CN108872591B - Biomarker for detecting juvenile dermatomyositis and application thereof - Google Patents
Biomarker for detecting juvenile dermatomyositis and application thereof Download PDFInfo
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Abstract
The invention discloses application of LIM zinc finger domain protein 1, namely LIMS1, or a fragment thereof in preparing a reagent for diagnosing Juvenile Dermatomyositis (JDM). The inventionThe high-density protein chip technology is adopted to screen the target antigen of the multiple myositis/dermatomyositis (PM/DM) related autoantibody, and 91 PM/DM related autoantibodies are determined. Adopting 91 PM/DM related autoantibodies to correspond to a target antigen to construct and prepare a PM/DM related autoantibody target antigen chip, and carrying out clinical amplification sample verification. The research result shows that the positive rate of the anti-LIMS 1(LIM zinc finger domain protein 1) antibody in the JDM group is 31.4 percent and is higher than the positive rate of the disease and health control group by 9.0 percent (P ═ 2.4 multiplied by 10)‑4) Meanwhile, the positive rate is 8.6 percent higher than that of the APM/ADM group (P is 3.3 multiplied by 10)‑4) The differences in the results were all significant. It was shown that the anti-LIMS 1 antibody can be used as a diagnostic and detection marker for juvenile dermatomyositis.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a biomarker for detecting juvenile dermatomyositis and application thereof.
Background
Idiopathic Inflammatory Myopathies (IIMs) are heterogeneous diseases characterized by proximal symmetric muscle weakness and multiple organ involvement, and mainly include clinical subtypes such as Polymyositis (PM), Dermatomyositis (DM), immune-mediated necrotizing myopathy (IMNM), sporadic inclusion body myositis (sIBM), and Juvenile Idiopathic Myositis (JIM). PM/DM is a striated muscle non-suppurative inflammatory myopathy autoimmune disease. The clinical characteristics are that serum creatinase is increased, electromyogram is changed in myogenesis, and muscle tissues such as limb girdle muscle, cervical muscle, pharyngeal muscle and the like are subjected to inflammation and degeneration change, so that symmetric muscle weakness and certain muscle atrophy are caused, and finally a plurality of systems and organs are involved, so that a patient dies. The disease can occur at any age, is bimodal, and has a peak at 5-14 years of age in children and 45-60 years of age in adults. The JIM subtype is most commonly referred to as Juvenile Dermatomyositis (JDM). Adult PM/DM (APM/ADM) is common among female patients, with a male to female ratio of about 1: 2.
autoantibodies are antibodies directed against tissues, organs, cells and cell components of the body, and are important weapons of the humoral immune system of the body to defend against pathogenic bacteria invasion and maintain the growth balance of the body. Serum autoantibody markers are produced during the pathogenesis of autoimmune diseases and often occur years or even more before the disease has a typical clinical presentation and any laboratory or imaging abnormalities. Evidence suggests that the onset of PM/DM is associated with autoimmunity: (1) histopathological examination showed that the patient's muscle tissue appeared to present T cells, macrophages, dendritic cells, B cells and plasma cells; (2) serum detection revealed the presence of autoantibodies to nuclear cytosolic components in PM/DM patients. Autoantibodies associated with myositis are classified into two categories based on their diagnostic value for disease: myositis-specific antibodies (MSAs) and myositis-associated antibodies (MAAs). MSAs have been reported to have anti-ARS antibodies (anti-Jo-1, PL-7, PL-12, EJ, OJ, KS, YRS, Zo antibodies), anti-Mi-2 antibodies, anti-MDA 5 antibodies, anti-TIF 1 antibodies, anti-NXP 2 antibodies, anti-SAE antibodies, anti-SRP antibodies, anti-HMGCR antibodies, anti-cN 1A antibodies; MAAs include anti-PM-Scl antibody, anti-Ku antibody, anti-SSA antibody, anti-Ro 50 antibody, anti-U1-RNP antibody, anti-SSB antibody, and AECA. MSAs may be associated with specific clinical subtypes, aid in predicting complications, aid in diagnosis, prognosis and prompt for clinical selection of the correct treatment strategy.
JDM is a chronic autoimmune inflammatory myopathy occurring in childhood, which is characterized by striated muscle and skin suppurative inflammation, and clinically manifested as proximal muscle weakness and various rashes, and organs such as digestive tract and lung can also be affected. The incidence of JDM is reported in foreign countries to be 2-4 people/million children, and the onset age is mostly 5-14 years. The etiology of the disease is unknown, and the disease is considered to be developed by the combined action of genetic susceptibility and environmental factors (such as infection). There is a certain difference in the order of appearance and incidence of clinical manifestations of JDM and ADM. The symptoms of children are similar to those of adults, but children often have urgent diseases, and adults often have hidden diseases. The infant usually suffers from skin lesions, then proximal muscle weakness occurs, and then general symptoms, often accompanied by fever, appear. Ectopic calcium deposits occur in children at higher rates than in adults, and are common in later stages of disease. JDM also exhibits visceral manifestations similar to ADM, but is more susceptible to gastrointestinal lesions, on the one hand ulceration, bleeding or perforation of the gastrointestinal tract due to vasculitis, and on the other hand is associated with diminished smooth muscle function. Heart diseases are less common than adults, and some children may have pericardial effusion and pleural effusion, and electrocardiogram may have changes such as conduction block. In the past, the prognosis of JDM was very poor, with mortality rates accounting for 1/3 in all children, and 1/3 remaining with lifelong disabilities. In recent years, JDM is becoming more and more deeply known and the number of treatment methods is increasing, the fatality rate of JDM is greatly reduced to 2% -3%, and the prognosis is also significantly improved. More and more clinicians are inclined to use non-invasive tests such as muscle nuclear magnetic scans instead of electromyography and muscle biopsies. It is worth mentioning that new advances in MSAs research have provided significant help for the diagnosis of JDM. However, the diagnostic value of the autoantibodies related to JDM reported at present is relatively low, and finding new markers remains the focus of research and a problem to be solved in clinic.
Disclosure of Invention
In order to solve the above problems, the present invention provides a use of LIM zinc finger domain protein 1, LIMs1, or a fragment thereof, in the preparation of an agent for diagnosing Juvenile Dermatomyositis (JDM).
In one embodiment of the invention, the diagnosis comprises: determining the level of antibodies reactive to LIMS1 or fragments thereof in a biological sample obtained from a patient exhibiting juvenile dermatomyositis; optionally, the step of (a) is carried out,
comparing the level of antibodies in the biological sample to adult polymyositis/dermatomyositis control data, other autoimmune disease control data, and healthy human control data, wherein a detectable increase in antibodies reactive to LIMS1 in the sample relative to the control data is indicative of a likelihood of developing juvenile dermatomyositis.
Wherein the biological sample is a serum sample.
Wherein the level of anti-LIMS 1 antibody is measured by the steps comprising:
a. contacting a biological sample from a patient with LIMS1 or a fragment thereof;
b. forming an antibody-protein complex between an antibody present in the biological sample and LIMS1 or a fragment thereof;
c. washing to remove any unbound antibody;
d. adding a detection antibody that is labeled and reactive with an antibody from the biological sample;
e. washing to remove any unbound labeled detection antibody; and
f. converting the label of the detection antibody to a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-LIMS 1 antibody in the patient.
Wherein said LIMS1 or fragments thereof are deposited on or immobilized on a solid support.
Wherein the solid support is in the form of a latex bead, a multi-well plate or a membrane strip.
Wherein the detection antibody is labeled by covalent attachment to an enzyme, a label with a fluorescent compound or metal, or a label with a chemiluminescent compound.
The present invention also provides a diagnostic kit for detecting and/or quantifying an anti-LIMS 1 antibody in a biological sample, comprising: a solid support, wherein said LIMS1 or fragment thereof is deposited on or immobilized on a solid support, and said anti-LIMS 1 autoantibody is useful as a biomarker for diagnosing juvenile dermatomyositis.
In one embodiment of the invention, the diagnostic kit further comprises a detection antibody that is labeled and reactive with an antibody from the biological sample.
Wherein the solid support is in the form of a latex bead, a multi-well plate, or a membrane strip.
The invention adopts the high-density protein chip technology to screen the target antigen of the PM/DM related autoantibody for 40 PM/DM patients, 30 autoimmune disease control patients and 20 healthy controls, and performs statistical analysis on the screening result to determine 91 PM/DM related autoantibodies. Then, a PM/DM related autoantibody target antigen protein chip is constructed and prepared by adopting 91 PM/DM related autoantibodies corresponding to target antigens, and serum of 100 APMs, 262 ADMs, 35 JDMs, 200 disease control patients (comprising 40 RA,40 SLE, 40 SS, 40 SSc and 40 chronic diseases) and 100 healthy controls is subjected to clinical expansion sample verification. The research result shows that the positive rate of the anti-LIMS 1 antibody in the JDM group is 31.4%, which is higher than the positive rate of 9.0% in the disease and healthy control group (P ═ 2.4X 10)-4) Meanwhile, the positive rate is 8.6 percent higher than that of the APM/ADM group (P is 3.3 multiplied by 10)-4) The differences in the results were all significant. It was shown that the anti-LIMS 1 antibody can be used as a diagnostic and detection marker for juvenile dermatomyositis.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
437 PM/DM patients enrolled in this study were sourced from Beijing cooperative hospitals and hospitals supported by industry fund topics. Of these, 131 male patients and 306 female patients were aged 2-79 years, with the mean age (44.07 ± 16.57) being the age of the patient. All PM/DM patients met the criteria for diagnosis of Bohan/Peter in 1975, and other autoimmune diseases such as Rheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), Sjogren's syndrome ((Sjogren's syndrome))
syndrome, SS), systemic sclerosis (SSc), and the like. 220 disease control groups, including 46 RA, group entry criteria: ACR differential diagnosis criteria in 1987; 47 SLE, entry criteria: 1997 ACR classificationA diagnostic criteria; 47 SS, group entry criteria: AECG differential diagnostic criteria; 50 cases of SSc, grouping criteria: 2013 SSc classification diagnostic criteria; 40 other chronic disease controls. 120 healthy control groups were obtained from Beijing coordination hospital examination center. Strictly according to the research requirements of proteomics, samples are reserved, peripheral venous blood is collected, serum is separated within 2 hours and then subpackaged, and the blood is frozen at minus 80 ℃ for standby.
EXAMPLE 1 high Density protein chip screening for PM/DM related autoantibodies
All protein antigens on the high-density protein chip are provided with GST tags at the N ends for protein purification. First, the quality of the chip is verified by hybridizing the rabbit anti-GST monoclonal antibody with the chip protein. Then, 40 sera of PM/DM patients, 30 control sera of autoimmune patients and 20 healthy control sera were selected and hybridized with 90 high density protein chips to identify candidate PM/DM associated autoantigens through signal collection and data analysis.
1.1 extraction of high Density protein chip Scan data
Scanning a high-density protein chip by adopting a GenePix 4000B chip scanner, obtaining a TIFF format gray image, automatically completing the segmentation and extraction of each protein point signal pixel by analysis software according to an operation manual of GenePix Pro 6.0 software and sample point matrix parameters on the high-density protein chip, manually checking the signal intensity of all protein points on the chip one by one, manually adjusting the position and size deviation of the protein signal points caused by avoiding artificial factors such as impurities, scratches and the like, finally completing the extraction of all protein antigen point signal intensities on each high-density protein chip, generating a data document, and further analyzing and using the data document.
1.2 evaluation of the detection quality of the high-Density protein chip
After the gray image of the anti-GST antibody hybridized high-density protein chip is used, the Signal intensity of each protein point of the chip is collected, and when the Signal-to-Noise Ratio (SNR) of two parallel points of a probe on the chip is simultaneously greater than 2, the protein point on the chip can be detected. The ratio of protein spots detectable on the chip and the correlation of signal intensity between two parallel spots of each protein were then calculated.
1.3 high Density protein chip for detecting serum autoantibodies
The serum samples for the high density protein chip detection comprise 40 cases of PM/DM patients, 30 cases of autoimmune disease control patients and 20 cases of normal control. The specific operation steps are as follows:
(1) taking out the high-density protein chip stored at-80 ℃, isolating air to balance to room temperature, immersing the high-density protein chip in 5ml of protein chip sealing liquid containing 3% BSA PBST, and sealing for 1 hour (h) at room temperature;
(2) mu.l serum samples were taken according to 1:1000 parts of the serum are diluted in lml 3% BSA PBST, uniformly mixed by shaking, centrifuged at 10000rpm by l Omin, and the supernatant is the diluted serum. And (3) after the protein chip is taken out from the sealing liquid, if the surface of the protein chip has air bubbles, sucking the sealing liquid in the incubation box by using an lml sample adding gun, washing the sealing liquid from the top end, sucking the redundant sealing liquid from one side by using absorbent paper to be dry as much as possible, and placing the sealing liquid in a wet box. Then, sucking 150 mul of diluted serum and adding the diluted serum onto a protein chip, slowly covering a cover glass for sealing without damaging protein antigens on the chip by a gun head so as to avoid generating bubbles, and incubating for 1h at room temperature;
(3) placing the incubated protein chip in 10ml of PBST lotion, carefully taking down the cover glass, placing the chip in a washing box, and rinsing for 10min each time for 3 times at room temperature PBST 40 rpm;
(4) alexa 647-labeled goat anti-human IgG antibodies were taken as follows: 1000 parts of the mixture is diluted in 3% BSA PBST, uniformly mixed by shaking, centrifuged at 10000rpm for 10min, and the supernatant is the diluted secondary antibody. The protein chip was removed from the washing box, and if there were air bubbles on the surface of the chip, the liquid in the incubation box was sucked up with a 1ml sample application gun and washed from the top, and the excess washing liquid was sucked up from one side with a water absorbent paper and placed in a wet box. Adding 150 mu l of diluted fluorescent secondary antibody to the chip, slowly adding a cover glass for sealing to avoid generating bubbles, and incubating the secondary antibody for 1h at room temperature in a dark place;
(5) placing the incubated protein chip in PBST lotion, carefully taking down the cover glass, placing the chip in a washing box, and washing for 10min each time for 3 times in dark at 40rpm of PBST;
(6) then distilled with double distilled water (ddH) at room temperature20) Rinsing the protein chip at 40rpm in dark for 3 times, each time for 10 min;
(7) taking out the protein chip, placing in a 50ml centrifuge tube, centrifuging at 1000rpm for 2min with the side of the chip with the antigen protein facing outwards, and spin-drying the residual ddH on the chip20；
(8) The GenePix 4000B chip scanner scans the protein chip, and the same scanning parameters are set for scanning the chip after each hybridization is completed.
1.4 determination of PM/DM related autoantibody target antigens
The signal intensity information of all protein spots on each high density protein chip collected by the GenePix Pro 6.0 software was imported into an Excel table. The foreground signal intensity (F635 mean) of each protein spot was divided by the background signal intensity (B635 mean) around the protein spot to serve as the signal value of the protein spot. I.e. Iij-F635 medium/B635 medium (Iij represents the signal value of protein i point in block j). The signal value of the protein antigen protein spot approaches 1, indicating that the lower the concentration of the corresponding autoantibody in the serum, the less easily the antibody is detected. Higher signal values indicate a greater ability of the autoantibody to bind to the target antigen protein in the serum sample. The average value of the signal intensity of two different spots of the same protein is taken as the signal intensity of the spot of the protein on the chip. When the average value is not less than 2, the protein spot is considered positive. Then, the information of the positive rate of the immunoreaction of each protein antigen on each serum and the high-density protein chip is counted. And performing data analysis on the PM/DM patient group, the autoimmune disease control group and the normal control group by adopting a common gene chip Significance Analysis (SAM) method in a high-throughput chip bioinformation analysis technology.
1.5 results
A total of 91 PM/DM associated target antigens were analytically determined and incorporated into the PM/DM associated autoantibody target antigen chiplet preparation, detailed in Table 1.
TABLE 1 proteins for preparing protein chips for PM/DM related autoantibodies target antigens
Example 2 construction of PM/DM related autoantibody target antigen protein chip and serum screening validation
The variety of autoantibodies produced by different patients with the same autoimmune disease is greatly different due to different genetic backgrounds and different environmental influence factors. In order to confirm the value of the PM/DM related autoantibodies screened from small samples by adopting a high-density protein chip in diagnosis of PM/DM patients of different age groups, the PM/DM related autoantibody target antigen screened from the high-density protein chip is prepared into a PM/DM related autoantibody target antigen protein chip for clinical amplification sample verification. The serum samples used for the PM/DM related autoantibody target antigen protein chip to carry out clinical large sample validation detection comprise 362 cases of APM/ADM, wherein 100 cases of APM, 262 cases of ADM, 35 cases of JDM, 200 cases of disease control patients and 100 cases of healthy controls. The method comprises the following specific steps:
(1) taking out the PM/DM related autoantibody target antigen protein chip stored at-80 ℃, standing for 30min (avoiding condensed water), isolating air, recovering the room temperature, cutting off the package, carefully taking out the protein chip, facing to a fluorescent lamp, placing a fence on the front surface of the chip according to the position of each Block on the chip, pressing the fence tightly with force, and then placing the chip into a chip hybridization wet box with the front surface upward;
(2) add 50. mu.l of PBST (0.1% Tween20, v/v) buffer containing 3% BSA to each pen in a small square matrix and block for 1 hour at 37 ℃;
(3) placing the protein chip in a 50ml centrifuge tube, enabling the surface of the chip with the protein antigen to face outwards, and centrifuging at 1000rpm for 2min to remove liquid in each small square matrix;
(4) take 1 μ l serum as 1: diluting in lml 3% BSA PBST at a ratio of 1000, shaking and uniformly mixing, centrifuging at 10000rpm for 10min, and obtaining supernatant which is diluted serum;
(5) respectively adding 50 mu l of diluted serum into each small square matrix of the PM/DM related autoantibody target antigen protein chip to avoid generating bubbles, and incubating for 1h at room temperature;
(6) soaking the protein chip in 10ml of PBST washing liquor, and slowly shaking and rinsing the protein chip on a decoloring shaking table for 3 times, 40 revolutions per minute and 10min each time;
(7) taking out the protein chip from the washing box, placing the chip in a 50ml centrifuge tube, centrifuging for 2min at 1000rpm with the surface of the chip with the protein antigen facing outwards, and centrifuging to remove liquid in each small square matrix;
(8) alexa 647-labeled goat anti-human IgG antibodies were taken as follows: diluting the mixture in a ratio of 1000 in 3% BSA PBST, uniformly mixing the mixture by shaking, centrifuging the mixture at 10000rpm for 10min, and obtaining supernatant which is diluted secondary antibody;
(9) adding 50 microliter of A1: 1000 diluted A1exa647 labeled goat anti-human IgG antibody into each small matrix of the protein chip, and incubating for 1 hour at 37 ℃ in the dark;
(10) carefully tearing off the 14-hole fence adhered to the protein chip, soaking the chip in 10ml of PBST washing liquor, slowly shaking the chip on a decoloring shaking table, and rinsing the chip for 3 times in a dark place at 40rpm for 10min each time;
(11) then using 10ml ddH2The protein chip was rinsed 3 times for 10min each time in 0 dark. Placing the protein chip in a 50ml centrifuge tube, enabling the surface of the chip with the protein probe to face outwards, and centrifuging at 1000rpm for 2min to remove residual liquid on the chip;
(12) protein chip scanning was performed with a chip scanner. The parameters are set as follows: the excitation light wavelength was 635nm, the photomultiplier detector (PMT) was set to 650, the Power supply (Power) was set to 90, and the scanning pixels were set to 5 μm.
The signal intensity information of all protein spots on each protein chip collected by the GenePix Pro 6.0 software was imported into an Excel table. The foreground signal intensity (F635 mean) of each protein spot was divided by the background signal intensity (B635 mean) around the protein spot to serve as the signal value of the protein spot. I.e., Iij-F635 medium-B635 medium (Iij represents the signal value of protein i point in block j). The closer the signal value Iij of the protein antigen protein spot approaches 0, the lower the corresponding level of autoantibodies in serum, the less easily the antibody is detected by the chip. A higher Iij signal value indicates a greater ability of the autoantibody to bind to the target antigen protein in the serum sample. The average value of the signal intensity of two different spots of the same protein is used as the signal intensity of the spot of the protein on the chip. The average signal intensities Iij (Averagenmamal) and Std of the target antigen were calculated for each protein spot of 100 healthy control groups included in the study, and Iij (Averagenmamal) +3Std was used as the positive response Cutoff value of the protein spot. Then, the positive rate information of each protein antigen immunoreaction on the chip of the serum and PM/DM related autoantibody target antigen protein of each research group is counted. The chi-square test was used to compare the positivity of autoantibody markers between different groups, and differences were considered significant when P < 0.01.
The detection results of 91 target antigens on the PM/DM related autoantibody target antigen chip on APM/ADM and JDM are detailed in Table 2.
TABLE 2 detection of PM/DM related autoantibody target antigens in different age groups PM/DM
The research result shows that the positive rate of the anti-LIMS 1(LIM zinc finger domain protein 1) antibody in the JDM group is 31.4 percent and is higher than that of the disease and healthy control groupThe sexual rate was 9.0% (P ═ 2.4 × 10)-4) Meanwhile, the positive rate is 8.6 percent higher than that of the APM/ADM group (P is 3.3 multiplied by 10)-4) The differences in the results were all significant. It was shown that the anti-LIMS 1 antibody can be used as a diagnostic and detection marker for juvenile dermatomyositis.
Claims (6)
- Use of LIMs zinc finger domain protein 1, LIMs1, or a fragment thereof, for the preparation of a detection reagent for the diagnosis of juvenile dermatomyositis, LIMs1 for the detection of LIMs1 autoantibodies in a serum sample.
- 2. The use of claim 1, wherein said diagnosing comprises: determining the level of autoantibodies reactive to LIMS1 or fragments thereof in a biological sample obtained from a patient exhibiting juvenile dermatomyositis; optionally, the step of (a) is carried out,comparing the level of autoantibodies in the biological sample to adult polymyositis/dermatomyositis control data, other autoimmune disease control data, and healthy human control data, wherein a detectable increase in autoantibodies reactive to LIMS1 in the sample relative to the control data is indicative of the likelihood of juvenile dermatomyositis.
- 3. The use of claim 1 or 2, wherein the level of anti-LIMS 1 antibody is measured by the steps comprising:a. contacting a biological sample from a patient with LIMS1 or a fragment thereof;b. forming an antibody-protein complex between autoantibodies present in the biological sample and LIMS1 or a fragment thereof;c. washing to remove any unbound antibody;d. adding a detection antibody that is labeled and reactive with autoantibodies from the biological sample;e. washing to remove any unbound labeled detection antibody; andf. converting the label of the detection antibody to a detectable signal; wherein the presence of the detectable signal indicates the presence of anti-LIMS 1 antibody in the patient.
- 4. The use of claim 3, wherein said LIMS1 or fragment thereof is deposited on or immobilized on a solid support.
- 5. The use of claim 4, wherein the solid support is in the form of a latex bead, a multi-well plate or a membrane strip.
- 6. The use of claim 3, wherein the detection antibody is labeled by covalent attachment to an enzyme, a fluorescent compound or a metal, or a chemiluminescent compound.
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