Fusion polypeptide for detecting procalcitonin, kit and application thereof

Abstract

The invention provides a fusion polypeptide, a kit and a detection method for procalcitonin detection. The provided SCFV antibodies fused to the large subunit and the small subunit of luciferase respectively can provide the best detection effect. When the fusion polypeptide is used for detecting the procalcitonin or the analogue thereof, excellent clinical comparison correlation is achieved, the detection time is shorter, the detection speed is higher, and a matched instrument is simple in structure.

Description

Fusion polypeptide, kit and application thereof for detecting procalcitonin Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a fusion polypeptide, a kit, a detection method and applications thereof for detecting procalcitonin. Background Art

[0002] Procalcitonin (PCT) is the propeptide of calcitonin. Under normal circumstances, its concentration in the blood is very low. In the past clinical practice, it was often used as an indicator of bacterial, fungal, and parasitic infections, as well as sepsis and multiple organ failure, and has been widely recognized by the academic community. However, due to the low content of PCT in the blood (normal range <50pg / ml), the immunological detection of human physiological or disease-related targets, especially the clinical detection of low-abundance targets, is currently the mainstream detection method, magnetic microparticle chemiluminescence detection, which requires magnetic separation and washing steps, long detection time, slow detection speed, and overly complex instruments. At the same time, magnetic bead labeling and enzyme labeling are required in the production process of reagents. The production process is complex, the cost is high, and the batch difference is difficult to control. The homogeneous wash-free detection method can eliminate the magnetic separation and washing process, greatly saving detection time. It is urgently needed in ICU wards or other POCT scenarios and even in the laboratory.

[0003] The level of procalcitonin in the blood is a core indicator for infection detection and has important clinical significance. Currently, there is no luciferase fusion antibody technology applied to the clinical sample detection of procalcitonin (PCT).

[0004] Summary of the Invention

[0005] The present invention provides a series of SCFV antibodies fused to the large and small subunits of luciferase, respectively, capable of providing optimal detection results. The fusion polypeptides or coupled polypeptides of the present invention exhibit excellent clinical correlation when detecting procalcitonin or its analogs, while also shortening detection time, increasing detection speed, and simplifying the supporting instrument structure.

[0006] In one aspect, the present invention provides a fusion polypeptide or a coupled polypeptide comprising a first antigen-binding region and a first peptide derived from luciferase, wherein the first antigen-binding region comprises at least one light chain variable region and at least one heavy chain variable region; the light chain variable region comprises: a LCDR1 as shown in at least one of SEQ ID NOs: 17-24 or an active variant thereof, a LCDR2 with an amino acid sequence of DAS, DTS, KAS, NDD or RAS or an active variant thereof, and / or a LCDR3 as shown in at least one of SEQ ID NOs: 25-32 or an active variant thereof; and / or the heavy chain variable region comprises: a HCDR1 as shown in at least one of SEQ ID NOs: 33-40 or an active variant thereof, and / or a HCDR2 as shown in at least one of SEQ ID NOs: 41-48 or an active variant thereof, and / or a HCDR3 as shown in at least one of SEQ ID NOs: 49-56 or an active variant thereof.

[0007] In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 17, a LCDR2 with the amino acid sequence of RAS, and a LCDR3 of SEQ ID NO: 25, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 33, a HCDR2 of SEQ ID NO: 41, and a HCDR3 of SEQ ID NO: 49. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 18, a LCDR2 with the amino acid sequence of NDD, and a LCDR3 of SEQ ID NO: 26, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 34, a HCDR2 of SEQ ID NO: 42, and a HCDR3 of SEQ ID NO: 50. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 19, a LCDR2 with the amino acid sequence of DTS, and a LCDR3 of SEQ ID NO: 27, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 35, a HCDR2 of SEQ ID NO: 43, and a HCDR3 of SEQ ID NO: 51. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 20, a LCDR2 of the amino acid sequence DAS, and a LCDR3 of SEQ ID NO: 28, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 36, a HCDR2 of SEQ ID NO: 44, and a HCDR3 of SEQ ID NO: 52. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 21, a LCDR2 of the amino acid sequence RAS, and a LCDR3 of SEQ ID NO: 29, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 37, a HCDR2 of SEQ ID NO: 45, and a HCDR3 of SEQ ID NO: 53. In some embodiments, the light chain variable region has LCDR1 shown in SEQ ID NO:22, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:30, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:46, and HCDR3 shown in SEQ ID NO:54.In some embodiments, the light chain variable region has a LCDR1 set forth in SEQ ID NO: 23, a LCDR2 with an amino acid sequence of DTS, and a LCDR3 set forth in SEQ ID NO: 31, and the heavy chain variable region has a HCDR1 set forth in SEQ ID NO: 39, a HCDR2 set forth in SEQ ID NO: 47, and a HCDR3 set forth in SEQ ID NO: 55. In some embodiments, the light chain variable region has a LCDR1 set forth in SEQ ID NO: 24, a LCDR2 with an amino acid sequence of KAS, and a LCDR3 set forth in SEQ ID NO: 32, and the heavy chain variable region has a HCDR1 set forth in SEQ ID NO: 40, a HCDR2 set forth in SEQ ID NO: 48, and a HCDR3 set forth in SEQ ID NO: 56.

[0008] In some embodiments, the first antigen binding region comprises a light chain variable region sequence shown in at least one of SEQ ID NOs: 1-8 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids, and / or a heavy chain variable region sequence shown in at least one of SEQ ID NOs: 9-16 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids.

[0009] In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 1 and the heavy chain variable region sequence set forth in SEQ ID NO: 9. In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 2 and the heavy chain variable region sequence set forth in SEQ ID NO: 10. In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 3 and the heavy chain variable region sequence set forth in SEQ ID NO: 11. In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 4 and the heavy chain variable region sequence set forth in SEQ ID NO: 12. In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 5 and the heavy chain variable region sequence set forth in SEQ ID NO: 13. In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 6 and the heavy chain variable region sequence set forth in SEQ ID NO: 14. In some embodiments, the first antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 7 and the heavy chain variable region sequence set forth in SEQ ID NO: 15. In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:16.

[0010] In some embodiments, the first peptide has the amino acid sequence shown in SEQ ID NO: 57 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids.

[0011] In some embodiments, the first peptide has the amino acid sequence shown in SEQ ID NO: 57: VEGYRLFDRKS.

[0012] In some embodiments, when the first peptide contacts a second peptide having an amino acid sequence shown in SEQ ID NO: 58, which is an active variant obtained by adding, deleting, replacing or modifying one or more amino acids, a detectable luminescent signal is generated in the presence of a substrate. Preferably, the first peptide associates with the second peptide.

[0013] In some embodiments, the second peptide has the sequence shown in SEQ ID NO: 58:

[0014] In some embodiments, the first antigen binding region is directly linked to the first peptide or linked via a first linker.

[0015] In some embodiments, the first linker is a polypeptide linker and / or a non-polypeptide linker.

[0016] In some embodiments, in the coupled polypeptide, the first linker is a flexible linker peptide or a rigid linker peptide.

[0017] In some embodiments, the first linker is a flexible linker peptide having the formula (G)nS(G)mS(G)lS, wherein n is selected from 1 to 5, m is selected from 0 to 5, and l is selected from 0 to 5. In some embodiments, n can be 1, 2, 3, 4, or 5, m can be 0, 1, 2, 3, 4, or 5, and l can be 0, 1, 2, 3, 4, or 5.

[0018] In some embodiments, the first linker comprises GGGS, GGGSGGS (SEQ ID NO: 84), GGGSGGGGS (SEQ ID NO: 85), GGGSGGGSGGGS (SEQ ID NO: 86), or GGGGSGGGGSGGGGS (SEQ ID NO: 87), preferably GGGGSGGGGSGGGGS (SEQ ID NO: 87).

[0019] In some embodiments, the first linker is a rigid linker peptide having the formula A(EAAAK)oA, wherein o is selected from 0 to 5, for example, 0, 1, 2, 3, 4 or 5.

[0020] In some embodiments, the first linker comprises AEAAAKA (SEQ ID NO:88), AEAAAKEAAAKA (SEQ ID NO:89) or AEAAAKEAAAKEAAAKA (SEQ ID NO:90).

[0021] In some embodiments, in the coupled polypeptide, the first linker is a chemical linker.

[0022] In some embodiments, the first linker comprises one or more of acrylic acid phosphoramidite, adenylation, azide, digoxigenin, cholesterol-TEG, amino modifier, alkyne, biotinylation, thiol modification, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), glycerol, C2 to C12 alkyl, and succinimide.

[0023] In some embodiments, the first antigen-binding region and the first peptide are linked via a polypeptide linker and a chemical linker. In some specific embodiments, the first antigen-binding region and the first peptide are linked via a GSlinker and an SMCC coupling agent, wherein one end of the SMCC coupling agent is coupled to a primary amino group of the first antigen-binding region and the other end is coupled to a free cysteine ​​of the first peptide.

[0024] In some embodiments, the first antigen-binding region and the first peptide are directly linked via 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0025] In another aspect, the present invention provides a fusion polypeptide or a coupled polypeptide comprising a second antigen-binding region and a second peptide derived from luciferase, wherein the second antigen-binding region binds to one or more of procalcitonin, preprocalcitonin, the N-terminus of procalcitonin, calcitonin, and a calcitonin peptide, wherein the second antigen-binding region comprises at least one light chain variable region and at least one heavy chain variable region; the light chain variable region comprises: a LCDR1 as shown in at least one of SEQ ID NOs: 17-24 or an active variant thereof, a LCDR2 having an amino acid sequence of DAS, DTS, KAS, NDD or RAS or an active variant thereof, and / or a LCDR3 as shown in at least one of SEQ ID NOs: 25-32 or an active variant thereof; and / or the heavy chain variable region comprises: a HCDR1 as shown in at least one of SEQ ID NOs: 33-40 or an active variant thereof, and / or a HCDR2 as shown in at least one of SEQ ID NOs: 41-48 or an active variant thereof, and / or a HCDR3 as shown in at least one of SEQ ID NOs: 49-56 or an active variant thereof.

[0026] In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 17, a LCDR2 with the amino acid sequence of RAS, and a LCDR3 of SEQ ID NO: 25, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 33, a HCDR2 of SEQ ID NO: 41, and a HCDR3 of SEQ ID NO: 49. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 18, a LCDR2 with the amino acid sequence of NDD, and a LCDR3 of SEQ ID NO: 26, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 34, a HCDR2 of SEQ ID NO: 42, and a HCDR3 of SEQ ID NO: 50. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 19, a LCDR2 with the amino acid sequence of DTS, and a LCDR3 of SEQ ID NO: 27, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 35, a HCDR2 of SEQ ID NO: 43, and a HCDR3 of SEQ ID NO: 51. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 20, a LCDR2 of the amino acid sequence DAS, and a LCDR3 of SEQ ID NO: 28, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 36, a HCDR2 of SEQ ID NO: 44, and a HCDR3 of SEQ ID NO: 52. In some embodiments, the light chain variable region comprises a LCDR1 of SEQ ID NO: 21, a LCDR2 of the amino acid sequence RAS, and a LCDR3 of SEQ ID NO: 29, and the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 37, a HCDR2 of SEQ ID NO: 45, and a HCDR3 of SEQ ID NO: 53. In some embodiments, the light chain variable region has LCDR1 shown in SEQ ID NO:22, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:30, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:46, and HCDR3 shown in SEQ ID NO:54.In some embodiments, the light chain variable region has a LCDR1 set forth in SEQ ID NO: 23, a LCDR2 with an amino acid sequence of DTS, and a LCDR3 set forth in SEQ ID NO: 31, and the heavy chain variable region has a HCDR1 set forth in SEQ ID NO: 39, a HCDR2 set forth in SEQ ID NO: 47, and a HCDR3 set forth in SEQ ID NO: 55. In some embodiments, the light chain variable region has a LCDR1 set forth in SEQ ID NO: 24, a LCDR2 with an amino acid sequence of KAS, and a LCDR3 set forth in SEQ ID NO: 32, and the heavy chain variable region has a HCDR1 set forth in SEQ ID NO: 40, a HCDR2 set forth in SEQ ID NO: 48, and a HCDR3 set forth in SEQ ID NO: 56.

[0027] In some embodiments, the second antigen-binding region comprises a light chain variable region sequence shown in at least one of SEQ ID NOs: 1-8 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids, and / or a heavy chain variable region sequence shown in at least one of SEQ ID NOs: 9-16 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids.

[0028] In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 1 and the heavy chain variable region sequence set forth in SEQ ID NO: 9. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 2 and the heavy chain variable region sequence set forth in SEQ ID NO: 10. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 3 and the heavy chain variable region sequence set forth in SEQ ID NO: 11. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 4 and the heavy chain variable region sequence set forth in SEQ ID NO: 12. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 5 and the heavy chain variable region sequence set forth in SEQ ID NO: 13. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 6 and the heavy chain variable region sequence set forth in SEQ ID NO: 14. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence set forth in SEQ ID NO: 7 and the heavy chain variable region sequence set forth in SEQ ID NO: 15. In some embodiments, the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:16.

[0029] In some embodiments, the second peptide has the amino acid sequence shown in SEQ ID NO: 58 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids.

[0030] In some embodiments, when the second peptide contacts an active variant of a first peptide having an amino acid sequence shown in SEQ ID NO: 57, which is obtained by adding, deleting, replacing or modifying one or more amino acids, a detectable luminescent signal is generated in the presence of a substrate. Preferably, the first peptide associates with the second peptide.

[0031] In some embodiments, the second antigen binding region is directly linked to the second peptide or linked via a second linker.

[0032] In some embodiments, the second linker is a polypeptide linker and / or a non-polypeptide linker.

[0033] In some embodiments, in the coupled polypeptide, the second linker is a flexible linker peptide or a rigid linker peptide.

[0034] In some embodiments, the second linker is a flexible linker peptide having the formula GSS(G)p(S)q(G)r(S)s(G)t(S)u, wherein p is selected from 1 to 5, q is selected from 0 to 5, r is selected from 0 to 5, s is selected from 0 to 1, t is selected from 0 to 5, and u is selected from 0 to 5. In some embodiments, p can be 1, 2, 3, 4, or 5, q can be 0, 1, 2, 3, 4, or 5, r can be 0, 1, 2, 3, 4, or 5, s can be 0 or 1, t can be 0, 1, 2, 3, 4, or 5, and u can be 0, 1, 2, 3, 4, or 5.

[0035] In some embodiments, the second linker comprises GSSG, GSSGGGS (SEQ ID NO: 91), GSSGGGSGGS (SEQ ID NO: 92), GSSGGGGSGGGS (SEQ ID NO: 93) or GSSGGGGSGGGGSGG (SEQ ID NO: 94), preferably GSSGGGGSGGGGSGG (SEQ ID NO: 94).

[0036] In some embodiments, in the coupled polypeptide, the second linker is a chemical linker.

[0037] In some embodiments, the second linker comprises one or more of acrylic acid phosphoramidite, adenylation, azide, digoxigenin, cholesterol-TEG, amino modifier, alkyne, biotinylation, thiol modification, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), glycerol, C2 to C12 alkyl and succinimide.

[0038] In some embodiments, the second antigen-binding region and the second peptide are linked via a polypeptide linker and a chemical linker. In some specific embodiments, the second antigen-binding region and the second peptide are linked via a GSlinker and an SMCC coupling agent, wherein one end of the SMCC coupling agent is coupled to a primary amino group of the second antigen-binding region and the other end is coupled to a free cysteine ​​of the second peptide.

[0039] In some embodiments, the second antigen-binding region and the second peptide are directly linked via 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0040] In yet another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the fusion polypeptide of the present invention.

[0041] In another aspect, the present invention provides an expression vector comprising the nucleotide sequence of the nucleic acid molecule of the present invention.

[0042] In yet another aspect, the present invention provides a host cell comprising the expression vector of the present invention.

[0043] In another aspect, the present invention provides a composition comprising a fusion polypeptide or coupled polypeptide comprising a first antigen-binding region and a first peptide, and a fusion polypeptide or coupled polypeptide comprising a second antigen-binding region and a second peptide.

[0044] In some embodiments, the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9, or comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10, or comprises the light chain variable region sequence shown in SEQ ID NO:3 and the heavy chain variable region sequence shown in SEQ ID NO:11, or comprises the light chain variable region sequence shown in SEQ ID NO:4 and the heavy chain variable region sequence shown in SEQ ID NO:12, or comprises the light chain variable region sequence shown in SEQ ID NO:5 and the heavy chain variable region sequence shown in SEQ ID NO:13, or comprises the light chain variable region sequence shown in SEQ ID NO:6 and the heavy chain variable region sequence shown in SEQ ID NO:14, or comprises the light chain variable region sequence shown in SEQ ID NO:7 and the heavy chain variable region sequence shown in SEQ ID NO:15, or comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:16.

[0045] In some embodiments, the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9, or comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10, or comprises the light chain variable region sequence shown in SEQ ID NO:3 and the heavy chain variable region sequence shown in SEQ ID NO:11, or comprises the light chain variable region sequence shown in SEQ ID NO:4 and the heavy chain variable region sequence shown in SEQ ID NO:12, or comprises the light chain variable region sequence shown in SEQ ID NO:5 and the heavy chain variable region sequence shown in SEQ ID NO:13, or comprises the light chain variable region sequence shown in SEQ ID NO:6 and the heavy chain variable region sequence shown in SEQ ID NO:14, or comprises the light chain variable region sequence shown in SEQ ID NO:7 and the heavy chain variable region sequence shown in SEQ ID NO:15, or comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:16.

[0046] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9.

[0047] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10.

[0048] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:3 and the heavy chain variable region sequence shown in SEQ ID NO:11.

[0049] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:6 and the heavy chain variable region sequence shown in SEQ ID NO:14.

[0050] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:4 and the heavy chain variable region sequence shown in SEQ ID NO:12, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9.

[0051] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:4 and the heavy chain variable region sequence shown in SEQ ID NO:12, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10.

[0052] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:7 and the heavy chain variable region sequence shown in SEQ ID NO:15, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:2 and the heavy chain variable region sequence shown in SEQ ID NO:10.

[0053] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:7 and the heavy chain variable region sequence shown in SEQ ID NO:15, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:5 and the heavy chain variable region sequence shown in SEQ ID NO:13.

[0054] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:4 and the heavy chain variable region sequence shown in SEQ ID NO:12, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:16.

[0055] In some embodiments, the first antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:16, and the second antigen binding region comprises the light chain variable region sequence shown in SEQ ID NO:1 and the heavy chain variable region sequence shown in SEQ ID NO:9.

[0056] In some embodiments, the first linker comprises GGGS, GGGSGGS (SEQ ID NO:84), GGGSGGGGS (SEQ ID NO:85), GGGSGGGSGGGS (SEQ ID NO:86), or GGGGSGGGGSGGGGS (SEQ ID NO:87).

[0057] In some embodiments, the second linker comprises GSSG, GSSGGGS (SEQ ID NO:91), GSSGGGSGGS (SEQ ID NO:92), GSSGGGGSGGGS (SEQ ID NO:93), or GSSGGGGSGGGGSGG (SEQ ID NO:94).

[0058] In some embodiments, the composition further comprises a substrate, wherein the substrate is selected from coelenterazine and / or its analogs. In some embodiments, the substrate is packaged together with the fusion polypeptide or packaged independently.

[0059] In some embodiments, the substrate includes but is not limited to at least one of Furimazine, coelenterazine-h (CTZ-h), coelenterazine-hh (CTZ-hh), and coelenterazine-hcp (CTZ-hcp).

[0060] In yet another aspect, the present invention provides a bioluminescence detection kit comprising the composition of the present invention.

[0061] The bioluminescence detection kit provided by the present invention is used for detecting one or more test substances selected from procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide.

[0062] In another aspect, the present invention also provides the use of the fusion polypeptide or coupled polypeptide, the nucleic acid molecule, the expression vector, the host cell or the composition in preparing a kit for detecting an analyte comprising one or more selected from procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide.

[0063] In another aspect, the present invention also provides the use of the fusion polypeptide or coupled polypeptide, the nucleic acid molecule, the expression vector, the host cell, the composition or the kit in detecting an analyte comprising one or more selected from procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide.

[0064] In another aspect, the present invention also provides a homogeneous detection method, which comprises incubating a sample to be tested with a fusion polypeptide or coupled polypeptide comprising a first antigen binding region and a first peptide, and a fusion polypeptide or coupled polypeptide comprising a second antigen binding region and a second peptide, and detecting a luminescent signal in the presence of a substrate.

[0065] In some embodiments, the test sample is suspected of containing one or more analytes selected from the group consisting of procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide.

[0066] In some embodiments, the incubation time is 3-5 minutes.

[0067] In some embodiments, the luminescent signal is detected within 0.01-100 seconds after the addition of the substrate solution containing the coelenterazine analog.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] (1) The present invention obtained a series of antibody combinations with high signal-to-noise ratio and sensitivity after pairing a batch of SCFV antibodies independently developed, and found that rSCFV18 fused to the N-terminus of the small subunit RB73 and rSCFV5 fused to the C-terminus of the large subunit can provide the best detection effect;

[0070] (2) By optimizing the connecting peptide between the SCFV antibody and the luciferase subunit, a better connecting peptide was obtained and combined to obtain a new fusion peptide, which increased the signal-to-noise ratio by about 5 times and further improved the sensitivity of the detection;

[0071] (3) The fusion polypeptide of the present invention has excellent clinical correlation in the detection of procalcitonin or its analogs, and can detect the concentration of procalcitonin in clinical body fluid samples within 5 minutes, which is much shorter than products based on other methods;

[0072] (4) The present invention also provides a homogeneous procalcitonin detection method that is as sensitive as conventional magnetic microparticle chemiluminescence detection. Furthermore, this method does not require magnetic separation, washing, or other detection steps, resulting in a shorter detection time, faster detection speed, and a simpler instrument structure. The reagent production process does not require magnetic bead labeling or enzyme labeling, resulting in a simple production process, low cost, and minimal batch-to-batch variability. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] FIG1 shows a schematic diagram of the principle of complementary luciferase dual antibody (SCFV) complementary target detection.

[0074] FIG2 shows a schematic diagram of the fusion of rSCFV18 and the small subunit.

[0075] FIG3 shows a schematic diagram of the fusion of SCFV5 and the large subunit.

[0076] FIG4 shows the concentration-RLU curves of the homogeneous detection of PCT standards using different combinations of enzyme-fused antibodies.

[0077] FIG5 shows the concentration-RLU curves of the homogeneous detection of PCT standards using different combinations of enzyme-fused antibodies.

[0078] FIG6 shows the correlation analysis results of the PCT clinical samples measured by the combination of rSCFV18-RB73 and OL-rSCFV5.

[0079] FIG7 shows the standard curve (RLU-concentration) of PCT detection using the enzyme-antibody combination after optimization of the linker peptide.

[0080] FIG8 shows the correlation analysis results of the enzyme-antibody combination for PCT measurement after the linker peptide is optimized.

[0081] FIG9 shows the correlation analysis results of PCT clinical samples detected by different substrates.

[0082] FIG10 shows the correlation analysis results of luciferase fusion antibody detection of PCT clinical samples. DETAILED DESCRIPTION

[0083] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0084] definition

[0085] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0086] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0087] The term "antibody" as used herein includes intact antibodies and any antigen-binding fragments (i.e., "antigen-binding regions," "antigen-binding polypeptides," or "immunobinders"), or single chains thereof. A single-chain antibody fragment (scFv) is composed of the heavy and light chain variable regions of an antibody, linked by a short peptide (linker) of 15-20 amino acids. scFvs retain their affinity for antigens and possess advantages such as low molecular weight, strong penetrating power, and low antigenicity.

[0088] The term "hypervariable region" or "HVR" of the present invention refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariable loop"). Typically, a natural four-chain antibody comprises six HVRs: three present in VH (H1, H2, H3) and three present in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions (CDRs)", which have the highest sequence variability and / or are involved in antigen recognition. It will be understood by those skilled in the art that, unless otherwise specified, "CDRs" and "complementarity determining regions" of a given antibody or region thereof (e.g., variable region) should be understood to encompass "CDRs" and "complementarity determining regions" as defined by any one of the methods known in the art. Based on the CDR sequences or heavy chain / light chain variable region sequences of the present invention, amino acid sequences with one or more amino acid shifts / extensions / losses obtained by the definition rules of other CDRs should also fall within the scope of protection of the present invention.

[0089] The term "fusion polypeptide" as used herein refers to the use of genetic expression of polynucleotides encoding proteins or protein synthesis methods to connect two or more proteins or fragments thereof through their respective peptide backbones. Preferably, the polypeptides or fragments thereof are from different sources. In some embodiments, the fusion polypeptide comprises the peptide and an antibody to the antigen to be detected. The fusion polypeptide or fragment thereof herein may contain conservative amino acid substitutions at one or more amino acid residues, for example, at essential or non-essential amino acid residues. "Conservative amino acid substitutions" are replacements of amino acid residues with amino acid residues having similar side chains.

[0090] As used herein, the term "coupled polypeptide" refers to the covalent attachment of two or more proteins or fragments thereof via a non-polypeptide linker using chemical coupling methods. Preferably, the polypeptides or fragments thereof are from different sources. In some embodiments, the coupled polypeptide comprises the peptide and an antibody to the antigen to be detected.

[0091] The term "luciferase" as used herein is a general term for enzymes that can produce bioluminescence in nature. In the present invention, luciferase refers to all enzymes that catalyze luminescence in the presence of substrates such as coelenterazine or its analogs, and combinations of enzyme fragments that still have catalytic activity after recombining their fragments, including but not limited to: Oplophorus luciferase, Nanoluc, Nanobit, Gaussia luciferase, Renilla luciferase, etc. Nanoluc is a new type of luciferase with improved stability and activity. Its activity is 150 times higher than that of firefly luciferase Fluc. The protein is stable and has a small molecular weight. NanoLuc Binary Technology (Nanobit) is a two-subunit system based on luciferase NanoLuc, which is used to detect protein interactions in living cells. Nanobit divides NanoLuc luciferase into two subunits, namely the large subunit (LgBiT) and the small subunit (SmBiT), wherein the N-terminal large subunit is about 158 ​​amino acids and the C-terminal small subunit is about 11 amino acids. These two subunits can be expressed as fusion proteins with two target proteins to be tested. When the interaction between the target proteins causes the two subunits to bind, an active luciferase is obtained, which can then react with the substrate to produce luminescence for the determination of molecular interactions between target molecules. In some embodiments, Nanobit large and small subunits are associated with the target molecule and operate the bioluminescent complex through the molecular interaction of the target molecule. If the target molecule participates in a sufficiently stable interaction, a bioluminescent complex is formed and a bioluminescent signal is generated. If the target molecule cannot participate in a sufficiently stable interaction, a bioluminescent complex will not be formed or will only be formed weakly, and the bioluminescent signal is undetectable or greatly reduced. In some embodiments, the amplitude of the detectable bioluminescent signal is proportional (e.g., directly proportional) to the amount, intensity, promotion and / or stability of the molecular interaction between the target molecules.

[0092] The term "coelenterazine" as used herein is a luciferin found in seven phyla of aquatic organisms that emits light and is a substrate for many luciferases and photoproteins. Coelenterazine and the system consisting of luciferases using it as a substrate are a common bioluminescent system. In addition to natural coelenterazine being a substrate for luciferase, coelenterazine analogs with characteristics such as different spectral properties and stability can also be used. Exemplary coelenterazine analogs include: Coelenterazine h (CTZ-h): (2-deoxy coelenterazine or 2,8-dibenzyl-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one); Coelenterazine hh (CTZ-hh): (dideoxy coelenterazine or 2,8-dibenzyl-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one); Furimazine (FZ): (8-benzyl-2-[(furan-2-yl)methyl]-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one); Coelenterazine-f (Coelenterazine f, CTZ-f): (8-Benzyl-2-(4-fluorophenyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one); JRW-0238: (8-Benzyl-2-(furan-2-ylmethyl)-6-(3-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one); JRW-1743: (6-(3-amino-2-fluorophenyl)-8-(2-fluorobenzyl)-2-(furan-2-ylmethyl) imidazo[1,2-α]pyrazine-3(7H)-one); JRW-1744: (6-(3-amino-2-fluorophenyl)-8-benzyl-2-(furan-2-ylmethyl)imidazo[1,2-α]pyrazine-3(7H)-one); other exemplary coelenterazine analogs include coelenterazine n, coelenterazine f, coelenterazine hcp, coelenterazine cp, coelenterazine c, coelenterazine e, coelenterazine fcp, coelenterazine I, coelenterazine icp, coelenterazine v, 2-methyl coelenterazine, and the like.

[0093] Among them, coelenterazine has the following structure:

[0094] An exemplary coelenterazine analog has the following structure:

[0095] The gene encoding procalcitonin (PCT), as used herein, is located on human chromosome 11. The gene consists of six exons and five introns, with a total length of 7,600 base pairs (bp), of which the coding region is 2,800 bp. PCT is a specific marker for severe bacterial and fungal infections and a reliable indicator of multiple organ failure associated with sepsis and inflammatory activity. MBP-PCT refers to PCT tagged with maltose-binding protein (MBP).

[0096] As used herein, the term "connector" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more connecters can be connected in series. When multiple connecters are present, each connecter can be the same or different. In general, the connecter provides flexibility or prevents / improves steric hindrance. The connecter is not typically cut; however, in some aspects, such cutting may be desirable. Therefore, in some embodiments, the connecter may include one or more protease cleavable sites, which may be located within the connecter sequence or flanking the connecter at either end of the connecter sequence.

[0097] In some embodiments, the linker is a polypeptide linker. In some embodiments, the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95 or at least about 100 amino acids. In some embodiments, the linker comprises one or more amino acids.

[0098] In some embodiments, the linker is a non-polypeptide linker. In some embodiments, the linker comprises an acrylic acid phosphoramidite (e.g., ACRYDITE™), adenylation, azide (NHS Ester), digoxigenin (NHS Ester), cholesterol-TEG, I-LINKER™, an amino modifier (e.g., amino modifier C6, amino modifier C12, amino modifier C6 dT, or Uni-Link™ amino modifier), an alkyne, a 5' hexynyl, a 5-octadiynyl dU, biotinylation (e.g., biotin, biotin (azide), biotin dT, biotin-TEG, dual biotin, PC biotin, or desthiobiotin), a thiol modification (thiol modifier C3 SS, dithiol, or thiol modifier C6 SS), tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), glycerol, a C2 to C12 alkyl group, a succinimide, or any combination thereof.

[0099] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0100] The reagents used in the following examples are as follows:

[0101] (1) PBSA formula (enzyme diluent):

[0102] Anhydrous KH2PO4 0.2g / L, anhydrous Na2HPO4 0.61g / L, KCl 0.2g / L, NaCl 8g / L, BSA 1g / L, PEG 20000 20g / L, TritonX-405 100μl / L, proclin 300 1ml / L, ethanol 1.37ml / L, pH 7.4.

[0103] (2) Obuffer formula (substrate working solution):

[0104] MES 100mM, CDTA 1mM, Tergitol 0.02%, KCl 150mM, Thiorea 35mM, proclin 300 0.10%, pH 6.37.

[0105] (3) Furimazine mother solution formula:

[0106] 10% Methanol, 15% DMSO, 75% Propylene Glycol, 2.5 mM Furimazine.

[0107] (4) CTZ-h mother liquor formula:

[0108] 10% methanol, 15% DMSO, 75% propylene glycol, 2.5 mM CTZ-h.

[0109] The drugs or culture media used in the following examples are as follows:

[0110] TritonX-405, purchased from Aladdin, catalog number T101476;

[0111] Proclin 300, purchased from Sigma, product number 48912-V;

[0112] MES (4-morpholineethanesulfonic acid), purchased from Aladdin, product number M163013-25g;

[0113] CDTA (1,2-cyclohexanediaminetetraacetic acid) was purchased from Aladdin, product number C193548-5g;

[0114] Tergitol, purchased from Sigma, product number 15S9-100ml;

[0115] polymyxin B (polymyxin B sulfate), purchased from Sigma, catalog number P1004-25MU;

[0116] Furimazine (a luminescent substrate of imidazopyrazinone) was purchased from Wuhan Costan Biotechnology Co., Ltd., catalog number CS15359;

[0117] CTZ-h (coelenterazine h) was purchased from Baiying Biotechnology, catalog number 21165;

[0118] CTZ-hcp (coelenterazine) was purchased from Baiying Biotechnology, catalog number 21154;

[0119] SB medium: 3.2% tryptone (OXOID, LP0042B), 2% yeast extract (OXOID, LP0021B), 0.5% NaCl (Sangon Biotech, A501218-0001), sterilized by high-pressure steam.

[0120] MBP-PCT (procalcitonin fused to the maltose binding protein MBP at the N-terminus, PCT; SEQ ID NO: 59) is prepared by fusing MBP to PCT (NP_001029124.1, AA26-141) via a linker peptide (NAEAAAKEAAAKALVPRGSGGGGS). After fusion expression in E. coli, the fusion protein was affinity purified using MBP protein purification resin. In the following examples, the concentration of MBP-PCT is calculated based on the effective concentration of PCT. For example, a 1 ng / ml MBP-PCT concentration means an effective PCT concentration of 1 ng / ml. The actual concentration of MBP-PCT required is 1 * 55.9 / 12.8 / 90% = 4.85 ng / ml (the estimated molecular weight of MBP-PCT is 55.9 kD, the estimated molecular weight of PCT is 12.8 kD, and the purity of MBP-PCT is calculated as 90%).

[0121] Unless otherwise specified, other reagents, instruments, etc. used in the present invention are commonly used and commercially available in the art and can be purchased on the market.

[0122] Referring to Figure 1 , the present invention fuses a series of independently developed PCT antibodies with the large and small subunits of luciferase, respectively, to obtain fusion combinations in which the luminescence value is linearly correlated with the PCT content. Furthermore, the connecting peptides between the antibody and enzyme fragments in the preferred combination are optimized and modified to screen for connecting peptides with higher signal-to-noise ratio and sensitivity.

[0123] Example 1 Screening of combinations of different SCFV antibodies fused to luciferase large and small subunits

[0124] (1) Expression of antibody-small subunit fusion polypeptide:

[0125] The rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22 and luciferase small subunit variant RB73 fusion expression vector pComb3X (AF268281.2, containing the OMPA secretion signal peptide and g3 deleted) was constructed by seamless cloning. The fusion sequence was OMPA-H6-GSG-rSCFVx (rSCFVx, respectively, rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22)-GGGSGGGGS (SEQ ID NO:85)-VEGYRLFDRKS (SEQ ID NO:57, RB73), see Figure 2. Correctly sequenced clones were inoculated at a 1:100 ratio into 200 ml of SB medium and cultured at 37°C, 250 rpm, for ~3 h. When the OD600 reached 1.0-1.5, IPTG was added to 1 mM, and the culture was continued at 30°C, 250 rpm, for 16-20 h. The cells were collected by centrifugation, resuspended in 10 ml PBS, and polymyxin B was added to 20,000 U / ml. The cells were shaken at room temperature for 1 hour and centrifuged at 15,000 g for 10 minutes. The supernatant was purified according to the operating procedures of Ni-NTA purification resin to purify the his-tagged protein.

[0126] (2) Expression of large subunit-antibody fusion polypeptide:

[0127] A seamless cloning approach was used to construct the fusion expression vector Abvec (FJ475055.1, with CH1, 2, and 3 deleted) for the large luciferase subunit OL and rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22. The fusion sequence was mouse IgG1 secretion signal peptide-OL-GSSGGGGSGGGS (SEQ ID NO: 93)-rSCFVx-GQAG-H6 (rSCFVx represented rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22, respectively), as shown in Figure 3. Plasmids were extracted from correctly sequenced clones, endotoxin-free, and filter-sterilized. Freestyle 293 suspension cells were transfected using PEI25000 as the transfection mediator according to standard protocols and cultured at 37°C, 125 rpm, and 7% CO2 for 9 days. The His-tagged protein was purified from the cell culture supernatant using Ni-NTA purification resin. The relevant sequences of rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22 are shown in Table 1 below.

[0128] Table 1

[0129] (3) Expression of antigen standards:

[0130] Preparation of PCT antigen standard: The procalcitonin (PCT) sequence is fused with the maltose binding protein (MBP) gene, induced and expressed in Escherichia coli, and purified. The product protein MBP-PCT is used as a procalcitonin standard. The protein concentration is estimated based on the actual effective concentration of PCT and calibrated using Roche's electrochemiluminescence clinical detection reagent. The PCT concentrations indicated in this patent are all calibrated concentrations.

[0131] (4) Test method:

[0132] Antibody-small subunit fusion peptides (rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22-RB73) were diluted to 20 nM in PBSA and 10 μl was added to each well. Large subunit-antibody fusion peptides (OL-rSCFV2 / 5 / 12 / 13 / 14 / 17 / 18 / 22) were diluted to 20 nM in PBSA and 10 μl was added to each well. Procalcitonin was diluted to 5 ng / ml in PBSA and 10 μl was added to each well. A control was replaced with 10 μl of PBSA. The antibody-enzyme-antigen mixture was gently mixed and incubated at 37°C for 6 min. 50 μl of Obuffer containing furimazine (furimazine concentration: 10 μM) was added to each well, and luminescence was read after 90 s. Pg luminescence value = (signal value of wells containing PCT - signal value of wells without PCT) / number of Pg added to PCT; signal-to-noise ratio = signal value of wells containing PCT / signal value of wells without PCT. The results of different combinations of enzyme-fused antibodies are shown in Table 2 below.

[0133] As can be seen from the results in Table 2, the luminescence values ​​and signal-to-noise ratios of different combinations of the above-mentioned SCFV antibodies fused with luciferase fragments were excellent.

[0134] Example 2

[0135] (1) The fusion expression and purification of antibody-small subunit fusion polypeptide and large subunit-antibody fusion polypeptide were the same as in Example 1.

[0136] (2) Test method:

[0137] The antibody-small subunit fusion peptide was diluted to 10 nM in PBSA, and 14 μl was added to each well. The large subunit-antibody fusion peptide was diluted to 10 nM in PBSA, and 14 μl was added to each well. The PCT antigen was serially diluted from 1 μg / ml in PBSA in a 10-fold increment, and 14 μl was added to each well. All three components were mixed and incubated at 37°C for 15 minutes. 50 μl of O buffer containing furimazine at a final concentration of 20 μM was added to each well, and luminescence was measured after 160 seconds. The signal-to-noise ratio (SNR) was calculated as the signal value of the well containing PCT / the signal value of the well without PCT. The SNR results for homogeneous detection of PCT using different combinations of enzyme-fused antibodies are shown in Table 3 below, and the standard curve is shown in Figure 4.

[0138] Table 3. Signal-to-noise ratio and sensitivity prediction of standard 1 for homogeneous detection of PCT using different combinations of enzyme-fused antibodies

[0139] From the results in Table 3 and Figure 4 , it can be seen that the luminescent signal values ​​of all combinations increased with increasing antigen amount, and there was a good linear relationship between the signal value and the antigen amount, and the detection sensitivity was high (<100 pg / ml).

[0140] Example 3

[0141] (1) The fusion expression and purification of antibody-small subunit fusion polypeptide and large subunit-antibody fusion polypeptide were the same as in Example 1.

[0142] (2) Test method:

[0143] Dilute the antibody-small subunit fusion peptide to 2.5-10 nM in PBSA, and add 14 μl to each well. Dilute the large subunit-antibody fusion peptide to 2.5-10 nM in PBSA, and add 14 μl to each well. Dilute the PCT antigen in PBSA in a 10-fold serial dilution from 1 μg / ml downward, and add 14 μl to each well. Mix all three components, incubate at 37°C for 10 minutes, add 50 μl of O buffer containing furimazine at a final concentration of 20 μM to each well, and measure luminescence after 120 seconds. Signal-to-noise ratio = signal value of wells containing PCT / signal value of wells without PCT. The signal-to-noise ratio results for homogeneous detection of PCT using different combinations of enzyme-fused SCFV antibodies are shown in Table 4 below, and standard curve 2 is shown in Figure 5.

[0144] Table 4. Signal-to-noise ratio and sensitivity prediction of standard 2 for homogeneous detection of PCT using different combinations of enzyme-fused antibodies

[0145] From the results in Table 4 and Figure 5 , it can be seen that the luminescence signal value of all combinations increases with the increase of antigen amount, and there is a good linear relationship between the signal value and the antigen amount. The estimated detection sensitivity can reach as low as 40 pg / ml.

[0146] Example 4

[0147] (1) The fusion expression and purification of antibody-small subunit fusion polypeptide and large subunit-antibody fusion polypeptide were the same as in Example 1.

[0148] (2) Test method:

[0149] In the same tube of PBSA, dilute the antibody-small subunit fusion peptide to 10 nM with PBSA, and dilute the large subunit-antibody fusion peptide to 10 nM with PBSA. Mix well and add 28 μl to each well. Add 10 μl of the assigned clinical plasma sample to each well. Mix these two components, incubate at 37°C for 6 minutes, and add 50 μl of O buffer containing a final concentration of 50 μM furimazine to each well. After 100 seconds, measure the luminescence value. The comparison results of enzyme fusion antibody combinations 4 and 7 in detecting PCT clinical samples are shown in Table 5 below. The correlation of homogeneous detection of PCT clinical samples by combination 7 is shown in Figure 6.

[0150] Table 5. Comparison of clinical samples for the detection of PCT using a combination of preferred enzyme-fused antibodies

[0151] From the results in Table 5 and Figure 6 , it can be seen that the clinical comparison correlation between combination 4 and combination 7 is high.

[0152] Example 5

[0153] (1) The fusion expression and purification of antibody-small subunit fusion polypeptide and large subunit-antibody fusion polypeptide were the same as in Example 1.

[0154] (2) Test method:

[0155] In the same tube of PBSA, the antibody-small subunit fusion polypeptide was diluted to 10 nM with PBSA, and the large subunit-antibody fusion polypeptide was diluted to 10 nM with PBSA. After mixing, 28 μl was added to each well. 10 μl of the assigned clinical plasma sample was added to each well. The two components were mixed, incubated at 37°C for 6 min, and 50 μl of Obuffer containing a final concentration of 200 μM Furimazine was added to each well. The luminescence value was measured after 180 s. The comparison results of clinical samples for PCT detection using different combinations of enzyme fusion antibodies are shown in Table 6 below.

[0156] Table 6. Comparison of clinical samples for the detection of PCT using a combination of preferred enzyme-fused antibodies

[0157] As can be seen from the results in Table 6, the clinical comparisons of different combinations of enzyme fusion antibodies have good correlation.

[0158] Example 6 Construction of Connector Peptide Optimization Mutants

[0159] In order to improve the complementary efficiency of the enzyme and enhance the signal-to-noise ratio, the connecting peptide between the antibody and the enzyme was optimized in this example. rSCFV18-RB73 was renamed R18 (wherein the connecting peptide was GGGSGGGGS (SEQ ID NO: 85), and then its connecting peptide was modified to form four mutants: R18-1 (SEQ ID NO: 76, wherein the connecting peptide was GGGS), R18-2 (SEQ ID NO: 77, wherein the connecting peptide was GGGSGGS (SEQ ID NO: 84)), R18-3 (SEQ ID NO: 78, wherein the connecting peptide was GGGSGGGSGGGS (SEQ ID NO: 86)), and R18-4 (SEQ ID NO: 79, wherein the connecting peptide was GGGGSGGGGSGGGGS (SEQ ID NO: 87)); OL-rSCFV18 was renamed R5 (wherein the connecting peptide was GSSGGGGSGGGS (SEQ ID NO: 93), and then its connecting peptide was modified to form four mutants: R5-1 (SEQ ID NO: 78, wherein the connecting peptide was GGGSGGGSGGGS (SEQ ID NO: 84)), and R5-2 (SEQ ID NO: 79, wherein the connecting peptide was GGGSGGGSGGGS (SEQ ID NO: 85)). NO:80, wherein the connecting peptide: GSSG), R5-2 (SEQ ID NO:82, wherein the connecting peptide: GSSGGGS (SEQ ID NO:91)), R5-3 (SEQ ID NO:82, wherein the connecting peptide: GSSGGGSGGS (SEQ ID NO:92)), R5-4 (SEQ ID NO:83, wherein the connecting peptide: GSSGGGGSGGGGSGG (SEQ ID NO:94)). The vector construction was constructed by seamless cloning. Except for the connecting peptide, the DNA sequences of other segments were consistent with the original R18 and R5. The expression of the R18 series proteins was carried out using the pComb3x vector for prokaryotic expression, and the expression of the R5 series proteins was carried out using the Abvec vector for eukaryotic expression. The purification method was referenced to Example 1.

[0160] Test method:

[0161] R18, R18-1, R18-2, R18-3, and R18-4 were diluted to 20 nM in PBSA, and 14 μl was added to each well. R5, R5-1, R5-2, R5-3, and R5-4 were diluted to 20 nM in PBSA, and 14 μl was added to each well. The large subunits were arranged horizontally and the small subunits were arranged vertically, for a total of 25 combinations and 50 test wells. PCT antigen protein was diluted to 0.25 ng / ml in PBSA, and 14 μl was added to each well. A background control was also added with 14 μl of PBSA. After gentle mixing, the three components were incubated at 37°C for 5 minutes. Then, 50 μl of O-buffer containing furimazine (final concentration 40 μM) was added, and the luminescence (RLU) value was measured after 100 seconds. Signal-to-noise ratio = RLU with PCT added / RLU without PCT added; RLU / pg = luminescence value generated after each pg of PCT antigen is bound by antibody enzyme. The results are shown in Table 7 below.

[0162] Table 7. Optimization of SCFV and enzyme fragment connecting peptide

[0163] From the results in Table 7, it can be seen that different linker peptide pairs all have high signal-to-noise ratios, among which the signal-to-noise ratio of the pairing of R18-4 and R5-4 is the highest.

[0164] Example 7

[0165] The expression of R18-4 and R5-4 proteins is shown in Example 6.

[0166] Test method:

[0167] R18-4 and R5-4 were diluted to 20 nM in PBSA-8, and 15 μl of each was added to each well. PCT antigen was diluted in PBSA starting from 25,000 pg / ml, and 14 μl was added to each well. After gentle mixing, the three components were incubated at 37°C for 5 minutes. Then, 50 μl of O buffer containing furimazine (final concentration 50 μM) was added. Luminescence (RLU) values ​​were measured after 30 seconds, 1 minute, 2 minutes, 5 minutes, and 8 minutes. Signal-to-noise ratio = RLU with PCT added / RLU without PCT added. Results are shown in Table 8 below. The standard curve (RLU-concentration) and correlation analysis for PCT determination using the enzyme-antibody combination after linker optimization are shown in Figures 7 and 8.

[0168] Table 8. PCT standard for large and small subunit fusion SCFV combination after optimized linker peptide

[0169] From the results in Table 8, Figures 7 and 8, it can be seen that the sensitivity of the PCT measurement of the combination of R18-4 and R5-4 after the optimization of the connecting peptide can reach 2 pg / ml; the correlation R2 between the standard concentration and the RLU value is greater than 0.99.

[0170] Example 8: Correlation of Luciferase Fusion Antibody Detection in Clinical Samples of PCT

[0171] The expression of R18-4 and R5-4 proteins is shown in Example 6.

[0172] Test method:

[0173] In the same tube of PBSA, R18-4 was diluted to 30 nM and R5-4 was diluted to 50 nM. After mixing, 30 μl of each was added to each well. 5 μl of the assigned clinical plasma sample was added to each well. After gently mixing the three components, the mixture was incubated at 37°C for 5 minutes. Then, 50 μl of Obuffer containing Furimazine / CTZ-h / CTZ-hcp (final concentration 250 μM) was added. After 90 seconds, the luminescence value (RLU) was measured. The results are shown in Table 9 below. Correlation analysis of PCT measurement using enzyme-fused antibody combinations with different substrates, Furimazine / CTZ-h / CTZ-hcp, is shown in Figure 9.

[0174] Table 9. Correlation of luciferase fusion antibodies with different substrates in detecting PCT in clinical samples

[0175] It can be seen from the results in Table 9 and Figure 9 that the correlation of the R18-4 and R5-4 combinations is relatively good when using different substrates. Among them, when FZ and CTZ-h are used as substrates, the correlation of the R18-4 and R5-4 combinations in measuring PCT in clinical plasma samples is good, and the correlation R2 can reach above 0.97; when CTZ-h is used as a substrate, the correlation coefficient R2 reaches 0.9927.

[0176] Example 9: Correlation of luciferase fusion antibody detection in clinical samples of PCT

[0177] In the same tube, R18-4 and R5-4 proteins were prepared at 90 nM and 75 nM, respectively, using enzyme diluent. 30 μl was added to each well. 52 randomly obtained clinical plasma samples with assigned PCT values ​​were added to each well. After gentle mixing, the samples were incubated at 37°C for 5 minutes. CTZ-h was diluted to 100 μM using substrate working solution Obuffer, and 50 μl was added to each well. The luminescence value was immediately measured using a bioluminescence detector. The correlation analysis results of luciferase fusion antibody detection of PCT clinical samples are shown in Figure 10.

[0178] As can be seen from the results in Figure 10, the clinical comparison correlation R2 of 52 randomly collected clinical plasma samples is 0.974, which is very good and meets the requirements of clinical diagnostic reagents.

[0179] Example 10: Lowest detection limit LLD and repeatability of PCT detection using luciferase fusion antibody

[0180] In the same tube, R18-4 and R5-4 proteins were diluted to 90 nM and 75 nM, respectively, using enzyme diluent. 30 μl was added to each well. 52 randomly obtained clinical plasma samples with assigned PCT values ​​were added to each well. After gentle mixing, the samples were incubated at 37°C for 5 minutes. CTZ-h was diluted to 50 μM using substrate working solution Obuffer and 50 μl was added to each well. Luminescence was immediately measured using a bioluminescence detector (GloMax 96). Luminescence values, repeatability, and minimum detection limit (LLD) are shown in Table 10 below. Samples with clinically assigned PCT values ​​of <0.02 ng / ml and 0.067 ng / ml were used for LLD calculations. Samples with clinically assigned PCT values ​​of 0.28 and 5.1 ng / ml were used for high- and low-value repeatability testing, respectively.

[0181] Table 10. Calculation of repeatability and minimum detection limit (LLD)

[0182] It can be seen from the results in Table 10 that the lowest detection limit LLD of PCT detection by luciferase fusion antibody is about 19 pg / ml, and the repeatability is good.

[0183] Example 11. Evaluation of the performance of luciferase-coupled PCT antibody reagents

[0184] In order to verify the target detection performance of the small subunit of the present invention after chemical coupling with the antibody, in this example, four SCFV antibodies of PCT were expressed in full-length IgG form by constructing eukaryotic expression vectors (accession NO.FJ475055.1, FJ475056.1), transiently transfected into 293F cell lines, and expressed as rIgG2 (whose heavy chain and light chain sequences are SEQ ID NO: 95-96, respectively), rIgG5 (whose heavy chain and light chain sequences are SEQ ID NO: 97-98, respectively), rIgG17 (whose heavy chain and light chain sequences are SEQ ID NO: 99-100, respectively), and rIgG22 (whose heavy chain and light chain sequences are SEQ ID NO: 101-102, respectively). Then, rIgG2, rIgG5, and rIgG22 were combined with synthetic RB73-2 (SEQ ID NO: 103) containing the small subunit of luciferase and RB73-4 (SEQ ID NO: 105) containing the small subunit of luciferase by SMCC. rIgG5 and rIgG17 were conjugated to an OLC (SEQ ID NO: 105) containing the large subunit of luciferase via SMCC using the standard protocol for sulfo-SMCC (Thermo, 22322). The conjugated antibodies were purified using molecular sieves or Protein G affinity purification.

[0185] Test plan: dilute the conjugated antibody to 1-3.3 nM with PBSA, add 16.5 μl to each well; dilute the PCT antigen with PBSA gradient to 0 / 0.001 / 0.01 / 0.1 / 1 / 10 / 100 ng / ml, add 5 μl to each well, mix gently, and incubate at 37°C for 5 min; add 50 μl of O buffer containing 50 μM CTZ-h, and immediately detect RLU using a bioluminescence detector 10 seconds after adding the substrate.

[0186] The results in Table 11 show that in PCT detection, the three combinations of conjugated antibody pairings all have excellent reactivity and linearity, especially the pairing of rIgG5-RB73-2 / 4 (3.3 nM) + rIgG17-OLC (3.3 nM). The linear correlation coefficient is R2 = 0.9987 in the PCT concentration range of 0.01 to 100 ng / mL, and the LLD is approximately 14.5 pg / ml, which fully meets the requirements of clinical test reagents.

[0187] Table 11. Preliminary evaluation of reagent performance of luciferase and PCT antibody chemical coupling products

[0188] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A fusion polypeptide or coupled polypeptide comprising a first antigen binding region and a first peptide derived from luciferase, wherein the first antigen binding region comprises at least one light chain variable region and at least one heavy chain variable region; The light chain variable region has: LCDR1 or an active variant thereof as shown in at least one of SEQ ID NOs: 17-24, LCDR2 or an active variant thereof having an amino acid sequence of DAS, DTS, KAS, NDD or RAS, and / or LCDR3 or an active variant thereof as shown in at least one of SEQ ID NOs: 25-32: and / or The heavy chain variable region has: HCDR1 or an active variant thereof shown in at least one of SEQ ID NOs:33-40, and / or HCDR2 or an active variant thereof shown in at least one of SEQ ID NOs:41-48, and / or HCDR3 or an active variant thereof shown in at least one of SEQ ID NOs:49-56.

2. The fusion polypeptide or coupled polypeptide according to claim 1, characterized in that: The light chain variable region has LCDR1 shown in SEQ ID NO:17, LCDR2 with an amino acid sequence of RAS, and LCDR3 shown in SEQ ID NO:25, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:33, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:49; or The light chain variable region has LCDR1 shown in SEQ ID NO:18, LCDR2 with an amino acid sequence of NDD, and LCDR3 shown in SEQ ID NO:26, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:34, HCDR2 shown in SEQ ID NO:42, and HCDR3 shown in SEQ ID NO:50; or The light chain variable region has LCDR1 shown in SEQ ID NO:19, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:27, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:35, HCDR2 shown in SEQ ID NO:43, and HCDR3 shown in SEQ ID NO:51; or The light chain variable region has LCDR1 shown in SEQ ID NO:20, LCDR2 with an amino acid sequence of DAS, and LCDR3 shown in SEQ ID NO:28, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:52; or The light chain variable region has LCDR1 shown in SEQ ID NO:21, LCDR2 with an amino acid sequence of RAS, and LCDR3 shown in SEQ ID NO:29, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:45, and HCDR3 shown in SEQ ID NO:53; or The light chain variable region has LCDR1 shown in SEQ ID NO:22, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:30, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:46, and HCDR3 shown in SEQ ID NO:54; or The light chain variable region has LCDR1 shown in SEQ ID NO:23, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:31, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:39, HCDR2 shown in SEQ ID NO:47, and HCDR3 shown in SEQ ID NO:55; or The light chain variable region has LCDR1 shown in SEQ ID NO:24, LCDR2 with an amino acid sequence of KAS, and LCDR3 shown in SEQ ID NO:32, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:48, and HCDR3 shown in SEQ ID NO:

56.

3. The fusion polypeptide or coupled polypeptide according to claim 1, characterized in that: The first antigen-binding region comprises a light chain variable region sequence shown in at least one of SEQ ID NOs: 1-8 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids, and / or a heavy chain variable region sequence shown in at least one of SEQ ID NOs: 9-16 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids; Preferably, the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 1 and the heavy chain variable region sequence shown in SEQ ID NO: 9; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 2 and the heavy chain variable region sequence shown in SEQ ID NO: 10; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 3 and the heavy chain variable region sequence shown in SEQ ID NO: 11; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 4 and the heavy chain variable region sequence shown in SEQ ID NO: 12; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 5 and the heavy chain variable region sequence shown in SEQ ID NO: 13; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 6 and the heavy chain variable region sequence shown in SEQ ID NO: 14; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 7 and the heavy chain variable region sequence shown in SEQ ID NO: 15; or the first antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 8 and the heavy chain variable region sequence shown in SEQ ID NO: The heavy chain variable region sequence shown in NO:

16.

4. The fusion polypeptide or coupled polypeptide according to claim 1, characterized in that: The first peptide has an amino acid sequence as shown in SEQ ID NO: 57 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids; Preferably, when the first peptide contacts an active variant of a second peptide having an amino acid sequence as shown in SEQ ID NO: 58, which is obtained by adding, deleting, replacing or modifying one or more amino acids, a detectable luminescent signal is generated in the presence of a substrate. Preferably, said first peptide is associated with said second peptide.

5. The fusion polypeptide or coupled polypeptide according to claim 1, characterized in that: The first antigen binding region is directly connected to the first peptide or connected via a first linker, Preferably, the first linker is a polypeptide linker and / or a non-polypeptide linker, More preferably, the first linker is of formula (G) n S(G) m S(G) l S, wherein n is selected from 1 to 5, m is selected from 0 to 5, and l is selected from 0 to 5, More preferably, the first linker is of formula A(EAAAK) o A rigid connecting peptide, wherein o is selected from 0 to 5, More preferably, the first linker comprises GGGS, GGGSGGS (SEQ ID NO: 84), GGGSGGGGS (SEQ ID NO: 85), GGGSGGGSGGGS (SEQ ID NO: 86) or GGGGSGGGGSGGGGS (SEQ ID NO: 87), More preferably, the first linker comprises AEAAAKA (SEQ ID NO: 88), AEAAAKEAAAKA (SEQ ID NO: 89) or AEAAAKEAAAKEAAAKA (SEQ ID NO: 90); Preferably, the first linker is a chemical linker, More preferably, the first linker comprises one or more of acrylic acid phosphoramidite, adenylation, azide, digoxigenin, cholesterol-TEG, amino modifier, alkyne, biotinylation, thiol modification, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), glycerol, C2 to C12 alkyl and succinimide.

6. A fusion polypeptide or coupled polypeptide comprising a second antigen binding region and a second peptide derived from luciferase, wherein the second antigen binding region binds to one or more of procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide, wherein the second antigen binding region comprises at least one light chain variable region and at least one heavy chain variable region; The light chain variable region has: LCDR1 or an active variant thereof as shown in at least one of SEQ ID NOs: 17-24, an amino acid sequence LCDR2 of DAS, DTS, KAS, NDD or RAS or an active variant thereof, and / or LCDR3 of at least one of SEQ ID NOs: 25-32 or an active variant thereof; and / or The heavy chain variable region has: HCDR1 or an active variant thereof shown in at least one of SEQ ID NOs:33-40, and / or HCDR2 or an active variant thereof shown in at least one of SEQ ID NOs:41-48, and / or HCDR3 or an active variant thereof shown in at least one of SEQ ID NOs:49-56.

7. The fusion polypeptide or coupled polypeptide according to claim 6, characterized in that: The light chain variable region has LCDR1 shown in SEQ ID NO:17, LCDR2 with an amino acid sequence of RAS, and LCDR3 shown in SEQ ID NO:25, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:33, HCDR2 shown in SEQ ID NO:41, and HCDR3 shown in SEQ ID NO:49; or The light chain variable region has LCDR1 shown in SEQ ID NO:18, LCDR2 with an amino acid sequence of NDD, and LCDR3 shown in SEQ ID NO:26, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:34, HCDR2 shown in SEQ ID NO:42, and HCDR3 shown in SEQ ID NO:50; or The light chain variable region has LCDR1 shown in SEQ ID NO:19, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:27, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:35, HCDR2 shown in SEQ ID NO:43, and HCDR3 shown in SEQ ID NO:51; or The light chain variable region has LCDR1 shown in SEQ ID NO:20, LCDR2 with an amino acid sequence of DAS, and LCDR3 shown in SEQ ID NO:28, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:36, HCDR2 shown in SEQ ID NO:44, and HCDR3 shown in SEQ ID NO:52; or The light chain variable region has LCDR1 shown in SEQ ID NO:21, LCDR2 with an amino acid sequence of RAS, and LCDR3 shown in SEQ ID NO:29, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:37, HCDR2 shown in SEQ ID NO:45, and HCDR3 shown in SEQ ID NO:53; or The light chain variable region has LCDR1 shown in SEQ ID NO:22, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:30, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:38, HCDR2 shown in SEQ ID NO:46, and HCDR3 shown in SEQ ID NO:54; or The light chain variable region has LCDR1 shown in SEQ ID NO:23, LCDR2 with an amino acid sequence of DTS, and LCDR3 shown in SEQ ID NO:31, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:39, HCDR2 shown in SEQ ID NO:47, and HCDR3 shown in SEQ ID NO:55; or The light chain variable region has LCDR1 shown in SEQ ID NO:24, LCDR2 with an amino acid sequence of KAS, and LCDR3 shown in SEQ ID NO:32, and the heavy chain variable region has HCDR1 shown in SEQ ID NO:40, HCDR2 shown in SEQ ID NO:48, and HCDR3 shown in SEQ ID NO:

56.

8. The fusion polypeptide or coupled polypeptide according to claim 6, characterized in that: The second antigen-binding region comprises a light chain variable region sequence shown in at least one of SEQ ID NOs: 1-8 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids, and / or a heavy chain variable region sequence shown in at least one of SEQ ID NOs: 9-16 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids; Preferably, the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 1 and the heavy chain variable region sequence shown in SEQ ID NO: 9; or the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 2 and the heavy chain variable region sequence shown in SEQ ID NO: 10; or the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO: 3 and the heavy chain variable region sequence shown in SEQ ID NO: 11 The second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:4 and the heavy chain variable region sequence shown in SEQ ID NO:12; or the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:5 and the heavy chain variable region sequence shown in SEQ ID NO:13; or the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:6 and the heavy chain variable region sequence shown in SEQ ID NO:14; or the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:7 and the heavy chain variable region sequence shown in SEQ ID NO:15; or the second antigen-binding region comprises the light chain variable region sequence shown in SEQ ID NO:8 and the heavy chain variable region sequence shown in SEQ ID NO:

16.

9. The fusion polypeptide or coupled polypeptide according to claim 6, characterized in that: The second peptide has an amino acid sequence as shown in SEQ ID NO: 58 or an active variant thereof obtained by adding, deleting, replacing or modifying one or more amino acids; Preferably, when the second peptide contacts an active variant of the first peptide having the amino acid sequence shown in SEQ ID NO: 57, which is obtained by adding, deleting, replacing or modifying one or more amino acids, a detectable luminescent signal is generated in the presence of a substrate. Preferably, said first peptide is associated with said second peptide.

10. The fusion polypeptide or coupled polypeptide according to claim 6, characterized in that: The second antigen binding region is directly connected to the second peptide or connected via a second linker, Preferably, the second linker is a polypeptide linker and / or a non-polypeptide linker, Preferably, the second linker is of the formula GSS(G) p (S) q (G) r (S) s (G) t (S) u A flexible connecting peptide, wherein p is selected from 1 to 5, q is selected from 0 to 5, r is selected from 0 to 5, s is selected from 0 to 1, t is selected from 0 to 5, and u is selected from 0 to 5, More preferably, the second linker comprises GSSG, GSSGGGS (SEQ ID NO:91), GSSGGGSGGS (SEQ ID NO:92), GSSGGGGSGGGS (SEQ ID NO:93) or GSSGGGGSGGGGSGG (SEQ ID NO:94); Preferably, the second linker is a chemical linker, More preferably, the second linker comprises one or more of acrylic acid phosphoramidite, adenylation, azide, digoxigenin, cholesterol-TEG, amino modifier, alkyne, biotinylation, thiol modification, tetraethylene glycol (TEG), hexaethylene glycol (HEG), polyethylene glycol (PEG), glycerol, C2 to C12 alkyl and succinimide.

11. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion polypeptide of any one of claims 1-10. An expression vector comprising the nucleotide sequence of the nucleic acid molecule according to claim 11 . A host cell comprising the expression vector according to claim 12 .

14. A composition comprising the fusion polypeptide or coupled polypeptide of any one of claims 1-5 and the fusion polypeptide or coupled polypeptide of any one of claims 6-10.

15. The composition according to claim 14, characterized in that The first antigen-binding region and / or the second antigen-binding region comprises a light chain variable region sequence as shown in SEQ ID NO: 1 and a heavy chain variable region sequence as shown in SEQ ID NO: 9, or comprises a light chain variable region sequence as shown in SEQ ID NO: 2 and a heavy chain variable region sequence as shown in SEQ ID NO: 10, or comprises a light chain variable region sequence as shown in SEQ ID NO: 3 and a heavy chain variable region sequence as shown in SEQ ID NO: 11, or comprises a light chain variable region sequence as shown in SEQ ID NO: 4 and a heavy chain variable region sequence as shown in SEQ ID NO: 12, or comprises a light chain variable region sequence as shown in SEQ ID NO: 5 and a heavy chain variable region sequence as shown in SEQ ID NO: 13, or comprises a light chain variable region sequence as shown in SEQ ID NO: 6 and a heavy chain variable region sequence as shown in SEQ ID NO: 14, or comprises a light chain variable region sequence as shown in SEQ ID NO: 7 and a heavy chain variable region sequence as shown in SEQ ID NO: 15, or comprises a light chain variable region sequence as shown in SEQ ID NO: 8 and a heavy chain variable region sequence as shown in SEQ ID NO: 16; and / or The first linker comprises GGGS, GGGSGGS (SEQ ID NO:84), GGGSGGGGS (SEQ ID NO:85), GGGSGGGSGGGS (SEQ ID NO:86) or GGGGSGGGGSGGGGS (SEQ ID NO:87); and / or The second linker comprises GSSG, GSSGGGS (SEQ ID NO:91), GSSGGGSGGS (SEQ ID NO:92), GSSGGGGSGGGS (SEQ ID NO:93) or GSSGGGGSGGGGSGG (SEQ ID NO:94).

16. The composition according to claim 14 or 15, characterized in that further comprising a substrate selected from coelenterazine and / or its analogs, Preferably, the substrate comprises at least one of Furimazine, coelenterazine-h (CTZ-h), coelenterazine-hh (CTZ-hh), and coelenterazine-hcp (CTZ-hcp).

17. A bioluminescent detection kit comprising the composition according to any one of claims 14 to 16.

18. Use of the fusion polypeptide or coupled polypeptide according to any one of claims 1 to 10, the nucleic acid molecule according to claim 11, the expression vector according to claim 12, the host cell according to claim 13, the composition according to any one of claims 14 to 16, or the kit according to claim 17 in detecting an analyte comprising one or more selected from procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide.

19. A homogeneous detection method, comprising incubating a sample to be tested with a fusion polypeptide or a coupled polypeptide according to any one of claims 1 to 5 and a fusion polypeptide or a coupled polypeptide according to any one of claims 6 to 10, and then detecting a luminescent signal in the presence of a substrate. Preferably, the sample to be tested contains one or more of the test substances selected from procalcitonin, preprocalcitonin, procalcitonin N-terminus, calcitonin, and calcium inhibitory peptide.

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