CN116068203A - Application of DDX5 in preparation of kit for diagnosing or detecting systemic lupus erythematosus - Google Patents

Abstract

The invention discloses an application of DDX5 in preparing a reagent kit for diagnosing or detecting systemic lupus erythematosus, which is used for detecting DDX5 in serum, can help to diagnose systemic lupus erythematosus, has wide clinical diagnosis application value and improves the positive rate of SLE detection. The invention has strong specificity, high sensitivity and low detection limit, and can respond quickly.

Description

Application of DDX5 in preparation of kit for diagnosing or detecting systemic lupus erythematosus

Technical Field

The invention belongs to the field of antigen detection, and particularly relates to application of DDX5 in preparation of a diagnostic or detection system lupus erythematosus kit.

Background

Fully human monoclonal autoantibody anti-DDX 5 antibodies were obtained in the peripheral blood of SLE patients and verified that their target protein was read-box RNA (DDX 5). DDX5 is an RNA-binding protein that plays an important role in maintaining cellular homeostasis, regulating cellular differentiation, and reprogramming somatic cells. At present, anti-double-stranded DNA antibodies and anti-Sm antibodies in serum are important indexes for diagnosing SLE, but the sensitivity and the accuracy of the anti-double-stranded DNA antibodies and the anti-Sm antibodies are low, and the level of the anti-double-stranded DNA antibodies and the anti-Sm antibodies and the activity and the clinical manifestation of SLE are not obviously related, so that a novel diagnosis marker of SLE with high performance needs to be searched. DDX5 is used as a diagnosis marker of SLE, the level of DDX5 in serum is detected by using a flow cytometry, the level of DDX5 in different autoimmune patients is compared, the specificity and sensitivity of DDX5 and common indexes for diagnosing SLE are compared, and the accuracy of DDX5 diagnosis alone and SLE diagnosis combined with other indexes is compared, so that the application value of DDX5 for diagnosing SLE and identifying different autoimmune diseases is evaluated. The most commonly used method in the existing serum antigen/antibody detection field is enzyme-linked immunosorbent assay (ELISA), which is a qualitative and quantitative detection method for carrying out immune reaction by combining soluble antigen or antibody onto a solid carrier and keeping the immune activity of the soluble antigen or antibody and utilizing the specific combination of antigen and antibody. ELISA is classified into direct assay, indirect assay, double antibody sandwich assay and competition assay. The direct method is characterized in that the enzyme-labeled antigen or antibody directly reacts with the corresponding antibody or antigen to be detected, but the experimental operation is inconvenient, the sensitivity is low, and the method is mainly used for detecting the antigen of which the secondary antibody is difficult to prepare. The indirect method refers to the generation of an anti-antibody by indirectly binding an enzyme to an antibody to be detected. In general, the anti-antibody has higher titer than the antibody, and one molecule of antibody can react with a plurality of molecules of anti-antibody, so that the detection antibody is mostly tested by adopting an enzyme-labeled anti-antibody through an indirect method so as to obtain higher sensitivity. The sandwich method is to react corresponding antigen and antibody successively, to perform multiple binding and multistage amplification, and has wide application range and high sensitivity. The competition method is mainly to compete enzyme-labeled antigen with antigen in the sample to be detected for corresponding quantitative antibody. The method has few operation steps and quick result, but the enzyme-labeled antigen is not easy to manufacture in time, has larger dosage and lower sensitivity, and is mainly used for detecting small-molecule antigens and hapten which are difficult to finish by a sandwich method. The ELISA results show that the substrate Optical Density (OD) value is within a certain range, or the positive value is more than 2.1 times greater than the negative control value, so that the ELISA results have certain credibility. The ELISA operation steps are complex, and factors influencing experimental results are more, for example, false positive results can appear if an ELISA plate is not cleaned, an ELISA antibody is adsorbed on a hole wall and the like, and false negative results can appear if a reagent is not balanced to room temperature, excessive washing and the like. In some experiments, if the concentration of the target protein in the sample is extremely low and the OD value obtained after detection by the ELISA is low and is not obvious, high-sensitivity ELISA can be tried, but the ELISA does not mean that the concentration of the sample can be detected, and the detectable rate and the OD value of the sample can be improved only to the greatest extent, so that the sample is positioned in a standard curve, and a more reliable result is obtained compared with the common ELISA.

The existing diagnostic method often needs antigen to be combined with solid phase, and the characteristics of difficult separation and difficult analysis of small molecule soluble antigen in serum bring huge obstacle to accurate detection, and meanwhile, due to different sources of antigen (natural or artificial synthesis) and different presentation modes of antigen (covalent coupling or crosslinking and the like), corresponding detection methods are also different, the repeatability of measurement results of different methods and even the consistency of trend of results are poor, so that the standardization of key index tests such as anti-double-stranded DNA (dsDNA) antibodies is challenging. Taking the detection of anti-dsDNA antibodies as an example, the detection of anti-dsDNA antibodies by ELISA requires binding of dsDNA to a solid phase, but this process can alter its antigenicity resulting in differences in performance characteristics between different assays, which prevents standardization and accuracy of the anti-dsDNA antibody ELISA detection.

The flow type liquid phase protein quantitative technology (CBA), namely cytokine microsphere detection technology, is a multiple protein quantitative detection method based on a flow type cell detection system, and can detect multiple indexes in a single sample at the same time. The basic principle of CBA is similar to ELISA detection in that each CBA microsphere is uniform in size, has a specific fluorescent intensity, is coated with a specific capture antibody suitable for a specific assay (such as other antibodies or soluble proteins), and provides a capture surface similar to an ELISA well plate. Compared with the traditional ELISA and Western Blotting (WB), CBA has the advantages of less sample and high sensitivity, and only 25-50 mu l sample is needed for each detection, but the sensitivity can reach pg/ml level; the experimental steps are simple and convenient, and the average time is only 5 hours; each microsphere has specific fluorescence intensity, and can simultaneously measure tens of proteins at most in one test tube, so that the experimental repeatability is high. The CBA quantifies the target protein according to the fluorescence intensity of the antibody, eliminates false positive results generated by enzyme-linked amplification in an ELISA sandwich method, and has no interference of enzyme-substrate background fluorescence. In addition, the accuracy of CBA is also higher, and each microsphere can provide a surface combined with specific protein, so that each microsphere is equivalent to a single ELISA plate which is coated well, and the specific accurate detection of single molecules can be achieved in the field of single microspheres.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and provides an application of DDX5 in preparing a reagent kit for diagnosing or detecting systemic lupus erythematosus.

Preferably, the kit is used to detect DDX5 in a biological sample, and up-regulation of DDX5 expression indicates that systemic lupus erythematosus is involved.

Preferably, the biological sample is serum.

It is a further object of the invention to provide a kit for diagnosing or detecting systemic lupus erythematosus, the kit comprising DDX5.

The invention also aims to provide a method for detecting DDX5 in serum for non-diagnostic purposes, which adopts the following technical scheme:

step (1), adding Dithiothreitol (DTT) solution into polystyrene microspheres, uniformly mixing, incubating for a period of time at room temperature in a dark place, adding coupling buffer solution, centrifuging, and discarding supernatant; repeatedly adding coupling buffer solution, centrifuging, discarding supernatant for multiple times, and then adding coupling buffer solution for resuspension to obtain microsphere heavy suspension;

preferably, after being uniformly mixed, the mixture is incubated for 1h at room temperature in a dark place;

preferably, the step of adding coupling buffer-centrifuging-discarding the supernatant is repeated 3 times.

Step (2), adding a solution of sodium 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester (sulfo-SMCC) into the DDX5 antibody solution, uniformly mixing, and incubating at room temperature in a dark place to obtain a DDX5 antibody-SMCC solution; adding DDX5 antibody-SMCC solution into microsphere heavy suspension, mixing, and incubating at room temperature in dark place;

preferably, the DDX5 antibody-SMCC solution is obtained after incubation for 1h at room temperature and in the dark;

preferably, the DDX5 antibody-SMCC solution is added into microsphere heavy suspension, and the mixture is incubated for 1h at room temperature in a dark place after being uniformly mixed.

Step (3), adding an N-ethylmaleimide (NEM) solution into the solution after the incubation in the step (2), uniformly mixing, and incubating at room temperature in a dark place; adding a storage buffer solution, centrifuging, and discarding a supernatant; repeatedly adding a storage buffer solution, centrifuging, discarding the supernatant for a plurality of times, and then adding the storage buffer solution for re-suspending to prepare a capture microsphere fluorescent probe;

preferably, the mixture is incubated for 15min at room temperature in a dark place after being uniformly mixed;

preferably, the steps of adding the storage buffer-centrifuging-discarding the supernatant are repeated 3 times.

Step (4), uniformly mixing the solution re-suspended in the step (3), PBS solution, a serum sample and an AF 488-labeled antibody solution, incubating at room temperature in a dark place, adding a washing buffer solution, centrifuging, discarding the supernatant, adding the washing buffer solution for re-suspension, and detecting by using a flow cytometer;

preferably, incubation is carried out at room temperature in the absence of light for 3h.

Compared with the prior art, the invention has the beneficial effects that:

the application of the DDX5 in preparing the reagent kit for diagnosing or detecting the systemic lupus erythematosus proves the dynamic relation between the expression quantity of the DDX5 and the systemic lupus erythematosus, and the provided reagent kit has good specificity and high sensitivity, can qualitatively measure the level of the DDX5 in human serum, and is beneficial to assisting early diagnosis of the systemic lupus erythematosus.

According to the invention, the functional microspheres marked by APC (advanced fluorescent powder) and the AF488 are marked, when FCM is used for detecting DDX5 in serum, the combination of the DDX5 and the functional microspheres and whether the combination is marked by the APC can be clearly seen from an image, and the result is visualized, wherein the detection limit is as low as 20pg/ml.

Drawings

FIG. 1 is a schematic flow chart of the preparation of a fluorescent probe according to the present invention;

FIG. 2 is a graph showing experimental results of a double positive control group in the example;

FIG. 3 is a graph showing experimental results of a single positive control group in the example;

FIG. 4 is DDX5 levels of healthy control HC serum samples in the examples;

FIG. 5 is serum DDX5 levels of SLE patients with systemic lupus erythematosus in the examples;

FIG. 6 shows the ROC curve analysis ELISA and the detection of DDX5 according to the invention for SLE diagnosis.

Detailed Description

The invention will be further described with reference to the accompanying drawings and examples.

Three control groups are set, namely a double positive serum sample, a single positive serum sample and a healthy control HC serum sample, and the serum sample of SLE patients with systemic lupus erythematosus is taken as an experimental group. Four groups of serum samples were tested using the following method:

1.1 instruments and reagents

CytoFLEX S flow cytometer (BECKMAN COULTER cat# B75408), centrifugal rapid pre-loaded desalting column (Biotyscience cat# BIOL-013), vortex, microcentrifuge, shaker, microcentrifuge tube, and lightproof iron box.

CBAFunctional Bead Conjugation Buffer Set (BD accession number: 558556), DTT (Pierce accession number: 20290), sulfo-SMCC (Pierce accession number: 22322), NEM (Pierce accession number: 23030), DMSO (Pierce accession number: 20684).

1.2 preparation of probes

All the used reagents are restored to room temperature, and the whole process of preparation and detection is protected from light.

(1) Activated microsphere

1) The microspheres were vortexed for 30s.

2) 75ul of microspheres were placed in a microcentrifuge tube and protected from light.

3) To the microcentrifuge tube, 1.9uLDTT solution (1M) was added and vortexed for 5s.

4) Incubate on a shaker at room temperature for 1h in the dark. (step 2.2 during the preparation of the sulfoSMCC solution)

5) 1ml of coupling buffer was added and vortexed for 5s.

6) Centrifuge at 900 Xg for 3min and aspirate the supernatant.

7) Repeating the steps 5) and 6) three times.

8) Add 20uL coupling buffer for resuspension.

(2) Protein modification

1) Mu.l of DDX5 antibody/PBS solution (0.165 mg/ml) was prepared.

2) A solution of sulfo-SMCC (2 mg/ml DI H2O) was prepared in advance.

3) To the antibody solution was added 2. Mu.l of sulfo-SMCC solution, which was vortexed for 5s.

4) Incubate on a shaker at room temperature for 1h in the dark. (preparation of chromatography column during incubation, step 3)

(3) Exchange buffer

1) Taking the desalting column, and removing the seal.

2) The stock solution was removed by centrifugation. Centrifuge at 1000 Xg for 2min and remove the stock.

3) And balancing the preassembled column. Add 350. Mu.l of coupling buffer, stand for 2min, centrifuge 1000 Xg for 2min. 200 μl of coupling buffer was added and the equilibration procedure repeated 2 times.

4) Sample treatment. Adding the modified antibody solution obtained in the step (2), standing for 30s, centrifuging at 1000 Xg for 2min, collecting a recovered sample, and preserving for later use.

(4) Protein binding

1) The DDX5 antibody/SMCC solution was transferred to microsphere heavy suspension and vortexed for 5s.

2) Incubate on a shaker at room temperature for 1h in the dark. (NEM solution was prepared during incubation, step 4.3)

3) NEM solutions (2 mg/ml DMSO) were prepared.

4) To the microcentrifuge tube was added 2. Mu.l NEM solution and vortexed for 5s.

5) Incubate on a shaker at room temperature for 15min in the dark.

6) To the microcentrifuge tube was added 0.5ml of storage buffer, and the supernatant was discarded by centrifugation at 900 Xg for 3 min.

7) Step 6) was repeated three times.

8) 0.5ml of storage buffer was added to resuspend. (final concentration 6X 10) ⁶ Microspheres/ml) were stored at 4℃protected from light.

The above process is shown in fig. 1.

1.3 flow cytometer detection

1) Mu.l of 1 XPBS solution was added to the microcentrifuge tube and 10. Mu.l of the capture microsphere heavy suspension was added and vortexed thoroughly.

2) Mu.l of serum samples were added.

3) 50 mu lAF 488-labeled antibody solution (2 ng/. Mu.l) was added. Vortex for 5s.

4) The tubes were incubated at room temperature in the dark for 3h.

5) 1ml of wash buffer (1 XPBS) was added, centrifuged for 200 Xg for 5min, the supernatant was aspirated, and 300. Mu.l of wash buffer was added to resuspend.

6) Vortex for 3-5 seconds, immediately check on machine.

Some of the results are shown in FIGS. 2-5, where the t-test analysis resulted in serum levels of DDX5 higher than HC (p < 0.05) in SLE patients.

The DDX5 levels of the control and experimental groups were measured by ELISA and compared with the present example, and the results are shown in fig. 6. From fig. 6, it can be seen that the specificity, sensitivity and AUC of the present invention all exhibit greater advantages compared to ELISA for detecting DDX5 levels to diagnose SLE.

Claims (9)

1. Application of DDX5 in preparing diagnosis or detection reagent kit for systemic lupus erythematosus.
2. 2. The use of claim 1, wherein the kit is for detecting DDX5 in a biological sample, and wherein up-regulation of DDX5 expression is indicative of having systemic lupus erythematosus.
3. 3. The use according to claim 2, wherein the biological sample is serum.
4. 4. A kit for diagnosing or detecting systemic lupus erythematosus, wherein the kit comprises DDX5.
5. 5. A method for detecting DDX5 in serum for non-diagnostic purposes, said method comprising the steps of:

   step (1), adding dithiothreitol DTT solution into polystyrene microspheres, uniformly mixing, incubating at room temperature in a dark place, adding coupling buffer solution, centrifuging, and discarding supernatant; repeatedly adding coupling buffer solution, centrifuging, discarding supernatant for multiple times, and then adding coupling buffer solution for resuspension to obtain microsphere heavy suspension;

   step (2), adding a sodium sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sodium salt sulfo-SMCC solution into the DDX5 antibody solution, uniformly mixing, and incubating at room temperature in a dark place to obtain a DDX5 antibody-SMCC solution; adding DDX5 antibody-SMCC solution into microsphere heavy suspension, mixing, and incubating at room temperature in dark place;

   step (3), adding an N-ethylmaleimide NEM solution into the solution after the incubation in the step (2), uniformly mixing, and incubating at room temperature in a dark place; adding a storage buffer solution, centrifuging, and discarding a supernatant; repeatedly adding a storage buffer solution, centrifuging, discarding the supernatant for a plurality of times, and then adding the storage buffer solution for re-suspending to prepare a capture microsphere fluorescent probe;

   and (4) uniformly mixing the solution subjected to the resuspension in the step (3), the PBS solution, the serum sample and the AF 488-labeled antibody solution, incubating at room temperature in a dark place, adding a washing buffer solution, centrifuging, discarding the supernatant, adding the washing buffer solution for resuspension, and detecting by using a flow cytometer.
6. 6. The method according to claim 5, wherein the number of times of repeating the addition of the coupling buffer-centrifugation-discarding of the supernatant in step (1) is 3.
7. 7. The method according to claim 5, wherein the incubation time at room temperature in the dark place in step (3) is 15min.
8. 8. The method according to claim 5, wherein the number of times of adding the storage buffer-centrifuging-discarding the supernatant repeatedly in step (3) is 3.
9. 9. The method according to claim 5, wherein the incubation time at room temperature in the dark place in step (4) is 3 hours.

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