Preparation of an anti-sickle cell anemia hemoglobin variant antibody and application of a typing detection kit

Abstract

The present invention discloses the preparation of antibodies against sickle cell hemoglobin variants and the application of a typing detection kit, belonging to the field of biomedicine. By comparing the sequences of different hemoglobins, the present invention analyzes the key mutation points of sickle cell hemoglobin. A prokaryotic expression system is used to recombinantly express the hemoglobin α chain (HBA) and hemoglobin β chain (including HBB, HBS, and HBC). The target proteins of different typings are then conjugated with KLH to prepare immunogens. Animals are then immunized and screened for antibodies targeting the different hemoglobin variants. Rabbit antibodies are obtained for HBB and HBS proteins, while mouse antibodies are obtained for HBA and HBC. DNA sequencing is used to analyze the antibody sequence information. The present kit has high sensitivity and specificity, and can rapidly distinguish hemoglobin typing, thus overcoming the shortcomings of existing detection technologies and assisting in clinical infection control and treatment.

Description

Technical Field

[0001] The present invention relates to the preparation of an anti-sickle cell anemia hemoglobin variant antibody and the application of a typing detection kit, belonging to the field of biomedicine. Background Art

[0002] Sickle cell disease (SCD) is an inherited genetic abnormality of hemoglobin (the oxygen-carrying protein in red blood cells). It is primarily caused by mutations in the beta-globin (HBB) gene responsible for hemoglobin production. It is characterized by sickle (crescent)-shaped red blood cells and the destruction of these abnormal red blood cells, leading to chronic anemia. Clinical manifestations include chronic hemolytic anemia, susceptibility to infection, and recurrent painful crises, leading to chronic ischemia and consequent organ and tissue damage. Treatment is primarily with blood transfusions and medication.

[0003] Hemoglobin mutations are common worldwide. While individuals with sickle cell trait do not necessarily develop sickle cell disease, they do have an increased risk of certain complications, such as hematuria. Mutated beta-globin causes sickle cell anemia. A lack of a beta chain causes beta-null thalassemia. A reduced amount of detectable beta-globin results in beta-plus thalassemia.

[0004] The two most common SCD genotypes are hemoglobin S (HBS) and hemoglobin SC (HBSC). Homozygosity for HBS is the most common and severe form of the disease. This gene mutation produces an abnormal hemoglobin, called hemoglobin S (HBS), which differs from normal adult hemoglobin (HBB). HBS is caused by a point mutation in the sixth codon of HBB, from GAG to GTG. When blood oxygen levels are low, HBS forms long polymers due to the hydrophobic interaction between valine and phenylalanine. Individuals with SCD have less flexible red blood cells, and these polymers cause rheological and biochemical changes that can lead to vascular occlusion.

[0005] The molecular basis of HBSC disease is similar to that of SCA; a point mutation in the gene, from GAG to AAG, replaces glutamic acid with lysine in the globin chain. HBSC tend to form amorphous aggregates within hemoglobin, resulting in morphological changes. Furthermore, in HBSC disease, alterations in the K-Cl cotransporter lead to red blood cell dehydration, increasing the intracellular hemoglobin concentration and making it denser than in red blood cells containing HbAA.

[0006] The clinical presentation of patients with SCD is broad, including acute painful episodes, pulmonary hypertension (PH), stroke, priapism, leg ulcers, acute chest syndrome (ACS), osteonecrosis, and cholelithiasis. PH, leg ulcers, and stroke are thought to be associated with the chronic hemolytic features of SCD, while acute painful crises, osteonecrosis, and ACS are associated with VO, potentially driving distinct subphenotypes. However, this dichotomy is not restrictive, as they often overlap and may not be useful for SCA and HbSC alone.

[0007] Immunochromatography is a rapid and convenient in vitro diagnostic technique that requires no instrumentation and is unaffected by false positives from gene silencing. However, false negatives can occur if target protein expression is weak or if key amino acids in the epitope recognized by the antibody used are altered. Therefore, there is a pressing market need for a rapid test product with high sensitivity and specificity that can distinguish hemoglobin typing. This invention aims to address this challenge, address the shortcomings of existing detection technologies, and assist in clinical infection control and treatment. Summary of the Invention

[0008] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide the preparation and application of antibodies against different types of sickle cell hemoglobin, aiming to solve the technical problem of the lack of rapid typing detection for sickle cell anemia in the prior art.

[0009] The first technical solution provided by the present invention is an anti-sickle cell anemia hemoglobin variant antibody, wherein the anti-sickle cell anemia hemoglobin variant antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are as shown in any one of groups (A) to (D):

[0010] (A) SEQ ID NO:5~7, SEQ ID NO:9~11;

[0011] (B) SEQ ID NO: 13~15, SEQ ID NO: 17~19;

[0012] (C) SEQ ID NO:21~23, SEQ ID NO:25~27;

[0013] (D) SEQ ID NO: 29~31, SEQ ID NO: 33~35.

[0014] In certain embodiments, the amino acid sequences of the light chain variable region and the heavy chain variable region are as shown in any one of the groups (E) to (H):

[0015] (E) SEQ ID NO:8, SEQ ID NO:12;

[0016] (F) SEQ ID NO:16, SEQ ID NO:20;

[0017] (G) SEQ ID NO:24, SEQ ID NO:28;

[0018] (H) SEQ ID NO:32, SEQ ID NO:36.

[0019] The second technical solution provided by the present invention is a polynucleotide encoding the anti-sickle cell anemia hemoglobin variant antibody described in the first technical solution.

[0020] The third technical solution provided by the present invention is a recombinant vector carrying the polynucleotide described in the second technical solution.

[0021] In certain embodiments, the recombinant vector uses plasmid pcDNA3.4 as an expression vector.

[0022] The fourth technical solution provided by the present invention is a recombinant cell expressing the anti-sickle cell anemia hemoglobin variant antibody described in the first technical solution, or containing the polynucleotide described in the second technical solution, or transformed with the recombinant vector described in the third technical solution.

[0023] In certain embodiments, the recombinant cell is hosted by a fungus, a bacterium, an animal cell, or a plant cell.

[0024] In certain embodiments, the fungus comprises yeast or mold, and the bacteria comprises Escherichia coli.

[0025] In certain embodiments, the animal cells include but are not limited to 293 cells.

[0026] The fifth technical solution provided by the present invention is a method for preparing an anti-sickle cell anemia hemoglobin variant antibody, which comprises culturing the recombinant cells described in the fourth technical solution to obtain a culture containing the antibody described in the first technical solution.

[0027] The sixth technical solution provided by the present invention is a biologically or chemically labeled product, wherein the product is an antibody labeled with a marker, and the antibody is derived from any one of the following:

[0028] (1) The anti-sickle cell anemia hemoglobin variant antibody described in the first technical solution;

[0029] (2) The culture of the recombinant cells described in the fourth technical solution.

[0030] In certain embodiments, the label includes but is not limited to enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge; the biotin includes but is not limited to biotin and its derivatives; the fluorescein includes but is not limited to AF350, AF488, AF532, AF546, AF555, AF56 8. AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, OregonGreen488, PacificBlue dye, PacificOrange dye, TexasRed, PerCP dye; the chemiluminescent type includes but is not limited to isoluminol and its derivatives, acridinium ester and its derivatives, terpyridine ruthenium and its derivatives, etc.; the isotope type includes but is not limited to iodine labeling; the colloidal labeling includes but is not limited to colloidal gold, colloidal carbon, colloidal selenium, etc.

[0031] The seventh technical solution provided by the present invention is a kit, which contains the anti-sickle cell anemia hemoglobin variant antibody described in the first technical solution or the biomarker or chemically labeled product described in the sixth technical solution.

[0032] In certain embodiments, the kit includes an enzyme-linked immunosorbent assay kit and an immunofluorescence assay kit.

[0033] In certain embodiments, the kit includes a test strip having a quality control line (C line) and multiple test lines (T lines), the test lines including an HbA line, an HbS line, and an HbC line, the HbA line being coated with a normal hemoglobin β chain-specific antibody having a light chain variable region amino acid sequence as shown in SEQ ID NO: 32 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 36, the HbS line being coated with a hemoglobin S type β chain-specific antibody having a light chain variable region amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 20, the HbC line being coated with a hemoglobin C type β chain-specific antibody having a light chain variable region amino acid sequence as shown in SEQ ID NO: 24 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 28, and the test strip is further sprayed with a labeled antibody, wherein the labeled antibody is a hemoglobin α chain-specific antibody having a light chain variable region amino acid sequence as shown in SEQ ID NO: 8 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 12.

[0034] The eighth technical solution provided by the present invention is the use of the anti-sickle cell anemia hemoglobin variant antibody described in the first technical solution, or the polynucleotide described in the second technical solution, or the expression recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the method described in the fifth technical solution, or the biomarker or chemically labeled product described in the sixth technical solution in the preparation of a product for in vitro detection of sickle cell anemia typing.

[0035] In certain embodiments, the product comprises a reagent, a kit, a detection chip, or a biosensor.

[0036] Compared to existing technologies, the present invention has the following advantages: By comparing the sequences of different hemoglobins, the present invention structurally analyzes the key mutations in sickle cell anemia hemoglobin. Using a prokaryotic expression system, the present invention recombinantly expresses the hemoglobin α chain (HBA) and hemoglobin β chain (including HBB, HBS, and HBC). The target proteins of different subtypes are conjugated with KLH to prepare immunogens. Animals are immunized and antibodies are screened for the corresponding hemoglobin variants. Antibodies are obtained from mouse-derived HBB, HBS, and HBC proteins, while antibodies are obtained from rabbit-derived HBA. DNA sequencing technology is used to analyze the antibody sequences, which contain heavy and light chain variable regions, respectively. Their amino acid sequences are shown in SEQ ID NOs. 5 to 36. The present kit offers high sensitivity, excellent specificity, and the ability to rapidly distinguish hemoglobin subtypes. This overcomes the shortcomings of existing detection technologies and aids in clinical infection control and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The protein structures of HBB, HBS, and HBC at the mutation points are shown.

[0038] Figure 2 This is the antibody gel map.

[0039] Figure 3 Figure 2 is the specificity verification result. A shows the HbF test results at dilution concentrations of 8µg / mL, 4µg / mL, 2µg / mL, 1µg / mL, and 500ng / mL. B shows the HbA2 test results at dilution concentrations of 8µg / mL, 4µg / mL, 2µg / mL, 1µg / mL, and 500ng / mL. C shows the HbA test results at dilution concentrations of 3µg / mL, 2µg / mL, 1µg / mL, 500ng / mL, and 250ng / mL. D shows the HbS test results at dilution concentrations of 5µg / mL, 2µg / mL, 1µg / mL, 800ng / mL, and 500ng / mL. E shows the mixed test results of HbC at dilution concentrations of 8µg / mL, 4µg / mL, 2µg / mL, 1µg / mL, and 500ng / mL and HbA at a dilution concentration of 500ng / mL. DETAILED DESCRIPTION

[0040] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0041] The raw materials used in the embodiment are:

[0042] 1. pET28a vector and pcDNA3.4 vector were purchased from Takara.

[0043] 2. Escherichia coli DH5α and Escherichia coli BL21 (DE3) were purchased from Takara.

[0044] 3. LB liquid medium: Prepared with 10 g / L peptone, 5 g / L yeast extract, and 5 g / L sodium chloride. The pH value needs to be adjusted to 7.3 ± 0.1.

[0045] LB solid medium: Prepared with 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 15 g / L agar powder. The pH value should be adjusted to 7.3 ± 0.1.

[0046] Serum-free DMEM medium was purchased from Gibco; fetal bovine serum was purchased from Sijiqing; and trypsin was purchased from Gibco.

[0047] 4. The implementation of the BALB / c mouse immunization process was entrusted to Jiangsu Dongkang Biological Co., Ltd.

[0048] 5. The implementation of the New Zealand white rabbit immunization process is entrusted to Jiangsu Dongkang Biological Co., Ltd.

[0049] Example 1 Immunogen Preparation

[0050] Hemoglobin is a specialized protein found within red blood cells, composed of globin and heme. Its primary function is to transport oxygen and carbon dioxide. Its content and function are crucial to human health. Clinically, hemoglobin levels are often measured to diagnose conditions such as anemia and hypoxia. It is a key indicator for assessing health, diagnosing anemia, and monitoring bleeding. To rapidly characterize sickle cell anemia, the present invention first compared the structures of various hemoglobin types and synthesized the target gene sequence. The α (HBA) and β (HBB) sites determine the structure of the two polypeptide chains in adult hemoglobin (HbA). A normal adult hemoglobin tetramer consists of two α chains and two β chains.

[0051] The amino acid sequence of HBA is shown in SEQ ID NO.1 (Uniprot: P69905), and the amino acid sequence of HBB is shown in SEQ ID NO.2 (Uniprot: P68871, PDB: 1A00). Meanwhile, the amino acid sequence of HBB variant HBS is shown in SEQ ID NO.3 (PDB: 2HBS), and the amino acid sequence of HBC is shown in SEQ ID NO.4 (PDB: 3S66). The protein structures of HBB, HBS, and HBC corresponding to the mutation points are compared as shown in the figure below. Figure 1 shown.

[0052] SEQ ID NO.1:

[0053] MVLSPADKTNVKAAWGKVGAHAGEYGAEALERMFLSFFPTTKTYFPHFDLSHGSAQVKGHGKKVADALTNAVAHVDDMPNALSALSDLHAHKLRVDPVNFKLLSHCLLVTLAAHLPAEFTPAVHASLDKFLASVSTVLTSKYR

[0054] SEQ ID NO.2:

[0055] MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

[0056] SEQ ID NO.3:

[0057] MVHLTPVEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

[0058] SEQ ID NO.4:

[0059] MVHLTPKEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVANALAHKYH

[0060] Structural analysis of the mutations revealed that the point mutations were located at one end of the α-helical structure, and the resulting amino acid structure changed significantly, making them more susceptible to forming antibody-binding epitopes. Therefore, the sequences of the four target genes, HBA, HBB, HBS, and HBC (HBA gene sequence from NCBI: NM_000517.6; HBB gene sequence from GenBank: AY894014.1; HBS gene sequence as SEQ ID NO. 37; HBC as SEQ ID NO. 38), were synthesized and then expressed in an E. coli expression system using a double enzyme digestion method (BamH I / Hind III) in a pET-28a expression vector. The four proteins were then expressed and purified, and the purified target proteins were conjugated to KLH using the glutaraldehyde method to enhance their immunogenicity.

[0061] SEQ ID NO.37:

[0062] atggtgcatctgaccccggtggaaaaaagcgcggtgaccgcgctgtggggcaaagtgaacgtggatgaagtgggcggcgaagcgctgggccgcctgctggtggtgtatccgtggacccagcgcttttttgaaagctttggcgatctgagcaccccggatgcggtgatgggcaacccgaaagtgaaagcgcatggcaaaaaagtgctgggcgcgtttagcgatggcctggcgcatctggataacctgaaaggcacctttgcgaccctgagcgaactgcattgcgataaactgcatgtggatccggaaaactttcgcctgctgggcaacgtgctggtgtgcgtgctggcgcatcattttggcaaagaatttaccccgccggtgcaggcggcgtatcagaaagtggtggcgggcgtggcgaacgcgctggcgcataaatatcat

[0063] SEQ ID NO.38:

[0064] atggtgcatctgaccccgaaagaaaaaagcgcggtgaccgcgctgtggggcaaagtgaacgtggatgaagtgggcggcgaagcgctgggccgcctgctggtggtgtatccgtggacccagcgcttttttgaaagctttggcgatctgagcaccccggatgcggtgatgggcaacccgaaagtgaaagcgcatggcaaaaaagtgctgggcgcgtttagcgatggcctggcgcatctggataacctgaaaggcacctttgcgaccctgagcgaactgcattgcgataaactgcatgtggatccggaaaactttcgcctgctgggcaacgtgctggtgtgcgtgctggcgcatcattttggcaaagaatttaccccgccggtgcaggcggcgtatcagaaagtggtggcgggcgtggcgaacgcgctggcgcataaatatcat

[0065] The specific operations are as follows:

[0066] The constructed and expressed HBA protein was diluted to 0.1 mg / mL with 50 mM PBS pH 7.4 solution. At the same time, 5 mg / mL KLH solution and 1% glutaraldehyde (prepared immediately before use) were prepared with 50 mM PBS pH 7.4 solution and sterilized by filtration.

[0067] The constructed and expressed HBB, HBS, and HBC proteins were diluted to 0.1 mg / mL with 50 mM Tris pH 8.0 solution. At the same time, 5 mg / mL KLH solution and 1% glutaraldehyde (prepared immediately before use) were prepared with 50 mM Tris pH 8.0 solution and sterilized by filtration.

[0068] Mix the above different protein solutions with KLH solution in a 1:1 volume ratio (total volume 2 mL) and gently invert to mix thoroughly. Add glutaraldehyde: Slowly add 1% glutaraldehyde solution dropwise to a final concentration of 0.08%. Stir thoroughly and incubate at 4°C for 2-4 hours. Terminate the reaction by adding 0.1M glycine. After 30 minutes, transfer the reaction solution into a dialysis bag (molecular weight cutoff 10 kDa) and dialyze against the corresponding buffer at 4°C to obtain the target antigen-KLH conjugated product. Detect free thiol groups using Ellman's reagent to verify the conjugation effect.

[0069] Example 2 Preparation and screening of antibodies

[0070] The HBA, HBB, HBS, and HBC obtained in Example 1 were used to immunize mice and rabbits.

[0071] Mouse Immunization Method: Healthy BALB / c female mice, 6-8 weeks old and weighing 18-22 g, were selected. The antigen-KLH conjugate product from Example 1 was emulsified with Freund's complete adjuvant at a 1:1 volume ratio until it did not spread when dropped into water. A primary immunization was administered subcutaneously with 100 μg of antigen per mouse in a total volume of 0.2-0.3 mL. Fourteen days after the primary immunization, a booster immunization was performed with two doses of 50 μg of antigen per mouse in a total volume of 0.2-0.3 mL emulsified with incomplete adjuvant as described above. A final immunization was performed 7-10 days after the booster immunization, with the same dose of the immunogen (without adjuvant) injected intravenously to boost antibody titers. Three to five days after the final immunization, a small amount of blood was collected from the retroorbital venous plexus for antibody titer determination by ELISA. Mice with titers ≥ 1:10,000 were sacrificed and blood was collected for serum collection for cell fusion.

[0072] Rabbit Immunization Method: Healthy New Zealand White rabbits, 3-4 months old and weighing 2-3 kg, were selected. The antigen-KLH conjugate was emulsified with Freund's complete adjuvant at a 1:1 volume ratio. Primary immunization was performed by subcutaneous injection at multiple sites in the back, neck, and groin. The antigen dose was 1 mg per rabbit, with each site ≤1 mL (complete adjuvant for the primary immunization, incomplete adjuvant for subsequent immunizations). Three booster immunizations were administered 21 days after the primary immunization. A final immunization was administered 7-10 days after the final booster immunization. The antigen (without adjuvant) was injected intravenously at a dose of 0.5 mg per rabbit, slowly into the marginal ear vein. Five to seven days after the final immunization, a small amount of blood (1-2 mL) was collected from the marginal ear vein for titer determination by ELISA. Rabbits with titers ≥1:100,000 were sacrificed and blood was collected for serum collection for cell fusion.

[0073] Cell fusion, screening, and cloning methods: After the final immunization, spleens from mice or rabbits with qualified titers verified by ELISA are fused with myeloma cells. The ratio of mouse splenocytes to myeloma cells is 10:1. Because rabbit splenocytes are numerous and heterologous fusion efficiency is low, a higher ratio (rabbit splenocytes to myeloma cells = 20:1) is often used to increase the probability of hybridoma generation. Spleen cells and myeloma cells are induced to fuse using PEG. Mouse splenocytes are treated with 50% PEG 4000 for 1-2 minutes, while rabbit splenocytes are treated with 50% PEG 6000 for 2-3 minutes (strict control is required to avoid cytotoxicity). Hybridomas are then selected using HAT medium, and positive clones are screened by ELISA. Cloning is performed by limiting dilution to ensure monoclonality. For mouse hybridomas, colonies are visible in approximately 7-10 days; for rabbit heterologous fusions, colonies form in approximately 10-14 days during HAT screening. Multiple cloning attempts are required to obtain single clones.

[0074] Example 3 Antibody Evaluation Pairing and Recombinant Expression

[0075] Cell supernatants from the positive clones obtained in Example 2 were subjected to affinity screening using BLI. Using a ForteBio BLItz instrument, the binding and dissociation processes between the antibody and antigen were monitored in real time by measuring the thickness of the biomolecular layer on the fiber-optic probe. The dissociation constant (KD = kd / ka) was calculated. The previously prepared HBA, HBB, HBS, and HBC proteins were diluted to 100 nM using the corresponding buffer. The Protein A probe was inserted into the pre-treated well (containing 10 mM Glycine-HCl, pH 2.0) and incubated for 30 seconds to remove residual antibody. The probe was then transferred to the equilibration well (20 mM PBS, pH 7.4) and incubated for 2 minutes to stabilize the probe baseline. The probe was immersed in the cell supernatant and incubated at room temperature for 5-10 minutes, monitoring the signal rise until saturation. After coupling was complete, the probe was moved to the equilibration well and incubated for 2 minutes to remove nonspecifically bound antibody. The probe was then sequentially immersed in antigen solutions of varying concentrations and incubated at room temperature for 3-5 minutes. The signal rise curve during the binding phase was recorded. After binding is completed, the probe is moved back to the equilibrium well and incubated for 5-10 minutes, and the signal decline curve in the dissociation stage is recorded.

[0076] The "pretreatment → antibody coupling → antigen binding → dissociation → regeneration" cycle was repeated once, and the above four antigens were replaced with: human hemoglobin HbA (α2β2), abnormal hemoglobin Hb S, and abnormal hemoglobin Hb C. The affinity was tested again, that is, anti-HBA antibodies and anti-HBB antibodies were incubated with human hemoglobin HbA (α2β2), anti-HBS antibodies were incubated with abnormal hemoglobin HbS, and anti-HBC antibodies were incubated with abnormal hemoglobin Hb C. Positive clones with poor affinity to the tetramer form of hemoglobin were excluded, and finally positive clones with better affinity were screened, and the KD value was calculated.

[0077] Positive clones with higher affinity were screened and their specific information is shown in Table 1.

[0078] Table 1 Affinity of positive clones

[0079]

[0080] The cell supernatant of the above-mentioned positive clones was further subjected to Protein A affinity chromatography to purify the secreted antibodies, which were paired using the ELISA method. The capture antibody was cross-paired with the detection antibody. To meet the detection requirements of the double antibody sandwich method, it was necessary to achieve a sandwich pairing between the normal hemoglobin β chain specific antibody (Anti-HBB mAb), the hemoglobin S type β chain specific antibody (Anti-HBS mAb), and the hemoglobin C type β chain specific antibody (Anti-HBC mAb) and the hemoglobin α chain specific antibody (Anti-HBA mAb). Therefore, an antibody pair with good sandwich reactivity was obtained. The results are shown in Table 2.

[0081] Table 2 Specific information of antibody pairs

[0082]

[0083] The purified antibodies were sent to Qingke Bio for sequencing, and the variable region sequences were obtained, as shown in Table 3. Based on the preferred codon characteristics of HEK293F cells, the coding codons were optimized and artificially synthesized for eukaryotic cell expression. The target DNA sequence was then combined with the mammalian expression vector pcDNA3.4 to construct a recombinant expression plasmid DNA. The target protein was induced to express using a eukaryotic expression system, and the antibody was purified using a Protein A / G column. Finally, SDS-PAGE electrophoresis was performed to confirm the antibody status ( Figure 2 ) for the subsequent development of detection kits.

[0084] Table 3 Amino acid sequences of the antibody pairs identified in the present invention

[0085]

[0086] Example 4 Development and Preparation of Immunochromatography Kit

[0087] Using the above-selected antibodies and a colloidal gold immunochromatography platform, we plan to develop a rapid typing detection kit for sickle cell anemia. The product is designed as a multiplex test format with different detection lines located on the same chromatographic test strip. The quality control line (C line) uses sheep anti-mouse IgG antibody, and different types of hemoglobin have different detection lines (T lines), namely, the HbA line is coated with a normal hemoglobin β chain-specific antibody (Anti-HBB mAb), the HbS line is coated with a hemoglobin S type β chain-specific antibody (Anti-HBSmAb), and the HbC line is coated with a hemoglobin C type β chain-specific antibody (Anti-HBC mAb). The labeled antibody is a hemoglobin α chain-specific antibody (Anti-HBA mAb). Therefore, typing detection can be achieved. When an appropriate amount of test sample treated with lysis buffer is added to the sample well of the test box, the sample will move forward along the test strip by capillary action. If the sample contains normal hemoglobin β chain or mutated hemoglobin S-type β chain and C-type β chain antigens, the antigen will form an immune complex with the corresponding specific antibody and be captured by the HbA line, HbS line and HbC line.

[0088] The specific preparation method is as follows:

[0089] Streaking process: The coating concentration of the quality control line is 2 mg / mL, the coating concentration of the HbA line is 1.5-2 mg / mL, the coating concentration of the HbS line is 1.5-2 mg / mL, and the coating concentration of the HbC line is 1.5-2 mg / mL. After streaking with a gold spray film streaker, the samples were dried at 37°C for 2 hours.

[0090] Labeling process: Add 4 ml of concentrated colloidal gold to a beaker and seal it with plastic wrap; add a clean rotor to the container and adjust the rotor position to the center of the container, and place it on a stirrer; add 6 ml of 0.01 MPB buffer (pH 7.4) to the concentrated colloidal gold and stir evenly; then add 0.6 mg of labeled antibody and stir evenly; react at 200 rpm for 15-30 minutes; after the reaction is completed, add 0.5 ml of 10% BSA and stir evenly. After blocking the reaction at 200 rpm while stirring for 10 minutes, centrifuge at 3000 rpm for 10 minutes at 2-8°C. After centrifugation, aspirate the supernatant with a pipette and discard it, then add 15 ml of preservation solution to re-dissolve it.

[0091] Gold spraying process: Use a gold spraying film scratcher, set the gold spraying amount to 3.0μL / cm±1.0μL / cm, select the appropriate gold spraying amount according to the situation, and dry in a 37℃ oven for 30min.

[0092] Sample pad treatment: Place the sample pad in a clean soaking box and pour in the sample pad treatment solution (0.01M PBS + 0.1mg / ml blocking agent, pH 7.4), ensuring that the sample pad is completely immersed in the solution. After soaking for 10 minutes, remove the sample pad and place it in a blast dryer at 37°C for 1 hour.

[0093] Diluent formula for chromatography: 0.01M PBS + 0.5% Triton-405, pH 7.4.

[0094] Example 5 Performance Verification of Immunochromatography Kit

[0095] The kit prepared in Example 4 was used to detect different types of hemoglobin. Fetal hemoglobin F, adult hemoglobin A, hemoglobin Hb A2, and abnormal hemoglobin Hb S and C were purchased.

[0096] The above hemoglobin was diluted into multiple concentration gradients (as shown in Table 4 below), and the kit prepared in Example 4 was repeatedly used for detection at each concentration gradient. The results were interpreted after 15 minutes of color development (as shown in Table 4). Figure 3 As shown in Figures A to E), the kit of the present invention has no cross-reaction to fetal hemoglobin F and hemoglobin Hb A2, and can normally detect adult hemoglobin A and abnormal hemoglobin HbS and C, with excellent specificity.

[0097] Table 4 Hemoglobin detection of different types and concentrations

[0098]

[0099] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An anti-sickle cell anemia hemoglobin variant antibody, characterized in that The anti-sickle cell anemia hemoglobin variant antibody comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region has a light chain CDR consisting of CDR-L1, CDR-L2, and CDR-L3, and the heavy chain variable region has a heavy chain CDR consisting of CDR-H1, CDR-H2, and CDR-H3, and the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are as shown in any one of the groups (B) to (C): (B) SEQ ID NO: 13~15, SEQ ID NO: 17~19; (C) SEQ ID NO:21~23, SEQ ID NO:25~27.

2. The anti-sickle cell anemia hemoglobin variant antibody according to claim 1, characterized in that The amino acid sequences of the light chain variable region and the heavy chain variable region are shown in any one of groups (F) to (G): (F) SEQ ID NO:16, SEQ ID NO:20; (G) SEQ ID NO:24, SEQ ID NO:

28.

3. A polynucleotide encoding the anti-sickle cell anemia hemoglobin variant antibody according to claim 1 or 2.

4. A recombinant vector carrying the polynucleotide according to claim 3.

5. A recombinant cell expressing the anti-sickle cell hemoglobin variant antibody according to claim 1 or 2, or containing the polynucleotide according to claim 3, or transformed with the recombinant vector according to claim 4.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell uses bacteria, fungi, animal cells or plant cells as hosts; the fungi are yeast or mold, and the bacteria are Escherichia coli.

7. A method for preparing an anti-sickle cell anemia hemoglobin variant antibody, characterized in that: The method comprises culturing the recombinant cell according to claim 5 or 6 to obtain a culture containing the anti-sickle cell anemia hemoglobin variant antibody according to claim 1 or 2.

8. A biologically or chemically labeled product, characterized in that The product is an antibody labeled with a marker, and the source of the antibody is any of the following: (1) The anti-sickle cell anemia hemoglobin variant antibody according to claim 1 or 2; (2) A culture of the recombinant cell according to claim 5 or 6.

9. The product according to claim 8, characterized in that The labels include enzymes, biotin, fluorescein, chemiluminescence, isotopes, colloids, latex microspheres, and magnetic beads; the enzymes include but are not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridges, alkaline phosphatase-anti-alkaline phosphatase bridges, and β-galactosidase-anti-β-galactosidase bridges; the biotin includes biotin and its derivatives; the fluorescein includes AF350, AF488, AF532, AF546, AF555, AF568, and AF59 4. AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, OregonGreen488, PacificBlue dye, PacificOrange dye, TexasRed, PerCP dye; the chemiluminescent type includes but is not limited to isoluminol and its derivatives, acridinium ester and its derivatives, terpyridine ruthenium and its derivatives; the isotope type includes iodine labeling; the colloidal labeling includes colloidal gold, colloidal carbon, and colloidal selenium.

10. A kit, characterized in that The kit contains the anti-sickle cell anemia hemoglobin variant antibody according to claim 1 or 2 or the biomarker or chemically labeled product according to claim 8 or 9.

11. The kit according to claim 10, characterized in that The kit includes an enzyme-linked immunosorbent assay kit and an immunofluorescence kit.

12. The kit according to claim 10 or 11, characterized in that The kit includes a test strip, which is provided with a quality control line and multiple detection lines, wherein the detection lines include an HbA line, an HbS line, and an HbC line. The HbA line is coated with a normal hemoglobin β chain-specific antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO: 32 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 36, the HbS line is coated with a hemoglobin S type β chain-specific antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO: 16 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO: 20, and the HbC line is coated with a hemoglobin C type β chain-specific antibody with a light chain variable region amino acid sequence as shown in SEQ ID NO: 24 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:

28. The test strip is also sprayed with a labeled antibody, which is a specific antibody for the hemoglobin α chain with a light chain variable region amino acid sequence as shown in SEQ ID NO: 8 and a heavy chain variable region amino acid sequence as shown in SEQ ID NO:

12.

13. Use of the anti-sickle cell hemoglobin variant antibody of claim 1 or 2, or the polynucleotide of claim 3, or the recombinant vector of claim 4, or the recombinant cell of claim 5 or 6, or the method of claim 7, or the biomarker or chemically labeled product of claim 8 or 9 in the preparation of a product for in vitro detection of sickle cell anemia typing.

14. The use according to claim 13, characterized in that The products include reagents, test kits, detection chips or biosensors.

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