CN109705221B - C peptide immunogen, monoclonal antibody pair thereof and application of antibody pair in C peptide magnetic particle chemiluminescence immunoassay reagent - Google Patents
C peptide immunogen, monoclonal antibody pair thereof and application of antibody pair in C peptide magnetic particle chemiluminescence immunoassay reagent Download PDFInfo
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- CN109705221B CN109705221B CN201811610541.0A CN201811610541A CN109705221B CN 109705221 B CN109705221 B CN 109705221B CN 201811610541 A CN201811610541 A CN 201811610541A CN 109705221 B CN109705221 B CN 109705221B
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Abstract
The invention provides a C peptide immunogen, which comprises a first complete antigen and a second complete antigen, wherein the first complete antigen is obtained by coupling a C peptide N-terminal amino acid sequence with a carrier protein, and the second complete antigen is obtained by coupling a C peptide C-terminal amino acid sequence with the carrier protein; the N-terminal amino acid sequence of the C peptide of the first complete antigen is a sequence from 1 to 12 bits in the full-length sequence of the C peptide, and the C-terminal amino acid sequence of the C peptide of the second complete antigen is a sequence from 18 to 31 bits in the full-length sequence of the C peptide. The antigen can be obtained by the existing synthetic method and is an available complete antigen, the preparation method is simple, and the obtained complete antigen has a stable structure; the monoclonal antibody pair obtained by animal immunization can be respectively and specifically combined with the N end and the C end of the C peptide, and the combination sites are far away, so that the situation that pairing cannot be carried out due to steric hindrance is avoided, and the requirements of developing C peptide magnetic particle chemiluminescence immunodiagnosis reagents and other immunological diagnosis reagents on the antibodies can be met.
Description
Technical Field
The invention relates to the technical field of immunodiagnosis, in particular to a C peptide immunogen and a monoclonal antibody pair thereof.
Background
The C peptide is a secretion product of islet cells and has a common precursor proinsulin with insulin, and as shown in FIG. 5, one molecule of proinsulin is cleaved into one molecule of insulin and one molecule of C peptide after enzyme digestion. C peptide is a straight-chain polypeptide consisting of 31 amino acids, has a slow clearance rate in vivo, is hardly taken up by the liver and is not influenced by exogenous insulin, and can reflect the level of endogenous insulin.
C-peptide detection can assist in determining diabetes typing; the C peptide detection is not interfered by the islet antibody, and the islet function of the insulin treatment patient can be judged; can identify the cause of hypoglycemia, and is helpful for diagnosis of islet cell tumor and judgment of operation effect; liver and kidney diseases can also be judged according to the ratio of C peptide to insulin in blood.
The currently and commonly used C peptide detection methods mainly comprise radioimmunoassay, enzyme-linked immunoassay, chemiluminescence immunoassay and the like. These techniques are based on the specific interaction of antigen and antibody, the specific antibody pair being the core material for these detection methods.
The C peptide is a straight-chain polypeptide consisting of 31 amino acids and belongs to a small molecule hapten. Firstly, the C peptide has low immunogenicity, and needs to be linked with macromolecules to synthesize complete antigens in the preparation of antibodies, common methods such as methods disclosed in Chinese patents CN201210555678.7 and CN201810058354.X all adopt fusion expression protein methods, the methods need gene synthesis, vector construction, prokaryotic expression, purification and the like, the operation is complex, and the structure of target protein is easily changed by carrier protein in the fusion protein. Secondly, the C peptide has only 31 amino acids, the middle part of the peptide chain has dominant epitopes, and the binding sites of the monoclonal antibody obtained by adopting the complete molecule and the antigen are mostly concentrated in the middle region, so that the monoclonal antibody can not be paired due to steric hindrance, and the application of the monoclonal antibody in the development of diagnostic reagents is influenced.
Disclosure of Invention
In order to solve the technical problems, the invention provides a C peptide immunogen which is easy to prepare and convenient for obtaining a monoclonal antibody pair.
The technical scheme of the invention is to provide a C peptide immunogen, which comprises a first complete antigen and a second complete antigen, wherein the first complete antigen is obtained by coupling a C peptide N-terminal amino acid sequence with a carrier protein, the second complete antigen is obtained by coupling a C peptide C-terminal amino acid sequence with a carrier protein, and the carrier protein of the first complete antigen and the carrier protein of the second complete antigen can be the same or different; the N-terminal amino acid sequence of the C peptide of the first complete antigen is a sequence from 1 to 12 bits in the full-length sequence of the C peptide, and the C-terminal amino acid sequence of the C peptide of the second complete antigen is a sequence from 18 to 31 bits in the full-length sequence of the C peptide.
The invention also provides a monoclonal antibody pair obtained by an animal immunization method by using the antigen.
The invention also aims to provide the application of the monoclonal antibody in the chemiluminescence immunoassay reagent of the C peptide magnetic particles.
The carrier protein includes Bovine Serum Albumin (BSA), Ovalbumin (OVA), and hemocyanin (KLH).
The carrier proteins in the first complete antigen and the second complete antigen are the same.
The N-terminal amino acid sequence of the C peptide in the first complete antigen is provided with a linking group GGC connected with a carrier protein, the specific sequence is shown as SEQ ID NO. 1, and the amino acid sequence shown as SEQ ID NO. 1: EAEDLQVGQVELGGC, the C-terminal amino acid sequence of the second complete antigen has a linking group CGG connected with carrier protein, the specific sequence is shown as SEQ ID NO. 2, SEQ ID NO. 2: CGGAGSLQPLALEGSLQ are provided. Wherein the linker group GGC or CGG provides a linker arm and provides a thiol group when coupled to a carrier protein.
The 14 short peptides for detecting the binding sites of the monoclonal antibody pair have the sequences shown in SEQ ID NO. 3-SEQ ID NO. 16.
SEQ ID NO:3：EAEDLQVGQVELGGGPG
SEQ ID NO:4：EAEDLQVGQVELGG
SEQ ID NO:5：EAEDLQVGQVEL
SEQ ID NO:6：EAEDLQVGQV
SEQ ID NO:7：EDLQVGQVELGGGPG
SEQ ID NO:8：LQVGQVELGGGPG
SEQ ID NO:9：VGQVELGGGPG
SEQ ID NO:10：GGPGAGSLQPLALEGSLQ
SEQ ID NO:11：GGPGAGSLQPLALEGS
SEQ ID NO:12：GGPGAGSLQPLALE
SEQ ID NO:13：GGPGAGSLQPLA
SEQ ID NO:14：PGAGSLQPLALEGSLQ
SEQ ID NO:15：AGSLQPLALEGSLQ
SEQ ID NO:16：SLQPLALEGSLQ
The invention has the advantages and beneficial effects that: the amino acid sequence can be obtained by the existing synthetic method, and can be coupled with carrier protein to form complete antigen, and the preparation method is simple; the monoclonal antibody pair obtained by animal immunization can be respectively and specifically bound with the N end and the C end of the C peptide, and the binding sites are far away, so that the situation that pairing cannot be carried out due to steric hindrance is avoided, the problem that the antibody pair with the far binding sites is difficult to obtain due to the dominant epitope in the middle of the C peptide when the full-length sequence of the C peptide is adopted is avoided, and the requirements of developing C peptide magnetic particle chemiluminescence immunodiagnosis reagents and other immunological diagnosis reagents on antibodies can be met.
Drawings
FIG. 1 shows the results of the detection of the binding site of the N-terminal monoclonal antibody and the full-length antigen of the C-peptide of the present invention.
FIG. 2 shows the results of the detection of the binding site of the C-terminal monoclonal antibody and the C-peptide full-length antigen.
FIG. 3 is a linear fit of a pair of monoclonal antibodies of the invention.
FIG. 4 is a graph showing the correlation test between the monoclonal antibody of the present invention and an Elecsys C-Peptide diagnostic kit.
FIG. 5 is a schematic representation of the amino acid sequence composition of proinsulin.
Detailed Description
The present invention will be further described with reference to the following embodiments.
Example 1
Polypeptide synthesis
The full-length sequence (1-31aa) EAEDLQVGQVELGGGPGAGSLQPLALEGSLQC (SEQ ID NO:17), the N-terminal amino acid sequence (1-12aa) EAEDLQVGQVELGGC, the C-terminal amino acid sequence (18-31aa) CGGAGSLQPLALEGSLQ, and GGC or CGG in the N-terminal amino acid sequence or the C-terminal amino acid sequence are synthesized respectively to provide a connecting arm and a sulfhydryl group when coupled with a carrier protein. In addition, short peptides EAEDLQVGQVELGGGPG (1-17aa), EAEDLQVGQVELGG (1-14aa), EAEDLQVGQVEL (1-12aa), EAEDLQVGQV (1-10aa), EDLQVGQVELGGGPG (3-17aa), LQVGQVELGGGPG (5-7aa), VGQVELGGGPG (7-17aa), GGPGAGSLQPLALEGSLQ (14-31aa), GGPGAGSLQPLALEGS (14-29aa), GGPGAGSLQPLALE (1-27aa), GGPGAGSLQPLA (14-25aa), PGAGSLQPLALEGSLQ (16-31aa), 13AGSLQPLALEGSLQ (18-31aa), 14SLQPLALEGSLQ (20-31aa) were synthesized.
Synthesis of the second, first and second complete antigens
Weighing 3mg BSA and dissolving in 600ul PBS pH7.4; weighing 0.5mg of sulf-SMCC, and dissolving in 100ul of PBS; then slowly dripping the dissolved SMCC solution into the BSA solution while lightly mixing, and then reacting for 0.5 hour at room temperature; adding the solution into a dialysis bag, dialyzing with PBS pH7.4, removing redundant SMCC, changing 3 times of dialysate every 2 hours, slowly dripping the dissolved 4mg of N-terminal amino acid sequence or C-terminal amino acid sequence into the half-conjugate while uniformly mixing, reacting at room temperature for 2 hours, and directly subpackaging and freezing after measuring the concentration.
Example 2
First, animal immunization
Strong female Balb/c mice, 6-8 weeks old, were selected for immunization. The first immunization adopts Freund's complete adjuvant, 100 mug/mouse and multi-point injection under the armpit; boosting, adopting Freund incomplete adjuvant, 50 mug/mouse, boosting 3 times, the boosting interval time of adjacent times is 2 weeks; the first immunization was 3 weeks apart from the first booster immunization. Blood is collected by eyeballs 1 week after the last boosting immunization, the enzyme label plate is coated by the C peptide full-length antigen, and the antiserum titer is measured by an indirect ELISA method. 3 days before fusion, 100 mu g antigen/mouse intraperitoneal injection impact
Cell fusion and monoclonal antibody preparation
Mice that had been previously impacted were sacrificed by cervical dislocation and sterilized by immersion in 75% ethanol. In a clean bench, the spleens of mice were aseptically removed, squeezed with a sterilized ground glass slide, and lightly ground to isolate splenocytes, which were counted. The SP2/0 cells and splenocytes were mixed at a ratio of 1:1, centrifuged at 300g for 5 minutes at room temperature, and the supernatant was discarded. 1ml of PEG was added and the PEG and cells were mixed well and homogeneously, this was completed within 3 minutes. 50ml of fresh DMEM medium is added, the mixture is centrifuged at room temperature and 300g for 5 minutes, and HAT medium is resuspended and gently mixed. 100 ul/well was added to a 96-well plate and placed in an incubator for culture.
HT medium was changed when cells grew to 1/3 basal area in 96-well plates, C peptide full-length antigen was coated, and positive clones were detected by indirect ELISA. After 3-4 times of subcloning, the amplification culture is ensured when the monoclonal is formed, and ascites cells are injected and the antibody is purified.
Example 3
C peptide full-length antigen is coated on a 96-well plate, the coating concentration is 2ug/ml, and the temperature is 4 ℃ for 2 hours; 1% casein was blocked at 37 ℃ for 1 small test, and washed three times with PBS pH7.4; respectively diluting synthetic short peptides SEQ ID NO 3-16 to 15ug/ml, and respectively adding 50 ul/hole; monoclonal antibody 50 ul/well, 1ug/ml, reaction at 37 ℃ for 1 hour, PBS pH7.4 washing three times; adding a diluted goat anti-mouse secondary antibody at a ratio of 1:2000, 100 ul/hole, reacting at 37 ℃ for 1 hour, and washing with PBS (phosphate buffer solution) pH7.4 for four times; the reaction was terminated after development for 5 minutes. Reading data and analyzing, wherein all short peptide sequence consensus sequences which can compete for reaction are the monoclonal antibody binding sites.
As shown in FIG. 1, the N-terminal monoclonal antibody can react with short peptides SEQ ID NO. 3 to 5 and SEQ ID NO. 7 to 8, and the amino acids at positions 5-12 of the consensus sequence are the binding site of the N-terminal monoclonal antibody; as shown in FIG. 2, the C-terminal monoclonal antibody can react with the short peptides SEQ ID NO 10 to 12 and SEQ ID NO 14 to 16, and the consensus sequence thereof is amino acids 20 to 27, i.e., the binding site of the C-terminal monoclonal antibody.
Example 4 application of peptide C monoclonal antibody in magnetic particle chemiluminescence immunodiagnostic reagent
Firstly, preparation of luminescent reagent
Taking about 0.1mgC peptide C-terminal or N-terminal monoclonal antibody, adding acridinium ester label according to the molar ratio of 1:5-1:15, uniformly mixing at room temperature in the dark for reaction for 2h, dialyzing and desalting PB buffer solution in the dark, wherein the volume of dialysate is 20 times larger than that of the label, changing the dialysate every 4 hours for at least 3 times, measuring the concentration, and diluting to 1-10 mug/mL by PBS-BSA for later use.
Preparation of two, solid phase reagents
Taking about 0.1mgC peptide N-terminal or C-terminal monoclonal antibody, adding biotin label according to the molar ratio of 1:5-1:15, mixing uniformly at room temperature for 2h, dialyzing and desalting with 20mM PB buffer solution, changing the volume of the dialysate which is 20 times larger than the volume of the label once every 4 hours, changing the dialysate at least 3 times, and diluting with PBS-BSA to 0.1-1mg/mL for later use after measuring the concentration.
And washing PB (magnetic bead) by about 0.5mL for three times, diluting to 1-10mg/mL, adding a biotin labeled antibody in an equal volume, uniformly mixing at room temperature for 2h, washing with PBS-BSA for three times, and diluting to 0.1-1mg/mL for later use.
Third, sample and calibrator detection
Diluting the C Peptide full-length antigen in a gradient manner, carrying out detection assignment by using an Elecsys C-Peptide diagnostic kit, selecting 5 points in the range of 0.1-20ng/mL as calibration points, and detecting a calibration CL120 chemiluminescence immunoassay by using a self-prepared luminescent reagent and a solid-phase reagent, wherein the test conditions are as follows: one-step method, 100. mu.L of luminescent reagent, 50. mu.L of solid phase reagent, 10. mu.L of sample (i.e., C peptide full length antigen), incubated at 37 ℃ for 15 min.
And (3) lowest detection line: taking 5% bovine serum as a blank sample, continuously measuring for 25 times, and testing conditions are as follows: one-step method, 100. mu.L luminescence reagent, 50. mu.L solid phase reagent, 10. mu.L sample (5% bovine serum blank sample), and incubation at 37 ℃ for 15 min. The test results were recorded, and the average value av, the standard deviation sd, and the lowest detection line LOB ═ av +2sd were calculated, respectively, and the results are shown in table 1, and the monoclonal antibody reached 0.04ng/ml for the lowest detection line.
Linear range: taking 20-25ng/mL clinical positive serum, diluting the clinical positive serum into 10 gradient samples by a 5% bovine serum gradient, and simultaneously detecting the 10 gradient samples and a blank sample by using the luminescent reagent and the solid phase reagent under the following test conditions: one step method, 100. mu.L luminescent reagent, 50. mu.L solid phaseReagent, 10. mu.L of sample (i.e., clinical positive serum), incubated at 37 ℃ for 15 min. Recording the test result, performing linear fitting and calculating R2Values, see FIG. 3, monoclonal antibody vs. the conjugated luminescent reagent R20.998, with a linear range of 0ng/ml to 21 ng/ml being preferred.
Correlation: 50 clinical positive serum samples are collected, the theoretical value covers the linear range of 0.1-20ng/mL, and the samples are tested by using a Roche Elecsys C-Peptide diagnostic kit and an electrochemiluminescence analyzer according to the conditions of the instruction. The same positive sample is detected by the self-prepared luminescent reagent and the solid phase reagent, and the test conditions are as follows: one step, 100. mu.L of luminescent reagent, 50. mu.L of solid phase reagent, 10. mu.L of sample, incubated at 37 ℃ for 15 min. Recording the test result, and calculating the linear fitting correlation curve equation and correlation coefficient R of the self-prepared reagent and the test result of the kit A2Referring to FIG. 4, the curve of linear fitting between the self-contained luminescent reagent (containing the monoclonal antibody of the present invention) and the Elecsys C-Peptide diagnostic kit is 1.068X-0.025, R2When the ratio is 0.997, the self-prepared luminescent reagent has better correlation with the value measured by an Elecsys C-Peptide diagnostic kit.
Materials, reagents and experimental equipment related to the embodiment of the invention are all commercial products conforming to the field of biological and pharmaceutical products unless specified otherwise.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, modifications and decorations can be made without departing from the core technology of the present invention, and these modifications and decorations shall also fall within the protection scope of the present invention. Any changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Sequence listing
<110> Meikang Biotechnology Ltd
<120> C peptide immunogen and monoclonal antibody pair thereof and application of antibody pair in C peptide magnetic particle chemiluminescence immunoassay reagent
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Claims (4)
1. A C peptide immunogen is characterized by comprising a first complete antigen and a second complete antigen, wherein the first complete antigen is obtained by coupling a C peptide N-terminal amino acid sequence with a carrier protein, the second complete antigen is obtained by coupling a C peptide C-terminal amino acid sequence with a carrier protein, and the carrier protein of the first complete antigen is the same as or different from the carrier protein of the second complete antigen; the N-terminal amino acid sequence of the C peptide of the first complete antigen is a sequence from 1 to 12 bits in the full-length sequence of the C peptide, and the C-terminal amino acid sequence of the C peptide of the second complete antigen is a sequence from 18 to 31 bits in the full-length sequence of the C peptide;
the leucine residue of the C-terminal amino acid sequence of the first complete antigen has a linker GGC connected with the carrier protein, as shown in SEQ ID NO:1, and the alanine residue of the C-terminal amino acid sequence of the second complete antigen has a linker CGG connected with the carrier protein, as shown in SEQ ID NO: 2.
2. Use of a monoclonal antibody produced from a C-peptide immunogen of claim 1 in the production of a C-peptide magnetic particle chemiluminescent immunoassay reagent.
3. The peptide C immunogen of claim 1, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin.
4. The C-peptide immunogen of claim 1, wherein the carrier protein in the first complete antigen and the second complete antigen are the same.
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| GB9716790D0 (en) * | 1997-08-07 | 1997-10-15 | Creative Peptides Sweden Ab | Recombinant DNA molecules comprising multimeric copies of a gene sequence and expression thereof |
| CN1219548C (en) * | 2001-05-18 | 2005-09-21 | 上海益众生物技术有限公司 | Join of C peptide and insulin for treating diabetes complication and C peptide-specific antibody |
| GB0218970D0 (en) * | 2002-08-14 | 2002-09-25 | Creative Peptides Sweden Ab | Fragments of Insulin c-peptide |
| EP2365334A1 (en) * | 2005-11-14 | 2011-09-14 | MetaMol Theranostics, LLC | Peptide sequence that promotes tumor invasion |
| CN102680713B (en) * | 2012-06-18 | 2014-07-23 | 王钊 | Competitive latex-particle-enhanced immunoturbidimetric assay kit for C-peptide and preparation method thereof |
| CN103074302B (en) * | 2012-12-19 | 2015-01-21 | 北京利德曼生化股份有限公司 | Anti-C-peptide monoclonal antibody and use thereof in detection of C-peptide content |
| CN105601750B (en) * | 2016-01-22 | 2019-05-31 | 美康生物科技股份有限公司 | A kind of gene recombinant human C peptide fusion protein and the preparation method and application thereof |
| CN108219002B (en) * | 2018-01-22 | 2021-05-14 | 三诺生物传感股份有限公司 | Recombinant C peptide immunogen and application thereof |
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