CN117447578A - Detection kit for ribosomal protein S26 antibody - Google Patents
Detection kit for ribosomal protein S26 antibody Download PDFInfo
- Publication number
- CN117447578A CN117447578A CN202311164315.5A CN202311164315A CN117447578A CN 117447578 A CN117447578 A CN 117447578A CN 202311164315 A CN202311164315 A CN 202311164315A CN 117447578 A CN117447578 A CN 117447578A
- Authority
- CN
- China
- Prior art keywords
- protein
- sr12s
- antigen
- radioactive
- rps26a
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 101000862491 Homo sapiens 40S ribosomal protein S26 Proteins 0.000 title claims abstract description 20
- 102100027337 40S ribosomal protein S26 Human genes 0.000 title claims abstract description 19
- 108091007433 antigens Proteins 0.000 claims abstract description 43
- 102000036639 antigens Human genes 0.000 claims abstract description 43
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 34
- 230000002285 radioactive effect Effects 0.000 claims abstract description 30
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 229920000936 Agarose Polymers 0.000 claims abstract description 9
- 239000006180 TBST buffer Substances 0.000 claims abstract description 8
- 101710120037 Toxin CcdB Proteins 0.000 claims abstract description 4
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 10
- 229930182817 methionine Natural products 0.000 claims description 10
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 6
- 150000001413 amino acids Chemical group 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 2
- 239000013641 positive control Substances 0.000 claims description 2
- 101150102970 RPS26A gene Proteins 0.000 abstract description 29
- 101100200729 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) RPS21A gene Proteins 0.000 abstract description 29
- 101100040012 Schizosaccharomyces pombe (strain 972 / ATCC 24843) rps2601 gene Proteins 0.000 abstract description 29
- 239000000427 antigen Substances 0.000 abstract description 22
- 239000013612 plasmid Substances 0.000 abstract description 14
- 239000002287 radioligand Substances 0.000 abstract description 13
- 238000000034 method Methods 0.000 abstract description 10
- 238000013518 transcription Methods 0.000 abstract description 9
- 230000035897 transcription Effects 0.000 abstract description 9
- 101150055163 RPS26 gene Proteins 0.000 abstract description 8
- 238000013519 translation Methods 0.000 abstract description 8
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 abstract description 5
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 abstract description 5
- 101710204410 Scaffold protein Proteins 0.000 abstract description 5
- 239000000243 solution Substances 0.000 abstract description 4
- 230000006872 improvement Effects 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 17
- 230000014509 gene expression Effects 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 9
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 206010012601 diabetes mellitus Diseases 0.000 description 6
- 238000003745 diagnosis Methods 0.000 description 6
- 238000003908 quality control method Methods 0.000 description 6
- 102210042925 HLA-A*02:01 Human genes 0.000 description 5
- 101001024703 Homo sapiens Nck-associated protein 5 Proteins 0.000 description 5
- 102100036946 Nck-associated protein 5 Human genes 0.000 description 5
- 239000002033 PVDF binder Substances 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 5
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 108020004705 Codon Proteins 0.000 description 3
- 108010000605 Ribosomal Proteins Proteins 0.000 description 3
- 102000002278 Ribosomal Proteins Human genes 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000006957 competitive inhibition Effects 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 102000008214 Glutamate decarboxylase Human genes 0.000 description 2
- 108091022930 Glutamate decarboxylase Proteins 0.000 description 2
- 241000282376 Panthera tigris Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 210000004153 islets of langerhan Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 102220468481 Annexin A2_S26E_mutation Human genes 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 241000208152 Geranium Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000287501 Phoenicopterus ruber Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 101150039863 Rich gene Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004248 Zinc Transporter 8 Human genes 0.000 description 1
- 108090000702 Zinc Transporter 8 Proteins 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000001610 euglycemic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 238000007410 oral glucose tolerance test Methods 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 210000004923 pancreatic tissue Anatomy 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a detection kit of a ribosomal protein S26 antibody, which comprises a radioactive antigen protein SR12S, a TBST buffer solution, protein A agarose and protein G agarose. According to the invention, two sections of RPS26 gene sequences are connected to two sides of a scaffold protein R12 through hinge genes to construct corresponding plasmids, the radioactive antigen protein SR12S obtained after transcription and translation can effectively carry radioactive signals, each protein contains two RPS26 antigen structural domains, and the capacity of capturing antibodies is greatly improved. The invention successfully establishes the detection of RPS26A by the first radioligand method based on the improvement.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a detection kit for a ribosomal protein S26 antibody.
Background
Type 1diabetes (type 1diabetes mellitus,T1DM) is one of diabetes, also known as insulin dependent diabetes mellitus, and patient pathology usually occurs in childhood or adolescence, but also occurs in any age group, including adult onset. The disease acts as an autoimmune disease, the autoimmune system attacks on the beta cells that produce insulin in the body, eventually resulting in the inability to produce insulin in the body, and the patient needs to inject exogenous insulin to control blood glucose in the body. At present, diagnosis of T1DM mainly depends on two aspects of clinic and immunity, namely, diagnosis of the immunity, namely, detection of islet autoantibodies, wherein positive islet autoantibodies are gold standards for determining the diagnosis of T1 DM. Currently known putative islet autoantibodies include ZnT8A (zinc transporter 8 auto-antibody), GADA (glutamic acid decarboxylase autoantibody, glutamate decarboxylase antibody), ICA (Islet cell antigen autoantibody, islet cell antibody), IA-2A (protein phosphatase-like IA-2 auto-antibody), and IAA (insmu Lin autoantibodies, insulin autoantibody). Because of the limitations of experimental conditions, unknown islet autoantibodies cannot be widely developed clinically at present and no mature kit exists, so that the possibility of missed diagnosis of T1DM is unavoidable depending on the current autoantibodies.
Ribosomal protein S26 (Ribosomal Protein S, RPS 26) encodes a ribosomal protein as a type 1diabetes inheritance susceptibility gene, which is a component of the 40S subunit, belonging to the S26E family of ribosomal proteins. The inventors have previously found through a letter analysis (as shown in fig. 1 and 2) that the ribosome presents a T1DM islet antigen-specific epitope polypeptide, suggesting that T1DM patients present autoantibodies against the protease. However, there is no detection method for the RPS26 autoantibody (RPS 26A).
Disclosure of Invention
It is an object of the present invention to provide a radioactive antigen protein SR12S. The radioactive antigen protein SR12S adopts S 35 The antigen protein SR12S is marked by methionine, and the amino acid sequence of the antigen protein SR12S is shown as SEQ ID NO. 1.
In one embodiment of the present invention, the gene of interest (two RPS26 gene sequences linked to both sides of the scaffold protein R12 gene by a hinge gene) is introduced into the vector pTnT TM Vector construction gave an SR12S plasmid, which was then translated by rapid transcription to obtain radiolabeled antigenic protein. Specifically, the plasmid structure includes: xhoI (ctcgag) +kozak sequence (gccac) +ATG+SR12S sequence of interest+stop codon+XbaI. Each of the radioactive antigen proteins SR12S obtained after transcription and translation comprises two RPS26 protein antigen domains, the middle of which is connected through a hinge protein (as shown in fig. 3), the ability to carry radioactive signals is effectively doubled, and the ability to capture antibodies is greatly improved by the two antigen domains.
The second object of the present invention is to provide a kit for detecting ribosomal protein S26 antibody.
The detection kit comprises: the above radioactive antigen proteins SR12S, TBST buffer, protein A agarose and protein G agarose.
Further, the detection kit also comprises a positive control and a negative control.
The invention also aims to provide an application of the detection kit in preparing a type 1diabetes diagnosis reagent.
According to the invention, two sections of RPS26 gene sequences are connected to two sides of a scaffold protein R12 through hinge genes to construct corresponding plasmids, the radioactive antigen protein SR12S obtained after transcription and translation can effectively carry radioactive signals, each protein contains two RPS26 antigen structural domains, and the capacity of capturing antibodies is greatly improved. The invention successfully establishes the detection of RPS26A by the first radioligand method based on the improvement.
Drawings
FIG. 1 shows the results of the expression profile analysis of 56 human tissues and human islets in the GTEx database and the Tiger database.
FIG. 2 shows the analysis results of the expression level of RPS26 gene by nPOD database.
FIG. 3 is a protein pattern diagram of the radioactive antigen protein SR12S of the present invention.
Fig. 4 is a graph of radiation readings detected by RPS26A at various simulated sample additions.
FIG. 5 shows the result of RPS26A threshold determination for healthy people.
Fig. 6 is a distribution of RPS26A in T1DM patients, T2DM patients, and healthy people.
FIG. 7 shows the results of a radioligand assay to detect competitive inhibition of RPS 26A.
Detailed Description
Since the detection of autoantibodies has extremely high reference significance for the diagnosis of clinical T1DM, the establishment of the detection of RPS26A has important significance for subsequent further studies. The conventional detection method has the problems of narrow detection range and poor sensitivity in the detection of the RPS 26A. Inventor laboratory owned S 35 Radioligand assay platform by adding radioactive amino acid raw material S during transcription and translation of plasmid 35 The labeled methionine is used to obtain radioactive protein antigen, and the principle of in-situ amino acid replacement does not change the spatial structure of any protein, so that the result has high specificity, wide detection range and high sensitivity. However, the application premise of the detection platform is that the protein sequence to be translated contains a certain content of methionine sequence and can be used for replacing radioactive methionine, and through the structural evaluation of the protein of the RPS26 by the inventor, the protein sequence does not contain methionine at all and cannot carry any radioactive signal.
To solve this problem, the inventors found 43 methionine-rich gene sequences of different animal origin by screening the gene library, and then calculated the methionine sequence content, excluding 25 sequences of lower content; then, the remaining 18 were plasmid synthesized and transcribed and translated, the 17 sequences failed in transcription and translation and cross-reacting with serum of a human healthy person were deleted, and the remaining last gene sequence, derived from the Geranium caribbeanum (Phoenicopterus ruber), gene name N337-12018, was methionine-rich. The inventor uses the protein as a scaffold protein (named as R12) for detecting RPS26A by a radioligand method. In order to enhance antibody antigen reaction, two sections of RPS26 gene sequences are connected to two sides of R12 through hinge genes to construct corresponding plasmids, radioactive antigen protein SR12S obtained after transcription and translation can effectively carry radioactive signals, each protein contains two RPS26 antigen domains, the capacity of capturing antibodies is greatly improved, and an antigen protein pattern diagram obtained after plasmid expression is shown in figure 3. The invention successfully establishes the detection of RPS26A by the first radioligand method based on the design.
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The expression profile analysis of 56 human tissues and human islets in the GTEx (Genotype-Tissue Expression) database and Tiger (Tumor Immunotherapy Gene Expression Resource) database shows that the RPS26 gene is used as a gene with broad-spectrum expression, and the expression abundance in the islets and pancreatic tissues is higher, as shown in figure 1.
Analysis in the diabetic pancreas donor database (the Network for Pancreatic Donors with Diabetes, nPOD) revealed that the expression level of RPS26 gene was significantly up-regulated in islet beta cell single cell sequencing of T1DM patients, as shown in fig. 2 (AAB is an autoantibody positive non-diseased population sample).
Further analysis of the bioinformatic epitope prediction (NetMHCpan 4.0) showed that the protein encoded by the gene contained a plurality of potential islet antigen-specific epitope polypeptides, suggesting that the protein encoded by the gene was a potential islet-specific autoantigen, as shown in Table 1.
TABLE 1 list of HLA-A0201 restricted epitope polypeptides
| allele | seq_num | start | end | length | peptide | score | percentile_rank |
| HLA-A*02:01 | 1 | 66 | 74 | 9 | KLYVKLHYC | 0.579451 | 0.21 |
| HLA-A*02:01 | 1 | 39 | 47 | 9 | FVIRNIVEA | 0.575528 | 0.21 |
| HLA-A*02:01 | 1 | 63 | 71 | 9 | VLPKLYVKL | 0.529093 | 0.24 |
| HLA-A*02:01 | 1 | 66 | 75 | 10 | KLYVKLHYCV | 0.330366 | 0.49 |
| HLA-A*02:01 | 1 | 62 | 71 | 10 | YVLPKLYVKL | 0.325179 | 0.5 |
It is therefore speculated that islet-specific autoantibodies against RPS26 may be present in type 1 diabetics.
Example 2
1. Experimental materials
1. Sample source: RPS26A detection positive quality control samples were from commercial antibodies purchased against RPS26 protein. Negative quality control serum specimens were obtained from healthy volunteers without a family history of diabetes. Of the Diabetes (DM) sera, 643 cases of type 1diabetes (T1 DM) serum and 71 cases of type 2 diabetes (T2 DM) serum. 382 healthy people from the recruited group [ age (33.5±5.3) years old; 190 men and 192 women ]; the sugar tolerance test (OGTT) has the advantages of fasting and 2 hours of euglycemic, and can eliminate chronic and endocrine diseases such as heart, brain, liver, kidney and the like, without the history of diabetes family diseases and autoimmune diseases.
2. Main reagents and instruments: TNT SP6 rapid transcription translation kit (L2080, promega); s is S 35 Methionine (NEG 709 A5 mci, perkinelmer); protein A agarose PA (17-5280-02, GE); protein G agarose PG (17061805, GE); 96-well PVDF plates (3504, corning); microscint-20 scintillation fluid (6031321, perkinelmer); TBST buffer (Tris-Base 2.424g,NaCl 8.70g,Tween-20 1.5mL,BSA 1.0g plus distilled water to 1000mL, pH 7.4); beta Counter liquid scintillation Counter (2450Microplate Counter,Perkin-Elmer); NAP Column (17-0853-02, GE); RPS26 (Ag 6706, proteontech); RPS26A (14909-1-AP, proteontech); plasmid vector pTnT TM Vector(L5610，Promega)。
2. Experimental method
1. Construction of SR12S plasmid: two sections of RPS26 genes are connected to two ends of a scaffold protein R12 through hinge genes, so that corresponding plasmids are constructed; each radioactive antigen protein SR12S obtained after transcription and translation comprises two RPS26 protein antigen domains, the middle is connected through a hinge protein, the capacity of carrying radioactive signals is effectively doubled, and the capacity of capturing antibodies is greatly improved by the two antigen domains.
Codon optimization according to the mammalian protein expression system (avoiding two restriction sites) is constructed in a plasmid vector pTnT TM Vector (L5610, promega) contains kozak sequences, xhoI and XbaI cleavage sites and the sequence of interest.
The plasmid structure comprises: xhoI (ctcgag) +kozak sequence (gccac) +ATG+SR12S sequence of interest+stop codon+XbaI.
The amino acid sequence of the antigen protein SR12S is shown as follows:
MTKKRRNNGRAKKGRGHVQPIRCTNCARCVPKDKAIKKFVIRNIVEAAAVRDISEASVFDA
YVLPKLYVKLHYCVSCAIHSKVVRNRSREARKDRTPPPRFRPAGAAPRPPPKPMPKPSTPP
GSSGGGSIKNILQPGSVDSQTEMVLVNAVYFKGMWEKAFKDEDTQAMPFRMTEQESTPV
QMMYQVGSFKVAEMASEKMKILELPYASGELSMLVLLPDDVSGLEEIENAITFEKLTEWTS
SSIMEERKIKVYLPRMKMEEKYNLTSVLMALGMTDLFPKPSTPPGSSGGGSMTKKRRNN
GRAKKGRGHVQPIRCTNCARCVPKDKAIKKFVIRNIVEAAAVRDISEASVFDAYVLPKLYVKLHYCVSCAIHSKVVRNRSREARKDRTPPPRFRPAGAAPRPPPKPM(SEQ ID NO.1)。
wherein the method comprises the steps ofPKPSTPPGSSGGGSIs a hinge sequence.
2. Rapidly transcribing and translating the plasmid to obtain the radiolabeled antigen
S 35 Thawing the mixture of methionine and TNT SP6, placing on ice, sequentially adding 40. Mu.L of TNT mixture, 1. Mu.L (1. Mu.g/. Mu.L) of constructed SR12S plasmid and 5. Mu. L S 35 Methionine, supplemented with 4. Mu.L nuclease-free water to a total reaction system of 50. Mu.L, after thoroughly mixing, placed in a 30℃water bath for 90min, and then removed and placed on ice to prepare NAP-5 columns.
1 NAP-5 column was taken out and placed on a test tube rack, both the upper and lower covers were opened, and after the equilibration solution was discarded, 1mL TBST buffer was added to equilibrate the NAP-5 column, and elution was performed 3 times. The reaction mixture was carefully applied to the NAP-5 column packing surface, after washing the reaction tube with 100. Mu. LTBST buffer, it was still added to the NAP-5 column, after the red liquid was slowly moved down to column 2/3, 500. Mu.L TBST buffer was added, the color change of the drop under the column was carefully observed, about 500. Mu.L of red column passing liquid was collected, 2. Mu.L of column passing antigen was taken out from this column passing liquid and mixed with 1mL of scintillation liquid in the scintillation tube, and the number of pulses per minute (CPM) was counted on a 96 well. Beta. Counter scintillation Counter.
3. Binding and detection of test sample and antigen
Each well is added with 5 mu L of sample serum or simulated sample, each sample and quality control serum are double-well, a proper amount of labeled antigen is taken, the labeled antigen is diluted to 20000CPM/60 mu L by using 6mL of TBST buffer solution, 60 mu L of diluted labeled antigen is added to each well, the CPM value of each well is required to be more than or equal to 20000, the labeled antigen and the serum are uniformly mixed and oscillated for 1 hour, and the mixture is stored in a refrigerator at 4 ℃ overnight. PVDF plates were incubated at 150. Mu. LTBST/well overnight in a refrigerator at 4 ℃. The next day, the liquid in PVDF plate is poured out, 25 mu L of protein A/G mixed agarose (62.5% PA and 20% PG are prepared according to 4:1) is added into each hole, 50 mu L of mixed liquid is sequentially taken out from each hole of a 96-hole plate and transferred onto a 96-hole PVDF filter plate, after the mixed liquid is uniformly mixed in a refrigerator at 4 ℃ for 1h, antigen-antibody complex is taken out, liquid is pumped out by a vacuum pump, 200 mu L of TBST buffer solution is firstly added into each hole of the PVDF filter plate to wash the precipitate, the liquid is pumped out by a vacuum pump to leave the precipitate, 150 mu L of buffer solution is added to repeatedly wash for 7 times, 60 mu L of scintillation liquid is added into each hole after the mixture is dried by an oven, and the mixture is counted on a 96-hole beta Counter for 1min.
Calculated as follows:
radioactivity Index (Index) = (specimen serum CPM-negative quality control CPM)/(positive quality control CPM-negative quality control CPM).
All data were counted using SPASS26 software, and the mean ± standard deviation for all data were normalized to the normal distributionThe expression is that single factor analysis of variance, analysis of variance trend test and the like are adopted. P (P)<0.05 is significant in difference and has statistical significance.
3. Experimental results
1. Validity judgment of capturing RPS26A by radioactive antigen protein SR12S
The SR12S plasmid was transcribed and translated to obtain SR12S antigen with radioactive signal, and the purchased RPS26A was used as a simulated sample, and the transcribed and translated radioactive SR12S antigen was used for capturing, and the radioactive readings at different simulated sample amounts were detected according to the above-described experimental method.
As shown in fig. 4, the radioactivity reading CPM decreases with decreasing loading of the simulated sample, and the trend is extremely pronounced through analysis of variance trend test P < 0.0001. Thus, the transcribed and translated SR12S labeled antigen has biological activity and can effectively capture RPS26A, and the carried radioactive signal changes along with the change of the antibody content.
In the subsequent experiments, the purchased antibody was used as a positive reference, and 0.6. Mu.g of antibody was selected and CPM about 5178 was used as the positive reference loading according to the signal to noise ratio (S/N) >15 (S/N: CPM value of different RPS26A loading amounts/CPM value of negative reference) and cost comprehensive consideration, as shown in Table 2.
TABLE 2 Signal-to-noise ratio (S/N) at different RPS26A additions
2. Detection of healthy human threshold of RPS26A by radioligand method
Serum of 192 healthy people is taken for measuring RPS26A by a radioligand method, a radioindex is calculated, 99% percentile is taken as a boundary value, a positive boundary value is calculated to be 0.12, and a positive judgment standard is more than or equal to 0.12, as shown in figure 5.
3. Radioligand method for detecting intra-batch-to-batch differences in RPS26A
The 3 sera were repeatedly tested 5 times in and between batches (n=5) according to RPS26A index low, medium, high in normal persons and patients, respectively, and the intrA-And inter-batch variation coefficients (coefficient of variance, CV) are shown in table 3. The result shows that the intra-batch CV of the detection index of the RPS26A of the radioligand method is 5.39-8.86%, the inter-batch CV is 9.23-11.37%, and the repeatability of the judgment of the yin-yang result is 100%.
TABLE 3 intra-lot variation for radioligand RPS26A detection
Distribution of RPS26A among different populations
The percentage of RPS26A in T1DM, T2DM and healthy people is 6.53% (42/643), 2.82% (2/71) and 0.53% (1/190), respectively, and the difference is obvious after single factor variance analysis P < 0.01. As shown in fig. 6.
5. Competitive inhibition of RPS26A positive samples
18 samples positive for RPS26A were taken for competitive inhibition experiments, and 2. Mu.g of commercial RPS26 protein without radioactive signal purchased was added to each well in the assay, and the results showed that the radioactivity index of the antibody was reduced to the negative range after the addition of RPS26 protein. Radioligand RPS26A detection was therefore effective in recognizing authentic RPS26A antibodies (fig. 7).
From the above results, the radioactive antigen SR12S constructed by the invention can effectively carry radioactive signals, and the RPS26A can be effectively identified by the detection of the RPS26A by a radioligand method, so that the CVs between batches in the batch are all in an effective range.
Claims (4)
1. A radioactive antigen protein SR12S is characterized in that the radioactive antigen protein SR12S adopts S 35 The antigen protein SR12S is marked by methionine, and the amino acid sequence of the antigen protein SR12S is shown as SEQ ID NO. 1.
2. A kit for detecting a ribosomal protein S26 antibody, comprising: the radioactive antigen protein SR12S, TBST buffer, protein a agarose and protein G agarose of claim 1.
3. The test kit of claim 2, further comprising a positive control and a negative control.
4. Use of the detection kit of claim 2 for the preparation of a type 1diabetes diagnostic reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311164315.5A CN117447578A (en) | 2023-09-11 | 2023-09-11 | Detection kit for ribosomal protein S26 antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311164315.5A CN117447578A (en) | 2023-09-11 | 2023-09-11 | Detection kit for ribosomal protein S26 antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117447578A true CN117447578A (en) | 2024-01-26 |
Family
ID=89582460
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311164315.5A Pending CN117447578A (en) | 2023-09-11 | 2023-09-11 | Detection kit for ribosomal protein S26 antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117447578A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4650756A (en) * | 1981-08-31 | 1987-03-17 | Sloan Kettering Institute For Cancer Research | Monoclonal antibodies to cell surface antigens of human renal cancer |
| CN101111514A (en) * | 2005-01-19 | 2008-01-23 | 法克斯因内特公司 | Compositions comprising pathogen-associated molecular patterns and antigens and their use to stimulate an immune response |
| CN104830874A (en) * | 2015-04-16 | 2015-08-12 | 南京医科大学第一附属医院 | Codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof |
-
2023
- 2023-09-11 CN CN202311164315.5A patent/CN117447578A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4650756A (en) * | 1981-08-31 | 1987-03-17 | Sloan Kettering Institute For Cancer Research | Monoclonal antibodies to cell surface antigens of human renal cancer |
| CN101111514A (en) * | 2005-01-19 | 2008-01-23 | 法克斯因内特公司 | Compositions comprising pathogen-associated molecular patterns and antigens and their use to stimulate an immune response |
| CN104830874A (en) * | 2015-04-16 | 2015-08-12 | 南京医科大学第一附属医院 | Codon optimized severe fever associated thrombocytopenia syndrome virus nucleoprotein gene and nucleic acid vaccine thereof |
Non-Patent Citations (1)
| Title |
|---|
| CHAOJIA CHEN等: "Ribosomal protein S26 serves as a checkpoint of T-cell survival and homeostasis in a p53-dependent manner", CELL MOL IMMUNOL, vol. 18, no. 7, 25 May 2021 (2021-05-25), pages 1844 - 1846, XP037495152, DOI: 10.1038/s41423-021-00699-4 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2395100B1 (en) | Novel diagnostic marker for type 1 diabetes mellitus | |
| CN114675040A (en) | Kit for detecting insulin receptor intracellular segment antibody | |
| CN113930435A (en) | Kit for detecting C peptide antibody by radioligand method | |
| Garnier et al. | Screening of ZnT8 autoantibodies in the diagnosis of autoimmune diabetes in a large French cohort | |
| CN111426850B (en) | Composition containing DNAJC8 protein fragment for detecting Alzheimer's disease autoantibody | |
| CN113999841A (en) | Protein scaffold OVAL100 and application thereof in radioligand method | |
| EP3044594B1 (en) | Identification of novel biomarkers of flares of systemic lupus erythematosus | |
| CN107024590A (en) | A kind of serum protein markers group for diagnosing 1 type and diabetes B | |
| CN117447578A (en) | Detection kit for ribosomal protein S26 antibody | |
| EP2787346B1 (en) | Biomarker for lymphocytic infundibuloneurohypophysitis, and use applications thereof | |
| CN117434270B (en) | Protein scaffold capable of carrying radioactive signals and application of protein scaffold in antibody detection | |
| CN117417427B (en) | Kit for detecting endogenous autologous insulin-mediated insulin antibody | |
| CN117402841A (en) | Detection kit for sulfite oxidase antibody | |
| CN110964827B (en) | SNP marker related to Chinese non-small cell lung cancer auxiliary diagnosis and application thereof | |
| KR100902282B1 (en) | A diagnostic composition for diabetes , a diagnostic kit comprising it and diagnostic methods of diabetes | |
| CN114675019B (en) | Kit for detecting insulin receptor extracellular domain antibody | |
| JP3620689B2 (en) | A method to detect insulin dependence in non-insulin dependent diabetes mellitus | |
| Brooks-Worrell et al. | Islet Autoimmunity is Highly Prevalent and Associated With Diminished Beta-Cell Function in Patients With Type 2 Diabetes in the Grade Study | |
| CN111518894B (en) | Reagent for detecting rs9273471 locus polymorphism and application thereof | |
| EP4039828A1 (en) | Biomarker composition for predicting therapeutic effect of mesenchymal stem cells on systemic lupus erythematosus | |
| Petrelli et al. | A multi-omics integration approach relying on circulating factors does not discern endotypes in childhood type 1 diabetes. | |
| WO2020037378A1 (en) | Detection of functional autoantibodies | |
| CN116024328A (en) | Application of miRNA in exosomes in preparation of differential diagnosis and early screening kit for neurodegenerative diseases | |
| CN115078721A (en) | Application of urine microtubule-associated protein CAMSAP1L1 and polypeptide fragment thereof in prostatic hyperplasia and prostatic cancer | |
| CN108169402A (en) | A kind of detection method of diabetes B sialoprotein marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |