Autoantibody ELISA Research Articles

Evaluation of Biochemical Characteristics and Performance of the 3 Screen ICA ELISA Kit.

We conducted a fundamental evaluation of the 3 Screen ICA ELISA kit, which can simultaneously measure three major anti-islet autoantibodies important in diagnosing and predicting type 1 diabetes, to assess its usefulness as a measuring reagent. In autoantibody-positive samples, the coefficient of variation for intra-assay variation ranged from 1.37% to 2.50%, inter-assay variation from 2.81% to 3.61%, and lot-to-lot variation from 2.01% to 8.61%, demonstrating good reproducibility. Additionally, interfering substances did not affect the autoantibody titers, and satisfying performance was observed in tests examining the sample freeze-thaw stability. Notably, even when the titer of GAD autoantibodies was below the cut-off value of the GAD autoantibody ELISA, the 3 Screen ICA signal was completely absorbed by recombinant GAD65 protein, indicating that the detection sensitivity for GAD autoantibody in the 3 Screen ICA ELISA is higher than that of the GAD autoantibody ELISA kit. Furthermore, in a study using IASP2020 samples from the Immunology and Diabetes Society, which aims to standardize anti-islet autoantibody assays, this kit achieved excellent results with a sensitivity of 96.0%, specificity of 100%, and accuracy of 98.57%. Measuring multiple anti-islet autoantibodies in combination is crucial for diagnosing and predicting type 1 diabetes. The ELISA kit used in this study is highly versatile and can be used in any measurement facility, making it extremely useful for routine testing.

Distinct Scleroderma Autoantibody Profiles Stratify Patients for Cancer Risk at Scleroderma Onset and During the Disease Course.

We examined whether an array of scleroderma autoantibodies associates with risk of cancer and could be useful tools for risk stratification. Scleroderma cancer cases and scleroderma controls without cancer from the Johns Hopkins Scleroderma Center and the University of Pittsburgh Scleroderma Center were studied. Sera were assayed by Lineblot and enzyme-linked immunosorbent assay (ELISA) for autoantibodies against centromere, topoisomerase 1, RNA polymerase (POLR) 3, PM/Scl, Th/To, NOR90, U3 RNP, Ku, Ro52, U1RNP, and RNPC3. Logistic regression models were constructed to examine whether distinct autoantibodies associated with overall cancer at any time and cancer-associated scleroderma (cancer occurring three years before and after scleroderma onset). The effects of having more than one autoantibody on cancer were further examined using random forest analysis. A total of 676 cases and 687 controls were studied. After adjusting for relevant covariates, anti-POLR3 (odds ratio [OR] 1.47, 95% confidence interval [CI] 1.03-2.11) and monospecific anti-Ro52 (OR 2.19, 95% CI 1.29-3.74) were associated with an increased overall cancer risk, whereas anticentromere (OR 0.69, 95% CI 0.51-0.93) and anti-U1RNP (OR 0.63, 95% CI 0.43-0.93) were associated with lower risk. When examining risk of cancer-associated scleroderma, these immune responses remained associated with increased or decreased risk: anti-POLR3 (OR 2.28, 95% CI 1.33-3.91), monospecific anti-Ro52 (OR 2.58, 95% CI 1.05-6.30), anticentromere (OR 0.39, 95% CI 0.20-0.74), and anti-U1RNP (OR 0.32, 95% CI 0.11-0.93). Anti-Ro52 plus anti-U1RNP or anti-Th/To was associated with decreased cancer risk compared with anti-Ro52 alone. These data suggest that five distinct scleroderma immune responses, alone or in combination, may be useful tools to stratify the risk of cancer for scleroderma patients. Further study examining cancer risk in autoantibody subgroups relative to the general population is warranted.

B-064 Analytical and Diagnostic Performances of Anti-Integrin Antibody System in Inflammatory Bowel Disease

Abstract Background Anti-integrin αvβ6 autoantibodies may have value in the diagnosis of Inflammatory Bowel Disease (IBD) and more specifically Ulcerative Colitis (UC). Our objective was to establish the analytical and diagnostic performance of this novel autoantibody system in distinguishing IBD from a group of Normal Healthy Volunteers (NHV) and UC from Crohn’s Disease (CD). Methods All testing was performed using a Tecan Evo200 equipped with 8-channel and 96-channel arms, plate washer, and plate reader. The ELISA assay used calibration controls and diluted serum samples added to ELISA plates coated with Integrin αvβ6 antigen. Anti-human IgG horseradish peroxidase complexed with Anti-integrin αvβ6 antibodies was detected using colorimetry. The analytical performance was evaluated by using repeatability and reproducibility with coefficient of variation (CV) calculated. The diagnostic performances were evaluated by testing specimens collected from consented subjects available from a biobank in autoimmune gastrointestinal diseases. This consisted of 60 NHV, and 187 IBD (91 CD and 96 UC). Sensitivity, specificity, diagnostic odds ratio (OR), positive and negative predictive values (PPV and NPV) were estimated. Results Intra-assay CVs ranged from 1.5% to 5.2% over dynamic range. Inter-assay CVs were below 10%. Median titers were below 5 U/mL for NHV and CD, and 26 U/mL for UC (Fig. 1). Cutoff of 5 U/mL was 100% specific for UC and CD. Sensitivity for UC and CD was 80% (77/96), and 15% (14/91), respectively. Diagnostic Odds Ratio yielded 22.3-fold (95% CI: 10.5–47.5) higher likelihood of UC than CD in the presence of Anti-integrin αvβ6 titers above cutoff, with PPV and NPV values of 84% (76%-90%) and 81% (74%–87%), respectively (50% pre-test). Conclusion We have established the analytical performance of an Anti-integrin αvβ6 autoantibody ELISA assay. These preliminary data suggests that the Anti-Integrin αvβ6 autoantibody system is highly specific for UC with high sensitivity and specificity.

Mitochondrial sourcing of interferogenic ligands and an autoantigen in human obesity-associated metaflammation.

Visceral adipose tissue (VAT) inflammation contributes to metabolic dysregulation in obesity. VAT recruitment and activation of plasmacytoid dendritic cells (pDCs) through toll-like receptor 9 (TLR9) recognition of self-DNA, leading to induction of type I interferons, arecrucial innate triggers for this VAT inflammation. It was hypothesized that mitochondrial DNA (mtDNA) can contribute to TLR9 activation in VAT-recruited pDCs in obesity, and this study aimed to identify the carrier protein for ligand access to TLR9 and to explorewhether this also provides for a source of autoantigensin this context. VAT samples, used for gene expression studies as well as adipose explant cultures, were collected from patients with obesity (n = 54) and lean patients (n = 10). Supernatants from human pDC cultures, treated with adipose explant culture supernatants, were used for interferon α ELISA. Venous plasma, from patients with (n = 114) and without (n = 45) obesity, was used for an ELISA for autoantibodies. MtDNA from VAT in obesity, in complex with mitochondrial transcription factor A protein (TFAM), acts as interferogenic ligands for pDCs. Humoral autoreactivity against TFAM is also induced in obesity. Interferogenic ligands and an autoantigen can be sourced from dysfunctional mitochondria in VAT of humans with obesity. Further therapeutic and prognostic potential for this immune mechanism in obesity warrants exploration.

Incidence of celiac disease autoimmunity and associations with maternal tuberculosis and pediatric Helicobacter pylori infections in 4-year-old Ethiopian children followed up in an HLA genotyped birth cohort.

The prevalence of celiac disease in the general population is mainly unknown in most of sub-Saharan African countries. The aim of this study was to determine the incidence of celiac disease autoimmunity (CDA) and its associations with latent Mycobacterium tuberculosis (LMTB) and Helicobacter pylori (HP) infections in Ethiopian children aged 4 years in an HLA genotyped cohort study. Of 1,389 recruited children between 2018 and 2022, 1,046 (75.3%) had been screened at least twice for celiac disease between the ages of 2 and 4 years using a tissue transglutaminase autoantibody (tTGA) ELISA kit. Tissue TGA-positive children were retested using radio-binding assays. CDA was defined as persistent-confirmed tTGA positivity in two consecutive samples. Associations of CDA with LMTB and HP were tested in a subpopulation of 752 children born to mothers who were previously tested for LMTB with IFN-γ and anti-HP antibodies in samples collected at a mean age of 49.3 ± 5.3 months. Screening detected 38 out of 1,046 (3.6%) IgA-tTGA-positive children. Ten (1.0%) were confirmed to be positive, with six (0.6%) children diagnosed with CDA. The incidence of CDA at 4 years of age was 1.2 per 1,000 person-years. LMTB was found in 4 of 6 (66.7%) mothers with CDA children compared with 340 of 734 (46.3%) mothers of children without CDA (p = 0.424), while HP was found in 3 of 6 (50.0%) CDA children compared with 315 of 746 (42.2%) children without CDA (p = 0.702). The incidence of CDA in Ethiopian children is lower than the pooled global incidence. Neither LMTB nor HP infections are associated with CD in Ethiopian children.

P489 Anti-granulocyte-macrophage colony-stimulating factor (Anti-GM-CSF) autoantibodies—the underrecognized cause of Cryptococcosis in non-HIV individuals in Thailand: Case series from a single tertiary care hospital

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM BackgroundCryptococcosis is an opportunistic fungal infection in immunocompromised patients. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the functions of phagocytes and alveolar macrophages, which are crucial in cryptococcal control. Anti-granulocyte-macrophage colony-stimulating factor (Anti-GM-CSF) autoantibodies have been found to be associated with cryptococcosis in non-HIV individuals but this syndrome has never been described in Thai population.MethodsWe report here the case series of patients hospitalized in a tertiary care hospital in Northern Thailand.ResultsThree apparently immunocompetent patients, 34, 38, and 45 years old, were presented with neurological manifestations. Brain computed tomography scans and lumbar punctures were performed and the results showed evidence of cryptococcal meningoencephalitis. Two of the patients also had pulmonary cryptococcosis. We performed Anti-GM-CSF autoantibody ELISA assays in the patient's sera and all of three serum samples revealed a high titer of anti-GM-CSF autoantibodies. The patients were treated with amphotericin B deoxycholate with or without flucytosine for induction antifungal therapy, followed by fluconazole consolidation treatment. All patients were cured and had favorable outcomes.ConclusionsAnti-GM-CSF autoantibodies syndrome is underrecognized in Thai patients and is a new entity of immunodeficiency associated with cryptococcal meningoencephalitis and disseminated cryptococcosis in Thai patients.

Abstract #1153940: Specific Detection of TSAb and TBAb in Serum of Suspected AITD Patients using Two Rapid Homogenous Bioassays

Two rapid homogenous bioassays, named as Turbo TSI and Turbo TBI, have been developed to either quantitatively detect Thyroid Stimulating Antibodies (TSAb) or qualitatively detect Thyroid Blocking Antibody (TBAb) in Autoimmune Thyroid Disease (ATID) patient sera. The two bioassays do not require cell culture, sample dilution, washing or cell lysis steps, resulting in a dramatically reduced turnaround time to 60 minutes. This study was designed to demonstrate the high specificity of the two bioassays in comparison with the TSHR Autoantibody (TRAb) ELISA Kit (Kronus).

Determination of anti‐ADAMTS‐13 autoantibody titers in ELISA: Influence of ADAMTS‐13 presentation and autoantibody detection

BackgroundImmune‐mediated thrombotic thrombocytopenic purpura (iTTP) is caused by inhibitory and/or clearing anti‐ADAMTS‐13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats, member 13) autoantibodies. To determine the presence and total level of anti‐ADAMTS‐13 autoantibodies, commercial and in‐house developed ELISAs are performed. However, different ELISA methods vary in relation to the presentation of recombinant (r)ADAMTS‐13 and the detection method of the anti‐ADAMTS‐13 autoantibodies. Currently, the influence of those different approaches on anti‐ADAMTS‐13 autoantibody titers is not known. ObjectivesTo assess the influence of different ADAMTS‐13 presentation‐ and autoantibody detection methods on anti‐ADAMTS‐13 autoantibody titers in ELISA. Materials/MethodsAnti‐ADAMTS‐13 autoantibody titers from 18 iTTP patients were determined using four different set‐ups of anti‐ADAMTS‐13 autoantibody ELISAs. The ELISAs varied in the used presentation of rADAMTS‐13 (directly coated full‐length rADAMTS‐13, directly coated rMDTCS and rT2C2, or antibody‐captured full‐length rADAMTS‐13) and the detection antibodies (polyclonal anti‐human IgG or monoclonal anti‐human IgG1‐4 antibodies). ResultsStrong correlations between the different anti‐ADAMTS‐13 autoantibody ELISA approaches were observed, when using polyclonal anti‐human IgG detection antibodies recognizing all IgG subclasses similarly, independent of the method of rADAMTS‐13 presentation. Anti‐ADAMTS‐13 autoantibody titers correlated less when using a mixture of monoclonal anti‐human IgG1–4, because not all IgG subclasses were recognized with similar affinities. ConclusionAnti‐ADAMTS‐13 autoantibody levels using different methods of rADAMTS‐13 presentation strongly correlate. However, the levels of anti‐ADAMTS‐13 autoantibodies are highly dependent on the detection antibody used, which should detect all IgG subclasses (IgG1–4) equally well.

Inhibitory anti ADAMTS13 antibodies with a new rapid fully automated CLiA assay

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder characterized by severe ADAMTS13 deficiency. The acquired form is associated with autoantibodies directed against ADAMTS13. Both noninhibitory and inhibitory autoantibodies can be detected by ELISA assay, while only inhibitory autoantibodies are detected by Bethesda assay. Due to its short TAT and good performance, chemiluminescence (CliA) ADAMTS13 activity (HemosIL Acustar) has proven to be a good choice in the diagnosis of TTP in emergency settings. Aim of this study was to analyse the performance of the CliA ADAMTS13 activity assay in detecting inhibitory ADAMTS13 antibodies using the Bethesda assay. A method comparison study was performed on 69 stored samples: 11 acute TTPs, 38 TTP follow-ups, 5 TTP relapses, 1 congenital TTP, 10 HUS, 4 suspected TTPs. We retrieved the results of tests previously run in ELISA for both activity and autoantibodies. At the same time, we reran new tests including ELISA and CliA activity, ELISA autoantibodies, and ELISA and CliA Bethesda assays on thawed frozen samples. Very good correlation was observed between ELISA and CliA activity assay results (r=0.96) and between archived ELISA and CliA activity results (r=0.93). Agreement between the anti-ADAMTS13 assays ranged from good (k=0.63) to very good (k=0.92). CliA and ELISA Bethesda assays showed very good agreement with samples run at the same time using ELISA ADAMTS13-autoantibody assay. Albeit more expensive, the CliA Bethesda assay identified inhibitory anti-ADAMTS13 within almost the same TAT as ELISA, but with better automation and limited operator involvement.

IgM and IgA in addition to IgG autoantibodies against FcɛRIα are frequent and associated with disease markers of chronic spontaneous urticaria.

IgG autoantibodies against the high-affinity IgE receptor, FcɛRIα, contribute the pathogenesis of autoimmune chronic spontaneous urticaria (CSU). However, it is not known whether such patients also exhibit IgM or IgA autoantibodies against FcɛRIα. To address this question we developed an ELISA to assess serum levels of IgG, IgM, and IgA autoantibodies against FcɛRIα and investigated whether their presence is linked to clinical features of CSU including the response to autologous serum skin testing (ASST). Serum samples of 35 CSU patients (25 ASST-positive) and 52 healthy control individuals were analyzed using a newly developed competitive ELISA for IgG, IgM, and IgA autoantibodies to FcɛRIα. One in four CSU patients (8/35, 24%) had elevated serum levels of IgG-anti-FcɛRIα compared with (3/52, 6%) healthy controls. More than half of patients had IgM (21/35, 60%) and IgA (20/35, 57%) vs (3/52, 5%) each in healthy controls. Elevated IgM, but not IgG or IgA, autoantibodies were significantly more frequent in ASST-positive CSU patients (18/25, 72%) compared with ASSTnegative patients (3/10, 33%, P=.022). Also, elevated levels of IgM-anti-FcɛRIα, but not of IgG or IgA against FcɛRIα, were linked to low blood basophil (r=.414, P=.021) and eosinophil (r=.623, P<.001) counts. Increased serum levels of IgM-anti-FcɛRIα are common in patients with CSU and linked to features of autoimmune CSU. The role and relevance of autoantibodies to FcɛRIα in CSU can and should be further characterized in future studies, and our novel assay can help with this.

P6288Autoantibodies in heart failure associate with disease severity and differentially expressed genes in apoptotic, fibrotic, and hypoxia pathways in cardiomyocytes

Abstract Background Previous studies suggest that autoantibodies against cardiac myosin lead to dilated cardiomyopathy (DCM). Anti-cardiac myosin antibodies cross-react with the beta adrenergic receptor (βAR) and signal cAMP-dependent protein kinase A (PKA) in cardiomyocytes leading to apoptosis, fibrosis, dilated cardiomyopathy and arrhythmias. Purpose To determine if cross-reactive anti-cardiac myosin/anti-βAR autoantibodies which signal cardiomyocytes through PKA might play a role to establish DCM by promoting remodeling, apoptosis, and fibrosis. Methods Forty-one adults with DCM were enrolled &lt;6 months from symptom onset and followed for 12 months. Serum and myocarditis/DCM-derived human mAb were analyzed by ELISA for autoantibodies, and a PKA assay measured anti-HCM/βAR antibody-mediated signaling of cardiomyocytes (ATCC primary heart cell line H9c2). The top 50 genes differentially expressed in the cardiomyocytes treated with sera or human mAb were identified and compared to genes differentially expressed in blood of DCM patients to identify shared disease-specific genes. Results Anti-HCM autoantibodies including autoantibody responses against 32 overlapping synthetic peptides of the S2 fragment of HCM were significantly elevated in patients whose ejection fraction did not improve over 1-year compared to those with improved ejection fraction. The human mAb confirmed our results with HCM, βAR, specific HCM peptides, and PKA signaling. RNA sequencing revealed differentially expressed genes in serum/mAb-treated cardiomyocytes compared to genes identified after RNA sequencing of peripheral blood of patients (n=10) with DCM for &gt;1 year from onset. A primary heart cell line (H9c2-ATCC) treated with myocarditis/DCM patient sera or human mAb revealed differentially expressed genes associated with cardiac hypertrophy and heart failure, and included inflammasome component NLRP3 and complement factor H. Ingenuity Pathway Analyses revealed 27, 7, and 1 differentially expressed genes related to apoptosis, fibrosis, and hypoxia, respectively. Gene expression of CASZ1, a transcription factor important in protection against DCM, was strongly correlated with PKA signaling (r=0.89). The KDM6B gene for lysine demethylase associated with hypoxia and apoptosis pathways and was shared between cardiomyocyte and peripheral blood analysis of DCM patients. Overall, 5 genes were shared in heart failure vs in vitro Ab-treated cardiomyocyte RNA sequencing analysis: CYP4F3, KDM6B, MBOAT7, SMAP2, and DDIT4, which affects phosphorylation of mTOR to promote autophagy and cell death, cardiac hypertrophy and dysfunction. Conclusions Significantly higher responses to cardiac myosin in patients with DCM were related to lack of left ventricular function improvement and to differential expression of genes promoting apoptosis, fibrosis and disease severity. These studies identify autoantibody-directed gene signaling as a potential novel therapeutic target in DCM. Acknowledgement/Funding National Heart, Lung, and Blood Institute, Bethesda, MD, USA

2418Th17 signature, autoimmunity and differentially expressed genes in cardiomyopathy and heart failure

Abstract Background Cardiomyopathy may occur due to viral infections or drug induced heart damage. Cardiac myosin released from damaged heart has been shown to be a damage associated molecular pattern which binds to TLR2 or TLR8 and can act as an adjuvant to induce a strong autoimmune response against the heart. The result is autoimmunity against the heart which can lead to apoptosis, fibrosis and heart failure. Purpose Immune biomarkers of the early stages of heart failure are needed to identify individuals who develop progressive heart failure, do not recover their ejection fraction and may be candidates for immunotherapies. Methods Forty-one patients with myocarditis and heart failure &lt;6 months after onset were followed for 12 months and compared to age matched controls. Peripheral blood mononuclear cells were analyzed by FACS analysis and serum analyzed by ELISA for autoantibodies and cytokines. Statistical analysis was determined by Mann Whitney test. Peripheral blood of 10 patients with dilated cardiomyopathy (DCM) vs 19 healthy controls were analyzed for gene expression by RNA sequencing and pathway analysis using Reactome. Results Autoantibodies against human cardiac myosin and the beta-adrenergic receptor were significantly elevated in our cohort and functionally acted on cardiomyocytes to activate protein kinase A. Concomitantly, a Th17+ immunophenotype was significantly elevated in blood as well as in cardiac biopsies. CD4+IL17+ T cells (p=0.0008) and Th17-promoting cytokines TGF beta (p&lt;0.0001), IL-6 (p&lt;0.0001), IL-23 (p=0.0001), GMCSF (p=0.0336) and GMCSF-secreting CD4+ T cells (p=0.0006) were significantly elevated in blood. A Th17 immunophenotype was significantly associated with heart failure primarily in males (p=0.029). Persistent heart failure (NYHA class III and IV) and non-recovery of left ventricular function were associated with significantly higher percentages of IL17A-producing T cells at baseline, 6 and 12 months after onset, and IL-17A (p=0.019) and elevated Th17-promoting cytokines IL-6 (p=0.0001) and TGF-beta (p=0.0076). Decreased T regulatory immunosuppressive cells were significantly (p=0.0006) decreased and correlated with elevated Th17 cytokines in heart failure. Overrepresentation analysis of differentially expressed genes (adj p&lt;0.05) in blood of patients with DCM &gt;1year were identified using Reactome which revealed significant (FDR = 1.52E-13) enrichment of neutrophil degranulation (48 genes). Conclusion Our study illustrates a strong Th17 signature in more severe heart failure early in disease with elevated anti-cardiac myosin autoantibodies in non-recovery of left ventricular function. We observed a strong correlation with Th17-related neutrophil degranulation pathways in later disease, which may be biomarkers of fibrosis progression and disease severity in patients with heart failure. Cardiomyopathy with a Th17 signature might be treated with preventive immunomodulatory therapies such as anti-IL17A. Acknowledgement/Funding National Heart Lung and Blood Institute, Bethesda, MD, USA

A new ELISA for autoantibodies to steroid 21-hydroxylase.

A new ELISA for autoantibodies to steroid 21-hydroxylase (21-OH Ab) is described. In the assay test sample autoantibodies form a bridge between 21-OH coated onto the plate well and liquid phase 21-OH-biotin. Bound 21-OH-biotin is detected by the addition of streptavidin peroxidase and colorogenic peroxidase substrate. Of 100 samples from patients with autoimmune Addison's disease, 86 (86%) were positive for 21-OH Ab ELISA whereas 84 (84%) were positive in an immunoprecipitation assay based on 125I-labeled 21-OH. Six (0.6%) of 928 healthy adult blood donors and 1 (2.0%) of 49 adult patients with type 1 diabetes mellitus (T1DM) were positive by ELISA. No samples from adult patients with Graves' disease (GD; n=50), celiac disease (n=29), systemic lupus erythematosis (n=9) or rheumatoid arthritis (n=20) were positive by ELISA. However, 2/51 (3.9%) children with GD, 3/69 (4.3%) children with Hashimoto's thyroiditis (HT) and 3/119 (2.5%) children with T1DM alone or associated with autoimmune thyroid disorders were ELISA positive. The new assay should be useful for screening patients known to be at increased risk of developing clinical autoimmune Addison's disease, in particular children with HT, GD and/or T1DM.

Serum anti-TOPO48 autoantibody as a biomarker for early diagnosis and prognosis in patients with esophageal squamous cell carcinoma

We previously reported a novel tumor associated antigen (TTA) with molecular weight around 48kDa that is a fragment derived from human DNA-topoiomerase I (TOP1). We termed the novel TAA as TOPO48 and termed autoantibody against the TAA as anti-TOPO48 autoantibody. The aim of this study is to further investigate the clinical applications of the autoantibody in patients with esophageal squamous cell carcinomas (ESCC). Serum levels of the anti-TOPO48 autoantibody in 112 ESCC patients, 112 age- and gender-matched healthy controls and 75 patients with esophageal benign tumors were determined by using a specific anti-TOPO48 autoantibody ELISA. Then, we statistically evaluated its clinical significance. We found that serum anti-TOPO48 autoantibody levels in ESCC patients were significantly higher than that in healthy controls and benign tumor patients (P=0.001). The percentage of sera with a positive level of anti-TOPO48 autoantibody in early stages was significantly higher than that in advanced stages of the cancer patients when the maximum level of healthy control sera was taken as a cut-off value (P=0.001). The area under ROC curve was 0.863 (95% CI=0.797-0.928) for healthy controls vs. early stage ESCC. In addition, patients with positive anti-TOPO48 autoantibody had significantly higher survival rate and longer survival time than that with negative anti-TOPO48 autoantibody in cancer patients (P=0.038, 0.025 and 0.047 for all stages, early stage and advanced stage, respectively). Our results suggest that anti-TOPO48 autoantibody may be a potentially useful biomarker for early diagnosis and prognosis of ESCC.

3 Screen islet cell autoantibody ELISA: A sensitive and specific ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8

3 Screen, a new ELISA for the combined measurement of autoantibodies to GAD65, to IA-2 and to ZnT8, has been developed and evaluated.In the assay serum samples were incubated (overnight; 2–8°C) in ELISA plate wells coated with GAD65, IA-2 and ZnT8 followed by a wash step and incubation with biotinylated GAD65, IA-2 and ZnT8 (1h; 2–8°C,). The assay was completed by addition of streptavidin-peroxidase and tetramethylbenzidine. Samples tested in the 3 Screen were also analysed in ELISAs and radiobinding assays for the three individual autoantibodies.129/132 (98%) samples from newly diagnosed T1DM children and 1/100 non-diabetic children controls were positive in 3 Screen. There was good agreement between 3 Screen and the individual autoantibody assays. Dilution of positive samples showed good linearity characteristics. In the 2015 Islet Autoantibody Standardization Program 3 Screen achieved 94% sensitivity, 95.6% specificity and 0.948 area under curve by ROC analysis.3 Screen provides a simple and sensitive method for combined measurement of three major diabetes associated autoantibodies in a single sample. The assay should be a useful tool for large scale population screening for individuals at risk of developing T1DM.

Validation of a panel of ADAMTS13 assays for diagnosis of thrombotic thrombocytopenic purpura: activity, functional inhibitor, and autoantibody test.

Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone). A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%. ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study.

Anti-transcription intermediary factor 1-gamma autoantibody ELISA development and validation

A quantitative anti-transcription intermediary factor 1-gamma (anti-TIF1-γ) ELISA may improve the detection of cancer-associated myositis (CAM). The aims of this study were the development and validation of a quantitative anti-TIF1-γ autoantibody ELISA in patients with myositis. We developed an ELISA using recombinant purified full-length human TIF1-γ. Patient serum was incubated with TIF1-γ-coated ELISA plates, and secondary antibody that bound human IgG was used to detect anti-TIF1-γ binding. Protein immunoprecipitation (IP) was used as the gold standard for the presence of anti-TIF1-γ. Serum samples from myositis patients with positive and negative anti-TIF1-γ by IP, from non-myositis autoimmune patients (SSc, SLE and RA) and from healthy controls were analysed. The ELISA's sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated. Agreement between the ELISA and IP results was determined using chi-squared and kappa tests. Test-retest reliability of the ELISA was assessed. We identified 55 myositis patients with and 111 controls without anti-TIF1-γ by IP. Anti-TIF1-γ positivity by ELISA showed strong agreement (93.9%) with IP results (κ = 0.87). The sensitivity, specificity, PPV, NPV and overall accuracy of the anti-TIF1-γ ELISA were 91%, 96%, 93%, 95% and 94%, respectively. The area under the curve (AUC) of a receiver operating characteristic (ROC) curve was 0.938. Test-retest reliability was strong (Pearson r = 0.913, P < 0.001). We developed a quantitative ELISA for detecting serum anti-TIF1-γ autoantibodies and validated the assay in myositis and other connective tissue disease patients. The availability of a validated, quantitative ELISA should improve the detection of anti-TIF1-γ autoantibodies and may improve the detection of CAM.

Application of a High Throughput Method of Biomarker Discovery to Improvement of the EarlyCDT®-Lung Test

BackgroundThe National Lung Screening Trial showed that CT screening for lung cancer led to a 20% reduction in mortality. However, CT screening has a number of disadvantages including low specificity. A validated autoantibody assay is available commercially (EarlyCDT®-Lung) to aid in the early detection of lung cancer and risk stratification in patients with pulmonary nodules detected by CT.Recent advances in high throughput (HTP) cloning and expression methods have been developed into a discovery pipeline to identify biomarkers that detect autoantibodies. The aim of this study was to demonstrate the successful clinical application of this strategy to add to the EarlyCDT-Lung panel in order to improve its sensitivity and specificity (and hence positive predictive value, (PPV)).Methods and FindingsSerum from two matched independent cohorts of lung cancer patients were used (n = 100 and n = 165). Sixty nine proteins were initially screened on an abridged HTP version of the autoantibody ELISA using protein prepared on small scale by a HTP expression and purification screen. Promising leads were produced in shake flask culture and tested on the full assay. These results were analyzed in combination with those from the EarlyCDT-Lung panel in order to provide a set of re-optimized cut-offs. Five proteins that still displayed cancer/normal differentiation were tested for reproducibility and validation on a second batch of protein and a separate patient cohort. Addition of these proteins resulted in an improvement in the sensitivity and specificity of the test from 38% and 86% to 49% and 93% respectively (PPV improvement from 1 in 16 to 1 in 7).ConclusionThis is a practical example of the value of investing resources to develop a HTP technology. Such technology may lead to improvement in the clinical utility of the EarlyCDT­-Lung test, and so further aid the early detection of lung cancer.

Development and Validation of a High Throughput System for Discovery of Antigens for Autoantibody Detection

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.

Comparison of Particle Agglutination Assay and ELISA for Anti-thyroid Autoantibodies

Comparison of Particle Agglutination Assay and ELISA for Anti-thyroid Autoantibodies