Autoantibodies In Sera Of Patients Research Articles

Autoimmunity as a candidate for the etiopathogenesis of Meniere's disease: detection of autoimmune reactions and diagnostic biomarker candidate.

Meniere's disease is an inner ear disorder that can manifest as fluctuating vertigo, sensorineural hearing loss, tinnitus, and aural fullness. However, the pathologic mechanism of Meniere's disease is still unclear. In this study, we evaluated autoimmunity as a potential cause of Meniere's disease. In addition we tried to find useful biomarker candidates for diagnosis. We investigated the protein composition of human inner ear fluid using liquid column mass spectrometry, the autoimmune reaction between circulating autoantibodies in patient serum and multiple antigens using the Protoarray system, the immune reaction between patient serum and mouse inner ear tissues using western blot analysis. Nine proteins, including immunoglobulin and its variants and interferon regulatory factor 7, were found only in the inner ear fluid of patients with Meniere's disease. Enhanced immune reactions with 18 candidate antigens were detected in patients with Meniere's disease in Protoarray analysis; levels of 8 of these antigens were more than 10-fold higher in patients than in controls. Antigen-antibody reactions between mouse inner ear proteins with molecular weights of 23–48 kDa and 63–75 kDa and patient sera were detected in 8 patients. These findings suggest that autoimmunity could be one of the pathologic mechanisms behind Meniere's disease. Multiple autoantibodies and antigens may be involved in the autoimmune reaction. Specific antigens that caused immune reactions with patient's serum in Protoarray analysis can be candidates for the diagnostic biomarkers of Meniere's disease.

HEp-2 Cell Classification Using Descriptors Fused into the Dissimilarity Space into the Dissimilarity Space

Autoimmune diseases are strictly connected with the presence of autoantibodies in patient serum. Detection of Antinucleolar Antibodies (ANAs) in patient serum is performed using a laboratory technique named Indirect Immunofluorescence (IIF) followed by manual evaluation on the acquired slides from specialized personnel. In this procedure, several limitations appear and several automatic techniques have been proposed for the task of ANA detection. In this work we present a method achieving state-of-the-art performance on a publicly available dataset. More precisely, two powerful and rotation invariant descriptors are incorporated into a two stage classification scheme where the feature vectors are represented and fused in the dissimilarity space. Then, in a second level dissimilarity vectors are classified using a linear SVM classifier. Evaluation on the HEp-2 cell contest dataset yields a 70.16% performance on cell-level classification. Furthermore we provide results in Image Level Classification where a 78.57% classification rate was achieved.

Mitotic cells recognition in HEp-2 images

Indirect immunofluorescence is the recommended method to detect autoantibodies in patient serum, which could cause connective tissue diseases. One step of the diagnostic procedure needs to distinguish between mitotic and interphase cells in HEp-2. Furthermore, information on mitotic cell staining, namely positive and negative, could be used to improve the ability to discriminate between staining patterns of interphase cells. Despite the growing efforts to develop computer-aided-diagnosis systems supporting the diagnostic procedure, mitotic cell recognition has received little attention. Indeed this task is made difficult since the traditional classification algorithms cannot cope with the high imbalance between the number of samples in the mitotic and interphase classes. In this paper we present a classification system employing a cascade structure that first discriminates between mitotic and interphase cells tackling class imbalance and then it recognizes the positive or negative staining of a mitotic cell. It uses a set of features specifically tailored for the task at hand. The approach has been successfully evaluated on a public reference dataset of HEp-2 cells applying eleven different classification paradigms and four classifiers. The results suggest that the proposed classification system can be used to recognize mitotic cells.

HEp-2 cells classification via sparse representation of textural features fused into dissimilarity space

Autoimmune diseases are proven to be connected with the occurrence of autoantibodies in patient serum. Antinuclear autoantibodies (ANAs) identification can be accomplished in a laboratory using indirect immunofluorescence (IIF) imaging. In this paper a system for automatic classification of staining patterns on HEp-2 fluorescence images is proposed. Our method utilizes two descriptors in order to encode gradient and textural characteristics of the depicted patterns. Along with distribution of SIFT features, we propose the new GoC-LBP descriptor based on co-occurrences of uniform Local Binary Patterns along directions dictated by the orientation of local gradient. At a second stage, the descriptors are fused while creating a dissimilarity representation of an image. A powerful classification scheme is incorporated, utilizing a discriminative sparse representation-based scheme for the classification. Experiments were conducted using a publicly available dataset, comparing the obtained performance to recently reported results of a relevant contest, demonstrating the effectives of the proposed method.

Serum autoantibodies positivity prevalence in patients with chronic HCV and impact on pegylated interferon and ribavirin treatment response

Prevalence of serum autoantibodies in chronic hepatitis C (HCV) patients is higher than that in the general population. Interferon may induce autoimmune manifestations in patients treated with peg-interferon and ribavirin. Effect of autoantibody seropositivity and treatment response are limited and controversial. To detect the prevalence of serum autoantibodies in patients with chronic HCV and impact on histopathology and treatment response. Retrospective study including 3673 Egyptian chronic HCV naïve patients enrolled in the Egyptian national programme for HCV treatment with pegylated interferon and ribavirin in the years 2007-2010. Antinuclear antibody (ANA) was determined by ELISA considered positive with a titre ≥ 1:40 by indirect immunofluorescence. ANA-positive patients pre treatment workup including serum aminotransferases, thyroid profile and liver biopsy, follow-up during treatment and sustained virological response (SVR) were assessed compared to ANA-negative patients. Serum ANA was positive in 1.6% of the studied patients. There were no statistically significant differences concerning the demographic, biochemical and histopathological data in ANA positive and negative patients. SVR was comparable between ANA-positive and ANA-negative patients (67.8% and 61.3% respectively). Follow-up treatment; ANA-positive patients' did not experience statistically significant haematological complications, flare-up of serum transaminases, thyroid dysfunction. No systemic autoimmune disorders developed during follow-up. ANA positivity is not a factor in chronic HCV disease progression and does not affect the treatment response. Pegylated interferon and ribavirin therapy is safe and effective in autoantibodies-positive chronic HCV patients with no need for further follow-up or worry during the treatment in absence of systemic autoimmune disorders.

Early Detection of NSCLC with scFv Selected against IgM Autoantibody

Survival of patients with lung cancer could be significantly prolonged should the disease be diagnosed early. Growing evidence indicates that the immune response in the form of autoantibodies to developing cancer is present before clinical presentation. We used a phage-displayed antibody library to select for recombinant scFvs that specifically bind to lung cancer-associated IgM autoantibodies. We selected for scFv recombinant antibodies reactive with circulating IgM autoantibodies found in the serum of patients with early stage lung adenocarcinoma but not matched controls. Discriminatory performance of 6 selected scFvs was validated in an independent set of serum from stage 1 adenocarcinoma and matching control groups using two independent novel methods developed for this application. The panel of 6 selected scFvs predicted cancer based on seroreactivity value with sensitivity of 0.8 and specificity of 0.87. Receiver Operative Characteristic curve (ROC) for combined 6 scFv has an AUC of 0.88 (95%CI, 0.76–1.0) as determined by fluorometric microvolume assay technology (FMAT) The ROC curve generated using a homogeneous bridging Mesa Scale Discovery (MSD) assay had an AUC of 0.72 (95% CI, 0.59–0.85). The panel of all 6 antibodies demonstrated better discriminative power than any single scFv alone. The scFv panel also demonstrated the association between a high score - based on seroreactivity - with poor survival. Selected scFvs were able to recognize lung cancer associated IgM autoantibodies in patient serum as early as 21 months before the clinical presentation of disease. The panel of antibodies discovered represents a potential unique non-invasive molecular tool to detect an immune response specific to lung adenocarcinoma at an early stage of disease.

Binding of circulating autoantibodies in breast cancer to native and peroxynitrite-modified RNA

Peroxynitrite (ONOO(-)) is a powerful oxidant and nitrosative agent and has in vivo existence. The half life of ONOO(-) at physiological pH is less than 1 s. It can react with nucleic acids, proteins, lipoproteins, saccharides, cardiolipin, etc., and can modify their native structures. Action of ONOO(-), synthesized in the authors' laboratory by a rapid quenched flow process, on structural changes of commercially available RNA was studied by ultraviolet (UV), fluorescence, and agarose gel electrophoresis. Compared to native RNA, the ONOO(-)-modified RNA showed hyperchromicity at 260 nm. Furthermore, the ethidium bromide (EtBr) assisted emission intensities of ONOO(-)-modified RNA samples were found to be lower than the emission intensity of native RNA-EtBr complex. Agarose gel electrophoresis of ONOO(-)-modified RNA showed a gradual decrease in band intensities compared to native RNA, an observation clearly due to the poor intercalation of EtBr with ONOO(-)-modified RNA. Native and ONOO(-)-modified RNA samples were used as an antigen to detect autoantibodies in sera of patients with clinically defined breast cancer. Both direct binding and inhibition enzyme-linked immunosorbent assay (ELISA) confirmed the prevalence of native and 0.8 mmol/L ONOO(-)-modified RNA specific autoantibodies in breast cancer patients. Moreover, the progressive retardation in the mobility of immune complexes formed with native or 0.8 mmol/L ONOO(-)-modified RNA and affinity purified immunoglobulin G (IgG) from sera of breast cancer patients supports the findings of the direct binding and inhibition ELISAs. The peroxynitrite treatment to RNA at a higher concentration appears to have damaged or destroyed the typical epitopes on RNA and thus there was a sharp decrease in autoantibodies binding to 1.4 mmol/L ONOO(-)-modified RNA. It may be interpreted that cellular nitrosative stress can modify and confer immunogenicity on RNA molecules. Higher concentrations of nitrogen reactive species can be detrimental to RNA. However, the emergence of native as well as 0.8 mmol/L ONOO(-)-modified RNA as a novel antigen/substrate for autoantibodies in breast cancer patients indicates that, in future, these molecules might find a place on the panel of antigens for early diagnosis of breast cancer.

Knowledge Extraction from HEp2 Dataset

Pattern recognition is the process of taking the raw data, extract the features and categorize the patterns based on the features. Pattern recognition has been widely used in the area of diseases diagnosis. Autoimmune diseases are proven to be connected with the occurrence of autoantibodies in patient serum. Antinuclear autoantibodies (ANAs) identification can be accomplished in a laboratory using indirect immunofluorescence (IIF) imaging. In this paper we present the results of image analysis image analysis, feature extraction and classification of HEp-2 cell images. The experimental studies are performed on images of HEp-2 cells (1). 

Serum autoantibodies for the diagnosis and management of autoimmune liver diseases

The spectrum of autoimmune liver diseases (AILD) includes primary biliary cirrhosis, primary sclerosing cholangitis and autoimmune hepatitis. The immunological mechanisms triggering the initiation and perpetuation of AILD remains unknown, while autoantigens are now recognized in most cases, and are generally nontraditional in their widespread distribution. Sensitive and specific methods for the detection of serum autoantibodies in patients affected by AILD represent a challenge for researchers and clinicians who desire to obtain an early and certain diagnosis as well as markers of disease control. To this regard, the use and interpretation of serum autoantibodies in AILD may be seen as paradigmatic for the large gaps in our knowledge based on the lack of true population-based studies. The present review article will critically discuss the available evidence on the use of autoantibody findings in the diagnosis or management of autoimmune liver disease.

Anti-ovarian antibodies in sera of patients with ovarian tumors

Aim of paperThe purpose of this study was to assess the presence of anti-ovarian autoantibodies in sera of patients with ovarian tumors. Materials and methodsThe study group consisted of 82 patients treated at the Division of Gynecological Surgery, Poznan University of Medical Sciences, Poland in 2007–2011. 46 patients with malignant ovarian tumors and 36 patients diagnosed with benign ovarian tumor were included into the study. Age-matched healthy control groups consisted of 15 women and 19 men. Anti-ovarian autoantibodies in serum were assessed with the use of indirect immunofluorescence. The presence of anti-ovarian autoantibodies in serum was correlated with clinical and histopathological features of the disease. ResultsSerum anti-ovarian autoantibodies were found in 59% (27/46) of patients with malignant ovarian tumors, compared to 55% (20/36) and 26% (4/15) of patients with benign ovarian tumors and healthy female controls respectively. The difference was statistically significant (P=0.04). All serum samples from the male controls were negative. Anti-ovarian antibodies occurred more often among advanced stage (III and IV stage according to FIGO, P=0.037) and grade 3 (P=0.049) ovarian cancers, however, there were no differences in median progression-free survival (P=0.388). The presence of anti-ovarian antibodies was neither influenced by histopathological type of the tumor, menopausal status, presence of ascites nor CA125 levels. Seropositivity for anti-ovarian antibodies was correlated positively with patients’ age and negatively with tumor size. ConclusionsAnti-ovarian autoantibodies develop with higher frequency in ovarian cancer comparing to healthy controls, however with similar proportion to benign ovarian tumors patients. The presence of antibodies against normal ovarian tissue correlates with ovarian cancer aggressiveness.

Detection of Anti–β1-AR Autoantibodies in Heart Failure by a Cell-Based Competition ELISA

Autoantibodies directed against the second extracellular loop of the cardiac β1-adrenergic receptor (β1-AR) are thought to contribute to the pathogenesis of dilated cardiomyopathy (DCM) and Chagas heart disease. Various approaches have been used to detect such autoantibodies; however, the reported prevalence varies largely, depending on the detection method used. We analyzed sera from 167 DCM patients (ejection fraction<45%) and from 110 age-matched volunteers who did not report any heart disease themselves, with an often used simple peptide-ELISA approach, and compared it with a novel whole cell-based ELISA, using cells expressing the full transgene for the human β1-AR. Additionally, 35 patients with hypertensive heart disease with preserved ejection fraction were investigated. The novel assay was designed according to the currently most reliable anti-TSH receptor antibody-ELISA used to diagnose Graves disease ("third-generation assay") and also detects the target antibodies by competition with a specific monoclonal anti-β1-AR antibody (β1-AR MAb) directed against the functionally relevant β1-AR epitope. Anti-β1-AR antibodies were detected in ≈60% of DCM patients and in ≈8% of healthy volunteers using the same cutoff values. The prevalence of these antibodies was 17% in patients with hypertensive heart disease. Anti-β1-AR antibody titers (defined as inhibition of β1-AR MAb-binding) were no longer detected after depleting sera from IgG antibodies by protein G adsorption. In contrast, a previously used ELISA conducted with a linear 26-meric peptide derived from the second extracellular β1-AR loop yielded a high number of false-positive results precluding any specific identification of DCM patients. We established a simple and efficient screening assay detecting disease-relevant β1-AR autoantibodies in patient sera yielding a high reproducibility also in high throughput screening. The assay was validated according to "good laboratory practice" and can serve as a companion biodiagnostic assay for the development and evaluation of antibody-directed therapies in antibody-positive heart failure.

Clinical significance of serum autoantibodies in patients with NAFLD: results from the nonalcoholic steatohepatitis clinical research network

Some studies have suggested that autoantibodies might define a subcategory and phenotype of nonalcoholic fatty liver disease (NAFLD) associated with advanced histological features. We evaluated the relationship between the presence of serum autoantibodies and liver histology in a large cohort of well-characterized patients with NAFLD. A total of 864 NAFLD patients participating in two prospective multicentre clinical studies underwent testing for serum autoantibodies within 24months of a liver biopsy. Liver histology was compared between the patients with and without ANA≥1:160 or ASMA≥1:40 or both. Autoantibodies were present in 182 patients (21%). There was no difference in age, gender, race, body mass index (BMI), homeostasis model assessment of insulin resistance (HOMA-IR), or history of diabetes between the two groups. Biopsies in subjects with autoantibodies were less likely to have moderate-to-severe steatosis (i.e., >33%) compared to controls (57.1 vs. 43.0%, P value=0.0006). Lobular inflammation (46.7 vs. 47.5%), ballooning degeneration (38.5 vs. 42.5%), and advanced fibrosis (33.2 vs. 29.3%) were not different between the two groups. Histologic evidence of 'definite' NASH did not differ significantly between the two groups (55.5 vs. 58.9%). After adjusting for age, gender, BMI, race, and diabetes, the presence of autoantibodies was independently associated with lower prevalence of moderate-to-severe steatosis [odds ratio (OR), 0.58; 95% confidence interval (CI), 0.41-0.82; P=0.01]. Autoantibodies are frequently positive in NAFLD in the absence of autoimmune hepatitis and their occurrence is not associated with more advanced histologic features.

Valosin-Containing Protein is a Novel Autoantigen in Patients with Glaucoma

The purpose of this study was to identify novel autoantigens that react with specific serum autoantibodies in patients with glaucoma. Sera from patients with glaucoma (n = 80) and healthy subjects without a known pathology (n = 20) were investigated by immunoblot performed with bovine optic nerve lysates and resolved by one- and two-dimensional electrophoresis. Proteins in the immunoreactive spots were selected and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) data analysis. All the sera from subjects were assayed using enzyme-linked immunosorbent assay to identify autoantibodies. We selected two prominent bands with molecular weights of 100 and 220 kDa by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and these two bands were only found in the glaucoma patients. Using one-dimensional electrophoresis and LC-MS/MS analyses, we identified these proteins to be valosin-containing protein (VCP) and fodrin, respectively, and using two-dimensional electrophoresis and LC-MS/MS analyses, VCP was identified to be a common target antigen. In patients with primary open angle glaucoma and normal tension glaucoma, the frequency of autoantibodies to recombinant human VCP was 42.0 and 23.3%, respectively (p < 0.002). In the enzyme-linked immunosorbent assay tests, autoantibody titers to recombinant human VCP were significantly higher than that in healthy controls (p < 0.025). VCP represents a potential candidate target for autoantibodies on the optic nerve in patients with glaucoma.

Prognostic relevance of Bmi-1 expression and autoantibodies in esophageal squamous cell carcinoma

BackgroundOverexpression of Bmi-1 has been observed in a variety of cancers, and it has been suggested to be an independent prognostic marker for the patients. The objective of this study was to determine the level of Bmi-1 expression or its autoantibodies in human esophageal squamous cell carcinoma (ESCC) and to correlate it with clinicopathologic data.MethodsWe first examined Bmi-1 expression in ESCC cell lines and tumor samples by RT-PCR and Western blot analysis. We then analyzed Bmi-1 protein expression in 171 clinicopathologically characterized ESCC cases by immunohistochemistry. In addition, we detected its autoantibodies in sera of patients with ESCC by ELISA.ResultsWe found that Bmi-1 expression was higher in the immortalized cells, cancer cell lines and most cancer tissue than in non-tumorous control tissue at both mRNA and protein level. In addition, Bmi-1 expression was observed in 64.3% (110 of 171) archive ESCC specimen by immunohistochemistry analysis, and the location of Bmi-1 in ESCC was in the nuclei instead of cytoplasm of tumor cells. There was a significant difference of Bmi-1 expression in patients categorized according to stage (P = 0.003) and pN classification (P = 0.047). Multivariate analysis suggested that Bmi-1 expression was an independent prognostic marker for ESCC patients. A prognostic significance of Bmi-1 was also found in the subgroup of T3~T4 and N1 tumor classification. Bmi-1 autoantibodies were detected in sera of 39.0% (62 of 159) ESCC patients. The correlations between anti-Bmi-1 antibodies and tumor stage (P = 0.040), or lymph node status (P < 0.001) were significant.ConclusionsOur results suggest that Bmi-1 protein is a valuable marker of ESCC progression. The presence of Bmi-1 autoantibodies in sera from patients with ESCC may have clinical utility in esophageal cancer diagnosis.

Anti-MDA5 and anti-TIF1-γ antibodies have clinical significance for patients with dermatomyositis

Myositis-specific autoantibodies are useful for diagnosing PM/DM. Recently, two new myositis-specific autoantibodies against melanoma differentiation-associated gene 5 (MDA5) and transcriptional intermediary factor 1-gamma (TIF1-gamma) were identified in DM. Here, we detected these autoantibodies in patient sera using new assays with recombinant MDA5 and TIF1-gamma, and associated clinical features with the presence of anti-MDA5 or anti-TIF1-gamma antibodies. We screened 135 Japanese patients with various CTDs, including 82 with DM. DM patients were classified as clinically amyopathic DM (CADM), cancer-associated DM or classical DM without cancer. Anti-MDA5 and anti-TIF1-gamma antibodies were detected by their ability to immunoprecipitate biotinylated recombinant proteins. Sera from 21 (26%) of 82 DM patients immunoprecipitated MDA5, and every anti-MDA5-positive patient had DM (except one patient with SSc). Sera from 20 (65%) of 31 CADM patients reacted with MDA5. Notably, anti-MDA5-positive DM patients had significantly more interstitial lung disease than anti-MDA5-negative DM patients (95 vs 32%, P < 0.001). Sera from 12 (15%) of 82 DM patients immunoprecipitated TIF1-gamma, and anti-TIF1-gamma antibodies were only detected in DM patients. Strikingly, 7 (58%) of 12 patients with cancer-associated DM had sera that reacted with TIF1-gamma. Anti-TIF1-gamma-positive DM patients had significantly more internal malignancies than anti-TIF1-gamma-negative DM patients (58 vs 9%, P < 0.001). Anti-MDA5 and anti-TIF1-gamma antibodies were confirmed to be serological DM subset markers. Anti-MDA5 and anti-TIF1-gamma antibodies were detected based on their ability to immunoprecipitate biotinylated recombinant MDA5 and TIF1-gamma, and were closely associated with life-threatening complications in DM.

Proteomics-based identification of a tumor-associated antigen and its corresponding autoantibody in gastric cancer

Identification of novel tumor-related antigens and autoantibodies will lead to early diagnosis of cancer and the development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens from the gastric cancer cell lines MkN-1, MkN-45 and KATOIII, and their related autoantibodies in sera of patients with gastric cancer using a proteomics-based approach. Proteins from the gastric cancer cell lines (MkN-1, MkN-45 and KATOIII) were separated by two-dimensional polyacrylamide gel electrophoresis, followed by Western blotting and antibody reaction with sera from patients with gastric cancer, healthy individuals and patients with other cancers. Positive spots were excised from Coomassie blue stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Sera from patients with gastric cancer yielded multiple spots, one of which was identified as the 78 kDa glucose-regulated protein (GRP78) by MALDI-TOF/TOF MS. Western blots against recombinant GRP78 showed reactivity in sera from 17/60 (28.3%) patients with gastric cancer and 0/20 (0.0%) of healthy individuals. Autoantibodies against GRP78 were found in 4/15 (26.7%) and 3/15 (20.0%) patients with esophageal and colon cancer, respectively. We identified for the first time an autoantibody against GRP78 in gastric cancer patients. The proteomic approach implemented in this study offers a powerful tool for identifying novel serum markers that may display clinical usefulness in cancer.

A Novel Method for Detecting p53 Autoantibodies in Sera of Patients with NSCLC.

Serum autoantibody detection is useful means for the early diagnosis and prognosis of cancer. So our objective was to synthesize peptide array to analyse p53 autoantibodies in the sera of patients with non small cell lung cancer (NSCLC). Cellulose-bound overlapping peptides (12 mers) derived from p53 wild type protein were synthesized using SOPTs synthesis technique by an AutoSpot robot -ASP SL (Intavis, Germany). The membrane was incubated with 1/400 dilutions of p53 monoclonal antibody (Sc-53394) to establish a new approach to detect p53 antibody, and the epitopes of the p53 monoclonal antibody is already known. We analysed the p53 autoantibodies from the sera of NSCLC and controls by peptide array and ELISA. We synthesized on cellulose membranes twelve-amino-acid overlapping peptides which included all of the sequences of the polypeptide chain of p53. The p53 autoantibody was positive in seven cases of thirty patients sera with NSCLC and was negative in sera of the controls, with the same result of ELISA CONCLUSIONS: The peptide array could be applied not only to detect the autoantibodies in the sera of patients with lung cancer, but also to map the epitopes of the autoantibodies which might be useful for the early diagnosis and prognosis of cancer.

Expression of a functional tagged human thyrotropin receptor in HeLa cells using recombinant vaccinia virus.

We report a new effective system for expression of FLAG and six histidine tagged TSH receptor in mammalian cells using recombinant vaccinia virus. HeLa cells infected with recombinant virus produced large amounts of human TSH receptor of approximately 150,000 functional molecules per cell. This value is one to two orders of magnitude higher than those in thyroid cells and is comparable with receptor number of the best stably transfected mammalian cell clones previously described. Vaccinia virus produced TSH receptor was able to bind TSH (Kd of 2.1 +/- 0.1 x 10(-10) M) and Graves'disease autoantibodies. TSH caused an increase of the intracellular cAMP level in infected HeLa cells in a concentration-dependent manner, demonstrating the coupling of expressed recombinant TSH receptor to the cAMP second messenger system of the cells. 6His-tagged recombinant TSH receptor was immobilized on Ni2+ nitrilotriacetic acid-agarose. Bound receptor was fully functional, interacting with both TSH and Graves' disease autoantibodies in patient sera. The solid phase bound TSH receptor technique provides a new and simple method for the diagnosis of autoimmune diseases.

Designed Glycopeptides with Different β-Turn Types as Synthetic Probes for the Detection of Autoantibodies as Biomarkers of Multiple Sclerosis

Circulating autoantibodies have been recognized as disease biomarkers of autoimmune diseases. We have previously disclosed a synthetic glycopeptide that is able to detect specific autoantibodies in sera of patients who are affected by multiple sclerosis (MS). This glycopeptide is characterized by a type I' beta-turn around the minimal epitope Asn(Glc) that allows an efficient exposure of this moiety to antibody interactions in the context of a solid-phase immunoenzymatic assay. With the aim of optimizing the glycopeptide-antibody interactions, we analyze a series of new glycopeptides based on different turn structures. Our results confirm the role of conformation in the recognition and binding of synthetic antigenic probes to MS autoantibodies. Glycopeptide 2, which is characterized by a type I beta-turn around the minimal epitope Asn(Glc), shows the highest antibody affinity (IC50 = 11.8 nM), and thus it appears to be a promising tool for the detection of specific autoantibodies as MS biomarker in patients' sera.

Uji kulit serum otolog modifikasi pada pasien Urtikaria Otoimun di RSUP Palembang

There is an urgent need to find reliable and relevant diagnostic tools for patients with autoimmune urticaria especially in a developing country such as Indonesia. The basophil histamine release assay (HRA) is currently the “gold standard” for detecting functional autoantibodies in serum of patients with chronic urticaria. However, this bioassay is difficult to standardize because it requires fresh basophils from healthy donors, is not widely commercially available, and is time consuming. The detection of patients with autoantibodies using in vivo autologous serum skin test (ASST) also poses challenges. Firstly, the ASST is at best around 80% sensitive and specific using in vitro HRA as the “gold standard”, and secondly, there is some divergence results obtained by different methods used to detect patients with autoantibodies, due to inconsistency in methods, results and interpretation of the ASST in the literatures. The purpose of this study was to find and develop a new method of ASST. This new method should be practical and with higher reliability (sensitivity and specificity) as a diagnostic tool for autoimmune urticaria. The method used in this study was to perform a preliminary study followed by advanced study through cross sectional designs. The results of this study showed that the new ASST, has a higher reliability than the former ASST methods reported in literatures. In addition, we also found that disease severity with cut-off value of < 12, could be used as an adjunct to detect patients with positive functional autoantibodies. Finally, we also report with pride that we have now the ability to perform HRA which was never done before.