Monoclonal antibody to afp and method of making and using same

Abstract

The application provides an AFP monoclonal antibody and a preparation method and application thereof, relates to the field of antibodies in the field of proteins, and the AFP monoclonal antibody has the following sequences: the heavy chain CDR1 sequence is SEQ ID NO.4; the heavy chain CDR2 sequence is SEQ ID NO.5; the heavy chain CDR3 sequence is SEQ ID NO.6; the light chain CDR1 sequence is SEQ ID NO.7; the light chain CDR2 sequence is SEQ ID NO.8; the light chain CDR3 sequence is SEQ ID NO.9; or the heavy chain CDR1 sequence is SEQ ID NO.12; the heavy chain CDR2 sequence is SEQ ID NO.13; the heavy chain CDR3 sequence is SEQ ID NO.14; the light chain CDR1 sequence is SEQ ID NO.15; the light chain CDR2 sequence is SEQ ID NO.16; and the light chain CDR3 sequence is SEQ ID NO.17.

Description

Technical Field

[0001] This invention relates to the field of antibodies within the protein field, specifically to an AFP monoclonal antibody, its preparation method, and its applications. Background Technology

[0002] Alpha-fetoprotein (AFP) is a glycoprotein belonging to the albumin family, named for its high concentration in fetal blood. AFP plays multiple roles, including transport, growth factor function, immune regulation, and induction of apoptosis.

[0003] Within about one month after birth, alpha-fetoprotein (AFP) is gradually replaced by albumin. In healthy adults (excluding pregnant women), AFP blood concentrations are very low, almost undetectable. Elevated AFP levels in adults are often a marker for hepatitis, liver cancer, and tumors such as pancreatic cancer, gastrointestinal cancer, colorectal cancer, and lung cancer. Therefore, AFP is a known and usable tumor marker, especially for liver cancer.

[0004] The most common methods for detecting serum biomarkers are immunoassay-based methods such as ELISA. The effectiveness of these methods is highly dependent on the antibody's affinity, specificity, and other properties. Therefore, to better utilize AFP as a disease biomarker, it remains necessary to develop monoclonal antibodies with improved detection performance. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides an AFP monoclonal antibody, its preparation method, and its application, solving the technical problem of developing monoclonal antibodies with good specificity and sensitivity and better detection performance.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] On one hand, the present invention provides an AFP monoclonal antibody, wherein the heavy chain CDR1 sequence of the AFP monoclonal antibody is SEQ ID NO.4; the heavy chain CDR2 sequence is SEQ ID NO.5; the heavy chain CDR3 sequence is SEQ ID NO.6; the light chain CDR1 sequence is SEQ ID NO.7; the light chain CDR2 sequence is SEQ ID NO.8; and the light chain CDR3 sequence is SEQ ID NO.9.

[0010] Alternatively, the heavy chain CDR1 sequence of the AFP monoclonal antibody is SEQ ID NO.12; the heavy chain CDR2 sequence is SEQ ID NO.13; the heavy chain CDR3 sequence is SEQ ID NO.14; the light chain CDR1 sequence is SEQ ID NO.15; the light chain CDR2 sequence is SEQ ID NO.16; and the light chain CDR3 sequence is SEQ ID NO.17.

[0011] Furthermore, the heavy chain variable region sequence of the AFP monoclonal antibody is SEQ ID NO.2; the light chain variable region sequence is SEQ ID NO.3.

[0012] Furthermore, the heavy chain variable region sequence of the AFP monoclonal antibody is SEQ ID NO.10; the light chain variable region sequence is SEQ ID NO.11.

[0013] On the other hand, the present invention provides a gene that encodes the aforementioned AFP monoclonal antibody.

[0014] On the other hand, the present invention provides a vector carrying the above-mentioned gene.

[0015] On the other hand, the present invention provides a host cell carrying the aforementioned vector.

[0016] On the other hand, the present invention provides a method for producing the above-mentioned AFP monoclonal antibody, the method comprising the step of culturing the above-mentioned host cells.

[0017] The host cell can be any suitable yeast, Escherichia coli, Bacillus subtilis, or cell-free expression system. Those skilled in the art can also select a suitable commercial vector based on the host and perform insertion operations according to the instructions.

[0018] When the antibody CDR region or variable region is known, those skilled in the art can routinely design the backbone region and other steps, and design the corresponding gene sequence according to the host preference.

[0019] On the other hand, the present invention provides the application of the above-mentioned AFP monoclonal antibody in the preparation of a kit for diagnosing tumors.

[0020] On the other hand, the present invention provides a non-diagnostic method for detecting AFP, wherein the method uses the above-mentioned AFP monoclonal antibody to detect AFP.

[0021] The non-diagnostic methods mentioned include, but are not limited to, scientific research methods and population health statistics.

[0022] (III) Beneficial Effects

[0023] This invention provides an AFP monoclonal antibody, its preparation method, and its applications. Compared with existing technologies, it has the following advantages:

[0024] Based on the results of screening, affinity testing, and actual testing, the AFP monoclonal antibody of the present invention has good specificity and sensitivity, and also has good potential for use as a detection reagent. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows the results of Western blotting identification of purified AFP protein.

[0027] Figure 2 Figure showing the results of quantitative purification of AFP protein by Coomassie Brilliant Blue staining.

[0028] Figure 3 The figure shows the serum antibody titer results of mice immunized with AFP antigen protein.

[0029] Figure 4 For identification of Western blot method Figure 3 Image of the results from the strongly positive wells of ELISA.

[0030] Figure 5 Figures showing the screening and identification results of the monoclonal antibody AFPH8: A represents the AFPH8 subclone ELISA screening results; B represents the AFPH8 subclone Western Blot identification results.

[0031] Figure 6 Figures showing the screening and identification results of the monoclonal antibody AFPE2: A represents the AFPE2 subclone ELISA screening results; B represents the AFPE2 subclone Western Blot identification results.

[0032] Figure 7 The comparison results of the detection sensitivity of the kit prepared for Example 5 of the present invention and the kit of the comparative example are shown in the following figure: A shows the comparison of OD values ​​of the kit of Example 5 of the present invention and the kit of the comparative example; B shows the color development results of the kit of Example 5 of the present invention and the kit of the comparative example after adding TMB substrate. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] This application provides an AFP monoclonal antibody, its preparation method, and its application. The AFP monoclonal antibody has good specificity and sensitivity, and also has good potential as a detection reagent.

[0035] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0036] Example 1 - Antigen Preparation:

[0037] The antigen gene (full-length AFP gene) was obtained as shown below: a 6×His tag was added to the end. The 6×His tag is a mouse-derived monoclonal antibody that recognizes the six consecutive histidine residues (HHHHHH) carried by the recombinant protein for the purification and detection of the target protein.

[0038]

[0039] Expression and purification of antigen proteins:

[0040] The above gene was cloned into the pET28 prokaryotic expression vector and expressed in the BL21 Escherichia coli strain.

[0041] A small-scale trial was conducted first. The specific procedure was as follows: the pET28-AFP plasmid, which had been verified by sequencing, was transformed into E. coli strain BL21, plated on LB agarose plates, and five clones of the strain were selected. The bacteria were incubated overnight with 15 ml of shaking solution (1 mM IPTG induction). After centrifugation, 2 ml of bacterial lysis buffer (MACS Buffer: 300 mM NaCl, 50 mM NaH2PO4 + 1% Triton X-100 + 1 mM lysozyme) was added, and the bacteria were lysed on ice for 1 hour. The supernatant and precipitate were then separated by centrifugation. The soluble protein in the supernatant will be purified and used as an immunogen for immunization and as an antigen in the screening of monoclonal antibodies.

[0042] Figure 1 The flowchart for identification and purification using Western blotting is shown. A small-scale soluble AFP-expressing strain was obtained, cultured in 1 L LB medium, and induced to express by 1 mM IPTG overnight. Bacteria were collected by centrifugation (5000 rpm, 15 min); 100 ml MACS Buffer was added, and the bacteria were sonicated on ice for 1 hour. Figure 1 (M step); centrifuge to collect the lysate supernatant ( Figure 1 The WL(S) step, where WL(S) is whole lysate (supernatant), is centrifuged at 1000 rpm for 30 min. 1 ml of Ni-NTA beads is added to the supernatant and the mixture is vortexed for 1 hour to allow the beads to bind to the protein. Figure 1 In the out and FT steps, out stands for ultrafiltration and FT stands for flow-through (protein flow through). Add MACS Buffer + 5mM imidazole and wash 3 times. Figure 1 (W1-W3 steps); Add the beads to the chromatography column and elute the protein three times with 10 ml Elution Buffer (MACS Buffer + 100 mM imidazole). Figure 1 (E1-E3 steps), use 10ml of elution buffer for each elution.

[0043] Figure 2 The image shows the results of quantitative purification of AFP protein using Coomassie Brilliant Blue staining. The protein indicated by the arrow is AFP protein. After purifying enough AFP antigen protein to immunize mice and screen for monoclonal antibodies, the protein was dialyzed into PBS Buffer and ultrafiltered to concentrate to 10 ml. The protein concentration for Coomassie staining identification was approximately 100 ug / ml.

[0044] Example 2 - Immunizing mice with purified protein:

[0045] Three Balb / c mice aged 8-12 weeks were immunized with purified AFP protein (15ug / mouse), designated as mouse 1, mouse 2, and mouse 3. The initial immunization was emulsified with Freund's complete adjuvant (antigen:adjuvant ratio 1:1). Subsequent immunizations were performed every month using Freund's incomplete adjuvant. Antibody titers in the immunized mice were measured using ELISA after the three immunizations.

[0046] See Figure 3 , Figure 3 The data shown are the antibody titers of three immunized mice, pre-immunized mice (blank control), and the antibody titer of the purified HIS monoclonal antibody of the control. The antibody titers (dilutions) of the three immunized Balb / C mice were all higher than 3.2 million times dilution, which was significantly higher than the purified HIS monoclonal antibody of the control (Thermo, USA).

[0047] Spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 3:1 (PEG method). The fused cells were seeded into eight 96-well plates and cultured in DMEM + 20% FBS + HAT medium. After about 8-10 days, approximately 30 clones were observed to have grown in each well. At this point, 100 μL of culture supernatant from each well was collected for indirect ELISA screening (antigen coating, incubation with hybridoma cell culture supernatant from the 96-well plate).

[0048] The positive wells of the ELISA were then subjected to Western blotting to determine whether the antibody recognized the target antigen, and whether the antibody's specificity and sensitivity were excellent. The results were as follows: Figure 4 As shown. Figure 4 In the diagram, lanes 1-16 represent Western blotting of cell culture supernatants from 16 positive wells to determine whether the protein recognizes AFP. Lane 22 represents anti-HIS monoclonal antibody (positive control, purified antibody, Thermo, USA).

[0049] Hybridoma cells from 16 wells with good antibody specificity and strong Western blotting signals were selected for subcloning. Approximately 1-2 cells per well were seeded into two 96-well plates. The same method and procedure were used until one positive well was identified (this is a single clone). After stable culture and screening, a total of 10 hybridoma cell lines were obtained.

[0050] Example 3 - Subclonal screening and identification of AFPH8 and AFPE2 monoclonal hybridoma strains:

[0051] AFPH8 monoclonal screening and identification:

[0052] The original clone 5H8 was a polyclonal sample with a good Western blotting signal in the screening process described above. Therefore, the single clone screened from the original well was named AFPH8.

[0053] Subcloning was performed on 5H8 wells selected for initial screening using Western blotting and ELISA, which showed good specificity and sensitivity. Cells from the original 5H8 wells were pipetted into a single-cell suspension and seeded into subcloning plates at 1-2 cells / well (Balb / C mouse peritoneal cells were pre-seeded as feeder cells and mouse feeder cells one day prior). The plates were then cultured in selection medium (DMEM + 20% FBS + penicillin antibody + 1X HAT) for 8 days. Single-cloning wells were labeled, and the cell count was observed to reach 200-500 cells / clone. ELISA selection was then performed (ELISA plates were coated with purified AFP protein). The selection results are shown below. Figure 5 As shown in Figure A, the red wells in A represent single-clone wells with high ELISA values.

[0054] Select wells with high ELISA scores and take 100 μL of culture supernatant as antigen for Western blotting to determine antibody specificity and sensitivity. The results are as follows: Figure 5 As shown in Figure B, all six selected monoclonal wells produced AFP monoclonal antibodies with excellent specificity and sensitivity. Subclone C12 was selected for subsequent antibody variable region sequencing and further research, while the remaining five subclones A3, H8, F7, C8, and C12 cells were expanded and cryopreserved for later use.

[0055] In B, - represents the lysate of the colon cancer cell line SW480; + represents the lysate of the colon cancer cell line SW480 + purified AFP protein; HIS is the control group, representing the anti-His monoclonal antibody (Thermo, USA).

[0056] AFPE2 monoclonal screening and identification:

[0057] The original clone 12E2 was a polyclonal sample with good Western blotting signal in the above screening, and the single clone screened from the original well was named AFPE2.

[0058] After the original fusion well 12E2 was identified as producing antibodies with good specificity and sensitivity, the cells in the original 12E2 well were pipetted into a single-cell suspension and seeded into 96-well subcloning plates at a rate of 1-2 cells / well (Balb / C mouse peritoneal cells were seeded into the plates one day in advance as feeder cells, and mouse feeder cells were also prepared). The plates were then cultured in selection medium (DMEM + 20% FBS + penicillin antibody + 1XHAT) for 8 days. Single-clone wells were labeled, and the cell count was observed to reach 200-500 cells / clone. ELISA screening was then performed (the ELISA plate was coated with purified AFP protein), and Western blotting identified wells producing antibodies but without single clones. Several wells with low clone counts were selected and seeded again at a rate of 1-2 cells / well in 96-well subcloning plates for a second subcloning process. Single-clone wells were then selected for ELISA screening. The screening results are as follows: Figure 6 As shown in Figure A, the red wells in A represent single-clone wells with high ELISA values.

[0059] Select wells with high ELISA scores and take 100 μL of culture supernatant as antigen for Western blotting to determine antibody specificity and sensitivity. The results are as follows: Figure 6 As shown in Figure B, all six identified subclones produced very good antibodies. Subclone F8 was selected for subsequent antibody variable region sequencing and further research, while the remaining subclones F11, E7, C8, H10, and G12 were expanded and cryopreserved for future use.

[0060] In B, - represents lysis buffer of colon cancer cell line SW480; + represents lysis buffer of colon cancer cell line SW480 + AFP purified protein; HIS represents anti-His monoclonal antibody (Thermo, USA).

[0061] Sequencing results of AFPH8 and AFPE2 monoclonal antibodies:

[0062] Approximately 2 × 10⁶ hybridoma cells were cultured. 7 Cells were collected by centrifugation (1500 rpm, 5 minutes) and 1 ml of Trizol reagent (Thermo) was added. The cells were then packaged on dry ice and sent to General Biotechnology (Anhui) Co., Ltd. (Chuzhou, Anhui) for total RNA extraction. RT-PCR was then performed using specific antibody V-region primers to amplify the heavy and light chain gene sequences of the antibody V-region. The amino acid sequences were determined based on the cDNA sequences. The CDR sequences were determined using a professional antibody analysis website (https: / / www.imgt.org / IMGT_vquest / input).

[0063] AFPH8 V region and CDR amino acid sequence:

[0064] Heavy chain V region amino acid sequence (115 amino acids)

[0065] Subtype: IgG1:

[0066] QVQLKESGPALVKPSQSLSLTCTVKGYSITSAYSWHWIRQFPGNKLEWMGYINFSGATNYNPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCSRDFLFSYWGQGTLVTVSA (SEQ ID NO.2)

[0067] AFPH8 light chain V region amino acid sequence (112 amino acids)

[0068] Subtype: Kappa

[0069] DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO.3)

[0070] The three CDR sequences of the AFPH8 heavy chain:

[0071] CDR1: GYSITSAYS (SEQ ID NO.4)

[0072] CDR2: YINFSGATNYNPSLKS (SEQ ID NO.5)

[0073] CDR3: SRDFLFSY (SEQ ID NO.6)

[0074] The three CDR sequences of the AFPH8 light chain:

[0075] CDR1: QSLLDSDGKTY (SEQ ID NO.7)

[0076] CDR2: LVSKLDS (SEQ ID NO.8)

[0077] CDR3: WQGTHFPRT (SEQ ID NO.9)

[0078] AFPE2 V region and CDR amino acid sequence:

[0079] Heavy chain V region amino acid sequence (121 amino acids)

[0080] Subtype: IgG1

[0081] EVQLQQSGPELVKPGASMRISCKASGYSFSGGTMSWVRQGHGKNLEWIGLINPYTAYTSYNQKFKDKATLTVDKSSSTAYMDLLLSLTSEDSAVYYCAKSTSYRYDVYFDVWGAGTTVTVSS (SEQ ID NO.10)

[0082] The amino acid sequence of the V region of the light chain (108 amino acids)

[0083] Subtype: Kappa

[0084] QIVLTQSPALMAASPGEKVTITCSVSSSIGTSNLHWYQQKSETSPRPWIYSSSTLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK (SEQ ID NO.11)

[0085] Heavy chain 3 CDR sequences:

[0086] CDR1: GYSFSGGT (SEQ ID NO.12)

[0087] CDR2: INPYTAYT (SEQ ID NO.13)

[0088] CDR3: AKSTSYRYDVYFDV (SEQ ID NO.14)

[0089] The light chain has three CDR sequences:

[0090] CDR1: SSSIGTSN (SEQ ID NO.15)

[0091] CDR2:WIYSSST (SEQ ID NO.16)

[0092] CDR3: QQWSSYPFT (SEQ ID NO. 17).

[0093] Example 4 - Monoclonal Antibody Affinity Assay:

[0094] The binding and dissociation constants of antigen-antibody interactions were determined using ForteBio Octet technology, and antibody affinity was measured. Based on the binding and dissociation curves of the antibody provided by this invention with AFP-His protein at different dilution concentration gradients (500 nM-31 nM), the affinity KD value of the monoclonal antibody of this invention was calculated.

[0095] AFPH8: 3.9 × 10 -11 (R2 =0.987);

[0096] AHPE2: 4.5×10 -11 (R 2 =0.985).

[0097] Example 5 - Comparison of serum AFP detection sensitivity, standard curve, and comparative kit:

[0098] AFPH8 monoclonal antibody (100 ng / well) was used to coat 98-well plates (F8 MAXISORP NUNC-IMMUNOMODULE, Thermo, USA) overnight with 0.05 M carbonate buffer (pH 9.6), followed by blocking with PBS buffer + 3% BSA overnight. For testing, add 25 μL of AFP serum of various concentrations, 75 μL of PBS buffer, and 3% BSA. Incubate at room temperature for 2 hours. Pour out the test samples and wash each well once with PBS buffer (pH=7.2). Then add HRP-AFPE2 labeled antibody (25 ng / well), shake and incubate at room temperature for 1 hour. Pour out the samples and wash the plates 5 times with PBS buffer (pH=7.2) for 10 minutes each time. After each wash, pat the ELISA plates dry on absorbent paper. Add 100 μL of HRP substrate TMB solution (Thermo, USA) and incubate for 20 minutes. Then add 100 μL of HCl (1N) to terminate the reaction. Read the OD value at 450 nm using a microplate reader.

[0099] Comparative example:

[0100] A commercially available American reagent kit was used as a control group for the reagent kit prepared in Example 5. The detection experiments performed were also conducted as in Example 5. The detection results of the reagent kit in Example 5 were compared with the detection results of the American reagent kit in the comparative example. The comparison data are as follows: Figure 7 As shown.

[0101] In summary, compared with existing technologies, it has the following beneficial effects:

[0102] Using the two antibodies of this invention to form a double-antibody sandwich ELISA kit, the detection sensitivity is 0.33 OD absorbance at 5 ng / ml, while the normal serum value is 0-7 ng / ml. Therefore, this kit can detect mildly elevated AFP protein in cancer. In contrast, the detection sensitivity of the comparative kit is approximately 25 ng / ml. Therefore, the double-antibody sandwich detection kit composed of the two antibodies AFPH8 and AFPE2 of this invention can be used more reliably for early cancer screening than the comparative kit.

[0103] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0104] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody against AFP, characterized in that, The heavy chain CDR1 sequence of the monoclonal antibody is SEQ ID NO.4, the heavy chain CDR2 sequence is SEQ ID NO.5, the heavy chain CDR3 sequence is SEQ ID NO.6, the light chain CDR1 sequence is SEQ ID NO.7, the light chain CDR2 sequence is SEQ ID NO.8, and the light chain CDR3 sequence is SEQ ID NO.9; Alternatively, the heavy chain CDR1 sequence of the monoclonal antibody is SEQ ID NO.12, the heavy chain CDR2 sequence is SEQ ID NO.13, the heavy chain CDR3 sequence is SEQ ID NO.14, the light chain CDR1 sequence is SEQ ID NO.15, the light chain CDR2 sequence is SEQ ID NO.16, and the light chain CDR3 sequence is SEQ ID NO.

17.

2. The anti-AFP monoclonal antibody according to claim 1, wherein the heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.2 and the light chain variable region sequence is SEQ ID NO.

3.

3. The anti-AFP monoclonal antibody according to claim 1, wherein the heavy chain variable region sequence of the monoclonal antibody is SEQ ID NO.10 and the light chain variable region sequence is SEQ ID NO.

11.

4. A gene, characterized by, The gene encodes a monoclonal antibody against AFP according to any one of claims 1-3.

5. A carrier, characterized in that, The vector carries the gene according to claim 4.

6. A host cell, characterized in that, The host cell carries the vector according to claim 5.

7. A method for producing an anti-AFP monoclonal antibody according to any one of claims 1-3, characterized in that, The method includes the step of culturing the host cell according to claim 6.

8. The use of the anti-AFP monoclonal antibody according to any one of claims 1-3 in the preparation of a kit for diagnosing colon cancer.

9. A non-diagnostic method for detecting AFP, wherein the method uses a monoclonal antibody against AFP according to any one of claims 1-3 to detect AFP.

10. The method according to claim 9, wherein the method is used to detect AFP levels in serum.

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