Detection Of Several New Kinds Of Autoantibodies And Their Clinical Diagnostic Value In Patients With SLE

Background:Systemic lupus erythematosus (SLE) is an autoimmune disease which has a variety of clinical manifestations, and can involve multiple systems and organs. SLE is a relatively common disease in the connective tissue diseases. It occurs at young female with the peak incidence between the ages of15-40years old. The prevalence ratio of male to female is about1:9. The global prevalence of SLE is about30-50in every ten thousand people. According to our country’s preliminary statistics of the epidemiology, the incidence of SLE in our country is about70to100in every hundred thousand people, and has a tendency to increase each year. SLE pathogenesis has not fully been elucidated. It may associated with factors such as heredity, environment, sex hormone and etc, but excessive activation of B cells and antibodies produced played an important role in the pathogenesis of SLE. These autoantibodies produced by body react with the corresponding antigen to form immune complex which deposits in the place such as skin, blood vessels, kidneys, nerve system, and result in tissue and organ damage. When involving neural spirit system called neuropsychiatric systemic lupus erythematosus (NPSLE) is a seriously manifestation of SLE, and its fatality rate is high. NPSLE has varied clinical manifestations, can occur in SLE at any time. NPSLE pathogenesis has not fully been elucidated, but in recent years a large number of scholars found that autoantibody may be associated with the pathogenesis of NPSLE. But now there have no exactly diagnostic criteria of NPSEL, and lack of early prediction and diagnosis of NPSLE laboratory diagnostic methods.Autoantibody of laboratory testing has become one of SLE diagnosis standard, anti-Sm antibody, anti-phospholipid antibody, anti-ribosomal antibody and anti-DNA antibody had been incorporated into diagnostic criteria of SLE which revised by American College of Rheumatology(ARA) in1997. But the SLE is a kind of autoimmune disease which has a variety of autoantibody participates in, now more new antibodies which may be involved in the pathogenesis of SLE have been found by more and more researchers.Anti-Smith antibody is a trademark of SLE, it was found in a SLE patient called Smith in1966by Tan. Anti-Smith antibody is a serum specificity symbol of SLE patients; its positive rate is21%-30%in SLE patients. Its diagnosis specificity to SLE is high, but low sensitivity. It has confirmed that the level of the anti-Smith antibody have no relationship with the degree of the disease activity and clinical manifestations, however, anti-Smith antibody has very great help in the diagnosis of SLE patients who has early and atypical clinical manifestations or the retrospective diagnosis after treatment. At present, the detection of anti-Smith antibody has three methods, immunodiffusion (ID), Immunoblotting test test (IBT), counter immune electrophoresis (CIE). The grassroots hospital use IBT as the main detection of anti-Smith antibody. But its diagnosis specificity to anti-Smith is high, but low sensitivity. Main reason is that IBT has low accuracy of result judgment. At present most of the IBT method to28KD,29KD and13.5KD band as a positive judgment standard. But Sm antigen has a variety of isomer composition peptides, because of the identity of Sm antibodies, so it has different affinity with different Sm polypeptides or antigenic determinant. With different stripe as positive judgment standard of anti-Smith antibody will affect positive rate of antibody detection.Anticardiolipin antibody(ACA) is one of the anti-phospholipid antibody, it can combined on the negatively charged cell membrane phospholipids such as red blood cells, platelets and endothelial cells, etc, it can cause thrombosis, thrombocytopenia and other damage by damaging red blood cells, platelets and endothelial cells. A lot of study has shown that the ACA play an important role in arteriovenous thrombosis, thrombocytopenia, and the mechanism of spontaneous abortion. ACA can appear in SLE, or other connective tissue diseases, but with the highest positive rate in patients with SLE, can reach30%to40%. Researcher has observed that ACA positive has relationship with the SLE patients who has thrombocytopenia, thrombosis, vascular inflammation and associated lesions in the nervous system.Anti-ribosomal P-protein autoantibody (ARPA) is a antibody for ribosomal P protein C terminal area, mainly aimed at60s ribosomal subunit protein P0, P1, P2phosphate, in recent years there is a lot of report find that the ARPA only found in patients with SLE, it’s a specific antibody for SLE, it’s diagnosis specificity to SLE is high, so it’s become another specificity antibody after anti-Smith antibody and anti-DNA antibody were found. Early people just found ARPA play an important role in SLE patients with the nervous system damage, and related with the emergence of severe depression, epilepsy, dementia, across the spinal cord injury, aseptic meningitis such clinical symptom of NPSLE. The titer of ARPA is related to the severity of psychosis, depression and clinical activity level. At present, we find ARPA also has relationship with the disease activity level of SLE, thrombocytopenia and kidney damage. So ARPA can be used as evaluation indexes of SLE disease activity degree.Anti-N-methyl-D-aspartic acid receptor antibody is an antibody aim at N-methyl-D-aspartic acid receptor (NMDA). The NMDA receptor is central excitability glutamate receptors. It has expressed in the whole brain nerve cells, and plays an important role in the formation of the neural synapse, the development of neural neurons and neurons migration. It also affects learning, memory and other physiological. After anti-NMDA receptor antibody through the blood brain barrier and combine with NMDA receptors causing neurons apoptosis and central nervous system abnormalities. Anti-NMDA receptor antibody also can combine with hippocampal neurons and cause cognitive dysfunction such as memory and learning. A number of studies have confirmed that NMDA receptor antibody in involved in the pathogenesis of NPSLE, and connected with clinical manifestations such as NPSLE patients with moodiness, short-term memory, learning ability, cognitive dysfunction.Anti-neuronal antibody common type is IgG and IgM, constitute by antibody which has a variety of antigen, it can be roughly divided into antibody of anti-nerve cell membrane antigen and anti-cytoplasmic antigen. Anti-neuronal can antibodies combine with nerve cell; promote the secretion of cytokines, reaction of inflammation reaction, and lead to a wide range of nerve tissue damage. Researchers find neurons antibody in serum and cerebrospinal fluid of SLE patients who with nervous system damage resistance, so that anti-neuronal antibodies may be involved in resistance to neurons NPSLE occurrence mechanism, as in-depth study of the anti-neuronal antibodies, we not found anti-neuron nuclear antibody (anti-Hu, anti-yukio okamoto, anti-Ri) play a role in the occurrence of NPSLE mechanism, however find anti-neuronal antibody (IgG) associated with the nervous mental symptoms of NPSLE.Now we use enzyme linked immunosorbent assay (ELISA) as detection method of ACA and ARPA, because of its simple and convenient operation, and can quantitatively detect antibody levels, so it is widely used in clinical laboratory. But anti-NMDA receptor antibody and anti-neuronal antibodies without a mature method for laboratory testing, some researchers use a synthesis peptide chain as antigen to establish ELISA method to detect anti-NMDA receptor antibody. However they use different positive control, such as strong positive specimens in preliminary experiment and monoclonal antibody. The former has low accuracy and the latter will increase the testing costs. The methods of anti-neuronal antibodies has immunofluorescence, immune radiation notation and so on, most of scholars adopt human neuroblastoma tumor cells as a source of neuronal antibody. The principle of immunofluorescence is to use fluorescent markers to check corresponding antibody. Because of the nonspecific immune fluorescence staining problem has not been completely resolved, low objectivity of results and complex technology application; it’s less use in clinical. And immune radiation notation also has insufficient, it’s a method which use isotope labeling antigen to track antibody, so it can cause the experimenter radiation damage. And it occur reaction and false positive reaction frequently, so it also nor the preferred detection method for clinical. So to build and perfect anti-NMDA receptor antibody and anti-neuronal antibodies (IgG) laboratory testing method and to study its correlation with the NPSLE has significance for the diagnosis and prevention of NPSLE.Objectives:1. Improve or establish the laboratory testing method of anti-NMDA receptor antibody and anti-neuronal antibodies (IgG).2. To analyze four antibodies:ACA, ARPA, anti-NMDA receptor antibody and anti-neuronal antibodies (IgG) in SLE patients, to study the correlation analysis between the level of these four antibodies and the disease activity of SLE, further evaluation of four kinds of antibodies in the diagnosis of SLE clinical application value.3. To establish immune double diffusion method to detect resistance to Sm antibody in SLE patients’ serum, evaluate the clinical commonly used method of western blot detection of Sm sensitivity and specificity of antibody.Methods:1. Use double antibody sandwich enzyme-linked immunosorbent kit detect level of ACA and ARPA in30cases of SLE patients (including21active SLE patients and9inactive SLE),30cases of healthy controls,2cerebrospinal fluid of NPSLE patients.2.Use a in vitro synthetic peptide antigen (Ac-WEYSvwL-SN-NH2) as antigen build the indirect enzyme-linked immunosorbent to measure the level of anti-NMDA receptor antibody in30cases of SLE patients (including21active SLE patients and9inactive SLE),30cases of healthy controls,2cerebrospinal fluid of NPSLE patients.3.Use neuroepithelial tumor cell lines (SK-N-MC) as antigen build the cell enzyme-linked immunosorbent to measure the level of anti-neuronal antibodies (IgG) in30cases of SLE patients (including21active SLE patients and9inactive SLE),30cases of healthy controls,2cerebrospinal fluid of NPSLE patients.4. Use rabbit thymus acetone powder extract as antigen to establish immunodiffusion test and immunoblotting test detect anti-Smith antibody in32cases of SLE patients (including22active SLE patient and10inactive SLE),30cases of healthy controls.The statistical treatment consisted of analyzing variance and multiple comparison of group means by using of SPSS13.0. The study makes a comparison among the groups of sample through independent sample T-test. If our dates were fit for test of homogeneity of variances, we dealed them with LSD. If our date were not fit for it, we analyzed all datas through Welch and Dunnett’s T3, crosstabs χ2test. Completely random design of the two sample rate compared with χ2test, comparison of two sample rate matching design with McNemar test, data correlation analysis with nonparametric Spearman rank correlation. Consistency check with Kappa test. It is significance when P value less than0.05.Results:1. The level and positive ratio of serum ACA, ARPA in SLE patients are significant higher than non-active phase and normal control group (P<0.05), and there are positive correlation to SLEDAI (P<0.05).The detection rate of ACA is high in SLE patients who has thrombocytopenia, skin rashes and proteinuria. The ARPA has relationship with SLE patients’skin rashes and proteinuria. In SLE patients, positive rate of combined assay of these two antibodies has no statistically significant between nether ACA nor ARPA alone (P>0.05).2. The level and positive ratio of serum anti-NMDA receptor antibody and anti-neuronal antibody in SLE patients are significant higher than normal control group (P<.05), but it has no significant difference between active phase and non-active phase(P>0.05), they have no relationship with the clinical manifestations of SLE patients such as thrombocytopenia, skin rashes, arthritis and proteinuria.(P>0.05).3. ARPA, anti-NMDA receptor antibody and anti-neuronal antibody are positive in serum and cerebrospinal fluid of2NPSLE patients. After they receive treatment (one use1.0g of methyl prednisolone intravenous infusion for3days, one use ammonia armor sphenoid ridge5mg and5mg dexamethasone intrathecal injection), ARPA is positive in serum of two patients, but the drop degree is decreased obviously. Anti-NMDA receptor antibody and anti-neuronal antibody are both negative in serum, oppositely, these three kinds of antibody in cerebrospinal fluid is still positive, but the drop degree is decreased obviously.4. Compare immunodiffusion test and Immunoblotting test test to detect anti-Smith antibodies in the serum of SLE patients, they are consistency is poorer, the positive ratio of two methods has statistically significant (P<0.05), Immunoblotting test test has higher sensitivity and low specificity than immunodiffusion test.Conclusion:1. ACA, ARPA, anti-NMDA receptor antibody and anti-neuronal antibody can become the serologic basis of SLE diagnosis, and ACA, ARPA are have relationship with severity of SLE, so they can become a judge index of SLE treatment;2. The ACA has relationship with SLE patients’ clinical manifestations: thrombocytopenia, skin rashes and proteinuria. The ARPA has relationship with SLE patients’ clinical manifestations:skin rashes and proteinuria. Anti-NMDA receptor antibody and anti-neuronal antibody have no relationship with the clinical manifestations of SLE patients such as thrombocytopenia, skin rashes, arthritis and proteinuria;3. ARPA, anti-NMDA receptor antibody and anti-neuronal antibody are positive in cerebrospinal fluid of2NPSLE patients before and after treatment, but the drop degree is decreased obviously. However, there are become negative in serum after treatment. It prompt that these three antibodies in cerebrospinal fluid is more useful in determining and forecasting the occurrence of NPSLE.4. In clinical diagnosis, we should be cautious to those who has insufficient diagnosis evidence but anti-Smith antibody positive, and should further confirmed by immunodiffusion test in order to avoid misdiagnosis.