CN117706089A - Biomarker for evaluating ionizing radiation damage based on MSD technology and detection method thereof - Google Patents
Biomarker for evaluating ionizing radiation damage based on MSD technology and detection method thereof Download PDFInfo
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Abstract
The invention discloses a biomarker for evaluating ionizing radiation damage based on an MSD technology and a detection method thereof. In particular, the use of biomarkers, which are MMP-2 proteins, SAA proteins and/or IGFBP-3 proteins, and/or substances detecting said biomarkers for the preparation of a product for detecting or assessing ionizing radiation damage is disclosed. The invention provides a method for evaluating ionizing radiation damage by detecting SAA, MMP-2 and IGFBP-3 protein expression levels in serum based on an MSD hypersensitive multifactorial electrochemiluminescence analysis technology, which has the characteristics of high sensitivity, high specificity and high precision, is good in reliability, is simple and convenient to operate, and can rapidly and accurately reflect the degree of ionizing radiation damage.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to a biomarker for evaluating ionizing radiation damage based on an MSD technology and a detection method thereof. In particular to a method for evaluating ionizing radiation damage judgment on SAA (serum amyloid A), MMP-2 (matrix metalloproteinase-2) and IGFBP-3 (insulin-like growth factor binding protein-3) in serum proteins by a hypersensitive multifactorial electrochemiluminescence analysis technology.
Background
Ionizing radiation is a physical factor capable of ionizing atoms or molecules, which has a remarkable damaging effect on living organisms. Ionizing radiation damage is a common and serious problem, both in radiotherapy, space exploration, nuclear accident, and the like. Upon exposure to ionizing radiation, a series of biochemical changes are triggered in the organism, which in turn cause various biological effects such as genetic mutations, cell death, inflammatory reactions, etc. Therefore, it is necessary to evaluate ionizing radiation damage effectively in time. Currently, the commonly used methods for evaluating ionizing radiation damage mainly include the use of radiation dosimeters, the measurement of biological effects, the detection of biological markers, and the like. 1) Use of a radiation dosimeter: a dosimeter is an instrument that measures the dose of ionizing radiation. By measuring the dose of ionizing radiation, the potential damage to the organism caused by the ionizing radiation can be assessed. Common radiation dosimeters include ionization chambers, detectors, dosimeters, and the like. These instruments can measure the dose rate and total dose of ionizing radiation to help assess potential damage caused by the radiation. 2) Measurement of biological effects: damage to organisms by ionizing radiation is mainly manifested by changes in physiological and biochemical reactions of cells, and dysfunction of tissues and organs, etc. By measuring the change in these biological effects, the effect of ionizing radiation on the organism can be quantitatively assessed. Common biological effect measurement methods include cell viability assays, chromosomal aberration assays, apoptosis assays, and the like. These methods can help assess the biological effects of ionizing radiation and make associated risk assessments. 3) Detection of biological markers: ionizing radiation damage can cause a range of changes in the organism, including damage to DNA and other molecules, alterations in cell cycle, increased apoptosis, and the like. By detecting these biological markers, the extent of the effect of ionizing radiation on the organism can be assessed. Common biological markers include DNA breaks, chromosomal aberrations, alterations in protein and enzyme activities, and the like. By analyzing the changes in these markers, the effect of ionizing radiation on the organism can be determined. Among them, changes in protein and enzyme activities are one of the biological markers commonly used in methods of assessing ionizing radiation damage. These protein molecular markers include: gamma-H2 AX,53BP1,MDC1,MRE11,ATM,DNA-PKcs, ATR, ATM, chk1, chk2, TBARS, MDA,4-HNE, TNF-alpha, IL-1, IL-6, GM-CSF, MCP-1, etc. By detecting changes in protein and enzyme activity, the extent of damage to cells and tissues by ionizing radiation can be revealed, thereby assessing the effect of the radiation. The common protein molecular marker detection method comprises the following steps: enzyme-linked immunosorbent assay (ELISA), mass spectrometry, immunohistochemistry (IHC), protein chip and the like. However, these methods have some disadvantages such as limited selection range of the markers, insufficient detection sensitivity, complicated evaluation process, and the like. Since damage to the body caused by ionizing radiation involves multiple levels and factors, there is a need for a more accurate and sensitive method to monitor and evaluate the condition of the body.
MSD hypersensitive multifactorial electrochemiluminescence analysis technology (Meso Scale Discovery) is an immunoassay technology which utilizes electrochemical stimulation to generate specific chemiluminescent reaction on the surface of an electrode. The method can detect a plurality of biomarkers in the same sample at the same time, and has the advantages of high sensitivity, low matrix interference, high signal stability, wide application range and the like. The MSD hypersensitive multifactor electrochemiluminescence analysis technology is a multifunctional biological analysis platform based on the electrochemiluminescence principle.
In view of the shortcomings of the traditional ionizing radiation damage assessment method in terms of accuracy and sensitivity, research and development of a new detection method for assessing damage caused by ionizing radiation to an organism are needed to comprehensively understand the reaction condition of the organism to the ionizing radiation, and the accuracy and reliability of the ionizing radiation damage assessment are improved.
Disclosure of Invention
The technical problem to be solved by the invention is to provide an application of a biomarker or a substance for detecting the biomarker in the preparation of a product for detecting or evaluating ionizing radiation damage. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description.
To solve the above technical problem, the present invention provides first any one of the following applications of a biomarker and/or a substance detecting the biomarker:
a1 Use in the manufacture of a product for detecting or assessing ionizing radiation damage;
a2 Use in the manufacture of a product for identifying exposure to ionizing radiation;
the biomarker is MMP-2 protein, SAA protein and/or IGFBP-3 protein.
Further, the MMP-2 protein, SAA protein and/or IGFBP-3 protein may be proteins in serum.
Further, the biomarker may be a biomarker for assessing ionizing radiation damage based on MSD technology.
Further, the application is based on MSD technology.
In such applications, the agent for detecting the biomarker may include reagents for detecting the amount of MMP-2 protein, SAA protein and/or IGFBP-3 protein expression.
In such applications, the agent may comprise an antibody, polypeptide, protein or nucleic acid molecule that binds MMP-2 protein, SAA protein and/or IGFBP-3 protein.
In the above application, the product may be a reagent or a kit.
The reagents or kits described herein may be electrochemiluminescence detection reagents or kits.
The reagents or kits described herein may be detection reagents or kits based on MSD technology.
In the above application, the biomarker may be a serum biomarker.
The product can be used for detecting the expression level of MMP-2 protein, SAA protein and/or IGFBP-3 protein in serum of a subject.
The invention also provides a kit for assessing ionizing radiation damage or identifying ionizing radiation exposure, which may comprise a substance for detecting the biomarker as described herein.
Further, the kit may be an MSD technology detection kit.
Further, the kit may include a capture antibody immobilized on the electrode, which may be an anti-MMP-2 antibody, an anti-SAA antibody, and/or an anti-IGFBP-3 antibody.
Further, the capture antibody may have a biotin tag.
Further, the kit may include a detection antibody bound to the electrochemiluminescent tag, which may be an anti-MMP-2 antibody, an anti-SAA antibody, and/or an anti-IGFBP-3 antibody.
The detection antibody may correspond to the capture antibody (for capturing the antigen to be detected), and is an antibody for detecting the antigen to be detected.
Further, the electrochemical luminescence label may be a ruthenium complex or an iridium complex.
Further, the ruthenium complex may be ruthenium terpyridyl or a ruthenium terpyridyl derivative, and may be particularly sulfo-tag.
Further, the kit may further comprise a streptavidin-coated plate, a standard, a sample diluent, an antibody diluent, a stop solution, a read plate solution, and/or a wash buffer.
Further, the streptavidin-coated plate may be a multi-index combinatorial assay plate (e.g., U-PLEX plate).
Further, the kit may further comprise a U-PLEX Linker (e.g., U-PLEX connector) having a biotin binding region, which can be coupled to a capture antibody having a biotin tag, and which can self-assemble onto a streptavidin-coated plate.
The U-PLEX Linker can carry capture antibodies with biotin labels to different areas of one well, and can realize the coating of multiple capture antibodies in the same well. Different capture antibodies were coupled to different U-PLEX Linker.
The biomarkers described herein or the substances detecting the biomarkers are also within the scope of the invention.
The ionizing radiation described herein may be X-ray radiation and/or carbon ion radiation.
The test sample of the kit described herein may be a serum sample.
The change in the expression level of the biomarkers described herein can be used as a basis for determining whether a subject is damaged or exposed to ionizing radiation.
Comparing the level of MMP-2 protein, SAA protein and/or IGFBP-3 protein in serum of a normal healthy individual to the level of MMP-2 protein, SAA protein and/or IGFBP-3 protein in serum of a normal healthy individual, and determining that the subject is at risk of being damaged or exposed to ionizing radiation when the expression of at least any one of the three proteins is increased.
The present invention also provides a method of preparing a kit for detecting multiple factors of SAA, MMP-2 and IGFBP-3, said method being based on MSD technology, said method comprising the steps of: 1) Preparing biotinylated conjugated antibodies, namely SAA antibody, MMP-2 antibody and IGFBP-3 antibody, respectively obtaining three biotinylated antibodies (also called capture antibodies); 2) Respectively coupling the biotinylated antibodies to different U-PLEX markers to obtain three biotinylated coupling antibodies with different U-PLEX markers; 3) Preparing a multiple coating solution: three biotinylation coupling antibodies with different U-PLEX Linker are mixed by vortex to obtain U-PLEX composite coating solution; 4) Coating an MSD plate with the U-PLEX composite coating solution; 5) Electrochemiluminescent labeled detection antibodies are prepared, which may be SAA antibodies, MMP-2 antibodies, and IGFBP-3 antibodies.
In the above method, the electrochemiluminescence label may be a label with a ruthenium complex or an iridium complex.
In the above method, the ruthenium complex may be terpyridyl ruthenium or a terpyridyl ruthenium derivative, and specifically may be sulfo-tag.
In the above method, the MSD sheet may be a graphite electrode sheet.
The invention provides a method for evaluating ionizing radiation damage based on MSD hypersensitive multifactorial electrochemiluminescence analysis technology to detect SAA, MMP-2 and IGFBP-3 combination in serum. The method comprises the following steps: 1. the establishment of a multi-factor MSD detection kit for detecting SAA, MMP-2 and IGFBP-3; 2. semi-quantitatively detecting a serum sample; 3. SAA, MMP-2 and IGFBP-3 are useful for evaluation of the effects of ionizing radiation detection. The invention realizes high sensitivity and high specificity of judging the ionizing radiation damage by carrying out joint evaluation on three proteins closely related to the ionizing radiation damage signals. The invention aims to provide a method capable of rapidly and accurately detecting body damage caused by ionizing radiation. According to the method, the change condition of the relative contents of serum protein SAA, MMP-2 and IGFBP-3 caused by ionizing radiation is detected, and an organism radiation damage assessment model is established, so that the quick assessment of the organism damage degree caused by the ionizing radiation is realized.
Compared with the prior art, the method has the following advantages:
1. high sensitivity: by adopting MSD hypersensitive multifactor electrochemiluminescence analysis technology, SAA, MMP-2 and IGFBP-3 with low concentration can be detected, and the accuracy of the evaluation result is improved.
2. High specificity: by simultaneously detecting multiple indicators to assess ionizing radiation damage, the effects of cells and tissues can be more fully reflected.
3. High flux: the MSD hypersensitive multifactor electrochemiluminescence analysis technology has high flux characteristics, can process a plurality of samples simultaneously, and improves the working efficiency.
4. High convenience: the method is simple and convenient to operate, does not need to carry out complex sample extraction and treatment, and can rapidly and conveniently carry out ionizing radiation damage assessment. The method combines the MSD technology and the specific serum protein biomarker, can accurately and stably evaluate the ionizing radiation damage in a short time after the emergency, and is particularly suitable for screening and testing the radiation condition of large-scale personnel.
5. High efficiency: the method provided by the invention adopts an MSD technology, has the characteristics of high sensitivity, high specificity and high precision, and can rapidly and accurately reflect the degree of ionizing radiation damage.
6. Reliability: the method of the invention uses SAA, MMP-2 and IGFBP-3 in serum as markers, and the markers are closely related to ionizing radiation injury and have higher reliability.
In summary, the present invention proposes a novel method based on MSD hypersensitive multifactorial electrochemiluminescence analysis technology for assessing the damage caused by ionizing radiation to the body by detecting levels of SAA, MMP-2 and IGFBP-3 in serum. By simultaneously analyzing a plurality of biomarkers, the reaction condition of the body to the ionizing radiation can be more comprehensively known, and the accuracy and the reliability of the ionizing radiation damage evaluation are improved. This would provide an innovative tool for ionizing radiation related health risk assessment and monitoring.
Drawings
FIG. 1 shows the concentrations of SAA, MMP-2 and IGFBP-3 in mouse serum.
FIG. 2 is a graph showing MSD measurements of SAA, MMP-2 and IGFBP-3 in mouse serum.
FIG. 3 is a ROC curve of SAA, MMP-2 and IGFBP-3 in mouse serum after ionizing radiation.
Fig. 4 is a sample dilution detection simulation curve.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 acquisition of biomarkers for assessing ionizing radiation injury
Kunming mice of about 6 weeks of age were selected and randomly divided into 2 groups of 6 animals each. Different doses of whole body radiation were received, respectively, wherein low LET radiation (X-rays) was generated by an X-RAD 225-type X-ray apparatus, with radiation doses of 0Gy and 1Gy. After irradiation, whole blood samples are obtained by adopting an eyeball blood sampling method at 24 hours, clotting is carried out at room temperature for 2 hours, centrifugation is carried out at 2000g for 20 minutes, serum is separated, and samples without hemolysis are selected to be stored at-80 ℃, or subsequent experiments are directly carried out.
Pre-experiments have found that protein IGFBP-3, MMP-2 and SAA are expressed in post-irradiated mouse serum to further verify the reliability of the results, we performed an exact result verification using Elisa kit (Irelett, china), which showed that protein SAA, MMP-2 and IGFBP-3 are expressed significantly (P < 0.05) in post-irradiated mouse serum (FIG. 1), and therefore, they can be used as potential biomarkers for assessing ionizing radiation damage. Wherein: protein SAA (serum amyloid A) is an apolipoprotein that binds to High Density Lipoprotein (HDL) and has multiple subtypes, some of which are normally stable. SAA is an acute phase response protein involved in cholesterol transport and metabolism, chemotaxis of inflammatory cells, degradation of extracellular matrix, and modulation of immune and signaling pathways. MMP-2 (matrix metalloproteinase-2) is a zinc-dependent endopeptidase capable of specifically degrading type IV collagen, which is the major component of the basement membrane. MMP-2 plays an important role in the processes of embryo development, tissue remodeling, angiogenesis, tumor metastasis and the like. IGFBP-3 (insulin-like growth factor-binding protein 3) is a protein capable of binding insulin-like growth factor (IGF), one of the most abundant members of the IGFBP family. IGFBP-3 may modulate the half-life and bioavailability of IGF in the blood and may also affect the binding between IGF and its receptor. IGFBP-3 also has biological activity and can affect the processes of cell proliferation, differentiation, apoptosis, migration and the like. IGFBP-3 is closely associated with Growth Hormone (GH) secretion and is also associated with a variety of metabolic and tumor-related diseases.
Example 2 construction of detection kit for detecting SAA, MMP-2 and IGFBP-3 multifactorial MSD
This example prepares a test kit for assessing ionizing radiation damage based on the biomarkers SAA, MMP-2 and IGFBP-3 obtained in example 1. The method comprises the following steps:
1. materials and compositions
SAA, MMP-2 and IGFBP-3 antibody pairs (accession numbers: ab241828, ab256748 and ab256673, abcam, U.S.A.), respectively;
conjugation buffer:10 XPBS (cat: E607016, industry, china), pH 7.9;
conjugation Storage buffer:1 XPBS (cat number: G420, soy Bao, china) (containing 0.05% NaN) 3 )，pH 7.4；
Sample diluent: 1 XPBS (containing 0.5% BSA (cat. No. A8020, soy, china));
wash (WS buffer): 0.05% PBST (cat# C100530, innovative, china);
filter filters (0.22 μm) (cat# SLGP033RB, millipore, USA);
1.5mL centrifuge tube and 15mL centrifuge tube;
protein quantification kit (cat# C100530, innovative, china);
biotin quantitative kit (Biotin Labeling Kit-NH2, cat# LK03, toosendan Chemie, japan);
amicon Ultra-0.5 centrifugal filter (cat# UFC503024, millipore, USA) with Ultra-30 filter membrane;
dimethyl sulfoxide (DMSO, cat# D8370, soy pal, china);
U-PLEX Development Pack,96-well 3-Assay (including U-PLEX Linker Set; U-PLEX 3-Assay,96-Well SECTOR Plate; U-PLEX Stop Solution; MSD GOLD Read Buffer B) (cat. Number: K15228N-2,Meso Scale Diagnostics, U.S.).
2. Test method
The antibodies were placed on ice and the other materials were equilibrated at room temperature for 30min before starting the test.
2-1, biotinylated conjugated antibodies
Biotinylated linking of the antibodies was performed according to the Biotin Labeling Kit-NH2 kit protocol.
1) 100. Mu.L of WS buffer and one of the pair of antibodies containing 100. Mu.g were added to the filter tube. Centrifuge at 8,000-10,000g for 10min.
2) mu.L of DMSO was added to the NH2-reactive biotin and dissolved by pipetting.
3) 100. Mu. L Conjugation buffer and 8. Mu.L of NH2-reactive biotin solution were added to the filter tube and mixed by blowing.
4) The filter tube was placed in an incubator and incubated at 37℃for 10 minutes.
5) mu.L of WS buffer was added to the filter tube and centrifuged at 8,000-10,000g for 10min to remove the filtrate.
6) 200. Mu.L of WS buffer was added to the filter tube and centrifuged at 8,000-10,000g for 10min, and the procedure was repeated once.
7) 100 mu L Conjugation Storage buffer was added to the filter tube and blown 10-15 times to recover the labeled product. The solution was transferred to a 0.5mL tube and stored at 0-5 ℃.
8) Calculating the concentration of the binding protein; the molar concentration of biotin conjugate (biotin quantification kit) was calculated.
9) Determination of the biotin protein binding ratio (table 1):
the molar concentration of biotin conjugate/molar concentration of conjugated protein.
TABLE 1 ratio of biotin protein binding
| Antibodies to | MMP2 | SAA | IGFBP3 |
| Binding ratio of biotin proteinRate of | 1.159 | 0.256 | 1.389 |
10 Biotin-conjugated antibodies can be stored at 2-8 degrees for one year or used directly in subsequent experiments.
2-2 preparation of Single biotinylated Capture antibody and U-PLEX Linker coupling reagent
The individual biotinylated antibodies were coupled to a unique Linker U-PLEX Linker, the biotinylated antibodies were diluted to 10. Mu.g/mL with a coating buffer, and the antibody identity was recorded. U-PLEX Linker 1 is labeled IGFBP1, U-PLEX Linker 3 is labeled SAA, and U-PLEX Linker 10 is labeled MMP2.
1) 200. Mu.L of biotinylated antibody (10. Mu.g/mL) was added to 300. Mu.L of labeled U-PLEX Linker, vortexed, and allowed to stand for 30min without shaking. ( And (3) injection: each U-PLEX Linker bottle matches one color cap and label. And centrifuging before uncapping, and slightly opening the bottle cap. The U-PLEX Linker was opened one at a time and the lid was closed immediately after use. Multiple experiments suggested that the same antibody used the same U-PLEX Linker. )
2) 200. Mu.L of Stop solution was added, vortexed, and room temperature was maintained for 30min.
Note that: at the end of step 1), the coating concentration of each U-PLEX Linker conjugated antibody solution was 10-fold and could be stored at 2-8 degrees. The storage time should not exceed 7 days.
2-3, preparation of multiple coating solutions
1) 600. Mu.L of each U-PLEX linker conjugated antibody solution was combined into 15mL tubes and mixed by vortexing. Up to 10U-PLEX Linker conjugated antibodies can be pooled (U-PLEX Linker conjugated antibody solutions sharing the same U-PLEX Linker are not used in combination when fewer than 10 antibodies are conjugated).
2) When less than 10 antibodies were bound, the solution was brought to 6mL by mixing with the stop solution to give a final 1-fold concentration. By vortex mixing.
Note that: at the end of step 2), the U-PLEX composite coating solution was 1X and could be stored at 2-8 degrees. The storage time should not exceed 7 days.
2-4, coated U-PLEX 96 well plate
1) To each well 50 μl of the composite coating solution was added. Seal the plate with an adhesive plate seal and shake at room temperature for 1 hour.
2) Plates were washed 3 times with at least 150 μl/well 1 x wash buffer or PBST.
The MSD sheet is now coated and ready for use. The plates can be stored in original bags with desiccant and sealed at 2-8deg.C for 7 days.
2-5, detection antibody electrochemiluminescence labeling (MSD GOLD) TM SULFO-TAG)
1) Pre-linking process
a. 1-2mg/mL of linker protein (dissolved in Conjugation buffer) was prepared. The protein solution may be concentrated or replaced with buffer using an equilibrated Conjugation Buffer column for Zeba spin desalination.
b.23 degree (20-25 degree) Balanced antibody protein
c. The amount of SULFO-TAG NHS-Ester required was calculated according to the following formula
The formula:
1000 Xprotein concentration (mg/mL)/protein MW (Da). Times.Change ratio. Times.protein volume (μL) =SULFO-TAG NHS-Ester amount (nmol)
The Challenge ratio is between 5:1 and 20:1
SULFO-TAG NHS-Ester (nmol) amount/SULFO-TAG NHS-Ester concentration (nmol/. Mu.L) =SULFO-TAG NHS-Ester volume (mu.L)
2) Linking process
a. The vial of SULFO-TAG NHS-Ester was gently tapped, centrifuged at 1000g for 1min to collect the lyophilizate, and 50. Mu.L of cold distilled water was added to 150nmol of the vial of SULFO-TAG NHS-Ester to prepare a stock solution with a concentration of 3 nmol/. Mu.L, gently vortexed, and stored on ice for a maximum of 10min before use.
Note that: for < 100. Mu.g of conjugated protein, 100. Mu.L of Conjugation Buffer was added to the above mother liquor to give a solution of SULFO-TAG NHS-Ester at a concentration of 1 nmol/. Mu.L.
b. Adding the calculated volume of SULFO-TAG NHS-Ester calculated in the pre-linking process c according to the (2-5. Electrochemiluminescence labeling of detection antibody step 1) to the protein solution in the remaining antibody pair of biotin-coupled protein, and immediately vortexing. The remaining SULFO-TAG NHS-Ester was discarded.
c.23℃for 2 hours (20-25 ℃). The test tube was covered with aluminum foil or placed in a dark place protected from light.
3) Post linking process
a. Preparing a Zeba spin desalting column, removing the bottom seal of the column, and releasing the cover without removing the cover, placing the column in a collection tube, removing a storage buffer, and washing three times with MSD Conjugation Storage Buffer, wherein each step is 2-8 degrees.
b. The coupling reagent was added to the center of the spin column in a drop-like fashion and the coupling protein was collected in a clean fresh collection tube with the binding protein present in the eluate. The column was discarded.
c. The binding protein was filtered through a 0.2um filter column.
d. The binding protein concentration (BCA, bradford or Lowry) was calculated without using OD280 absorbance measurements.
e. The absorbance of the MSD SULFO-TAG protein conjugate at 455nm was measured with a spectrophotometer. The measured value was divided by the path length (cm) and by the TAG extinction coefficient (15400M-1 cm-1) to give the MSD SULFO-TAG TAG concentration (mol/L).
f. Determination of the SULFO-TAG TAG from calculations in worksheets protein reference (Table 2)
(SULFO-TAG TAG: protein). MSD SULFO-TAG binding proteins may be sensitive to prolonged exposure to light and should be stored in dark or amber or opaque vials. Antibody conjugates are generally stable for at least 2 years, with the final retention time determined by the antibody shelf life.
TABLE 2 protein reference ratio of SULFO-TAG TAGs
| Antibodies to | IGFBP-3 | SAA | MMP-2 |
| SULFO-TAG TAG protein reference | 3.7209 | 1.7291 | 18.1329 |
SAA, MMP-2 and IGFBP-3 multifactor MSD detection kits have been constructed and can be used in subsequent experiments.
Example 3, SAA, MMP-2 and IGFBP-3 multifactor MSD detection methods
1. Sample pretreatment
Collecting a serum sample to be detected, and performing pretreatment to remove interfering substances;
blood collection is carried out by a conventional blood collection method, and if the sample contains visible impurities, the impurities are removed by centrifugation. Placed on ice. The assay is preceded by dilution with a quantity of sample diluent.
2. Semi-quantitative detection of SAA, MMP-2 and IGFBP-3
2-1, adding samples
1) To each well 25 μl of sample diluent was added. The plate was gently tapped on each side, the reagents were tiled, and no bubbles were generated.
2) To each well 25 μl of the prepared calibrator standard or sample was added. Sealing the plate with an adhesive plate seal. The culture was allowed to shake at room temperature for 1 hour.
2-2, washing and adding a detection antibody solution
1) Each detection antibody (100X) was taken in 60. Mu.L to 15mL centrifuge tubes, diluted with antibody diluent to a final detection antibody concentration of 1X and a final volume of 6mL.
2) Plates were washed 3 times with at least 150 μl/well of 1 x wash buffer.
3) To each well 50 μl of detection antibody solution was added. Sealing the plate with an adhesive plate seal. The culture was allowed to shake at room temperature for 1 hour.
2-3, washing and detection
1) Plates were washed 3 times with at least 150 μl/well of 1 x wash buffer.
2) 150 μ L MSD GOLD Read Buffer B was added to each well. Plates on MSD instrument were analyzed.
EXAMPLE 4 evaluation of Effect of SAA, MMP-2 and IGFBP-3 on detection of ionizing radiation
Kunming mice of about 6 weeks of age were selected and randomly divided into 4 groups of 6 animals each. The whole body irradiation was respectively received at different doses, wherein low LET rays (X-rays) were generated by an X-RAD 225 type X-ray apparatus, and high LET (carbon ion beam) was provided by a Lanzhou heavy ion accelerator cooled storage ring (HIRFL-CSR) biological irradiation shallow terminal. The irradiation dose is as follows: x-rays (0 Gy, 0.5Gy, 1Gy, and 4 Gy), and carbon ion beams (0 Gy, 0.1Gy, 0.5Gy, and 2 Gy). After irradiation, whole blood samples were obtained by an eyeball blood sampling method for 24 hours, coagulated for 2 hours at room temperature, centrifuged for 20 minutes at 2000g to separate serum, and samples without hemolysis were selected to be stored at-80 ℃ or subjected to subsequent experiments directly.
SAA, MMP-2 and IGFBP-3 OD values were determined in test mouse serum using the MSD kit established in example 2 and the method of example 3 (results are shown in FIG. 2). After detection of ionizing radiation, the OD values and relative amounts of SAA, MMP-2 and IGFBP-3 were increased to varying degrees in the serum of the mice. In the X-ray irradiated group, the relative content of IGFBP-3 was significantly higher at doses of 0.5Gy, 1Gy and 4Gy than in the control group (P < 0.05), while the relative content of MMP-2 and SAA was significantly higher at doses of 1Gy and 4Gy than in the control group (P < 0.01). The relative amounts of SAA, MMP-2 and IGFBP-3 were also higher in the carbon ion irradiated group than in the control group, but the differences were not significant (P > 0.05). It can be determined that all three protein molecules have strong radiation sensitivity and can be used as ionizing radiation markers of X rays.
The invention draws ROC curves (FIG. 3) according to experimental data, and calculates AUC values, sensitivities and specificities of various biomarkers in serum samples after irradiation.
As can be seen from FIG. 3, the AUC values of the three indicators IGFBP-3, SAA and MMP-2 in serum of the invention for determination of ionizing radiation injury after X-ray treatment are respectively: AUC values for ionizing radiation injury determinations after combination of the three indicators of 0.867, 0.817 and 0.925 were: 0.867, all significantly higher than 0.5, indicates that all three indicators have higher diagnostic value. Wherein the highest AUC value of MMP-2 indicates the best diagnostic effect. In addition, the progressive significance of the three indexes is respectively: there was a significant difference between 0.005, 0.014 and 0.001 (P < 0.05), the progressive significance of the combination was 0.007, indicating that they had different discrimination capability for ionizing radiation damage determination. According to the Youden index method, we can calculate that the corresponding sensitivities of the three indexes are respectively: 1.1 and 0.733, the combined sensitivity is 0.733; the specificity is respectively as follows: 0.625, 0.725 and 1, the specificity of the combination is 1. The sensitivity and specificity of the three indexes are different, and IGFBP-3 and SAA have the highest sensitivity, which means that the covering capacity of the IGFBP-3 and SAA on ionizing radiation injury is the widest; MMP-2 and the three marker combinations have the highest specificity, which means that the removal capacity of the combination is the strongest for non-ionizing radiation damage; IGFBP-3 has the lowest specificity, indicating that it is prone to false positives.
In summary, the specificity and sensitivity of IGFBP-3, SAA and MMP-2 in serum of the invention for use in ionizing radiation injury determination using ROC curve analysis can be concluded as follows: the IGFBP-3, SAA and MMP-2 in serum and the combination thereof have higher diagnostic value and distinguishing capability, and can be used as effective indexes for judging ionizing radiation injury. Among the three indicators, MMP-2 and the combination of the three indicators have the highest AUC value and specificity, which indicates that the diagnostic effect is the best, and can be used as the first-choice indicator.
After carbon ion radiation treatment, the AUC values of IGFBP-3, SAA and MMP-2 in serum are all greater than 0.7, the combined AUC is: 0.607, and significantly higher than the level of random guessing (auc=0.5), all three indicators have a certain diagnostic value. Of these three criteria, MMP-2 has the highest AUC (0.743), has the best diagnostic effect, and can maintain higher sensitivity and higher specificity. SAA and IGFBP-3 have similar AUC values (0.722 and 0.708), have comparable diagnostic effects, and their ROC curves are approximately coincident, indicating that they have no significant difference in their ability to recognize ionizing radiation damage at different thresholds.
The present study uses a linear simulation approach to establish a mathematical model between radiation dose and measurements of SAA, MMP-2 and IGFBP-3 before and after radiation (three data were randomly extracted for subsequent model data validation) and to arrive at the following simulation formula (equation 1). According to the modeling formula, a positive correlation is exhibited between the radiation Dose (Dose) and the measurements of SAA, MMP-2 and IGFBP-3 before and after radiation, i.e., the higher the measurement of these three indicators, the higher the radiation Dose. This indicates that all three indexes reflect the damage degree of ionizing radiation to human body. In addition, both the X-ray simulation and the carbon ion simulation showed a higher goodness of fit (R 2 All are larger than 0.8), which shows that the simulation formula can better fit the change of the data. Wherein the correlation coefficient of the carbon ion simulation is slightly higher than that of the X-ray simulation, and the influence of the carbon ion radiation on the human body is presumed to be more remarkable and consistent. Furthermore, the extent to which different types of radiation affect different indicators varies. SAA has the greatest effect on radiation dose (coefficient 0.272) for X-ray radiation and MMP-2 has the greatest effect on radiation dose (coefficient 0.423) for carbon ion radiation. This suggests that different types of radiation may have different regulatory effects on the expression or activity of different proteins.
Equation 1: mathematical model between radiation dose and measurements of SAA, MMP-2 and IGFBP-3 before and after radiation:
x-ray simulation: dose= -0.264+0.112 IGFBP-3+0.242 SAA+0.168 MMP-2
R 2 ＝0.892
Carbon ion simulation: dose= -0.899+0.254 IGFBP-3+0.216 SAA+0.423 MMP-2
R 2 ＝0.862
IGFBP-3＝OD IGFBP-3 /Mean(OD IGFBP-3Ctrl )
SAA＝OD SAA /Mean(OD SAA Ctrl )
MMP-2＝OD MMP-2 /Mean(OD MMP-2Ctrl )
To evaluate the accuracy of the model, the measurement data is used for verification according to the constructed linear simulated regression model. Three sets of detection data relative values (OD/Mean (OD) were randomly chosen that were not used to construct the linear regression model for verification of the model Ctrl ) IGFBP-3, SAA and MMP-2 are respectively: 1.411,1.431,1.168;1.855,2.805,1.697 and 1.611,2.934,1.884, the predicted values are: 0.5,1,1 is brought into a regression model, and calculated simulation values are respectively as follows: 0.436,0.908,0.943. Through correlation analysis, the pearson correlation coefficient is calculated to be 0.998, and the difference between the calculated value and the predicted value is small, so that the linear simulation curve can be well fit with the data. The simulation curve can well reflect the change rule of the data.
Example 5 determination of stability, sensitivity, dynamic Range of the kit
1. Experimental materials and reagents
Animal serum, SAA, MMP-2 and IGFBP-3 multifactor MSD detection kit
2. Experimental method
See example 4.
3. Results and analysis
3-1, sensitivity
For each factor assay, we measured the lower limit of detection (LLOD) (table 3). Serum samples have higher sensitivity even when diluted 1000-fold. The test results are shown in Table 3.
TABLE 3 serum dilution 1000X, detection values for each antigen
3-2, data repeatability detection
To evaluate the data reproducibility of the kit, we randomly selected 10 mouse serum samples for multiple measurements. We calculated the coefficient of variation (% CV) of the OD values of the individual biomarkers (SAA, MMP-2 and IGFBP-3) in each sample and averaged them. The coefficient of variation is an indicator for measuring the degree of dispersion of data, which is equal to the standard deviation divided by the average value, expressed in percentages. The smaller the coefficient of variation, the more stable the data, and the better the reproducibility. Our test results showed that the average% CV of OD values for each biomarker was below 10% in all samples, with most of the% CVs measured below 5% and only the detection of SAA in one well was slightly above 5% (7.5423%), indicating that the kit had better data reproducibility (see table 4).
Table 4, measured values and CV% values for each well (IGFBP-3, SAA, and MMP-2).
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3-3, sample dilution detection Linear analysis
To evaluate the linear range of different concentrations, we measured serum samples at different dilution factors and calculated the linear correlation between the measured values of IGFBP-3, SAA, and MMP-2 and the dilution factors. We found that the measured values and dilution factors of these three factors exhibited strong negative correlation in the 50-1000 fold dilution concentration range, R 2 The values are all higher than 0.85, and the fitting degree is stronger (figure 4).
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (10)
1. Use of a biomarker and/or a substance detecting said biomarker for any of the following:
a1 Use in the manufacture of a product for detecting or assessing ionizing radiation damage;
a2 Use in the manufacture of a product for identifying exposure to ionizing radiation;
the biomarker is MMP-2 protein, SAA protein and/or IGFBP-3 protein.
2. The use of claim 1, wherein the agent that detects the biomarker comprises an agent for detecting the amount of MMP-2 protein, SAA protein, and/or IGFBP-3 protein expressed.
3. The use of claim 2, wherein the agent comprises an antibody, polypeptide, protein or nucleic acid molecule that binds to MMP-2 protein, SAA protein and/or IGFBP-3 protein.
4. A kit for assessing ionizing radiation damage or identifying ionizing radiation exposure, characterized in that the kit comprises a substance for detecting the biomarker according to any of claims 1-3.
5. The kit of claim 4, comprising a capture antibody immobilized on an electrode, wherein the capture antibody is an anti-MMP-2 antibody, an anti-SAA antibody, and/or an anti-IGFBP-3 antibody.
6. The kit of claim 5, wherein the capture antibody has a biotin tag.
7. The kit of any one of claims 4-6, wherein the kit comprises a detection antibody that binds to an electrochemiluminescent tag, the detection antibody being an anti-MMP-2 antibody, an anti-SAA antibody, and/or an anti-IGFBP-3 antibody.
8. The kit of claim 7, wherein the electrochemiluminescent label is a ruthenium complex or an iridium complex.
9. The kit of any one of claims 5-8, further comprising a streptavidin-coated plate, a standard, a sample diluent, an antibody diluent, a stop solution, a read-plate solution, and/or a wash buffer.
10. A biomarker, or a substance that detects the biomarker, according to any of claims 1 to 3.
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