CN109061192B - Urine protein related to osteoarthritis and application thereof - Google Patents

Abstract

The invention provides an application of ADIRF protein in preparation of products for detection, auxiliary diagnosis or treatment of osteoarthritis, wherein the ADIRF protein is up-regulated in urine of an osteoarthritis patient; furthermore, the invention also provides an ELISA kit for detecting osteoarthritis, wherein the kit detects that a sample is a urine specimen, and has the advantages of non-invasive acquisition, large-scale repeated sampling and convenient storage. The urine protein related to osteoarthritis provided by the invention can be used as an early biomarker of osteoarthritis, so that a new direction is provided for further researching the pathological mechanism of osteoarthritis and exploring a drug target for early prevention and treatment of osteoarthritis.

Description

Urine protein related to osteoarthritis and application thereof

Technical Field

The invention relates to the technical field of biomedical detection, in particular to urine protein related to osteoarthritis and application thereof.

Background

Osteoarthritis (OA) is a chronic degenerative joint disease which is caused by a plurality of factors and is very common in middle-aged and elderly people, is widely distributed in the world, is more common in women than in men, is common in knee joints, finger joints, hip joints, shoulder joints, wrist joints, ankle joints and the like. Osteoarthritis is characterized by wear and degeneration of articular cartilage and periarticular hyperosteogeny, and is also frequently accompanied by synovitis in the acute phase. According to investigation, the prevalence rate of osteoarthritis in people over 60 years old reaches more than 50%, the prevalence rate of osteoarthritis in people over 75 years old reaches 80%, and the disability rate of osteoarthritis can reach 53%.

Knee osteoarthritis causes knee pain, swelling, stiffness, limited knee mobility, dysfunction and the like, seriously affects the life quality of patients, and finally indirectly affects the life expectancy of the patients. At present, due to the lack of fundamental understanding of the etiology of OA, the medical community still lacks effective pharmaceutical means for radical treatment, and joint replacement surgery often becomes the treatment modality that patients must ultimately choose. In order to overcome the difficulties in treating OA, the research is directed to elucidating the etiology and mechanism of OA and exploring effective solutions for treating OA.

The definition of proteome refers to that all proteins expressed by a genome, one or more cells or certain tissues, whether as small as a cell or as large as an organism, are studied from an overall level for all proteins involved in life activities. All these proteins are not a constant set but vary from time to time and place, since different proteins are always expressed or modified as the vital activity progresses and homeostasis regulates. Proteins are the carriers of life activities and are also components of most organism structures, and the formation of proteins is subject to a series of post-transcriptional modifications, so that the situation of proteome cannot be directly deduced from genome and transcriptome.

The samples for proteomics research can be tissues, cells, blood, joint fluid, urine and the like, and the research on the proteomics of urine in early years is usually limited to the diseases of the urinary system, and gradually extends to the diseases which are expressed by the systemic metabolic disorder. Since urine is rarely affected by proteins with high abundance, urine proteomics is more favorable for finding biomarkers of disease than blood and synovial fluid, and may be found in disease mechanisms.

At present, the research on urine proteomics by taking urine as a specimen in the field of orthopedic joints is not many, and the research on urine proteomics in the field shows a trend of increasing along with the appearance and development of high-throughput proteomics technologies such as iTRAQ technology and the like. As mentioned in the introduction, there is increasing evidence that the development and progression of osteoarthritis is related to metabolic factors, and many scholars have been trying to find biomarkers for osteoarthritis for years, and therefore, it is well suited to search for biomarkers for osteoarthritis and study the associated pathological mechanisms using urine proteomics.

Disclosure of Invention

In order to solve the problems, the invention aims to provide a urine proteomic biomarker ADIRF for early diagnosis of osteoarthritis and provides application of the urine proteomic biomarker ADIRF in preparation of products for detecting osteoarthritis.

The invention firstly provides the application of ADIRF protein in the preparation of products for detecting, assisting in diagnosis or treating osteoarthritis.

Preferably, the ADIRF protein is up-regulated in expression in the osteoarthritis sample.

Preferably, the osteoarthritis sample is a urine sample.

Preferably, the product comprises a reagent, a kit, a chip or a medicament.

Preferably, the product is capable of detecting the risk of having osteoarthritis by detecting the expression level of the ADIRF protein in a sample from the subject.

Preferably, the detecting comprises:

a) urine from the subject and a normal control is obtained,

b) optionally, isolating the urine protein,

c) determining the expression level of ADIRF protein in urine of the subject and a normal control,

d) comparing the expression level of the protein in urine of the subject and a normal control,

e) detecting whether the subject has osteoarthritis disease based on the comparison of step d).

Preferably, said determination in step c) is performed using a method selected from the group consisting of: mass spectrometry, ELISA, Western methods or combinations thereof.

Further, the present invention provides an ELISA kit for detecting osteoarthritis disease, which comprises a detection reagent for ADIRF protein.

Preferably, the detection reagent for the ADIRF protein comprises an antibody reagent specific for the ADIRF protein.

Preferably, the antibody specific to the ADIRF protein is a monoclonal antibody and/or a polyclonal antibody.

The ELISA kit for detecting osteoarthritis adopts the coating antibody as an anti-ADIRF monoclonal antibody, the coating antibody is used for capturing ADIRF in a sample to be detected (such as a urine sample), and the capture principle is specific binding of an antigen and an antibody. The requirement of the invention for the coating antibody is that the coating antibody can capture the antigenic determinant on the ADIRF, so that the ADIRF and the ADIRF can be effectively and specifically combined, therefore, the antibody which can be specifically combined with the antigenic molecule can be satisfied, and the coating antibody can be all adopted as the coating antibody, including the anti-ADIRF monoclonal antibody of species origin such as mice, rabbits, chickens, dogs or monkeys. Among them, a mouse anti-human ADIRF monoclonal antibody is preferably used mainly because the specificity of a single epitope recognized by a murine antibody is high, which facilitates the binding of an antigen to be detected from a sample.

The ELISA kit for detecting osteoarthritis adopts a detection antibody which is an anti-ADIRF polyclonal antibody, and the detection antibody is combined with an antigen molecule ADIRF on a coating antibody, so that the ADIRF in a sample to be detected is detected. The requirement of the invention for detecting the antibody is that the antibody can be combined with the antigenic determinant on the ADIRF, so that the antibody which can be combined with the antigenic molecule specifically can be adopted as the detection antibody, and the antibody comprises the species-derived anti-ADIRF polyclonal antibody of mice, rabbits, chickens, dogs, monkeys and the like. Among them, it is preferable that the rabbit anti-human coating antibody is commercially available or can be prepared by itself, and the preparation method is known to those skilled in the art.

In particular, the coating antibody and the detection antibody are both anti-ADIRF antibodies, but the same species of anti-ADIRF antibody cannot be used. The reason is that: the different species of anti-ADIRF antibodies are used to enable the detection antibody to bind to different antigenic sites from the coating antibody so as to detect different antigenic epitopes, and when the same species of antibody is used, the antibody with chromogenic reaction enzyme can be simultaneously combined with the capture and detection antibody, so that the chromogenic reaction has no specificity, and the amount of the antigen to be detected cannot be determined. Therefore, in selecting the coating antibody and the detection antibody, it is necessary to select an anti-ADIRF antibody from different species.

Preferably, the kit further comprises an enzyme label plate coated by the anti-ADIRF monoclonal antibody, a labeled anti-ADIRF polyclonal antibody, a protein standard ADIRF, a TMB substrate solution, a diluent, a washing buffer solution and a termination solution.

Further, the invention provides application of the kit in preparation of a kit for detecting or assisting in diagnosis of osteoarthritis.

Furthermore, the invention provides the use of an antibody specific for the ADIRF protein for the manufacture of a medicament for the treatment of osteoarthritis.

Preferably, the antibody specific for the ADIRF protein comprises a complete polyclonal antibody, a monoclonal antibody, any antigen binding fragment (i.e., "antigen binding portion") thereof, or a single chain.

Falling within the scope of the antibodies of the present disclosure are polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. These antibodies can be readily prepared using techniques well known in the art. Furthermore, antibodies useful in the present disclosure can be whole antibodies consisting of two full-length light chains and two full-length heavy chains, or functional fragments of a whole antibody molecule. "functional fragments" of said antibody molecules are intended to mean fragments which retain the binding function of the antibody, such as Fab, F (ab')2 and Fv.

Further, the invention also provides a method for screening a urine sample of an osteoarthritis patient for differentially expressed proteins, which comprises the following steps:

obtaining urine of osteoarthritis patients and a control group;

separating urine protein;

performing reductive alkylation treatment on the two groups of protein samples respectively to obtain two groups of protein sample solutions;

adding trypsin into the two groups of protein sample solutions respectively for enzymolysis;

carrying out iTRAQ labeling treatment on the two groups of samples subjected to enzymolysis respectively by using different labeling reagents, and mixing labeled osteoarthritis patient groups with sample solutions of a control group;

sixthly, performing liquid chromatogram separation on the mixed sample, and performing tandem mass spectrometry on the peptide segments separated by the liquid chromatogram;

performing protein identification on the mass spectrum analysis result by using protein identification software, and screening and analyzing to obtain differential expression proteins in samples of osteoarthritis patients and a control group according to the abundance level of the proteins;

the self-checking protein is analyzed and identified by bioinformatics and the biological function of the differential protein is explained;

ADIRF protein related to osteoarthritis is finally screened out.

Preferably, the step-of-self bioinformatics analysis includes differential protein GO enrichment analysis and KEGG signaling pathway enrichment analysis.

Advantageous effects

Compared with a control group, the ADIRF protein is up-regulated in urine of an osteoarthritis patient through research verification, and the application prospect of the ADIRF protein in an osteoarthritis early detection and auxiliary diagnosis reagent or kit and/or a therapeutic drug is researched; the invention further provides an ELISA kit for detecting osteoarthritis, wherein a detection sample of the kit is a urine specimen, and the kit has the advantages of non-invasive acquisition, large-scale repeated sampling and convenient storage. The urine protein related to osteoarthritis provided by the invention can be used as an early biomarker of osteoarthritis, so that a new direction is provided for further researching the pathological mechanism of osteoarthritis and exploring a drug target for early prevention and treatment of osteoarthritis.

Drawings

FIG. 1 shows that Western blot is used to detect the expression of ADIRF protein in urine sample.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

The experimental methods in the examples, in which specific conditions are not specified, are generally conventional in the art.

The inventor of the invention carries out iTRAQ experiments on 4 osteoarthritis samples and 4 control samples, screens by combining a bioinformatics method, and selects candidate protein ADIRF protein, and the ADIRF protein is not reported as a marker related to osteoarthritis in the existing research.

The ADIRF protein of the present invention is a known protein prior to the present invention, and its basic information is as follows:

ADIRF NCBI Reference Sequence:NP_006820.1；

ADIRF (adipogenesis regulatory factor) in the present invention is a protein encoded by ADIRF gene, and ADIRF-related diseases including vaginal sarcoma and vaginal leiomyosarcoma have been studied. Its associated pathways are peroxisome proliferator activated receptor alpha (PPARalpha) and developmental biology regulating lipid metabolism.

Adipogenesis modulators play a key role in adipocyte development: promote adipocyte differentiation and stimulate transcription initiation of major adipogenic factors such as PPARG and CEBPA early in preadipocyte differentiation. Its overexpression confers resistance to the anticancer chemotherapeutic agent cisplatin.

The invention identifies protein markers in the urine of osteoarthritis patients by using an iTRAQ combined mass spectrometry method. Isotope labeling relative and absolute quantification (iTRAQ) technique an in vitro isotope labeling relative and absolute quantification technique developed by AB SCIEX corporation. The technology utilizes a plurality of isotope reagents to mark the N terminal of protein polypeptide or lysine side chain groups, and protein expression quantity among up to 8 samples can be simultaneously compared through high-precision mass spectrometer tandem analysis, so that the technology is a high-throughput screening technology commonly used in quantitative proteomics in recent years. The iTRAQ quantitative proteomics is that polypeptide is formed after protease digestion, and the N-terminal of the polypeptide or lysine side chain group is labeled by iTRAQ isotope reagent. And performing liquid phase separation on the marked peptide fragments, and performing primary mass spectrometry and secondary mass spectrometry, wherein the same peptide fragment in different marked samples shows the same mass-to-charge ratio and other physical and chemical properties before secondary mass spectrometry. In the secondary mass spectrum, signal ions are expressed as peaks with different mass-to-charge ratios (114-121), and according to the height and the area of the peak, the protein can be identified and quantitative information of different treatments of the same protein can be analyzed.

The iTRAQ reagent comprises three parts: a reporter moiety, a peptide reaction moiety, an equilibrium moiety. (1) The report section has eight types: 113-121 (no 120), so that the iTRAQ reagent can label 8 groups of samples simultaneously. (2) Peptide reaction part: can be covalently linked with the N end of the peptide segment and the amino group of the lysine side chain to mark the peptide segment. (3) A balance part: ensuring that the mass-to-charge ratio of the same labeled peptide fragment is the same. Compared with traditional quantitative analysis by dielectrophoresis, iTRAQ has the following technical service advantages: (1) the sensitivity is high, the detection limit is low, and low-abundance proteins can be detected; (2) the separation capacity is strong, the analysis range is wide, and the iTRAQ can be used for separating and identifying any type of protein, including high molecular weight protein, acidic protein, basic protein, membrane protein and insoluble protein; (3) high flux: the 8 samples are analyzed simultaneously, so that the experimental flux is improved, and the proteins at multiple time points or processed differently can be analyzed simultaneously; (4) the result is reliable: the qualitative and quantitative analysis results are more reliable; (5) the automation degree is high: the liquid phase is used together with the mass spectrum, the automatic operation is realized, the analysis speed is high, and the separation effect is good.

The term "sample" as used herein includes a specimen or culture obtained from any source. The sample may be obtained from blood (including any blood product, such as whole blood, plasma, serum, or a particular type of blood cell), urine, saliva, and the like. Samples also include tissue samples. In one embodiment, the sample is from urine.

The term "expression level" as used herein refers to the measurable quantity of a given nucleic acid or protein as determined by methods known to those skilled in the art.

The term "control" as used herein refers to an individual or group of individuals who do not show any symptoms of OA and are not diagnosed with osteoarthritis or OA. Preferably, the control individual is not administered a drug that affects OA and is not diagnosed as having any other disease. More preferably, the control individuals have similar sex, age and weight index (BMI) as compared to the test sample.

The term "enzyme-linked immunosorbent assay (ELISA)" used in the present invention refers to a detection method that uses the characteristic that an antibody molecule can be specifically bound to an antigen molecule to separate free hetero-protein from a target protein bound to a solid carrier, and uses a special marker to qualitatively or quantitatively analyze the protein. The ELISA process comprises the following steps: the antigen is adsorbed on the solid phase carrier, the process is called coating, the antibody to be detected is added, then the corresponding enzyme-labeled antibody is added, the complex of the antigen-antibody to be detected-enzyme-labeled antibody is generated, and then the complex reacts with the substrate of the enzyme to generate the colored product. The amount of antibody was calculated by means of the light absorption of a spectrophotometer. The amount of antibody to be detected is directly proportional to the amount of colored product. The antibody can be coated to measure the antigen content. Four methods most commonly used for ELISA: directly determining the antigen; measuring the antibody by an indirect method; antigen determination by a double antibody sandwich method; competition methods measure antigen.

EXAMPLE 1 Collection of samples

Case-control studies are the method used in this study. The study subjects of the experimental group were 4 patients with severe osteoarthritis who were hospitalized in Beijing cooperative hospital for orthopedics in 2015 years, but who had not received surgery, and the study subjects of the control group were 4 relatively healthy patients without osteoarthritis, and the experimental group met the osteoarthritis diagnosis standard of the bone science division of the Chinese medical society. The age of the experimental group was 58.5. + -. 2.0 years (56-61 years), and the age of the control group was 57.6. + -. 2.1 years (55-61 years). According to the standard of adult weight judgment made by the national Committee for health and family planning of the people's republic of China in 2013, overweight people with BMI between 24 and 28 are selected in both groups. Protocol was agreed by Beijing coordination and the ethical Committee of Hospital, and written informed consent was obtained from each subject. The basic data of the experimental group and the control group are shown in Table 1.

The second morning mid-stream urine from the experimental and control groups was collected, first contained in a sterile wide-mouth container and transferred to a centrifuge tube, centrifuged at 4,000g for 10min (4 ℃), and the supernatant aliquoted into 50mL sterile tubes and stored at-80 ℃ for later use.

TABLE 1 basic data of the samples

Inclusion criteria for OA case group:

the overweight female patients with severe osteoarthritis (K-L grade 4) are diagnosed according to the medical history and the diagnosis standard and signed an informed consent form.

Exclusion criteria for the OA case group:

knee trauma operation history, knee joint infection, adult knee joint deformity, metabolic bone disease, unequal lower limb, knee joint tumor history, osteoporosis, liver and kidney diseases, hyperlipidemia, hypertension, diabetes, hyperthyroidism, hyperparathyroidism and other joint diseases such as patients taking estrogen and progestogen recently, gout, rheumatoid arthritis and the like are treated by antirheumatic or immunosuppressive treatment for changing the disease condition, and are treated by intra-articular injection in nearly 6 months. BMI less than 24 or greater than 28.

Inclusion criteria for control group:

overweight female patients without osteoarthritis were selected as the control group, and the gender, age and BMI of the control group were matched to the OA case group and signed an informed consent.

Exclusion criteria for control group:

knee trauma operation history, knee joint infection, adult knee joint deformity, metabolic bone disease, unequal lower limb, knee joint tumor history, osteoporosis, liver and kidney diseases, hyperlipidemia, hypertension, diabetes, hyperthyroidism, hyperparathyroidism and other joint diseases such as patients taking estrogen and progestogen recently, gout, rheumatoid arthritis and the like are treated by antirheumatic or immunosuppressive treatment for changing the disease condition, and are treated by intra-articular injection in nearly 6 months. BMI less than 24 or greater than 28.

Example 2 iTRAQ assay screening for differential proteins

Instruments and reagents used

Vortex oscillator (model: QL-901 manufactured by Qinlebel instruments, Haimen)

Centrifuge (Thermo, model: PICO17)

Ultrasonic cell disruptor (Nanjing Xiou Instrument manufacturing Co., Ltd., model: XO)

Enzyme-linked immunosorbent assay (Thermo, model: M mu Ltiska MK3)

Constant temperature incubation bath device (Shanghai Pudong Rongfeng scientific instrument Co., Ltd., model HH.S4)

Vacuum freeze drier (Thermo, model: SPD2010-230)

RIGOL L-3000 high performance liquid chromatography system (Beijing Puyuan smart electro-technology Co., Ltd.), mobile phase A: 98% ddH₂O, 2% acetonitrile (pH 10); mobile phase B: 98% acetonitrile, 2% ddH₂O (pH 10) high performance liquid chromatograph: (Thermo scientific EASY-nLC 1000 System (Nano HPLC)), mobile phase A: 100% ultrapure water, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid Mass Spectrometry System (Thermo, model: Q-active)

Urea (Bio-Rad, cat # 161-

Thiourea (Sigma-Aldrich, cat # T7875, USA)

CHAPS (Bio-Rad, cat # 161-

Protease Inhibitor Cocktail (Roche, Cat # 04693116001, USA)

Protein quantitative dye liquor (Thermo Scientific, cat # 23238, USA)

Bovine Serum Albumin (Bovine Serum Albumin, BSA) (Sigma-Aldrich, cat # A2058, USA)

DTT (Bio-Rad, cat # 161-

Iodoacetamide (Bio-Rad, cat # 163-

Disolution Buffer in kit (AB Sciex, PN: 4381664)

Pancreatin (Promega, cat # V5111, USA)

10K ultra-filtration tube (mileore, PN: UFC5010BK)

8 mark

Kit (AB Sciex, PN: 4390812, PN: 4381664)

Ziptip(Millipore，PN：ZTC18M096(2μg))

A chromatographic column: Durashell-C18, 4.6mm X250 mm, 5 μm,

(Agela, cat # DC952505-0)

Acetonitrile (Merck, cat # 100030, Germany)

Ammonia (Sigma-Aldrich, cat # 17837, USA)

Pre-column (Acclaim PepMap100 column, 2cm x 100 μm, C18, 5 μm)

Chromatographic column (EASY-Spray column, 12cm x 75 μm, C18, 3 μm)

Sample bottle (Thermo, 11190533)

Bottle cap (Thermo, 11150635)

Spray needle (Thermo, PN: ES542)

Quantitative experimental process of II and iTRAQ

1. Sample protein extraction

1) Samples were prepared according to a 1: 10(W/V) lysis buffer (7M urea, 2M thiourea, 0.1% CHAPS, pellet/50 mL Protease Inhibitor Cocktail) was added and vortexed to mix.

2) Ultrasound 60s (0.2s on, 2s off), amplitude 22%.

3) Extraction was carried out at room temperature for 30 minutes.

4)15,000g, centrifuged at 4 ℃ for 20min, the supernatant carefully removed, split-packed and frozen at-80 ℃.

2. Protein quantification (Bradford method)

1) The Bradford method [ Marion M. Bradford. analytical Biochemistry,1976,72:248-]The concentration of protein extracted from the sample is determined. The sample was diluted with lysis buffer (7M urea, 2M thiourea, 0.1% CHAPS) by a certain fold to a final concentration within the range of the standard, and 10. mu.L of each of the diluted sample and the standard (BSA dissolved in lysis buffer to a series of concentrations of standard protein) was takenRespectively reacting with 300 μ L of protein quantitative dye in dark for 20min, simultaneously measuring absorbance at 595nm of the standard and the sample with a microplate reader, and drawing a standard curve according to the relationship between the absorbance and the concentration of each tube of the standard (curve formula: y is 1.4337 × x2+0.0406 × x +0.03728, R is²＝0.98906)。

2) And calculating the protein concentration of each sample according to a curve formula.

3. Proteolysis (FASP)

1) After protein is quantified, 200 mu g of protein solution is put into a centrifuge tube;

2) adding 25mM DTT with final concentration, and reacting at 60 ℃ for 1 hour;

3) adding iodoacetamide with the final concentration of 50mM, and keeping the temperature for 10 minutes;

4) adding the protein solution after reductive alkylation into a 10K ultrafiltration tube, centrifuging for 20 minutes at 12,000 revolutions, and discarding the solution at the bottom of the collection tube;

5) adding 100 mu L of precipitation Buffer in an iTRAQ kit, centrifuging for 20 minutes at 12,000rpm, discarding the solution at the bottom of the collecting tube, and repeating for 3 times;

6) replacing a new collecting pipe, adding trypsin into the ultrafiltration pipe, wherein the total amount of trypsin is 4 mu g (the mass ratio of trypsin to protein is 1:50), the volume of the trypsin is 50 mu L, and the trypsin reacts at 37 ℃ overnight;

7) the next day, centrifuging for 20 minutes at 12,000 revolutions, and centrifuging the peptide fragment solution after enzymolysis and digestion at the bottom of a collecting pipe;

8) add 50. mu.L of digestion Buffer into the ultrafiltration tube, centrifuge again for 20 minutes at 12,000rpm, combine with the previous step, collect the bottom of the tube to obtain 100. mu.L of the sample after enzymolysis.

iTRAQ labeling

1) Taking the iTRAQ reagent out of the refrigerator, balancing to room temperature

The reagent was centrifuged to the bottom of the tube.

2) To each tube

Adding 150 μ L isopropanol into the reagent, vortex and shaking, and centrifuging to the bottom of the tube。

3) A50. mu.L sample (100. mu.g of the enzymatic hydrolysate) was transferred to a new centrifuge tube.

4) The iTRAQ reagent was added to the sample, vortexed, centrifuged to the bottom of the tube, and reacted at room temperature for 2 hours.

5) The reaction was stopped by adding 100. mu.L of water.

6) In order to detect the labeling efficiency and the quantitative accuracy, 1. mu.L of each of the 4 groups of samples was mixed, desalted with Ziptip and identified by MALDI-TOF-TOF (AB SCIEX 4800 Plus) to confirm that the labeling reaction was good;

7) the labeled sample was mixed, vortexed, and centrifuged to the bottom of the tube.

8) Vacuum freeze-drying and centrifugal drying.

9) And (4) freezing and storing the sample after being pumped and dried for later use.

5. Enzymolysis peptide fragment offline pre-separation and LC-MS/MS mass spectrometry

5.1 reverse phase chromatographic separation at high pH

1) The mixed labeled sample was dissolved in 100. mu.L of mobile phase A, centrifuged at 14000g for 20min, and the supernatant was collected for use.

2) The conditions of the system were checked by separating with 400. mu.g of enzymatically hydrolyzed BSA (column temperature 45 ℃ C., detection wavelength 214 nm).

3) 100 μ L of the prepared sample was sampled and separated at a flow rate of 0.7 mL/min.

5.2 nanoliter reverse phase chromatography-Q active for protein analysis

1. Experimental procedure

1) The fraction obtained by high pH reverse phase separation was redissolved with 20. mu.L of 2% methanol, 0.1% formic acid.

2) Centrifuge at 12,000rpm for 10 minutes and aspirate the supernatant.

3) The loading volume is 10 mu L, and the loading is carried out by a sandwich method.

4) The Loading Pump flow rate was 350nl/min, 15 minutes.

5) The separation flow rate was 300nl/min, and the separation gradient was as follows in Table 2:

TABLE 2 nanoliter reversed phase chromatographic separation gradient

2. Mass spectrometry parameter settings

a) Ion source parameters:

Spray voltage：2.3kv；

Capillary Temperature：320℃；

Ion Source：EASY-Spray source；

DP：100；

b)FμLl MS：

Resolution：70000FWHM；

FμLl Scan AGC target：3e6；

FμLl Scan Max.IT：20ms；

Scan range：300-1800m/z；

c)dd-MS2：

Resolution：17500 FWHM；

AGC target：1e5；

Maximum IT：120ms；

Intensity threshold：8.30E+03；

Fragmentation Methods：HCD；

NCE：32％；

Top N：20；

6. mass spectrometric data analysis

6.1 database

The database selection is based on the desired species, completeness of database annotation, and sequence reliability. The database chosen in this experiment was from UniProt (http:// www.uniprot.org /), in the version: uniprot. rat.201509. fasta.

6.2 search software

The mass spectrometry of iTRAQ is completed by Thermo Q-active type mass spectrometry, and the generated mass spectrometry original file is RAW processed by searching a database by adopting Mascot 2.5.1 software, and the quality control is carried out on the database searching result by adopting scaffold software.

7. Bioinformatics analysis

7.1 statistics of quantitative information on differential proteins

Two groups of samples to be compared were statistically analyzed using Perseus 1.3.0.4.(www.maxquant.org) software to screen for differential proteins with a P value of 0.05 or less and a protein ratio of 1.25 or less or a protein ratio of 0.75 or less.

7.2 differential protein functional Annotation

A comprehensive biological functional annotation was performed on all proteins using UniProt (http:// www.uniprot.org /), and functional information was obtained on all of the proteins. Including annotation information such as Gene Ontology (GO) and pathway.

7.3 differential protein functional enrichment assay

Functional enrichment analysis was performed on different groups of differential proteins using MetaCore software. Groups of differentially expressed proteins were subjected to Gene Ontology (GO) based enrichment analysis of biological processes, cellular components (cell μ Lar component) and molecular functions (molecular μ Lar function) using MetaCore. The analysis refers to annotating each protein with a functional annotation tool at a high throughput to obtain the distribution of the experimentally identified protein in various biological processes or molecular functions, comparing the distribution with the corresponding distribution of the overall protein to confirm which biological processes or molecular functions the experimentally identified protein is significantly enriched in (i.e., p value is less than 0.05), and obtaining protein molecules associated with certain specific functions by grasping the degree of coincidence between the overall identified protein and the expected functional distribution. And obtaining the results of the biological process, the cell components and the molecular function enrichment of the differential protein through MetaCore analysis.

7.4 differential protein pathway enrichment analysis

Pathway analysis was performed on differentially expressed proteins using MetaCore software to obtain all relevant pathway information and enriched pathways for the differentially expressed proteins.

8. Results

Based on the iTRAQ technology, 1413 proteins were identified from urine of the experimental group and the control group together, wherein the total difference proteins between the two groups were 394, 170 proteins were up-regulated in the OA experimental group, and 224 proteins were down-regulated in the OA experimental group. From GO analysis it can be seen that: the differential proteins are mainly located in extracellular vesicles, extracellular secretory bodies, extracellular matrix parts and cell membranes; the differential protein is related to the regulation and inhibition effects of peptidase, endopeptidase and the like, and is also related to molecular functions such as the combination of cell adhesion molecules and protein receptors; the differential protein is mainly involved in biological processes such as cell adhesion, regulation of injury repair, stress response and the like.

The inventors screened for a differential protein ADIRF whose expression is upregulated in the urine of osteoarthritis patients by Gene Onlogy and signaling pathway analysis, as well as in the literature. The ADIRF protein can be used as a urine biomarker of osteoarthritis, and provides a theoretical basis for diagnosis, treatment and prognosis of diseases.

Example 3 detection of ADIRF protein expression in osteoarthritis patients by western blot

The ADIRF protein expression of urine samples of 4 osteoarthritis patients and 4 control patients was further verified. The protein in the sample is provided by referring to example 2, and a Kangji micro BCA protein quantification kit (product number: CW2011) is adopted, and the specific steps are shown in the specification. Then, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed, and the specific steps were as follows:

the SDS-polyacrylamide gel electrophoresis:

1. protein sample denaturation:

a) the same mass of total protein extract was added to each gel well based on the BCA protein concentration assay. Protein samples and protein loading buffer (5 ×) were mixed at a ratio of 0.25 microliters protein loading buffer per 1 microliter protein sample.

b) Heating at 100 deg.C or boiling water bath for 3-5 min to fully denature protein.

c) Cooling to room temperature, and directly loading the mixture into SDS-PAGE gel sample loading holes.

2. Preparing a rubber plate:

a gel with the thickness of 0.75mm is prepared by adopting a minitype vertical plate electrophoresis device of Bio-Rad company, 5mL of 10% separation gel is prepared in a small beaker after a glass plate is installed according to the instruction, and the formula is as follows:

TABLE 3 Release glue formulation

Components	Dosage of
		30% acrylamide solution	1.7mL
Tris-HCL(1.5M，pH8.8)	1.3mL
		10％SDS	0.05mL
10％AP	0.05mL
		TEMED	0.002mL
Sterilization ddH2O	Make up to 5mL

And (3) pouring glue immediately after mixing, then adding 1mL of distilled water to cover, standing at room temperature for about 30min until the glue is polymerized, washing with distilled water for 2-3 times, and then sucking dry with filter paper. Then 2mL of 5% concentrated gum was prepared, the formulation was as follows:

TABLE 4 concentrated gum formulation

And (3) pouring glue immediately after mixing, inserting a sample comb to avoid generating bubbles, taking out the sample comb after the glue is solidified, and then washing sample holes by using distilled water and 1x protein electrophoresis buffer solution successively.

3. Sample loading and electrophoresis

And (3) placing the gel plate on an electrophoresis device, filling 1x protein electrophoresis buffer solution into the inner groove, and sequentially loading the 1x protein electrophoresis buffer solution into the outer groove in a way that the 1x protein electrophoresis buffer solution exceeds the platinum wire. Protein mass standard protein gradients were added to the terminal lanes. During electrophoresis, the blue dye reaches the position near the bottom end of the gel, and then the electrophoresis is stopped.

The western blot is:

1. and soaking the NC membrane, the filter paper and the sponge pad in a transfer buffer solution in advance. And taking out the gel after SDS-PAGE is finished, removing the concentrated gel, rinsing the gel in a Tris/glycine buffer solution for several seconds, and then soaking the gel in a transfer buffer solution for 15-30 min. Opening the electric transfer clamp, filling a special sponge pad soaked by transfer buffer solution on each side, respectively placing a piece of filter paper soaked by the transfer solution, wherein the filter paper has the same size as the sponge pad or the NC membrane, and the gel has the same size, flatly placing the gel on the filter paper on the cathode side, finally flatly placing the NC membrane on the gel, removing bubbles, and clamping the electric transfer clamp. Filling the electrophoresis tank with the electric transfer liquid, inserting the electric transfer clip, putting the electrophoresis tank into a refrigerator (the electric transfer liquid is put into the refrigerator for precooling before), connecting electrodes, switching on current, and enabling the NC film of the transfer clip to correspond to the anode of the electrophoresis tank.

2. And (3) sealing: rinse once with 1 xTBS. Adding a non-fat milk powder TBS sealing buffer solution containing 5%, and placing in a shaking incubator for sealing;

3. primary antibody hybridization: removing the blocking solution, adding a primary Antibody hybridization solution diluted by a primary Antibody (ADIRF Antibody, LSBio, LS-C140569) diluent, placing the mixture in a shaking incubator for hybridization overnight at 4 ℃, and hybridizing the mixture in the shaking incubator the next day;

4. recovering the primary antibody hybridization solution, and washing the membrane for 3 times by using TBST;

5. abandoning TBST, adding a secondary antibody (Goat Anti-Rabbit IgG, HRP Conjugated, CW0103) hybridization solution diluted by a blocking buffer solution, and placing the mixture in a shaking incubator for hybridization;

6. discarding the secondary antibody solution, and washing the membrane for 3 times by using TBST;

7. ECL chemiluminescence and image acquisition and analysis: according to a high-sensitivity chemiluminescence detection kit (Kangji brand CW0049B), the specific steps refer to the instruction book.

8. And (3) carrying out data standardization by taking beta-Actin as an internal reference, and observing the relative expression level of ADIRF protein in the osteoarthritis urine sample by taking a normal person as a reference sample.

Results figure 1 shows that the expression of ADIRF protein in the urine of osteoarthritis patients was up-regulated in the osteoarthritis group compared to the control group. The method is consistent with the results of proteomics experiments. It was suggested that ADIRF may be a key factor associated with the development of osteoarthritis.

Example 4 osteoarthritis-associated ADIRF protein detection kit Assembly

Based on the above results, the present inventors can establish an ELISA kit for diagnosing a urine sample of an osteoarthritis patient by detecting the expression level of ADIRF protein using ADIRF protein and/or an antibody thereof, comprising: a solid phase carrier, a marker and a substrate which can generate color reaction with the marker; the marker is enzyme-labeled ADIRF protein, enzyme-labeled ADIRF protein antibody, biotin-labeled ADIRF protein or biotin-labeled ADIRF protein antibody. The specific kit can comprise the following reagents:

1) an ELISA plate (solid phase carrier);

2) human ADIRF standard protein;

3) a label;

4) a sample diluent;

5) antibody diluent;

6) a wash solution;

7) a color-developing reagent;

8) and (4) stopping the solution.

As is known to those skilled in the art, the above components are merely illustrative, and the microplate may be primary-antibody-coated (for double-sandwich ELISA, competition methods) or uncoated (for direct ELISA); the human ADIRF standard protein can be an enriched or expressed, synthetic whole protein or a peptide fragment containing an antigenic determinant; the label can be horseradish peroxidase label or biotin label; the chromogenic reagent may be TMB, OPD, ABTS, or the like. The ELISA method may be a double sandwich method, a direct method or a competition method.

The following ELISA kit for osteoarthritis was assembled using the ELISA double sandwich method as an example:

reagents conventional in ELISA experiments:

coating buffer (carbonate buffer ph 9.6): na (Na)₂CO₃1.59g，NaHCO₃2.93g, distilled water was added to 1L.

Washing buffer (ph 7.4): 8.0g NaCl; 0.2g KH₂PO₄；2.9g Na₂HPO₄·12H₂O; 0.2g of KCl; 0.5mL of 0.05% Tween-20, plus ddH₂O to 1L.

Diluting with 0.1g Bovine Serum Albumin (BSA) and 100mL washing buffer;

the mouse-derived monoclonal antibody against human ADIRF protein used in the present invention is purchased from bioscience, Inc., Shanghai Yuanmu, and the polyclonal antibody against ADIRF rabbit is purchased from abcam.

The protein standard ADIRF is a human recombinant protein and is purchased from abcam.

2. Coating of ELISA plate:

the ELISA plate coated by the mouse monoclonal antibody of the anti-ADIRF is prepared by the following method: diluting the purified anti-ADIRF monoclonal antibody to a target concentration of 0.63 mu g/mL by using a carbonate coating buffer solution with the pH value of 9.6; uniformly mixing the diluted antibody solution, adding the mixture into micropores, keeping the volume at 100 mu L/hole, and standing overnight at 4 ℃; washing the plate for 3 times, 200 mu L/hole; adding 3% BSA blocking solution, 300. mu.L/well, and standing overnight at 4 ℃; washing the plate for 3 times, 200 mu L/hole; storing at-20 deg.C.

3. Preparation of enzyme-labeled antibody:

and (3) coupling the rabbit polyclonal antibody resisting ADIRF protein with HRP respectively to obtain the enzyme-labeled antibody. Adding a certain amount of enzyme-labeled antibody into the diluent, mixing well to make the final concentration 2 μ g/mL (depending on the specific conditions), and storing at 2-8 deg.C in dark.

4. Assembling the kit:

an ELISA kit for detecting osteoarthritis includes the components in table 5:

TABLE 5 ELISA test kit

The use method of the kit is as follows:

(1) respectively adding 50 mu L of ADIRF standard protein solution with the concentrations of 900pg/mL, 600pg/mL, 300pg/mL, 150pg/mL and 75pg/mL into an antibody-coated ELISA plate to draw a standard curve;

(2) blank holes (the blank reference hole is not added with the sample and the enzyme labeling reagent, the rest steps are operated the same) and sample holes to be detected are respectively arranged, 40 mu L of sample diluent is added into the sample holes to be detected on the enzyme labeling plate, and then 10 mu L of sample to be detected is added (the final dilution of the sample is 5 times). Adding a sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

(3) sealing the plate with a sealing plate film, and then incubating for 30 minutes at 37 ℃;

(4) carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;

(5) adding 50 mu L of enzyme-labeled reagent into each hole;

(6) incubating and washing, the steps are the same as the above;

(7) adding 50 mu L of TMB substrate solution A into each hole, adding 50 mu L of TMB substrate solution B into each hole, gently shaking and uniformly mixing, and developing for 15 minutes at 37 ℃ in a dark place;

(8) adding 50 mu L of stop solution into each hole to stop the reaction;

(9) and (4) adjusting to zero by using a blank hole, sequentially measuring the absorbance (OD value) of each hole at the wavelength of 450nm, and calculating the protein content in each sample.

The ELISA kit can be applied to the preparation of osteoarthritis disease detection kits.

The kit disclosed by the invention has the advantages of noninvasive acquisition of urine samples, large-scale repeated sampling and convenience in storage, and the ADIRF protein and the polypeptide fragment thereof are detected by utilizing the urine samples.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims (3)

1. Use of an ADIRF protein for the preparation of a product for the detection or the auxiliary diagnosis of osteoarthritis, said ADIRF protein being up-regulated in expression in an osteoarthritic urine sample compared to a healthy control.
2. 2. The use according to claim 1, wherein the product is capable of detecting the presence or absence of osteoarthritis by detecting the expression level of ADIRF protein in a sample from the subject.
3. 3. Use according to claim 1 or 2, wherein the product comprises a reagent, a kit or a chip.

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