ALP monoclonal antibody and its application

Abstract

The present invention discloses ALP monoclonal antibodies and applications thereof, belonging to the field of biotechnology. The ALP monoclonal antibodies of the present invention include monoclonal antibodies 1-G5-1 and monoclonal antibodies 7-H10-2; monoclonal antibody 1-G5-1, which comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO:1-3, and VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO:4-6; monoclonal antibody 7-H10-2, which comprises VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO:7-9, and VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO:10-12. The ALP monoclonal antibody combination of the present invention can bind to different epitopes of alkaline phosphatase to form a double antibody sandwich mode, thereby achieving highly sensitive and specific detection of ALP, which is beneficial to the early diagnosis of cancer using ALP as a biomarker.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an ALP monoclonal antibody and an application thereof. Background Art

[0002] Alkaline phosphatase (ALP) is a hydrolase widely distributed in prokaryotic and eukaryotic cells. It can remove the phosphate groups of various phosphorylated substrates under alkaline conditions and catalyze the hydrolysis of phosphate monoesters (Coleman J E. Annu. Rev. Biophys. Biomol. Struct., 1992, 21: 441-483.). Each ALP molecule is composed of two structurally similar monomers, each of which contains two zinc ions, one magnesium ion and five cysteine ​​residues (Guo JT, Yu HB, Cui TTJ Biomed. Mater. Res., 2021, 109(2): 214-226.). ALP has four isoenzymes in the human body, namely placental alkaline phosphatase, germ cell alkaline phosphatase, intestinal alkaline phosphatase and tissue nonspecific alkaline phosphatase (Millán J L. Purinergic Signal., 2006, 2: 335-341.).

[0003] ALP is widely present in human tissues and is involved in many processes such as cell cycle, growth, apoptosis and signal transduction. ALP in serum mainly comes from liver, bone and kidney tissues, and its activity changes are closely related to many diseases, such as osteoporosis, osteomalacia, biliary obstruction, cirrhosis and sepsis. At the same time, ALP is an important biomarker for some cancers, such as bone cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer. In addition, ALP can also hydrolyze adenosine monophosphate to generate adenosine, which can be used as a cell signaling molecule. Therefore, the sensitive detection of ALP is of great significance for clinical disease diagnosis and biomedical research.

[0004] The detection methods of ALP mainly include: colorimetry, electrochemical method, surface enhanced Raman scattering method and fluorescence analysis method, among which, colorimetry has the advantages of intuitive results and no need for complex and expensive instruments, but the sensitivity is low; electrochemical method has the advantages of good selectivity, low cost and portable instruments, but has the defects of relatively weak signal, complex synthesis of ALP substrate and the need for multiple tool enzymes; surface enhanced Raman scattering method has the advantages of high sensitivity and the ability to provide molecular specific fingerprint spectra, but the complex operation and unstable Raman signal are still urgent problems to be solved; fluorescence analysis method has the advantages of high sensitivity, good selectivity, real-time in situ, low background interference and in vivo imaging ability, but it still has certain limitations in detection specificity, fluorescence penetration and imaging resolution. Monoclonal antibodies have the characteristics of high purity, high sensitivity, strong specificity and less cross-reaction. Therefore, the development of a monoclonal antibody combination that can specifically recognize ALP will be conducive to promoting the development of ALP detection and cancer diagnosis technology. Summary of the invention

[0005] In view of the above prior art, the purpose of the present invention is to provide an ALP monoclonal antibody and its application.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, there is provided an ALP monoclonal antibody combination, comprising: monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2;

[0008] The monoclonal antibody 1-G5-1 comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6;

[0009] The monoclonal antibody 7-H10-2 comprises VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are shown in SEQ ID NOs: 7-9, and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NOs: 10-12.

[0010] Further, the amino acid sequence of the heavy chain variable region of monoclonal antibody 1-G5-1 is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14;

[0011] The amino acid sequence of the heavy chain variable region of monoclonal antibody 7-H10-2 is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

[0012] Further, the amino acid sequence of the heavy chain of monoclonal antibody 1-G5-1 is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is shown in SEQ ID NO: 18;

[0013] The amino acid sequence of the heavy chain of monoclonal anti-7-H10-2 is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

[0014] The second aspect of the present invention provides a gene encoding the monoclonal antibody 1-G5-1.

[0015] Preferably, the encoding gene includes: a DNA sequence shown in SEQ ID NO:21, which is used to encode the heavy chain of monoclonal antibody 1-G5-1; and a DNA sequence shown in SEQ ID NO:22, which is used to encode the light chain of monoclonal antibody 1-G5-1.

[0016] The third aspect of the present invention provides a gene encoding the monoclonal antibody 7-H10-2.

[0017] Preferably, the encoding gene includes: a DNA sequence as shown in SEQ ID NO: 23, which is used to encode the heavy chain of monoclonal antibody 7-H10-2; and a DNA sequence as shown in SEQ ID NO: 24, which is used to encode the light chain of monoclonal antibody 7-H10-2.

[0018] The fourth aspect of the present invention provides the use of the above-mentioned ALP monoclonal antibody combination in the following (1) or (2):

[0019] (1) Preparing products for detecting ALP;

[0020] (2) Preparation of cancer diagnosis kits or reagents.

[0021] In the above application, the products for detecting ALP include but are not limited to: ELISA detection kit, colloidal gold detection kit and chemiluminescence immunoassay kit, etc.

[0022] In the above application, the cancer uses ALP as a biomarker, including but not limited to: bone cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer.

[0023] In a fifth aspect, the present invention provides an ELISA kit for detecting ALP, wherein the ELISA kit contains the above-mentioned ALP monoclonal antibody combination.

[0024] Preferably, the ELISA kit uses monoclonal antibody 1-G5-1 as the capture antibody and monoclonal antibody 7-H10-2 as the detection antibody.

[0025] Beneficial effects of the present invention:

[0026] (1) The ALP monoclonal antibody 1-G5-1 and the monoclonal antibody 7-H10-2 developed and designed by the present invention have high affinity and specific recognition ability for alkaline phosphatase, providing a new antibody source for the detection of ALP.

[0027] (2) The ALP monoclonal antibody combination of the present invention can bind to different epitopes of alkaline phosphatase to form a double antibody sandwich model, thereby achieving highly sensitive and specific detection of ALP, which is beneficial for early diagnosis of cancer using ALP as a biomarker. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : SDS-PAGE detection results of the recombinant human ALP protein expressed in Example 1.

[0029] Figure 2 : ALP sensitivity test results; the ordinate in the figure refers to the OD value at 450nm.

[0030] Figure 3 : Specificity detection results of monoclonal antibody 1-G5-1; the ordinate in the figure refers to the OD value at 450nm.

[0031] Figure 4 : Specificity detection results of monoclonal antibody 7-H10-2; the ordinate in the figure refers to the OD value at 450nm. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] As mentioned above, ALP is an important biomarker for many cancers, and the sensitive detection of ALP is of great significance for clinical disease diagnosis and biomedical research.

[0034] Based on this, the present invention has developed and designed a monoclonal antibody capable of recognizing ALP. The present invention immunizes mice with exogenously expressed recombinant human ALP protein to obtain immune mice, and then uses lymphocytes of the immune mice to fuse with myeloma cells; then screens to obtain 10 hybridoma cell lines, and then prepares ascites antibodies and purifies them to obtain 10 monoclonal antibodies.

[0035] Since the specificity of monoclonal antibodies secreted by different hybrid cell lines to recognize ALP may vary, the present invention uses the 10 obtained monoclonal antibodies as capture antibodies and detection antibodies, respectively, for a total of 90 paired combinations. The 90 combinations were used to detect ALP samples, and the paired combinations with the best detection effect were screened out. It was found that monoclonal antibody 1-G5-1 was used as the capture antibody and monoclonal antibody 7-H10-2 was used as the detection antibody, and its detection effect on ALP was the best. Therefore, the combination of monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2 was used for specific recognition and detection of ALP, and the sequences and structures of the obtained monoclonal antibodies 1-G5-1 and monoclonal antibodies 7-H10-2 were analyzed, thereby proposing the present invention.

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0037] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0038] Example 1: Preparation of ALP monoclonal antibody

[0039] (1) Expression of recombinant human ALP protein in Escherichia coli;

[0040] Select the appropriate human ALP protein sequence (NP_112603.2alkaline phosphatase, germ cell type preproprotein [Homo sapiens]) from NCBI. Entrust Suzhou Jinweizhi Company to synthesize the expression vector: plasmid: pET-28a (+), 5' restriction site: Ndel, 3' restriction site: Xhol, expression strain: BL21 (DE3);

[0041] Mix 100ng of plasmid and 20ul competent cells, place on ice for 30min; heat shock for 45s, place on ice for 2min, culture at 37℃, 200rpm for 1h, centrifuge at 2000rpm, and keep a small amount of supernatant to coat Kana resistance plates. Culture overnight at 37℃ incubator; the next day, expand the culture with 1mM IPTG and induce at 37℃ for 4h; after successful expression, increase the volume of the culture, perform protein purification, and detect the protein expression and purification effect by SDS-PAGE. The results are as follows Figure 1 shown.

[0042] (2) Rapidly immunize mice with recombinant human ALP protein to obtain immune mice, use lymphocytes from the immune mice to fuse with myeloma cells, and then screen positive cells and perform subcloning.

[0043] The mouse immunization process is as follows:

[0044] First immunization: 100 μg recombinant human ALP protein and complete Freund's adjuvant were completely mixed in a volume ratio of 1:1 and injected subcutaneously into the hind foot pad of mice;

[0045] Second immunization: The second immunization was performed 2 weeks after the first immunization. 100 μg of recombinant human ALP protein and incomplete Freund's adjuvant were completely mixed in a volume ratio of 1:1 and injected subcutaneously into the hind foot pad of mice.

[0046] The third immunization: 2 weeks after the second immunization, 100 μg recombinant human ALP protein and PBS buffer were mixed in a volume ratio of 1:1 and injected intraperitoneally;

[0047] Ten days after the third immunization, periocular blood of mice was collected for titer testing;

[0048] Booster immunization: Three days before fusion, 100 μg of recombinant human ALP protein was mixed with PBS buffer at a volume ratio of 1:1 and then injected into the tail vein of mice.

[0049] The specific operation of the fusion experiment of lymphocytes and myeloma cells is as follows:

[0050] Myeloma cells and lymphocytes were mixed at a ratio of 1:5 to obtain a cell mixture; the cell mixture was placed in a 50 ml centrifuge tube, diluted with RPMI-1640 basal medium, and then centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the centrifuge tube was shaken to make the cells uniform, 1 ml of 50% PEG was slowly added, reacted for 90 seconds, and then 10 ml of RPMI-1640 medium was added to terminate PEG, the fused cells were placed in a 37° C. water bath to react for 10 min, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and RPMI-1640 complete medium was added to resuspend the cells;

[0051] The fused cells were added to a 96-well plate, 100 μl per well; then the cell culture plate was placed in a CO 2 The cells were cultured in an incubator and 10 days after fusion, the cells in the positive wells were screened for fusion.

[0052] The specific operation of the subcloning is as follows:

[0053] Blow up the cells in the positive wells, take 10 μl and count as N, add PBS buffer to the centrifuge tube according to the limiting dilution method, take 100 μl of cell suspension into the centrifuge tube, blow it evenly, take 30 μl, add 500 μl RPMI-1640 complete medium, blow it evenly and inoculate it into semi-solid culture medium, after 6-8 days, select the monoclonal cell lines visible to the naked eye and inoculate them into 96-well plates, and then screen out the positive monoclonal cell lines.

[0054] (3) Screening the strongly positive cell lines obtained after subcloning in step (2) to prepare ascites, and purifying the ascites. The specific operation of purifying the ascites is as follows:

[0055] The protein A agarose gel medium was loaded into a nickel ion affinity chromatography column, and the ascites and PBS were mixed in equal amounts at a volume ratio of 1:1 and then slowly loaded. After the antibody was bound, it was eluted with glycine elution buffer, and finally 10 monoclonal antibodies (monoclonal antibody 1-G5-1 to monoclonal antibody 1-G5-5, monoclonal antibody 7-H10-1 to monoclonal antibody 7-H10-5) were purified.

[0056] Example 2: Screening of a monoclonal antibody combination that specifically recognizes ALP

[0057] The 10 monoclonal antibodies obtained in Example 1 were used as capture antibodies and detection antibodies, respectively, for ELISA sandwich antibody pairing, wherein the detection antibody was labeled with HRP. The ELISA detection process was as follows: coating with capture antibody, washing the plate three times, blocking, washing the plate three times, adding ALP antigen for incubation, washing the plate, adding detection antibody, incubating, washing the plate, adding TMB colorimetric solution, incubating at room temperature in the dark for 10-15 minutes, and then terminating, and reading OD with an enzyme reader. 450 The test results are shown in Table 1.

[0058] Table 1: Screening of paired antibodies by double antibody sandwich ELISA

[0059]

[0060] Note: “-” in the table represents OD 450 Value < 0.5, “+” represents OD 450 The value is high or low, "4+" represents OD 450 Value>4.0, “3+” represents OD 450 Value>3.0, “2+” represents OD 450 Value>2.0, “1+” represents OD 450 Value>1.0.OD 450 Higher values ​​indicate better pairing.

[0061] Therefore, the monoclonal antibody 1-G5-1 was selected as the capture antibody, and the monoclonal antibody 7-H10-2 was selected as the detection antibody. This pairing combination was used as the monoclonal antibody combination that specifically recognizes ALP.

[0062] Example 3: Sequence and structural analysis of monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2

[0063] Further sequence and structural analysis was performed on monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2, wherein:

[0064] The monoclonal antibody 1-G5-1 includes: a heavy chain (Heavy Chain) and a light chain (Light Chain); the amino acid sequence of the heavy chain of the monoclonal antibody 1-G5-1 is shown in SEQ ID NO: 17, and the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO: 21, which are as follows:

[0065] Amino Acid Sequence:

[0066] MDRLTSSFLLLIVPAYVLSQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWI RQPSGKGLEWLAHIWWDDVKYYNPSLKSQLAISKDTSRNQVFLKVTSVDTADTATYYC TRTDYFYIDYWGPGTTLTVSS.

[0067] Note: The shaded area is the heavy chain variable region; the italic bold area is the CDR (complementarity determining cluster) region.

[0068] Nucleotide sequence:

[0069] ATGGACAGGCTTACTTCTTCATTCCTGCTGCTGATTGTCCCTGCATATGTCTTGTCCCAAGTTACTCTAAAAGAGTCTGGCCCTGGAATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCT GGCACACATTTGGTGGGATGATGTTAAGTATTATAATCCATCCCTGAAGAGCCAGCTCGCAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGGTCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTACTCGAACTGATTACTTCTACATTGACTACTGGGGCCCAGGCACCACTCTCACAGTCTCCTCA.

[0070] Note: The shaded area is the coding sequence of the heavy chain variable region.

[0071] The heavy chain variable region of monoclonal antibody 1-G5-1 comprises three CDR regions, namely: VHCDR1, VHCDR2 and VHCDR3, whose amino acid sequences are shown in SEQ ID NO: 1-3, and are specifically as follows:

[0072] VHCDR1:TSGMGVG; (SEQ ID NO:1)

[0073] VHCDR2:HIWWDDVKYYNPSLKS; (SEQ ID NO:2)

[0074] VHCDR3: TDYFYIDY; (SEQ ID NO: 3)

[0075] The amino acid sequence of the heavy chain variable region of monoclonal antibody 1-G5-1 is shown in SEQ ID NO.13, and is as follows:

[0076] QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWD DVKYYNPSLKSQLAISKDTSRNQVFLKVTSVDTADTATYYCTRTDYFYIDYWGPGTTLTV SS.

[0077] The amino acid sequence of the light chain of monoclonal antibody 1-G5-1 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 22, which are as follows:

[0078] Amino Acid Sequence:

[0079] MESQTQVFLSLLLWVSGTCGNIMMTQSPSSLAVSAGEKVTMSCKSSQSVFNSSNQK NYLAWYQQKPGQSPKLLIYWASTRESGVPYRFTGSGSGTDFTLTISSVQAEDLAVYYCH QFLSSWTFGGGTKLEIK.

[0080] Note: The shaded area is the light chain variable region; the italic bold area is the CDR (complementarity determining cluster) region.

[0081] Nucleotide sequence:

[0082] ATGGAATCACAGACTCAGGTCTTCCTCTCCCTGCTGCTCTGGGTATCTGGTACCTGTGGGAACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTTAACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCA GGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTTATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTGACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTTATTACTGTCATCAATTCCTCTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0083] Note: The shaded area is the coding sequence of the light chain variable region.

[0084] The light chain variable region of monoclonal antibody 1-G5-1 comprises three CDR regions, namely: VLCDR1, VLCDR2 and VLCDR3, whose amino acid sequences are shown in SEQ ID NO: 4-6, and are specifically as follows:

[0085] VLCDR1: KSSQSVFNSSNQKNYLA; (SEQ ID NO:4)

[0086] VLCDR2: WASTRES; (SEQ ID NO:5)

[0087] VLCDR3:HQFLSSWT; (SEQ ID NO:6)

[0088] The amino acid sequence of the light chain variable region of monoclonal antibody 1-G5-1 is shown in SEQ ID NO.14, and is as follows:

[0089] NIMMTQSPSSLAVSAGEKVTMSCKSSQSVFNSSNQKNYLAWYQQKPGQSPKLLIY WASTRESGVPYRFTGSGSGTDFTLTISSVQAEDLAVYYCHQFLSSWTFGGGTKLEIK

[0090] The monoclonal antibody 7-H10-2 includes: a heavy chain (Heavy Chain) and a light chain (Light Chain); the amino acid sequence of the heavy chain of the monoclonal antibody 7-H10-2 is shown in SEQ ID NO: 19, and the nucleotide sequence encoding the heavy chain is shown in SEQ ID NO: 23, which are as follows:

[0091] Amino Acid Sequence:

[0092] MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHW VKLSHGKSPEWVGGINPNNGGTSYNQKFQGKATLTVDKSSSTAYMELRSLTSEDSAVYY CVRSNWEIGMDYWGQGTSVTVSS.

[0093] Note: The shaded area is the heavy chain variable region; the italic bold area is the CDR (complementarity determining cluster) region.

[0094] Nucleotide sequence:

[0095] ATGGGATGGAGCTGGATCTTTCTCTTTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGCACTGGGTGAAGCTGAGCCATGGAAAGAGCCCTGAGTGGGTTGGAG GTATTAATCCTAACAATGGTGGTACTAGTTACAACCAGAAGTTCCAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAAGATTCTGCAGTCTATTACTGTGTAAGATCTAACTGGGAAATTGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0096] Note: The shaded area is the coding sequence of the heavy chain variable region.

[0097] The heavy chain variable region of monoclonal antibody 7-H10-2 comprises three CDR regions, namely: VHCDR1, VHCDR2 and VHCDR3, whose amino acid sequences are shown in SEQ ID NO: 7-9, and are specifically as follows:

[0098] VHCDR1:EYTMH; (SEQ ID NO:7)

[0099] VHCDR2:GINPNNGGTSYNQKFQG; (SEQ ID NO:8)

[0100] VHCDR3: SNWEIGMDY; (SEQ ID NO:9).

[0101] The amino acid sequence of the heavy chain variable region of monoclonal antibody 7-H10-2 is shown in SEQ ID NO.15, and is as follows:

[0102] EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKLSHGKSPEWVGGINPNNGGTSYNQKFQGKATLTVDKSSSTAYMELRSLTSEDSAVYYCVRSNWEIGMDYWGQGTSVTVSS

[0103] The amino acid sequence of the light chain of monoclonal antibody 7-H10-2 is shown in SEQ ID NO: 20, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO: 24, which are as follows:

[0104] Amino Acid Sequence:

[0105] MSPAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTH FPFTFGSGTKLEIK.

[0106] Note: The shaded area is the light chain variable region; the italic bold area is the CDR (complementarity determining cluster) region.

[0107] Nucleotide sequence:

[0108] ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCC AGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAG

[0109] Note: The shaded area is the coding sequence of the light chain variable region.

[0110] The light chain variable region of monoclonal antibody 7-H10-2 comprises three CDR regions, namely: VLCDR1, VLCDR2 and VLCDR3, whose amino acid sequences are shown in SEQ ID NO: 10-12, and are specifically as follows:

[0111] VLCDR1: KSSQSLLYSNGKTYLN; (SEQ ID NO:10)

[0112] VLCDR2:LVSKLDS; (SEQ ID NO:11)

[0113] VLCDR3: VQGTHFPFT; (SEQ ID NO: 12).

[0114] The amino acid sequence of the light chain variable region of monoclonal antibody 7-H10-2 is shown in SEQ ID NO.16, and is as follows:

[0115] DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSK LDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIK.

[0116] The monoclonal antibody 1-G5-1 and the monoclonal antibody 7-H10-2 obtained by the present invention have specific CDR regions, which are significantly different from the monoclonal antibodies reported previously; and the combination of the monoclonal antibody 1-G5-1 and the monoclonal antibody 7-H10-2 has excellent affinity and specific recognition performance for human ALP.

[0117] Example 4: Construction and methodological investigation of ELISA kit for detecting ALP

[0118] 1. Construction and detection of ELISA kit:

[0119] An ELISA detection kit was constructed using monoclonal antibody 1-G5-1 as the capture antibody and monoclonal antibody 7-H10-2 as the detection antibody. The specific detection steps are as follows:

[0120] (1) Coating: Dilute the capture antibody 2-9D to a working concentration of 2 μg / ml, add 100 μL per well to the coating plate, and coat overnight at 4°C;

[0121] (2) Blocking: Wash three times with PBST (containing 0.05% Tween), block with 5% skim milk powder, 200 μL per well, incubate at 37°C for 1 h;

[0122] (3) Adding antigen: Wash three times with PBST, pat dry and add 80 μL of AFP antigen; incubate at 37°C for 1 h;

[0123] (4) Adding secondary antibody: Wash 3-5 times with PBST, dilute the secondary antibody (HRP-labeled detection antibody 2-3D) 8000 times with 5% skim milk powder, add 50 μl to each well, and incubate at 37°C for 1 h;

[0124] (5) Wash with PBST 5 times, 3 min / time, pat dry and add colorimetric solution, incubate at room temperature in dark for 10 min, and read OD with a microplate reader 450 value.

[0125] 2. Methodological investigation:

[0126] (1) Sensitivity:

[0127] ALP protein with a concentration of 20 μg / ml, 2 μg / ml, 200 ng / ml, 20 ng / ml, 2 ng / ml, 200 pg / ml, and 20 pg / ml was used as an antigen, and the ELISA kit constructed above was used for detection, and the detection curve was drawn ( Figure 2 ).

[0128] The detection sensitivity of the ELISA kit of the present invention to ALP is 0.2 ng / ml.

[0129] (2) Specificity:

[0130] Elisa detected the specificity of two monoclonal antibodies. The antigen was recombinant human ALP protein, and the plating concentration gradient was set to 2μg / ml (positive control without human serum), 2μg / ml, 1μg / ml, 0.2μg / ml, 0.04μg / ml, 0.008μg / ml, 0.

[0131] The culture supernatant of 1-G5-1 and 7-H10-2 monoclonal cells and human serum were mixed evenly at a volume ratio of 1:1 and incubated for 0.5 h. The mixture was added to the ELISA plate and incubated at 37°C for 1 h. The sample volume per well was 100 μl.

[0132] The secondary antibody was goat anti-mouse labeled with HRP, and TMB was added for color development after incubation for 0.5 h.

[0133] Specificity testing mainly depends on whether there are interfering substances in human serum that bind non-specifically to antibodies. As long as the result is similar to the positive control, and the OD value is positively correlated with the antigen concentration, the specificity can be considered good. Figure 3 and Figure 4 As shown, the results show that the antibody titer does not decrease significantly after adding human serum, and is positively correlated with the antigen concentration, indicating that there are no substances in human serum that are non-specifically recognized by antibodies to affect the detection, and the 1-G5-1 and 7-H10-2 monoclonal antibodies have good specificity.

[0134] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. ALP monoclonal antibody combination, It is characterized in that include: monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2; The monoclonal antibody 1-G5-1 comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6; The monoclonal antibody 7-H10-2 comprises VHCDR1, VHCDR2 and VHCDR3 whose amino acid sequences are shown in SEQ ID NOs: 7-9, and VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in SEQ ID NOs: 10-12.

2. The ALP monoclonal antibody combination according to claim 1, It is characterized in that The amino acid sequence of the heavy chain variable region of monoclonal antibody 1-G5-1 is shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 14; The amino acid sequence of the heavy chain variable region of monoclonal antibody 7-H10-2 is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

3. The ALP monoclonal antibody combination according to claim 1, It is characterized in that The amino acid sequence of the heavy chain of monoclonal antibody 1-G5-1 is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is shown in SEQ ID NO: 18; The amino acid sequence of the heavy chain of monoclonal anti-7-H10-2 is shown in SEQ ID NO: 19, and the amino acid sequence of the light chain is shown in SEQ ID NO:

20.

4. A gene encoding the monoclonal antibody 1-G5-1 according to claim 1.

5. The coding gene according to claim 4, It is characterized in that The encoding gene includes: a DNA sequence shown in SEQ ID NO: 21, which is used to encode the heavy chain of monoclonal antibody 1-G5-1; and a DNA sequence shown in SEQ ID NO: 22, which is used to encode the light chain of monoclonal antibody 1-G5-1.

6. A gene encoding the monoclonal antibody 7-H10-2 according to claim 1.

7. The coding gene according to claim 6, It is characterized in that The encoding gene includes: a DNA sequence shown in SEQ ID NO: 23, which is used to encode the heavy chain of the monoclonal antibody 7-H10-2; and a DNA sequence shown in SEQ ID NO: 24, which is used to encode the light chain of the monoclonal antibody 7-H10-2.

8. Use of the ALP monoclonal antibody combination according to any one of claims 1 to 3 in the following (1) or (2): (1) Preparation of products for detecting ALP; (2) Preparing a cancer diagnosis kit or reagent, wherein the cancer is: bone cancer, pancreatic cancer, ovarian cancer, breast cancer and prostate cancer.

9. The use according to claim 8, It is characterized in that The products for detecting ALP include: ELISA detection kit, colloidal gold detection kit and chemiluminescence immunoassay kit.

10. An ELISA kit for detecting ALP, It is characterized in that The ELISA kit contains the monoclonal antibody combination according to any one of claims 1 to 3.

11. The ELISA kit according to claim 10, It is characterized in that The ELISA kit uses monoclonal antibody 1-G5-1 as a capture antibody and monoclonal antibody 7-H10-2 as a detection antibody.

Images