Monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide and application thereof

Abstract

The invention belongs to the field of antibody preparation and protein detection, and particularly relates to a monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide and application of the monoclonal antibody pair. The monoclonal antibody pair provided by the invention comprises a first antibody and a second antibody, the first antibody comprises a 14F8 antibody or a 8F1 antibody; the second antibody comprises a 6F2 antibody or a 9G1 antibody. According to the present invention, the amino acid sequences of the complementary determining regions CDR1, CDR2 and CDR3 of the light chain and heavy chain variable regions of the monoclonal antibody are determined, the IGFBP2 protein or IGFBP2 polypeptide can be specifically detected, the specificity is strong, the sensitivity is high, the IGFBP2 protein in human serum can be detected, and the basis is provided for the diagnosis and treatment of clinical tumor patients.

Description

Technical Field

[0001] The invention belongs to the field of antibody preparation and protein detection, and particularly relates to a monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide and an application thereof. Background Art

[0002] Insulin-like growth factor binding protein 2 (IGFBP2) is a member of the insulin-like growth factor binding protein (IGFBP) family. IGFBP is composed of an N-terminal domain, a linker, and a C-terminal domain. The special structure of IGFBP allows it to bind to integrins, heparin, and high-affinity insulin-like growth factor (IGF), playing an important role in cell proliferation, differentiation, and human growth and development. Studies have found that IGFBP2 is highly expressed in a variety of malignant tumors such as lung cancer, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, liver cancer, high-grade gliomas, and glioblastomas, and can promote tumor cell proliferation, migration, invasion, and epithelial-mesenchymal transition. In addition, IGFBP2 also plays an important role in neurodevelopment and neurodegenerative diseases. In terms of biological significance, IGFBP2 can be used as a tumor marker for clinical diagnosis and treatment. In the normal human body, the level of IGFBP2 is low, but it is often highly expressed in cancer patients. According to literature reports, the level of IGFBP2 in the serum of healthy people is 385.70±28.47 ng / mL, the level of IGFBP2 in patients with benign tumors is 464.15±67.8 ng / mL, and the level of IGFBP2 in patients with malignant tumors is 706.75±140.30 ng / mL (Fang Xiaolong. Detection and clinical significance of serum IGFBP2 levels in patients with malignant tumors [J]. Medical Animal Prevention, 2004, (12): 776-777). Other literature reports that the level of IGFBP2 in the serum of patients with high-grade gliomas is significantly higher than that in the serum of patients with low-grade gliomas and healthy controls (Lin Y, Jiang T, Zhou K, et al. Plasma IGFBP-2 levels predict clinical outcomes of patients with high-grade gliomas [J]. Neuro-oncology, 2009, 11 (5): 468-476).

[0003] Currently, many domestic and foreign biotechnology companies such as R&D, abcam, Invitrogen and other companies can purchase IGFBP2 protein detection kits, but the price is relatively expensive. There is no report on the capture antibody and detection antibody sequences in the kit. Therefore, it is necessary to independently develop a set of IGFBP2 protein detection antibody pairs and assemble them into a cost-effective detection kit to detect the changes in IGFBP2 levels in patient samples, thereby providing a basis for clinical diagnosis. Summary of the invention

[0004] The purpose of the present invention is to provide a monoclonal antibody pair for detecting IGFBP2 and its application, specifically detecting IGFBP2 protein or IGFBP2 polypeptide, especially detecting IGFBP2 in human serum, plasma and urine, and providing a basis for the diagnosis and treatment of clinical tumor patients.

[0005] The present invention provides a monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide, the monoclonal antibody pair comprising a first antibody and a second antibody; the first antibody comprising a 14F8 antibody or an 8F1 antibody; the second antibody comprising a 6F2 antibody or a 9G1 antibody; the light chain complementary determining region CDR1 of the 14F8 antibody comprises an amino acid sequence as shown in SEQ ID NO.2, the light chain complementary determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.3, and the light chain complementary determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.4; the heavy chain complementary determining region CDR1 of the 14F8 antibody comprises an amino acid sequence as shown in SEQ ID NO.6, the heavy chain complementary determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.7, and the heavy chain complementary determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.8; the light chain complementary determining region CDR1 of the 8F1 antibody comprises an amino acid sequence as shown in SEQ ID NO.2, the light chain complementary determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.3, and the light chain complementary determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO. NO.4; the heavy chain complementary determining region CDR1 of the 8F1 antibody comprises the amino acid sequence shown in SEQ ID NO.10, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.11, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.12; the light chain complementary determining region CDR1 of the 6F2 antibody comprises the amino acid sequence shown in SEQ ID NO.14, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.15, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.16; the heavy chain complementary determining region CDR1 of the 6F2 antibody comprises the amino acid sequence shown in SEQ ID NO.18, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.19, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.20; the light chain complementary determining region CDR1 of the 9G1 antibody comprises the amino acid sequence shown in SEQ ID NO.14, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in SEQ ID NO.15 is included in the light chain complementary determining region CDR3, and the amino acid sequence shown in SEQ ID NO.16 is included in the light chain complementary determining region CDR1 of the 9G1 antibody, and the amino acid sequence shown in SEQ ID NO.22 is included in the heavy chain complementary determining region CDR2 is included in the heavy chain complementary determining region CDR3 is included in the heavy chain complementary determining region CDR3 is included in the amino acid sequence shown in SEQ ID NO.24.

[0006] Preferably, the light chain variable region of the 14F8 antibody comprises the amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence that has at least 75% identity with the sequence as shown in SEQ ID NO.1 and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 14F8 antibody comprises the amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence that has at least 75% identity with the sequence as shown in SEQ ID NO.5 and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the light chain variable region of the 8F1 antibody comprises the amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence that has at least 75% identity with the sequence as shown in SEQ ID NO.1 and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 8F1 antibody comprises the amino acid sequence as shown in SEQ ID NO.9, or an amino acid sequence that has at least 75% identity with the sequence as shown in SEQ ID NO.10, and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide. NO.9, and has an amino acid sequence that is at least 75% identical to the sequence shown in SEQ ID NO.9, and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the light chain variable region of the 6F2 antibody includes the amino acid sequence shown in SEQ ID NO.13, or has at least 75% identity to the sequence shown in SEQ ID NO.13, and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 6F2 antibody includes the amino acid sequence shown in SEQ ID NO.17, or has at least 75% identity to the sequence shown in SEQ ID NO.17, and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the light chain variable region of the 9G1 antibody includes the amino acid sequence shown in SEQ ID NO.13, or has at least 75% identity to the sequence shown in SEQ ID NO.13, and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 9G1 antibody includes the amino acid sequence shown in SEQ ID NO.21, or has at least 75% identity to the sequence shown in SEQ ID NO.22, and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; The amino acid sequence has at least 75% identity to the sequence shown in NO. 21 and has the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide.

[0007] The present invention also provides a nucleic acid molecule, which encodes the monoclonal antibody pair described in the above technical solution.

[0008] Preferably, the sequence of the nucleic acid molecule encoding the light chain variable region of the 14F8 antibody includes the nucleotide sequence shown in SEQ ID NO.25; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 14F8 antibody includes the nucleotide sequence shown in SEQ ID NO.26; the sequence of the nucleic acid molecule encoding the light chain variable region of the 8F1 antibody includes the nucleotide sequence shown in SEQ ID NO.25; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 8F1 antibody includes the nucleotide sequence shown in SEQ ID NO.27; the sequence of the nucleic acid molecule encoding the light chain variable region of the 6F2 antibody includes the nucleotide sequence shown in SEQ ID NO.28; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 6F2 antibody includes the nucleotide sequence shown in SEQ ID NO.29; the sequence of the nucleic acid molecule encoding the light chain variable region of the 9G1 antibody includes the nucleotide sequence shown in SEQ ID NO.28; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 9G1 antibody includes the nucleotide sequence shown in SEQ ID NO.30.

[0009] The present invention also provides a biomaterial, which is a biomaterial for expressing the monoclonal antibody pair described in the above technical solution; the biomaterial includes a carrier or a cell.

[0010] The present invention also provides the use of the monoclonal antibody pair described in the above technical solution, the monoclonal antibody pair encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody pair expressed by the biological material described in the above technical solution in detecting IGFBP2 protein or IGFBP2 polypeptide.

[0011] The present invention also provides the use of the monoclonal antibody pair described in the above technical solution, or the monoclonal antibody pair encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody pair expressed by the biomaterial described in the above technical solution in one or more of the following:

[0012] (1) Preparation of products for detecting IGFBP2 protein or IGFBP2 polypeptide; (2) Preparation of products for diagnosing tumors; (3) Preparation of products for assisting in the treatment of tumors; (4) Preparation of products for distinguishing between benign and malignant tumors.

[0013] The present invention also provides a kit for detecting IGFBP2 protein or IGFBP2 polypeptide, the kit comprising a capture antibody and a detection antibody labeled with a tracer; the detection antibody is the first antibody in the monoclonal antibody pair described in the above technical solution; the capture antibody is the second antibody in the monoclonal antibody pair described in the above technical solution.

[0014] Preferably, the tracer comprises one or more of a radioactive isotope, an enzyme, a fluorescent substance, a luminescent substance, colloidal gold and a colored latex.

[0015] The present invention also provides a method for detecting IGFBP2 protein or IGFBP2 polypeptide, comprising mixing a sample to be detected, a detection antibody labeled with a tracer, and a capture antibody to form a complex; detecting the tracer signal in the complex to determine the concentration of the IGFBP2 protein or IGFBP2 polypeptide in the sample to be detected; the detection antibody is the first antibody in the monoclonal antibody pair described in the above technical solution; and the capture antibody is the second antibody in the monoclonal antibody pair described in the above technical solution.

[0016] Beneficial effects: The monoclonal antibody pair provided by the present invention includes a first antibody and a second antibody; the first antibody includes a 14F8 antibody or an 8F1 antibody; the second antibody includes a 6F2 antibody or a 9G1 antibody. The present invention clarifies the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the light chain and heavy chain variable regions of the monoclonal antibody, and can specifically detect IGFBP2 protein or IGFBP2 polypeptide, with high specificity and high sensitivity, and can detect IGFBP2 in human serum, providing a basis for the diagnosis and treatment of clinical tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 This is the SDS-PAGE test result after IGFBP2 protein purification;

[0019] Figure 2 The results are the determination results of the binding titer of serum antibodies of 6 mice to IGFBP2 protein;

[0020] Figure 3 The titer test results of 8 positive hybridomas are shown;

[0021] Figure 4 The SDS-PAGE test results after purification of some monoclonal antibodies in ascites; wherein, A is the purification result of 6F2 hybridoma supernatant, lane 1 is the starting sample of 6F2 hybridoma supernatant, lane 2 is the flow-through sample, and lanes 3-8 are the antibodies purified from 6F2 hybridoma supernatant; B is the purification result of 14F8 hybridoma supernatant, lane 1 is the starting sample of 14F8 hybridoma supernatant, lane 2 is the flow-through sample, and lanes 3-8 are the antibodies purified from 14F8 hybridoma supernatant; C is the purification result of 12F6 hybridoma supernatant, lane 1 is the starting sample of 12F6 hybridoma supernatant, lane 2 is the flow-through sample, and lanes 3-8 are the antibodies purified from 12F6 hybridoma supernatant;

[0022] Figure 5This is the standard curve for the one-step detection of IGFBP2 protein by the double antibody sandwich ELISA kit. DETAILED DESCRIPTION

[0023] The present invention provides a monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide, the monoclonal antibody pair comprising a first antibody and a second antibody;

[0024] The first antibody includes 14F8 antibody or 8F1 antibody; the second antibody includes 6F2 antibody or 9G1 antibody;

[0025] The light chain complementary determining region CDR1 of the 14F8 antibody comprises the amino acid sequence shown in SEQ ID NO.2, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.3, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.4; the heavy chain complementary determining region CDR1 of the 14F8 antibody comprises the amino acid sequence shown in SEQ ID NO.6, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.8; the light chain complementary determining region CDR1 of the 8F1 antibody comprises the amino acid sequence shown in SEQ ID NO.2, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.3, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.4; the heavy chain complementary determining region CDR1 of the 8F1 antibody comprises the amino acid sequence shown in SEQ ID NO.10, and the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO. The amino acid sequence shown in SEQ ID NO.11, and the heavy chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.12; the light chain complementary determining region CDR1 of the 6F2 antibody includes the amino acid sequence shown in SEQ ID NO.14, the light chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.15, and the light chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.16; the heavy chain complementary determining region CDR1 of the 6F2 antibody includes the amino acid sequence shown in SEQ ID NO.18, the heavy chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.19, and the heavy chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.20; the light chain complementary determining region CDR1 of the 9G1 antibody includes the amino acid sequence shown in SEQ ID NO.14, the light chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.15, and the light chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.16; the heavy chain complementary determining region CDR1 of the 9G1 antibody includes the amino acid sequence shown in SEQ ID NO. The heavy chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.22, the heavy chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.23, and the heavy chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.24. The CDR regions of the first antibody and the second antibody of the present invention are defined by the Kabat numbering scheme, which is based on the location of highly variable regions between sequences of the same domain type.

[0026] In the present invention, the light chain variable region of the 14F8 antibody preferably includes an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.1 and having the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 14F8 antibody preferably includes an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.5 and having the ability to bind to IGFBP2 protein or IGFBP 2 polypeptide. The light chain constant region of the 14F8 antibody of the present invention is preferably a κ chain, and the heavy chain constant region is preferably an IgG1 type. The light chain amino acid sequence of the 14F8 antibody of the present invention is preferably as shown in SEQ ID NO.32, specifically: DIQMT QSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISAATSLETGVPSRFSGSGSGKDYTLSITGLQTEDVATYYCQQYWSTPVTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the heavy chain amino acid sequence of the 14F8 antibody is preferably as shown in SEQ ID NO.33, specifically: QI QLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGIKWMGRINTSTGEPTYADEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRGFYYGNPWYFEYWG QGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0027] In the present invention, the light chain variable region of the 8F1 antibody preferably includes the amino acid sequence shown in SEQ ID NO.1, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.1 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 8F1 antibody preferably includes the amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.9 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide. The light chain constant region of the 8F1 antibody of the present invention is preferably a κ chain, and the heavy chain constant region is preferably an IgG1 type. The light chain amino acid sequence of the 8F1 antibody of the present invention is preferably as shown in SEQ ID NO.32, specifically: DIQMTQSSSY LSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISAATSLETGVPSRFSGSGSGKDYTLSITGLQTEDVATYYCQQYWSTPVTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the heavy chain amino acid sequence of the 8F1 antibody is preferably as shown in SEQ ID NO.34, specifically: QIQLVQSG PELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGIKWMGRINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRGVYYGNPWYFDYWGQGTT LTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISK TKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0028] In the present invention, the light chain variable region of the 6F2 antibody preferably includes the amino acid sequence as shown in SEQ ID NO.13, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO.13 and has the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 6F2 antibody preferably includes the amino acid sequence as shown in SEQ ID NO.17, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO.17 and has the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide. The light chain amino acid sequence of the 6F2 antibody of the present invention is preferably as shown in SEQ ID NO.35, specifically: DILLTQSPAILSVSPGERVSFSCRASQSIGTRIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCHQTNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the heavy chain amino acid sequence of the 6F2 antibody is preferably as shown in SEQ ID NO.36, specifically: QVQLQQSGAELVRPGTSVKISCKASGYAFTNYLLGWVKQRPGHGLEWIGDIYPGSGSTYY NEKFKGKATLTGDKSSSTVYMQLSSLTSEDSAVYFCARKEFLYYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPK VTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPP PKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0029] In the present invention, the light chain variable region of the 9G1 antibody preferably includes the amino acid sequence as shown in SEQ ID NO.13, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO.13 and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 9G1 antibody preferably includes the amino acid sequence as shown in SEQ ID NO.21, or an amino acid sequence that has at least 75% identity with the sequence shown in SEQ ID NO.21 and has the ability to bind to IGFBP2 protein or IGFBP2 polypeptide. The light chain amino acid sequence of the 9G1 antibody of the present invention is preferably as shown in SEQ ID NO.35, specifically: DILLTQSPAILSVSPGERVSFSCRASQSIGTRIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLTINSVESEDIADYYCHQTNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the heavy chain amino acid sequence of the 9G1 antibody is preferably as shown in SEQ ID NO.37, specifically: QVQLQQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIYPGSGTT YYNEKFKGKATLTGDKSSSTVYMQLSSSLTSEDSAVYFCARKDFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTP KVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIP PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0030] In the present invention, the at least 75% identity is preferably any value in the range of 75% to 99%, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% sequence identity.

[0031] In the present invention, the monoclonal antibody pair preferably consists of 14F8 antibody and 6F2 antibody, or preferably consists of 14F8 antibody and 9G1 antibody, or preferably consists of 8F1 antibody and 6F2 antibody, or preferably consists of 8F1 antibody and 9G1 antibody.

[0032] The present invention also provides a nucleic acid molecule, which encodes the monoclonal antibody pair described in the above technical solution.

[0033] In the present invention, the sequence of the nucleic acid molecule encoding the light chain variable region of the 14F8 antibody preferably includes the nucleotide sequence shown in SEQ ID NO.25; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 14F8 antibody preferably includes the nucleotide sequence shown in SEQ ID NO.26.

[0034] In the present invention, the sequence of the nucleic acid molecule encoding the 8F1 light chain variable region preferably includes the nucleotide sequence shown in SEQ ID NO.25; the sequence of the nucleic acid molecule encoding the 8F1 antibody heavy chain variable region preferably includes the nucleotide sequence shown in SEQ ID NO.27.

[0035] In the present invention, the sequence of the nucleic acid molecule encoding the light chain variable region of the 6F2 antibody preferably includes the nucleotide sequence shown in SEQ ID NO.28; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 6F2 antibody preferably includes the nucleotide sequence shown in SEQ ID NO.29.

[0036] In the present invention, the sequence of the nucleic acid molecule encoding the 9G1 light chain variable region preferably includes the nucleotide sequence shown in SEQ ID NO.28; the sequence of the nucleic acid molecule encoding the 9G1 antibody heavy chain variable region preferably includes the nucleotide sequence shown in SEQ ID NO.30.

[0037] The invention has no critical requirements on the origin of the nucleic acid molecule, and can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example by combining chemical synthesis and recombinant methods.

[0038] The present invention also provides a biomaterial, which can express the monoclonal antibody pair described in the above technical solution; the biomaterial includes a carrier or a cell.

[0039] In the present invention, the vector includes a basic vector and the nucleic acid molecule described in the above technical solution inserted into the basic vector, or a variant of the nucleic acid molecule described in the above technical solution inserted into the basic vector. The present invention has no strict requirements on the source of the vector, and any vector that can replicate and / or express polynucleotides in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells and bacterial cells (such as Escherichia coli (E. coli)) can be used, such as pBAD, pQE-12, pGEX, pBluescript, pET-series expression vectors, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, p TRE, pCAL-n-EK, pESP-1, pOP13CAT, pREP, pCEP4, pMC1neo, pXT1, pSG5, EBO-pSV2neo, pBPV-1, pFUSE, pRSVgpt, pRSVneo, pIZD35, pRc / CMV, pcDNA1, pcDNA3.1, pSPORT1, pGEMHE, pLXIN, pSIR, pIRES-EGFP, pEAK-10, pTriEx-Hygro, pCINeo, pAO815, pPIC9K and pPIC3.5K, etc. Variants of the nucleic acid molecules of the present invention may be naturally occurring variants, such as naturally occurring allelic variants, or may be non-naturally occurring variants, which may be prepared by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.

[0040] In the present invention, the cell preferably includes a host cell and the vector described in the above technical solution introduced into the host cell. The host cell described in the present invention preferably includes prokaryotic cells and eukaryotic cells, including but not limited to bacterial cells, such as Escherichia coli; fungal cells, such as Pichia pastoris, Saccharomyces cerevisiae, yeast cells; insect cells, such as Sf9 cells, Sf21 cells, Tn-368 cells, High Five cells; animal cells, such as CHO cells, COS, NSO, 293T, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), Expi293F, PERC.6 (human retinal cells); plant cells. The present invention has no strict requirements on the source of the host cell, and any cell known to those skilled in the art that can be used as a mammalian host cell can be used.

[0041] The present invention also provides the use of the monoclonal antibody pair described in the above technical solution or the monoclonal antibody pair encoded by the nucleic acid molecule described in the above technical solution or the monoclonal antibody pair expressed by the biomaterial described in the above technical solution in detecting IGFBP2 protein or IGFBP2 polypeptide or polypeptide. In the present invention, the method for detecting IGFBP2 protein or IGFBP2 polypeptide or polypeptide can be any known immunological detection technology, including radioimmunoassay, enzyme-labeled immunoassay, fluorescent-labeled immunoassay, chemiluminescent-labeled immunoassay, colloidal gold-labeled immunoassay technology, and colored latex-labeled immunoassay.

[0042] The present invention also provides the use of the monoclonal antibody pair described in the above technical solution, the monoclonal antibody pair encoded by the nucleic acid molecule described in the above technical solution, or the monoclonal antibody pair expressed by the biological material described in the above technical solution in one or more of the following: (1) preparing a product for detecting IGFBP2 protein or IGFBP2 polypeptide; (2) preparing a product for diagnosing tumors; (3) preparing a product for auxiliary treatment of tumors; (4) preparing a product for distinguishing benign tumors from malignant tumors.

[0043] In the present invention, the disease preferably includes one or more of lung cancer, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, liver cancer, high-grade glioma, glioblastoma and neurodegenerative diseases. The product of the present invention is preferably a kit, and more preferably a double antibody sandwich ELISA kit.

[0044] The present invention also provides a kit for detecting IGFBP2 protein or IGFBP2 polypeptide, the kit comprising a capture antibody and a detection antibody labeled with a tracer; the detection antibody is the first antibody in the monoclonal antibody pair described in the above technical solution; the capture antibody is the second antibody in the monoclonal antibody pair described in the above technical solution.

[0045] In the present invention, the tracer preferably includes one or more of radioisotopes, enzymes, fluorescent substances, luminescent substances, colloidal gold and colored latex, and more preferably radioisotopes, enzymes, fluorescent substances, luminescent substances, colloidal gold and colored latex; the radioisotopes include 3 H. 14 C. 32 P. 125 I. 131 I. 57 Co and 51Cr; the enzyme preferably includes one or more of β-galactosidase, β-glucosidase, alkaline phosphatase, horseradish peroxidase and malate dehydrogenase; the fluorescent substance preferably includes fluorescamine or fluorescein isothiocyanate; the luminescent substance preferably includes luminol, luminol derivatives, luciferin, lucigenin, acridinium ester, peroxy oxalate and dioxetanes; the colored latex is latex particles polymerized by polyethylene.

[0046] In the present invention, the kit preferably further comprises one or more of a microporous reaction plate, a sample treatment solution, a concentrated washing solution, a positive control, a negative control, a color developing solution and a stop solution. The capture antibody of the present invention is coated on a solid phase carrier, and the solid phase carrier can be a microporous plate, a magnetic bead, a latex particle, a membrane (nylon membrane, a nitrocellulose membrane or a PVDF membrane).

[0047] In the present invention, the capture antibody is preferably coated on the microporous reaction plate. The material of the microporous reaction plate of the present invention is preferably polystyrene. The present invention preferably adsorbs the capture antibody onto a solid support microporous reaction plate, blocks the microporous reaction plate with a blocking protein (e.g., skim milk powder, bovine serum albumin, etc.), allows the capture antibody to contact and incubate with the solid phase, removes the unreacted sample, and obtains a microporous reaction plate coated with the capture antibody.

[0048] In the present invention, the sample treatment solution preferably includes ethylenediaminetetraacetic acid, and more preferably a 4wt.% ethylenediaminetetraacetic acid solution; the pH value of the ethylenediaminetetraacetic acid solution is preferably 7. The concentrated washing solution of the present invention preferably includes a PBS solution of Tween-20, and more preferably a 20×PBS solution containing 2v / v% Tween-20. The positive control of the present invention preferably includes IGFBP2 protein or IGFBP2 polypeptide; the amino acid sequence of the IGFBP2 protein is preferably as shown in SEQ ID NO.31, specifically: ML PRVGCPALPLPPPPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ. The negative control of the present invention preferably includes PBS with 0.1v / v% Tween-20. The color developing solution of the present invention preferably includes color developing solution A and color developing solution B; the color developing solution A preferably includes sodium acetate, citric acid, hydrogen peroxide and distilled water, and further preferably includes 13.6g / 500mL, citric acid 1.6g / 500mL, 30% hydrogen peroxide 0.3mL / 500mL and the balance of distilled water; the color developing solution B preferably includes disodium ethylenediaminetetraacetate, citric acid, glycerol, tetramethylbenzidine, DMSO and distilled water, and further preferably includes disodium ethylenediaminetetraacetate 0.2g / 500mL, citric acid 0.95g / 500mL, glycerol 50mL / 500mL, tetramethylbenzidine 0.15g / 500mL, DMSO 3mL / 500mL and the balance of distilled water. The stop solution of the present invention preferably includes H2SO4.

[0049] The present invention also provides a method for detecting IGFBP2 protein or IGFBP2 polypeptide, comprising mixing a sample to be detected, a detection antibody labeled with a tracer, and a capture antibody to form a complex; detecting the tracer signal in the complex to determine the concentration of the IGFBP2 protein or IGFBP2 polypeptide in the sample to be detected; the detection antibody is the first antibody in the monoclonal antibody pair described in the above technical solution; and the capture antibody is the second antibody in the monoclonal antibody pair described in the above technical solution.

[0050] In the present invention, the method of mixing the sample to be detected, the detection antibody labeled with a tracer and the capture antibody to form a complex preferably includes: mixing the sample to be detected and the detection antibody labeled with a tracer to obtain a detection antibody-IGFBP2 antigen complex; mixing the detection antibody-IGFBP2 antigen complex and the capture antibody to obtain a detection antibody-IGFBP2 antigen-capture antibody complex; detecting the tracer signal in the detection antibody-IGFBP2 antigen-capture antibody complex to determine the concentration of IGFBP2 protein or IGFBP2 polypeptide in the sample to be detected; or, the method of mixing the sample to be detected, the detection antibody labeled with a tracer and the capture antibody to form a complex preferably includes: mixing the sample to be detected and the capture antibody to obtain a capture antibody-IGFBP2 antigen complex; mixing the capture antibody-IGFBP2 antigen complex and the detection antibody labeled with a tracer to obtain a capture antibody-IGFBP2 antigen-detection antibody complex; detecting the tracer signal in the capture antibody-IGFBP2 antigen-detection antibody complex to determine the concentration of IGFBP2 protein or IGFBP2 polypeptide in the sample to be detected. The present invention can determine the concentration of IGFBP2 in a sample to be detected based on the signal generated by the tracer marker on the detection antibody, and has strong specificity and high sensitivity.

[0051] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art.

[0052] To further illustrate the present invention, a monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] Preparation of monoclonal antibodies

[0055] 1. Preparation of recombinant protein IGFBP2

[0056] The gene sequence of human IGFBP2 was obtained from the GenBank sequence database, with the sequence number NM_000597.3. The gene sequence was synthesized by Jinweizhi Company and inserted between Nhe I and NotI of the pcDNA3.1 vector to obtain the pcDNA3.1-IGFBP2 recombinant vector. The pcDNA3.1-IGFBP2 recombinant vector was transferred into Expi293F cells (thermo) using the transfection reagent PEI (thermo). Expi293F cells were cultured using OPM medium (Cat. No.: P82019). Plasmid transfection and subsequent cell culture processes were performed according to the instructions of the OPM-CD 293 transient transfection system. On the third day after transfection, the culture supernatant was collected by centrifugation and purified using Ni-NTA (QIAGEN) to obtain the IGFBP2 recombinant protein, which was then tested by SDS-PAGE. The results showed that the IGFBP2 protein that can be used to immunize mice was successfully prepared in this example ( Figure 1 ).

[0057] 2. Mouse Antigen Immunization

[0058] The IGFBP2 recombinant protein prepared as above was used as an antigen to immunize 5 Balb / c mice, and a total of four immunizations were performed. In the first immunization, the antigen was mixed with equal volumes of Freund's adjuvant, fully ground into a water-in-oil emulsion, and 100 μL (40 μg antigen) of the mixed antigen was injected intraperitoneally; two weeks after the first immunization, the second immunization was carried out, 40 μg antigen was mixed with incomplete Freund's adjuvant at a volume ratio of 1:1, and 100 μL (40 μg antigen) of the mixed antigen was injected into the right hind calf muscle; the third immunization was carried out two weeks after the second immunization, and the dosage, method, and route of antigen injection were the same as those of the second immunization. After an interval of 2 weeks, 100 μL (40 μg) of antigen without adjuvant was injected intraperitoneally for booster immunization. Three days later, tail blood was collected and its titer was tested by ELISA. The specific detection process was as follows: the purified IGFBP2 recombinant protein was coated with carbonate buffer with a pH value of 9.6 at 4°C overnight, 100 ng / well; the next day, PBST was washed 3 times, each time for 3 minutes, and patted dry; 2% BSA was fully blocked and incubated at 37°C for 1 hour; PBST was washed 3 times, each time for 3 minutes, and patted dry ; The serum of 6 mice (5 immunized mice and 1 normal mouse) was diluted with PBS in multiple ratios, at 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, 1:109350, 100 μL was added to each well, incubated at 37°C for 1 hour, washed 3 times with PBST, 3 minutes each time, and patted dry; goat anti-mouse IgG-HRP was diluted 1:5000 and incubated at 37°C for 30 minutes. After washing 3 times with PBST and patting dry, TMB color development was performed for 10 minutes, 2M H2SO4 was terminated, and the absorbance value was measured at 450nm. According to the test results ( Figure 2 ), based on the titer and specificity data of serum antibodies, mouse No. 3 was selected for subsequent experiments.

[0059] 3. Cell fusion and hybridoma culture

[0060] Immunization was enhanced by intraperitoneal injection of antigens. The animals were killed 3 days later and spleen cells were obtained for cell fusion. A 37°C water bath was prepared in a clean bench. 5-6×10 7 Mouse myeloma cells (SP2 / 0) in logarithmic growth phase and in good growth condition and spleen cells were added to a 50mL centrifuge tube at a ratio of 1:10 and mixed. Centrifuge at 500g for 10min, aspirate the supernatant, flick the bottom of the centrifuge tube to loosen the cell pellet slightly; slowly drip 1mL of 45% PEG1450 solution pre-warmed to 37℃ within 90s; and shake the centrifuge tube gently continuously; the centrifuge tube was in a 37℃ water bath during the whole process. Then gradually add DMEM culture medium to the cell mixture, drip 1mL dropwise in the first minute, add 2mL in the second minute, add 3mL in the third minute, add 4mL in the fourth minute, add 5mL in the fifth minute, and shake while adding in a 37℃ water bath. Then incubate at 37℃ for 15min, centrifuge at 500g for 5min, and remove the supernatant. Add 5 mL of DMEM medium containing HAT (thermo, catalog number: 21060017), gently suspend the precipitated cells, and finally add DMED medium containing HAT to about 100 mL. Dispense into a 96-well cell culture plate with macrophages, 100 μL / well, and then place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0061] 4. Screening of positive hybridomas

[0062] ELISA was used to select positive clones. The growth of hybridoma cells was observed. After seven days, when the cell culture supernatant turned yellow, an appropriate amount of cell supernatant was taken for ELISA antibody detection. Based on the ELISA results, OD 450 The clones with high OD values, which are more than twice higher than the negative control, are plated on 96-well plates for the first subclone screening; ELISA is performed to detect antibodies 7 to 10 days later, and the OD values ​​are selected. 450 The clones with the highest OD values ​​were plated on 96-well plates for the second subclone screening, so that there was about 1 cell in each well; after culturing for 7 to 10 days, ELISA was performed to detect antibodies and the OD values ​​were selected. 450 The clones with the highest OD were plated in 96-well plates for the third subclone screening, with about 1 cell per well. The dilution ratio of cell supernatant antibody detection was 1:50. The sample incubation, washing, secondary antibody incubation, washing, color development and termination processes were the same as those in Example 1. After three screenings, a total of 8 clones with OD 450The positive clones with high values ​​were named as 1B6, 6F2, 6H6, 8F1, 9G1, 12F6, 14F8 and 14F10. The supernatants of the eight positive hybridomas were diluted 5 times, 25 times, 125 times, 625 times and 3125 times respectively, and ELISA was performed using 100 ng / well IGFBP2 protein. The results showed that the eight positive clones screened out could be detected after dilution of 625 times, and all had good binding ability with IGFBP2 protein ( Figure 3 ).

[0063] 5. Preparation of monoclonal antibodies from ascites and purification of monoclonal antibodies

[0064] Inject 300 μL of ascites adjuvant into the peritoneal cavity of 12-week-old Balb / c mice. After 2 weeks, culture the hybridoma cells until the cell activity is optimal and adjust the cell number to about 1×10 6 100 μL of positive hybridoma cells were inoculated into the peritoneal cavity of mice injected with ascites adjuvant, and ascites was collected 7 to 10 days later. The ascites was diluted with PBS with a pH value of 7.4, centrifuged and the supernatant was purified by protein G affinity chromatography (Cytiva), and tested by SDS-PAGE. Some of the results are shown in Figure 4 shown.

[0065] Example 2

[0066] Characterization of monoclonal antibodies

[0067] 1. Monoclonal antibody typing and identification

[0068] The antibody typing was identified by indirect ELISA. The IGFBP2 antigen was coated and blocked with 2% BSA, then incubated with the monoclonal antibody purified in step 5 of Example 1 at 37°C for 1 hour, and then incubated with different types of HRP-labeled rabbit anti-mouse (total IgG, IgG1, IgG2a, IgG2b, IgG3, IgM, IgG kappa chain, IgG lambda chain) secondary antibodies diluted 1:5000 at 37°C for 30 minutes. After washing with PBST three times, TMB was used for color development for 5 minutes. The detection results are shown in Table 1.

[0069] Table 1 Monoclonal antibody typing results

[0070]

[0071] 2. Identification of monoclonal antibody antigen epitopes

[0072] 2.1 Construction of IGFBP2 deletion mutant

[0073] By artificial synthesis, the IGFBP2 gene (NCBI sequence number: NM_002515.3) lacking amino acids 37-65, 66-94, 95-123, 124-152, 153-181, 182-210, 211-239, 240-268, 269-297, and 298-325 was synthesized into pCDNA3.1, and the restriction sites were Nhe I and Not I. I. The recombinant vectors were named pCDNA3.1-△37-65, pCDNA3.1-△66-94, pCDNA3.1-△95-123, pCDNA3.1-△124-152, pCDNA3.1-△153-181, pCDNA3.1-△182-210, pCDNA3.1-△211-239, pCDNA3.1-△240-268, pCDNA3.1-△269-297, and pCDNA3.1-△298-325, respectively. Each recombinant vector was transferred into cloning bacteria, extracted and sequenced, and then used for later use.

[0074] 2.2 Preparation of cell slides and antibody immunofluorescence staining of each deletion mutant

[0075] (1) 293T cell culture and transfection: DMEM high-glucose medium and FBS were prepared into 10% FBS-DMEM high-glucose medium at a volume ratio of 9:1. When the cells were confluent, they were subcultured at a ratio of 1:5-1:6 to a 10 cm culture dish with a 6 cm × 6 cm cell slide, and cultured overnight in a cell culture incubator at 37°C and 5% CO2. When the cell density reached 30%-40%, the above 10 recombinant plasmids and the control plasmid pCDNA3.1 were respectively transferred into the cells using PEI transfection reagent (the transfection reagent, plasmid dosage and experimental operation process were carried out according to the instructions of Thermo Company);

[0076] (2) Slide fixation and antibody staining

[0077] The cells grown for 48 h were washed twice with PBS, fixed with acetone at 4°C for 5 min, and then washed twice with PBS and dried. After the cell slides were dried, 11 types of 6 cm × 6 cm cell slides were cut into 0.25 cm × 0.25 cm cell slides and pasted on the same glass slide for later use.

[0078] The obtained cell slide was washed with PBST; 8 kinds of monoclonal antibodies purified in step 5 of Example 1 were added to the slide and incubated at room temperature for 1 hour; washed 3 times with PBST, each time for 5 minutes; FITC-labeled anti-mouse secondary antibody (manufacturer and product number: 515-095-003) was added and incubated at room temperature for 40 minutes; washed 3 times with PBST, each time for 5 minutes; the results were observed under a microscope and the experimental data were statistically analyzed. The results are shown in Table 2.

[0079] Table 2 Identification results of monoclonal antibody recognition epitopes

[0080]

[0081] Note: “-” means that the monoclonal antibody does not bind to the overexpressed protein lacking the nucleic acid sequence, and “+” means that the monoclonal antibody binds to the overexpressed protein lacking the nucleic acid sequence.

[0082] It can be seen from Table 2 that the recognition epitopes of monoclonal antibodies 12F6 and 14F10 are the same, and the recognition epitopes of the remaining antibodies are different.

[0083] Example 3

[0084] Screening of the best monoclonal antibody pairing in double antibody sandwich ELISA

[0085] Using the antibodies purified from ascites in step 5 of Example 1, a set of matrix format experiments was conducted to select the most suitable monoclonal antibody pairs for capture and detection in the sandwich ELISA method. That is, the 8 purified antibodies were used as capture antibodies to coat a 96-well plate, and the capture antibody coating amount was 200 ng / well. The IGFBP2 recombinant protein purified in step 1 of Example 1 was used as an antigen, and the antigen incubation amount was 40 ng / well. The antibody pairs were screened, and the 8 antibodies were labeled with horseradish peroxidase as detection antibodies (named HRP-1B6, etc.), and the detection antibody incubation amount was 200 μL / well (the detection antibody concentration was 1 mg / mL, and it was used after dilution 5000 times). The matrix format was used to pair with each antibody. The control group used the supernatant of overexpressed empty pCDNA3.1 as the capture antibody, and the supernatant of overexpressed empty pCDNA3.1 labeled with HRP as the detection antibody. According to the OD measured in each well 450 The capture antibody and detection antibody in sandwich ELISA were quickly screened based on the values. The results are shown in Table 3.

[0086] Table 3 Results of antibody pairing screening by double antibody sandwich ELISA

[0087]

[0088] Note: Positive well OD 450nm Value / negative well OD 450nmThe antibody pair corresponding to the maximum value (P / N) is the optimal antibody pair. The underlined bold values ​​correspond to the best paired coating antibody and enzyme-labeled antibody.

[0089] According to Table 3, it can be seen that monoclonal antibody 9G1 is used as a capture antibody and labeled monoclonal antibody 14F8 is used as a detection antibody, which is the best paired antibody; monoclonal antibody 9G1 is used as a capture antibody, labeled monoclonal antibody 8F1 is used as a detection antibody, monoclonal antibody 6F2 is used as a capture antibody, labeled monoclonal antibody 8F1 is used as a detection antibody, monoclonal antibody 6F2 is used as a capture antibody, and labeled monoclonal antibody 14F8 is used as a detection antibody, and its P / N value is also high, which can be used as an antibody pair for detecting GFBP2 protein. Therefore, the present application provides four pairs of antibody pairs that can be used to detect GFBP2 protein.

[0090] Example 4

[0091] Identification of anti-IGFBP2 monoclonal antibody sequence

[0092] The positive hybridomas 6F2, 8F1, 9G1, and 14F8 were grown to a certain scale (cell number > 3×10 6 After that, the cells were lysed with Trizol, and the total RNA of the hybridoma cell lysate was extracted using the Quick-RNA MicroPrep Kit. The RNA was separated by agarose gel electrophoresis, and then 5'RACE was performed using the Clontech SMARTer RACE 5' / 3'kit to obtain cDNA, which was used as a template. Max DNA Polymerase was used for PCR amplification to extract the antibody V region cDNA, which was then sequenced by a sequencing company. The Kabat method was used to mark the CDR region of the antibody amino acid sequence. The specific sequence information is shown in Table 4.

[0093] Table 4 Anti-IGFBP2 monoclonal antibody sequences

[0094]

[0095]

[0096]

[0097] Example 5

[0098] Double antibody sandwich ELISA kit for detecting IGFBP2

[0099] 1. Composition of the double antibody sandwich ELISA kit for detecting IGFBP2

[0100] (1) Microwell reaction plate coated with 100 ng / well capture antibody 6F2 or 9G1;

[0101] The preparation method of the microporous reaction plate coated with the capture antibody 6F2 or 9G1 is as follows: the capture antibody 6F2 or 9G1 is diluted to 1 μg / mL with 50 mM carbonate buffer (pH 9.6), and 0.1 mL / well is used to coat the polystyrene well plate, and the plate is kept at 4°C overnight. After patting dry, 0.3 mL of 0.25% casein (Sigma) blocking solution is added to each well, and the plate is kept at 4°C overnight to block non-specific binding sites. The plate strips are spun dry, vacuum dried for 12 to 24 hours, and the microporous reaction plate coated with the capture antibody 6F2 or 9G1 is obtained, and the plate is vacuum packed in an aluminum film bag and stored at 4°C for future use;

[0102] (2) Sample treatment solution: 4 wt.% EDTA (pH 7.0), i.e., weigh 40 g EDTA, dissolve in 1 L distilled water, adjust pH to 7.0 with NaOH, and store at 4°C;

[0103] (3) Enzyme conjugate: horseradish peroxidase-labeled detection antibody 8F1 or 14F8;

[0104] The preparation method of the enzyme conjugate is as follows: 5 mg horseradish peroxidase is dissolved in 1 mL distilled water, 0.2 mL freshly prepared 0.1 M sodium periodate is added for 30 min, placed in 1 mM sodium acetate buffer at pH 4.4, dialyzed at 4°C overnight, 20 μL 0.2 M carbonate buffer at pH 9.5 is added the next day, 10 mg detection antibody 8F1 or 14F8 dialyzed in 0.01 M carbonate buffer at pH 9.5 is added, and the mixture is heated to room temperature. Stir gently for 2-3 hours at a warm temperature and away from light, then add 0.1 ml of newly prepared 4 mg / ml sodium borohydride, keep at 4°C and away from light overnight, add an equal volume of saturated ammonium sulfate dropwise while stirring in an ice bath and away from light (adjust the pH value of ammonium sulfate to 7.0-7.2 with ammonia water before use), place at 4°C for 6 hours, centrifuge at 10000g, 4°C for 30 minutes, discard the supernatant, dissolve the precipitate in an appropriate amount of 10 mM phosphate buffer, dialyze with 10 mM phosphate buffer at 4°C overnight, and change the solution three times. Collect the conjugate, add a final concentration of 2% BSA and a concentration of 50% glycerol protective agent, and store at 4°C for use.

[0105] (4) Concentrated washing solution: 20×PBS containing 2% Tween-20, i.e., 1L of solution contains 4.56gNaH2PO4, 58.02gNa2HPO4·12H2O, 175.3gNaCl. After autoclaving at 121°C for 20min, add 20ml Tween-20 and mix well. Dilute 20 times before use.

[0106] (5) Positive control: recombinant IGFBP2 protein at a concentration of 20 ng / μL;

[0107] (6) Negative control: 10 mM pH 7.4 PBS containing 0.1% Tween-20, i.e., 1 L of the solution contains 4.56 g NaH2PO4, 58.02 g Na2HPO4·12H2O, and 175.3 g NaCl, sterilized by high pressure at 121°C for 20 min, and after 20-fold dilution, Tween-20 was added to make the final concentration 0.1%;

[0108] (7) Color developing solution: It is composed of color developing solution A and color developing solution B. When using, take equal amounts of the two and mix them evenly. The components of color developing solution A and B are as follows:

[0109] Color developing solution A: 13.6 g sodium acetate, 1.6 g citric acid, 0.3 mL 30% hydrogen peroxide, add distilled water to 500 mL, and store in a dark place;

[0110] Color development solution B: 0.2g disodium ethylenediaminetetraacetate, 0.95g citric acid, 50mL glycerol, 0.15g tetramethylbenzidine (TMB) dissolved in 3mL DMSO, add distilled water to 500mL, and store away from light. When using, take an equal amount of AB solution and mix well before use, and store away from light.

[0111] (8) Stop solution: 2M H2SO4.

[0112] 2. How to use the double antibody sandwich ELISA kit for detecting IGFBP2

[0113] (1) Sample addition:

[0114] ① One-step detection: Use sample treatment solution to dilute the sample to be tested at 1:100, mix it with horseradish peroxidase-labeled detection antibody 8F1 or 14F8 (antibody concentration is 1 mg / mL, 1:2500 dilution) in equal volumes, add 200 μL to the microwell reaction plate coated with capture antibody 6F2 or 9G1, set up 2 replicate wells for each sample, set up negative control and positive control at the same time, and incubate at 37°C for 1 hour;

[0115] ② Two-step detection: Use sample treatment solution to dilute the sample to be tested at 1:100, take 200μL in the microwell reaction plate coated with capture antibody 6F2 or 9G1, set up 2 duplicate wells for each sample, and set up negative control and positive control at the same time, incubate at 37℃ for 1h, wash the strips after 20-fold dilution with concentrated washing solution, wash the plate 4 times, add 100μl 1:5000 diluted enzyme conjugate (horseradish peroxidase labeled detection antibody 8F1 or 14F8) to each well, and incubate at 37℃ for 30min;

[0116] (2) Washing: Wash the strips 4 times after diluting the concentrated washing solution 20 times;

[0117] (3) Color development: After washing the plate, add color development solution (color development solution A and B are mixed in equal amounts and prepared before use), 100 μl / well, incubate at room temperature in the dark for 10 min, then add stop solution, 50 μl / well, to terminate the reaction.

[0118] 3. Determination of sensitivity and specificity of double antibody sandwich ELISA kit for detecting IGFBP2

[0119] (1) Sensitivity measurement

[0120] The purified IGFBP2 protein (concentration of 1 mg / mL) was diluted with sample treatment solution at different multiples to a final concentration of 10000 pg / mL, 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, and 156.25 pg / mL, respectively, as samples to be tested, and tested according to the one-step detection steps in step 2, and a standard curve was established; at the same time, the human IGFBP2 ELISA detection kit of R&D Company (Cat. No.: DGB200) was used as a control kit for comparison (refer to the instructions of R&D Company for the experiment), and the results are shown in Tables 5 and 6. Figure 5 shown.

[0121] Table 5 Sensitivity test results of double antibody sandwich ELISA kit for one-step detection of IGFBP2 protein

[0122]

[0123] According to Table 5 and Figure 5 It can be seen that the correlation coefficient of the standard curve established by the one-step detection of IGFBP2 protein using the double antibody sandwich ELISA kit of the present invention is 0.9996. When the IGFBP2 protein concentration is 156.25 pg / mL, it can still be detected. According to the instructions provided by the R&D company, the IGFBP2 ELISA detection kit can still detect when the IGFBP2 protein concentration is 312.5 pg / mL. The double antibody sandwich ELISA kit prepared in this application has good sensitivity in detecting IGFBP2 protein.

[0124] (2) Specificity determination

[0125] The gene sequences of human IGFBP1 (sequence number NM_000596.4), human IGFBP3 (sequence number NM_000598.5), human IGFBP4 (sequence number NM_001552.3), human IGFBP5 (sequence number NM_000599.4), and human IGFBP6 (sequence number NM_002178.3) were found in the GenBank sequence database, and the above gene sequences were synthesized by Jinweizhi Company and cloned into the pcDNA3.1 vector. The insertion sites of the gene sequence of human IGFBP6 were BamH I and Not I, and the insertion sites of the other four genes were Nhe I and Not I, and the recombinant vectors pCDNA3.1-IGFBP1, pCDNA3.1-IGFBP3, pCDNA3.1-IGFBP4, pCDNA3.1-IGFBP5, and pCDNA3.1-IGFBP6 were obtained;

[0126] After the constructed recombinant plasmid was sequenced correctly, the plasmid was transfected and purified in the manner described in Example 1. The purified IGFBP1 protein (concentration of 1 mg / mL), IGFBP3 protein (concentration of 1 mg / mL), IGFBP4 protein (concentration of 1 mg / mL), IGFBP5 protein (concentration of 1 mg / mL), and IGFBP6 protein (concentration of 1 mg / mL) were diluted to a concentration of 10 ng / mL using sample treatment solution, respectively, as samples to be tested, and tested according to the one-step detection step in step 2, and no obvious cross-reactivity was observed. Therefore, the double antibody sandwich ELISA kit for detecting IGFBP2 provided by the present invention has good specificity.

[0127] 4. Repeatability Experiment

[0128] Three different concentrations of IGFBP2 protein and three portions of human serum were taken and labeled as 1, 2, 3, 4, 5, and 6, respectively. Three replicates were performed in each group. The detection was performed according to the one-step method described in the method for using the ELISA kit in this example. The coefficient of variation was calculated and the intra-batch and inter-batch stability was evaluated. The results are shown in Table 6.

[0129] Table 6 Repeatability test results

[0130]

[0131] As shown in Table 6, when the established ELISA method was used for detection, when the test sample was protein, the intra-batch coefficient of variation was 0.9%-4.1%, when the test sample was serum, the intra-batch coefficient of variation was 7.0%-9.6%, and the overall intra-batch coefficient of variation was 0.9%-9.6%; when the test sample was protein, the inter-batch coefficient of variation was 1.5%-8.9%, when the test sample was serum, the inter-batch coefficient of variation was 8.5%-9.7%, and the overall inter-batch coefficient of variation was 1.5%-9.7%; both the intra-batch and inter-batch coefficients of variation were less than 10%, indicating that the method had good repeatability.

[0132] 5. Standard sample recovery experiment of double antibody sandwich ELISA kit for detecting IGFBP2 protein

[0133] (1) Take 1 μL of serum or cerebrospinal fluid samples from three healthy subjects, add a certain amount of IGFBP2 protein with a concentration of 100 ng / mL and an appropriate amount of sample treatment solution, make up the total volume to 100 μL, and then mix with 100 μL of 1:2500 diluted enzyme conjugate (horseradish peroxidase-labeled detection antibody 8F1, antibody concentration is 1 mg / mL), and the total system is 200 μL. The final concentration of serum or cerebrospinal fluid in the reaction system is 1:200, and the final concentration of IGFBP2 protein spiked in the same serum or cerebrospinal fluid sample is 10000 pg / mL, 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL and 0 pg / mL, and the final concentration of detection antibody 8F1 is 1:5000;

[0134] (2) Add the mixed sample to the polystyrene 96-well microplate coated with capture antibody 6F2, set up 2 replicate wells for each sample, and incubate at 37°C for 1 hour; wash the strips after diluting the concentrated washing solution 20 times, wash the plate four times for color development; add color development solution (color development solution A and B are mixed in equal amounts and prepared immediately before use), 100 μL / well, incubate at room temperature and away from light for 10 minutes, then add stop solution, 50 μl / well, to terminate the reaction. This experiment was repeated three times, the average value was taken and the recovery rate was calculated after calibration. The recovery rate calculation formula is: Recovery rate (%) = (sample protein concentration spiked with standard product / protein concentration spiked with standard product) × 100. The recovery rate results of each concentration of protein are shown in Table 7. The results of Table 7 were summarized and analyzed. The results show that this kit has a good recovery rate, as shown in Table 8.

[0135] Table 7 Recovery results of double antibody sandwich ELISA kit for detecting IGFBP2 protein

[0136]

[0137]

[0138] Table 8 Recovery analysis results of the double antibody sandwich ELISA kit for detecting IGFBP2 protein

[0139] Sample Type Average recovery rate Recovery rate range Serum (n=2) 103.97 88.05～121.5％ Cerebrospinal fluid (n=1) 108.22 102.38～122.86％

[0140] It can be seen from the above content that the monoclonal antibody pair provided by the present invention can specifically detect IGFBP2 protein or IGFBP2 polypeptide with strong specificity and high sensitivity, which provides a basis for the diagnosis and treatment of clinical tumor patients.

[0141] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide, characterized in that: The monoclonal antibody pair comprises a first antibody and a second antibody; The first antibody includes 14F8 antibody or 8F1 antibody; The second antibody includes 6F2 antibody or 9G1 antibody; The light chain complementary determining region CDR1 of the 14F8 antibody comprises the amino acid sequence shown in SEQ ID NO.2, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.3, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.4; The heavy chain complementary determining region CDR1 of the 14F8 antibody comprises the amino acid sequence shown in SEQ ID NO.6, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.7, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.8; The light chain complementary determining region CDR1 of the 8F1 antibody comprises the amino acid sequence shown in SEQ ID NO.2, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.3, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.4; The heavy chain complementary determining region CDR1 of the 8F1 antibody comprises the amino acid sequence shown in SEQ ID NO.10, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.11, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.12; The light chain complementary determining region CDR1 of the 6F2 antibody comprises the amino acid sequence shown in SEQ ID NO.14, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.15, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.16; The heavy chain complementary determining region CDR1 of the 6F2 antibody comprises the amino acid sequence shown in SEQ ID NO.18, the heavy chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.19, and the heavy chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.20; The light chain complementary determining region CDR1 of the 9G1 antibody comprises the amino acid sequence shown in SEQ ID NO.14, the light chain complementary determining region CDR2 comprises the amino acid sequence shown in SEQ ID NO.15, and the light chain complementary determining region CDR3 comprises the amino acid sequence shown in SEQ ID NO.16; The heavy chain complementary determining region CDR1 of the 9G1 antibody includes the amino acid sequence shown in SEQ ID NO.22, the heavy chain complementary determining region CDR2 includes the amino acid sequence shown in SEQ ID NO.23, and the heavy chain complementary determining region CDR3 includes the amino acid sequence shown in SEQ ID NO.

24.

2. The monoclonal antibody pair according to claim 1, characterized in that: The light chain variable region of the 14F8 antibody comprises an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.1 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 14F8 antibody comprises an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.5 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The light chain variable region of the 8F1 antibody comprises an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.1 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 8F1 antibody comprises an amino acid sequence as shown in SEQ ID NO.9, or an amino acid sequence having at least 75% identity with the sequence as shown in SEQ ID NO.9 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The light chain variable region of the 6F2 antibody comprises an amino acid sequence as shown in SEQ ID NO.13, or an amino acid sequence having at least 75% identity with the sequence as shown in SEQ ID NO.13 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 6F2 antibody comprises an amino acid sequence as shown in SEQ ID NO.17, or an amino acid sequence having at least 75% identity with the sequence as shown in SEQ ID NO.17 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The light chain variable region of the 9G1 antibody comprises an amino acid sequence as shown in SEQ ID NO.13, or an amino acid sequence having at least 75% identity with the sequence as shown in SEQ ID NO.13 and having the ability to bind to the IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 9G1 antibody comprises an amino acid sequence as shown in SEQ ID NO.21, or an amino acid sequence having at least 75% identity with the sequence shown in SEQ ID NO.21 and having the ability to bind to IGFBP2 protein or IGFBP2 polypeptide.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the monoclonal antibody pair according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that The sequence of the nucleic acid molecule encoding the light chain variable region of the 14F8 antibody includes the nucleotide sequence shown in SEQ ID NO.25; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 14F8 antibody includes the nucleotide sequence shown in SEQ ID NO.26; The sequence of the nucleic acid molecule encoding the 8F1 light chain variable region includes the nucleotide sequence shown in SEQ ID NO.25; the sequence of the nucleic acid molecule encoding the 8F1 antibody heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO.27; The sequence of the nucleic acid molecule encoding the light chain variable region of the 6F2 antibody includes the nucleotide sequence shown in SEQ ID NO.28; the sequence of the nucleic acid molecule encoding the heavy chain variable region of the 6F2 antibody includes the nucleotide sequence shown in SEQ ID NO.29; The sequence of the nucleic acid molecule encoding the 9G1 light chain variable region includes the nucleotide sequence shown in SEQ ID NO.28; the sequence of the nucleic acid molecule encoding the 9G1 antibody heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO.

30.

5. A biological material for detecting IGFBP2, characterized in that: The biological material is a biological material expressing the monoclonal antibody pair according to claim 1 or 2; The biological material includes carriers or cells.

6. Use of the monoclonal antibody pair according to claim 1 or 2, the monoclonal antibody pair encoded by the nucleic acid molecule according to claim 3 or 4, or the monoclonal antibody pair expressed by the biomaterial according to claim 5 in detecting IGFBP2 protein or IGFBP2 polypeptide.

7. Use of the monoclonal antibody pair according to claim 1 or 2, or the monoclonal antibody pair encoded by the nucleic acid molecule according to claim 3 or 4, or the monoclonal antibody pair expressed by the biomaterial according to claim 5 in one or more of the following: (1) Preparing a product for detecting IGFBP2 protein or IGFBP2 polypeptide; (2) Preparation of products for tumor diagnosis; (3) Preparation of products for adjuvant treatment of tumors; (4) Prepare products for distinguishing benign tumors from malignant tumors.

8. A kit for detecting IGFBP2 protein or IGFBP2 polypeptide, characterized in that: The kit includes a capture antibody and a tracer-labeled detection antibody; The detection antibody is the first antibody in the monoclonal antibody pair according to claim 1 or 2; The capture antibody is the second antibody in the monoclonal antibody pair according to claim 1 or 2.

9. The kit according to claim 8, characterized in that The tracer includes one or more of radioactive isotopes, enzymes, fluorescent substances and luminescent substances, colloidal gold, and colored latex.

10. A method for detecting IGFBP2 protein or IGFBP2 polypeptide, characterized in that: The sample to be detected, the detection antibody labeled with a tracer and the capture antibody are mixed to form a complex; the tracer signal in the complex is detected to determine the concentration of the IGFBP2 protein or IGFBP2 polypeptide in the sample to be detected; The detection antibody is the first antibody in the monoclonal antibody pair according to claim 1 or 2; the capture antibody is the second antibody in the monoclonal antibody pair according to claim 1 or 2.

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