CN116199783A

CN116199783A - ALP monoclonal antibody and application thereof

Info

Publication number: CN116199783A
Application number: CN202211654455.6A
Authority: CN
Inventors: 张祥伟
Original assignee: Shandong Narui Boen Biopharmaceutical Technology Co ltd
Current assignee: Shandong Narui Boen Biopharmaceutical Technology Co ltd
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-06-02

Abstract

The invention discloses an ALP monoclonal antibody and application thereof, and belongs to the technical field of biology. ALP monoclonal antibodies of the invention include monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2; a monoclonal antibody 1-G5-1 comprising VHCDR1, VHCDR2 and VHCDR3 having the amino acid sequences shown in SEQ ID NO 1-3, and VLCDR1, VLCDR2 and VLCDR3 having the amino acid sequences shown in SEQ ID NO 4-6; a monoclonal antibody 7-H10-2 comprising VHCDR1, VHCDR2 and VHCDR3 having the amino acid sequences shown in SEQ ID NOS 7-9 and VLCDR1, VLCDR2 and VLCDR3 having the amino acid sequences shown in SEQ ID NOS 10-12. The ALP monoclonal antibody combination can bind different epitopes of alkaline phosphatase to form a double-antibody sandwich mode, so that high-sensitivity and specificity detection of ALP is realized, and early diagnosis of cancers by taking the ALP as a biomarker is facilitated.

Description

ALP monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an ALP monoclonal antibody and application thereof.

Background

Alkaline phosphatase (Alkaline phosphatase, ALP) is a hydrolase widely distributed in prokaryotic and eukaryotic cells and is capable of removing the phosphate groups of various phosphorylated substrates under alkaline conditions, catalyzing the hydrolysis of phosphomonoesters (Coleman J E. Annu. Rev. Biophys. Biomol. Structure., 1992, 21:441-483.). Each ALP molecule consists of 2 structurally similar monomers, each containing 2 zinc ions, 1 magnesium ion and 5 cysteine residues (Guo J T, yu H B, cui t.j.biomed.mate.res., 2021, 109 (2): 214-226). ALP has 4 isozymes in humans, placental alkaline phosphatase, germ cell alkaline phosphatase, intestinal alkaline phosphatase, and tissue-nonspecific alkaline phosphatase, respectively (Milla n J L. Purinergic Signal.,2006, 2:335-341.).

ALP is widely found in human tissues and is involved in various processes such as cell cycle, growth, apoptosis and signal transduction. ALP in serum is mainly derived from liver, bone and kidney tissues, and its activity changes are closely related to various diseases such as osteoporosis, osteomalacia, biliary obstruction, cirrhosis, and sepsis. Meanwhile, ALP is an important biomarker for some cancers, such as bone cancer, pancreatic cancer, ovarian cancer, breast cancer, and prostate cancer. In addition, ALP hydrolyzes adenosine monophosphate to form adenosine, which can be used as a cell signaling molecule. Therefore, the sensitive detection of ALP is of great importance for clinical disease diagnosis and biomedical research.

The ALP detection method mainly comprises the following steps: colorimetric methods, electrochemical methods, surface-enhanced Raman scattering methods, fluorescence analysis methods and the like, wherein the colorimetric methods have the advantages of visual results, no need of complex expensive instruments and low sensitivity; the electrochemical method has the advantages of good selectivity, low cost and portability of the instrument, but has the defects of relatively weak signal, complex synthesis of ALP substrate, requirement of various tool enzymes and the like; the surface enhanced raman scattering method has the advantages of high sensitivity and capability of providing a molecular specific fingerprint spectrum, however, the complex operation and unstable raman signal are still the problems to be solved urgently; the fluorescence analysis method has the advantages of high sensitivity, good selectivity, real-time in situ, small background interference and living body imaging capability, but has certain limitations in detection specificity, fluorescence penetration and imaging resolution. The monoclonal antibody has the characteristics of high purity, high sensitivity, strong specificity and less cross reaction, so developing a monoclonal antibody combination capable of specifically recognizing ALP is beneficial to promoting the development of ALP detection and cancer diagnosis technologies.

Disclosure of Invention

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

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect of the invention there is provided an ALP monoclonal antibody combination comprising: 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 shown as SEQ ID NO. 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown as SEQ ID NO. 4-6;

the monoclonal antibody 7-H10-2 comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown as SEQ ID NO 7-9 and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown as SEQ ID NO 10-12.

Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1-G5-1 is shown as SEQ ID NO.13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14;

the amino acid sequence of the heavy chain variable region of the monoclonal antibody 7-H10-2 is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.

Furthermore, the amino acid sequence of the heavy chain of the monoclonal antibody 1-G5-1 is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain is shown as SEQ ID NO. 18;

the amino acid sequence of the heavy chain of the monoclonal antibody 7-H10-2 is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain is shown as SEQ ID NO. 20.

In a second aspect of the invention, there is provided a gene encoding monoclonal antibody 1-G5-1.

Preferably, the coding gene comprises: the DNA sequence shown in SEQ ID NO. 21 for encoding the heavy chain of monoclonal antibody 1-G5-1; the DNA sequence shown in SEQ ID NO. 22 is used for encoding the light chain of monoclonal antibody 1-G5-1.

In a third aspect of the invention, there is provided a gene encoding monoclonal antibody 7-H10-2.

Preferably, the coding gene comprises: the DNA sequence shown in SEQ ID NO. 23 for encoding the heavy chain of monoclonal antibody 7-H10-2; the DNA sequence shown in SEQ ID NO. 24 is used for encoding the light chain of monoclonal antibody 7-H10-2.

In a fourth aspect of the present invention, there is provided the use of an ALP monoclonal antibody combination as defined above in (1) or (2) as follows:

(1) Preparing an ALP detection product;

(2) Preparing a cancer diagnosis kit or reagent.

In the above applications, the ALP detecting products include, but are not limited to: ELISA detection kit, colloidal gold detection kit, chemiluminescent immunoassay kit, etc.

In the above applications, the cancer is characterized by ALP as a biomarker, including but not limited to: bone, pancreatic, ovarian, breast and prostate cancer.

In a fifth aspect of the present invention, there is provided an ELISA kit for detecting ALP, wherein the ELISA kit contains the ALP monoclonal antibody combination.

Preferably, the ELISA kit takes monoclonal antibody 1-G5-1 as a capture antibody and monoclonal antibody 7-H10-2 as a detection antibody.

The invention has the beneficial effects that:

(1) The ALP monoclonal antibody 1-G5-1 and the monoclonal antibody 7-H10-2 developed and designed by the invention have high affinity and specific recognition capability for alkaline phosphatase, and provide a new antibody source for ALP detection.

(2) The ALP monoclonal antibody combination can bind different epitopes of alkaline phosphatase to form a double-antibody sandwich mode, so that high-sensitivity and specificity detection of ALP is realized, and early diagnosis of cancers by taking the ALP as a biomarker is facilitated.

Drawings

Fig. 1: example 1 SDS-PAGE results of recombinant human ALP protein expression.

Fig. 2: ALP sensitivity detection results; the ordinate in the graph indicates the OD at 450 nm.

Fig. 3: specific detection results of monoclonal antibody 1-G5-1; the ordinate in the graph indicates the OD at 450 nm.

Fig. 4: specific detection results of monoclonal antibody 7-H10-2; the ordinate in the graph indicates the OD at 450 nm.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As previously mentioned, ALP is an important biomarker for a variety of cancers, and sensitive detection of ALP is of great importance for clinical disease diagnosis and biomedical research.

Based on this, the present invention developed and designed monoclonal antibodies capable of recognizing ALP. The invention utilizes exogenously expressed recombinant human ALP protein to immunize mice to obtain immunized mice, and utilizes lymphocytes and myeloma cells of the immunized mice to carry out cell fusion; then 10 hybridoma cell strains are obtained by screening, ascites antibodies are prepared and purified, and 10 monoclonal antibodies are obtained.

Because the specificity of the monoclonal antibodies secreted by different hybrid cell lines for recognizing ALP can be different, the obtained 10 monoclonal antibodies are respectively used as a capture antibody and a detection antibody, and 90 paired antibodies are combined. The 90 combinations are used for respectively detecting ALP samples, and pairing combinations with the best detection effect are screened out, so that the monoclonal antibody 1-G5-1 is used as a capture antibody, the monoclonal antibody 7-H10-2 is used as a detection antibody, and the detection effect on ALP is the best. Thus, the combination of the monoclonal antibody 1-G5-1 and the monoclonal antibody 7-H10-2 was used for specifically recognizing and detecting ALP, and the sequences and structures of the resulting monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2 were analyzed, thereby leading to the present invention.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and are commercially available. .

Example 1: ALP monoclonal antibody preparation

(1) Expressing recombinant human ALP protein in colibacillus;

suitable human ALP protein sequences (NP-112603.2alkaline phosphatase,germ cell type preproprotein[Homo sapiens) were selected in NCBI. Expression vectors were synthesized by the company Suzhou gold only: plasmid: pET-28a (+), 5' cleavage site: ndel, 3' cleavage site: xhol, expression strain: BL21 (DE 3);

mixing 100ng plasmid with 20ul competent cells, and standing on ice for 30min; heat shock for 45s, placing 2min on ice, culturing at 37℃and 200rpm for 1h, centrifuging at 2000rpm, and leaving a small amount of supernatant coated on Kana resistant plates. Culturing overnight in a 37 ℃ incubator; the next day of the expansion culture of the selected bacteria, 1mM IPTG, and induction for 4 hours at 37 ℃; after successful expression, the shaking volume is increased, protein purification is carried out, and the SDS-PAGE (SDS-PAGE) detection of the protein expression and purification effect is carried out, and the result is shown in figure 1.

(2) The recombinant human ALP protein is used for carrying out quick immunization on the mice to obtain immunized mice, lymphocytes and myeloma cells of the immunized mice are used for carrying out cell fusion, and then positive hole cells are screened out and subcloned.

The mice were immunized as follows:

primary immunization: 100 μg recombinant human ALP protein and complete Freund's adjuvant according to volume ratio 1:1 after complete mixing, subcutaneously injecting the hind foot pad of the mouse;

secondary immunization: a second immunization was performed 2 weeks after the completion of the first immunization, 100 μg of recombinant human ALP protein and incomplete freund's adjuvant in a volume ratio of 1:1 after complete mixing, subcutaneously injecting the hind foot pad of the mouse;

third immunization: a third immunization was performed 2 weeks after the completion of the second immunization, 100 μg of recombinant human ALP protein and PBS buffer in a volume ratio of 1:1, after being uniformly mixed, injecting into the abdominal cavity;

taking the blood around the eyes of the mice 10 days after the third immunization is completed for titer detection;

boosting: three days before fusion, 100 mug of recombinant human ALP protein and PBS buffer solution are mixed evenly according to the volume ratio of 1:1, and then the mice are injected by intravenous injection.

The specific procedure for the fusion experiments of lymphocytes with myeloma cells was as follows:

mixing myeloma cells and lymphocytes according to a number ratio of 1:5 to obtain a cell mixture; placing the cell mixture into a 50ml centrifuge tube, diluting with RPMI-1640 basic culture medium, centrifuging at 1000rpm for 5min, discarding the supernatant, shaking the centrifuge tube to make the cells uniform, slowly adding 1ml of 50% PEG, reacting for 90 seconds, adding 10ml of RPMI-1640 culture medium to stop PEG, placing the fused cells into a 37 ℃ water bath kettle to react for 10min, centrifuging at 1000rpm for 5min, discarding the supernatant, and adding the RPMI-1640 complete culture medium to resuspend the cells;

the fused cells were plated in 96-well plates, 100 μl per well; the cell culture plate is then placed in CO ₂ Culturing in an incubator, and fusing and screening positive hole cells after 10 days of fusion.

The subcloning was performed as follows:

blowing cells in positive holes, taking 10 mu l of count N, adding PBS buffer solution into a centrifuge tube according to a limiting dilution method, taking 100 mu l of cell suspension into the centrifuge tube, taking 30 mu l after blowing evenly, adding 500 mu l of RPMI-1640 complete culture medium, inoculating to a semisolid culture medium after blowing evenly, selecting macroscopic monoclonal cell strains after 6-8 days, inoculating to a 96-well plate, and screening out positive monoclonal cell strains.

(3) Screening the strong positive cell strain obtained after subcloning in the step (2), preparing the ascites, purifying the ascites, and specifically performing the following purification treatment on the ascites:

loading the protein A agarose gel medium into a nickel ion affinity chromatography column, mixing ascites and PBS in an equivalent volume ratio of 1:1, slowly loading the mixture, eluting the mixture by using glycine elution buffer solution after the antibody is combined, and finally purifying the mixture to obtain 10 monoclonal antibodies (monoclonal antibodies 1-G5-1-5 and 7-H10-1-7-H10-5).

Example 2: screening of monoclonal antibody combinations specifically recognizing ALP

Obtained in example 1The obtained 10 monoclonal antibodies are respectively used as a capture antibody and a detection antibody, and ELISA sandwich antibody pairing is carried out, wherein the detection antibody is marked by HRP. The ELISA detection flow is as follows: coating a capture antibody, washing a plate three times, closing, washing the plate three times, adding ALP antigen for incubation, washing the plate, adding a detection antibody, incubating, washing the plate, adding TMB color development liquid, incubating for 10-15min at room temperature in a dark place, stopping, and reading OD by an enzyme-labeled instrument ₄₅₀ Values. The detection results are shown in Table 1.

Table 1: screening of paired antibodies by double antibody sandwich ELISA method

Note that: the "-" in the table represents OD ₄₅₀ The value < 0.5, "+" number represents OD ₄₅₀ The value of "4+" represents OD ₄₅₀ The value > 4.0, "3+" represents OD ₄₅₀ The value > 3.0, "2+" represents OD ₄₅₀ The value > 2.0, "1+" represents OD ₄₅₀ The value is > 1.0.OD (optical density) ₄₅₀ The higher the value, the better the pairing effect.

Thus, the paired combination of monoclonal antibody 1-G5-1 as the capture antibody and monoclonal antibody 7-H10-2 as the detection antibody was selected as the monoclonal antibody combination specifically recognizing ALP.

Example 3: sequence and structural analysis of monoclonal antibody 1-G5-1 and monoclonal antibody 7-H10-2

Further sequence and structural analysis was performed on monoclonal antibodies 1-G5-1 and 7-H10-2, wherein:

monoclonal antibody 1-G5-1 includes: heavy (Heavy Chain) and Light (Light Chain); the amino acid sequence of the heavy chain of the monoclonal antibody 1-G5-1 is shown as SEQ ID NO. 17, the nucleotide sequence of the coding heavy chain is shown as SEQ ID NO. 21, and the specific steps are as follows:

amino acid sequence:

MDRLTSSFLLLIVPAYVLSQVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWI RQPSGKGLEWLAHIWWDDVKYYNPSLKSQLAISKDTSRNQVFLKVTSVDTADTATYYC TRTDYFYIDYWGPGTTLTVSS。

note that: the shaded region is the heavy chain variable region; the italic bold region is the CDR (complementarity determining region).

Nucleotide sequence:

ATGGACAGGCTTACTTCTTCATTCCTGCTGCTGATTGTCCCTGCATATGTCTTGTCCCAAGTTACTCTAAAAGAGTCTGGCCCTGGAATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGTTAAGTATTATAATCCATCCCTGAAGAGCCAGCTCGCAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGGTCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTACTCGAACTGATTACTTCTACATTGACTACTGGGGCCCAGGCACCACTCTCACAGTCTCCTCA。

note that: the shaded region is the coding sequence for the heavy chain variable region.

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

VHCDR1：TSGMGVG；(SEQ ID NO:1)

VHCDR2：HIWWDDVKYYNPSLKS；(SEQ ID NO:2)

VHCDR3：TDYFYIDY；(SEQ ID NO:3)

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

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWD DVKYYNPSLKSQLAISKDTSRNQVFLKVTSVDTADTATYYCTRTDYFYIDYWGPGTTLTV SS。

the amino acid sequence of the monoclonal antibody 1-G5-1 light chain is shown as SEQ ID NO. 18, the nucleotide sequence of the coded light chain is shown as SEQ ID NO. 22, and the specific steps are as follows:

amino acid sequence:

MESQTQVFLSLLLWVSGTCGNIMMTQSPSSLAVSAGEKVTMSCKSSQSVFNSSNQK NYLAWYQQKPGQSPKLLIYWASTRESGVPYRFTGSGSGTDFTLTISSVQAEDLAVYYCH QFLSSWTFGGGTKLEIK。

note that: the shaded region is the light chain variable region; the italic bold region is the CDR (complementarity determining region).

Nucleotide sequence:

ATGGAATCACAGACTCAGGTCTTCCTCTCCCTGCTGCTCTGGGTATCTGGTACCTGTGGGAACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTATGAGCTGTAAGTCCAGTCAAAGTGTTTTTAACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGTGTCCCTTATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTGACCATCAGCAGTGTACAAGCTGAAGACCTGGCAGTTTATTACTGTCATCAATTCCTCTCCTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA。

note that: the shaded region is the coding sequence for the light chain variable region.

The light chain variable region of monoclonal antibody 1-G5-1 comprises 3 CDR regions of: the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 shown in SEQ ID NO 4-6 are as follows:

VLCDR1：KSSQSVFNSSNQKNYLA；(SEQ ID NO:4)

VLCDR2：WASTRES；(SEQ ID NO:5)

VLCDR3：HQFLSSWT；(SEQ ID NO:6)

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

NIMMTQSPSSLAVSAGEKVTMSCKSSQSVFNSSNQKNYLAWYQQKPGQSPKLLIY WASTRESGVPYRFTGSGSGTDFTLTISSVQAEDLAVYYCHQFLSSWTFGGGTKLEIK

monoclonal antibody 7-H10-2 includes: heavy (Heavy Chain) and Light (Light Chain); the amino acid sequence of the heavy chain of the monoclonal antibody 7-H10-2 is shown as SEQ ID NO. 19, the nucleotide sequence of the coding heavy chain is shown as SEQ ID NO. 23, and the specific steps are as follows:

amino acid sequence:

MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHW VKLSHGKSPEWVGGINPNNGGTSYNQKFQGKATLTVDKSSSTAYMELRSLTSEDSAVYY CVRSNWEIGMDYWGQGTSVTVSS。

Nucleotide sequence:

ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATACACCATGCACTGGGTGAAGCTGAGCCATGGAAAGAGCCCTGAGTGGGTTGGAGGTATTAATCCTAACAATGGTGGTACTAGTTACAACCAGAAGTTCCAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAAGATTCTGCAGTCTATTACTGTGTAAGATCTAACTGGGAAATTGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

The heavy chain variable region of monoclonal antibody 7-H10-2 comprises 3 CDR regions of: the amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 shown in SEQ ID NO 7-9 are as follows:

VHCDR1：EYTMH；(SEQ ID NO:7)

VHCDR2：GINPNNGGTSYNQKFQG；(SEQ ID NO:8)

VHCDR3：SNWEIGMDY；(SEQ ID NO:9)。

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

EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKLSHGKSPEWVGGINPNNGGTSYNQKFQGKATLTVDKSSSTAYMELRSLTSEDSAVYYCVRSNWEIGMDYWGQGTSVTVSS

the amino acid sequence of the monoclonal antibody 7-H10-2 light chain is shown as SEQ ID NO. 20, the nucleotide sequence of the coded light chain is shown as SEQ ID NO. 24, and the specific steps are as follows:

amino acid sequence:

MSPAQFLFLLVLWIQETNGDVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLN WLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTH FPFTFGSGTKLEIK。

Nucleotide sequence:

ATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCAGGAAACCAACGGTGATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCTATCTCTTGCAAGTCAAGTCAGAGCCTCTTATATAGTAATGGAAAAACCTATTTGAATTGGTTATTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGAACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTACTGCGTGCAAGGTACACATTTTCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAG

The light chain variable region of monoclonal antibody 7-H10-2 comprises 3 CDR regions of: the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 shown in SEQ ID NO 10-12 are as follows:

VLCDR1：KSSQSLLYSNGKTYLN；(SEQ ID NO:10)

VLCDR2：LVSKLDS；(SEQ ID NO:11)

VLCDR3：VQGTHFPFT；(SEQ ID NO:12)。

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

DVVMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSK LDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPFTFGSGTKLEIK。

the monoclonal antibodies 1-G5-1 and 7-H10-2 obtained by the invention have specific CDR regions, and have obvious differences with the monoclonal antibodies reported in the prior art; and the combination of the monoclonal antibody 1-G5-1 and the monoclonal antibody 7-H10-2 has excellent affinity and specific recognition performance on human ALP.

Example 4: construction of ELISA kit for detecting ALP and methodological investigation

1. Construction and detection of ELISA kit:

the ELISA detection kit is constructed by taking monoclonal antibody 1-G5-1 as a capture antibody and monoclonal antibody 7-H10-2 as a detection antibody, and the specific detection steps are as follows:

(1) Coating: 2-9D capture antibody was diluted to a working concentration of 2. Mu.g/ml, added to the coating plate at 100. Mu.L per well, coated overnight at 4 ℃;

(2) Closing: PBST (containing 0.05% Tween) was washed 3 times, blocked with 5% skimmed milk powder, 200 μl per well, and incubated at 37deg.C for 1h;

(3) Adding an antigen: PBST is washed for 3 times, AFP antigen is added after the mixture is patted dry, and the addition amount is 80 mu L; incubating for 1h at 37 ℃;

(4) Adding a secondary antibody: PBST was washed 3-5 times, and the secondary antibody (HRP-labeled detection antibody 2-3D) was 8000-fold diluted with 5% nonfat dry milk, 50 μl per well, and incubated at 37deg.C for 1h;

(5) Washing with PBST for 5 times and 3 min/time, drying, adding color development liquid, incubating at room temperature in dark for 10min, and reading OD with enzyme-labeled instrument ₄₅₀ Values.

2. Methodology investigation:

(1) Sensitivity:

ALP proteins with concentrations of 20. Mu.g/ml, 2. Mu.g/ml, 200ng/ml, 20ng/ml, 2ng/ml, 200pg/ml and 20pg/ml were used as antigens, respectively, and the detection was performed using the ELISA kit constructed as described above, and a detection curve was drawn (FIG. 2).

The ELISA kit provided by the invention has the detection sensitivity of 0.2ng/ml for ALP.

(2) Specificity:

elisa detects the specificity of two monoclonal antibodies, the antigen is recombinant human ALP protein, the plating concentration gradient is set to 2 mug/ml (positive control without human serum), 2 mug/ml, 1 mug/ml, 0.2 mug/ml, 0.04 mug/ml, 0.008 mug/ml, 0.

The culture supernatant of the 1-G5-1 and 7-H10-2 monoclonal cells and human serum are evenly mixed according to the volume ratio of 1:1 and then incubated for 0.5H, an ELISA plate is added for continuous incubation for 1H at 37 ℃, and the loading amount of each hole is 100 mu l.

The secondary antibody is a goat anti-mouse marked with HRP, and TMB is added for color development after incubation for 0.5 h.

The specificity detection mainly shows whether the human serum has an interfering substance which is not specifically combined with the antibody, and the specificity can be considered to be good as long as the result is similar to a positive control, and then the OD value and the antigen concentration are positively correlated. The results are shown in fig. 3 and 4, and the results indicate that: after the human serum is added, the antibody titer is not obviously reduced, and the antibody titer is positively correlated with the antigen concentration, which indicates that no substance which is not specifically recognized by the antibody in the human serum affects the detection, and the specificity of the 1-G5-1 and 7-H10-2 monoclonal antibodies is better.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

An alp monoclonal antibody combination comprising: 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 shown as SEQ ID NO. 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown as SEQ ID NO. 4-6;

the monoclonal antibody 7-H10-2 comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences shown as SEQ ID NO 7-9 and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences shown as SEQ ID NO 10-12.
2. The ALP monoclonal antibody combination of claim 1, wherein 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 the monoclonal antibody 7-H10-2 is shown as SEQ ID NO.15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16.
3. The ALP monoclonal antibody combination of claim 1, wherein the heavy chain of monoclonal antibody 1-G5-1 has the amino acid sequence shown in SEQ ID No. 17 and the light chain has the amino acid sequence shown in SEQ ID No. 18;

the amino acid sequence of the heavy chain of the monoclonal antibody 7-H10-2 is shown as SEQ ID NO. 19, and the amino acid sequence of the light chain is shown as SEQ ID NO. 20.
4. The coding gene of the monoclonal antibody 1-G5-1.
5. The coding gene of claim 4, wherein the coding gene comprises: the DNA sequence shown in SEQ ID NO. 21 for encoding the heavy chain of monoclonal antibody 1-G5-1; the DNA sequence shown in SEQ ID NO. 22 is used for encoding the light chain of monoclonal antibody 1-G5-1.
6. The coding gene of the monoclonal antibody 7-H10-2.
7. The coding gene of claim 6, wherein the coding gene comprises: the DNA sequence shown in SEQ ID NO. 23 for encoding the heavy chain of monoclonal antibody 7-H10-2; the DNA sequence shown in SEQ ID NO. 24 is used for encoding the light chain of monoclonal antibody 7-H10-2.
8. Use of the ALP monoclonal antibody combination of any one of claims 1-3 in (1) or (2) as follows:

(1) Preparing an ALP detection product;

(2) Preparing a cancer diagnostic kit or reagent;

preferably, the ALP detection products include, but are not limited to: ELISA detection kit, colloidal gold detection kit and chemiluminescent immunoassay kit;

preferably, the cancers include, but are not limited to: bone, pancreatic, ovarian, breast and prostate cancer.
9. An ELISA kit for detecting ALP, comprising the monoclonal antibody combination of any one of claims 1-3.
10. The ELISA kit of claim 9, wherein the ELISA kit uses monoclonal antibody 1-G5-1 as a capture antibody and monoclonal antibody 7-H10-2 as a detection antibody.