • Additional modification useful in the present invention may include sequences containing amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
  • peptides and constructs for use in the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-protein sequence.
  • a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or
  • the present invention is directed to in vitro FEC assays to detect analytes of interest in samples where the presence or absence of the analyte is measured by detecting a glow luminescence signal.
  • the method typically involves contacting the sample with luminescence reporter components, as described above, which are capable of reconstituting an enzyme activity in the presence of an analyte of interest in a sample, which enzyme activity can act on a suitable substrate present in the reaction mixture to give rise to a luminescence signal.
  • luminescence reporter components as described above, which are capable of reconstituting an enzyme activity in the presence of an analyte of interest in a sample, which enzyme activity can act on a suitable substrate present in the reaction mixture to give rise to a luminescence signal.
  • active enzyme is produced which acts on the substrate in the reaction mixture to generate a luminescent signal. This signal is then detected.
  • the in vitro assay conditions are such that the signal generated includes glow luminescence, as opposed to flash luminescence.
  • the assay conditions used need not necessarily exclude the possibility that a flash luminescence signal is generated in addition to the detectable glow luminescence signal, and in some embodiments both types of signals may be generated in the presence of the analyte of interest.
  • Analytes of interest include those present in environmental or biological samples. Biological samples include whole blood, serum, saliva and urine.
  • Hn vitro is taken to mean in the present context that the assays are conducted outside of living cells, such as in cell-free assays.
  • Analytes of interest include antibodies, such as antibodies to pathogens, antigens, such as antigens derived from pathogens, clinical markers such as hormones and host molecules e.g. prostate-specific antigen and other disease-related diagnostic marker molecules.
  • pathogen includes viruses, bacteria and prions (e.g. Herpes Simplex Viruses and Hepatitis virus such as HBV and HCV).
  • Suitable luminescence reporter components are described in detail above.
  • the substrate used will depend on the type of enzyme activity reconstituted by the association of the luminescence reporter components in the presence of an analyte of interest and is selected so that the action of the reconstituted enzyme on the substrate generates a luminescent signal.
  • the substrate in the case of biologically-derived luciferases such as Ren ⁇ lla, the substrate is typically coelenterazine or an analog thereof, such as one of the analogs shown in Figure 3, with coelenterazine analogues f, h and n being particularly preferred.
  • the substrate is typically luciferin or an analog thereof.
  • Suitable chemiluminescent substrates can also be designed for non- bioluminescent enzymes such as beta-lactamase, for example, pro-luciferin (Fan and Wood, 2007, Assay Drug Dev. Technol. 5(1): 127-136).
  • beta-lactamase for example, pro-luciferin
  • Several patents disclose the synthesis and use of luminescent ⁇ -lactamase substrates, for example, those disclosed in; US Patents 5,955,604, 7,018,802 and published US Patent Application 20060292656.
  • ⁇ luco' was described in the literature in Yao et al., 2007, Angew. Chem. Int. Ed. 46: 1-5.
  • Bluco is a pro-luciferin in which the 6-hydroxy group of D-luciferin is coupled to a cephalosporin through an ether bond, ⁇ -lactamase acts to open the ⁇ -lactam ring resulting in spontaneous fragmentation allowing firefly luciferase to subsequently act on the luciferin resulting in light emission.
  • the amount of reporter component required for an in vitro assay is generally higher than mat found in vivo.
  • the concentration of reporter components, such as polypeptides, in the reaction mix may be at least 1 pM, such as at least 10 or 100 pM, or 1 or 10 nM.
  • Reducing agents the inclusion of one or reducing agents was found to be important for the generation of an extended glow luminescence signal.
  • Reducing agents as referred to herein are able to act reversibly on protein disulphide bonds and therefore include thiol reducing agents such as DTT and related compounds as well as 2-mercaptoethanol and related compounds and phosphines and related compounds.
  • the preferred total concentration of reducing agents is from about 1 to about 2% w/v. However, the optimum concentration can be determined empirically for a given system e.g. using the procedures described in the examples.
  • Denaturants one or more denaturants are preferably included to reduce the natural affinity of the reporter enzyme fragments for each other (spontaneous complementation). Denaturants include urea, thiourea and guanidine hydrochloride, with thiourea being particularly preferred.
  • the total concentration of denaturants may be from about 1 to about 2% w/v, such as from about 1.3 to about 1.6 w/v. However, again, the optimum concentration can be determined empirically for a given system e.g. using the procedures described in the examples.
  • detergents are preferably included to reduce protein-protein interactions, particularly self-association of reporter fragments.
  • Preferred detergents are nondenaturing zwitterionic detergents such as aminosulphobetaine-14, 3-[(3-Cholamidopropyl) dimethylammonio]-l -propane sulfonate (CHAPS) 5 [(3-[3-Cholamidopropyl) dimethylammonio]-2-hydroxy-l-propane sulfonate (CHAPSO) and other sulphobetaine derivatives of cholic acid, preferably at a total concentration of from about 0.1 to about 0.2% w/v.
  • the optimum concentration can be determined empirically for a given system e.g. using the procedures described in the examples.
  • salt this is preferably included at a concentration from from about 0.25 M to 0.5 M.
  • NaCl is a preferred example but other salts may be used.
  • the optimum concentration can be determined empirically for a given system e.g. using the procedures described in the examples.
  • Tris.HCl as a buffer. This was shown to give improved results compared with sodium or potassium phosphate buffers.
  • the concentration of Tris.HCl used is typically in the range of from about 20 mM to about 200 mM, such as about 50 mM.
  • the pH of the reaction mix is preferably from about 6.5 to about 8.0, such as from about 6.8 to about 7.5. The optimum concentration and pH can be determined empirically for a given system e.g. using the procedures described in the examples.
  • Metal chelators one or more metal chelators such as EDTA and variants thereof, e.g. EGTA. This is especially relevant where samples to be tested, such as blood samples, include metal ions. Typically total concentrations of metal chelators are from about 1 mM to 10 mM, such as from 7 mM to 9 mM, e.g. about 5 mM. The optimum concentration can be determined empirically for a given system e.g. using the procedures described in the examples.
  • Light emission resulting from the presence of analyte of interest can be detected using methods familiar to those skilled in the art, such as a luminometer.
  • the various assay conditions described above are preferably selected so as to provide for an extended period of glow luminescence, in the presence of an analyte of interest, of at least 5 minutes, more preferably at least 10 minutes.
  • Rates of light emission are relative to the integration time, fragment concentration and substrate concentration, among other factors. However, preferred rates of light emission are greater than 1 mRLU/min for kinetic determinations, such as greater than 2, 5, 10, 20 or 50 mRLU/min; greater than 1 RLU for endpoint determinations, such as greater than 2, 5 or 10 RLU. Preferred signal-to-noise ratios are greater man 2 for both kinetic and endpoint detection modalities, such as greater than 5 or 10.
  • the methods of the invention may be applied to both in vitro homogenous assays and in vitro heterogeneous assays, the latter being where at least one reporter fragment or reporter component is immobilized to a solid phase.
  • the assays are conducted in parallel in a plurality of reaction vessels, such as in different wells of a microliter plate.
  • assays may be conducted substantially simultaneously in at least 6 wells, such as in at least 10 or 12 wells.
  • the generation of an extended glow luminescence signal improves the ability to then read the luminescence signal in all of the wells before the signal has decayed.
  • multiple analytes are detected in the same reaction using different reporter components which generate distinct luminescent signals, e.g. a dual glow signal, such as a glow signal produced by firefly luciferase and a glow signal produced by Renilla luciferase. Simultaneous detection of two independent analytes within a single sample is known as multiplexing.
  • multiple analytes are detected in the same reaction using different reporter components, at least one of which can give rise to an extended glow luminescent signal and at least one of which gives rise to a non-luminescent signal, such as a colorimetric signal.
  • the present invention also provides reporter fragments and/or reporter components as kits together with other kit components required for the generatation of a glow luminescence signal in the present of an analyte of interest.
  • kits include the reporter components described above together with reagents for providing in vitro assayconditions which allow the reporter components to associate in the presence of analyte of interest in the sample, through the affinity of the interactor domains of the reporter components with the analyte, to produce a glow luminescent signal through the action of the resulting active enzyme on a substrate.
  • the reagents include one or more of the various components described in items (i) to (vi) above. Ih one embodiment, the reagents are provided as a concentrated reaction buffer which is diluted prior to use, e.g. a 2X to 2OX concentrate, such as a 5X to 1OX concentrate.
  • the present invention provides an assay reagent storage composition comprising (i) one or more reporter polypeptides at a total concentration of greater than about 10 nM and ( ⁇ ) one or more sugars and/or one or more inert proteins.
  • the sugars and/or inert proteins are used to stabilise/extend the half-life of reporter polypeptides during storage, e.g. at about 4 0 C, and optionally also to reduce reporter polypeptide aggregation.
  • Reporter polypeptides in the context of the storage compositions of the invention include both luminescent reporter polypeptides as described above and other types of reporter polypepides.
  • at least one reporter polypeptide or reporter polypeptide pair is a luminescent reporter polypeptide, such as a luciferase reporter polypeptides for an in vitro forced enzyme complementation assay. Ih a particular embodiment the luciferase is aRenilla luciferase.
  • the one or more reporter polypeptides are present in the compositon at a total concentration of greater than about 10 nM, such as greater than about 20, 30, 40 or 5OnM.
  • the one or more sugars are preferably selected from the group consisting of sucrose, trehalose and mixtures thereof. Sucrose is particularly preferred.
  • the total concentration of the one or more sugars is at least about 50 mM, such as at least about 100, 250 or 500 mM.
  • the total concentration of the one or more sugars may be about 5 M or less, such as about 2.5 M or less. Ih one embodiment the total concentration of the one or more sugars is from 1 M to 2.5 M.
  • the one or more inert proteins is selected from the group consisting of serum albumin, gelatin and mixtures thereof.
  • the term "inert protein” means that the protein does not react with the reporter polypeptides, analytes of interest or substrates.
  • the serum albumin is preferably bovine serum albumin.
  • gelatin is fish gelatin.
  • the total concentration of the one or more inert proteins is at least about 0.2 mg/ml, such as at least about 0.4 or 0.5 mg/ml.
  • FEC assays derived from alternative breakpoint positions of RLuc also give rise to operable fragments, for example, breakpoint between serine at position 91 and tyrosine at position 92 (also demonstrated in the invention).
  • operable fragments for example, breakpoint between serine at position 91 and tyrosine at position 92 (also demonstrated in the invention).
  • operative FEC assays FIG. 5 can be designed as functionally required.
  • the functionality of the engineered fragments in vitro relies principally on the reaction conditions and composition, without which glow luminescent detection and relevant FEC signal to noise ratios cannot be obtained.
  • Example 1 Homogeneous in vitro Renilla luciferase FEC assays for antibody detection using glow luminescence.
  • a codon optimised gene encoding full-length RLuc incorporating the C124A mutation (SEQ ID NOS 9 and 10) for enhanced folding and stability was synthesised by DNA2.0 (Menlo Park, U.S. A) for optimal E.coli expression.
  • Synthetic pJ36-RLuc plasmid (2 ⁇ g) was supplied on a filter disk that was purified using the QIAprep spin miniprep kit (Qiagen, Australia) by incubating the filter disk in appropriate buffer, applying the supernatant to a QIAprep spin column and completing the purification according to the manufacturer's instructions.
  • Purified plasmid was transformed into XLl -Blue competent cells (Stratagene, c/o Integrated Sciences, Australia) according to the manufacturer's instructions. Colonies were screened to confirm the presence of the plasmid and sequence integrity was checked by DNA sequencing (using primers RLucl and RLuc2 - Table 2).
  • RLuc was excised from pJ36-RLuc by digestion with Ndel and Xhol restriction endonucleases (New England Biolabs, c/o Genesearch Pty Ltd, Australia) and ligated with Ndel and Xhol digested pET-26b(+) vector (Novagen, Australia), a prokaryotic expression vector that allows for inducible expression of recombinant proteins in E. colL Ligated plasmids were transformed into BL21-Gold (DE3) competent cells (Stratagene) according to the manufacturer's instructions.
  • RLuC-(Xg 1 (residues 2-91) was amplified using forward primer RLuc3 and reverse primer RLuc4.
  • RLuc_(X229 (residues 2-229) incorporating the C124A mutation was amplified using forward primer RLuc3 and reverse primer RLuc7.
  • Amplification products were purified directly from the reaction tube using the QIAquick PCR purification kit (Qiagen) and were subsequently digested with NdeVKp ⁇ l and cloned into- each NdeVKpnl digested universal pET- ⁇ Pl and pET- ⁇ P2 plasmids encoding a C- terminal HSV-I and HSV-2 specific cassette [(G 4 S) 3 linker, truncated HSV antigen (Pl and P2), G 4 S linker and histidine tag (6H)], yielding pET-RLuc_ ⁇ 91 Pl (SEQ ID NOS 19 and 20), pET-RLuc_ ⁇ 229 Pl (SEQ ID NOS 21 and 22), pET-RLuc_ ⁇ 91 P2 (SEQ ID NOS 23 and 24) and pET-RLuc_ ⁇ 229 P2 (SEQ ID NOS 25 and 26) respectively.
  • Qiagen QIAquick PCR purification kit
  • Ligated plasmids were transformed into BL21-Gold (DE3) competent cells and selected colonies from each transformation were screened to confirm the presence of the cloned insert. Positive clones were again confirmed by DNA sequencing (using primers RLucl andRLuc2 - Table 2). Construction ofpET-KLuc_co 92 ProG and pET-RLuc_c ⁇ 23 ⁇ ProG
  • RLuc_ 92 to (residues 92-311) incorporating the C124A mutation was amplified using forward primer RLuc5 and reverse primer RLuc ⁇ .
  • RLuc_2 30 ⁇ (residues 230-311) was amplified using forward primer RLuc8 and reverse primer RLuc6.
  • Amplification products were digested with NdeVXho ⁇ and each were cloned into NdeVXhol digested universal pET- ⁇ ProG plasmid encoding an N-terminal protein-G subunit specific cassette [histidine tag (6H), G 4 S linker, protein-G subunit (ProG) and (G 4 S) 3 linker], yielding pET-RLuc_9 2 ⁇ ProG (SEQ ID NOS 27 and 28) and pET-RLuc_ 230 ⁇ ProG (SEQ ID NOS 29 and 30).
  • Ligated plasmids were transformed into BL21-Gold (DE3) competent cells and selected colonies from each transformation were screened to confirm the presence of the cloned insert by sequence confirmation (using primers RLucl and RLuc2 - Table 2).
  • universal ⁇ Pl/2 and ⁇ ProG fusion genes were synthesized by DNA2.0 (Menlo Park, U.SA) incorporating restriction endonuclease sites located between the enzyme fragment, linkers and binding moieties to enable the substitution of various domain sequences.
  • the universal ⁇ Pl/2 construct, pET- ⁇ Pl/P2 was designed to have Kpnl, BamHL and Spel sites between the ⁇ sequence, (G 4 S)S linker and antigen encoding moieties with Ndel and Xho ⁇ sites on either end for ligation into the NdeVXhol site of pET-26b(+).
  • the universal ⁇ ProG construct pET- ⁇ ProG
  • pET- ⁇ ProG was designed to have Bam ⁇ l, Spel and Nhel sites inserted between the ProG, (G 4 S)S linker and ⁇ encoding domains with Ndel and Xhol sites on either end for ligation into the NdeVXhol site of pET-26b(+).
  • the sequence encoding the C2 IgG-binding domain of Streptococcus strain G148 Protein G was obtained by back-translation of the published amino acid sequence (G ⁇ lich et al., 2002, Protein Eng. 15(10): 835-42) with an E. coli codon usage table, using Vector NTI (Thvitrogen).
  • the locations of flanking restriction endonuclease sites placed in between domains are indicated as underlined font in the attached sequences of the invention (SEQ ID NOS 19, 21, 23, 25, 27 and 29).
  • HSV-I specific antigen SEQ ID NOS 1, 2 and 3
  • gGl glycoprotein Gl
  • This region of gGl contains an immunodorninant epitope (residues 112-127), and two key amino acids within a second epitope known to confer an HSV type-1 specific response in humans.
  • the HSV-2 specific peptide (SEQ ID NOS 4, 5 and 6) is composed of residues 551-641 of glycoprotein G2 (gG2) and is comprised of two immunodominant epitopes (residues 561-578 and 626-640) known to confer an HSV type-2 specific response in humans.
  • the pellet from a 250 ml overnight induction was lysed in 10 ml/g (wet pellet weight) of lysis buffer (6 M GuHCl, 100 mM sodium phosphate, 200 mM L-arginine, 20 mM imidazole, 2 mM DTT pH 8). Following a 1-hour incubation at 4 0 C with shaking at 100 rpm, each suspension was sonicated in an ice bath for 5 cycles of 30 seconds on/30 seconds off (70% duty cycle) using a Branson 250 sonifier. Soluble protein was recovered by centrifugation at 18,514 g at 4 0 C for 30 min. Supernatant (10 ml) was passed through a 0.2 ⁇ m filter.
  • Immobilised metal affinity chromatography IMAC
  • ELuc_aP2 ELuc_aP2
  • BLucjaProG Immobilised metal affinity chromatography
  • Bound protein was refolded over a 20 CV gradient from 8 M urea, 100 mM sodium phosphate, 200 mM L-arginine, pH 8 to 100 mM sodium phosphate, 200 mM L- arginine, pH 8 at 1 ml/min.
  • Contaminating protein was washed off the column with 10 CV of 50 mM imidazole, 100 mM sodium phosphate, 300 mM NaCl, pH 7.5, followed by a second 10 CV wash of 100 mM imidazole, 100 mM sodium phosphate, 300 mM NaCl, pH 7.5.
  • Histidine-tagged proteins were eluted with 10 CV of 500 mM imidazole, 100 mM sodium phosphate, 300 mM NaCl, pH 7.5 and collected into 1 ml fractions (FIG. 7 and 8). Fractions from each chromatography run were assessed for purity by SDS-PAGE using a NuP AGE® Novex4-12% Bis-Tris gel (Invitrogen). Fractions containing the protein peak (2.5 ml) were pooled and buffer exchanged into 50% glycerol, 50 mM sodium phosphate, pH 7.0, using a PDlO column (GE Healthcare) and stored at —20 0 C. Protein was quantified using a Bradford assay (Pierce, c/o Quantum Scientific, Australia), prior to storage at —20 0 C.
  • the protocol was as follows: equilibration with 2 CV of 50 mM sodium phosphate, 150 mM NaCl, pH 7 at 0.6 ml/min; injection of 250 — 500 ⁇ l of sample at 0.6 ml/min; and isocratic flow of 1 CV of 50 mM sodium phosphate, 150 mM NaCl, pH 7 at 0.6ml/min. See FIG. 8 for exemplary results.
  • Luminescence from full-length RLuc is typically recorded as a flash emission that reaches maximal light intensity within seconds or milliseconds. Due to the speed of light emission, the reaction needs to be initiated and recorded in front of the light detection device where the time interval between substrate addition and detection is kept constant between samples.
  • An alternate detection method is glow luminescence, a steady state kinetic signal. The constant signal is preferable as the reaction can be initiated outside of the detection chamber yielding more sensitive and reproducible results.
  • RLuc FEC assays were performed in 96-well white Costar plates (Corning, Australia) using components from the Renilla Luciferase Assay Kit (Promega, Sydney, Australia) as indicated below.
  • Variable ⁇ (RLuc_ ⁇ 9iPl or RLucjot ⁇ Pl) and ⁇ (RLuc_G) 92 ProG or RLuc_ ⁇ 23 oProG) fragment concentrations (5nM - ⁇ 50 nM, as indicated in FIG. 9), were added to a final reaction volume of 200 ⁇ L containing 0.5 x PLB.
  • 0.5 x PBS pH 7.3, 0.5 M NaCl and His MAb analyte (5 nM ⁇ 25 nM, as indicated in FIG. 9).
  • Renilla luciferase in vitro assay buffer known as 1 x Matthew's buffer (Matthews et al., 1977, Biochemistry 16(1): 85-91), has a standard composition that consists of:
  • bovine serum albumin (BSA)
  • RLuc FEC assays were performed in 96-well white Costar plates as above using components from the Renilla Luciferase Assay Kit (Promega, Sydney, Australia) as indicated. Five nM ⁇ (RLuc_ ⁇ 229 Pl) and ⁇ (RLuc_ ⁇ 2 3 0 ProG) fragments were added to a final reaction volume of 100 ⁇ L containing assay buffer (refer to Table 4), 1 x PLB, 0.5 M NaCl, 5 mM EDTA 5 5mM DTT and control serum (1/100). Immediately, following the reaction setup, His MAb (5 nM) was added and the reactions were incubated for 10 rnin at RT without agitation.
  • FEC Native RLuc is inhibited by divalent metal cations (Matthews et al., 1977, Biochemistry 16(1): 85-91), a process that is reversible by metal chelation.
  • FEC is designed for the detection of analyte in human serum samples which contain high levels of metal cations, including for example, zinc (70-150 ⁇ g/dL), copper (70- 155 ⁇ g/dL), iron (60-180 ⁇ g/dL) and manganese (0.04-1.4 ⁇ g/dL) (Fundamentals of Clinical Chemistry. N.W. Teitz, Ed. Philadelphia, W.B. Saunders Company, 1987).
  • the assay was prepared as follows: 5 nM of each fragment (RLuc_ ⁇ 2 29Pl and RLuc_ ⁇ 2 3oProG) was added to a dilution series of EDTA (10 mM to 1 mM), in the presence of 50 mM Tris.HCl pH 7.0, 1 x PLB, 0.5 M NaCl, 5 mM DTT and control serum (1/100) in a final reaction volume of 100 ⁇ L. To the first set of wells, 5 nM His MAb was added and mixed, whereas the second set contained no His MAb.
  • the plate was incubated for 10 minutes at RT following which 1 ⁇ l Promega 100 x assay substrate was added per well and the kinetics of light emission were measured over 10 min with a 1 sec integration per well. Endpoint readings were subsequently taken (at the completion of the kinetic read) with a 5 sec integration using an LmaxII 384 luminometer (Molecular Devices, Melbourne, Australia). Signal to noise ratios were determined by comparing the rates (nVRT.TT-rm ' n "1 ) of reaction calculated over the first 50-100 Vmax points (using SoflMaxPro V5 software).
  • Electrostatic effects have an important function on both substrate catalysis and protein-protein interactions, and these effects can be modulated or modified using different salt concentrations.
  • Different sodium chloride (NaCl) concentrations were tested to determine their effect on forced enzyme complementation and on the resulting light emission (Table-5).
  • the assay was prepared as follows: 5 nM of each fragment (RLuc_ ⁇ 229Pl and RLuc_ ⁇ 2 3oProG) was added to a dilution series of NaCl (1 M, 0.75 M, 0.5 M, 0.25 M and 0 M), in the presence of 50 mM Tris.HCl pH 7.0, 1 x PLB, 5 mM DTT and control serum (1/100) in a final reaction volume of 100 ⁇ L.
  • reducing agents were shown to be essential for the generation of extended glow luminescence rather than flash emission (refer to Table 3). It is important to empirically determine the concentration of reducing agents in order to ensure that critical disulphide bonds present in proteins such as the target antibody are not reduced thus mamtaining analyte integrity, whilst ensuring that the extended glow luminescent signal is not impeded.
  • Various DTT concentrations were tested to determine their effect on light emission and the ability of the analyte to induce forced enzyme complementation (Table 7).
  • the final concentrations of DTT tested were 5 mM, 2 mM and 1 mM and the assay was prepared as foEows: 5 nM of each fragment (RLuc_ ⁇ 2 2 9 Pl and RLuc_ ⁇ 23 oProG) was added to 50 mM TrisJHCl pH 7.0, 1 x PLB, 0.25 M NaCl 5 5 mM EDTA and control serum (1/100) in a final reaction volume of 100 ⁇ L. Five nM His MAb was added to the first set of wells, while control wells did not contain His MAb.
  • the plate was incubated for 10 minutes at RT following which 1 ⁇ l Promega 100 x assay substrate was added to each well and the kinetics of light emission were measured over 10 ⁇ m * n with a 1 sec integration per well. Endpoint readings were subsequently taken (at the completion of the kinetic read) with a 5 sec integration using an LmaxII 384 luminometer.
  • Reactions were carried out in 50 mM Tris.HCl pH 7.0, 0.25 M NaCl, 5 mM EDTA, 2.5 mM DTT, control serum (1/100) and an in-house formulation of 1 x enhancer buffer (30 mM HEPES pH 8.0, 10% glycerol, 0.2% CHAPS) in a final reaction volume of 100 ⁇ L.
  • Detergents are widely used to reduce protein-protein interactions and nonspecific binding in immunoassays.
  • the use of ionic detergents in FEC to reduce self- association of fragments in the absence of analyte is critical to further increase the signal to noise ratio.
  • CHAPS nondenaturing zwitterionic detergent 3-[(3- Cholamidopropyl) dimethylarnmonio]-l-propanesulfonate
  • PB enhancer buffer C provides an optimal signal to noise ratio
  • PB enhancer buffer C could provide 2.3 fold and 1.3 fold increases in the signal to noise ratio with kinetic and endpoint readings respectively (Table 10), over that obtained with the Promega lysis buffer.
  • PB enhancer buffer C (3O mM HEPES pH 8, 0.2% CHAPS, 1% thiourea, 5% glycerol - final reaction concentration) in combination with 50 mM Tris.HCl pH 7.0, 0.25 M NaCl, 5 mM EDTA, and 2.5 mM DTT in the presence of a 1/100 dilution of serum can yield an excellent signal (37 mRLU-min "1 ) and negligible self-association/autoluminescence (0.4 mRLU-min "1 ) resulting in a signal to noise ratio of 96:1 and 14:1 with kinetic and endpoint reading respectively (exemplary results are shown in FIG. 10).
  • RLuc_oc 2 2 9 Pl and RLuc_oo 23 oProG were assayed in 50 mM Tris.HCl pH 7.0, 0.25 M NaCl, 5 mM EDTA, 2.5 mM DTT, 1 x PB enhancer buffer C and control serum (1/100) in a final reaction volume of 100 ⁇ L. Analyte was added (5 nM His MAb) and the plate was incubated at RT (10 min). Stock coelenterazine substrates (coelenterazine sampler kit, Sigma, Australia) were resuspended in 100% ethanol (500 ⁇ M substrate) and used at a final concentration of 5 ⁇ M.
  • CTMV ⁇ ison of Coelenterazin * Kinetic value KineticRatio Endpoint Value Endpoint Ratio
  • Example 2 Homogeneous in vitro Renilla Iucif erase FEC reagent storage buffer components.
  • Full-length RLuc is susceptible to loss of activity and prone to aggregation upon storage at a concentration of greater than 0.5 mg/mL at 4 0 C (Matthews et al. 5 1977, Biochemistry 16(1): 85-91). It is expected that splitting the full-length RLuc parental enzyme at amino acids 229/230 followed by addition of analyte interaction moieties, may increase the propensity of the recombinant fragments to lose activity and stability. Indeed, producing fragments from a monomeric enzyme can result in the exposure of the hydrophobic core resulting in aggregation upon storage.
  • a single point mutation, C124A, introduced into the ⁇ fragment of RLuc to enhance enzyme stability goes some way to addressing these concerns.
  • chemical components added to the fragment storage buffer are a benign way in which to tackle protein stability during storage without affecting the FEC process.
  • the procedures outlined herein describe the components used to extend the half-life of dilute concentrations of RLuc fragments stored at 4°C.
  • RLuc fragments used in this example include RLuc_oc229Pl and RLuc_2 30 ⁇ ProG, their synthesis and characterisation are described in example 1 of the invention.
  • Buffer additives known to increase the stability of dilute concentrations of proteins in solution can be composed of solvents, inert proteins and sugars. Protein degradation can be reduced upon storage by the inclusion of reducing agents, hydrophobic additives, protease inhibitors and antibacterial agents. Finally, lyopMlization can also be used for long-term protein storage. In this example, we focus on increasing the stability of working dilutions (100 nM) of both the ⁇ and ⁇ purified fragments. Sugars (trehalose and sucrose) and inert proteins (BSA and gelatin) were tested for their ability to retain fragment integrity and activity as determined by His MAb directed FEC following extended fragment storage at 4 0 C.
  • ⁇ and ⁇ fragment working dilutions were prepared by dissolving individual purified fragments into solutions of 1.5 M trehalose, 1.5 M sucrose, 2 mg/mL BSA (Pierce), 2 mg/mL fish gelatin (Sigma) and 50 mM sodium phosphate buffer pH 7.0. Assays were performed as described herein; 5 nM of each fragment (RLuc_ ⁇ 2 2 9 Pl and RLuc_c ⁇ 23oProG) from the various stabilising solutions were added to 50 mM Tris.HCl pH 7.0, 0.25 M NaCl, 5 mM EDTA, 2.5 mM DTT, control serum (1/100) and 1 x PLB, in a final reaction volume of 100 ⁇ L.
  • Analyte (5 nM His MAb) was added and the reactions were incubated for 10 minutes at RT. Substrate was then added (1 ⁇ l Promega 100 x assay substrate) to each well and the kinetics of light emission (10 min with 1 sec integration) and endpoint readings (5 sec integration) were read using an LmaxII 384 luminometer. Assays were repeated at various time intervals up to 6 days storage at 4°C.
  • Fragment dissolution in sucrose, BSA and fish gelatin produced excellent stabilisation of low concentration fragments stored for up to 6 days at 4 0 C, resulting in high signal to noise ratios (Table 12). Fragments stored in 50 mM sodium phosphate buffer pH 7.0 lost significant enzymatic activity resulting in a decrease of 80% and 76% in light emission for kinetic and endpoint readings, respectively. Stabilising solutions composed of half strength solutions mixed in combinations as follows; 0.75 M sucrose, 1 mg/mL BSA (diluent #1) and 0.75 M sucrose, 1 mg/mL fish gelatin (diluent #2) were also tested as above following storage at 4°C for up to 18 days (Table 13).
  • Example 3 Homogeneous in vitro Renilla luciferase FEC assays for polyclonal HSV-I and HSV-2 IgG specific patient antibody detection using glow luminescence.
  • Examples 1 and 2 describe the synthesis, purification and characterisation of RLuc fragments generated by splitting the full-length parental enzyme (RLuc) at amino acids 229/230.
  • the ⁇ fragments incorporating a single point mutation (C124A) were linked to a flexible linker [(G 4 S) 3 ], HSV-I or HSV-2 truncated antigen and polyhistidine tag at the breakpoint termini resulting in the generation of RLuc_ ⁇ 2 29 Pl and RLUCJX22 9 P2, for HSV-I and HSV-2 detection, respectively.
  • the enzyme fragments were used to detect disease specific IgG antibodies in patient serum samples (HSV-I and HSV-2 IgG specific patient polyclonal antibody binding to Pl and P2 truncated antigens respectively) by forced enzyme complementation in a homogeneous in vitro assay format.
  • RLuc FEC assays were performed in 96-well white Costar plates (Corning, Australia) using the Renilla Luciferase Assay Kit (Promega, Australia). Sera from five normal individuals (HSV-l/HSV-2 negative) and five individuals infected with HSV-I positive/HSV-2 negative and HSV-2 positive/HSV-1 negative (Brisbane, Australia) were confirmed using an existing commercial assay, HerpeSelectl and 2 ELISA IgG (Focus Diagnostics, USA).
  • FEC homogeneous assays were carried out in a 200 ⁇ L reaction mix containing 5 nM RLuc_ci229Pl and RLuc_23oCoProG, 0.5 x Promega lysis buffer (PLB) 5 0.5 x PBS pH 7.3, 0.5 M NaCl. Sera were added to a final concentration of 1/50 and reactions were incubated for 30 rnin at RT on a platform rocker. DTT was then added for a final concentration of 2.5 mM, followed by the addition of 0.5 ⁇ l of 100 x Promega assay substrate.
  • PLB Promega lysis buffer
  • Sera were added to a final concentration of 1/50 and reactions were incubated for 30 rnin at RT on a platform rocker.
  • DTT was then added for a final concentration of 2.5 mM, followed by the addition of 0.5 ⁇ l of 100 x Promega assay substrate.
  • RLuc FEC components of the invention including PB assay buffer (50 mM Tris ⁇ Cl pH 7.0, 0.25 M NaCl, 5 mM EDTA, 2.5 mM DTT), 5 x PB enhancer buffer C (150 mM HEPES pH 8, 1% CHAPS, 5% thiourea, 25% glycerol) and fragment stabilisation/storage buffer (0.75 M sucrose, 1 mg/mL BSA) will further improve the limit of detection and dynamic range of these results.
  • PB assay buffer 50 mM Tris ⁇ Cl pH 7.0, 0.25 M NaCl, 5 mM EDTA, 2.5 mM DTT
  • 5 x PB enhancer buffer C 150 mM HEPES pH 8, 1% CHAPS, 5% thiourea, 25% glycerol
  • fragment stabilisation/storage buffer (0.75 M sucrose, 1 mg/mL BSA
  • Example 3 describes the use RLuc enzyme fragments for the detection of disease specific IgG antibodies in patient serum samples (HSV-I IgG specific patient polyclonal antibody binding to Pl truncated antigen) by forced enzyme complementation in a homogeneous in vitro assay format.
  • LOD limit of detection
  • the limit of detection (LOD) required for antibody-based analytes in human serum is in the high picomolar (pM, 10 " 12 M) range, whilst the detection of antigens, for example, Hepatitis B surface antigen (HBsAg), is in the low femtomolar (fM, 10 '15 M) to attomolar (aM, 10 '18 M) range (Anderson et al. 5 2002, MoI.
  • scFv of a mouse monoclonal antibody (5S) against the 'a' epitope of the HBsAg S domain.
  • the ⁇ fragment was linked to a polyhistidine tag, flexible linker and second HBsAg scFv at the breakpoint termini of the ⁇ fragment generating RLuc_92C ⁇ Ell.
  • the Ell designated scFv was originally cloned and expressed from a mouse monoclonal antibody (125El 1) against the PreSl (21-47) fragment of the large HBsAg (Yang et al., 2005, Protein Expr. Purif. 41:341- 348). Together these two fragments should be capable of in vitro homogeneous FEC detection of HBsAg in human serum samples.
  • Codon optimised A5-scFv (SEQ ID NOS 31 and 32) and Ell-scFv (SEQ ID NOS 33 and 34) genes were synthesised by DNA2.0 (Menlo Park, U.S.A) for E.coli expression.
  • Synthetic pJ36-A5 and pJ36-Ell plasmid (2 ⁇ g) was supplied on a filter disk that was purified using the QIAprep spin miniprep kit (Qiagen, Australia) by incubating the filter disk in appropriate buffer, applying the supernatant to a QIAprep spin column and completing the purification according to the manufacturer's instructions.
  • Purified plasmid was digested with BamHVSpel and the A5-scFV BamHJJSpel insert was cloned into the BamHi/Spel digested universal pET- ⁇ plasmid encoding a C-terminal antigen specific cassette [(G 4 S)3 linker, antigen and G 4 S linker and histidine tag (6H)], yielding pET- ⁇ A5.
  • RLuc_ ⁇ 9 i (residues 2-91) was amplified using forward primer RLuc3 and reverse primer RLuc4 as previously described in example 1.
  • Amplification products were purified directly from the reaction tube using the QIAquick PCR purification kit (Qiagen) and were subsequently digested with NdeVKpnl and cloned into Nde ⁇ /Kpn ⁇ digested universal pET- ⁇ A5 destination plasmid encoding a C-terminal HBsAg scFv cassette [(G 4 S) 3 linker, HBsAg-A5, G 4 S linker and histidine tag], yielding pET- Rluc_ ⁇ 9 iA5 (SEQ ID NOS 35 and 36).
  • RLuc_92G0 (residues 92-311) incorporating the C124A mutation was amplified using forward primer RLuc5 and reverse primer RLuc6 as previously described in example 1.
  • Amplification products were purified directly from the reaction tube using the QIAquick PCR purification kit (Qiagen) and were subsequently digested with NdeVKpnl and cloned into NdeVKpnl digested universal pET- ⁇ Ell destination plasmid encoding a N-terminal HBsAg scFv cassette [histidine tag (6H), G 4 S linker, HBsAg- EIl and (G 4 S) 3 linker], yielding pET- Rluc_ ⁇ 92 El 1 (SEQ ID NOS 37 and 38).
  • Example 5 Application of glow luminescent homogeneous FEC assays for multiple analyte detection.
  • Example 3 describes the use of RLuc enzyme fragments for the detection of disease specific IgG antibodies in patient serum samples (HSV-I IgG specific patient polyclonal antibody binding to Pl truncated antigen) by forced enzyme complementation in a homogeneous in vitro assay format Simultaneous detection of two independent analytes within a single sample is known as multiplexing and in this instance can be applied to the detection of both HSV-I and HSV-2. Dual analyte detection can be performed with the use of glow luminescent RLuc FEC to detect HSV- 1 in combination with colorimetric ⁇ -lactamase FEC to detect HSV-2.
  • the reaction mix contains all the necessary components for generation of a colorimetric output signal from ⁇ -lactamase HSV-2 and for luminescent detection from RLuc HSV-I.
  • the HSV-2 positive sample will be detected by an increase in relative light units (RLU) while the HSV-2 positive sample will be detected by an increase in absorbance at OD4 9 2mn accompanying a colorimetric change; both signals are typically proportionate to the amount of analyte present in the sample.
  • This design will combine two independent homogeneous assays into one single well resulting in the differentiation of HSV-I and HSV-2 disease from a single patient sample in one single homogeneous assay reaction. Further potential to detect more than two disease markers is anticipated, as one can simply change between different wavelengths on the light filter used in the photomultiplier tube (PMT) of a luminometer and on the spectrophotometer for different enzyme substrates used. Table 14. Expected result for multiplexing signals
  • Example 6 Homogeneous in vitro ⁇ -lactamase FEC assays for glow luminescent detection.
  • split bioluminescent reporter enzymes As previously described in Example 1, 3 and 4, is the use of split reporter enzymes that do not traditionally catalyse chemiluminescent reactions, by combining these with synthetically produced luminescent substrates that generate glow luminescence.
  • the recently described bioluminogenic substrate for ⁇ -lactamase termed 'Bluco' is used in combination with split ⁇ -lactamase FEC in a homogeneous format to detect HSV-2 using glow luminescence in vitro, ⁇ -lactamase acts to open the ⁇ -lactam ring of the pro-luciferin substrate resulting in spontaneous fragmentation; Firefly luciferase subsequently acts on the luciferin producing light emission.
  • ⁇ -lactamase based FEC assay for HSV-2 specific IgG detection using glow luminescence ⁇ -lactamase FEC assays were performed in 96-well white Costar plates (Corning, Australia) using Bluco (Stanford University) and Firefly luciferase (Promega, Australia). Sera from normal individuals (HSV-l/HSV-2 negative) and individuals infected with HSV-2 positive/HSV-1 negative (Brisbane, Australia) were confirmed using an existing commercial assay, HerpeSelectl and 2 ELISA IgG (Focus Diagnostics, USA).
  • FEC homogeneous assays were carried out in a 200 ⁇ L reaction mix containing 2 nM BLaMl 82T-HSV-2 and 4 nM BL ⁇ N276D-ProG in 50 mM sodium phosphate buffer, 150 mM NaCl, 0.05% CHAPS, 5 mM o-phenanthroline (OP), 1 mM EDTA, pH7.6 containing 100 ⁇ M Bluco, 1.5 mM ATP and 1.5 mM MgCl. Sera were added to a final concentration of 1/100 and reactions were incubated for 60 min at RT followed by the addition of 100 nM Firefly luciferase.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne une méthode permettant de déterminer la présence d'une substance à analyser d'intérêt dans un échantillon, ladite méthode comprenant la mise en contact de l'échantillon avec : (i) une première paire de fragments rapporteurs purifiés comprenant un domaine d'interaction ayant une affinité pour la substance à analyser d'intérêt; et (ii) une seconde paire de fragments rapporteurs purifiés comprenant un domaine d'interaction ayant une affinité pour la substance à analyser d'intérêt et permettant de reconstituer une enzyme active au moment de l'association avec la première paire de fragments rapporteurs grâce aux affinités des domaines d'interaction des première et seconde paires de fragments rapporteurs avec la substance à analyser d'intérêt; dans des conditions de dosage in vitro qui permettent que la première et la seconde paire de fragments rapporteurs s'associent en présence de la substance à analyser d'intérêt dans l'échantillon grâce à l'affinité des domaines d'interaction avec la substance à analyser, afin de produire un signal luminescent par l'action de l'enzyme active reconstituée sur un substrat. La méthode permet également de détecter la présence ou l'absence, ou encore l'étendue, d'un signal luminescent.
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* Cited by examiner, † Cited by third party
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US9567390B2 (en) * 2008-01-23 2017-02-14 Department Of Biotechnology Humanized high affinity recombinant antibody against Hepatitis B surface antigen

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