BIOLUMINESCENT BIOSENSOR FOR DETECTING AND QUANTIFYING BIOMOLECULES

Abstract

A bioluminescent biosensor and use of such bioluminescent biosensor for providing a generic biosensor strategy allowing direct detection of biomolecules (e.g. antibodies) or ligands (e.g. small molecules) directly in solution.

Claims

1. A biosensor including a linker comprising a first end and a second end, wherein: the first end of the linker is fused to one terminus of a first luciferase domain via a first binding domain; and the second end of the linker is fused to a second luciferase domain via a second binding domain, wherein the biosensor optionally comprises a third luciferase domain fused to the other terminus of the first luciferase domain via a spacer, wherein the binding domains are configured to bind to a biomolecule, wherein the biosensor exists in two conformations, in which either the second or third luciferase domain binds to the first luciferase domain to form a complemented luciferase, and wherein a fluorophore is conjugated next to one of the second or third luciferase domains resulting in efficient Bioluminescence Resonance Energy Transfer (BRET) between the luciferase and the fluorophore in only one of the two conformations.

2. The biosensor according to claim 1, wherein the third luciferase domain is fused to the other terminus of the first luciferase domain via a spacer.

3. The biosensor according to claim 1, wherein the first luciferase domain is a large luciferase domain, preferably a large split luciferase fragment, and wherein the second and third luciferase domains are small luciferase domains, preferably small split luciferase fragments.

4. The biosensor according to claim 1, wherein the linker is a semi-flexible linker.

5. The biosensor according to claim 1, wherein the biomolecule comprises an antibody, antigen, protein or aptamer.

6. The biosensor according to claim 1, wherein the first binding domain is configured to form a conjugated system with the biomolecule and wherein the second binding domain is configured to bind to a ligand binding site of the biomolecule.

7. The biosensor according to claim 6, wherein the ligand binding site of the biomolecule comprises an antigen binding site of an antibody.

8. The biosensor according to claim 6, wherein the biomolecule is covalently bound to the first binding domain of the biosensor.

9. An In vitro biomolecule-detecting method, comprising the steps of: contacting a sample with the biosensor of claim 1; determining the change of the biosensor's luminescence in the presence of a sample; and determining the luminescence change in the presence of the sample to the quantitative and/or qualitative presence or absence of a biomolecule.

10. The In vitro biomolecule-detecting method according to claim 9, wherein in the absence of a biomolecule the biosensor is in a biomolecule-free state that at least some of the first luciferase domain complements with the second luciferase domain.

11. The In vitro biomolecule-detecting method according to claim 9, wherein binding between the binding domains of the biosensor and the biomolecule changes the equilibrium between the biomolecule-free state of the biosensor and a biomolecule-bound state of the biosensor such that the BRET between the luciferase and the fluorophore changed.

12. An In vitro ligand-detecting method, comprising the steps of: contacting the sample with the biosensor of claim 6; determining the change of the biosensor's luminescence in the presence of a sample; and determining the luminescence change in the presence of the sample to the quantitative and/or qualitative presence or absence of a ligand.

13. The In vitro ligand-detecting method according to claim 12, wherein in the absence of a ligand the biomolecule binds to the second binding domain disrupting the interaction of the second luciferase domain with the first luciferase domain.

14. The In vitro ligand-detecting method according to claim 12, wherein in the presence of a ligand the biosensor is in a ligand-bound state allowing that at least some of the first luciferase domain complements with the second luciferase domain.

15. A Kit of parts comprising the biosensor according to claim 1, wherein the biosensor comprises a first luciferase domain fused to a second luciferase domain via a semi-flexible linker containing two binding domains at the ends of the linker, and wherein the kit of parts further comprises a third luciferase domain.

Description

DESCRIPTION OF THE FIGURES

[0096] FIG. 1A shows an antibody sensor based on the antibody-induced disruption of an intramolecular complex between split luciferase fragments which results in a change of BRET efficiency. The sensor is labeled with a fluorophore (star). Binding of an antibody to the epitopes in the semi-flexible linker pulls the fluorophore and luciferase apart, changing the color of emission from red to blue.

[0097] FIG. 1B shows a schematic structure of the sensor sequence of NB-LUMABS-1

[0098] FIG. 2A shows luminescence spectra of 1 pM sensor in the absence (red line; .circle-solid.) and presence (blue line; .square-solid.) of 1.25 nM anti-HIV1-p17 antibody. (B) Antibody titration at a sensor concentration of 1 pM. Error bars represent meanSD (n=3).

[0099] FIG. 2B shows antibody titration at a sensor concentration of 1 pM. Error bars represent meanSD (n=3).

[0100] FIG. 3 shows the characterization of NB-LUMABS-2, -3, -4 (FIG. 3A), -5, -6, -7 (FIG. 3B) including the luminescence spectra of 1 pM sensor in the absence (red line; .circle-solid.) and presence (blue line; .square-solid.) of 1.25 nM anti HIV1-p17 antibody and the antibody titration at a sensor concentration of 1 pM. Error bars represent meanSD (n=2).

[0101] FIG. 4 shows the characterization of TRAS-NB-LUMSABS-1, -2 and -3 including the luminescence spectra of 100 pM sensor in the absence (red line; .circle-solid.) and presence (blue line; .square-solid.) of 1 M trastuzumab and the antibody titration at a sensor concentration of 100 pM. Error bars represent meanSD (n=3).

[0102] FIG. 5 shows pG-NB-LUMABS sensors allowing ratiometric bioluminescent detection of in principle any ligand. FIG. 5A shows the detection of CRP as an example of a protein biomarker. FIG. 5B shows the detection of a small molecule. Detection is based on competition between intermolecular binding of a peptide epitope (FIG. 5A) or small-molecule analogue (FIG. 5B) and the ligand to a ligand-specific biomolecule (e.g. monoclonal antibody). FIG. 5C shows a schematic overview of the pG-NB-LUMABS pET28a(+) vector, with a N-terminal His-Tag and a C-terminal Streptavidin tag for purification.

[0103] FIG. 6 shows the characterization of CTX-NB-LUMABS variants. Luminescence spectra of 100 pM sensor in the absence (red line; .circle-solid.) and presence (blue line; .square-solid.) of 8.3 M cetuximab. Antibody titration to 100 pM of sensor in PBS buffer (pH7.4, 1 mg/ml BSA). Error bars represent meanSEM (n=3). The red lines (.circle-solid.) represent fits to equation 1.

[0104] FIG. 7 shows a MTX-pG-NB-LUMABS for the detection of the small molecule methotrexate.

[0105] FIG. 8 shows the luminescence spectra of 2 nM pG-NB-LUMABS-17 in the presence and absence of 0.8 mM unpurified peptide 17 (FIG. 8A) and the emission ratio of 2 nM pG-NB-LUMABS-epitope 17 upon increasing concentration of peptide 17.

SENSOR DESIGN

[0106] FIG. 1 shows the general design of the sensor. In this example, split NanoLuc was chosen as a complementation reporter because the complemented complex is blue luminescent and a variety of SBiT variants are available with affinities between 0.7 nM and 190 M toward LBiT (Dixon, et al. ACS Chem. Biol., 2016, 11, 400; SEQ ID NO: 19).

[0107] In an exemplary design we focused on developing a sensor for the detection of the anti-HIV1-p17 antibody. Several well-characterized linear epitope sequences are available for this antibody, which has made it a popular choice for the development of new homogeneous antibody detection assays. The linker between the LBiT and SBiT1 has flexible blocks of (GGS).sub.10. The linker between the LBiT and SBiT2 initially has two epitopes (SEQ ID NO: 4, K.sub.d=42 nM) specific for the HIV1-p17-antibody that are separated by three flexible blocks of (GSG).sub.6 and two -helical blocks each having six EAAAK repeats (SEQ ID NO: 20). The linker was also used in some reported sensor protein based on the same switching strategy (see: Golynskiy, et al. ChemBioChem, 2010, 11, 2264; Banala, et al. ACS Chem. Biol., 2013, 8, 212; Arts, et al. Anal. Chem., 2016, 88, 4525; Van Rosmalen, et al. Anal. Chem., 2018, DOI: 10.1021/acs.analchem.8b00041; and Arts, et al. ACS Sens., 2017, 2, 1730). One cysteine residue was included adjacent to SBiT2 for fluorophore conjugation.

[0108] One variant (NB-LUMABS-1; SEQ ID NO: 6) was constructed in an exemplary embodiment containing SBiT1 with K.sub.d of 190 M (SEQ ID NO: 1) and SBiT2 with K.sub.d of 2.5 M (SEQ ID NO: 2), and a cysteine residue before the N-terminus of SBiT2. The sensor protein was expressed in E. coli BL21 (DE3) and purified using an N-terminal His-tag and a C-terminal Strep-tag. This two-step purification protocol ensures the isolation of full length protein only, without truncated version of the sensor lacking e.g. SBiT domain. The sensor protein was conjugated with synthetic fluorophore Cy3 via thiol-maleimide reaction.

[0109] Bioluminescent scan of NB-LUMABS-1 showed a higher Cy3 emission peak at 563 nm than the NanoLuc emission at 460 nm in the absence of anti-HIV1-p17 antibody, with an emission ratio of 1.3 which indicates very efficient BRET between NB and Cy3. Addition of anti-HIV1-p17 antibody resulted in a significant decrease of the Cy3 emission peak, indicating a large decrease of BRET efficiency. Titration experiments yielded apparent dissociation constants (K.sub.d,app) of 10.00.5 pM and a large dynamic range of 218% (FIG. 2).

Tuning Sensor Performance

[0110] To establish the influence of cysteine residue position, two variants were constructed containing either one cysteine residue after the C-terminus of SBiT2 (NB-LUMABS-2; SEQ ID NO: 7) or two cysteine residues both before the N-terminus of SBiT2 and after the C-terminus of SBiT2 (NB-LUMABS-3; SEQ ID NO: 8). NB-LUMABS-2 showed a relatively low emission ratio (red to blue) in the absence of anti-HIV1-p17 antibody. It suggests that Cy3 is farther away from the substrate binding site of luciferase. NB-LUMABS-3 also showed a low relatively low emission ratio (red to blue) at the antibody-free state. We assume that the conjugation with two Cy3 group might affect the assembly of SBiT2 with LBiT.

[0111] To establish the influence of LBiT-SBiT affinity, four variants were constructed containing different combination of SBiTs. NB-LUMABS-4 (SEQ ID NO: 9) contained SBiT1 with K.sub.d of 190 M (SEQ ID NO: 1) and SBiT2 with K.sub.d of 0.18 M (SEQ ID NO: 3). NB-LUMABS-5 (SEQ ID NO: 10) contained two same SBiTs with K.sub.d of 190 M (SEQ ID NO: 1). NB-LUMABS-6 (SEQ ID NO: 11) contained two same SBiTs with K.sub.d of 2.5 M (SEQ ID NO: 2). NB-LUMABS-7 (SEQ ID NO: 12) contained SBiT1 with K.sub.d of 2.5 M (SEQ ID NO: 2) and SBiT2 with K.sub.d of 0.18 M (SEQ ID NO: 3). NB-LUMABS-4 showed a high emission ratio in the absence of antibody, but it moderately decreased by antibody biding. NB-LUMABS-5 and -6 containing two same SBiTs in a single sensor protein showed low emission ratio at antibody-free state, which indicates that in these sensors SBiT1 already complements NanoLuc activity in a significant fraction of the sensor proteins. For NB-LUMABS-7, the emission ratio reached 1.8 at antibody-free state and it significantly decreased upon addition of antibody. The dynamic range (DR) reached 493%. Antibody titration experiments yielded apparent affinities between 10 and 15 pM for these four sensor variants, which are similar to that obtained for the NB-LUMABS-1 (FIG. 3).

TABLE-US-00001 TABLE 1 Properties of various NB-LUMABS (NB-LUMABS) variants targeting at anti-HIV1-p17 antibody. Sensor Cys K.sub.d,app name SBiT1 SBiT2 position DR (pM) NB- K.sub.d = 190 M K.sub.d = 2.5 M before 218% 10.0 0.5 LUMABS-1 SBiT2 NB- K.sub.d = 190 M K.sub.d = 2.5 M after 177% 13.7 1.3 LUMABS-2 SBiT2 NB- K.sub.d = 190 M K.sub.d = 2.5 M Before 182% 12.1 0.6 LUMABS-3 and after SBiT2 NB- K.sub.d = 190 M K.sub.d = 0.18 M before 138% 14.2 4.7 LUMABS-4 SBiT2 NB- K.sub.d = 190 M K.sub.d = 190 M before 252% 11.7 3.7 LUMABS-5 SBiT2 NB- K.sub.d = 2.5 M K.sub.d = 2.5 M before 160% 15.2 1.0 LUMABS-6 SBiT2 NB- K.sub.d = 2.5 M K.sub.d = 0.18 M before 493% 11.8 0.5 LUMABS-7 SBiT2

Tuning Sensor Selectivity

[0112] To challenge the modularity of our sensor design we tested whether the epitope sequences could be exchanged for epitope sequences targeting a different antibody. Sensors targeting therapeutic antibody trastuzumab (TRAS-NB-LUMABS-1, SEQ ID NO: 13; TRAS-NB-LUMABS-1, SEQ ID NO: 14; and TRAS-NB-LUMABS-3, SEQ ID NO: 15) were constructed by replacing the epitope sequences present in NB-LUMABS-1, -5 and -7 with a trastuzumab-binding mimitope QLGPYELWELS (SEQ ID NO: 5). Trastuzumab is used in the treatment of Her2-positive metastatic breast cancers. Population pharmacokinetics indicate interpatient variabilities in clearance and 10% patients with fast clearance, resulting in drug levels below the minimally effective concentration (Bruno, et al. Cancer Chemother. Pharmacol. 2005, 56, 361). The monovalent affinity of mimitope to trastuzumab was 294 nM. For these three sensors, efficient BRET was observed at the trastuzumab-free state (FIG. 4). Addition of 1 M trastuzumab to TRAS-NB-LUMABS-1 resulted in a moderate decrease in BRET, with a dynamic range of 37%. An increase of emission ratio was observed when adding large amount of trastuzumab. Possibly two molecules of antibody bind with one molecule of sensor, and thus the sensor restored as the closed state. TRAS-NB-LUMABS-2 kept in the closed state during antibody titration. We assume that the affinity between LBiT and SBiT2 is so high that the weak epitope affinity failed to lead to bivalent antibody binding. TRAS-NB-LUMABS-3 exhibited moderate change of emission ratio upon addition of trastuzumab, with a dynamic range of 36%. Titration of trastuzumab resulted in K.sub.d,app of 74.09.4 nM for TRAS-NB-LUMABS-1 and 85.76.3 nM for TRAS-NB-LUMABS-3.

[0113] Additionally, sensors targeting therapeutic antibody cetuximab (CTX-NB-LUMABS-1, SEQ ID NO: 25; CTX-NB-LUMABS-2, SEQ ID NO: 26; and CTX-NB-LUMABS-3, SEQ ID NO: 27) were constructed by respectively replacing the epitope sequences present in NB-LUMABS-1 and -2 with a cetuximab-binding meditope CVFDLGTRRLRC (monovalent K.sub.d of 61 nM; SEQ ID NO: 23) and NB-LUMABS-1 with a cetuximab-binding meditope CQFDLSTRRLKC (monovalent K.sub.d of 270 nM; SEQ ID NO: 24). Cetuximab is a clinically important anticancer therapeutic antibody. Since cetuximab binds to a discontinuous conformational epitope on the cancer marker EGFR, no linear epitope sequences are available. Nevertheless, disulfide-linked cyclic meditope peptides were identified with sufficient affinity to cetuximab and were used to construct a blue-green emitting LUMABS sensor for cetuximab with a relatively modest change in emission ratio (DR60%) (see: Van Rosmalen, et al. Anal. Chem., 2018, 90, 3592). As the presence of cysteine residues in the meditope peptides precludes the use of cysteine-maleimide chemistry to introduce the Cy3 dye, we instead introduced the non-canonical amino acid para-azidophenylalanine (pAzF) to allow site-specific conjugation with a DBCO-functionalized fluorophore via strain-promoted azide-alkyne click chemistry (SPAAC). In order to incorporate pAzF, a TAG amber stop codon was introduced either before the N-terminus (CTX-NB-LUMABS-1; SEQ ID NO: 25) or after the C-terminus of SBiT2 (CTX-NB-LUMABS-2; SEQ ID NO: 26). Co-expression with the orthogonal tRNA synthetase/tRNA pair for pAzF allowed successful incorporation of pAzF into the cetuximab sensor variants at the desired position. CTX-NB-LUMABS-1 showed bright Cy3 emission in the absence of cetuximab and a significant decrease in emission ratio upon antibody binding (FIG. 6). A dynamic range of 23312% was achieved, representing a 4-fold improvement compared to the blue-green CTX-LUMABS sensors (Van Rosmalen, et al. Anal. Chem. 2018, 90, 3592) while an overall K.sub.d of 34.73.7 nM obtained from fitting the titration data was found to be similar (FIG. 6). Introduction of Cy3 at the C-terminus of SBiT2 in CTX-NB-LUMABS-2 substantially decreased the BRET efficiency in the antibody-free state (FIG. 6), which is consistent with the results obtained for HIV-NB-LUMABS-2. We also constructed a sensor variant (CTX-NB-LUMABS-3) containing the cyclic cetuximab meditope with a weaker affinity (SEQ ID NO: 24). Titration experiments with CTX-NB-LUMABS-3 showed a K.sub.d of 18916 nM, enabling this sensor to reliably measure high cetuximab concentrations (FIG. 6). The lower affinity of CTX-NB-LUMABS-3 for cetuximab also resulted in a more rapid response compared to that of CTX-NB-LUMABS-1. These results demonstrate that the NB-LUMABS sensor format can be reconfigured to detect antibodies recognizing disulfide constrained cyclic peptides, yielding two cetuximab sensors whose combined responsive regime covers the clinically relevant cetuximab concentration range (25 nM-2.3 M).

[0114] These results further show that the framework developed for the exemplary anti-HIV1-p17 antibody can be used to easily develop sensor for other antibodies.

TABLE-US-00002 TABLE 2 Properties of various NB-LUMABS variants targeting at therapeutic antibodies. Fluorophore K.sub.d,app Sensor name SBiT1 SBiT2 position DR (nM) TRAS-NB- K.sub.d = 190 M K.sub.d = 2.5 M before SBiT2 37% 74.0 9.4 LUMABS-1 TRAS-NB- K.sub.d = 2.5 M K.sub.d = 0.18 M before SBiT2 0% LUMABS-2 TRAS-NB- K.sub.d = 190 M K.sub.d = 190 M before SBiT2 36% 85.7 6.3 LUMABS-3 CTX-NB- K.sub.d = 190 M K.sub.d = 2.5 M before SBiT2 233% 34.7 3.7 LUMABS-1 CTX-NB- K.sub.d = 190 M K.sub.d = 2.5 M after SBiT2 110% 20.7 3.4 LUMABS-2 CTX-NB- K.sub.d = 190 M K.sub.d = 2.5 M before SBiT2 88% 189 16 LUMABS-3

Experiments

Cloning and Mutagenesis

[0115] Synthetic DNA sequences encoding NB-LUMABS-1 (SEQ ID NO: 6) was ordered from GenScript (Piscataway, USA). NB-LUMABS-2 (SEQ ID NO: 7), NB-LUMABS-3 (SEQ ID NO: 10), NB-LUMABS-4 (SEQ ID NO: 9), NB-LUMABS-5 (SEQ ID NO: 10), NB-LUMABS-6 (SEQ ID NO: 11) and NB-LUMABS-7 (SEQ ID NO: 12) were constructed from NB-LUMABS-1 by site directed mutagenesis using specific primers (Table 3). The QuickChange site-directed mutagenesis kit (Agilent Technologies) was used in accordance with the manufacturer's instructions to introduce the mutations of interest.

[0116] To construct TRAS-NB-LUMABS-1 (SEQ ID NO: 13), TRAS-NB-LUMABS-2 (SEQ ID NO: 14) and TRAS-NB-LUMABS-3 (SEQ ID NO: 15), the gene encoding the semi-flexible liner including trastuzumab mimitope (QLGPYELWELSH) was cloned into the pET28a plasmids encoding NB-LUMABS-1, -5 or -7 via restriction-ligation approach using KpnI and SpeI. All cloning and mutagenesis results were confirmed by Sanger sequencing (StarSEQ GmbH).

[0117] To construct CTX-NB-LUMABS-1 (SEQ ID NO: 25), CTX-NB-LUMABS-2 (SEQ ID NO: 26) and CTX-NB-LUMABS-3 (SEQ ID NO: 27), pET28a(+) plasmids encoding CTX-LUMABS which were prepared according to the method disclosed by Van Rosmalen, et al. (Anal. Chem. 2018, 90, 3592) were digested with KpnI-HF and SpeI-HF restriction enzymes. The linkers including cetuximab meditope, i.e. CTX-NB-LUMABS-1 and -2 with meditope CVFDLGTRRLRC (SEQ ID NO: 23), and CTX-NB-LUMABS-3 with meditope CQFDLSTRRLKC (SEQ ID NO: 24) were then cloned into the pET28a(+) plasmids encoding NB-LUMABS-1 (SEQ ID NO: 6) by restriction-ligation approach with KpnI-HF and SpeI-HF. The cysteine residue was deleted and the TAG codon was introduced at desired position (Table 3) by site directed mutagenesis using specific primers. All cloning and mutagenesis results were confirmed by Sanger sequencing (StarSEQ GmbH).

TABLE-US-00003 TABLE 3 Construction of NB-LUMABS variants SBiT1-LBiT SBiT2-LBiT Sensor name interaction interaction Cys/TAG position NB-LUMABS-1 K.sub.d = 190 M K.sub.d = 2.5 M Cys before SBiT2 NB-LUMABS-2 K.sub.d = 190 M K.sub.d = 2.5 M Cys after SBiT2 NB-LUMABS-3 K.sub.d = 190 M K.sub.d = 2.5 M Cys before and after SBiT2 NB-LUMABS-4 K.sub.d = 190 M K.sub.d = 0.18 M Cys before SBiT2 NB-LUMABS-5 K.sub.d = 190 M K.sub.d = 190 M Cys before SBiT2 NB-LUMABS-6 K.sub.d = 2.5 M K.sub.d = 2.5 M Cys before SBiT2 NB-LUMABS-7 K.sub.d = 2.5 M K.sub.d = 0.18 M Cys before SBiT2 TRAS-NB- K.sub.d = 190 M K.sub.d = 2.5 M Cys before SBiT2 LUMABS-1 TRAS-NB- K.sub.d = 2.5 M K.sub.d = 0.18 M Cys before SBiT2 LUMABS-2 TRAS-NB- K.sub.d = 190 M K.sub.d = 190 M Cys before SBiT2 LUMABS-3 CTX-NB- K.sub.d = 190 M K.sub.d = 2.5 M TAG before SBiT2 LUMABS-1 CTX-NB- K.sub.d = 190 M K.sub.d = 2.5 M TAG after SBiT2 LUMABS-2 CTX-NB- K.sub.d = 190 M K.sub.d = 2.5 M TAG before SBiT2 LUMABS-3

Protein Expression and Purification

[0118] Proteins were expressed and purified using standard protocols. Briefly, the appropriate pET28a plasmid was transformed into E. coli BL21 (DE3). The cells were cultured in LB medium containing 30 g/ml kanamycin at 37 C. Protein expression was induced at an OD.sub.600 of 0.6 using 0.1 mM IPTG at 20 C. overnight. Cells were harvested and lysed by using the Bugbuster reagent (Novagen). Sensor proteins were purified using Ni-NTA affinity chromatography followed by Strep-Tactin purification according to the manufacturer's instructions. The protein concentration was determined by measuring the absorbance at 280 nm using extinction coefficient (calculated from the protein sequence). Protein purity was confirmed by SDS-PAGE. Purified proteins were stored at 80 C. until use.

[0119] The pET28a plasmids encoding CTX-NB-LUMABS was co-transformed into E. coli BL21 (DE3) together with a pEVOL vector encoding a tRNA/tRNA synthetase pair in order to incorporate para-azidophenylalanine (pAzF). The pEVOL vector was a gift from Peter Schultz (Addgene plasmid #31186). Cells were cultured in 2YT medium (16 g peptone, 5 g NaCl, 10 g yeast extract per liter) containing 30 g/mL kanamycin and 25 g/mL chloramphenicol. Protein expression was induced using 0.1 mM IPTG and 0.2% arabinose in the presence of 1 mM pAzF (Bachem, F-3075.0001). The sensor proteins were purified as described above.

[0120] The pET28a plasmids encoding the initial pG-NB-LUMABS was ordered from GenScript, without the epitope sequence. Additionally, two pUC57 vectors were additionally ordered from GenScript encoding the two epitope sequences (SEQ ID NO: 23 and SEQ ID NO: 24) in the semiflexible linker. These epitope sequences were cloned into the pET28a+ vector using restriction ligation cloning using KpnI and SpeI restriction enzymes, and ligated using T4 ligase. The vectors were amplified by transforming them in E. coli NovaBlue bacteria (Novagen) and subsequent culturing. The vectors were extracted using a QIAprep spin miniprep kit (Qiagen). The correct incorporation of the semiflexible linkers with epitopes was confirmed by Sanger sequencing (StarSeq, Germany). The newly obtained pET28a+ vectors, including the epitopes, were transformed into E. coli BL21(DE3) bacteria from Novagen, together with a pEVOL vector encoding the tRNA/tRNA synthetase for the incorporation of para-benzoylphenylalanine (pBPA) into the protein G. This pEVOL-pBpF vector was a gift from Peter Schultz (Addgene plasmid #31190). The bacteria were cultured in LB medium, after the addition of 30 g/mL kanamycin and chloramphenicol. The expression of the proteins was induced at an OD of 0.6 by 0.1 M IPTG (isopropyl--D-thiogalactopyranoside) and 0.2% arabinose, the unnatural amino acid pBPA (Bachem, F-2800.0001) was added simultaneously (1 mM). After overnight expression at 18 C., the cultures were centrifuged at 10 000g for 10 minutes. The cells were lysed for 20 minutes at room temperature using BugBuster (20 mL/L) and Benzonase (20 L/L). The lysed bacteria were centrifuged at 16 000g for 45 minutes at 4 C. Purification of the supernatant was done using nickel-affinity and Strep-Tactin column chromatography. The purity of the sensor proteins was confirmed using SDS-PAGE gels and Q-ToF-MS.

Fluorophore Labeling of Protein

[0121] Purified NB-LUMABS protein was reduced with 1 mM tris-carboxyethylphosphine (TCEP) in 50 mM Tris-HCl (pH 7.4) at room temperature for 20 min. Sulfo-cyanine3 maleimide (Lumiprobe) dissolved in water (10 mg/ml) was then added in a 30-fold molar excess into the protein solution and incubated for 1 hour at room temperature followed by overnight incubation at 4 C. Non-reacted dye was removed by using a PD10 desalting column and the conjugate was concentrated by using Amicon centrifuge filter of 10 kDa. The dye-to-protein ratio was determined by measuring absorbance at 280 nm and 553 nm and assuming extinction coefficients of 39,420 M.sup.1 cm.sup.1 and 162,000 M.sup.1 cm.sup.1 for the protein and dye, respectively. The Cy3-labeled NB-LUMABS protein was confirmed by Q-ToF LC-MS.

[0122] DBCO-Sulfo-Cy3 (Jena Bioscience, CLK-A140-1) was conjugated in a 40-fold molar excess to CTX-NB-LUMABS at room temperature overnight. Excess dye was removed by using Amicon centrifuge filter of 10 kDa. The dye-to-protein ratio was determined by measuring absorbance at 280 nm and 563 nm and using extinction coefficient (calculated from the protein sequence). The reaction product was analyzed by Q-TOF-MS.

[0123] Pure pG-NB-LUMABS (50 M, 800 L) in 50 mM TRIS pH 7 was bubbled with argon. 10 L 100 mM TCEP (tris-carboxyethylphospine), with a final concentration of 1 mM was added and flushed with argon at room temperature for 20 minutes. The sulfo-Cy3-maleimide from Lumiprobe (1 mg) was dissolved in 100 L MilliQ water. The dye was added to the protein solution (15 fold excess of dye) and flushed with argon. This was placed at room temperature for 1 hour and overnight at 4 C. The unreacted dye was removed using a PD-10 desalting column (GE Healthcare). The labeled protein was concentrated with a 3 kDa Amicon Ultra 0.5 mL centrifugal filter. The dye-to-protein ratio was measured using the NanoDrop (UV-VIS), absorption at 280 nm and 548 nm.

Photoconjugation of pG-NB-LUMABS

[0124] Photoconjugation reactions were performed using a Promed UVL-30 UV light source (49 watt). 2 M of antibody (HyTest) and 8 M of pG-NB-LUMABS in PBS buffer (pH 7.4) was used to test the photoconjugation efficiency. The mixture was placed under UV light (365 nm), on ice, for 30, 60, or 90 minutes. The samples were then run on a SDS-PAGE gel to determine the photoconjugation efficiency.

[0125] The excess of non-conjugated sensor protein was successfully removed using Size Exclusion Chromatography (SEC). Bioluminescence spectra show a substantial decrease in the Cy3 emission following conjugation of the pG-NB-LUMABS protein to its antibody, which is consistent with the anticipated binding of the peptide epitope to the antigen binding site, disrupting the LBit-Sbit2 interaction. Addition of the epitope peptide did result in an increase in BRET ratio, proving the feasibility of the sensor concept by showing the ability of the sensor to reversibly switch between the low- and high-BRET states as a result of competitive binding (FIG. 8B).

Sensor Characterization

[0126] Anti-HIV1-p17 antibody was obtained from Zeptometrix (clone 32/1.24.89). Trastuzumab (Herceptin, Roche) and cetuximab (Erbitux, Merck) were obtained via the Catherina hospital pharmacy in Eindhoven, the Netherlands. Antibody titrations to NB-LUMABS were performed in 100 L PBS (pH 7.4, 1 mg/ml BSA) in PerkinElmer flat white 96-well Optiplate using a sensor concentration of 1 pM. Sensor of 100 pM was used for trastuzumab and cetuximab titration. After incubation of sensor and antibody for 2 hours, NanoGlo substrate was added at a final dilution of 1000-fold.

[0127] Anti-CRP169 binding peptide was titrated to antibody-conjugated pG-NB-LUMABS using a sensor concentration of 2 nM. After incubation of sensor and peptide for 45 minutes, NanoGlo substrate was added at a final dilution of 500-fold.

[0128] Luminescence spectra were recorded between 398 nm and 653 nm on Tecan Spark 10M plate reader with a step size of 15 nm, a bandwidth of 25 nm and an integration time of 1000 ms. Titrations were fit to eq 1 to obtain apparent K.sub.d value.

[00001]$\begin{array}{cc}\mathrm{ER}=\frac{P1\left[\mathrm{analyte}\right]}{\mathrm{Kd},\mathrm{app}+\left[\mathrm{analyte}\right]}+P2& \left(\mathrm{Eq}.1\right)\end{array}$

[0129] P1 is the maximal change in emission ratio (ER) and P2 is the emission ration in absence of analyte. Dynamic range (DR) was calculated as:

[00002]$\begin{array}{cc}\mathrm{DR}=\frac{.Math.P1.Math.}{\mathrm{ER}\min }*100\%& \left(\mathrm{Eq}.2\right)\end{array}$

Signal Recording Using Digital Camera

[0130] Into a white 96-well plate, 200 L PBS buffer (pH 7.4, 1 mg/ml BSA) containing 100 pM CTX-NB-LUMABS, different concentrations of cetuximab and 1 L NanoGlo substrate was added. The plate was placed into a Styrofoam box in a dark room to exclude the surrounding light. For blood plasma measurements, samples were prepared in 200 L undiluted blood plasma with 5 nM sensor. The pictures were taken through a hole in the box using a SONY DSC-RX100 digital camera with exposure times of 10-30 s, F value of 1.8 and ISO value of 1600-6400. The images were analyzed by using ImageJ to calculate the ratio values between the average intensity in the blue and red color channel.