CN105646712A - Monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a monoclonal antibody with the biological activity of neutralizing human leukocyte differentiation antigen CD24 and the variable region sequence of the monoclonal antibody. The present invention uses the antigen peptide and hemocyanin conjugate CD24-KLH as the immunogen, immunizes and injects BALB/c mice, takes the spleen cells of the immunized mice and fuses them with myeloma cells, and obtains the antibody that can express anti-CD24 antibody Hybridoma cell line, which can be used to prepare anti-CD24 monoclonal antibody. The invention simultaneously clones the amino acid sequence of the variable region of the anti-CD24 antibody. The anti-CD24 antibody of the present invention can specifically bind to CD24, so it can be used to treat diseases related to excessive, abnormal and uncontrolled expression of CD24.

Description

Monoclonal Antibodies and Their Applications

The present invention is a divisional application with the application number 2014100277955, the application date is 2014-01-22, and the title of the invention is anti-CD24 monoclonal antibody, its variable region sequence and its application.

technical field

The invention belongs to the field of biotechnology, and specifically relates to a monoclonal antibody capable of specifically binding to human leukocyte differentiation antigen CD24, its variable region sequence and application thereof.

Background technique

Human leukocyte differentiation antigen CD24 (clusterofdifferentiation24) is a newly discovered and focused study of tumor markers.

Current research shows that CD24 molecule is a highly glycosylated sialoglycoprotein with an apparent molecular weight between 30 and 70 kDa. CD24 contains a core protein backbone structure consisting of 33 amino acid residues, with 16 potential O- or N-glycosylation sites, and is highly homologous to mouse heat-stable antigens; its mature polypeptide can pass through the carboxy-terminal phosphatidyl muscle Alcohol (glycosyl-phosphatidyl-inositol, GPI) is anchored on the surface of the cell membrane.

Under physiological conditions, CD24 molecules are only expressed at low levels on immature B cells, mature granulocytes, and a few epithelial cells and nerve cells; when the body is in a pathological state, significantly high levels can be detected on the surface of most malignant tumor cells The expression level of CD24 molecules is closely related to the occurrence and development of tumors.

P-selectin is expressed on the surface of activated platelets and endothelial cells, and CD24 is one of its ligands. The high expression of CD24 molecule enhances the adhesion of tumor cells to platelets and endothelial cells, and promotes tumor recurrence and metastasis. Of course, as a signal transduction molecule on the surface of the cell membrane, CD24 can mediate the proliferation, adhesion, metastasis and invasion of tumor cells through various mechanisms. The study further found that the expression of CD24 is closely related to the stemness of liver cancer cells, and CD24 may become a new surface marker of liver cancer stem cells.

It can be seen that CD24 is expected to become a new target for the study of tumorigenesis mechanism and the development of related diagnostic reagents.

Contents of the invention

The first object of the present invention is to provide a preparation method of anti-CD24 monoclonal antibody.

The second object of the present invention is to provide an anti-CD24 monoclonal antibody.

The third object of the present invention is to provide the amino acid sequence of the variable region of the anti-CD24 monoclonal antibody.

In the present invention, the conjugate of CD24 core polypeptide and hemocyanin KLH (CD24-KLH) is used as an immunogen to immunize and inject BALB/c mice, take the spleen cells of the immunized mice and fuse them with myeloma cells to obtain a viable A hybridoma cell line expressing anti-CD24 antibody, which can be used to prepare anti-CD24 monoclonal antibody.

The antibody heavy chain, light chain variable region amino acid sequence and hypervariable region cloned in the present invention are shown in SEQ ID No. 1-16.

The monoclonal antibody of the present invention can specifically bind to CD24.

Description of drawings

Figure 1 is the result of SDS-PAGE detection of the purified product of ProteinG affinity chromatography column. Swimming lane M is protein with standard molecular weight; Swimming lane 1 is ascites sample; Swimming lane 2 is sample collection solution; Swimming lane 3 is 20mM sodium phosphate buffer washing column collection solution; Swimming lanes 4-6 are 100mM glycine buffer (pH3.5) elution Collection solution (collect 1ml per minute, 3ml in total), lanes 7-10 are 100mM glycine buffer (pH2.7) elution collection solution (collect 1ml per minute, 4ml in total).

Figure 2 is the WesternBlot identification of the interaction between the purified antibody and CD24. Lane M is standard molecular weight protein; lane 1 is commercially available CD24-Fc (purchased from Beijing Sino Biological); lane 2 is commercially available Fc (purchased from Beijing Sino Biological).

Fig. 3 is the agarose electrophoresis result of PCR amplified antibody heavy and light chain variable region genes. Lane M is standard molecular weight DNA; Lane 1 is PCR amplified antibody heavy chain variable region gene product; Swimming lane 2 is PCR amplified antibody light chain variable region gene product.

Fig. 4 is the determination of the affinity of monoclonal antibodies G7, A7, E4, G5 and B4 by competitive ELISA.

detailed description

Embodiment 1 Preparation of anti-CD24 monoclonal antibody hybridoma cells

1. Immunogen

The CD24 core region consists of 33 amino acid residues, its molecular weight is about 3.2kD, and its immunogenicity is weak. The invention couples the antigenic peptide CD24 with the hemocyanin KLH, uses the conjugate CD24-KLH as the immunogen, and enhances the immunogenicity of the CD24 core region polypeptide. The CD24-KLH used in the present invention is synthesized by Sangon Biotech (Shanghai) Co., Ltd., with a purity of >90%.

2. Immunization of Animals

The immunized animals used in the present invention are BALB/c mice of SPF level, provided by the School of Comparative Medicine of Yangzhou University. The immune adjuvant was QuickAntibody, which was purchased from Beijing Kangbiquan Company. The immunization process was carried out according to the QuickAntibody manual.

The details are shown in Table 1.

Table 1. Immunization route and cycle

3. Cell Fusion

1) Cell preparation

A. Feeder layer cells

One day before the fusion, the peritoneal macrophages of ICR mice (SPF grade, purchased from the School of Comparative Medicine, Yangzhou University) were taken and resuspended in HAT medium (purchased from GIBCO, containing xanthine, aminopterin and thymidine ). Inoculated in 96-well plates, placed in a 37 ° C, 5% CO ₂ incubator for 24 hours.

B. Myeloma cells

The mouse myeloma cell line SP20-Ag14 (preserved in this laboratory) does not synthesize and secrete any mouse immunoglobulin heavy chain and light chain. This cell is resistant to 8-azaguanine, and it is selected in HAT Sexual medium cannot grow. One week before the fusion, the hybridoma cells were cultured in separate flasks with ordinary DMEM complete medium (containing 15% FBS, purchased from GIBCO), so that the cells could grow well. Prepare 250 ml of healthy cells in mid-logarithmic growth at a growth density of 2×l0 ⁶ cells/m1 for each fusion.

C. Immunized mouse spleen cells

The immunized mice were sacrificed, and the spleens were removed after infiltration with alcohol. Prepare a 10cm ² sterile Petri dish in advance, put it into a cell mesh, and add 10ml of serum-free DMEM medium. Place the spleen on a cell mesh and cut the spleen into pieces using sterile surgical scissors. After the inner core of the syringe squeezes and grinds the spleen, wash it with 5ml DMEM incomplete medium, and let the cells enter the 50ml centrifuge tube through the sieve. Collect spleen cell suspension for fusion.

2) Fusion

A. Prepare 20ml of serum-free DMEM medium and 1ml of PEG1450 (purchased from Sigma) and place them at 37°C for incubation.

B. Collect myeloma cells and spleen cells, centrifuge at 1500 rpm for 5 minutes at room temperature, wash twice with serum-free medium, and then suspend them in 10 ml serum-free medium. 5×10 ⁸ splenocytes and 1×10 ⁸ myeloma cells were thoroughly mixed, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. Tap the bottom of the tube lightly to loosen the cells.

C. Take out the medium and PEG preheated at 37°C, put the cells in a water bath at 37°C, add 1ml PEG slowly and dropwise within 1min at a uniform speed, and stop with serum-free medium in time after the addition. Discard the supernatant by centrifugation and resuspend the cells with 50ml HAT medium, and mix gently.

D. Add cells to five 96-well plates that have been plated with feeder cells, 100 μl per well. Place in a 37°C, 5% CO ₂ incubator for 4 days. According to the growth state of the cells, half-change the HAT medium for 1 to 3 times before screening to avoid yellowing of the medium in the wells.

E. After 7 days of fusion, replace the medium with HT medium. After 24 hours, the cell culture supernatant was aspirated, and positive clones were screened by indirect ELISA detection.

3) Monocloning of hybridomas

Select clone wells with higher positive values, and use the limiting dilution method to dilute the cells in the wells to 5, 10 and 50 cells per milliliter, and then inoculate 100 μl in each well in a 96-well plate. 37 ° C, 5% CO ₂ humidified culture for 7 to 10 days, the cell culture supernatant can be detected when there are clones visible to the naked eye. Observe under an inverted microscope, mark the wells where only a single clone grows, and take the supernatant for antibody detection. Cells from wells positive for antibody detection were taken for expansion and cultured, and frozen.

Preparation and purification of embodiment 2CD24 specific antibody

1. Ascites Collection

One week in advance, mice were intraperitoneally injected with 0.5 ml of paraffin oil (purchased from Sigma). One week after the sensitization, the vigorously growing hybridoma cells were centrifuged to discard the medium, resuspended with PBS or incomplete medium, adjusted the cell concentration to 2×10 ⁶ cells/ml, and injected 0.5ml of the cell suspension into the peritoneal cavity of the mouse middle. After 7 to 10 days, the abdominal cavity of the mice was obviously enlarged, and ascites was collected at this time. Centrifuge at 5000g for 20min at 4°C to remove cell debris and oil in ascites, then add an equal volume of glycerol and store at -20°C.

2. Purification of CD24-specific antibody

Antibodies were purified using ProteinG affinity chromatography columns. The specific steps are as follows: A. Wash the ProteinG affinity chromatography column with 10 times the column volume of water. B. Wash the ProteinG affinity chromatography column with 10 times column volume of 20 mM sodium phosphate buffer (pH7.0). C. Pump the desired purified sample into the chromatography column. D. Eluted with 100 mM glycine buffer (pH3.5, pH2.7), collected the eluted peaks; and neutralized the collection with 1MTris buffer (pH9.0). E. 10mM pH7.2 buffer dialysis desalting. F. Preliminary identification of the antibody by SDS-PGAGE electrophoresis. The results in Figure 1 show that the target bands appear at 50kD and 25kD.

3.Characteristic analysis of CD24-specific antibody

1) Identification of immunoglobulin subtypes

Using the mouse monoclonal antibody subtype identification kit of Wuhan Sanying Biotechnology Co., Ltd. to identify the antibody subtypes secreted by cell lines G7, A7, E4, G5, and B4, the results are: the immunoglobulin secreted by these five cell lines The heavy chain subtypes were all IgG1, and the light chain subtypes were all Kappa.

2) WesternBlot identification of specificity of monoclonal antibodies

The purified antibody was used as the primary antibody, and its reaction with CD24 was identified. Figure 2 shows that the obtained antibody can specifically bind to the CD24 part of the commercially available CD24-Fc fusion protein.

3) Determination of antibody affinity by competitive ELISA

According to the method of determining affinity by Friguet et al., an antigen competition binding antibody experiment was designed to determine the affinity constant. The antibody concentration was fixed at 10 pmol·L ^-1 , and the antigen concentration was changed to form a series of reaction systems. 50 μL antigens of 7 different concentrations (0, 1.95×10 ^-11 , 3.91×10 ^-11 , 7.81×10 ^-11 , 1.56×10 ^-10 , 3.13×10 ^-10 , 6.25×10 ^-10 mol·L ^-1 ) were mixed with 50 μL monoclonal antibodies G7, A7, E4, G5, and B4 respectively, and reacted overnight at 4°C. CD24 polypeptide was dissolved in 0.05 mol·L ^-1 carbonic acid buffer (pH 9.6) with a final mass concentration of 10 μg·mL ^-1 , added to a 96-well plate, 100 μL per well, and coated at 4°C for 15 hours. 5% skimmed milk powder blocked for 2h. The overnight reaction solution was added respectively, incubated at 37°C for 90 min, and washed three times with PBST and PBS respectively. Add 1:5000 dilution of HRP-labeled goat anti-mouse antibody. TMB chromogenic solution was used for color development, and 50 μL of 1mol·L ^-1 H ₂ SO ₄ was added to each well to terminate the reaction. The data was read on a microplate reader by the dual-wavelength method, and the "A450nm-A630nm" value of each monoclonal antibody was used. The initial concentration of the antigen is a ₀ , the initial concentration of the antibody is b ₀ , A ₀ and A _i are the absorbance values of the initial antibody and the antibody bound to the antigen respectively. B=(A ₀ -A _i )/A ₀ , where B is the antibody binding rate. K _D =(1-B)(a0-b ₀ B)/B. Measure the B value of each reaction system, draw a graph with B/(1-B) as the abscissa and (a ₀ -b ₀ *B) as the ordinate (Figure 4), and the slope is the affinity constant K _D . According to the linear regression results, the affinities of monoclonal antibodies G7, A7, E4, G5, and B4 were 0.8nM, 0.72nM, 0.84nM, 0.82nM, and 0.76nM, respectively.

Cloning of embodiment 3 hybridoma cell lines G7, A7, E4, G5, B4 heavy and light chain variable region genes

1. Extraction, amplification and preliminary identification of heavy and light chain variable region genes of anti-CD24 monoclonal antibody

1) Extraction of total RNA

Hybridoma cells in the logarithmic phase were collected, and total RNA was extracted with an RNA extraction kit (purchased from Sangon Biotech), dissolved in 20-50 μl RNase-free water, and stored at -70°C.

2) RT-PCR synthesis of the first strand of cDNA

The first strand of cDNA was synthesized by reverse transcription using total RNA as a template. The kit was purchased from Sangon Biotechnology Co., Ltd., and the reaction was carried out according to the product instructions.

3) PCR amplification of light and heavy chain variable region genes

The polymerase is PrimerSTAR high-fidelity polymerase. The primers used are degenerate primers designed in the literature based on the upstream and downstream gene sequences of the variable region of the hybridoma cell line antibody light and heavy chains, in which the light chain VLF (upstream primer) and VLB (downstream primer) and the heavy chain VHF1, VHF2 and VHB The sequences of the two pairs of primers are:

VLF:gggagctcgayattgtgmtsacmcarwctmca;

VLB:ggtgcatgcggatacagttggtgcagcatc;

VHF1: cttccggaattcsargtnmagctgsagtc

VHF2: cttccggaattcsargtnmagctgsagtcwgg

VHB: ggaagatctatagacagatgggggtgtcgttttggc

(Degenerate codon description: r=a, g; y=c, t; m=a, c; s=c, g; w=a, t)

The reaction volume was 50 μl, and the reaction conditions were: 94°C for 5 minutes; 94°C for 30 seconds, 58°C for 1 minute, 72°C for 1 minute, and 30 cycles; 72°C for 10 minutes. 5 μl of the final product was electrophoresed on a 100 g/L agarose gel for identification. Figure 3 shows that about 380 bp of heavy and light chain genes were obtained by PCR amplification. PCR products were purified using a gel recovery kit (purchased from Sangon Biotech).

4) Add adenine (A) to the 3′ end of the DNA product recovered by gel

Take 1 μl (5U/μl) rTaq enzyme, 4 μl ldNTP, 5 μl 10×PCR buffer and 40 μl purified electrophoresis product, the reaction conditions are: 94°C, 10min, 70°C for 40min, 4°C, 10min.

5) The PCR product is connected to the T vector

Take 4.5μl PCR product, 0.5μlpMD18-T vector, 5μlligationbuffersolutionI, and connect at 16°C for 4h.

6) Transformation and preliminary identification of ligation products

The ligation product was transformed into competent DH5α using the _CaCl2 conversion method. The specific operation is as follows:

A. Gently mix 200 μl of competent cells with 10 μl of DNA, and place on ice for 30 minutes.

B. Place in a circulating water bath at 42°C for 90 seconds, then transfer the centrifuge tube to an ice bath for 3 minutes.

C. Add 1ml non-resistant LB medium to each tube and incubate at 37°C for 1h.

D. Centrifuge at 4000 rpm for 10 min, and resuspend in 300 μl LB medium. Apply 300 μl of the bacterial suspension evenly on the Amp+ (100 μg/ml) resistant agar plate with a sterile-treated glass-coated rod.

E. After culturing in a 37°C incubator for 12 hours, observe the results.

F. Pick clones, add Amp+(100 μg/ml) LB medium and shake overnight.

G. Take 1 μl of bacterial liquid as a template, and carry out colony PCR verification with the original PCR step. Positive clones were selected and sent to Sangon for sequencing.

2. Gene sequencing and analysis of light and heavy chain variable regions of anti-CD24 monoclonal antibody

Sequencing results showed that the antibody variable region genes of five hybridoma cell lines were successfully obtained. Using the IMGT-VQUEST database (http://www.imgt.org/) to analyze the anti-CD24 monoclonal antibody light and heavy chain variable region genes, the analysis results show that: A7 hybridoma cell line antibody heavy and light chain variable region amino acid sequence and The sequence of the hypervariable region is shown in SEQIDNo.1-8; the amino acid sequence of the heavy and light chain variable region of the G7 hybridoma cell strain antibody and its hypervariable region sequence are shown in SEQIDNo.9-16; the antibody heavy and light chain of the E4 hybridoma cell strain The amino acid sequence of chain variable region and its hypervariable region sequence are shown in SEQ ID No.17-24; the amino acid sequence of G5 hybridoma cell line antibody heavy and light chain variable region and its hypervariable region sequence are shown in SEQ ID No.25-32; The amino acid sequence of the heavy and light chain variable region and the hypervariable region sequence of the antibody of the B4 hybridoma cell strain are shown in SEQ ID No.33-40.

sequence listing

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<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(8)

<223>VH4CDR2

<400>27

IleMetProAspSerSerThrIle

15

<210>28

<211>6

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(6)

<223>VH4CDR3

<400>28

AlaArgGlnGlyAspTyr

15

<210>29

<211>112

<212>PRT

<213> Artificial sequence

<220>

<221>V_region

<222>(1)..(112)

<223>VL4

<400>29

AspIleValMetThrGlnThrProLeuSerLeuSerValThrIleGly

151015

GlnProAlaSerIleSerCysLysSerSerGlnSerValLeuHisSer

202530

AspGlyLysThrTyrLeuAsnTrpLeuLeuGlnArgProGlyGlnSer

354045

ProLysArgLeuIleTyrValLeuSerLysLeuAspSerGlyValPro

505560

AspArgPheThrGlySerGlySerGlySerGlyThrAspPheThrLeuLysVal

65707580

SerArgValGluAlaGluAspLeuGlyValTyrTyrCysTrpGlnGly

859095

GlyHisPheProTyrThrPheGlyGlyGlyThrLysLeuGluIleLys

100105110

<210>30

<211>11

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(11)

<223>VL4CDR1

<400>30

GlnSerValLeuHisSerAspGlyLysThrTyr

1510

<210>31

<211>3

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(3)

<223>VL4CDR2

<400>31

ValLeuSer

1

<210>32

<211>9

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(9)

<223>VL4CDR3

<400>32

TrpGlnGlyGlyHisPheProTyrThr

15

<210>33

<211>113

<212>PRT

<213> Artificial sequence

<220>

<221>V_region

<222>(1)..(112)

<223>VH5

<400>33

GlnValLysLeuGluGluSerGlyGlyGlyLeuValGlnProGlyGly

151015

SerLeuLysIleSerCysAlaAlaSerGlyPheAspPheTyrArgTyr

202530

SerMetSerTrpValArgGlnAlaProGlyLysGlyLeuGluTrpIle

354045

GlyGluIleAsnProAspTrpSerThrIleAsnTyrThrProSerLeu

505560

ArgAspLysPheIleIleSerArgAspAsnAlaLysAsnThrLeuTyr

65707580

LeuGlnMetSerLysValArgTyrGluAspThrSerLeuTyrTyrCys

859095

AlaHisGlnGlyAspTyrTrpGlyGlnGlyThrSerValThrValSer

100105110

Ser

<210>34

<211>8

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(8)

<223>VH5CDR1

<400>34

GlyPheAspPheTyrArgTyrSer

15

<210>35

<211>8

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(8)

<223>VH5CDR2

<400>35

IleAsnProAspTrpSerThrIle

15

<210>36

<211>6

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(6)

<223> VH5CDR3

<400>36

AlaHisGlnGlyAspTyr

15

<210>37

<211>112

<212>PRT

<213> Artificial sequence

<220>

<221>V_region

<222>(1)..(112)

<223>VL5

<400>37

AspIleValMetThrGlnThrProLeuSerLeuSerValThrIleGly

151015

GlnProAlaSerIleSerCysLysSerSerGlnAsnGluLeuHisSer

202530

AspGlyLysThrTyrLeuAsnTrpLeuLeuGlnArgProGlyGlnSer

354045

ProLysArgLeuIleTyrAlaValAlaLysLeuAspSerGlyValPro

505560

AspArgPheThrGlySerGlySerGlySerGlyThrAspPheThrLeuLysVal

65707580

SerArgValGluAlaGluAspLeuGlyValTyrTyrCysTrpGlnLeu

859095

LeuHisPheProTyrThrPheGlyGlyGlyGlyThrLysLeuGluIleLys

100105110

<210>38

<211>11

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(11)

<223>VL5CDR1

<400>38

GlnAsnGluLeuHisSerAspGlyLysThrTyr

1510

<210>39

<211>3

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(3)

<223>VL5CDR2

<400>39

Ala Val Ala

1

<210>40

<211>9

<212>PRT

<213> Artificial sequence

<220>

<221>D-segment

<222>(1)..(9)

<223>VL5CDR3

<400>40

TrpGlnLeuLeuHisPheProTyrThr

15

<210>41

<211>33

<212>PRT

<213>Homosapiens

<400>41

SerGluThrThrThrGlyThrSerSerAsnSerSerGlnSerThrSer

151015

AsnSerGlyLeuAlaProAsnProThrAsnAlaThrThrLysAlaAla

202530

Gly

Claims (7)

1. A variable region sequence of an anti-CD24 monoclonal antibody, characterized in that its heavy chain and light chain variable regions comprise the amino acid sequences of CDR1~3 regions shown in SEQIDNo.10~12 and SEQIDNo.14~16 respectively . 2. An anti-CD24 monoclonal antibody containing the variable region sequence according to claim 1, characterized in that, the structure of the monoclonal antibody includes a heavy chain and a light chain shown in SEQ ID No.9 and 13 amino acid sequence. 3. A genetically engineered antibody, characterized in that the heavy chain and light chain variable region sequences it contains are consistent with the variable region sequence described in claim 1; the genetically engineered antibody includes but is not limited to: human - a murine chimeric antibody; or a humanized antibody; or a functional fragment Fab of an antibody; or a single chain antibody; or a functional fragment VH-L of an antibody in which the variable region of the heavy chain is fused to the entire light chain; or It is an antibody functional fragment formed by the arrangement, series or combination of one or more CDRs of the heavy chain and light chain; or it is obtained by connecting, splicing, or fusing the above-mentioned antibodies and antibody functional fragments with various other proteins or polypeptides Functional fusion protein of antibody-like. 4. An antibody conjugate, characterized in that the monoclonal antibody as claimed in claim 2 or the genetically engineered antibody as claimed in claim 3 is used as a targeting moiety and coupled with a radionuclide or a chemical drug or toxin. 5. The monoclonal antibody of claim 2 or the genetically engineered antibody of claim 3, characterized in that it selectively binds to CD24 or inhibits the binding of CD24 to a CD24 ligand or neutralizes CD24. 6. The monoclonal antibody as claimed in claim 2 or the genetically engineered antibody as claimed in claim 3 or the antibody conjugate as claimed in claim 4, characterized in that it is used for the preparation and treatment of diseases related to abnormal or excessive expression of CD24 and out of control medicine. 7. A method for preparing an anti-CD24 monoclonal antibody, characterized in that the conjugate of the polypeptide in the core region of CD24 and hemocyanin KLH is used as the antigen.