Recombinant rabbit monoclonal antibody of acth, preparation method and application thereof

Abstract

The application relates to the field of antibody technology, in particular to an ACTH recombinant rabbit monoclonal antibody, a preparation method and application thereof. The ACTH recombinant rabbit monoclonal antibody is screened by using phage display technology. The monoclonal antibody has high specificity, high detection sensitivity and good affinity, and solves the problems of low content of adrenocorticotropic hormone in samples and great detection difficulty. Through identification, the monoclonal antibody has good stability and high accuracy when repeatedly detected.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to an ACTH recombinant rabbit monoclonal antibody, a preparation method and an application thereof. Background Art

[0002] Adrenocorticotropic hormone (ACTH) is one of the key hormones secreted by the hypothalamus and pituitary gland. ACTH secretion is influenced by feedback from the hypothalamic hormone corticotropin-releasing factor (CRF) and cortisol. Elevated ACTH levels are typical in primary adrenal insufficiency, while low ACTH levels are often seen in adrenal insufficiency secondary to pituitary dysfunction. ACTH testing is valuable in differentiating adrenal insufficiency from adrenal hypersecretion and secondary hypertension. ACTH testing can also help identify the cause of excessive cortisol secretion in Cushing's syndrome. When excessive cortisol secretion is caused by adrenal cortical lesions or hyperplasia, ACTH levels are characteristically low. However, when the cause is ectopic pituitary ACTH production or excessive ACTH secretion, ACTH levels are elevated.

[0003] Hypertension is a common disease worldwide, particularly in spring and winter. It can lead to lesions in the heart, kidneys, and cerebral blood vessels, making it a major health hazard. Adrenal hyperplasia (AH) refers to secondary hypertension of the adrenal glands, with the underlying pathology being adrenal hyperplasia. Diagnosis is based on increased adrenal hormone secretion.

[0004] Therefore, the preparation of ACTH monoclonal antibodies with strong specificity, high sensitivity and good affinity is of great significance for improving the diagnosis rate of essential hypertension. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide an ACTH recombinant rabbit monoclonal antibody, a preparation method and an application thereof. The present invention provides an ACTH recombinant rabbit monoclonal antibody with strong specificity, high sensitivity and good affinity, as well as a preparation method and an application of the antibody.

[0006] The ACTH recombinant rabbit monoclonal antibody provided by the present invention comprises the following amino acid sequence:

[0007] (1) The CDR region of its heavy chain comprises at least one of the amino acid sequences shown in SEQ ID NO: 1, 2 or 3, and the CDR region of its light chain comprises at least one of the amino acid sequence shown in SEQ ID NO: 4, EAS or SEQ ID NO: 6; or

[0008] (2) An amino acid sequence that has one or more amino acids substituted, added or deleted from the amino acid sequence shown in (1), but has the same or similar function as the amino acid sequence shown in Ⅰ; or

[0009] (3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in (1) or (2).

[0010] In some embodiments, the ACTH recombinant rabbit monoclonal antibody provided by the present invention is

[0011] The amino acid sequence of the CDR region of the heavy chain comprises at least one of the sequences shown in SEQ ID NO: 1, 2 or 3;

[0012] The amino acid sequence of the CDR region of the light chain comprises at least one of the amino acid sequence of SEQ ID NO: 4, EAS, or the sequence shown in SEQ ID NO: 6.

[0013] In the present invention, the ACTH recombinant rabbit monoclonal antibody comprises:

[0014] I. At least one of the four FR regions of its heavy chain has the amino acid sequence shown in SEQ ID NOs: 7, 8, 9 and 10; and at least one of the four FR regions of its light chain has the amino acid sequence shown in SEQ ID NOs: 11, 12, 13 and 14.

[0015] II. An amino acid sequence that has one or more amino acids substituted, added or deleted from the amino acid sequence shown in I, but has the same or similar function as the amino acid sequence shown in I; or

[0016] III. An amino acid sequence having at least 80% homology to the amino acid sequence shown in I or II.

[0017] In the present invention, the sequence with at least 80% homology is an amino acid sequence obtained by replacing, adding or deleting one or more amino acids on the basis of the original sequence, and the multiple ones are 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0018] In some embodiments, among the three CDR regions of the ACTH recombinant rabbit monoclonal antibody heavy chain, the amino acid sequence of the CDR1 region is GIDLSSYT (SEQ ID NO: 1), the amino acid sequence of the CDR2 region is ISTANRI (SEQ ID NO: 2), and the amino acid sequence of the CDR3 region is ARSLTL (SEQ ID NO: 3);

[0019] In some embodiments, among the three CDRs of the ACTH recombinant rabbit monoclonal antibody light chain, the amino acid sequence of the CDR1 region is QSVYNNNN (SEQ ID NO: 4), the amino acid sequence of the CDR2 region is EAS, and the amino acid sequence of the CDR3 region is LGIYDCKRADCNA (SEQ ID NO: 6).

[0020] In some embodiments, the amino acid sequences of the four FR regions of the heavy chain of the ACTH recombinant rabbit monoclonal antibody are shown in SEQ ID NOs: 7, 8, 9, and 10, respectively;

[0021] In some embodiments, the amino acid sequences of the four FR regions of the ACTH recombinant rabbit monoclonal antibody light chain are shown in SEQ ID NOs: 11, 12, 13, and 14, respectively.

[0022] In some specific embodiments, the heavy chain variable region of the ACTH recombinant rabbit monoclonal antibody comprises the CDR region shown in SEQ ID NO: 1, 2 or 3 and the FR region shown in SEQ ID NO: 7, 8, 9 and 10.

[0023] In an embodiment of the present invention, the light chain variable region of the monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 4, the CDR region shown in EAS or SEQ ID NO: 6, and the FR region shown in SEQ ID NOs: 11, 12, 13 and 14.

[0024] In some specific embodiments, the heavy chain variable region of the ACTH recombinant rabbit monoclonal antibody has the amino acid sequence shown in SEQ ID NO: 15; the light chain variable region has the amino acid sequence shown in SEQ ID NO: 16.

[0025] The ACTH recombinant rabbit monoclonal antibody provided by the present invention further comprises a constant region, wherein the constant region of the heavy chain is of rabbit IgG subtype; and the constant region of the light chain is of κ1 type.

[0026] The present invention utilizes phage display technology to screen and obtain the recombinant rabbit monoclonal antibody against ACTH. This antibody exhibits strong specificity, high sensitivity, and good affinity. This antibody addresses the challenges of low levels of adrenocorticotropic hormone (ACTH) in samples and the difficulty of detecting it. Repeated testing using this monoclonal antibody has demonstrated excellent stability and high accuracy.

[0027] The present invention provides a biomaterial comprising at least one of the following:

[0028] 1) A nucleic acid encoding the ACTH recombinant rabbit monoclonal antibody;

[0029] 2), an expression vector comprising the nucleic acid;

[0030] 3) transforming or transfecting host cells with the expression vector;

[0031] 4) A conjugate prepared by coupling the ACTH recombinant rabbit monoclonal antibody with a solid medium or a semi-solid medium;

[0032] 5) The chemically labeled or biologically labeled recombinant rabbit monoclonal antibody against ACTH;

[0033] 6) A conjugate prepared by coupling the chemically labeled or biologically labeled ACTH recombinant rabbit monoclonal antibody with a solid medium or a semi-solid medium.

[0034] The present invention does not limit the nucleic acid sequence encoding the antibody; any nucleic acid encoding the antibody of the present invention is within the scope of protection of the present invention. In some specific embodiments, the nucleotide sequence comprises a nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotides to the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18, and having the same or similar functions as the nucleotide sequence of SEQ ID NO: 17 and SEQ ID NO: 18. More specifically, the nucleic acid comprises: a nucleic acid encoding a heavy chain variable region having a nucleotide sequence as shown in SEQ ID NO: 17; and a nucleic acid encoding a light chain variable region having a nucleotide sequence as shown in SEQ ID NO: 18.

[0035] The present invention provides a method for preparing the ACTH recombinant rabbit monoclonal antibody, which is characterized by comprising: culturing the host cell to induce the expression of the ACTH recombinant rabbit monoclonal antibody.

[0036] In some specific embodiments, the expression vector is RGFc-PCMV3 or RCL-PCMV3.

[0037] In some specific embodiments, the host cell is selected from Escherichia coli, yeast, insect cells or mammalian cells. Preferably, the host cell is a mammalian HEK293 cell.

[0038] The chemical label is an isotope, an immunotoxin, and / or a chemical drug; the biological label is biotin, avidin, or an enzyme label. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. The immunotoxin is preferably aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, abrin, mistletoe lectin, modeccin, PAP, herbivorin, gelonin, or luffa toxin.

[0039] The solid phase medium or semi-solid medium refers to any support to which the recombinant antibody or labeled recombinant antibody described in the present invention can be attached, including but not limited to nitrocellulose membrane, polyvinylidene fluoride (PVDF) membrane, iPDMS chip, microplate, polystyrene plate, microparticles, microcarriers, gel, etc.

[0040] The ACTH recombinant rabbit monoclonal antibody, biological material and / or host cell-expressed ACTH recombinant rabbit monoclonal antibody of the present invention are used in the preparation of reagents or kits for detecting hypertension-related diseases.

[0041] In some specific embodiments, the indicator for detecting hypertension-related diseases is detecting the level of adrenocorticotropic hormone.

[0042] Experiments have shown that the recombinant rabbit monoclonal antibody against ACTH provided by the present invention can be used as a detection antibody for the detection of adrenocorticotropic hormone (ACTH) with good sensitivity. The recombinant rabbit monoclonal antibody against ACTH provided by the present invention is suitable for immunological detection of ACTH. In some embodiments, the detection of ACTH utilizes a magnetic microparticle chemiluminescence assay.

[0043] The present invention provides a detection reagent or a detection kit, which comprises the ACTH recombinant rabbit monoclonal antibody of the present invention, the biological material and / or the ACTH recombinant rabbit monoclonal antibody expressed by the host cell.

[0044] The detection reagent or detection kit further includes acceptable adjuvants, buffers, auxiliary materials or carriers.

[0045] In some embodiments, the kit is suitable for magnetic particle chemiluminescence of adrenocorticotropic hormone.

[0046] The present invention also provides a method for detecting hypertension-related diseases, which comprises detecting a sample using the detection reagent or detection kit of the present invention.

[0047] The present invention utilizes phage display technology to screen and obtain the recombinant rabbit monoclonal antibody against ACTH, whose heavy chain variable region has the amino acid sequence shown in SEQ ID NO:15; and whose light chain variable region has the amino acid sequence shown in SEQ ID NO:16. This monoclonal antibody exhibits strong specificity, high detection sensitivity, and good affinity, addressing the challenges of low levels of adrenocorticotropic hormone (ACTH) in samples and the difficulty of detecting it. Repeated testing using this monoclonal antibody has demonstrated good stability and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Agarose gel electrophoresis diagram of extracted spleen RNA;

[0049] Figure 2 Agarose gel electrophoresis of the VL gene PCR product;

[0050] Figure 3 Agarose gel electrophoresis diagram of VH gene PCR products;

[0051] Figure 4 Agarose gel electrophoresis diagram of scFv gene PCR product;

[0052] Figure 5 Agarose gel electrophoresis diagram of the antibody library recombination rate bacterial liquid PCR identification;

[0053] Figure 6 This is the SDS-PAGE electrophoresis diagram of the recombinant antibody purification;

[0054] Figure 7 This is a correlation analysis between the detection concentrations of Antibody 1 and Antibody 2 in the present invention and 25 clinical samples, wherein: Figure 7 a is the correlation analysis of antibody 1, Figure 7 b is the correlation analysis of antibody 2;

[0055] Figure 8 Correlation analysis between the antibodies of the present invention and antibodies from reference manufacturers;

[0056] Figure 9 It is the deviation between the test result and the theoretical concentration. DETAILED DESCRIPTION

[0057] The present invention provides ACTH recombinant rabbit monoclonal antibodies, preparation methods, and applications thereof. Those skilled in the art can refer to the contents herein and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0058] The sequence shown in SEQ ID NO: 1 is GIDLSSYT;

[0059] The sequence shown in SEQ ID NO: 2 is ISTANRI;

[0060] The sequence shown in SEQ ID NO: 3 is ARSLTL;

[0061] The sequence shown in SEQ ID NO:4 is QSVYNNNN;

[0062] The sequence shown in SEQ ID NO:6 is LGIYDCKRADCNA;

[0063] The sequence shown in SEQ ID NO:7 is QQQLEESGGRLVTPGTPLTLTCTAS;

[0064] The sequence shown in SEQ ID NO:8 is MNWVRQAPGKGLEWIGL;

[0065] The sequence shown in SEQ ID NO:9 is YYASWAKGRFTISKTSTTVDLKIISPTTEDT ATYFC;

[0066] The sequence shown in SEQ ID NO: 10 is WGPGTLVTVSS;

[0067] The sequence shown in SEQ ID NO: 11 is ALVMTQTPPSVSAAVGSTVTINCQAS;

[0068] The sequence shown in SEQ ID NO: 12 is LAWYQQKPGQPPKLLIY;

[0069] The sequence shown in SEQ ID NO: 13 is KLASGVSSRFKGSGSGTQFTLTISDVQCD DAATYYC;

[0070] The sequence shown in SEQ ID NO: 14 is FGGGTKLEIK;

[0071] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0072] The present invention will be further described below in conjunction with the embodiments:

[0073] Example 1 Preparation for phage screening platform provided by the present invention:

[0074] 1. Animals immunized with recombinant immunogens:

[0075] New Zealand white rabbits were immunized four times with the prepared antigen ACTH-BSA. The immunization cycle was 30 days, with multiple subcutaneous injections at the back. For the first immunization, 2 mg of the immunizing antigen was emulsified with an equal volume of Freund's complete adjuvant. For the subsequent three immunizations, 1 mg of the immunizing antigen was emulsified with an equal volume of Freund's incomplete adjuvant. On the 10th day after the third immunization, blood was collected from the ear vein. After standing at 37°C for 1 hour, the blood was centrifuged at 6000 rpm for 10 minutes. The supernatant (antiserum) was collected and analyzed for immune efficacy using ELISA (see Example 2).

[0076] 2. Antiserum titer detection

[0077] Prepare the test plate, add RaIgGFc antibody to 0.05mol / LCB (pH9.6) coating buffer, and the coating concentration is 4μg / ml. The test serum is serially diluted starting from 1:200, 50μl / well, and an unimmunized rabbit serum control is set at the same time. Incubate at 37℃ for 30min; wash 5 times with PBST, pat dry, add ACTH enzyme conjugate at working concentration, 50μl / well, and incubate at 37℃ for 30min; wash 5 times with PBST, pat dry, add substrate A and color developer B, 50μl / well each, react in the dark for 5min, add 50ul of stop solution, and measure the signal value. The test results are shown in Table 1. After the titer reaches 1 / 64K, the last booster immunization is carried out. Three days later, the animals are sacrificed, spleen cells are extracted, and total RNA of spleen tissue is routinely extracted by Trizol method (such as Figure 1 cDNA was synthesized by reverse transcription.

[0078] Table 1 Antiserum titer detection

[0079]

[0080] Example 2: scFv gene splicing and phage screening construction

[0081] 1. scFv gene splicing:

[0082] PCR was used to amplify the light chain variable region and heavy chain variable region of the antibody, respectively. The PCR reaction program is shown in Table 2:

[0083] Table 2

[0084]

[0085] The PCR products were recovered from 1% agarose gel. Figure 2 and Figure 3 The amplified light chain variable region and heavy chain variable region were spliced ​​into scFv using overlap-PCR method, and the product was recovered through 1% agarose gel and stored at -20℃. Figure 4 shown.

[0086] 2. Construction and screening of phage single-chain antibody library:

[0087] The phagemid vector pcomb3XSS and the purified ScFv fragment were digested with SfiI to construct a recombinant plasmid. The recombinant plasmid was electroporated into TG1 competent cells to construct a rabbit-derived immune single-chain antibody library (the antibody library recombinant rate was determined by bacterial liquid PCR). Figure 5), and prepare a primary phage single-chain antibody library; the primary phage single-chain antibody library was enriched and screened for three rounds to obtain a specific phage single-chain antibody library with high affinity and strong specificity; 558 monoclonal strains were picked, monoclonal phage supernatants were prepared, and positive clones were identified by Phage-ELISA to obtain positive sequences.

[0088] After library construction and screening, multiple variable region sequences were identified. The diagnostic performance evaluation results of some of the sequences and samples are shown in Table 3:

[0089] Antibody 1:

[0090] Heavy chain variable region amino acid sequence:

[0091] QQQLEESGGRLVTPGTPLTLTCTASGIDLSSYTMNWVRQAPGKGLEWIGLISTANRIYYASWAKGRFTISKTSTTVDLKIISPTTEDTATYFCARSLTLWGPGTLVTVSS (SEQ ID NO: 15)

[0092] Heavy chain variable region nucleotide sequence:

[0093] CAGCAGCAGCTGGAGGAGTCCGGGGGTCGCCTGGTCACGCCGGGGACACCCCTGACACTCACCTGCACAGCCTCTGGAATCGACCTCAGTAGCTATACTATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGACTCATTAGTACTGCTAATAG GATATACTACGCGAGCTGGGCGAAAGGCCGCTTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCATTAGTCCGACAACCGAGGACACGGCCACCTATTTCTGTGCCAGATCCTTGACTCTGTGGGGCCCAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO:17)

[0094] CDR-H1 GIDLSSYT (SEQ ID NO:1);

[0095] CDR-H2 ISTANRI (SEQ ID NO:2);

[0096] CDR-H3 ARSLTL (SEQ ID NO:3);

[0097] FR-H1 QQQLEESGGRLVTPGTPLTLTCTAS (SEQ ID NO: 7);

[0098] FR-H2 MNWVRQAPGKGLEWIGL (SEQ ID NO: 8);

[0099] FR-H3 YYASWAKGRFTISKTSTTVDLKIISPTTEDTATYFC (SEQ ID NO: 9);

[0100] FR-H4 WGPGTLVTVSS (SEQ ID NO: 10);

[0101] The amino acid sequence of the variable region of the heavy chain of the antibody is:

[0102] ALVMTQTPPSVSAAVGSTVTINCQASQSVYNNNNLAWYQQKPGQPPKLLIYEASKLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGIYDCKRADCNAFGGGTKLEIK (SEQ ID NO: 16)

[0103] The nucleotide sequence of the variable region of the heavy chain of the antibody is:

[0104] GCGCTTGTGATGACCCAGACTCCACCCTCCGTGTCTGCAGCTGTGGGAAGCACAGTCACCATCAATTGCCAGGCCAGTCAGAGTGTTTATAATAACAACAACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACGAAGCGTCCAAACTGGCATCTGGGGTCTCATCGCGGTTCAAGGGCAGTGGATCTGGGACCCAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCATTTATGATTGTAAGCGTGCTGATTGTAATGCTTTCGGCGGAGGAACCAAGCTGGAGATCAAA (SEQ ID NO: 18)

[0105] CDR-H1 QVYSSY (SEQ ID NO: 1);

[0106] CDR-H2 YIHYSN (SEQ ID NO: 2);

[0107] CDR-L3 LGIYDCKRADCNA(SEQ ID NO:6);

[0108] FR-L1 ALVMTQTPPSVSAAVGSTVTINCQAS(SEQ ID NO:11);

[0109] FR-L2 LAWYQQKPGQPPKLLIY(SEQ ID NO:12);

[0110] FR-L3 KLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYC(SEQ ID NO:13);

[0111] FR-L4 FGGGTKLEIK(SEQ ID NO:14);

[0112] 抗体2:

[0113] 重链可变区氨基酸序列:

[0114] QSVKESGGRLVTPGTPLTLTCTVSGIDLSRYSFHWVRQAPGKGLEYIGTISNVGKTYFANWVKGRFTISKASTTVDLKLTSLTAPDTATYFCVRGFTLWGHGTLVTVSS(SEQ ID NO:19)

[0115] 重链可变区核苷酸序列:

[0116] CAGTCGGTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGTCTCTGGAATCGACCTCAGTCGCTACAGCTTCCATTGGGTCCGCCAGGCTCCAGGGAAGGGTCTGGAATACATCGGAACCATTAGCAATGTTGGTAAGACATACTTCGCGAACTGGGTTAAAGGCCGATTCACCATCTCCAAAGCCTCGACCACGGTGGATCTGAAGCTGACCAGTCTAACAGCGCCAGACACGGCCACATATTTCTGTGTCAGAGGCTTTACCTTGTGGGGCCACGGCACCCTGGTCACCGTCTCCTCAGGCCAGGCCGG(SEQ ID NO:20)

[0117] CDR-H1 GIDLSRYS(SEQ ID NO:21);

[0118] CDR-H2 ISNVGKT (SEQ ID NO: 22);

[0119] CDR-H3 VRGFTL (SEQ ID NO: 23);

[0120] FR-H1 QSVKESGGRLVTPGTPLTLTCTVS (SEQ ID NO: 24);

[0121] FR-H2 FHWVRQAPGKGLEYIGT (SEQ ID NO: 25);

[0122] FR-H3 YFANWVKGRFTISKASTTVDLKLTSLTAPDTATYFC (SEQ ID NO: 26);

[0123] FR-H4 WGHGTLVTVSS (SEQ ID NO: 27);

[0124] Amino acid sequence of the variable region of its heavy chain:

[0125] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWMGFIYPDSNTGDTKYVDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAADGYSSSSYMEYWGQGTQVTVSS (SEQ ID NO: 21)

[0126] Nucleotide sequence of the variable region of the heavy chain:

[0127] GCCATCGATATGACCCAGACTCCAGCCTCTGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAATAACTGGTTATCCTGGTATCAGCAGAAACCAGGACAGCCTCCCAAGGTCCTTATCTACGAAGCATCCAAACTGGCATCTGGGGTCCCATCGCGGTTCAAAGCCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGTGACGTACAGTGTGACGATGCTGCCACTTACTACTGTCTAGGTAGTTATGATTGTAAAACTGCTGATTGTGCGGCTTTCGGCGGAGGGACCAAGGTGGAGATCAAA (SEQ ID NO: 29)

[0128] CDR-L1 QSVYNNW (SEQ ID NO:30);

[0129] CDR-L2 EAS;

[0130] CDR-L3 LGSYDCKTADCAA (SEQ ID NO:32);

[0131] FR-L1AIDMTQTPASVSAAVGGTVTIKCQSS (SEQ ID NO:33);

[0132] FR-L2 LSWYQQKPGQPPKVLIY (SEQ ID NO:34);

[0133] FR-L3 KLASGVPSRFKASGSGTQFTLTISDVQCDDAATYYC (SEQ ID NO: 5);

[0134] FR-L4 FGGGTKVEIK (SEQ ID NO:31);

[0135] Table 3

[0136]

[0137] The results show that antibody 1 is more effective than antibody 2, so subsequent experiments will be conducted based on this.

[0138] Example 4: Expression of recombinant monoclonal antibodies and antibody purification

[0139] 1. Construction of Stable Cell Lines

[0140] The heavy and light chain variable regions of Antibody 1 were amplified by PCR to generate the variable region genes. The heavy and light chain antibody genes were homologously recombined with the KasI / BamHI-digested RGFc-PCMV3 and RCL-PCMV3 vectors, respectively, to generate expression vectors for the rabbit full antibody sequence. The recombinant full antibody plasmids were transformed into competent DH5α cells, and positive clones were selected for sequencing and plasmid extraction. The extracted plasmids were then transfected into HEK293 cells.

[0141] After 48 hours of transfection, the antibody expression was detected by ELISA, and then the feed was added. After 7 days of transfection, the cell supernatant was collected to obtain the target protein supernatant.

[0142] 2. Antibody SPA purification and SDS-PAGE identification column balance

[0143] Set the flow rate to 6.4 mL / min, and rinse the chromatography column with 0.02 mol / L PBS pH 7.4 equilibrium buffer. Set the flow rate to 3.8 mL / min for sample loading and purification. Take 10 μL of the sample in the flow-through tube and add it to 200 μL of G250 dye solution. When the test turns blue, place the flow-through tube in a 250 mL conical flask to start collecting the flow-through. Rebalance: Set the flow rate to 6.4 mL / min, and rinse the chromatography column again with 0.02 mol / L PBS pH 7.4 equilibrium buffer until no protein flows through. Dissociation: Place 10 4 mL centrifuge tubes on the dissociation tube rack, and add 200 μL of 1 mol / L Tris (pH 8.5), set the constant flow pump to a flow rate of 6.4 mL / min, dissociate the target protein with dissociation buffer 0.2 mol / L Lysine + 0.15 mol / L NaCl pH 2.7, take a sample of 10 μL through the tube and add it to 200 μL of G250 dye solution. Start manual collection when the color turns blue, collect 4 mL in each tube, take a sample of 10 μL through the tube and add it to 200 μL of G250 dye solution. Stop collection when the color turns colorless. The collected proteins are combined and detected by SDS-PAGE. Figure 6 As shown in the figure, it shows that the purified antibody has high purity and high concentration.

[0144] Example 5: Application of the kit for preparing the ACTH recombinant rabbit monoclonal antibody prepared by the present invention

[0145] The ACTH recombinant rabbit monoclonal antibody prepared in this invention is used as a magnetic bead goat anti-rabbit complex-bound antibody in a kit, and is used together with other components to detect antigens in samples. Specific performance evaluation is as follows:

[0146] 1. Preliminary evaluation of antibody-coated magnetic beads

[0147] Antibodies 1 and 2 obtained in the previous examples were applied to the kit components, and 25 clinical samples within the detection range were randomly selected for detection. The detected concentration values ​​were correlated with the hospital assigned clinical samples. The results were as follows: Figure 7 As shown. Among them, Figure 7 a is the correlation analysis of antibody 1, Figure 7 b is the correlation analysis of antibody 2.

[0148] The results showed that antibody 2 had poor correlation and had abnormal test values, so no further evaluation was performed.

[0149] 2. Accelerated thermal stability test of antibody 1

[0150] Antibody-coated magnetic beads were subjected to accelerated thermal stability testing at 37°C for 7 days. Five clinical samples were tested on the machine. The test results are shown in the following table:

[0151] Table 4 Stability evaluation results of Antibody 1

[0152]

[0153] 3. Accuracy determination:

[0154] 13 clinical samples with a certain mainstream commercial kit were used for testing. The ACTH concentration in the samples was calculated by the system and the correlation analysis was performed with the reference manufacturer. The results are shown in the figure. Figure 8 .

[0155] According to the test results, the linear equation is: y=8E-06x+2.0213, and the correlation coefficient R 2 =0.9829, indicating that the ACTH recombinant rabbit monoclonal antibody provided by the present invention has a high accuracy in detecting clinical samples of adrenocorticotropic hormone.

[0156] 4. Recovery performance evaluation:

[0157] Three high-value samples were selected and added to three low-value / matrix samples at a ratio of 1:9 to prepare recovery samples. The volume of the added high-value sample should not exceed 10% of the total volume. Each recovery sample was tested three times, and the average value was calculated to calculate the recovery rate. The recovery rates of the three recovered samples in the first batch were 92.3%, 94.4%, and 96.7%, respectively. The recovery rates of the three recovered samples in the second batch were 103.6%, 102.8%, and 103.7%, respectively. The recovery rates of the three recovered samples in the third batch were 102.4%, 103.0%, and 103.8%, respectively. The deviations were all less than 10%, and the recovery accuracy met the requirements.

[0158] 5. Linear analysis:

[0159] Select clinical high-value samples with ACTH concentration close to the expected linear range, 1 clinical low-value sample with concentration close to 0 or the lowest value that can be obtained clinically, and mix them in different proportions to obtain 9 linear samples with different concentrations. Finally, calculate the deviation between the test results and the theoretical concentration ( Figure 9 The results showed that the linear regression coefficient r>0.99 and the linear sample deviation was less than 10%.

[0160] 6. Precision assessment:

[0161] The precision evaluated in this study was intra-batch precision. Three concentration levels (high, medium, and low) were tested using the same batch of reagents. Each sample in each batch was repeated 20 times, and the mean, SD, and coefficient of variation (CV) were calculated. The intra-batch coefficient of variation (CV) for all three levels was less than 5%, as shown in Table 5, demonstrating good reproducibility of the kit.

[0162] Table 5 Precision results of ACTH rabbit monoclonal antibody

[0163]

[0164] 7. Sensitivity performance evaluation:

[0165] We selected 5 clinical samples close to 0 values, repeated 3 times for each sample for 4 days, and obtained 60 data points. We performed data verification and result analysis according to the EP17 method. The LoB values ​​of clinical 0 value samples of the 3 batches of kits were all less than 0.15nmol / L, which were all within the blank limit, indicating high sensitivity.

[0166] 8. Affinity determination analysis:

[0167] ACTH biotinylated recombinant rabbit monoclonal antibodies 1 and 2 were serially diluted to six concentrations, namely 2.08, 4.17, 8.33, 16.67, 33.33, and 66.67 nM. Binding and dissociation experiments (60 s binding, 300 s dissociation) were performed using an Octet RED96e sensor immobilized with ACTH and the biotinylated antibodies at different concentrations. Affinity measurements were then performed, and data were processed to obtain the KD values ​​of 2.76E-9 for Antibody 1 and 6.32E-8 for Antibody 2. This indicates that the ACTH biotinylated recombinant rabbit monoclonal antibody 1 provided by the present invention has a high affinity.

[0168] The above results indicate that the recombinant monoclonal antibody prepared by the present invention has achieved a level highly consistent with the detection results of mainstream manufacturers in the current market, and exhibits advantages such as high precision and specificity. When used as a detection antibody in a test kit, the performance of each detection item meets the requirements of the test kit according to the evaluation, which plays an important role in subsequent applications and clinical diagnosis.

[0169] The above results indicate that the recombinant monoclonal antibody prepared by the present invention has achieved a level highly consistent with the detection results of mainstream manufacturers in the current market, and exhibits advantages such as high precision. When used as a detection antibody in a test kit, the performance of each detection item meets the requirements of the test kit according to the evaluation, which plays an important role in subsequent applications and clinical diagnosis.

[0170] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. ACTH recombinant rabbit monoclonal antibody, characterized by: The amino acid sequence of the heavy chain CDR1 region is GIDLSSYT, the amino acid sequence of the CDR2 region is ISTANRI, and the amino acid sequence of the CDR3 region is ARSLTL; The amino acid sequence of the light chain CDR1 region is QSVYNNNN, the amino acid sequence of the CDR2 region is EAS, and the amino acid sequence of the CDR3 region is LGIYDCKRADCNA.

2. The ACTH recombinant rabbit monoclonal antibody according to claim 1, wherein The amino acid sequences of the four FR regions of its heavy chain are shown in SEQ ID NOs: 7, 8, 9, and 10, respectively; The amino acid sequences of the four FR regions of its light chain are shown in SEQ ID NOs: 11, 12, 13 and 14, respectively.

3. The ACTH recombinant rabbit monoclonal antibody according to claim 1 or 2, characterized in that The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO: 15; The amino acid sequence of its light chain variable region is shown in SEQ ID NO:

16.

4. The ACTH recombinant rabbit monoclonal antibody according to claim 3, wherein The constant region of the heavy chain is of rabbit IgG subtype; the constant region of the light chain is of κ1 type.

5. Biomaterial, characterized in that Include at least one of the following: 1) A nucleic acid encoding the ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4; 2), an expression vector comprising the nucleic acid; 3), transforming or transfecting the host cell with the expression vector; 4) A conjugate prepared by coupling the ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4 with a solid medium or a semi-solid medium; 5) The chemically labeled or biologically labeled ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4; 6) A conjugate prepared by coupling the chemically labeled or biologically labeled ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4 with a solid medium or a semi-solid medium.

6. The biomaterial according to claim 5, wherein The nucleic acid comprises: The nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 17; The nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO:

18.

7. The method for preparing the ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4, characterized in that: include: Cultivate the host cell according to claim 5 to induce the expression of the ACTH recombinant rabbit monoclonal antibody.

8. Use of at least one of the following i to iii in the preparation of a reagent or kit for detecting hypertension-related diseases: i. The ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4; ii. The biomaterial according to claim 5 or 6; iii. Prepare ACTH recombinant rabbit monoclonal antibody by the preparation method according to claim 7.

9. A detection reagent or a detection kit, characterized in that: Include at least one of the following ①~③:

1. The ACTH recombinant rabbit monoclonal antibody according to any one of claims 1 to 4; ②. The biomaterial according to claim 5 or 6; ③. Prepare ACTH recombinant rabbit monoclonal antibody by the preparation method described in claim 7.

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