C-peptide recombinant rabbit monoclonal antibody, preparation method and application thereof

Abstract

The present invention relates to the field of antibody technology, and more specifically to a recombinant rabbit monoclonal antibody to C-peptide, a preparation method, and its application. The present invention utilizes phage display technology to screen and obtain the recombinant rabbit monoclonal antibody to C-peptide described herein, which exhibits strong specificity, high sensitivity, and good affinity. The antibody provided by the present invention, combined with a specifically paired antibody, employs a double-antibody sandwich method to rapidly and sensitively detect C-peptide concentrations in patient serum. This detection method exhibits strong specificity and high accuracy, providing a reliable basis for clinical diagnosis and treatment.

Description

Technical Field

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

[0002] C-peptide is a byproduct of insulin biosynthesis, when proinsulin is cleaved into insulin. C-peptide is a single-chain polypeptide composed of 31 amino acids (AA33-63) with a molecular weight of approximately 3021 daltons. C-peptide plays a key role in building the disulfide bonds of the proinsulin molecule and the structure of the two insulin chains (A and B).

[0003] Proinsulin is stored in the secretory granules of the Golgi complex of pancreatic cells and is produced by the cleavage of preproinsulin. During the cleavage of proinsulin, insulin and C-peptide are secreted in equal amounts and released into the peripheral circulation. Although it is the other half of insulin, C-peptide is rarely taken up by the liver, resulting in a longer half-life than insulin, approximately 35 minutes. C-peptide concentrations in peripheral blood are 5 to 10 times that of insulin and fluctuate less than insulin. C-peptide is excreted through the kidneys, and urinary C-peptide concentrations are 20 to 50 times higher than those in serum. The secretory fragment of C-peptide in urine does not change. Therefore, C-peptide is often used as a measure of pancreatic islet function. A normal C-peptide value indicates that the body's pancreatic cells are currently secreting sufficient amounts of insulin.

[0004] However, due to its small molecular weight, C-peptide is not easily recognized by antibodies. Therefore, the specificity, sensitivity, and affinity of C-peptide detection antibodies screened by traditional antibody screening techniques are not sufficient to meet the needs of more accurate clinical diagnosis of diabetes. Summary of the Invention

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

[0006] The C-peptide 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 sequences shown in SEQ ID NO: 4, QVS 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 (1); or

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

[0010] In the present invention, the C-peptide recombinant rabbit monoclonal antibody comprises:

[0011] 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.

[0012] 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

[0013] III. An amino acid sequence having at least 80% homology to the amino acid sequence shown in I or II.

[0014] 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.

[0015] In some embodiments, the three CDR regions of the heavy chain of the C-peptide recombinant rabbit monoclonal antibody have the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively;

[0016] In some embodiments, the three CDR regions of the light chain of the C-peptide recombinant rabbit monoclonal antibody have the amino acid sequences shown in SEQ ID NO: 4, QVS and SEQ ID NO: 6, respectively.

[0017] In some embodiments, the four FR regions of the heavy chain of the C-peptide recombinant rabbit monoclonal antibody have the amino acid sequences shown in SEQ ID NOs: 7, 8, 9, and 10, respectively;

[0018] In some embodiments, the four FRs of the light chain of the C-peptide recombinant rabbit monoclonal antibody have amino acid sequences as shown in SEQ ID NOs: 11, 12, 13, and 14, respectively.

[0019] In some specific embodiments, the heavy chain variable region of the C-peptide 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.

[0020] In an embodiment of the present invention, the light chain variable region of the monoclonal antibody comprises a CDR region shown in SEQ ID NO: 4, QVS or SEQ ID NO: 6 and a FR region shown in SEQ ID NO: 11, 12, 13 and 14.

[0021] In some specific embodiments, the heavy chain variable region of the C-peptide 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.

[0022] The C-peptide 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.

[0023] The present invention utilizes phage display technology to screen and obtain the C-peptide recombinant rabbit monoclonal antibody of the present invention. The antibody has strong specificity, high sensitivity and good affinity.

[0024] The present invention provides a biomaterial comprising at least one of the following:

[0025] 1) A nucleic acid encoding the C-peptide recombinant rabbit monoclonal antibody;

[0026] 2) An expression vector comprising the nucleic acid;

[0027] 3) transforming or transfecting host cells with the expression vector;

[0028] 4) A conjugate prepared by coupling the C-peptide recombinant rabbit monoclonal antibody with a solid medium or a semi-solid medium;

[0029] 5) The chemically labeled or biologically labeled recombinant rabbit monoclonal antibody against the C-peptide;

[0030] 6) A conjugate prepared by coupling the chemically labeled or biologically labeled C-peptide recombinant rabbit monoclonal antibody to a solid medium or a semi-solid medium.

[0031] 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.

[0032] The present invention provides a method for preparing the C-peptide recombinant rabbit monoclonal antibody, which is characterized by comprising: culturing the host cell to induce the expression of the C-peptide recombinant rabbit monoclonal antibody.

[0033] In some specific embodiments, the expression vector is RGFc-PCMV3 or RCL-PCMV3.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] The invention relates to use of the C-peptide recombinant rabbit monoclonal antibody, biological material and / or the C-peptide recombinant rabbit monoclonal antibody expressed by host cells in the preparation of a reagent or kit for detecting diabetes.

[0038] In some specific embodiments, the indicator for detecting diabetes is detecting C-peptide levels.

[0039] The present invention provides a detection reagent or a detection kit, which comprises the C-peptide recombinant rabbit monoclonal antibody of the present invention, the biomaterial and / or the C-peptide recombinant rabbit monoclonal antibody expressed by the host cell.

[0040] The detection reagent or detection kit further includes acceptable adjuvants, buffers, auxiliary materials or carriers.

[0041] The present invention also provides a double-antibody sandwich detection kit, comprising the aforementioned recombinant rabbit monoclonal antibody against C-peptide and a 112# antibody that specifically binds to the C-peptide recombinant rabbit monoclonal antibody. The heavy chain of the 112# antibody has the amino acid sequence shown in SEQ ID NO: 5, and the light chain of the 112# antibody has the amino acid sequence shown in SEQ ID NO: 27.

[0042] Experiments showed that compared with other antibodies, the 112# antibody and the C-peptide recombinant rabbit monoclonal antibody were used to detect C-peptide in samples. It was found that the 112# antibody and the C-peptide recombinant rabbit monoclonal antibody had strong positive and negative coating reactivity, and could quickly and sensitively detect the concentration of C-peptide in patient serum. The cross-reactivity between the test results and 10 ng / mL proinsulin was lower than 0.25 ng / mL. After repeated tests, the results proved that the double-antibody sandwich detection kit had strong specificity and high accuracy, providing a reliable basis for clinical diagnosis and treatment.

[0043] In some specific embodiments, the 112# antibody is a coating antibody, and the C-peptide recombinant rabbit monoclonal antibody is a labeled antibody. In this case, the double-antibody sandwich detection kit has a stronger detection specificity.

[0044] In some specific embodiments, the double antibody sandwich detection kit is suitable for magnetic particle chemiluminescence of C-peptide.

[0045] In some specific embodiments, the double antibody sandwich detection kit further comprises a coating buffer, a washing solution, a blocking solution and / or a color developing solution.

[0046] The present invention also provides a method for detecting diabetes, which comprises using the detection reagent, detection kit or double antibody sandwich detection kit of the present invention to detect a sample.

[0047] The present invention utilizes phage display technology to screen and obtain the recombinant rabbit monoclonal antibody against C-peptide described herein. This antibody exhibits strong specificity, high sensitivity, and good affinity. The antibody provided herein, combined with a specifically paired antibody, employs a double-antibody sandwich assay for rapid and sensitive detection of C-peptide concentrations in patient serum. Test results indicate a cross-talk ratio with 10 ng / mL proinsulin of less than 0.25 ng / mL. This detection method demonstrates strong specificity and high accuracy, providing a reliable basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Correlation analysis between the 230# antibody of the present invention and the antibody of the reference manufacturer;

[0049] Figure 2 This is a correlation analysis between the 270# antibody of the present invention and the antibody of the reference manufacturer. DETAILED DESCRIPTION

[0050] The present invention provides C-peptide recombinant rabbit monoclonal antibodies, preparation methods, and applications thereof. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. 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 alter 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.

[0051] The sequences involved in the present invention are as follows:

[0052] The sequence shown in SEQ ID NO: 1 is GFSFSSYT;

[0053] The sequence shown in SEQ ID NO: 2 is IASSGNI;

[0054] The sequence shown in SEQ ID NO: 3 is GGTDTSL;

[0055] The sequence shown in SEQ ID NO:4 is QSVYSNR;

[0056] The sequence shown in SEQ ID NO:6 is LGSYDCRSVDCMA;

[0057] The sequence shown in SEQ ID NO:7 is QQLKESGGRLVTPGTPLTLTCTAS;

[0058] The sequence shown in SEQ ID NO:8 is MGWVRQAPGKGLEWIGS;

[0059] The sequence shown in SEQ ID NO:9 is YYARWAKGRFTISKTSTTVDLKITSPTTED TATYFCVR;

[0060] The sequence shown in SEQ ID NO: 10 is WGHGTLVTVSS;

[0061] The sequence shown in SEQ ID NO: 11 is DPVLTQTPASVSAAVGGTVTIKCQSS;

[0062] The sequence shown in SEQ ID NO: 12 is LSWYQQKPGQPPRRLIY;

[0063] The sequence shown in SEQ ID NO: 13 is TLTSGVSSRFKGSGSGTQFTLTISDVQCD DAATYYC;

[0064] The sequence shown in SEQ ID NO: 14 is FGGGTEVVVK.

[0065] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0066] The present invention will be further described below in conjunction with the embodiments:

[0067] Example 1 Preparation for phage screening platform provided by the present invention:

[0068] 1. Animals immunized with recombinant immunogens:

[0069] New Zealand white rabbits were immunized four times with the prepared antigen CP-(19-31) BSA. The immunization cycle lasted 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. Blood was collected from the ear vein on the 10th day after the third immunization. The blood was incubated at 37°C for 1 hour and then centrifuged at 6000 rpm for 10 minutes. The supernatant (antiserum) was collected and analyzed for ELISA.

[0070] 2. Antiserum titer and antibody titer detection

[0071] Prepare the assay plate by adding RaIgGFc antibody to 0.05 mol / L CCB (pH 9.6) coating buffer at a coating concentration of 4 μg / ml. Dilute the test serum serially starting at 1:200, using 50 μl / well. Also include a non-immune rabbit serum control. Incubate at 37°C for 30 min. Wash five times with PBST, pat dry, and add 50 μl / well of the working concentration of CP-(19-31) enzyme conjugate. Incubate at 37°C for 30 min. Wash five times with PBST, pat dry, and add 50 μl / well of substrate A and chromogen B, each of which is 50 μl / well. Incubate in the dark for 5 min. Add 50 μl of stop solution, and measure the signal.

[0072] The rabbit serum titer results are shown in Table 1.

[0073] Table 1 Antiserum titer detection

[0074]

[0075] The evaluation results of the interaction between the selected phage rabbit scFv and CP antigen are shown in Table 2.

[0076] Table 2

[0077]

[0078] From the results in Table 2 , two phage rabbit scFv monoclonal antibodies with the highest readouts for interaction with CP antigen were selected for competition evaluation of free CP in calibrators and clinical samples. The results are shown in Table 3 .

[0079] Table 3 Evaluation table of scFv monoclonal antibodies of phage rabbit

[0080]

[0081] The clones with an interaction with CP antigen greater than 10,000 and an interaction with KLH less than 5,000 were considered positive. The results in Tables 1 to 3 show that antibody 1# (i.e., antibody 270#) was more effective than antibody 2# (i.e., antibody 230#), and subsequent experiments were conducted based on this.

[0082] Example 2: Expression of recombinant monoclonal antibodies of the present invention

[0083] 1. Construction of full-length antibodies

[0084] The antibody variable region genes were amplified by PCR.

[0085] The nucleotide sequence of the heavy chain variable region of antibody #270 is as follows:

[0086] CAGCAGCTGAAGGAGTCCGGGGGTCGCCTGGTCACGCCTGGGACACCCCTGACACTCACCTGCACAGCCTCTGGATTCTCCTTCAGTAGTTATACAATGGGCTGGGTCCCGCCAGGCTCCAGGGAAGGGCCTGGAATGGATCGGAAGCATTGCTTCTAGTGGTAACATATACTAC GCGAGGTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAATCACCAGTCCGACAACCGAGGACACGGCCACTTATTTCTGTGTCAGAGGAGGAACTGATACGAGTTTGTGGGGCCACGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 17);

[0087] The amino acid sequence of the heavy chain variable region of antibody #270 is as follows: QQLKESGGRLVTPGTPLTLTCTASGFSFSSYTMGWVRQAPGKGLEWIGSIASSGNIYYARWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVRGGTDTSLWGHGTLVTVSS (SEQ ID NO: 15);

[0088] CDR-H1 GFSFSSYT (SEQ ID NO:1);

[0089] CDR-H2 IASSGNI (SEQ ID NO:2);

[0090] CDR-H3 GGTDTSL (SEQ ID NO:3);

[0091] FR-H1 QQLKESGGRLVTPGTPLTLTCTAS (SEQ ID NO:7);

[0092] FR-H2 MGWVRQAPGKGLEWIGS (SEQ ID NO:8);

[0093] FR-H3 YYARWAKGRFTISKTSTTVDLKITSPTTEDTATYFCVR (SEQ ID NO:9);

[0094] FR-H4 WGHGTLVTVSS (SEQ ID NO:10);

[0095] The nucleotide sequence of the light chain variable region of antibody #270 is as follows:

[0096] GACCCTGTGCTGACCCAGACTCCAGCCTCCGTGTCTGCAGCTGTGGGAGGCACAGTTACCATCAAGTGCCAGTCCAGTCAGAGTGTTTATAGTAACCGCTTGTCCTGGTACCAGCAGAAACCAGGCCAGCCTCCCAGACGCCTGATCTACCAGGTATCCACTCTGACATCTGGG GTCTCATCGCGGTTTAAAGGCAGTGGATCTGGGACACAGTTCACTCTCACCATCAGCGACGTGCAGTGTGACGATGCTGCCACTTACTACTGTCTAGGCAGTTATGATTGTCGTAGTGTTGATTGTATGGCTTTCGGCGGAGGGACCGAGGTGGTCGTCAAA (SEQ ID NO: 18);

[0097] The amino acid sequence of the light chain variable region of antibody #270 is as follows:

[0098] DPVLTQTPASVSAAVGGTVTIKCQSSQSVYSNRLSWYQQKPGQPPRRLIYQVSTLTSGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCRSVDCMAFGGGTEVVVK (SEQ ID NO: 16);

[0099] CDR-L1 QSVYSNR (SEQ ID NO:4);

[0100] CDR-L2 QVS;

[0101] CDR-L3 LGSYDCRSVDCMA (SEQ ID NO:6);

[0102] FR-L1 DPVLTQTPASSVSAAVGGTVTIKCQSS (SEQ ID NO:11);

[0103] FR-L2 LSWYQQKPGQPPRRLIY (SEQ ID NO:12);

[0104] FR-L3 TLTSGVSSRFKGSGSGTQFTLTISDVQCDDAATYYC (SEQ ID NO: 13);

[0105] FR-L4 FGGGTEVVVK (SEQ ID NO:14);

[0106] The amino acid sequence of the heavy chain variable region of antibody #230 is as follows:

[0107] QEQLEESGGGLVTPGGTLTLTCTASGIDLSSNAISWVRQAPGKGLEWIGIISAGGSTWYANWAKGRFTISKTSTTVDLKTTSPTTEDTATYFCARYPSYRSDSSLWGQGTLVTVSS (SEQ ID NO: 19)

[0108] The nucleotide sequence of the heavy chain variable region of antibody #230 is as follows:

[0109] CAGGAGCAGCTGGAGGAGTCCGGAGGAGGCCTGGTAACGCCTGGAGGAACCCTGACACTCACCTGCACAGCCTCTGGAATCGACCTCAGTAGCAATGCAATTAGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAATGGATCGGAATCATTAGTGCGGGTGGTTCCACATGGTACGCGAACTGGGCGAAAGGCCGATTCACCATCTCCAAAACCTCGACCACGGTGGATCTGAAAACCACCAGTCCGACAACCGAGGACACGGCCACTTATTTCTGCGCCAGATATCCTAGTTATAGATCTGATTCTAGCTTGTGGGGCCAAGGCACCCTGGTCACCGTCTCTTCA (SEQ ID NO:2);

[0110] The amino acid sequence of the variable region of the antibody light chain at position 230 is as follows:

[0111] AQVMTQTPSSVSAAVGGTVTISCQSSQSVFNNNFLSWYQQKPGQPPKLLIYKASTLASGVPSRFSGSGSGTQFTLTISDLECDDAATYFCAGGLPGEIYPFGGGTKVVVE (SEQ ID NO:21);

[0112] The nucleotide sequence of the variable region of the antibody light chain at position 230 is as follows:

[0113] GCCCAAGTGATGACCCAGACTCCATCGTCCGTGTCTGCAGCTGTGGGAGGCACAGTCACCATCAGTTGCCAGTCCAGTCAGAGTGTGTTTAATAACAACTTCTTATCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCCCATCGCGGTTCAGTGGCAGTGGATCTGGGACACAATTCACTCTCACCATCAGCGACCTGGAGTGTGACGATGCTGCCACTTACTTCTGTGCAGGCGGTTTGCCTGGTGAAATTTATCCTTTCGGCGGAGGGACCAAGGTGGTCGTCGAA (SEQ ID NO:22);

[0114] The amino acid sequences of the CDR and FR regions of the heavy and light chains of the 230# antibody are shown in Table 4 below.

[0115] Table 4

[0116]

[0117] 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.

[0118] After 48 hours of transfection, the antibody expression was detected by ELISA, and then the feed was added. 7 days after transfection, the cell supernatant was collected to obtain the target protein supernatant, which was then purified.

[0119] Example 3: Application of the kit for preparing the CP recombinant rabbit monoclonal antibody of the present invention

[0120] The CP 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 in conjunction with other components to detect antigens in samples. Specific performance evaluations are as follows:

[0121] 1. Specificity determination:

[0122] Using 112# antibody (antibody to CP positions 1-9, a known antibody) for coating, 270# antibody and 230# (antibody to CP positions 26-32aa) for labeling, and commercially available kits, the magnetic microparticle method was used to detect a clinical sample and proinsulin 340 (its actual concentration was 10 ng / ml, and C-PS0 was used as a blank control). The signal results are shown in Table 5.

[0123] Among them, the heavy chain amino acid sequence of antibody #112 is:

[0124] QSVKESGGRLVTPGGTLTLTCTASGFSLSRYWMSWVRQAPGKGLEWIGAIGVSSRQYYANWAKSRATITRNTNLNTVTLEMTSLTAADTATYFCARRFVGFSLNRWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:5);

[0125] The amino acid sequence of the light chain is:

[0126] DVVMTQTPSSVSATLGGTVTINCQTSRSISNYLIWYQQKPGQPPKLLIYGASKLASGVPSRFRGSGSGTEFTLTISDMKAEDAATYYCQSGYYSAGATFGAGTKVEIKRDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDITVTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCEVVQGSASPIVQSFNRGDC (SEQ ID NO:27).

[0127] Table 5

[0128]

[0129] The detected concentrations calculated according to the calibrator are shown in Table 6 below.

[0130] Table 6

[0131]

[0132] Table 5 shows that antibodies 270# and 230#, respectively, combined with 112#, can detect CP in the positive control, and the signal value can reach the level of commercially available kits, and the signal value of the reaction with 10 ng / mL proinsulin 340 is weak; Table 6 shows that the detection rates with 10 ng / mL proinsulin 340 are 0.03 ng / mL and 0.01 ng / mL, respectively, indicating that the cross-reaction rate of the CP recombinant rabbit monoclonal antibody provided by the present invention with 10 ng / mL proinsulin 340 is lower than the detection standard of 0.25 ng / mL, indicating that the two antibodies have good specificity.

[0133] 2. Accuracy measurement and analysis:

[0134] 394 clinical samples were selected using the 270# (enzyme conjugate) and 112# (magnetic beads) combination kit and the 230# (enzyme conjugate) and 112# (magnetic beads) combination kit, and mainstream commercial kits were also used for testing. Correlation analysis was performed with the reference manufacturer, and the results are shown in Figure 1 and Figure 2 .

[0135] According to the test results, the linear equation obtained for the 270# antibody is: y = 0.9862x +0.1007, and the correlation coefficient R 2 = 0.9798; the linear equation obtained for antibody 230# is: y = 1.0024x +0.0024, and the correlation coefficient R 2 = 0.9903, indicating that the combination of the CP recombinant rabbit monoclonal antibody 270# and its paired antibody 112# provided by the present invention has a high accuracy in detecting clinical samples of stimulating C-peptide.

[0136] 3. Affinity determination analysis:

[0137] CP recombinant rabbit monoclonal antibody 230# and 270# (CP, 26-32 aa site antibody) were serially diluted to four concentrations, and affinity was measured using Biacore 2000. The affinity of 230# antibody was KD=9.86E-08, and the affinity of 270# antibody was KD=9.86E-09, indicating that the affinity of the CP recombinant rabbit monoclonal antibody 270# provided by the present invention reached a relatively high level.

[0138] 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.

[0139] 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. C-peptide recombinant rabbit monoclonal antibody, characterized in that The amino acid sequences of the three CDR regions of its heavy chain are GFSFSSYT, IASSGNI, and GGTDTSL; The amino acid sequences of the three CDRs of its light chain are QSVYSNR, QVS and LGSYDCRSVDCMA, respectively.

2. The C-peptide recombinant rabbit monoclonal antibody according to claim 1, 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.

3. The C-peptide recombinant rabbit monoclonal antibody according to claim 2, characterized in that: The constant region of the heavy chain is of rabbit IgG subtype; the constant region of the light chain is of κ1 type.

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

5. The biomaterial according to claim 4, wherein The nucleic acid encoding the heavy chain variable region has the nucleotide sequence shown in SEQ ID NO: 17; The nucleic acid encoding the light chain variable region has the nucleotide sequence shown in SEQ ID NO:

18.

6. The method for preparing the C-peptide recombinant rabbit monoclonal antibody according to any one of claims 1 to 3, characterized in that: include: Cultivate the host cell according to claim 4 to induce the expression of the C-peptide recombinant rabbit monoclonal antibody.

7. Use of at least one of the following items i to iii in the preparation of a reagent or kit for detecting diabetes: i. The C-peptide recombinant rabbit monoclonal antibody according to any one of claims 1 to 3; ii. The biomaterial according to claim 4 or 5; iii. Prepare C-peptide recombinant rabbit monoclonal antibody by the preparation method according to claim 6.

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

1. The C-peptide recombinant rabbit monoclonal antibody according to any one of claims 1 to 3; ②. The biomaterial according to claim 4 or 5; ③. The C-peptide recombinant rabbit monoclonal antibody is prepared by the preparation method described in claim 6.

9. A double antibody sandwich detection kit, characterized in that: The invention comprises the C-peptide recombinant rabbit monoclonal antibody according to any one of claims 1 to 3, and a 112# antibody that specifically cooperates with the C-peptide recombinant rabbit monoclonal antibody, wherein the amino acid sequence of the heavy chain of the 112# antibody is shown in SEQ ID NO: 5, and the light chain amino acid sequence of the 112# antibody is shown in SEQ ID NO: 27.

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