CN115850492A - Monoclonal antibody and polynucleotide for resisting glypican-3, and preparation method and application thereof - Google Patents

Abstract

The application relates to the technical field of tumor immunodiagnosis, in particular to a monoclonal antibody and a polynucleotide for resisting phosphatidylinositol proteoglycan-3, and a preparation method and application thereof; the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain region CDR1 with an amino acid sequence shown as SEQ ID NO. 1, a heavy chain region CDR2 with an amino acid sequence shown as SEQ ID NO. 2 and a heavy chain region CDR3 with an amino acid sequence shown as SEQ ID NO. 3; the light chain variable region comprises a light chain region CDR1 with an amino acid sequence shown as SEQ ID NO. 4, a light chain region CDR2 with an amino acid sequence shown as SEQ ID NO. 5 and a light chain region CDR3 with an amino acid sequence shown as SEQ ID NO. 6; the dissociation constant KD (M) of the monoclonal antibody to a GPC3 antigen is 1.21E-11, belongs to a high-affinity N-terminal antibody, and is suitable for detection of clinical GPC3 serum samples.

Description

Monoclonal antibody and polynucleotide for resisting glypican-3, and preparation method and application thereof

Technical Field

The application relates to the technical field of tumor immunodiagnosis, in particular to a monoclonal antibody and a polynucleotide for resisting glypican-3, and a preparation method and application thereof.

Background

Primary liver cancer is one of the most common malignant tumors in the world, and ranks as the fifth most malignant tumor in the world. Among them, the most common primary tumor of liver is hepatocellular carcinoma (HCC), and since liver cancer is usually in middle and late stages when symptoms appear, recurrence and metastasis rate after resection are high, early diagnosis of liver cancer is of great significance for prolonging survival time of patients and reducing death rate of liver cancer.

At present, two means of imaging examination and tumor marker detection are mainly used for early diagnosis and screening of liver cancer, and imaging examination has certain lag, and examination depends on the technology and experience of an operator, so that the method is not suitable for large-scale application and is not beneficial to early diagnosis and treatment of liver cancer, and therefore the early diagnosis of liver cancer still needs to be realized by a high-sensitivity serum liver cancer specific marker. The most common tumor marker of the current primary liver cancer is alpha-fetoprotein (AFP), but the positive rate of diagnosing HCC by AFP is only 50%, the sensitivity is obviously reduced in small liver cancer with the diameter less than 3cm, the positive rate is less than 40%, and the missed diagnosis is easily caused; secondly, the increase of the number of benign liver diseases, reproductive teratoma, lung cancer and other patients can also cause misdiagnosis. Therefore, the search for a new liver cancer diagnosis marker which has high specificity, high sensitivity and easy detection has a far-reaching clinical significance.

Glypican-3 (Glypican-3, GPC3) is a cell surface protein belonging to the heparan sulfate proteoglycan family. GPC 3is highly expressed in fetal liver, but not in liver tissue of normal adults, but is recovered and expressed in liver cell liver cancer, has a close relationship with the occurrence and development of liver cancer, has high detection rate in early liver cancer occurrence, and is increased along with the development of liver cancer, so GPC3 can replace AFP to serve as a liver cancer diagnosis marker. However, because the existing antibodies against cancer markers have great differences in affinity and specificity, and are mostly difficult to meet the requirements of practical application, taking HCC as an example, if AFP is replaced by GPC3 to serve as a liver cancer diagnostic marker, because the affinity between a GPC3 monoclonal antibody and a target antigen is low, the specificity between the GPC3 monoclonal antibody and the antigen is not high, and the detection accuracy is affected, so how to provide a GPC3 monoclonal antibody with high affinity with the target antigen is a technical problem to be solved at present.

Disclosure of Invention

The application provides a monoclonal antibody and a polynucleotide for resisting glypican-3, and a preparation method and application thereof, which aim to solve the technical problem of low affinity between a GPC3 monoclonal antibody and a target antigen in the prior art.

In a first aspect, the present application provides a monoclonal antibody against glypican-3, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain region CDR1, a heavy chain region CDR2 and a heavy chain region CDR3, the amino acid sequence of the heavy chain region CDR1 is shown in SEQ ID NO. 1, the amino acid sequence of the heavy chain region CDR2 is shown in SEQ ID NO. 2, and the amino acid sequence of the heavy chain region CDR 3is shown in SEQ ID NO. 3;

the light chain variable region comprises a light chain region CDR1, a light chain region CDR2 and a light chain region CDR3, wherein the amino acid sequence of the light chain region CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the light chain region CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain region CDR 3is shown as SEQ ID NO. 6.

Alternatively, the amino acid sequence of the heavy chain region is shown in SEQ ID NO 7.

Alternatively, the amino acid sequence of the light chain region is shown in SEQ ID NO 8.

In a second aspect, the present application provides a polynucleotide encoding the monoclonal antibody of the first aspect.

Optionally, the polynucleotide includes a first coding region to encode the heavy chain variable region of the monoclonal antibody, the nucleotide sequence of the first coding region is shown in SEQ ID NO 9.

Optionally, the polynucleotide further comprises a second coding region for encoding the light chain variable region of the monoclonal antibody, and the nucleotide sequence of the second coding region is shown as SEQ ID NO. 10.

In a third aspect, the present application provides an expression vector comprising the polynucleotide of the second aspect.

In a fourth aspect, the present application provides a host cell comprising a polynucleotide according to the second aspect or an expression vector according to the third aspect.

In a fifth aspect, the present application provides a method for preparing a monoclonal antibody against glypican-3, the method comprising:

culturing the host cell of the third aspect, and then expressing to obtain a fusion protein;

constructing a hybridoma cell line from the fusion protein;

and (3) culturing the hybridoma cell line in vivo to obtain the monoclonal antibody of the anti-phosphatidylinositol proteoglycan-3.

In a sixth aspect, the present application provides a use of a monoclonal antibody against glypican-3, the use comprising using the monoclonal antibody of the first aspect in the preparation of a detection reagent for liver cancer detection.

Compared with the prior art, the technical scheme provided by the embodiment of the application has the following advantages:

the monoclonal antibody against glypican-3 provided in the embodiment of the application is designed by designing the complementarity determining regions of the heavy chain variable region and the complementarity determining regions of the light chain variable region of the monoclonal antibody, respectively, and since the binding properties of the antibody and the antigen can be described by the complementarity determining regions, the heavy chain variable region or the light chain variable region can be partitioned into 4 framework regions by using the designed amino acid sequences of the six complementarity determining regions, and at the stage when β folds formed in the framework regions are close to each other in a spatial structure, the designed six complementarity determining regions form the sites for antigen binding, and the matching strength between the para-position of the antibody and the epitope of the antigen is enhanced, so that the affinity between the antibody and the antigen is improved, and therefore, the affinity between the GPC3 monoclonal antibody and the target antigen can be improved by designing the complementarity determining regions of the heavy chain variable region and the complementarity determining regions of the light chain variable region.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and together with the description, serve to explain the principles of the invention.

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without inventive exercise.

Fig. 1 is a schematic flow chart of a method provided in an embodiment of the present application;

FIG. 2 is a SDS-PAGE pattern of expression purified GPC 3-554A protein provided in the examples herein, wherein 1 represents the total sample of cell expression supernatant, 2 represents the effluent of the total sample loading, and 3 represents the eluted protein of interest;

FIG. 3is a graph showing a comparison between the flow cytometry results of 4 antibodies and the hepatoma cell line HepG2 provided in the examples herein;

fig. 4 is a schematic diagram of a standard curve provided in the embodiment of the present application.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are illustrative of the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, 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 invention belongs. If there is a conflict, the present specification will control.

Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.

The inventive thinking of the application is that:

because the GPC3 gene codes and generates precursor core protein of about 70kDa, the precursor protein can be cut by furin (furin) to generate soluble amino terminal (N terminal) peptide of about 40kDa capable of entering blood and membrane-bound carboxyl terminal (C terminal) peptide of about 30-kDa containing 2 Heparan Sulfate (HS) sugar chains, GPC3 protein is attached to a cell membrane through Glycosyl Phosphatidyl Inositol (GPI) anchor, and GPC 3is highly expressed in fetal liver and not expressed in liver tissue of normal adults, but is restored and expressed in liver cell liver cancer, therefore, GPC3 has close relation with the occurrence and development of liver cancer, the detection rate is higher in the early stage of liver cancer occurrence, and the detection rate is increased along with the development of liver cancer.

Since the existing antibodies against cancer markers have great differences in affinity and specificity, and are mostly difficult to meet the requirements of practical application, taking GPC3 as an example, it is reported abroad that GPC3 has a low content in peripheral blood, and the increase of GPC3 concentration in liver cancer patients is small, so that the antibodies are not easy to be distinguished from normal controls, especially cirrhosis controls. The reason for this is that the monoclonal antibody of GPC3 has low specificity and low affinity with the target antigen, which results in unsatisfactory detection accuracy and limits its application in tumor detection.

In order to solve the technical problems, the embodiment of the invention provides the following general ideas:

in one embodiment of the present application, there is provided a monoclonal antibody against glypican-3, the monoclonal antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain region CDR1, a heavy chain region CDR2 and a heavy chain region CDR3, the amino acid sequence of the CDR1 of the heavy chain region being represented by SEQ ID No. 1, the amino acid sequence of the CDR2 of the heavy chain region being represented by SEQ ID No. 2, and the amino acid sequence of the CDR3 of the heavy chain region being represented by SEQ ID No. 3;

the light chain variable region comprises a light chain region CDR1, a light chain region CDR2 and a light chain region CDR3, wherein the amino acid sequence of the light chain region CDR1 is shown in SEQ ID NO. 4, the amino acid sequence of the light chain region CDR2 is shown in SEQ ID NO. 5, and the amino acid sequence of the light chain region CDR 3is shown in SEQ ID NO. 6.

In some alternative embodiments, the amino acid sequence of the heavy chain region is as set forth in SEQ ID NO 7.

In the examples of the present application, the amino acid sequence of the heavy chain region is controlled such that a part of the monoclonal antibody can be smoothly expressed in the entire heavy chain region on the basis of the complementarity determining region of the heavy chain region.

In some alternative embodiments, the amino acid sequence of the light chain region is as set forth in SEQ ID NO 8.

In the examples of the present application, the amino acid sequence of the light chain region is controlled such that a portion of the monoclonal antibody can be successfully expressed in the entire light chain variable region based on the complementarity determining region of the light chain variable region.

Next, a polynucleotide provided in an embodiment of the present application is described, the polynucleotide being for encoding the monoclonal antibody according to the first aspect.

Since the polynucleotides described in the examples of the present application and the monoclonal antibodies encoded by the polynucleotides are the monoclonal antibodies provided in the examples of the present application, the amino acid sequence and composition information of the monoclonal antibodies are not repeated herein. All polynucleotides encoding the monoclonal antibodies of the embodiments of the present application are within the scope of the present application.

In some alternative embodiments, the polynucleotide includes a first coding region to encode the heavy chain variable region of the monoclonal antibody, the nucleotide sequence of the first coding region being set forth in SEQ ID NO. 9.

In the embodiment of the present application, the first coding region is controlled to encode the heavy chain variable region, so that the nucleotides corresponding to the amino acid sequences of the three complementarity determining regions in the heavy chain variable region can be synchronously expressed while the first coding region is expressed, thereby smoothly expressing the heavy chain variable region.

In some alternative embodiments, the polynucleotide further comprises a second coding region encoding the light chain variable region of the monoclonal antibody, the nucleotide sequence of the second coding region being set forth in SEQ ID NO. 10.

In the embodiment of the present application, the second coding region is controlled to encode the light chain variable region, so that the nucleotides corresponding to the amino acid sequences of the three complementarity determining regions in the light chain variable region can be synchronously expressed while the second coding region is expressed, thereby smoothly expressing the light chain variable region.

Next, an expression vector provided in the embodiments of the present application, which includes the polynucleotide, is described.

Since the polynucleotide included in the expression vector described in the examples is the polynucleotide provided in the examples, the nucleotide sequence and composition information of the polynucleotide are not repeated herein. All expression vectors that include the polynucleotides of the embodiments of the present application are intended to be within the scope of the present application.

Next, a host cell containing the polynucleotide or the expression vector provided in the examples of the present application is described.

Since the host cell described in the embodiments of the present application includes the polynucleotide or expression vector provided in the embodiments of the present application as described above, the nucleotide sequence and composition information of the polynucleotide or expression vector will not be described herein. All host cells that include the polynucleotides or expression vectors of the embodiments are intended to be within the scope of the present application.

Next, a method for producing an anti-glypican-3 monoclonal antibody provided in the examples of the present application will be described, the method comprising:

s1, culturing the host cell of the third aspect, and expressing to obtain fusion protein;

s2, constructing a hybridoma cell line by using the fusion protein;

s3, culturing the hybridoma cell line in vivo to obtain the monoclonal antibody of the anti-phosphatidylinositol proteoglycan-3.

Since the preparation method described in the examples of the present application is the monoclonal antibody provided in the examples of the present application, the amino acid sequence and composition information of the monoclonal antibody will not be described herein again. All methods of preparation of monoclonal antibodies including the examples of the present application are within the intended scope of the present application.

Next, the application of the monoclonal antibody against glypican-3 provided in the embodiment of the present application, which includes the use of the monoclonal antibody in the preparation of a detection reagent for liver cancer detection, will be described.

Since the monoclonal antibodies included in the applications described in the examples of the present application are the monoclonal antibodies provided in the examples of the present application, the amino acid sequences and composition information of the monoclonal antibodies will not be described herein again. All methods of preparation of monoclonal antibodies including the examples of the present application are within the intended scope of the present application.

Example 1

As shown in FIG. 1, construction of the vector:

1. the gene is synthesized into NM-004484 218-1807bp (refer to NP-001158089.1, glypican-3isofomm 1, 25-554 AA), wherein the synthesized gene comprises the steps of adding V5H vector cloning enzyme cutting sites NheI/BamHI at both ends of the synthesized gene.

2. And carrying out double enzyme digestion on the synthesized gene sequence and the vector V5H by endonuclease NheI/BamHI respectively, carrying out agarose gel electrophoresis on the product, cutting gel and recovering to obtain a double enzyme digestion product.

3. The double digestion products were ligated overnight in a water bath at 16 ℃ with T4 ligase, transformed into E.coli DH 5. Alpha. Strain and screened for positive clones.

The inserted fragment is completely consistent with the fragment in the published complete sequence through sequencing identification, and is inserted into the cloning site of an expression vector in the correct direction, and the recombinant plasmid V5H-25-554AA is amplified and extracted.

Example 2

Example 2 is compared to example 1, with example 2 differing from example 1 in that:

as shown in fig. 1, expression of the fusion protein was purified:

1. the HEK293 cells meeting the requirements are processed at 2X 10 ⁵ And (4) paving the cells/hole in a 6-hole plate for culture, and after 24 hours of culture, performing transfection when the cell fusion degree is 80-90%.

2. The cell suspension was washed 3 times with PBS and serum-free DMEM high-sugar medium was added. The recombinant expression plasmid V5H-25-554AA was transfected according to the Lipofectamine TM2000 transfection reagent instructions at 37 ℃ and 5% by volume CO ₂ Culturing for 72h at the rotation speed of 120rpm under the condition.

3. The cell culture fluid was collected, centrifuged at 4500g for 15min, the cells were removed, and the supernatant was collected.

4. 1mL of Ni-NTA Agarose affinity filler was packed in a column, the Ni-NTA affinity column was equilibrated to 10 column volumes with an equilibration buffer (50 mM PB, 0.3M NaCl, 10mM imidazole, pH 8.0), and the centrifuged cell culture supernatant was passed through the Ni-NTA affinity column at 1mL/min, and the collected flow-through solution was stored at 4 ℃.

5. The column was washed with 10 volumes of washing buffer (50 mM PB, 0.3M NaCl, 20mM imidazole, pH 8.0) and the flow-through was collected and stored at 4 ℃. After 4 to 5 column volumes of elution buffer (50 mM PB, 0.3M NaCl, 250mM imidazole, pH 8.0) were washed, the eluate was collected and dialyzed overnight at 4 ℃ against a dialyzate (50 mM PB, pH 7.8,0.3M NaCl, 5% by mass glycerol) to obtain GPC3 25-554A protein, and a small amount of the protein was subjected to SDS PAGE, the results of which are shown in FIG. 2.

Example 3

Example 3is compared to example 2, with example 3 differing from example 2 in that:

as shown in FIG. 1, hybridoma cell lines were established:

1. 1.0mg/mL of the GPC 3-554A protein prepared in example 2 as an antigen was mixed with 1mL of complete Freund's adjuvant (purchased from Sigma-aldrich Co.) in a well emulsified state, and then BALB/c mice 6 to 8 weeks old were immunized subcutaneously with 100. Mu.g of GPC 3-554A protein antigen per mouse.

2. After 3 weeks GPC3 25-554A protein antigen was mixed with incomplete Freund's adjuvant by emulsification, mice were immunized subcutaneously with 50. Mu.g of GPC 3-554A protein antigen per mouse, followed by 2 weeks following, 50. Mu.g of GPC 3-554A protein antigen by subcutaneous booster immunization.

3. After 1 week of the 4 th booster immunization, the mice were coated with GPC3 25-554A protein, and the mouse antiserum titer was measured by ELISA, and the booster immunization was continued until the mouse antiserum titer reached>10 ⁵ . After 3 weeks of the last booster immunization, 20. Mu.g of GPC 3-554A protein antigen was immunized in the spleen for future use.

4. 4 days after the mice were boosted intraperitoneally, the spleens were aseptically removed, lymphocytes were separated by filtration through a 100-mesh filter, fused with the myeloma cell line SP2/0, selectively cultured for 3 days with hypoxanthine, aminopterin, and thymidine (HAT), supplemented with HT medium, and cultured for another 1 week.

5. GPC3 25-554A protein antigen was coated, positive clones were screened by ELISA, subcloned 3 times by limiting dilution, and continued to culture for 2 months, to finally obtain stable hybridoma cell lines (clone numbers: 1G3,4C8, 10F9 and 9D5, respectively).

6. The results of indirect ELISA screening for hybridoma cell lines of different clone numbers are shown in Table 1.

TABLE 1 ELISA screening results for hybridoma cell lines of different clone numbers

Coating \ antibody strain	1G3	4C8	10F9	9D5
					GPC3 25-554AA antigens	2.892	1.9	1.932	1.512
Blank space	0.057	0.089	0.076	0.078

Example 4

Example 4 is compared to example 3, with example 4 differing from example 3 in that:

as shown in fig. 1, ascites production and antibody purification:

1. f1 mice, 8-10 weeks old, were intraperitoneally injected with 500 μ L of norphytane (purchased from Sigma-aldrich). The 4 hybridoma cells were cultured separately and fresh viable cells were collected. According to 1X 10 ⁶ Injecting the cell/mouse into abdominal cavity of mouse, and collecting ascites after 7-10 daysCentrifuging at 10000g for 10min, and collecting supernatant. Protein G affinity columns (from GE) were equilibrated for 5 column volumes with PBS (0.01M PB,0.15M NaCl, pH 7.4).

2. The ascites supernatant was mixed with 2-fold volume of PBS (0.01M PB,0.15M NaCl, pH 7.4), filtered through a 0.22 μ M filter, and the resulting ascites supernatant was subjected to protein G affinity column filtration, and washed with 5 column volumes of PBS.

3. Eluting with elution buffer (0.1M Glycine HCl, pH 2.8), adding 1/10 volume of neutralization buffer (1M NaH) to the eluate ₂ PO ₄ pH 9.0).

4. The solution was dialyzed against PBS (0.01M PB,0.15M NaCl, pH 7.4) with two fluid changes with a time interval of more than 5 hours to obtain a dialysate.

5. The dialyzed solution was centrifuged at 10000g for 10min, and the supernatant was filtered through a 0.22 μm filter to obtain a purified solution corresponding to the anti-GPC 3 monoclonal antibody produced by each clone.

Example 5

Example 5 is compared with example 4, the difference between example 5 and example 4 being:

antibody epitope classification:

1. antigen coating: adopting carbonate buffer solution as coating solution, wherein the concentration of the GPC3 recombinant protein of the coating antigen is 0.5 mu g/mL, adding the coating solution and the coating antigen on a 96-well enzyme label plate according to 100 mu L per well, and standing overnight at 4 ℃; washing, namely after the coated plate is returned to the room temperature, pouring back to remove the coating solution, adding 300 mu L of washing solution into each hole, shaking lmin each time, washing for 3-4 times, and patting dry;

2. and (3) sealing: adding 200 μ L of calf serum with mass concentration of 10% as sealing liquid into each well, and keeping the temperature at 37 deg.C for 1h; washing, pouring off the confining liquid after the temperature is returned to the room temperature, washing for three times, shaking lmin each time, and patting dry;

3. diluting the GPC3 antibody with a buffer solution to a concentration of 5. Mu.g/mL, adding 100. Mu.L per well, and setting a blank control well (PBS) and a negative well (negative serum), standing at 37 ℃ for 30min; washing for 3 times, shaking lmin each time, and patting dry;

4. adding enzyme-labeled secondary antibody, adding 100 μ L of HRP enzyme-labeled goat anti-mouse IgG diluted with 1; washing for 3 times, shaking for 1min each time, and patting to dry;

5. developing, adding 100 μ L of substrate developing solution into each well, and reacting at 37 deg.C under heat preservation and light shielding conditions for 15min; the reaction was terminated by adding 50. Mu.L of a stop solution to each well

6. Measuring the OD450nm value of the solution after termination, reading the optical density value of each hole by using an enzyme-labeling instrument with the detection wavelength of 450nm, setting the OD450nm value of the negative control hole as N, setting the positive control hole as P, and setting the positive result as that P/N is more than or equal to 2.1, wherein the results are shown in Table 2.

TABLE 2 Indirect ELISA epitope Classification results Table

Coating \ antibody strain	1G3	4C8	10F9	9D5
					GPC3 25-554AA antigen	2.892	1.9	1.932	1.512
Externally purchased GPC 3N terminal antigen (25-358 AA)	2.413	2.318	0.783	0.115
					Purchased GPC 3C terminal antigen (359-554 AA)	0.033	0.115	0.096	1.661

In the table, antibodies 1G3,4C8 and 10F9 are antibodies against the N-terminal epitope of GPC3, and antibody 9D5 is an antibody against the C-terminal epitope of GPC 3.

Example 6

Example 6 is compared with example 5, the difference between example 6 and example 5 being:

monoclonal antibody affinity determination against GPC 3:

the affinity of each monoclonal antibody against GPC3 with recombinant human GPC3 was determined by a BIACORE3000 biomacromolecule interactometer (available from GE corporation), and the results are shown in Table 3. The AW mab-5 with the highest affinity was selected as a monoclonal antibody against GPC3 for detection, for subsequent detection and further development.

TABLE 3 dissociation constants of monoclonal antibodies against GPC3 and target antigen

Antibody strains	Dissociation constant KD (M)
		9D5	8.66E-09
10F9	7.58E-08
		4C8	2.78E-10
1G3	1.21E-11

Example 7

Example 7 is compared with example 6, which differs from example 6 in that:

evaluation of binding Activity:

1. HepG2, huH-7 and LO2 cell lines at 1X 10 per ml ⁶ Cells were suspended in FACS buffer (1% by mass FBS/PBS). The suspension was dispensed into Multiscreen-HV Filter Plate (Milloprene) at 100. Mu.L per well and the supernatant removed after centrifugation. 2.

2. An anti-GPC 3 antibody diluted to an appropriate concentration was added and reacted on ice for 30min.

3. Cells were washed once with FACS buffer.

4. FITC-labeled anti-mouse IgG antibody was added and the reaction was carried out on ice for 30min.

5. After the reaction, the cells were centrifuged at 500rpm for 1 minute, and the supernatant was removed.

6. Cells were suspended in 400 μ L FACS buffer and used for flow cell counting.

The results showed that the GPC3 antibodies 1G3,4C8 and 10F9 strongly bound to the hepatoma cell lines HepG2 and HuH-7,9D5 to a lesser extent, and that neither of the 4 cell lines bound to the normal hepatoma cell line LO2, indicating that these antibodies specifically recognized hepatoma.

The flow cytometry results of the 4-strain antibody and the hepatoma cell line HepG2 are shown in FIG. 3.

Example 8

Example 8 is compared with example 7, with the difference between example 8 and example 7 being that:

cloning of the variable regions of monoclonal antibody 1G 3:

1. RNA from the hybridoma of GPC3 antibody 1G3 was extracted using RNeasy Plus Universal Mini Kit (QIAGEN), and the variable region of the anti-GPC 3 antibody was amplified by RT-PCR.

2. The 5' -end gene fragment was amplified from 1. Mu.g of total RNA using SMART RACE cDNA amplification kit (CLONTECH) and synthetic oligonucleotides, and reverse transcription was performed at 42 ℃ for 1h and 30min to obtain a PCR mixture, wherein the synthetic oligonucleotides were as follows:

the synthetic oligonucleotide complementary to the mouse IgG1 constant region sequence is shown in SEQ ID NO: 11: GGG CCA GTG GAT agaacag ATG;

the synthetic oligonucleotide complementary to the mouse kappa chain constant region sequence is shown in SEQ ID NO 12: GCT CAC TGG ATG GTG GGA AGA TG.

3. A PCR mixture (50. Mu.L) was subjected to PCR using a PCR solution containing 5. Mu.L of 10 XDavantage 2PCR buffer, 5. Mu.L of 10 XDUART Mix, 0.2mM dNTPs (dATP, dGTP, dCTP and dTTP), 1. Mu.L of Advantage 2Polymerase Mix (all from CLONTECH), 2.5. Mu.L of reverse transcription reaction product and 10pmol of synthetic oligonucleotide, the PCR procedure comprising: the reaction product was obtained in 5 cycles of 94 ℃ 30s,94 ℃ 5s and 72 ℃ 3min, 5 cycles of 94 ℃ 5s,70 ℃ 10s and 72 ℃ 3min, and 25 cycles of 94 ℃ 5s,68 ℃ 10s and 72 ℃ 3 min.

4. The reaction products were heated at 72 ℃ for 7min, and then each PCR product was purified from agarose gel using QIAquick gel extraction kit (QIAGEN), cloned into pGEM-T Easy vector (Promega), and the nucleotide sequence was determined.

The results of the measurement showed that the nucleotide sequence of the first coding region (heavy chain variable region) of monoclonal antibody 1G3 was shown by SEQ ID NO. 9, and the translated amino acid sequence thereof was shown by SEQ ID NO. 7.

The nucleotide sequence of the second coding region (light chain variable region) of monoclonal antibody 1G 3is shown in SEQ ID NO. 10, while the translated amino acid sequence thereof is shown in SEQ ID NO. 8.

Example 9

Example 9 is compared to example 8, with example 9 differing from example 8 in that:

coupling of antibody and magnetic beads:

1. washing: mu.L (1 mg/mL) of magnetic beads were added to 1mL of reaction buffer (0.05M MES,0.5M NaCl, pH 5.5) and washed three times with shaking for 1min each time.

2. And (3) activation: after the magnetic beads are washed, the reaction buffer is absorbed, 200 mu L of activating agent EDC (with the mass concentration of 1 mg/mL) and 200 mu L of NHS (with the mass concentration of 1 mg/mL) are added, the mixture is ready for use, and the shaking reaction is carried out for 60min to complete the activation.

3. Protein coupling: after the activation, 1mL of coupling solution (0.15M sodium phosphate, 0.15M NaCl, pH 7.5) is sucked and washed with shaking for three times, 1min each time, after the washing is completed, 200 μ L of coupling solution is added, 1G3 to 30 μ G of monoclonal antibody is added, and the reaction is carried out with shaking at room temperature for 2h.

4. And (3) sealing: the coating solution was aspirated off, 1mL of blocking solution (a mixture of 20mg/mL BSA, 20mg/mL glycine, and a coupling solution) was added, and the reaction was performed with shaking at room temperature for 1 hour.

5. And (3) storage: after blocking, washing with 100. Mu.L of blocking solution for 3 times, shaking and washing for 5min each time, after washing, diluting to 300. Mu.L with stock solution (BSA with mass concentration of 20mg/mL, glycine with mass concentration of 20mg/mL, coupling solution and Proclin 300 with mass concentration of 0.05%), and storing in a refrigerator at 4 ℃.

Example 10

Example 10 is compared to example 9, with example 10 differing from example 9 in that:

chemiluminescence detection clinical samples:

a chemiluminescence double-antibody sandwich method is established by taking GPC3 antigen 25-554AA as a raw material to prepare standard products S1-S6, an antibody 4C8 of an alkaline phosphatase marked GPC3 antibody and magnetic beads coupled with GPC3 antibody 1G3 magnetic beads as main components of a kit, and a standard product curve is shown in figure 4.

Serum samples of different individuals are collected through multiple hospitals in different regions for a total of 500 cases, wherein 200 cases are marked for normal physical examination, 250 cases are marked for liver disease patient samples (220 cases are liver cancer patient samples, and 30 cases are hepatitis and liver cirrhosis samples), and 50 cases are marked for other cancer patient samples.

The samples were tested according to the established chemiluminescence method in the following steps:

1. adding 30 mu L of serum sample to be tested and high and low concentration calibrator into a reaction cup;

2. adding 50 mu L of magnetic microsphere solution coupled by the monoclonal antibody 1G3 in the kit;

3. adding 50 mu L of alkaline phosphatase labeled antibody 4C8 solution in the kit;

4. incubating each group at 37 deg.C for 15min, and cleaning in magnetic environment for 3 times;

5. adding a luminescent substrate to each group, and detecting the intensity of the optical signal;

6. and the GPC3 concentration of the sample to be detected can be automatically calculated according to the detection light intensity of the sample by the corrected working curve of the calibrator.

The clinical detection result statistics show that the concentration of 220 HCC serum GPC 3is 4.0 +/-20.01 ng/mL, and 15 cases are higher than 20ng/mL; the concentration of 30 parts of hepatitis cirrhosis serum GPC 3is 0.5 +/-2 ng/mL; other cancer samples: 0.4 +/-0.5 ng/m; normal samples: 0.3 +/-0.4 ng/m.

The GPC3 concentration in each group is in non-normal distribution, and the detection specificity is 96% and the sensitivity is 65.6% when the CUTOFF value is 1.1ng/mL calculated by SPSS software.

One or more technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

(1) The monoclonal antibody of the anti-phosphatidylinositol proteoglycan-3 provided by the embodiment of the application is determined by a BIACORE3000 biomacromolecule interaction instrument, has a dissociation constant KD (M) of 1.21E-11 to a GPC3 antigen, and belongs to a high-affinity antibody.

(2) The monoclonal antibody against glypican-3 provided in the examples of the present application belongs to the GPC3 protein N-terminal antibody as determined by epitope Elisa. It has been reported that GPC-3 protein enters blood circulation mainly in the N-terminal form, and therefore the antibody of the present invention is suitably applied to detection of clinical serum samples of GPC 3.

(3) The monoclonal antibody for resisting glypican-3 provided by the embodiment of the application can effectively identify a liver cancer cell line by matching with a flow cytometry detection technology.

(4) The monoclonal antibody of the anti-glypican-3 provided by the embodiment of the application is used for detecting a clinical sample by coupling the monoclonal antibody to magnetic beads and then by a chemiluminescence test method, and in the detection process of a liver cancer sample, the sensitivity reaches 65.6%, the specificity reaches 96%, and the monoclonal antibody is superior to antibodies of the same type or detection indexes of other liver cancer markers.

(5) The monoclonal antibody for resisting glypican-3 provided by the embodiment of the application has high sensitivity and good specificity, can realize large-batch rapid detection, is low in use cost and easier to popularize and apply, and can play a great role in diagnosis and treatment of tumor diseases in clinical application and detection.

It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrases "comprising 8230; \8230;" 8230; "does not exclude the presence of additional like elements in a process, method, article, or apparatus that comprises the element.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. The monoclonal antibody of anti-glypican-3 is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain region CDR1, a heavy chain region CDR2 and a heavy chain region CDR3, the amino acid sequence of the heavy chain region CDR1 is shown as SEQ ID NO. 1, the amino acid sequence of the heavy chain region CDR2 is shown as SEQ ID NO. 2, and the amino acid sequence of the heavy chain region CDR 3is shown as SEQ ID NO. 3;

the light chain variable region comprises a light chain region CDR1, a light chain region CDR2 and a light chain region CDR3, wherein the amino acid sequence of the light chain region CDR1 is shown as SEQ ID NO. 4, the amino acid sequence of the light chain region CDR2 is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain region CDR 3is shown as SEQ ID NO. 6.

2. The monoclonal antibody of claim 1, wherein the heavy chain region has the amino acid sequence set forth in SEQ ID NO 7.

3. The monoclonal antibody of claim 1, wherein the amino acid sequence of the light chain region is set forth in SEQ ID NO 8.

4. A polynucleotide encoding the monoclonal antibody of any one of claims 1-3.

5. The polynucleotide of claim 4, wherein the polynucleotide comprises a first coding region encoding the heavy chain variable region of the monoclonal antibody, and the nucleotide sequence of the first coding region is set forth in SEQ ID NO. 9.

6. The polynucleotide of claim 4, further comprising a second coding region encoding the light chain variable region of the monoclonal antibody, wherein the nucleotide sequence of the second coding region is set forth in SEQ ID NO. 10.

7. An expression vector comprising the polynucleotide of any one of claims 4-6.

8. A host cell comprising the polynucleotide of any one of claims 4 to 6 or the expression vector of claim 7.

9. A method for producing a monoclonal antibody against glypican-3, the method comprising:

culturing the host cell of claim 8 and expressing to obtain a fusion protein;

constructing a hybridoma cell line from the fusion protein;

and (3) culturing the hybridoma cell line in vivo to obtain the monoclonal antibody against the phosphatidylinositol proteoglycan-3.

10. Use of a monoclonal antibody against glypican-3, which comprises using the monoclonal antibody according to any one of claims 1 to 3 for the preparation of a detection reagent for liver cancer detection.

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