CN115746134A - Galectin-3 immunoassay - Google Patents

Abstract

The present application provides antibodies, or antigen-binding portions thereof, and antibody combinations for binding to human galectin-3. The present application also provides kits for detecting galectin-3 content in a sample from a human subject and uses thereof.

Description

Galectin-3 immunoassay

Technical Field

The present application relates generally to the field of biological detection, and in particular provides antibodies, or antigen-binding portions thereof, and antibody combinations for binding to human galectin-3. The present application also provides kits for detecting galectin-3 content in a sample from a human subject and uses thereof.

Background

Galectin-3 (Galectin-3, gal-3) is a galactose binding protein having a specific amino acid sequence and is widely present in animals ranging from low to high. Galectins (Galectins) are a large family of proteins, and 15 members of this family have been identified and classified into three groups based on their structural differences and the number of carbohydrate recognition domains conserved in their polypeptide chains: (1) The prototypical galectins (Galectin-1, 2, 5, 7, 10, 11, 13, 14 and 15), which comprise one CRD; (2) Tandem repeat galectins (Galectin-4, 6, 8, 9 and 12) which are linked by a linking region to two CRDs; (3) A chimeric Galectin (Galectin-3) comprising a CRD linked to an extended non-lectin N-terminal domain. Galectin-3 is one of the most studied members of the Galectin family. The human Galectin-3 gene is located in chromosome 14q21-22, has a total length of about 17kb, comprises 251 amino acid residues, has a molecular weight of about 29.31KD, and comprises 6 exons and 5 introns. Human Galectin-3 has 3 domains: (1) 1-12 amino acid NH2- (amino) terminus, which determines cellular localization and is also required for the highly conserved ser6 residue to participate in Galectin-3 anti-apoptotic activity; (2) Collagen protein repetitive sequences rich in glycine, tyrosine and proline, and functions as substrates of Matrix Metalloproteinases (MMPs); (3) Is a carbohydrate binding site with a spherical structure at the COOH- (carboxyl) terminus that interacts with extracellular matrix and glycoproteins.

Galectin-3 is widely distributed in tumor cells, epithelial cells, fibroblasts, macrophages and other inflammatory cells, has small expression in heart, kidney, liver, brain and pancreas, and has expression in cytoplasm, nucleus and cell surface, mainly cytoplasm. The biological function of Galectin-3 is related to intracellular localization. Cells are predominantly located in the cytoplasm when they are in a resting state and predominantly distributed within the nucleus when they are in a proliferating state. The secretion of Galectin-3 is independent of endoplasmic reticulum and golgi apparatus, and is transferred to the outside of cells through a non-classical mechanism similar to cellular exocytosis, and soluble Galectin-3 can enter the systemic circulation.

Galectin-3 is a substrate of MMPs, and after MMP-2 or MMP-9 proteolytically cleaves Galectin-3, a 22kU carbohydrate recognition domain and a 9kU amino acid residue fragment are generated. Galectin-3 can more easily play a role in the metastasis and invasion processes of tumors. In the research of breast cancer, it is reported in literature that decomposed Galectin-3 up-regulates the pFAK pathway by activating PKC or inducing GTP enzyme, enhances the chemotaxis of vascular endothelial cells, promotes the migration and chemotaxis of the vascular endothelial cells, and thus promotes the formation of new vessels.

Galectin-3 is involved in a number of physiological and pathological processes in the body including cell growth, cell adhesion, inflammatory responses, immune regulation, apoptosis and metastasis. Expression is significantly increased in many cancers such as melanoma, lung, breast, colorectal, head and neck, and non-hodgkin's lymphoma. Galectin-3 is involved in the formation of fibrosis in the organs such as kidney, lung and liver. Galectin-3 is closely related to rheumatic immune diseases and bronchial asthma. Galectin-3 is a proinflammatory signaling factor, is expressed in a variety of inflammatory factors, and is involved in macrophage phagocytosis, cell adhesion and the like. Heart failure is currently the most studied disease associated with Galectin-3, heart Failure (HF) is a severe manifestation or late stage of various heart diseases, with an increasing trend of morbidity, and a high mortality and rehospitalization rate, which are currently one of the major cardiovascular diseases. Heart failure is a complex clinical syndrome involving all systems of the body, the pathophysiological mechanisms of which are mainly related to disturbances in hemodynamics and abnormal activation of the neuroendocrine system.

At present, researches prove that the occurrence and the development of heart failure are closely related to the replacement of normal myocardial cells by myofibrils, and contemporaneous researches show that Galectin-3 (Galectin-3 and Gal-3) promote myocardial fibrosis and participate in processes such as myocardial inflammatory change, ventricular remodeling and the like. The fact that the expression level of Galectin-3 of a heart failure patient is continuously increased relative to that of a non-heart failure patient can be used as an index for evaluating heart failure. Myocardial fibrosis is primarily caused by excessive accumulation of collagen fibers within the heart muscle, which can lead to ventricular remodeling. Galectin-3 is obviously expressed in fibrotic tissues (heart and liver fibrosis model). Galectin-3 can reflect the degree of fibrosis.

The content of galectin-3in body fluids such as blood or serum can reflect disease conditions of human body, such as chronic heart failure, etc., so that accurate quantification of galectin-3 can be used for identifying or predicting diseases, evaluating the severity of diseases, staging or predicting disease consequences, monitoring the effect of drugs, etc. clinical or scientific needs.

It has been difficult to clearly distinguish galectin-3 from other galectins or protein fragments thereof by the assay methods so far, because galectin-3 shares a high degree of sequence similarity with other 14 mammalian galectins, particularly in the conserved Carbohydrate Recognition Domain (CRD), and galectin-3 is a substrate of MMPs, and can be cleaved by them. Another factor impeding the development of specific, reproducible detection assays for galectin-3 is the propensity of galectin-3 to bind to different proteins, carbohydrates, nucleic acids and lipids.

Summary of The Invention

In a first aspect, the present application provides an antibody or antigen-binding portion thereof capable of binding to at least a portion of the amino acid sequence of human galectin-3, said at least a portion of the amino acid sequence of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, or SEQ ID NO 10, or any combination thereof:

MLITILGTVKPN (1 st to 12 th of SEQ ID NO: 1; SEQ ID NO: 3),

MLITILGTVKPANS (1 st to 14 th of SEQ ID NO: 1; SEQ ID NO: 4),

IALDFQRNG (position 15 to position 25 of SEQ ID NO: 1; SEQ ID NO: 5),

VAFHFNPRFN (position 26 to position 35 of SEQ ID NO: 1; SEQ ID NO: 6),

TKLDNNW (position 45 to position 62 of SEQ ID NO: 1; SEQ ID NO: 7),

GREERSVFPFESGK (positions 63 to 78 of SEQ ID NO: 1; SEQ ID NO: 8),

VFPFESGKPFKIQVLVEP (70 th to 88 th of SEQ ID NO: 1; SEQ ID NO: 9),

DHFKVAVNDAHL (positions 89 to 100 of SEQ ID NO: 1; SEQ ID NO: 10).

In some embodiments, at least a portion of the amino acid sequence of human galectin-3 is located in a C-terminal portion of the human galectin-3, wherein the C-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 1:

MLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI(SEQ ID NO:1)。

in some embodiments, the antibody comprises HCDR1 of SEQ ID NO. 13, HCDR2 of SEQ ID NO. 14, HCDR3 of SEQ ID NO. 15, LCDR1 of SEQ ID NO.16, LCDR2 of SEQ ID NO. 17, and LCDR3 of SEQ ID NO. 18.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 13 through SEQ ID NO 18.

In some embodiments, the antibody comprises HCDR1 of SEQ ID NO. 19, HCDR2 of SEQ ID NO. 20, HCDR3 of SEQ ID NO. 21, LCDR1 of SEQ ID NO. 22, LCDR2 of SEQ ID NO. 23 and LCDR3 of SEQ ID NO. 24.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 19 through SEQ ID NO 24.

In some embodiments, the antigen binding portion is Fab, F (ab)' ₂ Or a single chain Fv.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.

In some embodiments, the antibody or antigen-binding portion thereof can be used for diagnosing heart failure, but is not limited to such use.

In some embodiments, diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

In some embodiments, the antibodies, or antigen-binding portions thereof, may be used to aid in the diagnosis of tumors, rheumatoid arthritis, or chronic kidney disease, but are not limited to such use.

In a second aspect, the present application provides an antibody or antigen-binding portion thereof capable of binding to at least a portion of the amino acid sequence of human galectin-3, the at least a portion of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12 or both SEQ ID NO. 11 and SEQ ID NO. 12:

MADNFSLEDLDSGGNPQGWPG (positions 1 to 24 of SEQ ID NO: 2; SEQ ID NO: 11),

AWGNQPAGGYPGASYPGAYPGGQACPPGAYPG (25 th to 56 th of SEQ ID NO: 2; SEQ ID NO: 12).

In some embodiments, at least a portion of the amino acid sequence of human galectin-3 is located in an N-terminal portion of the human galectin-3, wherein the N-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 2:

MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG(SEQ ID NO:2)。

in some embodiments, the antibody comprises HCDR1 shown in SEQ ID NO. 25, HCDR2 shown in SEQ ID NO. 26, HCDR3 shown in SEQ ID NO. 27, LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, and LCDR3 shown in SEQ ID NO. 30.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 25 to SEQ ID NO 30.

In some embodiments, the antibody comprises HCDR1 shown in SEQ ID NO. 31, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 33, LCDR1 shown in SEQ ID NO. 34, LCDR2 shown in SEQ ID NO. 35, and LCDR3 shown in SEQ ID NO. 36.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 31 through SEQ ID NO 36.

In some embodiments, the antigen binding portion is Fab, F (ab)' ₂ Or a single chain Fv.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.

In some embodiments, the antibody or antigen-binding portion thereof can be used for diagnosing heart failure, but is not limited to such use.

In some embodiments, diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

In some embodiments, the antibodies, or antigen-binding portions thereof, may be used to aid in the diagnosis of tumors, rheumatoid arthritis, or chronic kidney disease, but are not limited to such use.

In a third aspect, the present application provides an antibody combination comprising an antibody or antigen-binding portion thereof according to the first aspect and an antibody or antigen-binding portion thereof according to the second aspect.

In a fourth aspect, the present application provides a kit for detecting galectin-3 content in a sample from a human subject, comprising an antibody or antigen-binding portion thereof of the first aspect, or an antibody or antigen-binding portion thereof of the second aspect, or an antibody combination product of the third aspect, wherein the antibody or antigen-binding portion thereof is labeled with a detectable label.

In some embodiments, the detectable label comprises an enzyme or a chromogenic substance.

In some embodiments, the enzyme is alkaline phosphatase or horseradish peroxidase.

In some embodiments, the chromogenic substance is a chemiluminescent compound or a fluorescent substance.

In some embodiments, the chemiluminescent compound is ruthenium terpyridyl or acridinium ester.

In some embodiments, the fluorescent species is a fluorescent pigment, a fluorescent microsphere, a time resolved fluorescent microsphere, or a quantum dot, among others.

In some embodiments, the sample is derived from whole blood, serum, or plasma.

In some embodiments, antibodies that are not labeled with a detectable label are pre-attached to a solid surface.

In some embodiments, the solid surface is selected from the group consisting of nitrocellulose membranes, magnetic microparticles, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, plastics, and glass.

In some embodiments, the kit further comprises a standard and/or a quality control.

In some embodiments, the kit further comprises instructions.

In some embodiments, the kit may be used for diagnosing heart failure, but is not limited to such use.

In some embodiments, diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

In some embodiments, the antibodies or antigen-binding portions thereof can be used to aid in the diagnosis of tumors, rheumatoid arthritis, chronic kidney disease, but are not limited to such use.

In a fifth aspect, the present application provides the use of an antibody or antigen-binding portion thereof of the first aspect or an antibody or antigen-binding portion thereof of the second aspect or an antibody combination product of the third aspect in the preparation of a kit for detecting the amount of human galectin-3in an ex vivo sample.

In some embodiments, the ex vivo sample is derived from whole blood, serum, or plasma.

In a sixth aspect, the present application provides a method for detecting the amount of human galectin-3in an ex vivo sample in vitro, the method using the antibody or antigen binding portion thereof of the first or second aspect or the antibody combination product of the third aspect;

wherein the antibody or antigen-binding portion thereof of the first aspect is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of the first aspect is labeled with a detectable label, or the antibody or antigen-binding portion thereof of the second aspect is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of the first aspect is labeled with a detectable label; and

wherein the antibody or antigen-binding portion thereof detects human galectin-3 content by an immunological method.

In some embodiments, the ex vivo sample is derived from whole blood, serum, or plasma.

In some embodiments, the immunological method is selected from the group consisting of an enzyme-linked immunization method, an immunofluorescence method, a chemiluminescence method, an immunochromatography method, an immunoprecipitation method, and a combination thereof.

In some embodiments, the immunological method is an enzyme-linked immunosorbent assay.

In a seventh aspect, the present application provides the use of an antibody or antigen-binding portion thereof according to the first or second aspects or an antibody combination product according to the third aspect for detecting the amount of human galectin-3in an ex vivo sample.

In some embodiments, the ex vivo sample is derived from whole blood, serum, or plasma.

As non-limiting embodiments, the present application includes the following embodiments:

embodiment 1. An antibody or antigen-binding portion thereof, said antibody or antigen-binding portion thereof being capable of binding to at least a portion of the amino acid sequence of human galectin-3, said at least a portion of the amino acid sequence of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10, or any combination thereof.

Embodiment 2. The antibody or antigen-binding portion thereof of embodiment 1, wherein at least a portion of the amino acid sequence of human galectin-3 is located at a C-terminal portion of human galectin-3, wherein the C-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 1.

Embodiment 3. The antibody or antigen-binding portion thereof of embodiment 1, wherein the antibody comprises HCDR1 of SEQ ID NO 13, HCDR2 of SEQ ID NO 14, HCDR3 of SEQ ID NO 15, LCDR1 of SEQ ID NO 16, LCDR2 of SEQ ID NO 17, and LCDR3 of SEQ ID NO 18; or alternatively

The antibody comprises HCDR1 shown in SEQ ID NO. 19, HCDR2 shown in SEQ ID NO. 20, HCDR3 shown in SEQ ID NO. 21, LCDR1 shown in SEQ ID NO. 22, LCDR2 shown in SEQ ID NO. 23 and LCDR3 shown in SEQ ID NO. 24.

Embodiment 4. An antibody or antigen binding portion thereof, said antibody or antigen binding portion thereof being capable of binding to at least a portion of the amino acid sequence of human galectin-3, said at least a portion of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12 or both SEQ ID NO. 11 and SEQ ID NO. 12.

Embodiment 5 the antibody or antigen-binding portion thereof of embodiment 4, wherein at least a portion of the amino acid sequence of human galectin-3 is located in an N-terminal portion of the human galectin-3, wherein the N-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 2.

Embodiment 6. The antibody or antigen-binding portion thereof of embodiment 4, wherein the antibody comprises HCDR1 of SEQ ID NO. 25, HCDR2 of SEQ ID NO. 26, HCDR3 of SEQ ID NO. 27, LCDR1 of SEQ ID NO. 28, LCDR2 of SEQ ID NO. 29, and LCDR3 of SEQ ID NO. 30; or

The antibody comprises HCDR1 shown in SEQ ID NO. 31, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 33, LCDR1 shown in SEQ ID NO. 34, LCDR2 shown in SEQ ID NO. 35 and LCDR3 shown in SEQ ID NO. 36.

Embodiment 7. The antibody or antigen-binding portion thereof of any one of embodiments 1 to 6, wherein the antigen-binding portion is a Fab, F (ab)' 2, or single chain Fv.

Embodiment 8. The antibody or antigen binding portion thereof of any one of embodiments 1 to 6, wherein the antibody is a monoclonal antibody.

Embodiment 9. The antibody or antigen binding portion thereof of embodiment 8, wherein the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.

Embodiment 10. The antibody or antigen-binding portion thereof of any one of embodiments 1 to 9, which is useful for diagnosing heart failure, or for aiding in the diagnosis of a tumor, rheumatoid arthritis, or chronic kidney disease.

Embodiment 11 the antibody or antigen-binding portion thereof of embodiment 10, wherein the diagnosis of heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

Embodiment 12. An antibody combination comprising the antibody or antigen-binding portion thereof of any one of embodiments 1 to 3 and the antibody or antigen-binding portion thereof of any one of embodiments 4 to 6.

Embodiment 13. A kit for detecting galectin-3 content in a sample from a human subject comprising the antibody or antigen-binding portion thereof of any one of embodiments 1 to 11 or the antibody combination of embodiment 12, wherein the antibody or antigen-binding portion thereof is labeled with a detectable label.

Embodiment 14. The kit of embodiment 13, wherein the detectable label comprises an enzyme or a chromogenic substance.

Embodiment 15 the kit of embodiment 13, wherein the enzyme is alkaline phosphatase or horseradish peroxidase.

Embodiment 16 the kit according to embodiment 13, wherein the color-developing substance is a chemiluminescent compound or a fluorescent substance.

Embodiment 17. The kit of embodiment 16, wherein the chemiluminescent compound is ruthenium terpyridyl or acridinium ester.

Embodiment 18 the kit of embodiment 16, wherein the fluorescent substance is a fluorescent pigment, a fluorescent microsphere, a time-resolved fluorescent microsphere, or a quantum dot.

Embodiment 19 the kit of embodiment 13, wherein the sample is derived from whole blood, serum or plasma.

Embodiment 20. The kit of embodiment 13, wherein the antibody not labeled with a detectable label is pre-attached to a solid surface.

Embodiment 21. The kit of embodiment 20, wherein the solid surface is selected from the group consisting of nitrocellulose membranes, magnetic microparticles, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, plastics, and glass.

Embodiment 22 the kit of embodiment 13, wherein the kit further comprises a standard and/or a quality control.

Embodiment 23. The kit of embodiment 13, wherein the kit further comprises instructions.

Embodiment 24. Use of the antibody or antigen-binding portion thereof of any one of embodiments 1-11 or the antibody combination product of embodiment 12 in the preparation of a kit for detecting the amount of human galectin-3in an ex vivo sample.

Embodiment 25 the use of embodiment 24, wherein the ex vivo sample is derived from whole blood, serum or plasma.

Embodiment 26. A method for the in vitro detection of the amount of human galectin-3in an ex vivo sample using the antibody or the antigen-binding portion thereof according to any one of embodiments 1 to 11 or the antibody combination product of embodiment 12;

wherein the antibody or antigen-binding portion thereof of any one of embodiments 1 to 3 is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of any one of embodiments 4 to 6 is labeled with a detectable label, or the antibody or antigen-binding portion thereof of any one of embodiments 4 to 6 is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of any one of embodiments 1 to 3 is labeled with a detectable label; and

wherein the antibody or antigen-binding portion thereof detects human galectin-3 content by an immunological method.

Embodiment 27 the method of embodiment 26, wherein the ex vivo sample is derived from whole blood, serum or plasma.

Embodiment 28. The method of embodiment 26, wherein the immunological method is selected from the group consisting of an enzyme-linked immunosorbent assay, an immunofluorescence assay, a chemiluminescent assay, an immunochromatography assay, an immunoprecipitation assay, and a combination thereof.

Embodiment 29 use of the antibody or antigen-binding portion thereof of any one of embodiments 1-11 or the antibody combination product of embodiment 12 for detecting the amount of human galectin-3in an ex vivo sample.

Embodiment 30 the use of embodiment 29, wherein the ex vivo sample is derived from whole blood, serum or plasma.

Drawings

FIG. 1 shows a schematic diagram of the B cell in vitro cloning technology to obtain an immunoglobulin antibody specifically recognizing galectin-3.

Fig. 2 is a schematic view of a test strip/kit, wherein 1: a sample pad; 2: a bonding pad; 3: a detection line (T-line); 4: a quality control line (line C); 5: NC membranes (nitrocellulose membranes); 6: a water absorbent pad; 7: PVC bottom plate.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1

MLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI

SEQ ID NO:2

MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG

3 (1 st to 12 th of SEQ ID NO: 1): MLITILGTVKPN

SEQ ID NO:4 (positions 1 to 14 of SEQ ID NO: 1): MLITILGTVKPANA

5 (positions 15 to 25 of SEQ ID NO: 1): IALDFQRGND

6 (position 26 to position 35 of SEQ ID NO: 1): VAFHFNPRFRP

SEQ ID NO:7 (positions 45 to 62 of SEQ ID NO: 1): TKLDNNW

SEQ ID NO:8 (positions 63 to 78 of SEQ ID NO: 1): GREEERQSVFPFESGK

SEQ ID NO:9 (positions 70 to 88 of SEQ ID NO: 1): VFPFESGKPFKIQVLVEP

10 (positions 89 to 100 of SEQ ID NO: 1): DHFKVAVNDAHL

11 (position 1 to position 24 of SEQ ID NO: 2):

MADNFSLHDALSGSGNPNPQGWPG

12 (position 25 to position 56 of SEQ ID NO: 2)

AWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG

13 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:

EGLVQPGYFSSYATQGLL

14 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:

AYSAIDAGSVSGP

15 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:

TSRDSVDSLLADNSVSGP

16 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:

HSYGTARGVQGLTEV

17 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:

READATVYYCTIRVCL

18 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:

PAYTPPTATLLVE

19 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:

PGLSEPSYYTAELGVVEE

20 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:

ASANYVANSYYAST

21 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:

VVTLLIADPGATATE

22 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:

FVYDPATYLTAAAPQYP

SEQ ID NO. 23 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:

RTYSAGWDPEETPSAT

24 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:

SESLAPYGYILATGPASTVS

SEQ ID NO. 25 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:

LLEGLVYFSQPATGSYGQ

26 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:

SGPYASAVIDAGS

SEQ ID NO 27 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:

PTDSRSLSLLVADNSDGSV

28 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:

HGVQSTARTEGLVYG

29 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:

ADAREAILDATVRVCYYCT

30 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:

LVTPTPTALVAYTLE

31 is the amino acid sequence of HCDR1 of an antibody capable of binding to human galectin-3:

EPGYVVYTAELLSEPSGE

32 is the amino acid sequence of HCDR2 of an antibody capable of binding to human galectin-3:

AYASTVASYANNSY

33 is the amino acid sequence of HCDR3 of an antibody capable of binding to human galectin-3:

VIADPGVTATLLATE

34 is the amino acid sequence of LCDR1 of an antibody capable of binding to human galectin-3:

FYPVYLTAADPATYAPQ

35 is the amino acid sequence of LCDR2 of an antibody capable of binding to human galectin-3:

GWSADPERTYSAETPT

36 is the amino acid sequence of LCDR3 of an antibody capable of binding to human galectin-3:

TVYGYILATGPSESLAPASS

SEQ ID NO:37 is a primer HC-For: acctatactgtcagcacatta

38 is primer HC-Rev: ggatacagttggtgcagcatc

39 is a primer LC-For: gayattgtgmtsacmcarwct

40 is a primer LC-Rev: acctatactgtcagcacatta

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

Definition of

The term "heart failure" or "HF" as used herein refers to a complex clinical syndrome that impairs the ability of the ventricles to fill or eject blood. Any structural or functional cardiac disorder can cause HF, with most HF patients having impaired Left Ventricular (LV) myocardial function. Syndromes of HF include dyspnea (shortness of breath), fatigue, and fluid retention.

The term "subject" as used herein refers to a human or non-human organism. Preferred subjects herein are human subjects.

The term "antigen-binding portion" or "antigen-binding site" or "target-binding site" of an antibody, as used herein, means one or more fragments of a binding protein (e.g., an antibody or receptor), such as an immunoglobulin variable domain (e.g., VH or VL), that retain the ability to specifically bind to an antigen or target. The antigen-binding portion of an antibody can be a fragment of a full-length antibody. The term "antigen-binding portion" encompasses examples of binding fragments that include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) A F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment comprising a single variable domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, the VL and VH of the Fv encoded by separate genes can be joined by a synthetic linker using recombinant methods into a single protein chain in which the VH and VL regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such scfvs are also encompassed within the term "antigen-binding portion", as are other forms of single chain antibodies, such as diabodies and "linear antibodies" that comprise a pair of tandem Fv fragments (VH-CH 1-VH-CH 1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding sites. Not every amino acid of the antigen-binding portion can bind to an antigen. For example, the variable domain of an antibody comprises Complementarity Determining Regions (CDRs) and Framework Regions (FRs).

The term "CDR" as used herein means complementarity determining regions within an immunoglobulin variable region sequence. There are three CDRs in the variable regions of the heavy and light chains, respectively, which are designated CDR1, CDR2, and CDR3 for the heavy and light chain variable regions, respectively. The term "CDR set" refers to a set of three CDRs present in a single variable region capable of binding antigen. The exact boundaries of these CDRs have been defined according to different systems. The system described by Kabat (Kabat et al (1971) Ann. NY Acad. Sci.190:382-391 (Kabat et al (1987) Sequences of Proteins of Immunological Interest, fourth edition US Govt. Printing of Off.No.165-492 (Kabat et al (1991) Sequences of Proteins of Immunological Interest, fifth edition NIH publication No. 91-3242) provides not only a clear residue numbering system applicable to any antibody variable region, but also precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. The amino acid residues of the CDR regions are more variable (e.g., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of the antibody. Chothia and coworkers (Chothia and Lesk (1987) J.mol.biol.196:901-917; chothia et al (1989) Nature 342. These subsections are designated as L1 (LCDR 1), L2 (LCDR 2) and L3 (LCDR 3) or H1 (HCDR 1), H2 (HCDR 2) and H3 (HCDR 3), where "L" and "H" designate the light and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (1995) FASEB J.9:133-139 and MacCallum (1996) J.mol.biol.262 (5): 732-45. Still other CDR boundary definitions may not strictly follow one of the systems herein, but still overlap with the Kabat CDRs. The antibodies herein, or antigen binding portions thereof, can utilize CDRs defined according to any of these systems.

The term "antibody" as used herein may also refer to antigen binding portions thereof.

The term "a-th to b-th of SEQ ID NO: X" or "amino acids a to b of SEQ ID NO: X" as used herein refers to the amino acid sequence of SEQ ID NO: X from the N-terminus to the C-terminus of amino acids a to b.

As used herein, the term "a-th to b-th bases of SEQ ID NO: X" when the sequence is a nucleic acid sequence, may also refer to a-th to b-th bases from 5 'to 3' of the nucleic acid sequence of SEQ ID NO: X.

Galectin-3 and antibody against the same

Galectin-3 (GenBank accession No.: NC — 000014.7 (gene) and NP — 002297.2 (protein)) is one of the 15 mammalian beta galactoside-binding lectins or "galectins" that specifically bind galactose. Galectin-3 is known in the literature as LGALS3, MAC-2 antigen, carbohydrate Binding Protein (CBP) -35, laminin binding protein, galactose-specific lectin 3, mL-34, L-29, hL-31, ε BP, and IgE-binding protein. Galectin-3 consists of a carboxy-terminal Carbohydrate Recognition Domain (CRD) and an amino-terminal tandem repeat (Liu, F. -T. (2000) Role of Galectin-3in inflammation. Mutations and pathway 1. M.Caron and D.Seve, eds. Harwood Academic Publishers, amsterdam, the Netherlands, p.51; liu, F. -T. Et al (1995) am.J.Pathol.147: 1016). Galectin-3 is normally distributed in the epithelium of many organs and in various inflammatory cells, including macrophages, as well as dendritic cells and kupffer cells (Flotte, TJ. Et al (1983) am. J. Pathol.111: 112).

Galectin-3 has been shown to play a role in a number of cellular processes, including cell-cell adhesion, cell-matrix interactions, phagocytosis, cell cycle, apoptosis, angiogenesis, and mRNA splicing. Galectin-3 has been shown to act by both intracellular and extracellular effects (Sano, H. et al (2000) The Journal of Immunology,165 2156-2164). It is a component of heterogeneous nuclear ribonucleoprotein (hnRNP) (lane, j.g. et al (1998) Biochemistry 27. On the other hand, galectin-3 secreted from monocytes/macrophages (Sato, s. Et al (1994) j.biol.chem.269: 4424) and epithelial cells (Lindstedt, r.g. et al (1993) j.biol.chem.268: 11750) has been shown to act as extracellular molecules in activating different types of cells such as monocytes/macrophages (Liu, f. -t. (1993) Immunol Today 14 486), mast cells, neutrophils and lymphocytes (Hsu, d.k., s.r. et al (1996) am.j.pathol.148: 1661). Galectin-3 has been shown to act as a novel chemoattractant for monocytes and macrophages (Sano, h. Et al (2000) The Journal of Immunology,2000, 165 2156-2164). Galectin-3 is implicated in diseases such as cancer, inflammation and Heart Failure (HF). Quantification of galectin-3 is particularly suitable for use in assays for diagnosing and detecting the severity and predicting the outcome of HF, as disclosed in international patent publication No. wo 2005/040817.

The present inventors have found that galectin-3 can be reliably distinguished from other mammalian galectins by targeting the C-terminal and/or N-terminal domains of galectin-3. By using the antibodies disclosed herein in conjunction with immunoassays known in the art, such as ELISA, galectin-3 levels in the sample can be reliably and reproducibly detected and correlated with, for example, the presence, severity and stage of HF in the subject.

In a first aspect, the present application provides an antibody or antigen-binding portion thereof capable of binding to at least a portion of the amino acid sequence of human galectin-3, said at least a portion of the amino acid sequence of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, or SEQ ID NO 10, or any combination thereof:

MLITILGTVKPN (1 st to 12 th of SEQ ID NO: 1; SEQ ID NO: 3),

MLITILGTVKPANS (1 st to 14 th of SEQ ID NO: 1; SEQ ID NO: 4),

IALDFQRNG (position 15 to position 25 of SEQ ID NO: 1; SEQ ID NO: 5),

VAFHFNPRFN (position 26 to position 35 of SEQ ID NO: 1; SEQ ID NO: 6),

TKLDNNW (positions 45 to 62 of SEQ ID NO: 1; SEQ ID NO: 7),

GREERSVFPFESGK (positions 63 to 78 of SEQ ID NO: 1; SEQ ID NO: 8),

VFPFESGKPFKIQVLVEP (70 th to 88 th of SEQ ID NO: 1; SEQ ID NO: 9),

DHFKVAVNDAHL (positions 89 to 100 of SEQ ID NO: 1; SEQ ID NO: 10).

In some embodiments, at least a portion of the amino acid sequence of human galectin-3 is located in a C-terminal portion of the human galectin-3, wherein the C-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 1:

MLITILGTVKPNANRIALDFQRGNDVAFHFNPRFNENNRRVIVCNTKLDNNWGREERQSVFPFESGKPFKIQVLVEPDHFKVAVNDAHLLQYNHRVKKLNEISKLGISGDIDLTSASYTMI(SEQ ID NO:1)。

letters indicate standard amino acid codes. Although these amino acid sequences describe linear epitopes, epitopes resulting from secondary and tertiary structures at the N-terminus and comprising amino acids from non-contiguous stretches of the sequence can also be used. Moreover, one of skill in the art will appreciate that the two C-terminal and/or N-terminal epitopes used to bind to any two antibodies disclosed herein should be separated by at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, or any distance that does not affect the binding of the two antibodies to the epitope. The distance of the two epitopes can be routinely determined by the person skilled in the art.

In some embodiments, the antibody comprises HCDR1 of SEQ ID NO. 13, HCDR2 of SEQ ID NO. 14, HCDR3 of SEQ ID NO. 15, LCDR1 of SEQ ID NO.16, LCDR2 of SEQ ID NO. 17, and LCDR3 of SEQ ID NO. 18.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 13 through SEQ ID NO 18.

In some embodiments, the antibody comprising the amino acid sequence set forth in SEQ ID NO 13 to SEQ ID NO 18 is a first antibody.

In some embodiments, the antibody comprises HCDR1 of SEQ ID NO. 19, HCDR2 of SEQ ID NO. 20, HCDR3 of SEQ ID NO. 21, LCDR1 of SEQ ID NO. 22, LCDR2 of SEQ ID NO. 23, and LCDR3 of SEQ ID NO. 24.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 19 through SEQ ID NO 24.

In some embodiments, the antibody comprising the amino acid sequence set forth in SEQ ID NO 19 to SEQ ID NO 24 is a second antibody.

In some embodiments, the antigen binding moiety is Fab, F (ab)' ₂ Or a single chain Fv.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.

Monoclonal antibody technology (e.g., hybridoma, recombinant, and phage display technologies) to prepare monoclonal antibodies is known in the art.

Hybridoma monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone, directed against only a particular epitope of an antigen. B-cells with the ability to secrete specific antibodies and myeloma cells with the ability to immortalize are fused into B-cell hybridomas. By culturing a single hybridoma having such a characteristic into a cell population, a monoclonal antibody, which is a specific antibody against one epitope, can be produced.

BALB/C female mice were selected for immunization using galectin-3 containing recombinant full-length sequences or C-and N-terminal domains or polypeptide fragments as an immunogen.

Subcutaneous multiple injections were first mixed with complete adjuvant and antigen 1, followed by injections in mice mixed with incomplete adjuvant and antigen 1. B lymphocytes are stimulated to produce the corresponding antibodies. The lymphocytes are then fused with a mouse or rat myeloma cell line using a PEG polyethylene glycol fusion agent to form a hybridoma cell line. HAT selective medium is generally used. In HAT medium, unfused myeloma cells die because they lack hypoxanthine-guanine-phosphoribosyltransferase and cannot synthesize DNA using a salvage pathway. Unfused lymphocytes, while possessing hypoxanthine-guanine-phosphoribosyl transferase, are not themselves able to survive long term in vitro and die. Only the fused hybridoma cells can survive and proliferate in HAT medium because they obtain hypoxanthine-guanine-phosphoribosyltransferase and have the property that myeloma cells can proliferate indefinitely. Screening was performed using either full-length or truncated lactalbumin-3 antigen containing an N-terminus and a C-terminus, and the specificity of the hybridoma cell supernatants was detected by enzyme-linked immunosorbent assay.

In some embodiments, the antibodies or antigen-binding portions thereof can be used to diagnose heart failure.

In some embodiments, diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

In some embodiments, the antibodies, or antigen-binding portions thereof, are useful in aiding diagnosis of tumors, rheumatoid arthritis, chronic kidney disease, but are not limited to such use.

In a second aspect, the present application provides an antibody or antigen-binding portion thereof capable of binding to at least a portion of the amino acid sequence of human galectin-3, the at least a portion of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12 or both SEQ ID NO. 11 and SEQ ID NO. 12:

MADNFSLEDLDSGGNPQGWPG (positions 1 to 24 of SEQ ID NO: 2; SEQ ID NO: 11),

AWGNQPAGGYPGASYPGAYPGGQACPPGAYPG (25 th to 56 th of SEQ ID NO: 2; SEQ ID NO: 12).

In some embodiments, at least a portion of the amino acid sequence of human galectin-3 is located at an N-terminal portion of the human galectin-3, wherein the N-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 2:

MADNFSLHDALSGSGNPNPQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPG(SEQ ID NO:2)。

in some embodiments, the antibody comprises HCDR1 shown in SEQ ID NO. 25, HCDR2 shown in SEQ ID NO. 26, HCDR3 shown in SEQ ID NO. 27, LCDR1 shown in SEQ ID NO. 28, LCDR2 shown in SEQ ID NO. 29, and LCDR3 shown in SEQ ID NO. 30.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 25 through SEQ ID NO 30.

In some embodiments, the antibody comprising the amino acid sequence set forth in SEQ ID NO:25 to SEQ ID NO:30 is a third antibody.

In some embodiments, the antibody comprises HCDR1 shown in SEQ ID NO. 31, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 33, LCDR1 shown in SEQ ID NO. 34, LCDR2 shown in SEQ ID NO. 35, and LCDR3 shown in SEQ ID NO. 36.

In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO 31 through SEQ ID NO 36.

In some embodiments, the antibody comprising the amino acid sequence set forth in SEQ ID No. 31 to SEQ ID No. 36 is a fourth antibody.

In some embodiments, the antigen binding portion is Fab, F (ab)' ₂ Or a single chain Fv.

In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the antibody is a murine monoclonal antibody or a rabbit monoclonal antibody.

In some embodiments, the antibody or antigen-binding portion thereof can be used for diagnosing heart failure, but is not limited to such use.

In some embodiments, diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

In some embodiments, the antibodies, or antigen-binding portions thereof, are useful in aiding diagnosis of tumors, rheumatoid arthritis, chronic kidney disease, but are not limited to such use.

In a third aspect, the present application provides an antibody combination product comprising an antibody or antigen-binding portion thereof according to the first aspect and an antibody or antigen-binding portion thereof according to the second aspect.

In some embodiments, the antibody combination product comprises an antibody comprising the amino acid sequence set forth in SEQ ID NO 13 to SEQ ID NO 18 and an antibody comprising the amino acid sequence set forth in SEQ ID NO 25 to SEQ ID NO 30.

In some embodiments, the antibody combination product comprises an antibody comprising the amino acid sequence set forth in SEQ ID NO 13 to 18 and an antibody comprising the amino acid sequence set forth in SEQ ID NO 31 to 36.

In some embodiments, the antibody combination product comprises an antibody comprising the amino acid sequence set forth in SEQ ID NO 19 to SEQ ID NO 24 and an antibody comprising the amino acid sequence set forth in SEQ ID NO 25 to SEQ ID NO 30.

In some embodiments, the antibody combination product comprises an antibody comprising the amino acid sequence set forth in SEQ ID NO 19 to SEQ ID NO 24 and an antibody comprising the amino acid sequence set forth in SEQ ID NO 31 to SEQ ID NO 36.

In some embodiments, an antibody combination product comprises any combination of first through fourth antibodies, e.g., an antibody combination product comprises a first antibody and a third antibody; or a first antibody and a fourth antibody; or a second antibody and a fourth antibody; or a second antibody and a third antibody; or a first antibody, a second antibody, and a fourth antibody; or a first antibody, a second antibody and a third antibody; or a first antibody, a third antibody, and a fourth antibody; or a second antibody, a third antibody and a fourth antibody; or a first antibody, a second antibody, a third antibody and a fourth antibody.

In some embodiments, the antibody combination product comprises a combination of a first antibody or a second antibody and a third antibody or a fourth antibody.

The present application also provides an antibody combination comprising a plurality/plurality of antibodies of the first aspect or antigen-binding portions thereof.

The present application also provides an antibody combination comprising a plurality/plurality of antibodies of the second aspect or antigen-binding portions thereof.

The present application also provides an antibody combination comprising an antibody or antigen-binding portion thereof according to the first aspect and an antibody or antigen-binding portion thereof according to the second aspect.

The present application also provides an antibody combination comprising a plurality/plurality of antibodies of the first aspect or antigen-binding portions thereof and an antibody of the second aspect or antigen-binding portion thereof.

The present application also provides an antibody combination comprising an antibody or antigen-binding portion thereof according to the first aspect and a plurality/plurality of antibodies or antigen-binding portions thereof according to the second aspect.

The present application also provides an antibody combination comprising a plurality/plurality of antibodies of the first aspect or antigen-binding portions thereof and a plurality/plurality of antibodies of the second aspect or antigen-binding portions thereof.

Reagent kit

One of the antibodies or an antigen-binding portion thereof in the kit is used as a capture antibody and may be immobilized on a solid surface for capturing galectin-3. And the other antibody, or antigen binding portion thereof, serves as a detection antibody and is linked to a detectable label.

In a fourth aspect, the present application provides a kit for detecting galectin-3 content in a sample from a human subject, comprising an antibody or antigen-binding portion thereof according to the first aspect or an antibody or antigen-binding portion thereof according to the second aspect or an antibody combination product according to the third aspect, wherein the antibody or antigen-binding portion thereof is labeled with a detectable label.

In some embodiments, the kit comprises an antibody or antigen-binding portion thereof of the first aspect.

In some embodiments, the kit comprises an antibody or antigen-binding portion thereof according to the second aspect.

In some embodiments, the kit comprises the antibody combination product of the third aspect.

In some embodiments, the detectable label comprises an enzyme or a chromogenic substance.

In some embodiments, the enzyme is alkaline phosphatase or horseradish peroxidase.

In some embodiments, the chromogenic substance is a chemiluminescent compound or a fluorescent substance.

In some embodiments, the chemiluminescent compound is ruthenium terpyridyl or acridinium ester.

In some embodiments, the fluorescent species is a fluorescent pigment, a fluorescent microsphere, a time resolved fluorescent microsphere, or a quantum dot.

In some embodiments, the sample is derived from whole blood, serum, or plasma.

In some embodiments, the sample is derived from blood.

In some embodiments, the sample is derived from a bodily fluid.

The body fluid may be treated or untreated. Methods for obtaining a body fluid from a subject are known to those skilled in the art.

In some embodiments, the sample is derived from urine, gastric juice, bile, saliva, sweat, and spinal fluid, stool, or muscle biopsy.

In some embodiments, the antibody not labeled with a detectable label is pre-attached to a solid surface.

In some embodiments, the solid surface is selected from the group consisting of nitrocellulose membranes, magnetic microparticles, fluorescent microspheres, time-resolved fluorescent microspheres, quantum dots, plastics, and glass.

In some embodiments, the kit further comprises a standard and/or a quality control.

In some embodiments, the kit further comprises instructions.

In some embodiments, the kit may be used for diagnosing heart failure, but is not limited to such use.

In some embodiments, diagnosing heart failure comprises assessing the risk of heart failure, diagnosing the presence of heart failure, determining the severity of heart failure, or prognosing a prediction of heart failure.

In some embodiments, the antibodies, or antigen-binding portions thereof, are useful in aiding diagnosis of tumors, rheumatoid arthritis, chronic kidney disease, but are not limited to such use.

In some embodiments, the antibody of the first aspect is labeled with a detectable label.

In some embodiments, the antibody of the second aspect is labeled with a detectable label.

In some embodiments, the antibody of the first aspect is labeled with a detectable label and the antibody of the second aspect is not labeled with a detectable label.

In some embodiments, the antibody of the second aspect is labeled with a detectable label and the antibody of the first aspect is not labeled with a detectable label.

In some embodiments, the kit is prepared by coupling magnetic microspheres with an antibody to galectin-3 to form a solid phase conjugate, labeling the other antibody with an enzyme (e.g., horseradish peroxidase (HRP) or alkaline phosphatase (ALP)) that catalyzes a chemiluminescent reaction, forming a solid phase-coated antibody-antigen to be detected-enzyme-labeled antibody complex after immunoreaction with the corresponding antigen in a sample to be detected, washing, adding a luminescent agent, catalyzing and decomposing the enzyme to emit light, and reading with a chemiluminescence apparatus.

In some embodiments, the kit uses a fluorescent material as a marker to establish an immunochromatographic test strip, one antibody of galectin-3 is coated on a nitrocellulose membrane, the fluorescent material is used as the marker, the fluorescent material and the other antibody are coupled through a covalent bond to form a fluorescent probe, and after the fluorescent probe reacts with an antigen, an antigen-antibody complex containing a labeled fluorescent signal can be formed. The fluorescence signal on the test strip is then read using an immunofluorescence analyzer.

In a fifth aspect, the present application provides the use of an antibody or antigen-binding portion thereof of the first aspect or an antibody or antigen-binding portion thereof of the second aspect or an antibody combination product of the third aspect in the preparation of a kit for detecting the amount of human galectin-3in an ex vivo sample.

The application also provides the use of the antibody of the first aspect or an antigen-binding portion thereof in the preparation of a kit for detecting the amount of human galectin-3in an ex vivo sample.

The application also provides the use of the antibody or antigen-binding portion thereof of the second aspect in the preparation of a kit for detecting the amount of human galectin-3in an ex vivo sample.

The application also provides the use of the antibody combination product of the third aspect in the preparation of a kit for detecting the content of human galectin-3in an ex vivo sample.

In some embodiments, the antibody combination product comprises any combination of an antibody or antigen-binding portion thereof of the first aspect and an antibody or antigen-binding portion thereof of the second aspect, or any number or kind of combinations of an antibody or antigen-binding portion thereof of the first aspect and any number or kind of antibodies or antigen-binding portions thereof of the second aspect.

In some embodiments, the ex vivo sample is derived from whole blood, serum, or plasma.

In some embodiments, the ex vivo sample is derived from blood.

In some embodiments, the ex vivo sample is derived from a bodily fluid.

The body fluid may be treated or untreated. Methods of obtaining a bodily fluid from a subject are known to those skilled in the art.

In some embodiments, the ex vivo sample is derived from urine, gastric juice, bile, saliva, sweat, and spinal fluid, stool, or muscle biopsy.

Method for detecting content of human galectin-3in vitro sample

According to the above description, the amount of galectin-3in the sample can be quantified using an antibody capable of binding to the C-terminal and/or N-terminal portion of human galectin-3.

In a sixth aspect, the present application provides a method for detecting the amount of human galectin-3in an ex vivo sample in vitro, the method using the antibody or antigen binding portion thereof of the first or second aspect or the antibody combination product of the third aspect;

wherein the antibody or antigen-binding portion thereof of the first aspect is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of the first aspect is labeled with a detectable label, or the antibody or antigen-binding portion thereof of the second aspect is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of the first aspect is labeled with a detectable label; and

wherein the antibody or antigen-binding portion thereof detects human galectin-3 content by an immunological method.

In some embodiments, the ex vivo sample is derived from whole blood, serum, or plasma.

In some embodiments, the ex vivo sample is derived from a bodily fluid.

The body fluid may be treated or untreated. Methods of obtaining a bodily fluid from a subject are known to those skilled in the art.

In some embodiments, the ex vivo sample is derived from urine, gastric juice, bile, saliva, sweat, and spinal fluid, stool, or muscle biopsy.

In some embodiments, the ex vivo sample is derived from blood.

In some embodiments, the immunological method is selected from the group consisting of an enzyme-linked immunosorbent assay, an immunofluorescence assay, a chemiluminescence assay, an immunochromatography assay, an immunoprecipitation assay, and a combination thereof.

In some embodiments, the immunological method is an enzyme-linked immunoassay.

In some embodiments, galectin-3 may be detected and quantified using a "sandwich" assay. In this method, typically, an antibody is immobilized on a solid surface in order to bind and capture galectin-3. The antibody is thus referred to herein as a capture antibody. The second antibody is detectably labeled with a chromogenic substance (e.g., a fluorophore or a chemiluminescent compound) or an enzyme, such that binding of the second antibody to the galectin-3-complex indicates that galectin-3 has been captured. The intensity of the signal is proportional to the concentration of galectin-3in the sample. The second antibody is therefore also referred to herein as a detection antibody or a labeled antibody.

Such assay methods may be referred to as two-site immunoassays, "sandwich" methods, or "sandwich immunoassays. The capture and detection antibodies can be contacted with the test sample simultaneously or sequentially as is known in the art. The Sequential method, sometimes referred to as the "forward" method, can be accomplished by incubating the capture antibody with the sample and thereafter adding the labeled detection antibody at a predetermined time. Alternatively, the labeled detection antibody can be first incubated with the sample, and the sample can then be contacted with the capture antibody (sometimes referred to as the "reverse" method). Such assays may be performed in many specific formats known to those skilled in the art, including through the use of different high throughput clinical laboratory analyzers or with point of care (point of care) or home test equipment.

The most commonly used enzyme immunoassay is ELISA. ELISA is a technique that uses a labeled (e.g., enzyme-linked) form of an antibody to detect and measure the concentration of an antigen. There are different ELISA formats known to the person skilled in the art. Standard techniques for ELISA known in the art are described in "Methods in Immunodiagnosis", 2 nd edition, rose and bigzi, eds. John Wiley & Sons,1980; campbell et al, "Methods and Immunology", w.a. benjamin, inc.,1964; and Oelleric, m. (1984, j. Clin. Chem. Clin. Biochem.22.

In a "sandwich ELISA," an antibody (e.g., anti-galectin-3) is attached to a solid phase (i.e., a microtiter plate) and contacted with a biological sample containing an antigen (e.g., galectin-3). The solid phase is then washed to remove unbound antigen. The labeled antibody (e.g., enzyme-linked) is then bound to the bound antigen, thereby forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to an antibody are alkaline phosphatase, horseradish peroxidase, luciferase, urease and β -galactosidase. The enzyme-linked antibody reacts with the substrate to produce a colored reaction product that can be measured. This measurement can be used to deduce the concentration of galectin-3 present in the sample, for example by comparing the measurement to a galectin-3 standard curve.

The immunofluorescence method is based on the principle of antigen-antibody reaction, firstly, the known antigen or antibody is marked with fluorescein to prepare a fluorescent marker, and then the fluorescent antibody (or antigen) is used as a molecular probe to examine the corresponding antigen (or antibody) in cells or tissues. The antigen-antibody complex formed in the cell or tissue contains fluorescein, the specimen is observed by using a fluorescence microscope, the fluorescein emits bright fluorescence under the irradiation of exciting light, and the cell or tissue where the fluorescence is positioned can be seen, so that the property and the location of the antigen or the antibody can be determined, and the content can be measured by using a quantitative technology.

Chemiluminescence immunoassay (CLIA) is a detection and analysis technique for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, drugs and the like by combining a chemiluminescence assay technique with high sensitivity and a high specificity immunoreaction. Is a latest immunoassay technology developed after radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and time-resolved fluoroimmunoassay. Chemiluminescent immunoassays are carried out by directly labeling an antigen or antibody with a chemiluminescent agent. Among the chemiluminescent materials commonly used for labeling are acridinium ester compounds, which are efficient luminescent labels that emit light by activating the luminescent reagent, with intense direct luminescence occurring within one second, being rapid blinking. The acridinium ester is used as a marker for immunoassay, the chemical reaction is simple and rapid, no catalyst is needed, a competition method is adopted for detecting small molecular antigens, a sandwich method is adopted in a macromolecule antibody principle, the nonspecific binding is less, and the background is low; binding to macromolecules does not reduce the amount of light produced, thereby increasing sensitivity.

Immunoprecipitation is a method for purifying and enriching a target protein by using an antibody-specific reaction. After the antibody is combined with corresponding protein in a sample, the sample is incubated with agarose or Sepharose beads coupled with protein A/G (protein A/G) or secondary antibody, a bead-protein A/G or secondary antibody-target protein compound is obtained by centrifugation, the precipitate is re-suspended in electrophoresis loading buffer solution after being washed, boiled, under the action of high temperature and reducing agent, the antigen is dissociated from the antibody, and supernatant is collected by centrifugation, wherein the supernatant comprises the antibody, the target protein and a small amount of hybrid protein.

The marker used in the present application may be selected from any marker generally known in the art. Preferably the label is one which allows for more accurate quantitation. Examples of labels include, but are not limited to, fluorescent moieties, enzymes, electrochemically active species, radioisotopes, chemiluminescent molecules, latex particles or gold particles, detectable ligands, and the like. In a preferred embodiment, the label is an enzyme or a fluorescent molecule. Methods for attaching labels to antibodies are well known in the art and include covalent and non-covalent attachment.

In some embodiments, the antibody may be labeled with a fluorescent compound. When a fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can then be detected by the emitted fluorescence. Among the most commonly used fluorescent labeling compounds are Cy3 and Cy5 (water-soluble fluorescent dyes of the cyanine dye family- "Cy" dyes), fluorescein isothiocyanate, rhodamine, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

In some embodiments, the detection antibody is detectably labeled by linking the antibody to an enzyme. Whereby the enzyme will react with its substrate upon contact with its substrate, which reaction can be detected, for example, by spectrophotometric, fluorimetric or visual measurements. Enzymes that can be used to detectably label the antibodies of the invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

Detection can also be achieved using radiolabeled antibodies. The antibodies can then be detected by using radioimmunoassay. Can be detected by such methods as the use of gamma counter or scintillation counter or by autoradiographyAnd measuring the radioactive isotope. Isotopes particularly useful for the purposes of the present application are ³ H、 ¹³¹ I、 ³⁵ S、 ¹⁴ C, and preferably ¹²⁵ I。

The antibody may also be detectably labeled by coupling it to a chemiluminescent moiety. The presence of chemiluminescent antibody is then determined by detecting the presence of luminescence generated during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, luciferin, isoluminol, imidazole, acridinium salts and oxalate esters.

In a seventh aspect, the present application provides the use of an antibody or antigen-binding portion thereof according to the first or second aspects or an antibody combination product according to the third aspect for detecting the amount of human galectin-3in an ex vivo sample.

In some embodiments, the ex vivo sample is derived from whole blood, serum, or plasma.

In some embodiments, the ex vivo sample is derived from blood.

There are numerous advantages and benefits to the present application, for example:

the recombinant galectin-3 polypeptide is used for preparing and screening diagnostic antibodies, and has high yield and high sensitivity.

The content of the galectin-3 protein in the serum of the heart failure patient is determined by an immunological technique, and the index can be used as a key basis for the prognosis recovery or the early disease detection of the patient and has higher detection sensitivity and specificity.

Examples

The following examples are provided merely to illustrate some embodiments of the present application and are not intended to be limiting in any way. In addition, the methods in the examples will be performed according to conventional protocols in the art, unless otherwise specified.

Example 1: obtaining of murine monoclonal antibodies

A Balb/c mouse immunized by the human galectin-3 is used for obtaining hybridoma cells expressing the antibody, and the hybridoma cells are detected and screened to obtain the antibody specifically recognizing the galectin-3. The specific method is shown in figure 1.

Six Balb/c mice are immunized by adopting the recombinant galectin-3 full-length antigen, and female Balb/c mice of 6-8 weeks are selected. The antigen was used at a dose of 100. Mu.g/mouse. Mixing the antigen and the complete adjuvant 1 in a mixing ratio, emulsifying, and performing subcutaneous multipoint injection; the immunization cycle was six times. Every two needles are spaced by one week.

Fusing with mouse myeloma cells when the titer reaches more than 1 10000, detecting the absorbance OD value of cell supernatant by using an ELISA indirect method, selecting cells with positive holes according to the OD value result, cloning the cells by adopting a limiting dilution method, inoculating 1 cell per hole into a culture dish, and proliferating the cells to form a monoclonal cell line.

Screening to obtain 46 mouse monoclonal antibody hybridoma cells. Antibodies were generated using in vivo induction. Male Balb/c mice over 6 weeks old were selected and sensitized with Freund's incomplete adjuvant (500. Mu.L/mouse). Inoculating cell strain for about 7 days to reach density of 2X10 ⁶ . The abdominal distension can be seen obviously in about 7 days; ascites is extracted and filtered for sterilization, the aperture of the filter membrane is 0.45 mu m, and the obtained product is subpackaged and frozen at-20 ℃.

And (3) expanding culture and freezing storage: expanding and culturing the selected cell strain with strong specificity in time, and using 10% of DMSO frozen stock solution; and (5) freezing and storing the mixture according to the volume of 1 ml/tube until the mixture is stored in liquid nitrogen.

Example 2: identification of antibodies

The 46 mouse monoclonal antibodies obtained in example 1 were further characterized. The capture antibody and detection antibody pairs were identified using a checkerboard method. Briefly, all 46 antibodies were plated at a concentration of 2ng/mL (PBS adjusted antibody concentration) in a volume of 100. Mu.L per well to a 384-well microtiter plate as capture antibodies, each of which was plated in 47 wells, and after 18 hours, 100. Mu.L of PBS containing 4% BSA was added per well for blocking, and then the standard product Galectin-3 (purchased from BBI Solution) was added in a volume of 100. Mu.L per well to the 384-well microtiter plate, and after PBST was washed 5 times, the corresponding wells were detected with biotin-labeled antibodies other than itself as detection antibodies (concentration of 2ng/mL, 100. Mu.L per well).

The obtained 46 antibodies were detected by ELISA, and the antibody with weak positive signal was deleted. Two pairs (4) of mouse monoclonal antibodies specific for human Galectin-3 were obtained.

Expanding the selected cell lines with strong specificity in time, and using 10% DMSO to freeze the stock solution; and (5) freezing and storing the mixture according to the volume of 1 ml/tube until the mixture is stored in liquid nitrogen. Specific mouse antibodies were isolated and purified using a protein a column (purchased from LIFE TECH). Four antibodies, G3-5-5, G3-12-1, G3-2-1 and G3-2-3, were obtained, i.e., the first through fourth antibodies of the present application.

RNA from positive hybridoma cells was isolated using an RNA miniprep kit (purchased from Qiagen) and reverse transcribed to cDNA using reverse transcriptase (purchased from Biosystems). PCR was then performed using the following mouse antibody specific primers. Amplification products of mouse antibody Ig heavy and light chains were obtained and sequenced.

HC-For：acctattactgtcagcacatta(SEQ ID NO:37)

HC-Rev：ggatacagttggtgcagcatc(SEQ ID NO:38)

LC-For：gayattgtgmtsacmcarwct(SEQ ID NO:39)

LC-Rev：acctattactgtcagcacatta(SEQ ID NO:40)

By sequencing, G3-5-5 (first antibody) comprises the heavy chain variable region of HCDR1 shown in SEQ ID NO:13, HCDR2 shown in SEQ ID NO:14 and HCDR3 shown in SEQ ID NO:15, and the light chain variable region comprising LCDR1 shown in SEQ ID NO:16, LCDR2 shown in SEQ ID NO:17 and LCDR3 shown in SEQ ID NO: 18.

G3-12-1 (second antibody) comprises the heavy chain variable region of HCDR1 shown in SEQ ID NO:19, HCDR2 shown in SEQ ID NO:20 and HCDR3 shown in SEQ ID NO:21, and the light chain variable region comprising the LCDR1 shown in SEQ ID NO:22, LCDR2 shown in SEQ ID NO:23 and LCDR3 shown in SEQ ID NO: 24.

G3-2-1 (third antibody) comprises the heavy chain variable region of HCDR1 shown in SEQ ID NO:25, HCDR2 shown in SEQ ID NO:26 and HCDR3 shown in SEQ ID NO:27, and the light chain variable region comprising LCDR1 shown in SEQ ID NO:28, LCDR2 shown in SEQ ID NO:29 and LCDR3 shown in SEQ ID NO: 30.

G3-2-3 (fourth antibody) comprises the heavy chain variable region of HCDR1 shown in SEQ ID NO:31, HCDR2 shown in SEQ ID NO:32 and HCDR3 shown in SEQ ID NO:33, and the light chain variable region comprising LCDR1 shown in SEQ ID NO:34, LCDR2 shown in SEQ ID NO:35 and LCDR3 shown in SEQ ID NO: 36.

Example 3: determination of epitopes

And (4) performing bioinformatics analysis on the Galectin-3, predicting binding sites, synthesizing 88 corresponding short overlapping peptides, coupling the short peptides to carrier protein, and detecting by using an enzyme-linked immunosorbent assay.

The short peptide fusion protein was coated at a concentration of 5. Mu.g/ml (PBS adjusted antibody concentration), 100. Mu.L per well was plated on a 96-well plate, reacted overnight at 4 ℃ and blocked for 2 hours by adding 130. Mu.L of BSA containing 1% per well.

The primary antibody, secondary antibody, tertiary antibody, and fourth antibody obtained in example 2 at 4ng/ml were labeled, and 50. Mu.L per well was added to 96 wells; PBST was washed 5 times. An antibody other than itself labeled with biotin was used as the detection antibody. The corresponding wells were tested in an amount of 50 μ L per well. The specific antigen-binding epitope of the antibody is obtained according to the interaction between the fusion short peptide and the antibody and the binding firmness.

The first and second antibodies were detected to be capable of binding to non-contiguous epitopes on the C-terminal portion of human galectin-3. The third and fourth antibodies are capable of binding to non-contiguous epitopes on the N-terminal portion of human galectin-3.

Example 4 fluorescent immunochromatographic assay for quantitative detection of human galectin-3

As shown in fig. 2, the fluorescence immunochromatographic kit comprises a sample pad, a conjugate pad, a nitrocellulose membrane, a water absorbent pad, and a polyvinyl chloride (PVC) substrate.

In this example, the first antibody G3-5-5 of human galectin-3 and the secondary antibody are immobilized on a nitrocellulose membrane in advance to form a test line or a control line, and after a sample containing an analyte is added, the analyte is bound to the labeled antibody on the binding pad and is further captured by the monoclonal antibody immobilized on the T line by chromatography. Excess labeled antibody was captured by the secondary antibody on the C-line, and the signal intensity of the label on the T/C-line was read by a specific detection instrument and substituted into a pre-drawn standard curve.

Preparing fluorescent particles of the fourth antibody G3-2-3 coupled with human galectin-3:

dispersing the surface aminated fluorescein particles in a phosphate buffer, adding glutaraldehyde to react for 4-8 hours, centrifuging to remove glutaraldehyde, redispersing the fluorescein particles in a phosphate buffer, adding a fourth antibody G3-2-3 to human galectin-3, reacting for 4-12 hours, washing to obtain fluorescein particle-antibody complexes, and storing in a phosphate buffer containing 0.1% BSA and 0.1% Tween 20.

Assembling the fluorescence immunochromatographic test paper:

the fluorescein particle-antibody compound is coated on a binding pad, the first antibody G3-5-5 and the second antibody are respectively connected to an antibody bearing membrane in a linear mode to form a T line and a C line, and the sample pad, the binding pad, the antibody bearing membrane and the absorbent paper are mutually overlapped and adhered on a lining plate with an adhesive.

Preparation of human galectin-3 calibration sample:

human galectin-3 protein was prepared in standard buffer (50 mM Tris-HCl, 1% BSA, 0.9% NaCl, pH 7.4) at concentrations of 0, 5, 20, 40, 60, 100ng/mL, and was dispensed at 1mL per bottle and stored at 4 ℃ until use.

Detection of human galectin-3in ex vivo samples:

the operating procedure for use of the kit described herein is as follows: restoring the reagent and the sample to be detected to room temperature, and starting an immunofluorescence analyzer according to the instruction; and vertically adding the sample to be detected into the sample adding hole of the detection card corresponding to the sample pad by using a dropper, and incubating for 15 minutes. After incubation, the signal of the detection card is detected by using an immunofluorescence analyzer, and the content of the human galectin-3in the sample is in direct proportion to the relative fluorescence signal.

The antibody immobilized on the nitrocellulose membrane may be the first antibody or the second antibody, or may be the third antibody or the fourth antibody. Similarly, the antibody coupled to the fluorescent particle may be the third antibody or the fourth antibody, or may be the first antibody or the second antibody.

Using the same method as above, a fluorescence immunochromatographic kit combining different antibodies was also prepared, and the prepared kit was detected.

In this example, the third antibody G3-2-1 of human galectin-3 and the secondary antibody were also immobilized on a nitrocellulose membrane in advance to form a test line or a control line, and fluorescent microparticles to which the second antibody G3-12-1 of human galectin-3 was coupled were prepared to prepare a fluorescent immunochromatographic kit and detect it.

Example 5: evaluation of human galectin-3 fluorescence immunochromatography

The method and the prepared test paper in the embodiment 4 are adopted to detect the human galectin-3 calibration substance, and a standard curve is drawn. Similarly, the measurement of the sample to be tested was carried out, and the concentration of human galectin-3in the sample was calculated from the luminescence intensity of the sample.

Detection of sensitivity: the sensitivity of the human galectin-3 fluorescence immunochromatography reagent was calculated with reference to the recommended experimental protocol of CLSI EP17-A document, and the sensitivity was 1.5ng/mL.

And (3) linear detection: the linear analysis of the calibrator was performed, a linear correlation coefficient was calculated for the combination of the first and fourth antibodies, r =0.9996, and the linear range of the kit for detection of human galectin-3 samples was 1.5ng/mL to 80ng/mL. A linear correlation coefficient was calculated for the combination of the third antibody and the second antibody, r =0.9987, and the linear range of the kit for detection of human galectin-3 samples was 2ng/mL to 100ng/mL.

And (3) measuring the precision: two human galectin-3 samples with the concentrations of 15ng/mL and 60ng/mL are taken, each concentration of each sample is respectively carried out 3 parallels, three batches of kits are used for detection, the intra-batch difference and the inter-batch difference of the kits are calculated, and the result shows that the intra-batch difference and the inter-batch difference of the kits are both less than 10%.

Example 6 magnetic particle chemiluminescence assay for quantitative determination of human galectin-3

The magnetic particle chemiluminescence kit comprises a magnetic particle coating object, an enzyme marker, chemiluminescence substrate liquid, analysis buffer liquid, washing liquid, a standard substance and a quality control substance.

In this example, a first antibody G3-5-5 of human galectin-3 was coated on magnetic particles, a fourth antibody G3-2-3 of human galectin-3 was labeled on alkaline phosphatase, a sample containing an analyte treated with an assay buffer was added, the analyte was bound to the magnetic particle-coated substance and the enzyme-labeled substance to form an immune complex, and after washing with a washing solution, a chemiluminescent substrate solution was added, and the intensity of luminescence was measured by a chemiluminescent detector and substituted into a standard curve drawn in advance.

Preparing a magnetic particle coating material of a first antibody G3-5-5 coated with human galectin-3:

dispersing the surface-carboxylated magnetic microparticles in a phosphate buffer, adding a first antibody G3-5-5 to human galectin-3, mixing for 30 minutes, adding carbodiimide, reacting at 4 ℃ for 12-15 hours, separating with a magnetic frame, washing to obtain magnetic microparticle-antibody complexes, and storing in a phosphate buffer containing 0.5% BSA and 0.1% Tween 20.

Preparation of an enzyme marker for the fourth antibody G3-2-3 of human galectin-3:

adding glutaraldehyde to the alkaline phosphatase solution, reacting for 4-8 hr, dialyzing to remove glutaraldehyde, adding the fourth antibody G3-2-3 to human galectin-3, reacting for 4-12 hr, and storing in a phosphate buffer containing 50% glycerol, 0.1% BSA and 0.1% Tween 20.

Preparing a chemiluminescent substrate solution: the chemiluminescent substrate solution was Tris buffer containing 10% 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxyethane (AMPPD).

Preparation of assay buffer: the assay buffer was a phosphate buffer containing 1% BSA and 0.1% Tween 20.

Preparing a washing solution: the washing solution was phosphate buffered saline containing 1% Tween 20.

Preparing a standard substance and a quality control substance: the standard sample was prepared by dispensing human galectin-3 protein in standard buffer (50 mM Tris-HCl, 1% BSA, 0.9% NaCl, pH 7.4) at concentrations of 0, 5, 20, 40, 60, and 100ng/mL, 1mL per bottle, and storing at 4 ℃ until use.

The quality control product is prepared by preparing human galectin-3 protein into 10 and 50ng/mL respectively with standard buffer solution, subpackaging 1mL per bottle, and storing at 4 deg.C for use.

Assembling the magnetic particle chemiluminescence kit:

magnetic particle coating matter, enzyme label matter, chemiluminescence substrate liquid, analysis buffer liquid, washing liquid, standard substance and quality control substance are assembled into the human galectin-3 magnetic particle chemiluminescence kit.

Detection of human galectin-3in ex vivo samples:

the magnetic particle chemiluminescence kit described in the application is used in the following operating procedures: taking 50 mu L of sample to be detected or standard substance/quality control substance, 50 mu L of analysis buffer solution, 50 mu L of magnetic particle coating substance and 50 mu L of enzyme labeling substance to react together in a reaction cup, incubating for 10 minutes at 37 ℃, after the reaction is finished, carrying out magnetic separation and cleaning on the reactant for 3 times on a magnetic plate, adding 100 mu L of chemiluminescent substrate solution after the cleaning is finished, and uniformly mixing to carry out detection.

The antibody coated on the magnetic particle may be a first antibody or a second antibody, or a third antibody or a fourth antibody. Similarly, the antibody conjugated to alkaline phosphatase may be the third antibody or the fourth antibody, or may be the first antibody or the second antibody.

Using the same method as above, a magnetic particle chemiluminescence kit combining different antibodies was also prepared, and the prepared kit was detected.

Example 7: evaluation of performance of human galectin-3 magnetic particle chemiluminescence kit

The method and the kit prepared in the embodiment 6 are adopted to detect the human galectin-3 calibration substance, and a standard curve is drawn. Similarly, the measurement of the sample to be tested was carried out, and the concentration of human galectin-3in the sample was calculated from the luminescence intensity of the sample.

And (3) detection of sensitivity: the sensitivity of the human galectin-3 fluorescence immunochromatographic reagent was calculated with reference to the protocol recommended by the CLSI EP17-A document, and the sensitivity was 1.0ng/mL.

And (3) linear detection: the linear analysis of the calibrator was performed, a linear correlation coefficient was calculated for the combination of the first antibody and the fourth antibody, r =0.9996, and the linear range of the detection of the kit on the human galectin-3 sample was 1.0ng/mL to 100ng/mL. A linear correlation coefficient was calculated for the combination of the third antibody and the second antibody, r =0.9997, and the linear range of the kit for detection of human galectin-3 samples was 1.0ng/mL to 100ng/mL.

And (3) measuring the precision: taking two human galectin-3 samples with the concentrations of 15ng/mL and 60ng/mL, respectively carrying out 3 parallels on each concentration of each sample, detecting by using three batches of kits, and calculating the intra-batch difference and the inter-batch difference of the kits, wherein the results show that the intra-batch difference and the inter-batch difference of the kits are both less than 5%.

Sequence listing

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Claims (10)

1. An antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof is capable of binding to at least a portion of the amino acid sequence of human galectin-3, said at least a portion of the amino acid sequence of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 or SEQ ID NO 10, or any combination thereof.

2. The antibody or antigen-binding portion thereof of claim 1, wherein at least a portion of the amino acid sequence of human galectin-3 is located at a C-terminal portion of human galectin-3, wherein the C-terminal portion comprises the amino acid sequence set forth in SEQ ID No. 1.

3. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody comprises HCDR1 of SEQ ID NO 13, HCDR2 of SEQ ID NO 14, HCDR3 of SEQ ID NO 15, LCDR1 of SEQ ID NO 16, LCDR2 of SEQ ID NO 17, and LCDR3 of SEQ ID NO 18; or alternatively

The antibody comprises HCDR1 shown in SEQ ID NO. 19, HCDR2 shown in SEQ ID NO. 20, HCDR3 shown in SEQ ID NO. 21, LCDR1 shown in SEQ ID NO. 22, LCDR2 shown in SEQ ID NO. 23 and LCDR3 shown in SEQ ID NO. 24.

4. An antibody or antigen-binding portion thereof, which antibody or antigen-binding portion thereof is capable of binding to at least a portion of the amino acid sequence of human galectin-3, said at least a portion of human galectin-3 comprising the amino acid sequence set forth in SEQ ID NO. 11 or SEQ ID NO. 12 or both SEQ ID NO. 11 and SEQ ID NO. 12.

5. The antibody or antigen-binding portion thereof of claim 4, wherein at least a portion of the amino acid sequence of human galectin-3 is located at an N-terminal portion of human galectin-3, wherein the N-terminal portion comprises the amino acid sequence set forth in SEQ ID NO. 2.

6. The antibody or antigen-binding portion thereof of claim 4, wherein the antibody comprises HCDR1 of SEQ ID NO 25, HCDR2 of SEQ ID NO 26, HCDR3 of SEQ ID NO 27, LCDR1 of SEQ ID NO 28, LCDR2 of SEQ ID NO 29, and LCDR3 of SEQ ID NO 30; or alternatively

The antibody comprises HCDR1 shown in SEQ ID NO. 31, HCDR2 shown in SEQ ID NO. 32, HCDR3 shown in SEQ ID NO. 33, LCDR1 shown in SEQ ID NO. 34, LCDR2 shown in SEQ ID NO. 35 and LCDR3 shown in SEQ ID NO. 36.

7. An antibody combination comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 3 and the antibody or antigen-binding portion thereof of any one of claims 4 to 6.

8. A kit for detecting galectin-3 content in a sample from a human subject, comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 6 or the antibody combination of claim 7, wherein the antibody or antigen-binding portion thereof is labeled with a detectable label.

9. Use of the antibody or antigen-binding portion thereof of any one of claims 1-6 or the antibody combination product of claim 7 for the preparation of a kit for detecting the amount of human galectin-3in an ex vivo sample.

10. A method for detecting the amount of human galectin-3in an ex vivo sample using the antibody or antigen binding portion thereof of any one of claims 1 to 6 or the antibody combination of claim 7;

wherein the antibody or antigen-binding portion thereof of any one of claims 1 to 3 is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of any one of claims 4 to 6 is labeled with a detectable label, or the antibody or antigen-binding portion thereof of any one of claims 4 to 6 is for capturing human galectin-3 and the antibody or antigen-binding portion thereof of any one of claims 1 to 3 is labeled with a detectable label; and

wherein the antibody or antigen-binding portion thereof detects human galectin-3 content by an immunological method.

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