Monoclonal antibody for specifically recognizing specific metabolic fragment of human amyloid protein and application

Abstract

The invention provides a monoclonal antibody capable of specifically recognizing a specific metabolic fragment of human amyloid protein and application of the monoclonal antibody, and belongs to the technical field of genetic engineering. The invention provides a hybridoma cell strain AB5H, and the preservation number of the hybridoma cell strain AB5H is CCTCC (China Center For Type Culture Collection) NO: C201255. The hybridoma cell strain AB5H disclosed by the invention can secrete a monoclonal antibody which has relatively high activity and can specifically recognize human beta amyloid protein A beta 40; the monoclonal antibody is high in titer, can be used as a capture antibody, and is used for preparing a human Abeta40 protein quantitative detection kit and quantitatively detecting the content of Abeta40 in cerebrospinal fluid and blood, so that the detection result deviation caused by the fact that the content of Abeta40 in peripheral blood is easily influenced by metabolism, sample treatment conditions and methods and individual differences is overcome, and the detection accuracy is improved. The variation degree of a detection result is reduced, so that a judgment boundary value of a blood A beta detection result is conveniently obtained, and AD early diagnosis is realized.

Description

Technical Field

[0001] The present invention belongs to the field of gene engineering technology, and in particular relates to a monoclonal antibody specifically recognizing a specific metabolic fragment of human amyloid protein and its application. Background Art

[0002] Alzheimer's disease (AD) is the most prominent primary neurodegenerative disease of the central nervous system. As its etiology and pathogenesis are not yet fully understood, early diagnosis plays an important role in the prevention and control of AD. Plaques with amyloid β-protein (Aβ) as the main component deposited around neurons are one of the typical pathological changes in AD patients, among which Aβ protein mainly includes two forms, Aβ40 and Aβ42. Compared with the use of Aβ42 and Aβ40 change levels alone, the use of Aβ42 / Aβ40 ratio can more effectively judge the test results, and has a more suggestive effect on the prevention, development and treatment of AD diseases.

[0003] In recent years, the screening of blood-related markers has made the early diagnosis and prediction of AD more convenient, but peripheral blood Aβ42 is easily affected by metabolism, sample processing and individual factors, and has a large variation. There is currently no clear reference threshold, which brings confusion to the result judgment. This requires more sensitive and specific monoclonal antibodies to simultaneously detect Aβ40, better judge the blood Aβ test results, and provide important guarantees for the establishment of a relatively stable AD blood biomarker detection system. Summary of the invention

[0004] The purpose of the present invention is to provide a monoclonal antibody that specifically recognizes a specific metabolic fragment of human amyloid protein and its application. The monoclonal antibody of the present invention can specifically recognize human β-amyloid protein Aβ40 and is expected to be used for the early diagnosis and prediction of AD.

[0005] The invention provides a hybridoma cell strain, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C201255.

[0006] The present invention also provides the use of the hybridoma cell line described in the above scheme in preparing a monoclonal antibody that specifically recognizes human amyloid protein Aβ40.

[0007] The present invention also provides a monoclonal antibody, which is secreted and produced by the hybridoma cell line described in the above scheme.

[0008] The present invention also provides a monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody includes: a heavy chain complementary determining region CDR1 as shown in SEQ ID NO.1, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO.2, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of the monoclonal antibody includes: a light chain complementary determining region CDR1 as shown in SEQ ID NO.8, a light chain complementary determining region CDR2 with an amino acid sequence of LVF, and a light chain complementary determining region CDR3 as shown in SEQ ID NO.9.

[0009] Preferably, the heavy chain variable region of the monoclonal antibody has at least 90% sequence identity with the amino acid sequence selected from SEQ ID NO.13; the light chain variable region of the monoclonal antibody has at least 90% sequence identity with the amino acid sequence selected from SEQ ID NO.14.

[0010] The present invention also provides a nucleic acid molecule encoding the monoclonal antibody described in the above scheme.

[0011] The present invention also provides a recombinant vector into which the nucleic acid molecule described in the above scheme is inserted.

[0012] The present invention also provides a host cell comprising the nucleic acid molecule or the recombinant vector described in the above scheme.

[0013] The present invention also provides the use of the hybridoma cell line, the monoclonal antibody, the nucleic acid molecule, the recombinant vector or the host cell in the above scheme in preparing products for diagnosis or auxiliary diagnosis of Alzheimer's disease.

[0014] The present invention also provides a product for diagnosing or assisting in the diagnosis of Alzheimer's disease, comprising the monoclonal antibody described in the above scheme.

[0015] The present invention provides a hybridoma cell line AB5H, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C201255. The hybridoma cell line AB5H of the present invention can secrete a highly active monoclonal antibody that specifically recognizes human beta amyloid protein Aβ40; the monoclonal antibody has a high titer and can be used as a capture antibody to prepare a human Aβ40 protein quantitative detection kit to achieve quantitative detection of the content of Aβ40 in cerebrospinal fluid and blood, thereby overcoming the deviation of the detection result caused by the fact that the peripheral blood Aβ content is easily affected by metabolism, sample processing conditions and methods, and individual differences, reducing the degree of variation of the detection result, facilitating the determination of the blood Aβ detection result, and achieving early diagnosis of AD and monitoring of drug treatment effects.

[0016] Biological Deposit Description

[0017] The hybridoma cell line AB5H was deposited in China Center for Type Culture Collection on April 13, 2012. The deposit address is: Wuhan University, near Luonan 1st Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCC NO: C201255. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 The results of SDS-PAGE detection of purified monoclonal antibody AB5H;

[0020] Figure 2 Agarose gel electrophoresis shows the light and heavy chain variable region genes of monoclonal antibody AB5H;

[0021] Figure 3 To detect the recognition of APP / PS1 / tau triple transgenic mouse brain tissue by monoclonal antibody AB5H by immunohistochemistry;

[0022] Figure 4 The double antibody sandwich ELISA calibration curve for Aβ40 detection established using monoclonal antibody AB5H;

[0023] Figure 5 The application of monoclonal antibody AB5H in calibrating human plasma detection; where, *: p = 0.045 < 0.05. DETAILED DESCRIPTION

[0024] The present invention provides a hybridoma cell strain AB5H. The hybridoma cell strain is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C201255.

[0025] In the present invention, the hybridoma cell line AB5H is established and preserved by the Institute of Life Sciences and Bioengineering of Beijing Jiaotong University, its immunogen is a KLH conjugate containing human amyloid protein Aβ25-40 peptide (GSNKGAIIGLMV GGVV, SEQ ID NO.31), and the antibody molecule subtype secreted by it is IgG1.

[0026] The hybridoma cell line of the present invention produces a highly active monoclonal antibody that specifically recognizes human amyloid protein Aβ40 (human β-amyloid protein 1-40), which can specifically recognize biomarkers in AD blood and is expected to be used for early diagnosis and prediction of AD.

[0027] The present invention also provides the use of the hybridoma cell line described in the above scheme in preparing a monoclonal antibody that specifically recognizes human amyloid protein Aβ40.

[0028] The present invention also provides a monoclonal antibody, which is secreted and produced by the hybridoma cell line described in the above scheme.

[0029] In the present invention, the monoclonal antibody specifically recognizes human amyloid protein Aβ40. In the present invention, the monoclonal antibody is obtained by using human Aβ25-40 coupled protein as an immunogen, and has the characteristics of simple immunogen preparation method and the obtained antibody being able to recognize natural protein; the monoclonal antibody can recognize natural Aβ40 oligomers, and the reaction titer is higher than 256000, and can be used for early diagnosis of AD and monitoring of treatment effects.

[0030] The present invention also provides a monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody comprises: a heavy chain complementary determining region CDR1 as shown in SEQ ID NO.1, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO.2 and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO.3.

[0031] In the present invention, the amino acid sequence of the heavy chain complementary determining region CDR1 shown in SEQ ID NO.1 is specifically: GFNFSDTY; the amino acid sequence of the heavy chain complementary determining region CDR2 shown in SEQ ID NO.2 is specifically: IDPGRGNT; the amino acid sequence of the heavy chain complementary determining region CDR3 shown in SEQ ID NO.3 is specifically: APRYFYFDY.

[0032] In the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody preferably also includes: a heavy chain framework region FR1 as shown in SEQ ID NO.4, a heavy chain framework region FR2 as shown in SEQ ID NO.5, a heavy chain framework region FR3 as shown in SEQ ID NO.6 and a heavy chain framework region FR4 as shown in SEQ ID NO.7.

[0033] In the present invention, the amino acid sequence of the heavy chain framework region FR1 shown in SEQ ID NO.4 is specifically: VQAQQSGAELVKPGASVKLSCTAS; the amino acid sequence of the heavy chain framework region FR2 shown in SEQ ID NO.5 is specifically: MHWVKQRPEQGLEWIGR; the amino acid sequence of the heavy chain framework region FR3 shown in SEQ ID NO.6 is specifically: KYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYC; the amino acid sequence of the heavy chain framework region FR4 shown in SEQ ID NO.7 is specifically: WAKAPLSQ.

[0034] In the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody includes: a light chain complementary determining region CDR1 as shown in SEQ ID NO.8, a light chain complementary determining region CDR2 with an amino acid sequence of LVF, and a light chain complementary determining region CDR3 as shown in SEQ ID NO.9.

[0035] In the present invention, the amino acid sequence of the light chain complementary determining region CDR1 shown in SEQ ID NO.8 is specifically: QSHCHSDGKTY; the amino acid sequence of the light chain complementary determining region CDR3 shown in SEQ ID NO.9 is specifically: WQRTHFPLT.

[0036] In the present invention, the amino acid sequence of the light chain variable region of the monoclonal antibody preferably also includes: a light chain framework region FR1 as shown in SEQ ID NO.10, a light chain framework region FR2 as shown in SEQ ID NO.11, a light chain framework region FR3 as shown in SEQ ID NO.12 and a light chain framework region FR4 as shown in SEQ ID NO.13.

[0037] In the present invention, the amino acid sequence of the light chain framework region FR1 shown in SEQ ID NO.10 is specifically: DVVMTQTPLTLSVTIGQPASIFCKSS; the amino acid sequence of the light chain framework region FR2 shown in SEQ ID NO.11 is specifically: LNWLLQRPGQFPKRLIY; the amino acid sequence of the light chain framework region FR3 shown in SEQ ID NO.12 is specifically: KLDFGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC; the amino acid sequence of the light chain framework region FR4 shown in SEQ ID NO.13 is FGDGTKL.

[0038] In the present invention, the heavy chain variable region of the monoclonal antibody preferably has at least 90% sequence identity with the amino acid sequence selected from SEQ ID NO.14, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; the light chain variable region of the monoclonal antibody has at least 90% sequence identity with the amino acid sequence selected from SEQ ID NO.15, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0039] In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.14; the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID NO.15.

[0040] In the present invention, the amino acid sequence shown in SEQ ID NO.14 is specifically: VQAQQSGAELVKPGASVKLSCTASGFNFSDTYMHWVKQRPEQGLEWIGRIDPGRG NTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAPRYFYFDYWAKAPLSQ.

[0041] In the present invention, the amino acid sequence shown in SEQ ID NO.15 is specifically: DVVMTQTPLTLSVTIGQPASIFCKSSQSHCHSDGKTYLNWLLQRPGQFPKRLIYLVF KLDFGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQRTHFPLTFGDGTKL.

[0042] The present invention also provides a nucleic acid molecule encoding the monoclonal antibody described in the above scheme.

[0043] In the present invention, the heavy chain variable region gene of the monoclonal antibody preferably includes: a heavy chain complementary determining region CDR1 as shown in SEQ ID NO.16, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO.17 and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO.18.

[0044] In the present invention, the nucleotide sequence of the heavy chain complementary determining region CDR1 shown in SEQ ID NO.16 is specifically: GGCTTC AAC TTC AGT GAC ACC TAT; the nucleotide sequence of the heavy chain complementary determining region CDR2 shown in SEQ ID NO.17 is specifically: ATT GAT CCT GGAAGG GGTAATACT; the nucleotide sequence of the heavy chain complementary determining region CDR3 shown in SEQ ID NO.18 is specifically: GCC CCC CGG TAC TTT TAC TTT GAC TAC.

[0045] In the present invention, the heavy chain variable region gene of the monoclonal antibody preferably also includes: a gene encoding a heavy chain framework region FR1 as shown in SEQ ID NO.19, a gene encoding a heavy chain framework region FR2 as shown in SEQ ID NO.20, a gene encoding a heavy chain framework region FR3 as shown in SEQ ID NO.21, and a gene encoding a heavy chain framework region FR4 as shown in SEQ ID NO.22.

[0046] In the present invention, the nucleotide sequence of the gene encoding the heavy chain framework region FR1 shown in SEQ ID NO.19 is specifically: GTC CAG GCG CAG CAG TCT GGG GCA GAG CTT GTG AAG CCA GGG GCC TCA GTC AAGTTG TCC TGC ACA GCT TCT; the nucleotide sequence of the gene encoding the heavy chain framework region FR2 shown in SEQ ID NO.20 is specifically: ATG CAC TGG GTGAAG CAGAGG CCT GAA CAG GGC CTG GAG TGG ATT GGAAGG; the nucleotide sequence of the gene encoding the heavy chain framework region FR3 shown in SEQ ID NO.21 is specifically: AAATAT GAC CCGAAG TTCCAG GGCAAG GCC ACT ATA ACA GCA GAC ACA TCC TCC AAC ACA GCC TAC CTGCAG CTCAGCAGC CTGACATCT GAG GACACT GCC GTC TAT TAC TGT; The nucleotide sequence of the gene encoding the heavy chain framework region FR4 shown in NO.22 is specifically: TGG GCC AAG GCA CCA CTC TCA CAG.

[0047] In the present invention, the light chain variable region gene of the monoclonal antibody preferably includes: a gene encoding a light chain complementary determining region CDR1 as shown in SEQ ID NO.23, a gene encoding a light chain complementary determining region CDR2 with a nucleotide sequence of TTG GTG TTT, and a gene encoding a light chain complementary determining region CDR3 as shown in SEQ ID NO.24.

[0048] In the present invention, the nucleotide sequence of the gene encoding the light chain complementary determining region CDR1 shown in SEQ ID NO.23 is specifically: CAGAGC CAT TGT CATAGT GAT GGAAAGACATAT; the nucleotide sequence of the gene encoding the light chain complementary determining region CDR3 shown in SEQ ID NO.24 is specifically: TGG CAAAGGACA CAT TTT CCG CTCACG.

[0049] In the present invention, the light chain variable region gene of the monoclonal antibody preferably also includes: a gene encoding the light chain framework region FR1 as shown in SEQ ID NO.25, a gene encoding the light chain framework region FR2 as shown in SEQ ID NO.26, a gene encoding the light chain framework region FR3 as shown in SEQ ID NO.27, and a gene encoding the light chain framework region FR4 as shown in SEQ ID NO.28.

[0050] In the present invention, the nucleotide sequence of the gene encoding the light chain framework region FR1 shown in SEQ ID NO.25 is specifically: GAT GTT GTG ATG ACC CAG ACT CCA CTC ACT TTG TCG GTT ACC ATT GGA CAA CCAGCC TCC ATT TTT TGCAAG TCAAGT; the nucleotide sequence of the gene encoding the light chain framework region FR2 shown in SEQ ID NO.26 is specifically: TTG AAT TGG TTG TTA CAG AGG CCAGGC CAG TTT CCAAAG CGC CTAATTTAT; the nucleotide sequence of the gene encoding the light chain framework region FR3 shown in SEQ ID NO.27 is specifically: AAA CTG GACTTT GGA GTC CCT GAC AGG TTC ACT GGC AGT GGA TCA GGG ACA GAT TTC ACA CTGAAAATC AGC AGA GTG GAG GCT GAG GAT TTG GGA GTT TAT TATTGC; The nucleotide sequence of the gene encoding the light chain framework region FR4 shown in NO.28 is specifically: TTC GGT GAT GGG ACC AAG CTG.

[0051] In the present invention, the heavy chain variable region gene preferably has at least 90% sequence identity with the nucleotide sequence selected from SEQ ID NO.29, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; the light chain variable region gene preferably has at least 90% sequence identity with the nucleotide sequence selected from SEQ ID NO.30, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0052] In one embodiment of the present invention, the nucleotide sequence of the heavy chain variable region gene is shown as SEQ ID NO.29; the nucleotide sequence of the light chain variable region gene is shown as SEQ ID NO.30.

[0053] In the present invention, the nucleotide sequence shown in SEQ ID NO. 29 is specifically: GTC CAG GCG CAG CAG TCTGGG GCA GAG CTT GTG AAG CCA GGG GCC TCA GTC AAG TTG TCC TGCACA GCT TCT GGCTTC AAC TTC AGT GAC ACC TATATG CAC TGG GTG AAGCAG AGG CCT GAA CAG GGC CTG GAGTGG ATT GGAAGG ATT GAT CCT GGAAGG GGT AAT ACTAAA TAT GAC CCG AAG TTC CAG GGCAAG GCC ACT ATAACA GCA GAC ACA TCC TCC AAC ACA GCC TAC CTGCAG CTC AGC AGCCTGACA TCT GAG GAC ACT GCC GTC TAT TAC TGT GCC CCC CGG TAC TTT TACTTT GACTACTGG GCCAAG GCACCACTC TCACAG.

[0054] In the present invention, the nucleotide sequence shown in SEQ ID NO.30 is specifically: GAT GTT GTGATGACC CAGACT CCA CTC ACT TTG TCG GTT ACC ATT GGA CAA CCA GCC TCC ATTTTT TGC AAG TCAAGT CAG AGC CAT TGT CAT AGT GAT GGA AAG ACA TATTTG AAT TGG TTG TTA CAG AGGCCA GGC CAG TTT CCAAAG CGC CTAATTTAT TTG GTG TTT AAA CTG GAC TTT GGA GTC CCTGAC AGG TTC ACT GGCAGT GGA TCA GGG ACA GAT TTC ACA CTG AAAATC AGC AGA GTG GAGGCTGAG GAT TTG GGA GTT TAT TAT TAT TGC TGG CAA AGG ACA CAT TTT CCG CTCACG TTC GGTGAT GGGACCAAG CTG.

[0055] In the present invention, the heavy chain variable region gene and the light chain variable region gene can be used to express the amyloid protein Aβ40 with specific recognition of the carboxyl terminus GGVV after recombination. In the present invention, the heavy chain variable region gene and the light chain variable region gene are cloned from the hybridoma cell line AB5H described in the above scheme.

[0056] In the present invention, the cloning step preferably includes: extracting total RNA of hybridoma cell line AB5H; synthesizing cDNA from the total RNA by reverse transcription; using cDNA as a template and a primer pair for PCR amplification to obtain the heavy chain variable region gene and the light chain variable region gene. In the present invention, the method for extracting total RNA of hybridoma cell line AB5H is preferably Trizol.

[0057] The present invention also provides a recombinant vector into which the nucleic acid molecule described in the above scheme is inserted.

[0058] In the present invention, the method for constructing the recombinant vector is preferably: cloning the nucleic acid molecule described in the above scheme into an expression vector to obtain a recombinant vector.

[0059] The present invention also provides a host cell comprising the nucleic acid molecule or the recombinant vector described in the above scheme.

[0060] In the present invention, the method for constructing the host cell is preferably: transfecting / transforming the corresponding receptor cell with the recombinant vector described in the above scheme to obtain the host cell; the host cell can highly express the nucleic acid molecule encoding the monoclonal antibody; the receptor cell includes bacteria, mammals or yeast.

[0061] The present invention also provides a product for diagnosing or assisting in the diagnosis of Alzheimer's disease, comprising the monoclonal antibody described in the above scheme.

[0062] The product for diagnosing or assisting in diagnosing Alzheimer's disease of the present invention can be used for specific detection of human cerebrospinal fluid and blood samples with high sensitivity.

[0063] In the present invention, the product preferably includes a reagent or a kit; the kit preferably includes a double antibody sandwich ELISA kit; the double antibody sandwich ELISA kit uses the monoclonal antibody of the present invention as the coating antibody.

[0064] In the present invention, the preferred components also include: xylene, EDTA (pH 9.0) antigen repair solution, 3% hydrogen peroxide, BSA, hematoxylin staining solution, hematoxylin differentiation solution, hematoxylin blueing solution, neutral gum, secondary antibody: HRP-labeled goat anti-mouse, and histochemical kit DAB color developer.

[0065] The double antibody sandwich ELISA kit of the present invention is a quantitative detection kit for human Aβ40 protein, with a linear range of 7.8 to 250 pg / mL, and can be used for the detection of cerebrospinal fluid and blood samples.

[0066] The present invention also provides the use of the hybridoma cell line, the monoclonal antibody, the nucleic acid molecule, the recombinant vector or the host cell in the above scheme in preparing products for diagnosis or auxiliary diagnosis of Alzheimer's disease.

[0067] In the present invention, the diagnosis or auxiliary diagnosis of Alzheimer's disease preferably includes early diagnosis or early auxiliary diagnosis of Alzheimer's disease; the early stage here means that there are no clinical symptoms, but the biomarkers have changed.

[0068] In the present invention, the application preferably comprises the following steps:

[0069] The monoclonal antibody is matched with the relevant detection antibody to establish a double antibody sandwich ELISA calibration curve for Aβ40 protein detection, which is used to detect the content of Aβ40 in the test sample; the relevant antibodies preferably include: 6E10-Bio or 4G8-Bio mouse monoclonal antibodies. The unit of Aβ40 content is pg / mL. There is currently no completely unified diagnostic threshold for different races and populations, but the linear range of the detection method of the present invention can cover the reported human cerebrospinal fluid and blood Aβ1-40 protein content.

[0070] The monoclonal antibody of the present invention can quantitatively detect the content of Aβ40 in cerebrospinal fluid and blood. Since the content of Aβ42 in peripheral blood varies greatly under different individuals, different metabolic states, and multiple freeze-thaw conditions, if the ratio of Aβ42 / Aβ40 is analyzed, the deviation of the test results caused by the fact that the Aβ content in peripheral blood is easily affected by metabolism, sample processing conditions and methods, and individual differences can be overcome, the degree of variation of the test results can be reduced, and the judgment threshold of the blood Aβ test results can be obtained. The technical solution of the present invention can provide technical support for physical examinations of healthy elderly people in my country and early diagnosis and screening of AD.

[0071] The present invention also provides the use of the monoclonal antibody or the encoding gene or the recombinant vector or the host cell described in the above scheme in assisting the research and development of products for preventing Alzheimer's disease.

[0072] In the present invention, the sample to be tested of the product is preferably a blood sample, cerebrospinal fluid or brain tissue protein extract; the blood sample is preferably plasma.

[0073] In the present invention, the product preferably also includes a reagent for detecting the content of Aβ40 protein; the reagent is preferably AB5H monoclonal antibody or conjugated biotin, horseradish enzyme, magnetic beads and other labeling substances.

[0074] The present invention also provides a method for diagnosing Alzheimer's disease by double antibody sandwich ELISA, comprising the following steps:

[0075] The double antibody sandwich ELISA test was performed using the sample to be tested as the antigen and the monoclonal antibody described in the above scheme as the coating antibody.

[0076] To further illustrate the present invention, a monoclonal antibody specifically recognizing a specific metabolic fragment of human amyloid protein and its application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0077] The experimental methods without specific conditions in the following implementations can be operated according to the conditions described in common molecular cloning manuals. The experimental materials used in the following implementations, unless otherwise specified, were purchased from conventional biochemical reagent companies.

[0078] Example 1 Preparation of specific anti-human β-amyloid protein Aβ40 monoclonal antibody

[0079] 1. Materials

[0080] 1. Recombinant Aβ25-40 protein was synthesized by the company (Shanghai Sangon Biotechnology Co., Ltd.)

[0081] 2. Freund's complete adjuvant (Sigma, Germany)

[0082] 3. Freund's incomplete adjuvant (Sigma, Germany)

[0083] 4. Pristane (Sigma, Germany)

[0084] 5. PEG 4000 (polyethylene glycol) (Sigma, Germany)

[0085] 6. Fetal bovine serum (Hangzhou Tianhang Biotechnology Co., Ltd.)

[0086] 7. HAT (Sigma, Germany)

[0087] 8. HT (Sigma, Germany)

[0088] 9. Hepes (Biological Industries)

[0089] 10. Penicillin-Streptomycin (Biological Industries)

[0090] 11. L-Glutamine (Biological Industries)

[0091] 12. BCA kit (Pierce, USA)

[0092] 2. Methods

[0093] 1. Preparation of Immunogen

[0094] Preparation of immunogen: Human amyloid β-protein 25-40 polypeptide GSNKGAIIGLMV GGVV (SEQ ID NO. 31) coupled protein was used as the immunogen. When synthesizing Aβ25-40 polypeptide, cysteine ​​C was added to the N-terminus for coupling with carrier protein KLH.

[0095] 2. Immunization of Mice

[0096] Female BALB / c mice aged 6 to 8 weeks were selected and immunized with Aβ25-40 for a total of 5 times: the first immunization was performed by intraperitoneal injection of 100 μg of Aβ25-40 coupling protein and an equal volume of Freund's complete adjuvant for each mouse after full emulsification. The second immunization was performed one month later, and 60 μg of immunogen was fully emulsified with an equal volume of Freund's incomplete adjuvant for each mouse and intraperitoneal injection. Serum was collected from the corner of the eye one week after the injection to detect the titer. The third immunization was performed one month later, and the fourth booster immunization was performed two months later. The methods were the same as the second immunization. Finally, shock immunization was performed, and 100 μg of immunogen was injected intraperitoneally into each mouse. Splenocytes were taken 3 to 4 days after the injection and fused with Sp2 / 0-AG14 cells.

[0097] 3. Preparation of Feeder Cells

[0098] 6-8 week old female retired BALB / c mice were killed by cervical dislocation. The abdominal cavity was flushed several times with incomplete RPMI 1640 medium under sterile conditions. The flushing solution was centrifuged at 1000 rpm for 10 min and the supernatant was discarded. The cells were resuspended with complete RPMI 1640 medium and the cells were counted. The cell concentration was adjusted to 2×10 5 / mL, added into 96-well plate, 100 μL / well, and incubated in a 37°C, 5% CO2 incubator.

[0099] 4. Cell Fusion

[0100] Take mice 3-4 days after shock immunization, bleed their eyes, and collect pre-fusion serum. Kill mice by cervical induction and remove spleen aseptically, prepare single cell suspension, mix spleen cells with Sp2 / 0-AG14 cells in logarithmic growth phase at a ratio of 4:1, centrifuge at 1000rpm for 10min, and discard supernatant. Gently shake to loosen the cells. Preheat 50% PEG at 37℃, and take 1mL and drip it into the cells, drip it for 1min, and let it stand for 1-2min. Preheat incomplete RPMI 1640 medium at 37℃, drip 1mL at 2min; drip 2mL at 2min; drip 3mL at 2min; drip 5mL, 6mL and 7mL at 2min, shake while adding. After the dripping is completed, centrifuge at 1000rpm for 10min, and discard supernatant. Resuspend the cells with 40mL-60mL of complete RPMI 1640 medium and inoculate them into 96-well plates containing feeder cells, 100μL / well, 5×HAT screening. Incubate in a 37°C, 5% CO2 incubator and observe cell growth status day by day. On the 5th day after fusion, use 1×HAT to replace the cell medium, and on the 14th day, use 1×HT to replace the cell medium. Calculate the clone growth rate according to the following formula.

[0101] Cloning growth rate (%) = (cell clone growth well / inoculation well) × 100%

[0102] 5. Screening of positive clones

[0103] When the cell clones grow to 1 / 3 of the visual field, carefully aspirate the cell supernatant, use Aβ25-40-BSA, Aβ40 oligomers, Aβ42 oligomers and activated BSA as coating antigens, and detect the antibodies in the supernatant by indirect ELISA method (for specific methods and judgment criteria, refer to "Example 3 Indirect ELISA"). The clone wells where only the coating proteins are Aβ25-40-BSA protein and Aβ40 oligomer protein and have signals are positive wells.

[0104] Aβ25-40-BSA protein, Aβ1-40 oligomer protein (DAEFRH DSGYEV HHQKLV FFAEDVGSNKGAIIGLMV GGVV, SEQ ID NO.32), Aβ1-42 oligomer protein (DAEFRH DSGYEV HHQKLVFFAEDVGSNKGAIIGLMV GGVVIA, SEQ ID NO.33) and activated BSA protein were used for coating, and positive clones were screened from 600 original clones. The OD value detection results of some original clone wells are shown in Table 1. Note: Clone well AB5H can recognize Aβ25-40-BSA protein and Aβ40 oligomer protein and is a positive well.

[0105] Table 1 OD 450nm detection of some original clone wells

[0106]

[0107] 6. Establishment of hybridoma cell lines

[0108] The positive clones screened out are subcloned to obtain monoclonal cells that divide and grow to ensure that the properties of the antibodies produced are the same.

[0109] Subcloning: Use limiting dilution method to clone the antibody secretion positive wells repeatedly until the antibody positive rate of supernatant in all monoclonal wells is 100%.

[0110] The cloned positive clones were transferred into 24-well plates, 6-well plates and T25 cell culture flasks in sequence, and stably cultured for 90 days to establish strains.

[0111] 7. Preparation and purification of specific monoclonal antibodies against human amyloid protein Aβ40

[0112] 1.5×10 6 Hybridoma cells AB5H were inoculated into the CELLine1000 bioreactor at 10 cells / mL. After the cells were collected, the monoclonal antibody AB5H was purified by Protein A affinity chromatography using the AKTAexplore 100 protein purification system. The purified antibody was detected by SDS-PAGE. Figure 1 As shown, M is a protein molecular weight marker, mAb is a monoclonal antibody AB5H, and heavy chain and light chain bands are displayed.

[0113] 8. Identification of specific anti-human microtubule-associated protein monoclonal antibody subtypes

[0114] According to the mouse IgG isotype identification kit of Sigma Company, the IgG subtype of the specific monoclonal antibody AB5H secreted by the hybridoma cell line is IgG1.

[0115] Example 2 Cloning of the variable region gene of the specific anti-human amyloid β-protein 1-40 monoclonal antibody

[0116] 1. Materials

[0117] 1. GoldScript cDNA Synthesis Kit (Invitrogen Trading Co., Ltd.)

[0118] 2. 10mM each dNTP (NEB, USA)

[0119] 3. Q5 Reaction Buffer Pack (NEB, USA)

[0120] 4. Q5 High-Fidelity DNA Polymerase (NEB, USA)

[0121] 2. Methods

[0122] Hybridoma cells in the logarithmic growth phase were taken, and the total RNA of the hybridoma cell line was extracted using the Trizol reagent method, and 1% agarose gel electrophoresis was performed. The heavy chain variable region gene and light chain variable region gene of the anti-human microtubule-associated protein monoclonal antibody gene were amplified by the RT-PCR method. Primers were designed according to the conserved sequences of the light chain and heavy chain of mouse IgG, respectively. According to the instructions of the GoldScript cDNA synthesis kit, total RNA was used as a template, and the first chain of cDNA was synthesized using Oligo(d)T primer and SuperScriptII reverse transcriptase.

[0123] The specific reverse transcription reaction is as follows: 2μg total RNA, 1μL 0.5μg / μl Oligo(d)T12-18, 1μL 10mM dNTP mix, add DEPC-treated water to 10μL. Incubate at 65℃ for 5min and place on ice for 1min. Continue to add 9μL of 2× reaction mixture (2μL 10×RT buffer, 4μL 25mM MgCl2, 2μL 0.1M DTT, 1μL recombinant RNase inhibitor) to the reaction solution and mix gently, centrifuge briefly. Incubate at 42℃ for 2min. Then add 1μL GoldScript RT to the tube, incubate at 42℃ for 50min, incubate at 70℃ for 15min, terminate the reaction, and cool on ice. After the reverse transcription is completed, add 1μL RNaseH and incubate at 37℃ for 20min so that the product contains only the first chain of cDNA.

[0124] The variable region genes of antibody heavy chain (VH) and light chain (VL) were amplified using the universal primers of monoclonal antibody light and heavy chain variable regions and cDNA first strand as template. The amplified products VH and VL were identified by 1% agarose gel electrophoresis. Figure 2 In the figure, M is the DL2000 DNA molecular weight marker, 1 is the monoclonal antibody AB5H light chain variable region gene, and 2 is the monoclonal antibody AB5H heavy chain variable region gene.

[0125] Among them, the PCR amplification reaction was carried out according to the conventional method: using the above product as a template, the heavy chain and light chain primers were used to amplify the antibody heavy chain variable region gene and light chain variable region gene respectively. The reaction system is: cDNA 2μL, upstream and downstream primers (10mmol / L) 1μL each, 10mmol / L dNTP 4μL, 5×Q5 Buffer 10μL, Q5 DNA polymerase 0.5μL, add sterile water to 50μL. Mix well, centrifuge briefly and react in the PCR instrument. The PCR reaction conditions are: 98℃ pre-denaturation for 30s, cycle parameters are 98℃ denaturation for 10s, 55℃ annealing for 40s, 72℃ extension for 30s, a total of 30 cycles, and finally 72℃ extension for 10min.

[0126] After being recovered and purified using a PCR product recovery kit (Omega), it was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing analysis. The results are as follows:

[0127] The nucleotide sequence of the heavy chain variable region gene is specifically: GTC CAG GCG CAG CAG TCT GGG GCA GAGCTT GTG AAG CCA GGG GCC TCA GTC AAG TTG TCC TGC ACA GCT TCTGGC TTC AAC TTCAGT GAC ACC TATATG CAC TGG GTG AAG CAG AGG CCTGAA CAG GGC CTG GAG TGG ATTGGAAGG ATT GAT CCT GGAAGG GGT AATACTAAA TAT GAC CCGAAG TTC CAG GGCAAGGCCACTATAACAGCA GACACA TCC TCC AAC ACA GCC TAC CTGCAG CTC AGC AGC CTG ACA TCTGAGGAC ACT GCC GTC TAT TAC TGT GCC CCC CGG TAC TTT TAC TTT GAC TACTGG GCCAAGGCACCACTC TCACAG (as shown in SEQ ID NO.29).

[0128] The nucleotide sequence of the light chain variable region gene is specifically: GAT GTT GTGATGACC CAGACT CCA CTC ACTTTG TCG GTT ACC ATT GGA CAA CCA GCC TCC ATT TTT TGC AAGTCA AGT CAG AGC CATTGT CAT AGT GAT GGA AAG ACA TAT TTG AAT TGGTTG TTA CAG AGG CCA GGC CAG TTTCCA AAG CGC CTAATT TAT TTG GTGTTT AAA CTG GAC TTT GGA GTC CCT GAC AGG TTC ACTGGC AGT GGA TCAGGG ACA GAT TTC ACA CTG AAAATCAGC AGA GTG GAG GCT GAG GATTTGGGA GTT TAT TAT TGC TGG CAA AGG ACA CAT TTT CCG CTC ACG TTC GGTGATGGGACCAAG CTG (as shown in SEQ ID NO.30).

[0129] Example 3 Indirect ELISA

[0130] 1. Materials

[0131] 1. 96-well detachable ELISA plate (purchased from Corning)

[0132] 2. HRP-labeled goat anti-mouse IgG (Beijing Langbolide Biotechnology Co., Ltd.)

[0133] 3. TMB (Surmodics)

[0134] 2. Methods

[0135] 1. Coating: Take 400 ng / ml Aβ1-40 protein as the coating antigen, dissolve it in coating buffer (pH 9.6, 0.05 mol / L carbonate buffer), add it to a 96-well ELISA plate, 50 μL per well, and incubate overnight at 4°C.

[0136] 2. Use PBST (NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.44 g, Tween-20 500 μL, add deionized water to 1 L, adjust the pH to 7.2-7.4) to wash the plate 3 times, each time for 3 min.

[0137] 3. Blocking: Add 10% FBS, 200 μL per well, and incubate at 37°C for 2 h.

[0138] 4. Wash the plate 3 times with PBST, 3 min each time.

[0139] 5. Add 50 μL of monoclonal antibody AB5H diluted in 5% FBS to each well of a 96-well plate. Incubate at room temperature for 1.5 hours. Set up a negative control (using antibody diluent 5% FBS).

[0140] 6. Wash the plate 3 times with PBST, 3 min each time.

[0141] 7. Add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:3000 to each well and incubate at 37°C for 30 min.

[0142] 8. Wash the plate 3 times with PBST, 3 min each time.

[0143] 9. Add substrate TMB, 100 μL per well.

[0144] After 10 and 20 min, add 0.5 M stop solution (H2SO4) (add 178.3 mL of deionized water and add 21.7 mL of concentrated sulfuric acid (98%) dropwise), and detect the OD value at a wavelength of 450 nm.

[0145] 3. Judgment Criteria

[0146] The positive judgment criteria are as follows:

[0147] (OD value of positive well - OD value of blank control) / (OD value of negative well - OD value of blank control)≥2.1

[0148] The maximum dilution detected is the titer of the antibody.

[0149] IV. Results

[0150] The Aβ40 oligomer protein was used for coating, and the antibody titer in the supernatant of the cell reactor is shown in Table 2. Description: Monoclonal antibody AB5H can recognize recombinant Aβ40 oligomer protein, and the reaction titer is greater than 256,000.

[0151] Table 2 Titer of monoclonal antibody AB5H

[0152] Dilution ratio of monoclonal antibody AB5H OD450nm 1:4000 2.82 1:8000 2.77 1:16000 2.73 1:32000 2.57 1:64000 2.32 1:128000 1.9 1:256000 1.39 Negative control (5% FBS) 0.06

[0153] Example 4 Immunohistochemistry

[0154] 1. Materials

[0155] 1. Xylene (Sinopharm Group Chemical Reagent Co., Ltd.)

[0156] 2. EDTA (pH 9.0) antigen retrieval solution (Servicebio)

[0157] 3. 3% hydrogen peroxide (Servicebio)

[0158] 4. BSA (bioshark)

[0159] 5. Hematoxylin stain (Servicebio)

[0160] 6. Hematoxylin differentiation solution (Servicebio)

[0161] 7. Hematoxylin blueing solution (Servicebio)

[0162] 8. Neutral gum (Sinopharm Chemical Reagent Co., Ltd.)

[0163] 9. Secondary antibody: HRP-labeled goat anti-mouse (Servicebio)

[0164] 10. Histochemistry kit DAB colorimetric reagent (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.)

[0165] 2. Methods

[0166] Dewax the paraffin sections to water: put the sections into xylene I for 10 min - xylene II for 10 min - xylene III for 10 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 85% alcohol for 5 min - 75% alcohol for 5 min - distilled water to wash.

[0167] Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 9.0) in a microwave oven for antigen repair, boil for 8 minutes on medium heat, keep warm for 8 minutes, and then turn to medium-low heat for 10 minutes. During this process, prevent excessive evaporation of the buffer and do not dry the slides. After natural cooling, place the slides in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0168] Block endogenous peroxidase: Place the slices in 3% hydrogen peroxide solution, incubate at room temperature in the dark for 25 minutes, place the slides in PBS (pH 7.4) and wash on a decolorizing shaker 3 times, 5 minutes each time. Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue, and block at room temperature for 30 minutes.

[0169] Add primary antibody: Gently shake off the blocking solution, add primary antibody prepared in PBS at a ratio of 1:100 on the slices, and place the slices flat in a humidified box and incubate overnight at 4°C (add a small amount of water in the humidified box to prevent evaporation of the antibody).

[0170] Adding secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. After the slices are slightly dried, add HRP-labeled goat anti-mouse secondary antibody to cover the tissue in the circle and incubate at room temperature for 50 minutes. DAB color development: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle, control the color development time under a microscope, and rinse the slices with tap water to stop the color development. Counterstaining of cell nuclei: Counterstaining with hematoxylin for about 3 minutes, washing with tap water, differentiation with hematoxylin differentiation solution for a few seconds, washing with tap water, blueing with hematoxylin blueing solution, and washing with running water. Dehydration and sealing: Place the slices in 75% alcohol for 5 minutes-85% alcohol for 5 minutes-absolute ethanol I for 5 minutes-absolute ethanol II for 5 minutes-xylene I for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene and dry them slightly, and seal them with neutral gum. Microscope examination, image acquisition and analysis. Hematoxylin stains the cell nucleus in blue, and DAB shows positive expression in brown-yellow.

[0171] 3. Results

[0172] like Figure 3 As shown, the red arrow indicates the Aβ40 signal, indicating that the monoclonal antibody AB5H can specifically recognize the β-amyloid protein deposits outside the brain tissue cells and in the vascular walls of APP / PS1 transgenic AD model mice.

[0173] Example 5 Establishment and optimization of human Aβ40 double antibody sandwich ELISA detection method

[0174] 1. Materials

[0175] 1. Aβ monoclonal antibody 6E10; Aβ monoclonal antibody 4G8 (both biotin-conjugated) (Thermo Fisher, USA)

[0176] 2. Streptavidin-HRP (BioLegend, USA)

[0177] 2. Test methods

[0178] 1. Basic reaction process of double antibody sandwich ELISA

[0179] The basic reaction steps of the ELISA are as follows:

[0180] Monoclonal antibody coating: Use 1 μg / well monoclonal antibody AB5H as capture antibody, dilute with PBS, 50 μL / well, incubate overnight at 4°C;

[0181] Wash the plate: 300 μL / well 1×PBST (500 μL Tween-20 added to 1 L PBS (pH 7.4)), wash the plate 3 times, 3 min each time;

[0182] Blocking: add 200 μL of 5% skim milk (2.5 g skim milk dissolved in 50 mL 1× PBST) to each well and incubate at 37°C for 2 h;

[0183] Washing: 300 μL / well 1× PBST, wash 3 times, 3 min each time;

[0184] Add standard and biotin-conjugated dual detection antibody: 6E10-Bio

[0185] and 4G8-Bio dual detection antibody 1:4000 dilution, 50 μL per well; then the standard was serially diluted to 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.6 pg / mL, 7.8 pg / mL, 25 μL per well. Incubate overnight at 25°C;

[0186] Washing: 300 μL / well 1×PBST, wash 5 times, 3 min each time;

[0187] Add enzyme-labeled secondary antibody: dilute HRP-streptavidin antibody 1:1000 with 1% casein, 100 μL per well, incubate at 25°C for 1 h;

[0188] Washing: 300 μL / well 1× PBST, wash 5 times, 3 min each time;

[0189] Substrate color development: add 100 μL / well of color development solution and develop for 15 min in the dark;

[0190] Stop the reaction: add 50 μL of 0.5 mol / L sulfuric acid to each well and measure the OD 450nm reaction value.

[0191] The data of each test was processed to calculate the P / N value of the reaction. The reaction condition with the largest P / N value was used as the basis for judging the optimal reaction condition of ELISA. The calculation formula of the P / N value is as follows:

[0192] P / N value = mean OD 450nm value of positive control wells / mean OD 450nm value of negative control wells.

[0193] 2. Establishment of the calibration curve of double antibody sandwich ELISA for detecting Aβ1-40 protein

[0194] Aβ40 oligomers were diluted to 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, 15.6 pg / mL, and 7.8 pg / mL, and tested according to the reaction flow of test method 1. The obtained calibration curve is shown in the figure. Figure 4As can be seen from the figure, when the concentration of Aβ40 oligomers is between 7.8 and 250 pg / mL, the linear relationship is good. Linear regression equation y = 0.0118x-0.0183, R 2 =0.9972. Therefore, the detection range of the calibration curve is 7.8-250 pg / mL, and the detection limit is 7.8 pg / mL.

[0195] 3. Identification of the specificity of the double antibody sandwich ELISA detection method

[0196] Since the monoclonal antibody AB5H is specific to Aβ40 and does not recognize Aβ42, the results are shown in Table 3. Using Aβ40 and Aβ42 as different calibrants, there is a signal only when the calibrant is Aβ40 oligomer, and there is no obvious cross reaction with other substances. This shows that the established double antibody sandwich ELISA detection method has good specificity.

[0197] Table 3 Specificity detection of monoclonal antibody AB5H

[0198]

[0199] Example 6 Detection of plasma samples

[0200] 1. Collection and processing of peripheral blood

[0201] Plasma: When collecting blood, fill the entire blood collection tube (EDTA-Na2 is used as an anticoagulant), gently invert and mix 10-12 times; centrifuge at 4°C, 1500g for 15 minutes; transfer the upper plasma to another test tube. Package according to test requirements and store at -80°C.

[0202] 2. Sample testing

[0203] Thaw the recombinant Aβ40 standard and plasma on ice in advance. Use 1% casein to dilute the standard to 250pg / mL, 125pg / mL, 62.5pg / mL, 31.25pg / mL, 15.6pg / mL and 7.8pg / mL in sequence, and dilute the detection antibody at a ratio of 1:4000 with 1% casein. Add 50μL of Aβ40 standard and plasma to the coated ELISA plate per well; then add 50μL of detection antibody working solution per well. Tap the border of the ELISA plate to mix, react at room temperature, add 100μL of HRP-labeled streptavidin (1% casein diluted 1:2000) to each well, develop color, stop, and measure the OD value at a wavelength of 450nm with an ELISA reader.

[0204] When only blood Aβ42 was tested, it was not possible to distinguish between the AD and normal groups, so Aβ40 testing was added. The results of calibrating Aβ42 on plasma samples from normal people and AD patients are shown in Table 4. Figure 5 The scatter plot of Aβ42 / Aβ40 in plasma samples of normal people and AD patients is shown in Table 4 and Figure 5 It can be seen that the double antibody sandwich ELISA detection method can identify the content of Aβ40 protein in plasma, and can distinguish normal people from AD patients by jointly analyzing the results of the content of Aβ42 protein in plasma. According to this system, the initial blood Aβ detection threshold is as follows: Aβ42 / Aβ40 ratio less than 0.08 or 0.1 is high risk, indicating AD; Aβ42 / Aβ40 ratio 0.1 to 0.16 (inclusive) is medium risk, indicating MCI; Aβ42 / Aβ40 ratio greater than 0.16 (inclusive) is low risk, indicating routine observation. This set of detection thresholds has not been reported yet.

[0205] Table 4 Double antibody sandwich ELISA method for detecting plasma

[0206]

[0207] 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 hybridoma cell line, characterized in that: The hybridoma cell line is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C201255.

2. Use of the hybridoma cell line according to claim 1 in preparing a monoclonal antibody that specifically recognizes human amyloid protein Aβ40.

3. A monoclonal antibody, characterized in that The monoclonal antibody is secreted and produced by the hybridoma cell line according to claim 1.

4. A monoclonal antibody, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody includes: a heavy chain complementary determining region CDR1 as shown in SEQ ID NO.1, a heavy chain complementary determining region CDR2 as shown in SEQ ID NO.2, and a heavy chain complementary determining region CDR3 as shown in SEQ ID NO.3; the amino acid sequence of the light chain variable region of the monoclonal antibody includes: a light chain complementary determining region CDR1 as shown in SEQ ID NO.8, a light chain complementary determining region CDR2 with an amino acid sequence of LVF, and a light chain complementary determining region CDR3 as shown in SEQ ID NO.

9.

5. The monoclonal antibody according to claim 4, characterized in that: The heavy chain variable region of the monoclonal antibody has at least 90% sequence identity with the amino acid sequence selected from SEQ ID NO.13; the light chain variable region of the monoclonal antibody has at least 90% sequence identity with the amino acid sequence selected from SEQ ID NO.

14.

6. A nucleic acid molecule encoding the monoclonal antibody according to any one of claims 3 to 5.

7. A recombinant vector, characterized in that: The nucleic acid molecule according to claim 6 is inserted.

8. A host cell, characterized in that Comprising the nucleic acid molecule according to claim 6 or the recombinant vector according to claim 7.

9. Use of the hybridoma cell line according to claim 1, the monoclonal antibodies according to claims 3 to 5, the nucleic acid molecule according to claim 6, the recombinant vector according to claim 7 or the host cell according to claim 8 in the preparation of products for diagnosis or auxiliary diagnosis of Alzheimer's disease.

10. A product for diagnosing or assisting in the diagnosis of Alzheimer's disease, characterized in that: The monoclonal antibody comprises the monoclonal antibody according to any one of claims 3 to 5.

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