Autoantibodies In Samples Research Articles

Evaluation of a Ten-Antigen Immunodot Test in Autoimmune Hepatitis and Primary Biliary Cholangitis: Lessons Learned for a Tertiary Care Academic Hospital.

Autoimmune diseases of the liver and biliary tract require timely and accurate diagnosis. This study evaluates the D-tek panel (D-Tek, Mons, Belgium) of 10 immunodot antigens for its effectiveness in diagnosing autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC). We retrospectively analysed serum samples from 111 patients who had undergone routine testing, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assays (ELISA), to confirm or exclude autoimmune liver or biliary tract disease. The panel tested for M2/nPDC, M2/OGDC-E2, M2/BCOADC-E2, M2/PDC-E2, gp210, sp100, LKM1, LC1, SLA, and F-actin antigens. Results showed that all positive IIF+ELISA results were confirmed by the immunodot panel, except for two samples from patients who had never been diagnosed with AIH. The immunodot test identified over 20 additional autoantibodies in samples initially negative by IIF, corroborated by laboratory imaging and medical history. The immunodot technique proved to be a quick, sensitive, and specific method with high overall accuracy. This study suggests that the immunodot technique may be an effective screening and confirmatory method for autoimmune liver diseases, potentially improving diagnostic efficiency and accuracy in clinical practice.

Autoantibodies to ACE2 and immune molecules are associated with COVID-19 disease severity

BackgroundIncreased inflammation caused by SARS-CoV-2 infection can lead to severe coronavirus disease 2019 (COVID-19) and long-term disease manifestations. The mechanisms of this variable long-term immune activation are poorly defined. One feature of this increased inflammation is elevated levels of proinflammatory cytokines and chemokines. Autoantibodies targeting immune factors such as cytokines, as well as the viral host cell receptor, angiotensin-converting enzyme 2 (ACE2), have been observed after SARS-CoV-2 infection. Autoantibodies to immune factors and ACE2 could interfere with normal immune regulation and lead to increased inflammation, severe COVID-19, and long-term complications.MethodsHere, we deeply profiled the features of ACE2, cytokine, and chemokine autoantibodies in samples from patients recovering from severe COVID-19. We measured the levels of immunoglobulin subclasses (IgG, IgA, IgM) in the peripheral blood against ACE2 and 23 cytokines and other immune molecules. We then utilized an ACE2 peptide microarray to map the linear epitopes targeted by ACE2 autoantibodies.ResultsWe demonstrate that ACE2 autoantibody levels are increased in individuals with severe COVID-19 compared with those with mild infection or no prior infection. We identify epitopes near the catalytic domain of ACE2 targeted by these antibodies. Levels of autoantibodies targeting ACE2 and other immune factors could serve as determinants of COVID-19 disease severity, and represent a natural immunoregulatory mechanism in response to viral infection.ConclusionsThese results demonstrate that SARS-CoV-2 infection can increase autoantibody levels to ACE2 and other immune factors. The levels of these autoantibodies are associated with COVID-19 disease severity.

B-073 The Clinical Utility of Reporting AC-2 Pattern With Anti-DFS70 Antibody

Abstract Background The dense fine speckled (DFS) pattern (AC-2) is often observed in antinuclear antibody (ANA) indirect immunofluorescence (IIF) assay, and the related anti-DFS70 antibody is known to be negatively associated with a systemic autoimmune rheumatic disease (SARD). Identifying the AC-2 pattern is challenging, as it could be generally confused with the AC-1, AC-4, AC-5, or AC-29 patterns displaying stained metaphase plates or speckled nuclei. Since the anti-DFS70 antibody is not included in routine autoantibody tests, the clinical utility of the AC-2 pattern and anti-DFS70 antibody is not adequately applied in the laboratory and clinical practice. We evaluated the prevalence of anti-DFS70 and other autoantibodies in samples presenting AC-2 pattern in ANA IIF assay. Additionally, we analyzed the association of anti-DFS70 antibodies and clinical features in patients with AC-2 patterns. Method We retrospectively reviewed the ANA IIF images of 1839 samples made by an automated ANA analyzer classified as AC-1, AC-2, AC-4, AC-5, and AC-29 patterns. AC-1, AC-2, AC-4, AC-5, and AC-29 patterns comprised 60.5% of all ANA positives (1839/3039). Among them, the AC-2 pattern was identified in 18.5% (341/1839) by two experts, and only 197 with residual samples were available for further evaluation. Those 197 samples were tested for anti-DFS70 antibodies with enzyme-linked immunosorbent assay (ELISA) and line immunoblot assay (LIA). They were also tested for 22 other autoantibodies (dsDNA, nucleosomes, histones, SS-A/Ro-52, SS-B, RNP/Sm, Sm, Mi-2α, Mi-2β, Ku, CENP-A, CENP-B, Sp100, PML, Scl-70, PM/Scl100, PM/Scl75, RP11, RP155, gp210, PCNA) with LIA. Clinical information from 197 patients was obtained from a retrospective review of electronic medical records. Results Anti-DFS70 antibody prevalence by ELISA and LIA was 32.0% (63/197) and 19.8% (39/197), respectively. The agreement percentage between the two assays was 81.7%, with a kappa value of 0.53 (95% confidence interval: 0.40–0.66). Of the 69 DFS70+ samples (as per ELISA or LIA), the coexistence of other autoantibodies was found in 81.2% (56/69). Of the 128 DFS70- samples, 90.6% (116/128) revealed other autoantibodies. The three most frequent autoantibodies (except anti-DFS70) in 197 patients with AC-2 patterns were anti-SS-A/Ro-52 (30.5%, 60/197), anti-nucleosomes (27.4%, 54/197), and anti-histones (20.3%, 40/197). There was no significant difference in the prevalence of the other 22 autoantibodies between DFS70+ and DFS70− patients. The anti-DFS70 prevalence in the SARD group was 33.3% (50/150), and in the non-SARD was 40.4% (19/47) without significant difference. However, of the 69 DFS70+ patients, the isolated anti-DFS70 was found more frequently in the non-SARD group (36.8%, 7/19) than in the SARD group (12.0%, 6/50) (P = 0.0346). Conclusion The anti-DFS70 positive rate in AC-2 pattern samples is lower than a third, and the prevalence is affected by different detection assays. Along with the AC-2 pattern, just reporting the presence of anti-DFS70 is not valuable for SARD exclusion. Therefore, it is important to co-examine other autoantibodies, such as anti-SS-A, anti-nucleosomes, and anti-histones, to confirm the isolated anti-DFS70.

An ELISA for quantifying GPIHBP1 autoantibodies and making a diagnosis of the GPIHBP1 autoantibody syndrome

BackgroundAutoantibodies against GPIHBP1, the endothelial cell transporter for lipoprotein lipase (LPL), cause severe hypertriglyceridemia (“GPIHBP1 autoantibody syndrome”). Affected patients have low serum GPIHBP1 and LPL levels. We report the development of a sensitive and specific ELISA, suitable for routine clinical use, to detect GPIHBP1 autoantibodies in serum and plasma. MethodsSerum and plasma samples were added to wells of an ELISA plate that had been coated with recombinant human GPIHBP1. GPIHBP1 autoantibodies bound to GPIHBP1 were detected with an HRP-labeled antibody against human immunoglobulin. Sensitivity, specificity, and reproducibility of the ELISA was evaluated with plasma or serum samples from patients with the GPIHBP1 autoantibody syndrome. ResultsA solid-phase ELISA to detect and quantify GPIHBP1 autoantibodies in human plasma and serum was developed. Spiking recombinant human GPIHBP1 into the samples reduced the ability of the ELISA to detect GPIHBP1 autoantibodies. The ELISA is reproducible and sensitive; it can detect GPIHBP1 autoantibodies in samples diluted by >1000-fold. ConclusionWe have developed a sensitive and specific ELISA for detecting GPIHBP1 autoantibodies in human serum and plasma; this assay will make it possible to rapidly diagnose the GPIHBP1 autoantibody syndrome.

Concentration and Subclass Distribution of Anti-ADAMTS13 IgG Autoantibodies in Different Stages of Acquired Idiopathic Thrombotic Thrombocytopenic Purpura.

The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying deficiency of the ADAMTS13 protease is caused by autoantibodies, predominantly of the IgG isotype. Certain HLA-DR-DQ haplotypes were associated with the risk of developing TTP. To investigate the development of the ADAMTS13-specific antibody response during the course of the disease, we analyzed the concentration, subclass distribution, and inhibitory potential of anti-ADAMTS13 IgG autoantibodies in samples of TTP patients drawn during the first acute phase, in remission, and during relapse. Additionally, we compared the anti-ADAMTS13 IgG levels between patients carrying and not carrying risk and protective HLA-DR-DQ haplotypes. We determined the anti-ADAMTS13 IgG concentration and subclass distribution in 101 antibody-positive samples of 81 acquired TTP patients by ELISA methods. The presence and semi-quantitative amount of anti-ADAMTS13 inhibitors were determined in 97 of 100 deficient samples, and the specific inhibitory potential of anti-ADAMTS13 autoantibodies was determined in 49 selected samples, by mixing ADAMTS13-activity assays. HLA-DR-DQ typing and haplotype prediction were performed in 70 of the above patients. We found that IgG1 and IgG4 were the predominant subclasses, present in almost all samples. While IgG1 was the dominant subclass in almost half of the samples taken during the first acute episode, IgG4 was dominant in all samples taken during or following a relapse. The inhibitory potential of the samples correlated with levels of the IgG4 subclass. Anti-ADAMTS13 antibodies of IgG4-dominant samples had higher specific inhibitory potentials than IgG1-dominant samples, independently of disease stage. Interestingly, we found that patients carrying the protective DR7-DQ2 and DR13-DQ6 haplotypes had higher anti-ADAMTS13 IgG levels. Our results indicate that IgG4 becomes the dominant subtype at some point of the disease course, apparently before the first relapse, parallel to the increase in inhibitory potential of the anti-ADAMTS13 autoantibodies. Furthermore, we found an association between the genetic background and the antibody response in TTP.

Screening for Multiple Autoantibodies in Plasma of Patients with Breast Cancer.

Autoantibodies have potential as circulating biomarkers for early cancer detection. This study aimed to screen for known autoantibodies in human plasma using an Autoantibody Profiling System (APS) and quantify the levels in plasma of donors with/without breast cancer. Plasma from nine female donors diagnosed with breast cancer (test group) and nine matched donors with no personal history of cancer (reference group) were screened with an APS containing probes for 30 autoantibodies. Autoantibody levels ≥1.5 times the mean concentration of the group were considered elevated, and test/reference ratios ≥1.3 were considered higher in the test group compared to the reference group. Twenty percent of the probes detected elevated levels of autoantibodies against proteins involved in different cancer mechanisms. Amongst these, the levels of autoantibodies against interleukin 29 (IL29), osteoprotegerin (OPG), survivin (SUR), growth hormone (GRH) and resistin (RES) were significantly higher in the cancer group compared to the reference group (p<0.05), whereas the level of autoantibody against cytotoxic T-lymphocyte associated antigen-4 (CTLA4) was not significantly different between the two groups (p=0.38). Disease-relevant autoantibodies were detected in the plasma of patients with breast cancer and donors without breast cancer. This means that identifying the type and level of autoantibodies in samples will be important in determining their significance in the disease process. A microtiter plate-based array system could be a fast and inexpensive screening method for identifying and quantifying autoantibodies in human plasma.

Tetraspanin 7 autoantibodies in type 1 diabetes.

Autoantibodies to pancreatic beta cell proteins are markers of asymptomatic type 1 diabetes. The aim was to determine whether autoantibodies to the beta cell protein tetraspanin 7 would improve the ability to identify autoimmunity against pancreatic beta cells. Full length and external domain fragments of tetraspanin 7 were expressed as luciferase-tagged fusion proteins and used in immunoprecipitation assays to measure autoantibodies in samples from 363 patients with type 1 diabetes at onset of disease, 503 beta cell autoantibody negative first-degree relatives of patients, and 212 relatives with autoantibodies to insulin, glutamic acid decarboxylase, insulinoma antigen 2 or zinc transporter 8. Antibody binding was observed against the full length and external domains of tetraspanin 7, and was strongest against the full length protein. Autoantibodies that could be inhibited by untagged tetraspanin 7 were detected in 5 (1%) of 503 autoantibody negative relatives, 3 (3.2%) of 94 autoantibody negative patients, 95 (35.3%) of 269 autoantibody positive patients, 1 (1%) of 98 single autoantibody positive relatives and 25 (21.9%) of 114 multiple autoantibody positive relatives. Progression to diabetes did not differ between multiple autoantibody positive relatives with and without tetraspanin 7 autoantibodies. Tetraspanin 7 is an autoantigen in type 1 diabetes. Tetraspanin 7 autoantibodies are a marker of type 1 diabetes, but provide minor additional value to existing autoantibodies in identifying beta cell autoimmunity.

Multiplexed immunoassay for the rapid detection of anti-tumor-associated antigens antibodies

TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors).