Monoclonal antibody pair for detecting igfbp2 protein or igfbp2 polypeptide and application thereof

Abstract

The application 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 thereof. The monoclonal antibody pair provided by the application comprises a first antibody and a second antibody; the first antibody comprises 14F8 antibody or 8F1 antibody; and the second antibody comprises 6F2 antibody or 9G1 antibody. The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain and the heavy chain variable region of the monoclonal antibody are determined, the IGFBP2 protein or IGFBP2 polypeptide can be specifically detected, the specificity is high, the sensitivity is high, the IGFBP2 protein in human serum can be detected, and a basis is provided for the diagnosis and treatment of clinical tumor patients.

Description

TECHNICAL FIELD

[0001] The present application 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 thereof. BACKGROUND

[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 enables it to bind to integrins, heparin and high-affinity insulin-like growth factor (IGF), and plays an important role in cell proliferation, differentiation and human growth and development. Studies have found that IGFBP2 is highly expressed in various malignant tumors such as lung cancer, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, colorectal cancer, liver cancer, high-grade glioma and glioblastoma, and can promote tumor cell proliferation, migration, invasion and epithelial-mesenchymal transition. In addition, IGFBP2 also plays an important role in neural development and neurodegenerative diseases. Biologically, IGFBP2 can be used as a tumor marker for clinical diagnosis and treatment. In normal human body, the content of IGFBP2 is low, but it is often highly expressed in cancer patients. According to the literature, the content of IGFBP2 in healthy people is 385.70±28.47 ng / mL, the content of IGFBP2 in patients with benign tumors is 464.15±67.8 ng / mL, and the content of IGFBP2 in patients with malignant tumors is 706.75±140.30 ng / mL (Fang Xiaolong. Detection of serum IGFBP2 level in patients with malignant tumor and its clinical significance [J]. Medical Animal Prevention and Control, 2004, (12): 776-777); another literature reported that the level of IGFBP2 in serum of patients with high-grade glioma was significantly higher than that in serum of patients with low-grade glioma 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] At present, many biological companies at home and abroad such as R&D, abcam, Invitrogen and the like can purchase IGFBP2 protein detection kits, but the price is relatively high, and there is no report about the sequences of the capture antibody and the detection antibody in the kit, therefore, it is necessary to independently develop a set of IGFBP2 protein detection antibody pairs to assemble a cost-effective detection kit for detecting the change level of IGFBP2 in patient samples, so as to provide a diagnosis basis for the clinic. SUMMARY

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

[0005] The application 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 comprises 14F8 antibody or 8F1 antibody; the second antibody comprises 6F2 antibody or 9G1 antibody; the light chain complementarity determining region CDR1 of the 14F8 antibody comprises an amino acid sequence as shown in SEQ ID NO.2, the light chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.3, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.4; the heavy chain complementarity determining region CDR1 of the 14F8 antibody comprises an amino acid sequence as shown in SEQ ID NO.6, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.7, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.8; the light chain complementarity determining region CDR1 of the 8F1 antibody comprises an amino acid sequence as shown in SEQ ID NO.2, the light chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.3, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.4; the heavy chain complementarity determining region CDR1 of the 8F1 antibody comprises an amino acid sequence as shown in SEQ ID NO.10, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.11, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.12; the light chain complementarity determining region CDR1 of the 6F2 antibody comprises an amino acid sequence as shown in SEQ ID NO.14, the light chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.15, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.16; the heavy chain complementarity determining region CDR1 of the 6F2 antibody comprises an amino acid sequence as shown in SEQ ID NO.18, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.19, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.20; the light chain complementarity determining region CDR1 of the 9G1 antibody comprises an amino acid sequence as shown in SEQ ID NO.14, the light chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.15, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.16; the heavy chain complementarity determining region CDR1 of the 9G1 antibody comprises an amino acid sequence as shown in SEQ ID NO.22, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as shown in SEQ ID NO.23, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as shown in SEQ ID NO.24.

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

[0007] The present application also provides a nucleic acid molecule encoding the monoclonal antibody pair described in the above technical solution.

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

[0009] The present application also provides a biological material, which is a biological material expressing the monoclonal antibody pair of the above technical solution; and the biological material comprises a vector or a cell.

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

[0011] The present application also provides the use of the monoclonal antibody pair of the above technical solution or the monoclonal antibody pair encoded by the nucleic acid molecule of the above technical solution or the monoclonal antibody pair expressed by the biological material of the above technical solution in one or more of the following aspects:

[0012] (1) preparing a product for detecting IGFBP2 protein or IGFBP2 polypeptide; (2) preparing a product for diagnosing tumors; (3) preparing a product for assisting in treating tumors; and (4) preparing a product for distinguishing between benign tumors and malignant tumors.

[0013] The present application also provides a kit for detecting IGFBP2 protein or IGFBP2 polypeptide, which comprises a capture antibody and a tracer-labeled detection antibody; the detection antibody is the first antibody in the monoclonal antibody pair of the above technical solution; and the capture antibody is the second antibody in the monoclonal antibody pair of the above technical solution.

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

[0015] The application further provides a method for detecting IGFBP2 protein or IGFBP2 polypeptide, wherein a sample to be detected, a tracer-labeled detection antibody and a capture antibody are mixed to form a complex; a tracer signal in the complex is detected to determine the concentration of 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 technical solution; and the capture antibody is the second antibody in the monoclonal antibody pair described in the technical solution.

[0016] Beneficial effects: the monoclonal antibody pair provided by the application comprises a first antibody and a second antibody; the first antibody comprises 14F8 antibody or 8F1 antibody; and the second antibody comprises 6F2 antibody or 9G1 antibody. The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the light chain and the heavy chain variable region of the monoclonal antibody are determined, the IGFBP2 protein or IGFBP2 polypeptide can be specifically detected, the specificity is high, the sensitivity is high, the IGFBP2 in human serum can be detected, and the basis for the diagnosis and treatment of clinical tumor patients is provided. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0018] Figure 1 SDS-PAGE detection results of IGFBP2 protein after purification;

[0019] Figure 2 Determination results of the binding titers of serum antibodies of 6 mice and IGFBP2 protein;

[0020] Figure 3 Titer detection results of 8 positive hybridomas;

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

[0022] Figure 5A standard curve for detecting IGFBP2 protein by a two-antibody sandwich ELISA kit one-step method. DETAILED DESCRIPTION

[0023] The present application 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 comprises 14F8 antibody or 8F1 antibody; and the second antibody comprises 6F2 antibody or 9G1 antibody;

[0025] The light chain complementarity determining region CDR1 of the 14F8 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 2, the light chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 3, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 4; the heavy chain complementarity determining region CDR1 of the 14F8 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 6, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 7, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 8; the light chain complementarity determining region CDR1 of the 8F1 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 2, the light chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 3, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 4; the heavy chain complementarity determining region CDR1 of the 8F1 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 10, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 11, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 12; the light chain complementarity determining region CDR1 of the 6F2 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 14, the light chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 15, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 16; the heavy chain complementarity determining region CDR1 of the 6F2 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 18, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 19, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 20; the light chain complementarity determining region CDR1 of the 9G1 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 14, the light chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 15, and the light chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 16; the heavy chain complementarity determining region CDR1 of the 9G1 antibody comprises an amino acid sequence as set forth in SEQ ID NO. 22, the heavy chain complementarity determining region CDR2 comprises an amino acid sequence as set forth in SEQ ID NO. 23, and the heavy chain complementarity determining region CDR3 comprises an amino acid sequence as set forth in SEQ ID NO. 24. The CDR regions of the first and second antibodies of the present application are defined by the Kabat numbering scheme, which defines the location of hypervariable regions among sequences of the same domain type.

[0026] In the present application, the light chain variable region of the 14F8 antibody preferably comprises the amino acid sequence as shown in SEQ ID NO. 1, or an amino acid sequence having at least 75% identity compared with the sequence shown in SEQ ID NO. 1 and having the ability of binding to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 14F8 antibody preferably comprises the amino acid sequence as shown in SEQ ID NO. 5, or an amino acid sequence having at least 75% identity compared with the sequence shown in SEQ ID NO. 5 and having the ability of binding to IGFBP2 protein or IGFBP2 polypeptide. The light chain constant region of the 14F8 antibody of the present application is preferably a Kappa chain, and the heavy chain constant region is preferably of IgG1 type. The light chain amino acid sequence of the 14F8 antibody of the present application is preferably as shown in SEQ ID NO. 32, in particular: DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISAATSLETGVPSRFSGSGSGKDYTLSITGLQTEDVATYYCQQYWSTPVTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; and the heavy chain amino acid sequence of the 14F8 antibody of the present application is preferably as shown in SEQ ID NO. 33, in particular: QIVLSQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGIKWMGRINTSTGEPTYADEFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRGFYYGNPWYFEYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0027] In the present application, the light chain variable region of the 8F1 antibody preferably comprises the amino acid sequence as shown in SEQ ID NO. 1, or an amino acid sequence having at least 75% identity to the sequence shown in SEQ ID NO. 1 and having the ability of binding to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 8F1 antibody preferably comprises the amino acid sequence as shown in SEQ ID NO. 9, or an amino acid sequence having at least 75% identity to the sequence shown in SEQ ID NO. 9 and having the ability of binding to IGFBP2 protein or IGFBP2 polypeptide. The light chain constant region of the 8F1 antibody of the present application is preferably a Kappa chain, and the heavy chain constant region is preferably of IgG1 type. The light chain amino acid sequence of the 8F1 antibody of the present application is preferably as shown in SEQ ID NO. 32, specifically: DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISAATSLETGVPSRFSGSGSGKDYTLSITGLQTEDVATYYCQQYWSTPVTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; and the heavy chain amino acid sequence of the 8F1 antibody of the present application is preferably as shown in SEQ ID NO. 34, specifically: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGIKWMGRINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARRGVYYGNPWYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0028] In the present application, the light chain variable region of the 6F2 antibody preferably comprises an amino acid sequence as shown in SEQ ID NO. 13, or an amino acid sequence having at least 75% identity to the sequence shown in SEQ ID NO. 13 and having the ability of binding to IGFBP2 protein or IGFBP2 polypeptide; the heavy chain variable region of the 6F2 antibody preferably comprises an amino acid sequence as shown in SEQ ID NO. 17, or an amino acid sequence having at least 75% identity to the sequence shown in SEQ ID NO. 17 and having the ability of binding to IGFBP2 protein or IGFBP2 polypeptide. The light chain amino acid sequence of the 6F2 antibody of the present application is preferably as shown in SEQ ID NO. 35, specifically: DILLTQSPAILSVSPGERVSFSCRASQSIGTRIHWYQQRTNGSPRLLIKYASESISGIPSR FSGSGSGTDFTLTINSVESEDIADYYCHQTNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSG GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; the heavy chain amino acid sequence of the 6F2 antibody of the present application is preferably as shown in SEQ ID NO. 36, specifically: QVQLQQSGAELVRPGTSVKISCKASGYAFTNYLLGWVKQRPGHGLEWIGDIYPGSGSTYY NEKFKGKATLTGDKSSSTVYMQLSSLTSEDSAVYFCARKEFLYYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0029] In the present application, the light chain variable region of the 9G1 antibody preferably comprises an amino acid sequence as shown in SEQ ID NO. 13, or an amino acid sequence having at least 75% identity to the sequence shown in SEQ ID NO. 13 and having the ability to bind to an IGFBP2 protein or IGFBP2 polypeptide; and the heavy chain variable region of the 9G1 antibody preferably comprises an amino acid sequence as shown in SEQ ID NO. 21, or an amino acid sequence having at least 75% identity to the sequence shown in SEQ ID NO. 21 and having the ability to bind to an IGFBP2 protein or IGFBP2 polypeptide. The light chain amino acid sequence of the 9G1 antibody of the present application is preferably as shown in SEQ ID NO. 35, specifically: DILLTQSPAILSVSPGERVSFSCRASQSIGTRIHWYQQRTNGSPRLLIKYASESISGIPSR FSGSGSGTDFTLTINSVESEDIADYYCHQTNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSG GASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; and the heavy chain amino acid sequence of the 9G1 antibody of the present application is preferably as shown in SEQ ID NO. 37, specifically: QVQLQQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIYPGSGTT YYNEKFKGKATLTGDKSSSTVYMQLSSLTSEDSAVYFCARKDFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0030] In the present application, the at least 75% identity is preferably 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 application, 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 application also provides a nucleic acid molecule encoding the monoclonal antibody pair described in the above technical solution.

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

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

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

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

[0037] The present application does not have strict requirements for the source of the nucleic acid molecule, which can be synthesized, for example, by standard chemical synthesis methods and / or recombinant methods, or produced semi-synthetically, for example, by combinatorial chemical synthesis and recombinant methods.

[0038] The present application also provides a biological material capable of expressing the monoclonal antibody pair described in the above technical solution; the biological material comprises a vector or a cell.

[0039] In the present application, the vector includes a basic vector and the nucleic acid molecule of the above technical solution inserted into the basic vector, or a variant of the nucleic acid molecule of the above technical solution inserted into the basic vector. The present application does not have strict requirements for the source of the vector, and any vector that can replicate and / or express polynucleotides in eukaryotic or prokaryotic cells, including mammalian cells (for example, human, monkey, rabbit, rat, hamster or mouse cells), plant cells, yeast cells, insect cells and bacterial cells (such as E. coli) can be used, such as pBAD, pQE-12, pGEX, pBluescript, pET-series expression vector, pCAI-n, pPOW3.0, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, 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. The variant of the nucleic acid molecule of the present application can be a naturally occurring variant, such as a naturally occurring allelic variant, or can be a non-naturally occurring variant, which can be prepared by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.

[0040] In the present application, the cell preferably includes a host cell and the vector of the above technical solution introduced into the host cell. The host cell of the present application preferably includes prokaryotic cells and eukaryotic cells, including but not limited to bacterial cells such as E. 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 application does not have strict requirements for 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 application also provides the application of the monoclonal antibody pair in the above technical solution or the monoclonal antibody pair encoded by the nucleic acid molecule in the above technical solution or the monoclonal antibody pair expressed by the biological material in the above technical solution in detecting IGFBP2 protein or IGFBP2 polypeptide or polypeptide. In the application, 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, colored latex-labeled immunoassay.

[0042] The application also provides the application of the monoclonal antibody pair in the above technical solution or the monoclonal antibody pair encoded by the nucleic acid molecule in the above technical solution or the monoclonal antibody pair expressed by the biological material 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 assisting in treating tumors; (4) preparing a product for distinguishing between benign tumors and malignant tumors.

[0043] In the application, 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 disease. The product in the application is preferably a kit, and further preferably a double antibody sandwich ELISA kit.

[0044] The application also provides a kit for detecting IGFBP2 protein or IGFBP2 polypeptide, which comprises a capture antibody and a tracer-labeled detection antibody; the detection antibody is the first antibody in the monoclonal antibody pair in the above technical solution; and the capture antibody is the second antibody in the monoclonal antibody pair in the above technical solution.

[0045] In the application, the tracer preferably includes one or more of a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, colloidal gold and colored latex, and further preferably a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, colloidal gold and colored latex; the radioisotope includes 3 H, 14 C, 32 P, 125 I, 131 I, 57 Co and 51one or more of beta-galactosidase, beta-glucosidase, alkaline phosphatase, horseradish peroxidase and malate dehydrogenase; the fluorescent substance preferably includes fluorescamine or fluorescein isothiocyanate; the luminescent substance preferably includes one or more of luminol, luminol derivatives, luciferin, lucigenin, acridinium ester, peroxyoxalic acid ester and dioxetanes; and the colored latex is polyvinyl polymerized latex microparticles.

[0046] In the present application, the kit preferably further includes one or more of a micro-well reaction plate, a sample processing solution, a concentrated washing solution, a positive control, a negative control, a color developing solution and a termination solution. The capture antibody of the present application is coated on a solid support, which can be a micro-well plate, magnetic beads, latex microparticles, a membrane (nylon membrane, nitrocellulose membrane or PVDF membrane).

[0047] In the present application, the capture antibody is preferably coated on the micro-well reaction plate. The material of the micro-well reaction plate of the present application is preferably polystyrene. In the present application, the capture antibody is preferably adsorbed on a solid support micro-well reaction plate, the micro-well reaction plate is blocked with a blocking protein (e.g., skimmed milk powder, bovine serum albumin, etc.), the capture antibody is contacted with the solid phase and incubated, the unreacted sample is removed, and a micro-well reaction plate coated with the capture antibody is obtained.

[0048] In the present application, the sample processing solution preferably comprises ethylenediaminetetraacetic acid, further preferably a 4 wt.% ethylenediaminetetraacetic acid solution; the pH value of the ethylenediaminetetraacetic acid solution is preferably 7. The concentrated washing solution of the present application preferably comprises a Tween-20 PBS solution, further preferably a 20x PBS solution containing 2 v / v% Tween-20. The positive control of the present application preferably comprises an IGFBP2 protein or IGFBP2 polypeptide; the amino acid sequence of the IGFBP2 protein is preferably as shown in SEQ ID NO. 31, specifically: MLP R VGCPALPLPPPPLLPLLLLLLGASGGGGGARAEVLFRCPPCTPERLAACGPPPVAPPAAVAAVAGGARMPCAELVREPGCGCCSVCARLEGEACGVYTPRCGQGLRCYPHPGSELPLQALVMGEGTCEKRRDAEYGASPEQVADNGDDHSEGGLVENHVDSTMNMLGGGGSAGRKPLKSGMKELAVFREKVTEQHRQMGKGGKHHLGLEEPKKLRPPPARTPCQQELDQVLERISTMRLPDERGPLEHLYSLHIPNCDKHGLYNLKQCKMSLNGQRGECWCVNPNTGKLIQGAPTIRGDPECHLFYNEQQEARGVHTQRMQ. The negative control of the present application preferably comprises a 0.1 v / v% Tween-20 PBS. The color developing solution of the present application preferably comprises color developing solution A and color developing solution B; the color developing solution A preferably comprises sodium acetate, citric acid, hydrogen peroxide and distilled water, further preferably comprising 13.6 g / 500 mL, citric acid 1.6 g / 500 mL, 30% hydrogen peroxide 0.3 mL / 500 mL and the rest distilled water; the color developing solution B preferably comprises ethylenediaminetetraacetic acid disodium salt, citric acid, glycerol, tetramethylbenzidine, DMSO and distilled water, further preferably comprising ethylenediaminetetraacetic acid disodium salt 0.2 g / 500 mL, citric acid 0.95 g / 500 mL, glycerol 50 mL / 500 mL, tetramethylbenzidine 0.15 g / 500 mL, DMSO 3 mL / 500 mL and the rest distilled water. The termination solution of the present application preferably comprises H2SO4.

[0049] The application also provides a method for detecting IGFBP2 protein or IGFBP2 polypeptide, wherein a sample to be detected, a tracer-labeled detection antibody and a capture antibody are mixed to form a complex; a signal of the tracer in the complex is detected to determine the concentration of 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 application, the mixing of the sample to be detected, the tracer-labeled detection antibody and the capture antibody to form a complex preferably comprises: mixing the sample to be detected and the tracer-labeled detection antibody 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 signal of the tracer 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 mixing of the sample to be detected, the tracer-labeled detection antibody and the capture antibody to form a complex preferably comprises: 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 tracer-labeled detection antibody to obtain a capture antibody-IGFBP2 antigen-detection antibody complex; and detecting the signal of the tracer 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 application can determine the concentration of IGFBP2 in the sample to be detected based on the signal generated by the tracer label on the detection antibody, and has high specificity and sensitivity.

[0051] Unless otherwise defined, scientific and technical terms used in connection with the application have the meanings commonly understood by a person of ordinary skill in the art.

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

[0053] Example 1

[0054] Preparation of monoclonal antibody

[0055] 1. Preparation of recombinant protein IGFBP2

[0056] The gene sequence of human IGFBP2 was searched from the GenBank sequence database with the sequence number NM_000597.3, and the gene sequence was synthesized by Jinweizhi Company and inserted into pcDNA3.1 vector between Nhe I and Not I to obtain pcDNA3.1-IGFBP2 recombinant vector. The pcDNA3.1-IGFBP2 recombinant vector was transferred into Expi293F cells (thermo) by using transfection reagent PEI (thermo), and the Expi293F cells were cultured using OPM medium (item number: P82019). The plasmid transfection and subsequent cell culture process were performed according to the instructions of OPM-CD 293 transient transfection system. The culture supernatant was collected by centrifugation at the third day after transfection, and IGFBP2 recombinant protein was obtained by using Ni-NTA (QIAGEN) for purification, and SDS-PAGE detection was performed. The results showed that the IGFBP2 protein for immunizing mice was successfully prepared in the embodiment. Figure 1

[0057] 2, Mouse antigen immunization

[0058] Five Balb / c mice were immunized with the IGFBP2 recombinant protein prepared above as an antigen, and a total of four immunizations were performed. At the first immunization, the antigen was mixed with Freund's adjuvant in the same volume, and the mixture was ground into a water-in-oil emulsion liquid, and 100 μL (40 μg of antigen) of the mixed antigen was injected intraperitoneally. At the second immunization, two weeks after the first immunization, 40 μg of antigen was mixed with incomplete Freund's adjuvant at a volume ratio of 1:1, and 100 μL (40 μg of antigen) of the mixed antigen was injected into the right hind leg muscle. The third immunization was performed two weeks after the second immunization, and the dose, method and route of injection of the antigen were the same as those of the second immunization. Two weeks later, 100 μL (40 μg) of antigen without adjuvant was injected intraperitoneally for booster immunization, and mouse tail blood was collected 3 days later for ELISA detection of titer. The specific detection process is as follows: the purified IGFBP2 recombinant protein was coated with carbonate buffer at pH 9.6 at 4°C overnight, 100 ng / well; the next day, PBST was washed 3 times, each for 3 min, and dried; 2% BSA was added to the well for blocking, and incubated at 37°C for 1 h; PBST was washed 3 times, each for 3 min, and dried; the serum of 6 mice (5 immunized mice and 1 normal mouse) was diluted with PBS, and diluted at 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, 1:109350, 100 μL was added to each well, and incubated at 37°C for 1 h, and washed with PBST for 3 times, each for 3 min, and dried; goat anti-mouse IgG-HRP was diluted at 1:5000, and incubated at 37°C for 30 min. PBST was washed 3 times, and dried, and TMB was colored for 10 min, and 2M H2SO4 was used for termination, and the absorbance value was measured at 450 nm. According to the detection results, the titer of the serum of the immunized mice was 1:109350, and the titer of the serum of the normal mouse was 1:450.​Figure 2 ), the valence and specificity data of the serum antibodies were integrated, and No. 3 mouse was selected for subsequent experiments.

[0059] 3. Cell fusion and culture of hybridoma

[0060] The animals were killed three days after intraperitoneal injection of antigen for reinforcement immunization, and the spleen cells were taken for cell fusion. A 37°C water bath was prepared in a clean bench, 5-6x10 7 The mouse myeloma cells (SP2 / 0) and spleen cells in the logarithmic growth phase and in good growth state were added into a 50 mL centrifuge tube at a ratio of 1:10 in number, and mixed. After centrifugation at 500 g for 10 min, the supernatant was aspirated, and the bottom of the centrifuge tube was tapped to slightly loosen the cell precipitate. A 1 mL solution of 45% PEG1450 preheated to 37°C was slowly dropped in 90 s, and the centrifuge tube was constantly and gently shaken. The whole process was in a 37°C water bath. Then, DMEM medium was gradually added to the cell mixture, 1 mL was added dropwise at the 1st min, 2 mL at the 2nd min, 3 mL at the 3rd min, 4 mL at the 4th min, and 5 mL at the 5th min, while shaking in the 37°C water bath. Then, the mixture was incubated at 37°C for 15 min, centrifuged at 500 g for 5 min, and the supernatant was removed. 5 mL of DMEM medium containing HAT (thermo, item number: 21060017) was added to suspend the precipitate, and finally, DMED medium containing HAT was added to about 100 mL. The mixture was dispensed into a 96-well cell culture plate on which macrophages had been plated, 100 μL / well, and then the culture plate was placed in a 37°C, 5% CO2 incubator for culture.

[0061] 4. Screening of positive hybridoma

[0062] ELISA was used to select positive clones. The growth of the hybridoma cells was observed, and after seven days, when the cell culture supernatant turned yellow, an appropriate amount of cell supernatant was aspirated for ELISA detection of antibodies. According to the ELISA results, the clones with OD 450 values higher than twice those of the negative control were selected, plated in a 96-well plate, and subjected to the first subcloning screening. After 7-10 days, ELISA detection of antibodies was performed again, and the clones with high OD 450 values were selected, plated in a 96-well plate, and subjected to the second subcloning screening, so that there was about 1 cell per well. After 7-10 days of culture, ELISA detection of antibodies was performed again, and the clones with high OD 450 values were selected, plated in a 96-well plate, and subjected to the third subcloning screening, so that there was about 1 cell per well. The dilution ratio of the cell supernatant for antibody detection was 1:50, and the sample incubation, washing, secondary antibody incubation, washing, color development, and termination processes were the same as those in Example 1. After 3 screenings, 8 clones with high OD 450The positive clone cells with high value were named: 1B6, 6F2, 6H6, 8F1, 9G1, 12F6, 14F8, 14F10. The supernatants of the eight positive hybridomas were diluted by 5 times, 25 times, 125 times, 625 times and 3125 times, respectively, and ELISA detection was performed using 100 ng / well of IGFBP2 protein coated. The results showed that the eight positive clones selected could be detected after being diluted by 625 times, and all had good binding capacity with IGFBP2 protein. Figure 3

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

[0064] 300 μL of ascites adjuvant was injected into the abdominal cavity of 12-week-old Balb / c mice, and two weeks later, the hybridoma cells were cultured to the best cell activity state, and the cell number was adjusted to about 1 x 10 6 μL of the ascites adjuvant was injected into the abdominal cavity of 12-week-old Balb / c mice, and two weeks later, the hybridoma cells were cultured to the best cell activity state, and the cell number was adjusted to about 1 x 10 Figure 4 μL of the ascites adjuvant was injected into the abdominal cavity of 12-week-old Balb / c mice, and two weeks later, the hybridoma cells were cultured to the best cell activity state, and the cell number was adjusted to about 1 x 10

[0065] Example 2

[0066] Identification of monoclonal antibodies

[0067] 1. Subtype identification of monoclonal antibodies

[0068] The identification of antibody subtype was performed by indirect ELISA. After coating IGFBP2 antigen and blocking with 2% BSA, the purified monoclonal antibodies of step 5 in Example 1 were incubated 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 by 1:5000 at 37°C for 30 min. After PBST washing for 3 times, TMB was developed for 5 min, and the detection results are shown in Table 1.

[0069] Table 1. Results of subtype identification of monoclonal antibodies

[0070]

[0071] 2. Identification of antigenic epitopes of monoclonal antibodies

[0072] 2.1 Construction of IGFBP2 deletion mutants

[0073] ​IGFBP2 gene (NCBI sequence number is NM_002515.3) with deletion of 37-65th, 66-94th, 95-123th, 124-152th, 153-181th, 182-210th, 211-239th, 240-268th, 269-297th, 298-325th amino acids were synthesized on pCDNA3.1 by artificial synthesis method, and the insertion enzyme cutting sites were Nhe I and Not I. The recombinant vectors were named as 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. The recombinant vectors were transferred into cloning bacteria, and were stored after large-scale extraction and sequencing.

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

[0075] (1) 293T cell culture and transfection: 10% FBS-DMEM high glucose culture medium was prepared by mixing DMEM high glucose culture medium and FBS at a volume ratio of 9:1. When the cells were fully grown, they were subcultured at a ratio of 1:5-1:6 into 10 cm culture dishes containing 6 cm x 6 cm cell climbing sheets, and were cultured overnight in a 37°C, 5% CO2 cell incubator. When the cell density was 30%-40%, the above-mentioned 10 kinds of recombinant plasmids and control plasmid pCDNA3.1 were respectively transferred into the cells by PEI transfection reagent (the transfection reagent, plasmid dosage and experimental operation process were operated according to the instructions of thermo company).

[0076] (2) Climbing sheet fixation and antibody staining

[0077] The 48h-old cells were washed with PBS for 2 times, fixed with acetone at 4°C for 5min, washed with PBS for 2 times, dried, and then cut into 0.25cm x 0.25cm cell climbing sheets, which were pasted on the same glass slide, and were ready for use.

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

[0079] Table 2 Identification results of the epitopes recognized by the monoclonal antibodies

[0080]

[0081] Note: "-" represents that the monoclonal antibody does not bind to the overexpressed protein with the deletion of the nucleic acid sequence, and "+" represents that the monoclonal antibody binds to the overexpressed protein with the deletion of the nucleic acid sequence.

[0082] According to Table 2, it can be seen 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 pair of monoclonal antibodies by double antibody sandwich ELISA method

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

[0086] Table 3 Antibody pairing results screened by double antibody sandwich ELISA method

[0087]

[0088] Note: The positive well OD 450nm value / the negative well OD 450nmThe antibody pair corresponding to the maximum value (P / N) is the optimal antibody pair. The underlined and bolded value corresponds to the optimal pair of coating antibody and enzyme-labeled antibody.

[0089] As can be seen from Table 3, the monoclonal antibody 9G1 as a capture antibody and the labeled monoclonal antibody 14F8 as a detection antibody are the best pair of antibodies; the monoclonal antibody 9G1 as a capture antibody and the labeled monoclonal antibody 8F1 as a detection antibody, the monoclonal antibody 6F2 as a capture antibody and the labeled monoclonal antibody 8F1 as a detection antibody, the monoclonal antibody 6F2 as a capture antibody and the labeled monoclonal antibody 14F8 as a detection antibody have high P / N values and can be used as antibody pairs for detecting GFBP2 protein. Therefore, the present application provides four pairs of antibodies that can be used for detecting GFBP2 protein.

[0090] Example 4

[0091] Identification of sequences of anti-IGFBP2 monoclonal antibodies

[0092] After the positive hybridoma 6F2, 8F1, 9G1 and 14F8 were grown to a certain scale (cell number > 3 x 10 6 After the positive hybridoma 6F2, 8F1, 9G1 and 14F8 were grown to a certain scale (cell number > 3 x 10 Max DNA Polymerase for PCR amplification, and the antibody V region cDNA was extracted and sent to a sequencing company for sequencing. The CDR region of the antibody amino acid sequence was labeled using the Kabat method, and 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 double antibody sandwich ELISA kit for detecting IGFBP2

[0100] (1) Coating microtiter plates with 100 ng / well of capture antibody 6F2 or 9G1;

[0101] The method for preparing the microtiter plates coated with capture antibody 6F2 or 9G1 is as follows: dilute the capture antibody 6F2 or 9G1 to 1 μg / mL with 50 mM carbonate buffer (pH 9.6) and coat 0.1 mL / well of the polystyrene plates at 4°C overnight. After tapping dry, add 0.3 mL of 0.25% casein (Sigma) blocking solution to each well and incubate at 4°C overnight to block the non-specific binding sites. Shake off the plate and vacuum dry for 12-24 hours to obtain the microtiter plates coated with capture antibody 6F2 or 9G1, which are vacuum packaged with aluminum foil bags and stored at 4°C for later use;

[0102] (2) Sample treatment solution: 4 wt.% ethylenediaminetetraacetic acid (pH 7.0), i.e. weigh 40 g of ethylenediaminetetraacetic acid and dissolve in 1 L of distilled water, adjust the 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 method for preparing the enzyme conjugate is as follows: dissolve 5 mg of horseradish peroxidase in 1 mL of distilled water, add 0.2 mL of freshly prepared 0.1 M sodium periodate and incubate for 30 min, then place in 1 mM sodium acetate buffer at pH 4.4 and dialyze at 4°C overnight. The next day, add 20 μL of 0.2 M carbonate buffer at pH 9.5, add 10 mg of the detection antibody 8F1 or 14F8 dialyzed in 0.01 M carbonate buffer at pH 9.5, gently stir at room temperature in the dark for 2-3 h, then add 0.1 mL of freshly prepared 4 mg / mL sodium borohydride, incubate at 4°C in the dark overnight, and dropwise add an equal volume of saturated ammonium sulfate (the ammonium sulfate is adjusted to pH 7.0-7.2 with ammonia before use), place at 4°C for 6 h, centrifuge at 10,000 g at 4°C for 30 min, discard the supernatant, dissolve the precipitate in an appropriate amount of 10 mM phosphate buffer, dialyze in 10 mM phosphate buffer at 4°C overnight, and change the solution three times. Collect the conjugate, add a protective agent with a final concentration of 2% BSA and a concentration of 50% glycerol, and store at 4°C for later use.

[0105] (4) Concentrated washing solution: 20x PBS containing 2% Tween-20, i.e. 1 L of solution contains 4.56 g of NaH2PO4, 58.02 g of Na2HPO4-12H2O, 175.3 g of NaCl, 20 mL of Tween-20, and high-pressure sterilization at 121°C for 20 min; dilute 20-fold for 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. 4.56 g NaH2PO4, 58.02 g Na2HPO4-12H2O, 175.3 g NaCl per 1 L solution, autoclaved at 121 °C for 20 min, after 20-fold dilution, add Tween-20 to a final concentration of 0.1%;

[0108] (7) Chromogenic solution: composed of chromogenic solution A and chromogenic solution B, use equal amounts of the two mixed and used; the composition of chromogenic solution A and B is as follows:

[0109] Chromogenic solution A: sodium acetate 13.6 g, citric acid 1.6 g, 30% hydrogen peroxide 0.3 mL, distilled water to 500 mL, store in the dark;

[0110] Chromogenic solution B: disodium ethylenediaminetetraacetate 0.2 g, citric acid 0.95 g, glycerol 50 mL, 0.15 g tetramethylbenzidine (TMB) is dissolved in 3 mL DMSO, distilled water to 500 mL, store in the dark. Use equal amounts of AB liquid mixed and used, store in the dark.

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

[0112] 2. Method for using the double antibody sandwich ELISA kit for detecting IGFBP2

[0113] (1) Sample addition:

[0114] ① One-step detection: using sample treatment solution, dilute the sample to be tested with sample treatment solution at 1:100, mix with horseradish peroxidase-labeled detection antibody 8F1 or 14F8 (antibody concentration 1 mg / mL, 1:2500 dilution) at equal volume, add 200 μL to the microplate coated with capture antibody 6F2 or 9G1, set 2 duplicate holes for each sample, set negative and positive controls at the same time, incubate at 37 °C for 1 h;

[0115] ② Two-step detection: using sample treatment solution, dilute the sample to be tested at 1:100, take 200 μL to the microplate coated with capture antibody 6F2 or 9G1, set 2 duplicate holes for each sample, set negative and positive controls at the same time, incubate at 37 °C for 1 h, dilute the concentrated washing solution 20-fold to wash the plate, wash 4 times, then add 100 μl of 1:5000 diluted enzyme conjugate (horseradish peroxidase-labeled detection antibody 8F1 or 14F8) to each well, incubate at 37 °C for 30 min;

[0116] (2) Washing: dilute the concentrated washing solution 20-fold to wash the plate 4 times;

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

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

[0119] (1) Sensitivity determination

[0120] The purified IGFBP2 protein (concentration of 1 mg / mL) was diluted with a sample treatment solution to different multiples, so that the final concentration was 10,000 pg / mL, 5,000 pg / mL, 2,500 pg / mL, 1,250 pg / mL, 625 pg / mL, 312.5 pg / mL, and 156.25 pg / mL, respectively, as the samples to be detected, and detection was performed according to the one-step detection procedure of step 2, and a standard curve was established; at the same time, a human IGFBP2 ELISA detection kit from R&D Company (item number: DGB200) was used as a control kit for comparison (the experiment was performed according to the instructions of the R&D Company), and the results are shown in Table 5 and Figure 5 .

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

[0122]

[0123] According to Table 5 and Figure 5 , it can be seen that the correlation coefficient of the standard curve established by using the double antibody sandwich ELISA kit for detecting IGFBP2 protein by one-step method is 0.9996. When the concentration of IGFBP2 protein 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 be detected when the concentration of IGFBP2 protein is 312.5 pg / mL. The double antibody sandwich ELISA kit prepared in the present application has good sensitivity in detecting IGFBP2 protein.

[0124] (2) Specificity determination

[0125] The gene sequence of human IGFBP1 (sequence number NM_000596.4), the gene sequence of human IGFBP3 (sequence number NM_000598.5), the gene sequence of human IGFBP4 (sequence number NM_001552.3), the gene sequence of human IGFBP5 (sequence number NM_000599.4), and the gene sequence of human IGFBP6 (sequence number NM_002178.3) were obtained from the GenBank sequence database, and were synthesized by a company named Jinweizhi, and were cloned into pcDNA3.1 vectors. The insertion site of the gene sequence of human IGFBP6 was BamH I and Not I, and the insertion sites of the other four genes were Nhe I and Not I, to obtain the recombinant vectors pCDNA3.1-IGFBP1, pCDNA3.1-IGFBP3, pCDNA3.1-IGFBP4, pCDNA3.1-IGFBP5, and pCDNA3.1-IGFBP6.

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

[0127] 4. Reproducibility experiment

[0128] Three different concentrations of IGFBP2 protein and three human serum samples were taken, and were labeled as 1, 2, 3, 4, 5, and 6, respectively. Each group was repeated three times. The samples were detected according to the one-step method described in the ELISA kit use method of the application, the coefficient of variation was calculated, and the batch and batch stability was evaluated. The results are shown in Table 6.

[0129] Table 6. Results of reproducibility experiment

[0130]

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

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

[0133] (1) 1 μL of serum or cerebrospinal fluid sample of each of 3 healthy subjects, a certain amount of IGFBP2 protein with a concentration of 100 ng / mL, and a proper amount of sample treatment solution were taken, and the total volume was made up to 100 μL, and then 100 μL of 1:2500 diluted enzyme conjugate (horseradish peroxidase-labeled detection antibody 8F1, antibody concentration 1 mg / mL) was added for mixing, and the total system was 200 μL. The final concentration of serum or cerebrospinal fluid in the reaction system was 1:200, the final concentration of IGFBP2 protein added in the same serum or cerebrospinal fluid sample was 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 was 1:5000.

[0134] (2) The mixed sample was added to a polystyrene 96-well microreaction plate coated with capture antibody 6F2, 2 replicate wells were set for each sample, and incubation was performed at 37°C for 1 h; the plate strip was washed after 20-fold dilution of the concentrated washing solution, and color development was performed by washing the plate four times; 100 μL / well of color developing solution (color developing solution A and B were mixed in equal amounts and prepared immediately before use) was added, and color development was performed at room temperature for 10 min in the dark, 50 μL / well of stop solution was then added to terminate the reaction. The experiment was repeated three times, and the average value was taken to calculate the recovery rate after calibration. The calculation formula of the recovery rate was: recovery rate (%) = (sample protein concentration added with the standard sample / the standard sample protein concentration added) x 100, and the recovery rate results of each concentration of protein are shown in Table 7. The results of Table 7 were analyzed in summary, and the results showed that the kit had a good recovery rate, and the details are shown in Table 8.

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

[0136]

[0137]

[0138] Table 8: Results of recovery analysis of the double antibody sandwich ELISA kit for detecting IGFBP2 protein

[0139] Sample Type Average Recovery Recovery Range Serum (n=2) 103.97 88.05～121.5％ Cerebrospinal Fluid (n=1) 108.22 102.38～122.86％

[0140] According to the above, the monoclonal antibody pair provided by the application can specifically detect IGFBP2 protein or IGFBP2 polypeptide, has high specificity and sensitivity, and provides a basis for the diagnosis and treatment of clinical tumor patients.

[0141] Although the above embodiment has made a detailed description of the application, it is only a part of the embodiments of the application, not all the embodiments, and other embodiments can be obtained according to the embodiment without creativity, which all belong to the protection scope of the application.

Claims

1. A monoclonal antibody pair for detecting IGFBP2 protein or IGFBP2 polypeptide, characterized in that, The monoclonal antibody pair includes a first antibody and a second antibody; The first antibody includes either 14F8 antibody or 8F1 antibody; The second antibody includes either 6F2 antibody or 9G1 antibody; The amino acid sequence of the light chain complementarity-determining region CDR1 of the 14F8 antibody is shown in SEQ ID NO.2, the amino acid sequence of the light chain complementarity-determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO.

4. The amino acid sequence of the heavy chain complementarity-determining region CDR1 of the 14F8 antibody is shown in SEQ ID NO.6, the amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO.

8. The amino acid sequence of the light chain complementarity-determining region CDR1 of the 8F1 antibody is shown in SEQ ID NO.2, the amino acid sequence of the light chain complementarity-determining region CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO.

4. The amino acid sequence of the heavy chain complementarity-determining region CDR1 of the 8F1 antibody is shown in SEQ ID NO.10, the amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO.

12. The amino acid sequence of the light chain complementarity-determining region CDR1 of the 6F2 antibody is shown in SEQ ID NO.14, the amino acid sequence of the light chain complementarity-determining region CDR2 is shown in SEQ ID NO.15, and the amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO.

16. The amino acid sequence of the heavy chain complementarity-determining region CDR1 of the 6F2 antibody is shown in SEQ ID NO.18, the amino acid sequence of the heavy chain complementarity-determining region CDR2 is shown in SEQ ID NO.19, and the amino acid sequence of the heavy chain complementarity-determining region CDR3 is shown in SEQ ID NO.

20. The amino acid sequence of the light chain complementarity-determining region CDR1 of the 9G1 antibody is shown in SEQ ID NO.14, the amino acid sequence of the light chain complementarity-determining region CDR2 is shown in SEQ ID NO.15, and the amino acid sequence of the light chain complementarity-determining region CDR3 is shown in SEQ ID NO.

16. The amino acid sequence of the heavy chain complementarity-determining region (CDR1) of the 9G1 antibody is shown in SEQ ID NO.22, the amino acid sequence of the heavy chain complementarity-determining region (CDR2) is shown in SEQ ID NO.23, and the amino acid sequence of the heavy chain complementarity-determining region (CDR3) is 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 includes an amino acid sequence as shown in SEQ ID NO.1, or has at least 75% identity with the sequence shown in SEQ ID NO.1, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 14F8 antibody includes an amino acid sequence as shown in SEQ ID NO.5, or has at least 75% identity with the sequence shown in SEQ ID NO.5, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide. The light chain variable region of the 8F1 antibody includes an amino acid sequence as shown in SEQ ID NO.1, or has at least 75% identity with the sequence shown in SEQ ID NO.1, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 8F1 antibody includes an amino acid sequence as shown in SEQ ID NO.9, or has at least 75% identity with the sequence shown in SEQ ID NO.9, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide. The light chain variable region of the 6F2 antibody includes an amino acid sequence as shown in SEQ ID NO.13, or has at least 75% identity with the sequence shown in SEQ ID NO.13, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 6F2 antibody includes an amino acid sequence as shown in SEQ ID NO.17, or has at least 75% identity with the sequence shown in SEQ ID NO.17 and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide. The light chain variable region of the 9G1 antibody includes an amino acid sequence as shown in SEQ ID NO.13, or has at least 75% identity with the sequence shown in SEQ ID NO.13, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide; The heavy chain variable region of the 9G1 antibody includes an amino acid sequence as shown in SEQ ID NO.21, or has at least 75% identity with the sequence shown in SEQ ID NO.21, and has an amino acid sequence capable of binding to IGFBP2 protein or IGFBP2 polypeptide.

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

4. The nucleic acid molecule according to claim 3, characterized in that, The sequence encoding the variable region of the light chain of the nucleic acid molecule for the 14F8 antibody includes the nucleotide sequence shown in SEQ ID NO. 25; the sequence encoding the variable region of the heavy chain of the nucleic acid molecule for the 14F8 antibody includes the nucleotide sequence shown in SEQ ID NO.

26. The sequence encoding the 8F1 light chain variable region of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.25; the sequence encoding the 8F1 antibody heavy chain variable region of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.

27. The sequence encoding the variable region of the light chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO. 28; the sequence encoding the variable region of the heavy chain of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.

29. The sequence encoding the 9G1 light chain variable region of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.28; the sequence encoding the 9G1 antibody heavy chain variable region of the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO.

30.

5. A biomaterial for detecting IGFBP2, characterized in that, The biomaterial is a biomaterial expressing the monoclonal antibody pair as described in claim 1 or 2; The biomaterials include carriers or cells.

6. The use of the monoclonal antibody pair of claim 1 or 2, or the monoclonal antibody pair encoded by the nucleic acid molecule of claim 3 or 4, or the monoclonal antibody pair expressed by the biological material of claim 5, in the preparation of products for detecting IGFBP2 protein or IGFBP2 polypeptide.

7. A kit for detecting IGFBP2 protein or IGFBP2 polypeptide, characterized in that, The kit includes capture antibodies and tracer-labeled detection antibodies; The detection antibody is the first antibody in the monoclonal antibody pair described in claim 1 or 2; The capture antibody is the second antibody in the monoclonal antibody pair of claim 1 or 2.

8. The reagent kit according to claim 7, characterized in that, The tracer includes one or more of the following: radioactive isotopes, enzymes, fluorescent and luminescent substances, colloidal gold, and colored latex.

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