Antigenic peptides of lipoprotein(a), antibodies thereto and uses

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof to lipoprotein(a) which specifically binds to one or more KIV antigen peptides selected from the group consisting of SEQ ID NO: 1-3. Using such an antibody it is possible to accurately detect the lipoprotein(a) content in a sample without the need to use a standard to establish a curve. Furthermore, the present invention also relates to the corresponding antigen peptides, nucleic acid molecules, fusion proteins and methods.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and more particularly to an antigenic peptide of lipoprotein (a) and an antibody that specifically binds to it. Background Technology

[0002] Lipoprotein(a) is primarily synthesized in the liver. Detecting lipoprotein(a) in conjunction with patient symptoms and other relevant diagnostic results (e.g., triglycerides, cholesterol, high-density lipoprotein, low-density lipoprotein, homocysteine, etc.) can aid in the diagnosis of cardiovascular diseases. In other words, accurate lipoprotein(a) detection is beneficial for the auxiliary diagnosis of cardiovascular diseases. Like other lipoproteins, lipoprotein(a) consists of a fatty core containing cholesterol, triglycerides, phospholipids, etc. Surrounding this fatty core are two protein chains: apoB and apo(a), which represents the characteristics of lipoprotein(a), linked by disulfide bonds. Therefore, the detection of lipoprotein(a) can only target apo(a).

[0003] Apo(a) is a protein composed of many small "rings" (also known as pie-like structures). Based on differences in amino acid sequence, its pie-like structure can be classified into KIV-1 to KIV-10 types. Except for KIV-2 type, all other types have only one pie-like structure. However, the number of repeating pie-like structures varies among different apo(a) of KIV-2 type, ranging from 3 to 40 copies, resulting in apo(a) molecular weights ranging from 187,000 to 662,000 in different individuals. Therefore, the heterogeneity of the relative molecular mass of KIV-2 reduces the accuracy of lipoprotein(a) immunoassay methods. Specifically, if the antibody recognizes a multi-copy KIV-2 pie-like region, then if the apo(a) particle size of the test subject is smaller than that of the calibrator apo(a) particle, the obtained lipoprotein(a) detection result will be lower; conversely, the result will be higher.

[0004] To overcome the difficulty in accurately reflecting lipoprotein (a) levels, both Roche and Denka Seiken used calibrators with five different particle sizes. On the one hand, the accuracy of this method strongly depends on the curve established by the calibrators; on the other hand, the preparation of these calibrators is very cumbersome and costly.

[0005] To avoid the introduction of multiple gradient calibrators, theoretically, antibodies capable of recognizing a single copy number of the ring pie region (i.e., the non-KIV-2 region) on each apo(a) could be used to detect the particle concentration of lipoprotein(a).

[0006] In response, some researchers have attempted to obtain polyclonal antibodies by using recombinant KIV-1 pie rings as antigens for immunization. However, due to the high similarity between the antigens used and the KIV-2 pie rings, the obtained polyclonal antibodies still exhibit affinity for the KIV-2 pie rings. Therefore, it is impossible to obtain truly non-KIV-2 specific antibodies, which cannot accurately reflect the level of lipoprotein(a).

[0007] In addition, some researchers have tried to use natural lipoprotein(a) as an immunogen to screen for non-KIV-2 specific antibodies. However, due to the high copy number of KIV-2 and its high similarity to other types of KIV rings, the efficiency of using natural lipoprotein(a) as an immunogen to screen for non-KIV-2 specific antibodies is low, thus failing to efficiently obtain antibodies that accurately reflect lipoprotein(a) levels.

[0008] Therefore, in the field of lipoprotein(a) detection, there is a strong demand for rapid and convenient acquisition of non-KIV-2 specific antibodies. Summary of the Invention

[0009] To facilitate the screening of non-KIV-2 specific antibodies, the inventors investigated the structure of apo(a) in lipoprotein(a). In a first aspect, the present invention provides an antigenic peptide having an amino acid sequence as shown in one or more of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0010] Using the antigenic peptide of the present invention and following conventional antibody preparation methods, antibodies that specifically bind to the KIV-6 / 7, KIV-8, or KIV-9 regions but not the KIV-2 region can be obtained by immunizing only a few animals. Furthermore, such antibodies can be used for the detection of lipoprotein(a) and exhibit high comparability with commercially available products. Moreover, the antibodies obtained using the antigenic peptide of the present invention meet the performance requirements of reagents regarding the degree of bias.

[0011] In one specific embodiment, the amino acid sequence of the antigenic peptide of the present invention is shown in SEQ ID NO:1.

[0012] In one specific embodiment, the amino acid sequence of the antigenic peptide of the present invention is shown in SEQ ID NO:2.

[0013] In a preferred embodiment, the amino acid sequence of the antigenic peptide of the present invention is shown in SEQ ID NO:3.

[0014] In one specific embodiment, the antigenic peptide of the present invention may also be any combination of two or more antigenic peptides having the above-described amino acid sequence, used as an immunogen. For example, an antigenic peptide having amino acid SEQ ID NO:1 and an antigenic peptide having amino acid SEQ ID NO:3 may be used together as an immunogen.

[0015] It should be noted that by selecting the antigenic peptide shown in SEQ ID NO:3, this invention further improves the efficiency of screening for non-KIV-2 specific anti-lipoprotein(a) antibodies. As demonstrated in the following examples section, when antigens prepared using the antigenic peptide shown in SEQ ID NO:3 were used to immunize six animals, all six resulting immune sera were positive for KIV-9 but negative for KIV-2. Furthermore, when using antibodies screened by the antigenic peptide shown in SEQ ID NO:3 to detect lipoprotein(a), the results are highly comparable to commercially available products.

[0016] In a second aspect, the present invention provides a fusion protein comprising the antigenic peptide of the present invention.

[0017] In some embodiments, the fusion protein of the present invention further comprises a selection of affinity-tagged peptides.

[0018] In some embodiments, the fusion protein of the present invention further includes a protein carrier.

[0019] In some embodiments, the fusion protein of the present invention further includes a polypeptide that targets the fusion protein to a predetermined location.

[0020] In a third aspect, the present invention provides a nucleic acid molecule that encodes the antigenic peptide of the present invention or the fusion protein of the present invention.

[0021] In a fourth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the third aspect of the present invention.

[0022] In a fifth aspect, the present invention provides a host cell comprising the nucleic acid molecule of the third aspect of the present invention or the expression vector of the fourth aspect of the present invention.

[0023] In a sixth aspect, the present invention provides a method for obtaining a non-KIV-2 specific anti-lipoprotein (a) antibody, the method utilizing the antigenic peptide of the present invention as an antigen.

[0024] In one variation of the method for obtaining non-KIV-2 specific anti-lipoprotein (a) antibodies, the method utilizes the fusion protein of the antigenic peptide and protein carrier of the present invention as the antigen.

[0025] In a seventh aspect, the present invention provides a method for preparing antibodies, the method using the antigenic peptide of the present invention as an immunogen to produce antibodies.

[0026] In a variation of the method for preparing antibodies, the method utilizes the fusion protein of the antigenic peptide and the protein carrier of the present invention as an immunogen to produce antibodies.

[0027] In an eighth aspect, the present invention provides an antibody or an antigen-binding fragment thereof, which specifically binds to one or more KIV antigen peptides selected as shown in SEQ ID NO:1 to 3.

[0028] Alternatively, the antibody or antigen-binding fragment thereof of the present invention is generated using the antigenic peptide of the first aspect of the present invention.

[0029] It is understood that the antigenic peptide shown in SEQ ID NO:1 is located on the KIV-6 and KIV-7 regions. When screening using the antigenic peptide shown in SEQ ID NO:1, antibodies that specifically bind to the corresponding antigenic epitopes on KIV-6 and KIV-7 can be obtained. The antigenic peptide shown in SEQ ID NO:2 is located on the KIV-8 region. When screening using the antigenic peptide shown in SEQ ID NO:2, antibodies that specifically bind to the corresponding antigenic epitope on KIV-8 can be obtained. The antigenic peptide shown in SEQ ID NO:3 is located on the KIV-9 region. When screening using the antigenic peptide shown in SEQ ID NO:3, antibodies that specifically bind to the corresponding antigenic epitope on KIV-9 can be obtained.

[0030] In some embodiments, the antibody or antigen-binding fragment of the present invention does not specifically bind to the KIV-2 antigenic peptide as shown in SEQ ID NO:4.

[0031] In a preferred embodiment, the antibody or its binding fragment of the present invention specifically binds to the KIV antigen peptide as shown in SEQ ID NO:3.

[0032] In some embodiments, the antibodies of the present invention may be selected from monoclonal antibodies, polyclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and single-chain antibodies.

[0033] In a specific embodiment, the antibody of the present invention is a monoclonal antibody.

[0034] In a specific embodiment, the antibody of the present invention is a polyclonal antibody.

[0035] In specific implementations, the antibody may be of mouse, rabbit, or sheep origin.

[0036] In a ninth aspect, the present invention provides a nucleic acid molecule that encodes the antibody of the present invention or an antigen-binding fragment thereof.

[0037] In a tenth aspect, the present invention provides a kit for detecting lipoprotein(a), comprising the antibody of the present invention or an antigen-binding fragment thereof.

[0038] In some embodiments, the kit includes the antibody or antigen-binding fragment of the present invention, and said antibody or antigen-binding fragment is coated on a solid support.

[0039] In other embodiments, the kit includes the antibody or antigen-binding fragment of the present invention, and said antibody or antigen-binding fragment carries a detectable marker.

[0040] In some other embodiments, the kit includes a first antibody or antigen-binding fragment thereof of the present invention and a second antibody or antigen-binding fragment thereof of the present invention, wherein the KIV antigen peptide bound by the first antibody or antigen-binding fragment thereof is different from the KIV antigen peptide of the second antibody or antigen-binding fragment thereof.

[0041] In some other embodiments, the kit includes the antibody of the present invention or an antigen-binding fragment thereof, wherein the antibody is not coated on a solid support and does not carry a detectable marker.

[0042] In this invention, the terms "first" and "second" are used for descriptive purposes only to distinguish the substances being defined, and do not in any way limit the order or importance of the substances.

[0043] In an eleventh aspect, the present invention provides a method for detecting lipoprotein (a), comprising the following steps:

[0044] a) Contacting a sample derived from a subject with the antibody or antigen-binding fragment of the present invention; and

[0045] b) Determine whether lipoprotein(a) is present in the sample or determine the level of lipoprotein(a) present in the sample.

[0046] In some embodiments, the antibody or its antigen-binding fragment is coated on a solid support.

[0047] In other embodiments, the antibody or its antigen-binding fragment carries a detectable marker.

[0048] In some other embodiments, the antibody or its antigen-binding fragment is not coated on a solid support and does not carry a detectable marker.

[0049] In an exemplary embodiment, an antibody or its antigen-binding fragment is used in a double-antibody sandwich assay.

[0050] In a twelfth aspect, the present invention provides the use of an antibody or an antigen-binding fragment thereof in the preparation of a kit for detecting lipoprotein(a).

[0051] In a thirteenth aspect, the present invention provides a composition comprising one or more KIV antigenic peptides selected as shown in SEQ ID NO:1 to 3, and a KIV-2 antigenic peptide as shown in SEQ ID NO:4.

[0052] In a fourteenth aspect, the use of the antibody or antigen-binding fragment thereof of the eighth aspect of the invention is provided in the preparation of a kit for a method of evaluating cardiovascular disease, the method comprising the steps of:

[0053] a) Contacting a sample derived from a subject with the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6; and

[0054] b) Determine whether lipoprotein(a) is present in the sample or determine the level of lipoprotein(a) present in the sample.

[0055] By studying the apo(a) chain in lipoprotein(a), this invention is the first to discover and confirm the antigenic peptide sequences shown in SEQ ID NO: 1 to 3 located in the apo(a) chain. Using these antigenic peptide sequences, antibodies that specifically bind to KIV-6 / 7, KIV-8, or KIV-9 regions with only a single copy can be produced more conveniently and rapidly. These antibodies do not specifically bind to the KIV-2 region. When such antibodies are used to detect lipoprotein(a), the accuracy of the detection results can be improved, and this invention significantly improves the efficiency of screening such antibodies. Furthermore, the method of this invention avoids the introduction of multiple concentrations of standards during lipoprotein(a) detection, reducing the cost of detection reagents. Attached Figure Description

[0056] Figure 1 The results of comparing the lipoprotein (a) detection kit prepared with antibody #A1 with commercial reagents are shown.

[0057] Figure 2 The relationship between particle size and absolute deviation is shown when microspheres coated with antibody #A1 are used;

[0058] Figure 3 The absolute deviations for reference materials ranging from 0 to 300 nmol / L are shown when a lipoprotein (a) detection kit is prepared using antibody #A1. For concentrations ≤100 nmol / L, the evaluation criteria are based on absolute deviation.

[0059] Figure 4The relative deviations for reference materials ranging from 0 to 300 nmol / L are shown when a lipoprotein (a) detection kit is prepared using antibody #A1. The evaluation of reference materials with concentrations greater than 100 nmol / L is based on relative deviation.

[0060] Figure 5 The results of comparing the lipoprotein (a) detection kit prepared with antibody #B4 with commercial reagents are shown.

[0061] Figure 6 The relationship between particle size and absolute deviation is shown when microspheres coated with antibody #B4 are used;

[0062] Figure 7 The absolute deviations for reference materials ranging from 0 to 300 nmol / L are shown when a lipoprotein (a) detection kit is prepared using antibody #B4. The evaluation criteria for concentrations ≤100 nmol / L are absolute deviations.

[0063] Figure 8 The relative deviations for reference materials ranging from 0 to 300 nmol / L are shown when a lipoprotein (a) detection kit is prepared using antibody #B4. The evaluation of reference materials with concentrations greater than 100 nmol / L is based on relative deviation.

[0064] Figure 9 The results of comparing the lipoprotein (a) detection kit prepared with antibody #C3 with commercial reagents are shown.

[0065] Figure 10 The relationship between particle size and absolute deviation is shown when microspheres coated with antibody #C3 are used;

[0066] Figure 11 The absolute deviations for reference materials ranging from 0 to 300 nmol / L are shown when a lipoprotein (a) detection kit is prepared using antibody #C3. The evaluation criteria for concentrations ≤100 nmol / L are absolute deviations.

[0067] Figure 12 The relative deviations for reference materials ranging from 0 to 300 nmol / L are shown when a lipoprotein (a) detection kit is prepared using antibody #C3. The evaluation of reference materials with concentrations greater than 100 nmol / L is based on relative deviation. Detailed Implementation

[0068] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0069] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0070] As used herein, the term "antibody" refers to immunoglobulin molecules, including but not limited to monoclonal antibodies, polyclonal antibodies, bispecific antibodies, chimeric antibodies, humanized antibodies, and single-chain antibodies; it also includes antibodies that are recombinantly or synthetically produced / synthesized. Antibodies, if they contain a mass greater than or equal to about 10... 4 M -1 Preferably greater than or equal to about 10 5 M -1 More preferably greater than or equal to about 10 6 M -1 And more preferably greater than or equal to about 10 7 M -1 When the Ka atom binds to an antigenic peptide, it is defined as "immune-specific" or specifically bound.

[0071] As used herein, the term "monoclonal antibody" refers to a highly homogeneous antibody produced from a single B cell clone that targets only a specific antigenic epitope; that is, the individual antibodies constituting a population are identical, except for the possibility of naturally occurring mutations that may exist in small amounts. Monoclonal antibodies are highly specific against a single antigenic site. Monoclonal antibodies are advantageous because they can be obtained through hybridoma cell line culture and are largely unaffected by contamination from other immunoglobulins.

[0072] As used herein, the term "polyclonal antibody" refers to the blood supernatant collected after titer determination, obtained by directly injecting an antigen into an animal for immunization without the need for hybridoma cell preparation. Optionally, the supernatant may be further purified to obtain the polyclonal antibody.

[0073] As used herein, the term "antigen-binding fragment" generally includes at least a portion (e.g., six CDRs) of the antigen-binding region of the parent antibody, the light chain, and / or the variable region of the heavy chain, which retains at least some binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments.

[0074] Preferably, the antigen-binding fragment refers to the antigen-binding region, light chain and heavy chain variable regions, or the six CDRs of the antibody.

[0075] The antibodies of the present invention also include antibody derivatives. In this document, “antibody derivatives” refers to antibodies that may include conserved amino acid substitutions (also known as “conserved variants”) whose biological activity is substantially unchanged compared with that of the parent antibody.

[0076] Polyclonal antibodies can be prepared using any of a variety of techniques known to those skilled in the art. In one such technique, an immunogen containing the antigenic peptide of the present invention (e.g., an antigenic peptide conjugated with a protein carrier) is first injected into a suitable animal, preferably followed by one or more booster immunizations according to a predetermined schedule, and then blood is collected from the animal periodically. Polyclonal antibodies specific to the antigenic peptide of the present invention can then be purified from these serums using purification techniques such as Protein A affinity purification.

[0077] Similarly, monoclonal antibodies can be prepared using any of a variety of techniques known to those skilled in the art. For example, these methods may include: preparing an immortalized cell line capable of producing antibodies with the desired specificity. This cell line can be produced, for example, from spleen cells obtained from an immunized animal. The spleen cells are then immortalized, for example, by fusing myeloma cell fusion couples (such as couples homologous to the immunized animal). For example, spleen cells and myeloma cells can be bound for a few minutes using a membrane fusion promoter (such as polyethylene glycol or a nonionic detergent), and then plated at low density on a selective medium that supports hybrid cells but does not support myeloma cell growth. After a sufficient period, typically about 1–2 weeks, the hybrid colonies are observed. Individual colonies are selected and their binding activity to the antigenic peptide is tested. Hybridomas with high reactivity and specificity are preferred. Monoclonal antibodies can be isolated from the supernatant from the growth of hybridoma colonies. Alternatively, various techniques can be used to increase the yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host. The monoclonal antibodies are then harvested from the ascites or blood. Contaminants can be removed from antibodies using conventional techniques such as chromatography, gel filtration, precipitation, or extraction. For example, antibodies can be purified using standard techniques, such as chromatography with immobilized Protein G or Protein A.

[0078] The test kit of the present invention can take many forms, such as test strips, test kits containing various reagents required for testing, microfluidic chips, etc., and can be manufactured according to standard procedures known to those skilled in the art.

[0079] The kits of the present invention may, as needed, include containers, chips, instructions for use, buffers, immunomodulators and / or other materials, structures and / or reagents required for diagnosis / testing.

[0080] The kit of the present invention is illustrated in the example of using latex to increase the turbidity of the transmission. However, it should not be construed as the kit of the present invention being limited to this.

[0081] The kit of the present invention includes a non-KIV-2 specific antibody derived from an animal immunized with the antigenic peptide of the present invention, and may be present in a manner conventional in the art, for example, in a dissolved or dried form in a container, coated on a solid support (e.g., a membrane, plate, bead (such as magnetic beads), microspheres (such as carboxyl microspheres, amino microspheres, chloromethyl microspheres, physically adsorbed microspheres, etc.), or in a dissolved or dried form in a chip chamber, but the present invention is not limited thereto.

[0082] The antibody of the present invention can be used at a concentration of about 10 to 100 μg / ml.

[0083] Other reactants required for diagnosis / detection in the kit of the present invention include, but are not limited to, anti-lipoprotein (a) antibodies, anti-human antibodies, etc., other than the antibodies of the present invention. These other reactants may be present in a manner conventional in the art, for example, in a dissolved or dried form in a container, coated on a solid support, or in a dissolved or dried form in the chamber of a chip, but the present invention is not limited thereto.

[0084] Other materials required for testing in the kit of the present invention include, but are not limited to, materials for sampling, materials for control, and / or materials for observing the testing process or results.

[0085] Other reagents required for detection in the kit of the present invention include, but are not limited to, detergents, color developers and / or terminators.

[0086] In one embodiment, the antibody in the kit of the present invention is detectably labeled. Any labeling agent and labeling method known to those skilled in the art can be used. Commonly used labeling agents may include enzymes (such as horseradish peroxidase, β-galactosidase, alkaline phosphatase, etc.), radioactive isotopes (such as... 32 P or 125 I) Biotin, digoxigenin, colloidal metals (such as colloidal gold), fluorescent dyes (such as fluorescein, rhodamine, Texas red, etc.), chemiluminescent compounds, or bioluminescent compounds (such as dioxane, luminol, or acridine onione, etc.). Any labeling steps well known in the art can be used, such as covalent coupling of enzymes or biotin groups, iodination, phosphorylation, biotinylation, etc.

[0087] In some implementations, one or more of the other reactants required for detection may also be detectably labeled.

[0088] The present invention also includes a method for detecting the presence of lipoprotein (a) in a subject, the method comprising: contacting a biological sample from the subject with at least one antibody or an antigen-binding fragment thereof of the present invention to determine the presence in the biological sample of a molecule that is naturally present in the sample in a soluble form and has an antigenic determinant that can react with the at least one antibody or an antigen-binding fragment thereof, the contact being performed under conditions and for a time sufficient to detect the binding of the antibody or the antigen-binding fragment thereof to the antigenic determinant.

[0089] In one embodiment, the antibody or its antigen-binding fragment is detectably labeled. In another embodiment, the antibody or its antigen-binding fragment is not detectably labeled, and the detection of the binding of the antibody or its antigen-binding fragment to the antigenic determinant is indirect.

[0090] The present invention also includes a method for detecting the presence of lipoprotein(a) in a subject, the method comprising: contacting a biological sample from the subject with at least one antibody or antigen-binding fragment of the present invention conjugated to microspheres, the contact being performed under conditions and time sufficient to cause the antibody or antigen-binding fragment to specifically bind an antigenic peptide and thereby form a complex that produces turbidity, and thereby detecting the presence of lipoprotein(a).

[0091] The present invention also includes a method for detecting the presence of lipoprotein(a) in a subject, the method comprising: contacting a biological sample from the subject with at least one immobilized first antibody or antigen-binding fragment thereof specific to an antigen peptide of the present invention to determine the presence of the molecule in the sample, the contact being performed under conditions and time sufficient to cause the first antibody or antigen-binding fragment thereof to specifically bind the antigen peptide and thereby form an immune complex; removing components of the sample that do not specifically bind the first antibody; and contacting the immune complex with at least one second antibody or antigen-binding fragment thereof specific to an antigen peptide of the present invention, wherein the antigen-binding site of the second antibody or antigen-binding fragment thereof does not competitively inhibit the antigen-binding site of the immobilized first antibody or antigen-binding fragment thereof, the contact being performed under conditions and time sufficient to detect the specific binding of the second antibody or antigen-binding fragment thereof to the antigen peptide, and thereby detecting the presence of lipoprotein(a). In one embodiment, the immobilized first antibody or antigen-binding fragment thereof is selected from antibodies or antigen-binding fragments thereof that specifically bind to the antigen peptide shown in SEQ ID NO:3. In one embodiment, the second antibody or antigen-binding fragment thereof is selected from antibodies or antigen-binding fragments thereof that specifically bind to the antigen peptide shown in SEQ ID NO:1 or 2.

[0092] The present invention also discloses a method for detecting lipoprotein(a) in a subject, the method comprising: detecting lipoprotein(a) in a biological sample obtained from the subject; contacting the biological sample with an antibody or an antigen-binding fragment of the present invention under conditions and time sufficient to form an antibody / antigen complex; and indicating the presence of the complex.

[0093] In this invention, biological samples include serum and plasma.

[0094] Detection methods include, but are not limited to, autoradiography, turbidimetry, fluorescence microscopy, direct and indirect enzymatic reactions, and radioisotope or non-radioisotope methods. These methods particularly include immunoturbidimetry, latex-enhanced transmission turbidimetry, Western blotting, overlap assays, RIA (radioimmunoassay) and IRMA (immunoradioimmunoassay), GIA (colloidal gold immunoassay), EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescence immunoassay), and CLIA (chemiluminescent immunoassay).

[0095] Based on the study of the complete amino acid sequence of apo(a) (UniProtKB / Swiss-Prot:P08519.1), the inventors discovered that the CYHGDGQSYRGSFSTT fragment in KIV-6 can serve as an antigenic peptide to screen for non-KIV-2 specific lipoprotein(a) antibodies, and this peptide is also present in KIV-7; in addition, it was found that the CYRGDGQSYRGTLSTT fragment in KIV-8 can serve as an antigenic peptide to screen for non-KIV-2 specific lipoprotein(a) antibodies; and the CYHGDGRSYRGISSTT fragment in KIV-9 can serve as an antigenic peptide to screen for non-KIV-2 specific lipoprotein(a) antibodies.

[0096] When used as an antigen, the present invention also provides fusion proteins containing the antigenic peptides of the present invention. The antigenic peptides of the present invention can be coupled to protein carriers (also known as carrier proteins) commonly used in the field of immunology to form fusion proteins, thereby eliciting a more complete immune response. Exemplary protein carriers may be KLH, BSA, or OVA.

[0097] The antigenic peptide of the present invention can exist in multiple copies.

[0098] This invention also relates to nucleic acids encoding antigenic peptides for, for example, the production of the antigenic peptides of this invention or fusion proteins containing antigenic peptides. Nucleic acids encoding the antigenic peptides of this invention include, but are not limited to: the coding sequence of the antigenic peptide itself; the coding sequence of the antigenic peptide and additional coding sequences; the coding sequence of the antigenic peptide (and optionally additional coding sequences) and non-coding sequences, which may additionally include, but are not limited to, one or more regulatory nucleic acid sequences, which may be regulated or adjustable promoters, enhancers, other transcriptional regulatory sequences, repressor-binding sequences, translational regulatory sequences, or any other regulatory nucleic acid sequences. Therefore, nucleic acids encoding antigenic peptides encompass nucleic acids that include only the coding sequence of the peptide as well as nucleic acids that include additional coding and / or non-coding sequences.

[0099] This invention also includes equivalent expression vectors. Suitable nucleic acid sequences can be inserted into any of a variety of known expression vectors suitable for a chosen host cell using various methods. Generally, the nucleic acid sequence is inserted into an appropriate restriction endonuclease site using methods known in the art. Standard techniques used for cloning, isolation, amplification, and purification, involving enzymatic reactions such as DNA ligases, DNA polymerases, and restriction endonucleases, as well as various separation techniques, are all terms known and commonly used by those skilled in the art.

[0100] The antigenic peptides of the present invention can be produced using various expression systems, such as expression vectors and host cells in prokaryotic and eukaryotic expression systems. The following description uses a mammalian expression system as an example. Host cells may include the COS-7 cell line of monkey kidney fibroblasts and other cell lines capable of expressing compatible vectors, such as C127, 3T3, CHO, HeLa, and BHK cell lines. The mammalian expression vector should contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcriptional sequences. DNA sequences derived, for example, from SV40 splicing and polyadenylation sites can be used to provide the desired non-transcriptional genetic elements. The construct can be introduced into host cells using various methods familiar to those skilled in the art, including, but not limited to, calcium phosphate transfection, DEAE-glucan-mediated transfection, or electroporation.

[0101] The antigenic peptide of the present invention may be an unmodified polypeptide or a polypeptide that has been post-translationally modified, for example by glycosylation, phosphorylation, fatty acylation (including glycosylphosphatidylinositol anchoring modification, etc.), phospholipase cleavage (such as hydrolysis mediated by phosphatidylinositol-specific phospholipase C, etc.), protease cleavage, dephosphorylation, or any other type of protein post-translational modification, such as modifications involving the formation or breaking of covalent chemical bonds.

[0102] The embodiments of the present invention will be described in detail below with reference to the examples. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are conventional products that can be obtained commercially.

[0103] Example 1: Prediction of antigen peptide sequences

[0104] The complete amino acid sequence of apo(a) (UniProtKB / Swiss-Prot:P08519.1) was obtained from Genebank. The antigen peptide was predicted based on the inferred three-dimensional structure of KIV. The specific sequence is shown in Table 1 below.

[0105] Table 1

[0106]

[0107] Example 2 Preparation of Antigen Solution

[0108] First, antigenic peptides from the KIV-6 / 7, KIV-8, and KIV-9 regions were synthesized using a polypeptide synthesis method.

[0109] Subsequently, following the specific procedures for carboxyl and amino group coupling described in Bioconjugate Techniques, Elsevier Academic Press, 2nd edition, 2008, pp. 755-763, Chapter 19, the protein carrier KLH was first activated using EDC, and then the antigenic peptides of the KIV-6 / 7, KIV-8, and KIV-9 regions were coupled to the protein carrier respectively. The prepared solution can then be used as the antigen.

[0110] Example 3: Preparation of polyclonal antibodies specifically recognizing KIV-6 / 7

[0111] Immune steps :

[0112] 1) Take 3 adult New Zealand rabbits and label them #A1 to #A3 respectively;

[0113] First immunization: Intradermal injection at multiple points including both sides of the back, the soles of the feet, around the lymph nodes, and behind the ears. The total number of injections per animal is about 8 to 10 points, and the total amount of antigen injected is 10 to 100 μg.

[0114] Second immunization: 10-20 days later, the second immunization is administered by subcutaneous injection at multiple sites, with a total antigen injection volume of 10-100ug.

[0115] Third immunization: The third immunization is administered 10 to 15 days later, via subcutaneous injection, with a total antigen injection volume of 10 to 100 μg.

[0116] Fourth immunization: 7-10 days later, a fourth booster immunization will be administered, via subcutaneous injection, with a total antigen injection volume of 10-100ug.

[0117] Serum obtained after immunizing animals using the above method was tested for potency.

[0118] 1. Immobilize the known antigen onto the plastic well plate, and then wash away any excess antigen.

[0119] 2. Add test samples of different dilution gradients to each well. If the test sample contains the primary antibody to be tested, it will specifically bind to the antigen on the plastic well plate.

[0120] 3. Wash away excess test sample, add secondary antibody containing horseradish peroxidase, and bind it with the primary antibody to be tested;

[0121] 4. Wash away excess unbonded secondary antibody, add enzyme substrate to make the enzyme color, and measure the absorbance (OD450 value) in the plastic tray using an ELISA reader to assess the content of the colored final product, which can then be used to measure the content of the antibody to be tested.

[0122] KIV-6 / 7 antigenic peptide, KIV-2 antigenic peptide, and natural lipoprotein (a) were selected as antigens. Serum that were positive for both KIV-6 / 7 antigenic peptide and natural Lp(a) but negative for KIV-2 antigenic peptide were selected as target antibodies. The results are shown in Table 2 below.

[0123] Table 2

[0124]

[0125] Note: "Potency" is the highest dilution that satisfies the signal value / blank value ≥ 2.1; "Blank" is the average of two repeated measurements of OD450; NC is the negative control; in the "Coating" column, A is KIV-6 / 7 antigenic peptide, B is KIV-2 antigenic peptide and C is lipoprotein (a).

[0126] As shown in Table 2, the polyclonal antibody #A1 has high titer against KIV-6 / 7 antigenic peptides and high titer against lipoprotein(a), but low titer against KIV-2 antigenic peptides.

[0127] The polyclonal antibody #A1 was purified using Protein A affinity according to the purification steps described in the Protein Laboratory Manual (Mark Page and Robin Thorpe, Purification with Protein A or Protein G, Humana Press, 2nd Edition, 2002, Chapter 42: 993-994).

[0128] Example 4: Preparation of a lipoprotein (a) detection kit using polyclonal antibody #A1

[0129] Reagent 1: 0.2M phosphate buffer solution, pH 7.5, 1% BSA;

[0130] Reagent 2: Prepared by chemically coupling the purified polyclonal antibody #A1, which was finally prepared in Example 3, onto carboxyl microspheres using the two-step coupling process described below:

[0131] 1. Take 1 ml of commercial latex microspheres with a particle size of 100-200 nm (concentration 10%) and add them to 0.05 M pH 4.5-7.5 MES buffer. Mix thoroughly for 10 min.

[0132] 2. Add 3 times the molar ratio of carboxyl group activator EDC to the above solution within 5 seconds, and activate for 15-30 minutes;

[0133] 3. Centrifuge at 5000–1000g to remove the supernatant, and resuspend the precipitate in 0.05–0.2M potassium phosphate buffer solution with pH 6.5–8.0;

[0134] 4. Add the saturated amount of antibody to the activated microspheres and stir at medium speed for 2-4 hours;

[0135] 5. After the reaction in step 4, add a blocking agent (0.05-0.2M potassium phosphate buffer solution at pH 8.0, 3g / L BSA, 5g / L arginine and 2g / L Tween 80) to the solution;

[0136] The solution obtained in step 5 was sonicated (power 50%) for 10 minutes. The absorbance was below 10,000. The resulting solution was used as reagent 2.

[0137] Example 5: Methodological Comparison of Polyclonal Antibody #A1 with Commercial Reagents

[0138] To evaluate the effectiveness of the lipoprotein (a) detection kit based on polyclonal antibody #1 prepared in Example 4, it was compared with the methodology of the Roche Cobas 701 system, which is traceable to nmol / L.

[0139] The determination parameters of Roche reagents on the cobas 701 were performed according to the instructions of the Roche Lp(a) kit.

[0140] The process of using the reagent kit of this invention on the Mindray BS-2000 biochemical analyzer is as follows:

[0141] 1) Mix 2 μL of sample with 180 μL of R1 and incubate at 37°C;

[0142] 2) Add 45 μL of R2, mix well, and incubate at 37°C;

[0143] 3) Measurements were performed based on the following parameters.

[0144] Main wavelength: 605nm, secondary wavelength: 800nm, blank time: 18-18, endpoint time: 32-33. Results are as follows. Figure 1 As shown.

[0145] Depend on Figure 1 It can be seen that the lipoprotein (a) detection kit based on polyclonal antibody #A1 and the Roche Cobas 701 system R 2 At 0.9887, the two are highly comparable.

[0146] Example 6 Performance evaluation of polyclonal antibody #A1

[0147] To further investigate whether the lipoprotein (a) detection kit based on polyclonal antibody #1 prepared in Example 4 can meet the reagent performance requirements, the detection method of the kit of the present invention in Example 5 was followed, and 80 international nmol / L reference materials for traceability were used for detection. The test was repeated twice, and the average value was taken. At the same time, the absolute deviation and relative deviation (ratio to the target value of the reference material) were calculated.

[0148] Figure 2 Each point corresponds to a reference material. The distribution of these points shows that the absolute deviation is independent of particle size. Figure 3 It can be seen that when the sample concentration is between 0 and 50 nmol / L, the absolute deviation is within 5 nmol / L; when the sample concentration is between 50 and 100 nmol / L, the absolute deviation is within 10 nmol / L. Figure 4 It can be seen that when the sample concentration is greater than 100 nmol / L, the relative deviation is within 10%. Therefore, the absolute and relative deviations of the detection kit prepared using polyclonal antibody #A1 meet the requirements of reagent performance.

[0149] Example 7: Preparation of polyclonal antibodies that specifically recognize KIV-8

[0150] First, following the immunization steps in Example 3, four New Zealand rabbits were immunized using the KIV-8 antigen solution prepared in Example 2. Then, the serum obtained from the immunized animals was tested according to the titer detection method in Example 3, and the results are shown in Table 3 below.

[0151] Table 3

[0152]

[0153] Note: "Potency" is the highest dilution that satisfies the signal value / blank value ≥ 2.1; "Blank" is the average of two repeated measurements of OD450; NC is the negative control; in the "Coating" column, A is KIV-8 antigenic peptide, B is KIV-2 antigenic peptide and C is lipoprotein (a).

[0154] As shown in Table 3, polyclonal antibodies #B2 and #B4 have high titers against KIV-8 antigenic peptide and lipoprotein(a), but low affinity for KIV-2 antigenic peptide.

[0155] Similarly, according to the protein experiment manual, the polyclonal antibody #B4 was purified using Protein A affinity.

[0156] Example 8: Preparation of a lipoprotein (a) detection kit using polyclonal antibody #B4

[0157] Reagents 1 and 2 for lipoprotein (a) detection were prepared using polyclonal antibody #B4 as described in Example 4.

[0158] Example 9: Methodological Comparison of Polyclonal Antibody #B4 with Commercial Reagents

[0159] To evaluate the effectiveness of the lipoprotein (a) detection kit based on polyclonal antibody #1 prepared in Example 4, its methodology was compared with that of the nmol / L Roche reagent cobas 701 system, according to the method described in Example 5. The results are as follows: Figure 5 As shown.

[0160] Depend on Figure 5 It can be seen that the lipoprotein (a) detection kit based on polyclonal antibody #B4 and the Roche Cobas 701 system R 2 At 0.9924, the two are highly comparable.

[0161] Example 10 Performance evaluation of polyclonal antibody #B4

[0162] To further investigate whether the lipoprotein (a) detection kit based on polyclonal antibody #B4 prepared in Example 8 could meet the reagent performance requirements, the test was performed according to the method in Example 6, and the results are as follows. Figures 6-8 As shown.

[0163] Figure 6 Each point corresponds to a reference material. The distribution of these points shows that the absolute deviation is completely independent of particle size. Figure 7 It can be seen that when the sample concentration is between 0 and 50 nmol / L, the absolute deviation is within 5 nmol / L; when the sample concentration is between 50 and 100 nmol / L, the absolute deviation is basically within 10 nmol / L. Figure 8It can be seen that when the sample concentration is greater than 100 nmol / L, the relative deviation is generally within 10%. Therefore, the absolute and relative deviations of the test kit prepared using polyclonal antibody #B4 can meet the requirements for reagent performance traceability.

[0164] Example 11 Preparation of polyclonal antibodies that specifically recognize KIV-9

[0165] First, following the immunization steps in Example 3, six New Zealand rabbits were immunized using the KIV-9 antigen solution prepared in Example 2. Then, the serum obtained from the immunized animals was tested according to the titer detection method in Example 3, and the results are shown in Table 4 below.

[0166] Table 4

[0167]

[0168]

[0169] Note: "Potency" is the highest dilution that satisfies the requirement of signal value / blank value ≥ 2.1; "Blank" is the average of two repeated measurements of OD450; in the "Coating" column, A is KIV-9 antigenic peptide, B is KIV-2 antigenic peptide and C is lipoprotein (a).

[0170] As shown in Table 4, the polyclonal antibody #C3 has high titer against KIV-9 antigenic peptide and high titer against lipoprotein(a), but low titer against KIV-2 antigenic peptide.

[0171] Similarly, according to the protein experiment manual, the polyclonal antibody #C3 was purified by Protein A affinity.

[0172] Example 12: Preparation of a lipoprotein (a) detection kit using polyclonal antibody #C3

[0173] Reagents 1 and 2 for lipoprotein (a) detection were prepared using polyclonal antibody #C3 as described in Example 4.

[0174] Example 13: Methodological Comparison of Polyclonal Antibody #C3 and Commercial Reagents

[0175] To evaluate the effectiveness of the lipoprotein (a) detection kit based on polyclonal antibody #C3 prepared in Example 12, it was compared with the Roche Cobas 701 system (traceable to nmol / L) following the method described in Example 5. The results are as follows: Figure 9 As shown.

[0176] Depend on Figure 9 It can be seen that the lipoprotein (a) detection kit based on polyclonal antibody #C3 and the Roche Cobas 701 system R2 At 0.991, the two are highly comparable.

[0177] Example 14 Performance evaluation of polyclonal antibody #C3

[0178] To further investigate whether the lipoprotein (a) detection kit based on polyclonal antibody #C3 prepared in Example 12 could meet the reagent performance requirements, the detection was performed according to the method in Example 6, and the results are as follows: Figures 10-12 As shown.

[0179] Figure 10 Each point corresponds to a reference material. The distribution of these points shows that the absolute deviation is independent of particle size. Figure 11 It can be seen that when the sample concentration is between 0 and 50 nmol / L, the absolute deviation is within 5 nmol / L; when the sample concentration is between 50 and 100 nmol / L, the absolute deviation is within 10 nmol / L. Figure 12 It can be seen that when the sample concentration is greater than 100 nmol / L, the relative deviation is within 10%. Therefore, the absolute and relative deviations of the detection kit prepared using polyclonal antibody #C3 meet the requirements of reagent performance.

[0180] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent transformations made using the contents of the present invention under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention. sequence list <110> Shenzhen Mindray Bio-Medical Electronics Co., Ltd. <120> Antigenic peptides of lipoprotein(a), their antibodies and uses <130> <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> artificial synthesis <400> 1 Cys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Ser Phe Ser Thr Thr 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> artificial synthesis <400> 2 Cys Tyr Arg Gly Asp Gly Gln Ser Tyr Arg Gly Thr Leu Ser Thr Thr 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> artificial synthesis <400> 3 Cys Tyr His Gly Asp Gly Arg Ser Tyr Arg Gly Ile Ser Ser Thr Thr 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> artificial synthesis <400> 4 Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr 1 5 10 15

Claims

1. An antigenic peptide of a lipoprotein (a), the amino acid sequence of which is shown in one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

2. A fusion protein comprising at least one of the antigenic peptide and affinity tag polypeptide of claim 1, a protein carrier, or a polypeptide that targets the fusion protein to a predetermined location.

3. A nucleic acid molecule encoding the antigenic peptide of claim 1 or the fusion protein of claim 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A method for obtaining a non-KIV-2 specific anti-lipoprotein (a) antibody, said method using the antigenic peptide of claim 1 or a fusion protein of the antigenic peptide of claim 1 coupled with a protein carrier as an antigen.

7. Use of the antigenic peptide of claim 1 or the fusion protein of claim 2 in the preparation of a kit for a method of assessing cardiovascular disease, the method comprising the steps of: a) Contacting a sample derived from a subject with an antibody or antigen-binding fragment thereof generated by the antigenic peptide of claim 1 or the fusion protein of claim 2; as well as b) Determine whether lipoprotein(a) is present in the sample or determine the level of lipoprotein(a) present in the sample.

8. The use as described in claim 7, wherein, The antibody or its antigen-binding fragment is coated on a solid support, the antibody or its antigen-binding fragment carries a detectable marker, or the antibody or its antigen-binding fragment is not coated on a solid support and does not carry a detectable marker.

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