Preparation method and application of natural alpha fetoprotein

Abstract

The invention relates to the technical field of antigens and antibodies, in particular to a preparation method and application of natural alpha-fetoprotein, the natural alpha-fetoprotein is secreted and expressed by tumor cells HUH-7, cultured by a hollow fiber column and purified by immunoaffinity purification, amino acid sequences of an antibody AFP 20 # light chain variable region and a heavy chain variable region used for coupling NHS filler are shown as SEQ ID NO: 1-2, and the amino acid sequences of the antibody AFP 20 # light chain variable region and the heavy chain variable region are shown as SEQ ID NO: 1-2. The antibody can specifically recognize the alpha fetoprotein, the alpha fetoprotein purified by the antibody is high in recovery rate and purity, the protein stability, the matrix effect and the reaction rate can meet the use requirements of a calibration product and a quality control product, and the antibody makes a contribution to the application of the alpha fetoprotein in the in-vitro diagnosis industry.

Description

Technical Field

[0001] This invention relates to the field of antigen and antibody technology, and in particular to a method for preparing and applying natural alpha-fetoprotein. Background Technology

[0002] Alpha-fetoprotein (AFP) is a tumor-associated glycoprotein with a molecular weight of 68 kDa. It belongs to the albumin family and is primarily synthesized by fetal hepatocytes and the yolk sac. AFP is classified within the albumin gene superfamily, a family characterized by a U-shaped tertiary domain formed by cysteine ​​amino acid residues linked by disulfide bonds and folded into a loop. AFP is closely related to the development and progression of liver cancer and various other tumors, exhibiting high concentrations in multiple tumors and serving as a positive indicator for various cancers. Currently, it is mainly used clinically as a serum biomarker for primary liver cancer, aiding in its diagnosis and monitoring of treatment efficacy.

[0003] Currently, there is considerable research on alpha-fetoprotein (AFP) isolation methods both domestically and internationally, primarily including salting out, gel immunosorbent assay (GISA), molecular sieving, and affinity chromatography. Previous studies have shown that AFP loss during multi-step purification processes and the irreversible binding of some AFP to the purification column significantly reduce AFP recovery rates. Summary of the Invention

[0004] In view of this, the present invention provides a method for preparing and applying natural alpha-fetoprotein. Based on the principle of antigen-antibody reaction, an immunoaffinity purification column is prepared using the antibody AFP 20#, which can specifically bind to alpha-fetoprotein. This column is used to purify alpha-fetoprotein from serum, tissue, cell supernatant, and other samples to obtain high-purity alpha-fetoprotein. This alpha-fetoprotein can be used in pharmaceuticals, immunogens, quality control materials for clinical diagnostic reagents, standards, or positive substances.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides an antibody, which:

[0007] The amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO: 3 or 10; and / or

[0008] The amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO: 4 or 11; and / or

[0009] The amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO: 5 or 12; and / or

[0010] The amino acid sequence of the light chain variable region CDR1 is shown in SEQ ID NO: 6 or 13; and / or

[0011] The amino acid sequence of the light chain variable region CDR2 is YAS or STS; and / or

[0012] The amino acid sequence of the light chain variable region CDR3 is shown in SEQ ID NO: 7 or 14.

[0013] In some specific embodiments of the present invention, the heavy chain variable region of the antibody has:

[0014] (1) An amino acid sequence as shown in SEQ ID NO: 1 or 8; or

[0015] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (1), and whose function is the same as or similar to that of (1); or

[0016] (3) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (1) or (2).

[0017] In some specific embodiments of the present invention, the light chain variable region of the antibody has:

[0018] (4) An amino acid sequence as shown in SEQ ID NO: 2 or 9; or

[0019] (5) An amino acid sequence obtained by substituting, deleting, or adding one or more residues as shown in (4), and whose function is the same as or similar to that of (4); or

[0020] (6) An amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to the amino acid sequence shown in (4) or (5);

[0021] The "multiple" can be 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0022] In some specific embodiments of the present invention, the antibody described above may also be a single-chain antibody, such as a mouse monoclonal antibody.

[0023] The present invention also provides a nucleic acid molecule that encodes the aforementioned antibody.

[0024] The present invention also provides an expression vector comprising the above-described nucleic acid molecules.

[0025] The present invention also provides a host cell comprising the above-described nucleic acid molecule or the above-described expression vector.

[0026] The present invention also provides the use of any of the following in screening antigens or in preparing products for screening antigens:

[0027] i) The above-mentioned antibodies;

[0028] ii) The above-mentioned nucleic acid molecules;

[0029] iii) The aforementioned expression vectors;

[0030] iv) The aforementioned host cells.

[0031] In some specific embodiments of the present invention, the antigen used above includes alpha-fetoprotein, and the product includes an immunoaffinity column.

[0032] The present invention also provides a product for preparing screening antigens, wherein the antigen may be alpha-fetoprotein having the above-mentioned antibodies.

[0033] The present invention also provides an immunoaffinity column having the above-mentioned antibodies.

[0034] In some specific embodiments of the present invention, the above-described immunoaffinity column or the immunoaffinity column described in the above-described application further includes a carrier and / or alpha-fetoprotein;

[0035] The carrier can be at least one of cross-linked agarose, dextran, and polyacrylamide.

[0036] The present invention also provides a method for preparing the above-mentioned immunoaffinity column, which is based on the preparation of the above-mentioned antibody.

[0037] In some specific embodiments of the present invention, the preparation method of the above-mentioned immunoaffinity column includes: coupling the above-mentioned antibody to a carrier by chemical bonds to obtain the immunoaffinity column.

[0038] The present invention also provides a method for preparing alpha-fetoprotein, which is based on the above-mentioned antibody preparation.

[0039] In some specific embodiments of the present invention, the method for preparing the above-mentioned alpha-fetoprotein includes: separating the alpha-fetoprotein from the sample using the above-mentioned antibody, the above-mentioned product, or the above-mentioned immunoaffinity column.

[0040] In some specific embodiments of the present invention, the above-mentioned method for preparing alpha-fetoprotein can also be combined with SDS-PAGE protein gel electrophoresis or high performance liquid chromatography (HPLC) for detection to test the content of alpha-fetoprotein in the sample.

[0041] The samples include tumor cell supernatant, natural tissue, serum and / or recombinant cell supernatant.

[0042] The present invention also provides a method for purifying proteins, wherein the proteins may be proteins from tumor cell expression supernatants, serum proteins, tissue proteins, or recombinantly expressed proteins, which are purified based on the above-mentioned products or immunoaffinity columns.

[0043] The aforementioned alpha-fetoprotein can be used as an immunogen, a substitute for clinical samples, and as a quality control and calibrator in in vitro diagnostic reagents.

[0044] The AFP antibody used in this invention can specifically recognize alpha-fetoprotein. By coupling the antibody to NHS-activated packing material, high-purity alpha-fetoprotein can be purified by immunoaffinity. The purified protein has a high recovery rate, and its stability, matrix effect, and reaction rate can meet the requirements for calibrators and quality control products, thus contributing to the application of alpha-fetoprotein in the in vitro diagnostic industry.

[0045] The alpha-fetoprotein isolated using the antibody provided by this invention has a purity >90%. Identification has shown that this monoclonal antibody can be used as a positive control, as its potency and stability meet usage requirements. It can be used as an immunogen, a quality control for clinical diagnostic reagents, a calibrator, and a positive substance, providing a reliable basis for clinical diagnosis and treatment. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below.

[0047] Figure 1 Electrophoresis image of AFP 20# antibody;

[0048] Figure 2 Electrophoresis image of AFP 35# antibody;

[0049] Figure 3 The HPLC chromatograms of AFP 20# and AFP35# antibodies are shown. The green peak represents AFP 20# antibody, the red peak represents AFP35# antibody, and the blue peak represents the marker. The size of the marker from right to left is 1340 kDa, 670 kDa, 300 kDa, 150 kDa, 45 kDa, 17 kDa, and 1 kDa.

[0050] Figure 4 Electrophoresis image of antigen 1;

[0051] Figure 5 The HPLC chromatogram of AFP antigen 1 is shown, in which the green peak represents BSA, the blue peak represents AFP antigen 1, and the red peak represents the marker. The size of the marker from right to left is 670 kDa, 300 kDa, 150 kDa, 45 kDa, and 17 kDa. Detailed Implementation

[0052] This invention discloses a method for preparing natural alpha-fetoprotein and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0053] In practice, based on the principle of antigen-antibody specific reaction, AFP-specific antibodies are coupled to NHS-activated gel packing material. Alpha-fetoprotein (AFP) reversibly binds to the gel and is then separated and purified on an immunoaffinity column, yielding high-purity AFP protein with high recovery rate. This method is simple to operate, produces high-purity AFP with virtually no contamination from other proteins.

[0054] In the examples, the protein amino acid sequence information involved is as follows.

[0055] AFP 20# heavy chain variable region amino acid sequence:

[0056] QVQLQQSGAELARPGASVKLSCKASGYTFTGYWMQWVQQRPGQGLEWIGTIYPGDDYAKYTQKFKGKATLTADKSSNTAYMQLNSLTSEDSAVYYCARNYYGSSSSMDFWGQGTSVTVSS (SEQ ID NO: 1);

[0057] AFP 20# light chain variable region amino acid sequence:

[0058] DIVLTQSPATLSVTPGDSVSLSCRASQSINNNLHWYQQKSRESPRLLIKYASQSISGIPSRFSGSGSGTDFTLTINSVETEDFGMYFCQQSYSWYTFGGGTKLELKR (SEQ ID NO: 2);

[0059] AFP 20# heavy chain CDR1: GYTFTGYW (SEQ ID NO: 3), CDR2: IYPGDDYA (SEQ ID NO: 4), CDR3: ARNYYGSSSSMDF (SEQ ID NO: 5);

[0060] AFP 20# light chain CDR1: QSINNN (SEQ ID NO: 6), CDR2: YAS, CDR3: QQSYSWYT (SEQ ID NO: 7);

[0061] AFP 35# heavy chain variable region amino acid sequence:

[0062] EVQLKQSGPELVKPGASVKISCKASGYSFTGYYMHWVKQSHVKSLEWIGRINPYNGITKYNQNFNDKASLTVNKSSRTAYMELHSLTSEDSAFYYCARSLIHHYDRGAMDYWGQGTSVTVSS (SEQ ID NO: 8);

[0063] AFP 35# light chain variable region amino acid sequence:

[0064] EMVLTQSPAIMSASLGEEITLTCSASSSVSYMHWFQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYPLTISSVEAEDAADYYCHQWSSYTFGSGTKLEIK (SEQ ID NO: 9);

[0065] AFP 35# heavy chain CDR1: GYSFTGYY (SEQ ID NO: 10), CDR2: INPYNGIT (SEQ ID NO: 11), CDR3: ARSLIHHYDRGAMDY (SEQ ID NO: 12);

[0066] AFP 35# light chain CDR1: SVSY (SEQ ID NO: 13), CDR2: STS, CDR3: HQWSSYT (SEQ ID NO: 14).

[0067] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0068] The present invention will be further illustrated below with reference to the embodiments.

[0069] Example 1: Preparation, purification and subtype identification of anti-alpha-fetoprotein antibodies

[0070] 1.1 Materials and Methods

[0071] 1.1.1 Material Sources: SPA purification packing material was from GE; NHS activated agarose chromatography packing material was purchased from BioToolomics; the alpha-fetoprotein (AFP) detection kit was from Zhengzhou Antu Bioengineering Co., Ltd.; and the AnneXin kit was from Immunotech, France.

[0072] 1.1.2 Establishment of hybridoma cell lines and preparation of monoclonal antibodies

[0073] The immunogen used was alpha-fetoprotein (AFP) (purchased from Shanghai Bosen Biotechnology Co., Ltd.). Two mice were immunized with 200 μg / mL, 100 μg / mL, and 100 μg / mL immunization doses. Serum from the three immunizations was collected and its titer was detected using an enzyme immunoassay plate coated with AFP. The results are shown in Table 1. The serum titer met the fusion requirements.

[0074] Table 1: Results of serum titer (OD) values ​​in 3-immune mice

[0075]

[0076] Cell fusion: Mice were euthanized by dislocation and disinfected by immersion in 75% alcohol for 5 min. Myeloma cells (NS1) in logarithmic growth phase were resuspended by pipetting and centrifuged in a centrifuge tube, washing twice with serum-free DMEM medium. The spleen was dissected, ground, and centrifuged to extract myeloma cells and spleen cells. 20 mL of serum-free DMEM medium was added to each cell to resuspend the cells, and cell counting was performed. The spleen cells / myeloma cells were mixed at a ratio of (5-10) / 1, centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 1 mL of 50% PEG was added to the mixed cells, and after standing, 10 mL of serum-free DMEM medium was added. The cells were centrifuged at 1000 rpm for 7 min. The supernatant was discarded, and 10 mL of 1% HAT medium was added to resuspend the cells. HAT medium was added to each plate at a rate of 10 mL. The fused cell suspension was placed in 96-well plates at 100 µL / well and incubated at 37°C in a 5% CO2 incubator for 6–8 days. Clonal evaluation and screening were then performed. The cells were evaluated by enzyme immunoassay plate coated with alpha-fetoantigen (AFP) and the highly reactive monoclonal cells AFP 20# and AFP 35# were selected. The OD values ​​are shown in Table 2.

[0077] Table 2: OD value detection results of AFP 20# and AFP 35# cell lines

[0078]

[0079] Ascites preparation: Cells were injected intraperitoneally into BALB / c mice that had been pretreated with paraffin for 2-4 weeks. Each mouse was injected with 0.4 mL, approximately (5-10) × 10⁻⁶ cells. 5 Cells. Starting from day 6 post-inoculation, the abdominal condition of BALB / c mice was observed daily. When the color deepened and the abdomen became significantly distended, ascites fluid was extracted using a 20 mL disposable syringe needle and collected in a 50 mL centrifuge tube. The extracted ascites fluid was centrifuged at 3500 rpm for 5 min, and the supernatant was collected in a 50 mL centrifuge tube. The ascites fluid was labeled with its name and batch number and stored at -20℃ for later use.

[0080] 1.1.3 Antibody purification

[0081] The extracted ascites fluid was purified using SPA affinity chromatography. AFP 20# and AFP 35# ascites fluid were diluted 10-fold and filtered through a 0.45 μm filter membrane. The chromatography column packed with Protein A packing material was equilibrated with ultrapure water and buffer (0.02 M PBS, pH 7.4) sequentially. The filtered antibody supernatant was loaded onto the column and equilibrated again. Finally, the target protein antibody was eluted with dissociation buffer (0.2 M Gly + 1.5 M NaCl, pH 2.7), and the dissociation peak was collected.

[0082] After purification, antibodies AFP 20# and AFP 35# were subjected to SDS analysis, and the results are as follows: Figure 1 and Figure 2 As shown, the reduced bands were treated with reducing agent / DTT. HPLC analysis of AFP 20# and AFP 35# antibodies. Figure 3 As shown, the peaks are dominated by monomers with a molecular weight of 150 KD.

[0083] 1.1.4 Gene Acquisition

[0084] Total RNA was extracted from hybridoma cells using an RNA extraction kit (Sangon Biotech, Shanghai). Using Oligo-dT primers, the extracted RNA was used as a template for first-strand synthesis using NEB's ProtoScript kit. ® First Strand cDNA Synthesis Kit (Cat No. E6300S) was used to reverse transcribe cDNA. cDNA was then used as a template to amplify the scFv heavy and light chain gene fragments. PCR conditions were: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 30 cycles; followed by a final extension at 72℃ for 10 min. The light and heavy chain variable regions, purified by gel excision, were ligated into T-vectors and analyzed to obtain the variable region sequences. The sequences were then spliced ​​together using OverLap PCR after digestion with the SfiI restriction site to form the scFv gene fragment.

[0085] AFP 20#, the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 2, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 1.

[0086] AFP 35#, the amino acid sequence of its light chain variable region is shown in SEQ ID NO: 9, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 8.

[0087] Example 2: Preparation of Immunoaffinity Columns

[0088] 2.1 Pretreatment of AFP 20# and 35# antibodies: Prepare antibody antigens at a ratio of 1:2 mg / mL to 1:5 mg / mL (1 mL of packing material requires 2 to 5 mg of antibody), dialyze twice using a 14 KD dialysis bag at a ratio of 1:50, and dialyze to a 0.2 M NaHCO3 + 0.5 M NaCl pH 8.3 solution, changing the solution every 2 h.

[0089] 2.2 Packing material washing and coupling: After shaking the NHS-activated sephrose packing material, take the required volume and wash it three times with an equal volume of pre-cooled ultrapure water (centrifuged at 4000 r for 4 min); wash it three times with an equal volume of pre-cooled 1 mM HCl; and wash it twice with an equal volume of pre-cooled 0.2 M NaHCO3 + 0.5 M NaCl pH 8.3. After washing, aspirate the supernatant, add an equal volume of antibody antigen to each tube, and shake at 4℃ for 12–16 h.

[0090] 2.3 Packing Material Sealing and Storage: After shaking, allow to settle naturally for 0.5 h, remove the supernatant, and wash three times with 0.02 M PBS (pH 7.4). Add twice the volume of 1 M ethanolamine (pH 8.3) solution to the packing material, shake at 4°C for 12 h to seal the packing material. Wash four times alternately with equal volumes of pre-cooled 0.1 M Tris-HCl + 0.5 M NaCl (pH 8.0) and 0.1 M NaAc + 0.5 M NaCl (pH 4.0). After washing, wash four more times with ultrapure water. Then store with an equal volume of 20% ethanol for later use.

[0091] 2.4 Column packing: The cross-linked packing material is packed into the chromatography column to prepare an alpha-fetoprotein purification column with a specification of 1~300mL.

[0092] Example 3: Culture and purification of alpha-fetoprotein cell supernatant

[0093] 3.1 Cell Culture: Generally, HUH7 tumor cells are cultured in vitro using cubic flasks, which have a small culture volume and low production capacity. In this example, three hollow fiber columns were connected in parallel for large-scale in vitro culture, increasing the production capacity tenfold compared to cubic flasks. The hollow fiber membrane used in this example has a pore size of 20-30 kDa. The internal circulation of the hollow fiber columns uses a medium containing fetal bovine serum (FBS) to continuously provide nutrients. The external circulation of the fiber columns provides growth space for the cells. Small molecules in the internal circulation medium can permeate through the hollow fiber membrane for cell utilization, while metabolic waste products such as lactic acid and glucose from the external circulation can be removed through the hollow fiber membrane. The internal circulation medium is changed daily, and the external circulation antigen is harvested, thus achieving continuous culture and long-term harvesting of AFP antigen. The culture conditions were 5% CO2, 37℃, DMEM medium + 10% FBS, and a cell culture density of 2 × 10⁶ cells / mL.7 The culture period was 90 days, and the cell supernatant was harvested every 7 days. After centrifuging the harvested cell supernatant at 4000 rpm for 20 min, it was purified by immunoaffinity.

[0094] 3.2 Protein Purification: Cell supernatant was washed and concentrated to 5-fold volume using a 30 KD hollow fiber column. The concentration buffer was 0.02 M PBS, pH 7.4. After concentration, the protein was filtered through a 0.45 μm membrane and purified using a pre-conjugated AFP 20# antibody and an AFP 35# immunoaffinity column. The equilibration buffer was 0.02 M PBS, pH 7.4, and the dissociation buffer was 0.2 M GLy + 0.15 M NaCl, pH 2.7. The purified antibody was concentrated to a concentration of 1 mg / mL for subsequent performance evaluation. The antigen purified using the AFP 20# immunoaffinity column was named AFP antigen 1, and the antigen purified using the AFP 35# immunoaffinity column was named AFP antigen 2. The antigen purification yields of AFP 20# and 35# immunoaffinity columns were calculated and are shown in Table 3. The purification yield of AFP 20# immunoaffinity column was 11.44 mg / L, and the purification yield of AFP 35# immunoaffinity column was 5.94 mg / L, which was less than twice that of AFP 20# column. Therefore, AFP 20# antibody-linked packing material is preferred for alpha-fetoprotein purification.

[0095] Table 3: Analysis of AFP antigen purification data

[0096]

[0097] Example 4: Evaluation of AFP antigen activity, stability, and manufacturer-consistent cross-reactivity

[0098] 4.1 Evaluation of antigen activity

[0099] The alpha-fetoprotein (AFP) activity was detected using a kit (purchased from Zhengzhou Antu Biotechnology Co., Ltd.) according to the instructions. The AFP test results prepared in this example are shown in Table 4. The AFP antigen 1 activity was 2,876,900 ng / mL, and the antigen 2 activity was 1,748,000 ng / mL.

[0100] Table 4: Results of AFP antigen activity assay

[0101]

[0102] 4.2 Stability Evaluation

[0103] The antigen was added to a certain amount of diluent according to the calculated potency, and prepared at three levels (Q1, Q2, Q3) with target activity values ​​of 10-20 ng / mL, 70-90 ng / mL, and 160-180 ng / mL, respectively. After lyophilization, the potency change after reconstitution and 14 days of accelerated heat treatment at 37°C was evaluated. The stability data are shown in Table 5. The results showed that the activity change of AFP antigen 1 at 2-8°C and after 14 days of accelerated heat treatment was within 10% compared to -20°C (control), and the stability met the requirements for use as a quality control and calibrator. The potency change of AFP antigen 2 at 2-8°C was within 10% compared to -20°C (control), but the activity change after 14 days of accelerated heat treatment was greater than 10%, indicating that AFP antigen 1 was superior to AFP antigen 2.

[0104] Table 5: Results of AFP antigen stability test

[0105]

[0106] 4.3 Manufacturer Consistency

[0107] Based on the calculated potency, the self-made antigen was added to a certain amount of diluent to prepare three levels (Q1, Q2, and Q3) at low, medium, and high levels (antigen activity target values ​​of 10±1, 75±1, and 177±1, respectively). The prepared samples were tested using reagent kits from Antu, Roche, Abbott, Beckman Coulter, and Siemens on different instrument models to assess manufacturer consistency. The variance rate was calculated, and a variance rate of 15%±5% met the performance requirements. The evaluation results are shown in Table 6. The results indicate that the variance rates for the three levels (Q1, Q2, and Q3) of AFP antigen 1 and antigen 2 prepared by different manufacturers were 19.6%, 9.3%, and 6.11%, respectively, meeting the performance requirements.

[0108] Table 6: Consistency Evaluation Results of AFP Antigens from Various Manufacturers

[0109]

[0110] 4.4 Antigen Cross-Detection

[0111] The antigen activity was adjusted to 100-160 ng / mL, and reactivity was measured on 20 tumor markers (CA125, CA15-3, CA19-9, CA242, CA50, CA72-4, CEA, Cyfra21-1, Ferritin, fPSA, HE4, NSE, PGI, PGII, ProGRP, SCCA, Tg, tPSA, β2-Microglobulin, β-HCG). All kits were purchased from Zhengzhou Antu Biotechnology Co., Ltd. The test results were required to contain no or minimal tumor markers. Control product II contained no other markers besides AFP antigen. After preparation as a quality control product, other markers contained in the antigen should not significantly affect the quality control product. The cross-detection results of antigens 1 and 2 are shown in Table 7. The two antigens showed low detection values ​​on the other 20 tumor markers besides AFP, with no significant cross-detection, and both met the requirements for use as quality control products.

[0112] Table 7: Results of AFP Antigen Cross-Detection

[0113]

[0114] In summary, antigen 1 obtained by immunoaffinity purification with AFP20# antibody showed higher yield and better stability than antigen 2 obtained by immunoaffinity purification with AFP35# antibody. Further analysis of AFP antigen 1 purified by AFP20# antibody immunoaffinity column using SDS and HPLC revealed the following results: Figure 5 As shown, the electrophoretic position of AFP (alpha-fetoprotein) is around 69 kDa. Figure 4 HPLC results show a single main peak and high purity, reaching 90%. The AFP 20# antibody can be used to conjugate magnetic beads, enzyme immunoassay plates, packing materials, and other carriers for the evaluation, detection, and purification of alpha-fetoprotein. The purified alpha-fetoprotein can be applied to biomedicine, quality control products, or calibrators, contributing to the in vitro diagnostic application of alpha-fetoprotein.

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antibody, characterized in that: The amino acid sequence of its heavy chain variable region CDR1 is shown in SEQ ID NO: 3; The amino acid sequence of its heavy chain variable region CDR2 is shown in SEQ ID NO: 4; The amino acid sequence of its heavy chain variable region CDR3 is shown in SEQ ID NO: 5; The amino acid sequence of its light chain variable region CDR1 is shown in SEQ ID NO: 6; Its light chain variable region CDR2 has the amino acid sequence YAS; The amino acid sequence of its light chain variable region CDR3 is shown in SEQ ID NO:

7.

2. The antibody as described in claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; and / or The antibody is a single-chain antibody.

3. A nucleic acid molecule, characterized in that, Encoding the antibody as described in claim 1 or 2.

4. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 3.

5. A host cell, characterized in that, Includes the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. Any of the following applications in screening antigens or preparing products for screening antigens: i) The antibody according to claim 1 or 2; ii) The nucleic acid molecule according to claim 3; iii) The expression vector as described in claim 4; iv) The host cell as described in claim 5.

7. The application as described in claim 6, characterized in that, The antigen includes alpha-fetoprotein, and the product includes an immunoaffinity column.

8. An immunoaffinity column, characterized in that, It has the antibody as described in claim 1 or 2.

9. The method for preparing the immunoaffinity column as described in claim 8, characterized in that, Antibody preparation based on the method described in claim 1 or 2.

10. A method for preparing alpha-fetoprotein, characterized in that, Antibody preparation based on the method described in claim 1 or 2.

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