AFP detection kit and application thereof

Abstract

The application provides an AFP detection kit and application thereof, relates to the technical field of tumor marker detection, and the kit is a double-antibody sandwich ELISA kit. The kit contains two AFP monoclonal antibodies, AFPH8 and AFPE2. The amino acid sequences of the CDR of the heavy chain of AFPH8 are shown in SEQ ID NO. 4-6, the amino acid sequences of the CDR of the light chain of AFPH8 are shown in SEQ ID NO. 7-9, the amino acid sequences of the CDR of the heavy chain of AFPE2 are shown in SEQ ID NO. 12-14, and the amino acid sequences of the CDR of the light chain of AFPE2 are shown in SEQ ID NO. 15-17. Experiments show that the kit has high sensitivity and can detect slightly elevated AFP in initial serum.

Description

Technical Field

[0001] This invention relates to the field of tumor marker detection technology, specifically to an AFP detection kit and its application. Background Technology

[0002] AFP (alpha-fetoprotein) is an important tumor marker. Its protein level is elevated in the serum of about 60% of patients with primary liver cancer. The level of AFP can also be used as an auxiliary indicator for the diagnosis of other tumor diseases besides liver cancer, thus making it an important and traditional tumor marker.

[0003] Currently, the core raw material for AFP tumor marker detection kits used in clinical practice, monoclonal antibodies, is costly to produce and has low detection sensitivity, which is not conducive to early screening for cancer in the public. As a result, many patients are diagnosed with late-stage liver cancer as soon as they are diagnosed.

[0004] Therefore, independently developing high-quality monoclonal antibodies to replace existing test kits, reducing prices and improving test quality such as increasing test sensitivity, is an effective way to achieve widespread early screening of tumor markers in my country, improve the early detection rate of cancer patients, and reduce mortality. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides an AFP detection kit and its application, which solves the technical problems of high production cost and low availability of existing AFP tumor marker kits. Furthermore, the AFP detection kit of this invention has excellent detection sensitivity.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, this invention first requires the development of high-quality monoclonal antibodies that recognize AFP as the core raw material for the kit. Using the full-length AFP gene expressed in *E. coli* as an antigen, Balb / c mice are immunized to prepare anti-AFP monoclonal antibodies, which are then used to develop a double-antibody sandwich ELISA kit (enzyme-linked immunosorbent assay kit) for detecting AFP levels in human serum. After fusing spleen cells from immunized mice with myeloma cell line (SP2 / 0), ELISA and Western blotting methods are used for screening and identification, resulting in 10 high-quality monoclonal antibodies. After studies on antibody specificity, sensitivity, and amino acid sequence determination of the variable region (V region), two of these monoclonal antibodies (AFPH8 and AFPE2) are used as the coating antibody and detection antibody in the kit (AFP double-antibody sandwich detection kit). Since the two monoclonal antibodies originate from the same immunized mouse fusion, the antigen preparation, immunization method, hybridoma fusion, and monoclonal antibody screening methods are the same; only the monoclonal antibody strains are different.

[0009] This invention is achieved through the following technical solution:

[0010] On one hand, the present invention provides an AFP detection kit, which is a double-antibody sandwich ELISA kit containing two AFP monoclonal antibodies, AFPH8 and AFPE2; the amino acid sequence of the AFPH8 heavy chain CDR is shown in SEQ ID NO. 4-6, and the amino acid sequence of the AFPH8 light chain CDR is shown in SEQ ID NO. 7-9; the amino acid sequence of the AFPE2 heavy chain CDR is shown in SEQ ID NO. 12-14, and the amino acid sequence of the AFPE2 light chain CDR is shown in SEQ ID NO. 15-17.

[0011] Furthermore, the heavy chain variable region sequence of AFPH8 is SEQ ID NO.2, and the light chain variable region sequence is SEQ ID NO.3; the heavy chain variable region sequence of AFPE2 is SEQ ID NO.10, and the light chain variable region sequence is SEQ ID NO.11.

[0012] Furthermore, in the kit, AFPH8 is a coating antibody coated on a solid-phase carrier, and AFPE2 is an enzyme-labeled antibody.

[0013] Furthermore, AFPE2 in the kit is an HRP-labeled antibody.

[0014] Furthermore, the kit also includes HRP substrate, control, diluent, washing solution, and stop solution.

[0015] Common components of an ELISA kit, such as substrates, controls, diluents, washes, standards, and stop solutions, can be selected or used by those skilled in the art based on common knowledge in the field.

[0016] On the other hand, the present invention provides the application of the above-mentioned kit in the preparation of reagents for diagnosing tumors.

[0017] Furthermore, the tumor is liver cancer or other tumors.

[0018] Furthermore, the reagent is used to detect the level of AFP in serum.

[0019] (III) Beneficial Effects

[0020] This invention provides an AFP detection kit and its application. Compared with the prior art, it has the following advantages:

[0021] The kit of the present invention can detect 20 pg of AFP protein, while the upper limit of normal AFP in serum is 7 ng / ml (the kit of the present invention uses 25 μl of serum for detection, which is 175 pg). Therefore, the ELISA kit of the present invention can detect mildly elevated AFP protein in serum with high sensitivity, which is beneficial for early warning screening of AFP-positive tumors. Attached Figure Description

[0022] 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.

[0023] Figure 1 The image shows the results of Western blotting identification of purified AFP protein.

[0024] Figure 2 Figure showing the results of quantitative purification of AFP protein by Coomassie Brilliant Blue staining.

[0025] Figure 3 The figure shows the serum antibody titer results of mice immunized with AFP antigen protein.

[0026] Figure 4 For identification of Western blot method Figure 3 Image of the results from the strongly positive wells of ELISA.

[0027] Figure 5Figures 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.

[0028] 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.

[0029] Figure 7 A schematic diagram illustrating the experimental principle and workflow for detecting the tumor marker AFP protein in serum using ELISA (double antibody sandwich assay).

[0030] Figure 8 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

[0031] 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.

[0032] This invention provides an AFP detection kit and its application, which solves the technical problems of high production cost and low popularity of existing AFP tumor marker kits. Furthermore, the AFP detection kit of this invention has excellent detection sensitivity.

[0033] 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.

[0034] Example 1 - Antigen Preparation:

[0035] 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.

[0036]

[0037] Expression and purification of antigen proteins:

[0038] The above gene was cloned into the pET28 prokaryotic expression vector and expressed in the BL21 Escherichia coli strain.

[0039] 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, and five strain clones 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.

[0040] 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.

[0041] 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.

[0042] Example 2 - Immunizing mice with purified protein:

[0043] 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.

[0044] 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).

[0045] 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).

[0046] 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).

[0047] 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.

[0048] Example 3 - Subclonal screening and identification of AFPH8 and AFPE2 monoclonal hybridoma strains:

[0049] AFPH8 monoclonal screening and identification:

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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).

[0054] AFPE2 monoclonal screening and identification:

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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).

[0059] Sequencing results of AFPH8 and AFPE2 monoclonal antibodies:

[0060] 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).

[0061] AFPH8 V region and 3 CDR amino acid sequences:

[0062] Heavy chain V region amino acid sequence (115 amino acids)

[0063] Subtype: IgG1:

[0064] QVQLKESGPALVKPSQSLSLTCTVKGYSITSAYSWHWIRQFPGNKLEWMGYINFSGATNYNPSLKSRISITRDTSKNQFFLHLNSVTTEDTATYYCSRDFLFSYWGQGTLVTVSA (SEQ ID NO.2)

[0065] AFPH8 light chain V region amino acid sequence (112 amino acids):

[0066] Subtype: Kappa

[0067] DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO.3)

[0068] Heavy chain 3 CDR sequences:

[0069] CDR1: GYSITSAYS (SEQ ID NO.4)

[0070] CDR2: YINFSGATNYNPSLKS (SEQ ID NO.5)

[0071] CDR3: SRDFLFSY (SEQ ID NO.6)

[0072] The three CDR sequences of the AFPH8 light chain:

[0073] CDR1: QSLLDSDGKTY (SEQ ID NO.7)

[0074] CDR2: LVSKLDS (SEQ ID NO.8)

[0075] CDR3: WQGTHFPRT (SEQ ID NO.9)

[0076] AFPE2 V region amino acid sequence and CDR:

[0077] Heavy chain V region amino acid sequence (121 amino acids)

[0078] Subtype: IgG1

[0079] EVQLQQSGPELVKPGASMRISCKASGYSFSGGTMSWVRQGHGKNLEWIGLINPYTAYTSYNQKFKDKATLTVDKSSSTAYMDLLLSLTSEDSAVYYCAKSTSYRYDVYFDVWGAGTTVTVSS (SEQ ID NO.10)

[0080] The amino acid sequence of the light chain V region (108 amino acids):

[0081] Subtype: Kappa

[0082] QIVLTQSPALMAASPGEKVTITCSVSSSIGTSNLHWYQQKSETSPRPWIYSSSTLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPFTFGSGTKLEIK (SEQ ID NO.11)

[0083] Heavy chain 3 CDR sequences:

[0084] CDR1: GYSFSGGT (SEQ ID NO.12)

[0085] CDR2: INPYTAYT (SEQ ID NO.13)

[0086] CDR3: AKSTSYRYDVYFDV (SEQ ID NO.14)

[0087] The light chain has three CDR sequences:

[0088] CDR1: SSSIGTSN (SEQ ID NO.15)

[0089] CDR2:WIYSSST (SEQ ID NO.16)

[0090] CDR3: QQWSSYPFT (SEQ ID NO. 17).

[0091] Example 4 - Monoclonal Antibody Affinity Assay:

[0092] 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.

[0093] AFPH8: 3.9 × 10 -11(R 2 =0.987);

[0094] AHPE2: 4.5×10 -11 (R 2 =0.985).

[0095] Example 5 - Composition and sensitivity testing of the ELISA kit:

[0096] according to Figure 7 As shown, an ELISA kit was constructed using monoclonal antibodies AFPH8 and AFPE2 as coating antibodies and HRP-labeled antibodies, respectively, to detect AFP in serum via ELISA.

[0097] The AFPH8 coating volume was 100 ng / well, and the coating solution was 0.05 M carbonate buffer (pH=9.6). After coating overnight, the wells were blocked overnight with PBS buffer + 3% BSA.

[0098] The detection system contains 25 μL of AFP serum at various concentrations, 75 μL of PBS buffer, and 3% BSA.

[0099] Incubate at room temperature for 2 hours, pour out the test sample, rinse with PBS buffer (pH=7.2), add 25 ng / well of HRP-AFPE2 labeled antibody, incubate at room temperature with shaking for 1 hour, pour out the sample, and wash the plate 5 times with PBS buffer (pH=7.2) on a shaker for 10 minutes each time.

[0100] Add 100 μL of HRP substrate TMB solution and incubate for 20 minutes. Take a picture of the blue plate. Then add 100 μL of HCl (1N) to terminate the reaction. Read the OD value at 450 nm using a microplate reader.

[0101] Comparative example:

[0102] A commercially available 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 reagent kit in the control group. The comparison data are as follows: Figure 8 As shown.

[0103] Performance test results:

[0104] Because the AFPH8 and AFPE2 antibodies are excellent, they can be sensitively detected at 20 pg of AFP protein using traditional ELISA methods. The upper limit of normal AFP levels in serum is 7 ng / ml (the kit in Example 5 of this invention uses 25 μl of serum for detection, and the detection value is 175 pg). Therefore, the double-antibody sandwich ELISA kit of this invention can very sensitively detect slightly elevated AFP protein in serum, which is beneficial for early warning screening of AFP-positive tumors.

[0105] like Figure 8 As shown in the figure, A represents a comparison of OD values ​​between the kit of Example 5 of the present invention and the kit of the comparative example, where HEPG2 is an AFP-positive liver cancer cell line and SW480 is an AFP-negative colon cancer cell line. The culture supernatants of both cell lines were serially diluted 1:5. The detection sensitivity of the kit of Example 5 of the present invention is 0.16 μL, while the detection sensitivity of the kit of the comparative example is 4 μL, indicating that the detection sensitivity of the kit of Example 5 of the present invention is 25 times higher than that of the kit of the comparative example.

[0106] B indicates that the colorimetric intensity of the kit in Example 5 of this invention is significantly better than that of the comparative kit after the addition of TMB substrate, which also shows that the kit in Example 5 of this invention has higher detection sensitivity.

[0107] In summary, compared with existing technologies, it has the following beneficial effects:

[0108] The kit of the present invention can detect 20 pg of AFP protein, while the upper limit of normal AFP in serum is 7 ng / ml (the kit of the present invention uses 25 μl of serum for detection, which is 175 pg). Therefore, the ELISA kit of the present invention can detect mildly elevated AFP protein in serum with high sensitivity, which is beneficial for early warning screening of AFP-positive tumors.

[0109] 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.

[0110] 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. An AFP detection kit, characterized in that, The kit is a double-antibody sandwich ELISA kit, which contains two AFP monoclonal antibodies, AFPH8 and AFPE2. The amino acid sequences of the AFPH8 heavy chain CDR are shown in SEQ ID NO. 4-6, and the amino acid sequences of the AFPH8 light chain CDR are shown in SEQ ID NO. 7-9. The amino acid sequences of the AFPE2 heavy chain CDR are shown in SEQ ID NO. 12-14, and the amino acid sequences of the AFPE2 light chain CDR are shown in SEQ ID NO. 15-17.

2. The kit according to claim 1, wherein the AFPH8 heavy chain variable region sequence is SEQ ID NO.2 and the light chain variable region sequence is SEQ ID NO.3; the AFPE2 heavy chain variable region sequence is SEQ ID NO.10 and the light chain variable region sequence is SEQ ID NO.

11.

3. The kit according to claim 1 or 2, wherein AFPH8 is a coated antibody coated on a solid-phase carrier and AFPE2 is an enzyme-labeled antibody.

4. The kit according to claim 3, wherein AFPE2 in the kit is an HRP-labeled antibody.

5. The kit according to claim 1, wherein the kit further comprises HRP substrate, control, diluent, washing buffer, and stop solution.

6. The use of the kit according to any one of claims 1-5 in the preparation of reagents for diagnosing tumors.

7. The application according to claim 6, wherein the tumor is liver cancer.

8. The application according to claim 6, wherein the reagent is used to detect the level of AFP in serum.

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