Quantification Of Autoantibodies Research Articles

PB0805 Comparison of Three ELISA Assays for the Detection and Quantification of Autoantibodies against Complement Factor H

PB0805 Comparison of Three ELISA Assays for the Detection and Quantification of Autoantibodies against Complement Factor H

Abstract 3296: Tumor antigens Fetuin-A and Secreted Protein Acidic and Rich in Cysteine (SPARC) Autoantibodies as diagnostic and prognostic biomarkers in prostate cancer

Abstract Introduction: Improvements in blood-based biomarkers for distinguishing between indolent and aggressive prostate cancer are critical in enhancing the management of the disease. To address this, we have focused on the quantification of autoantibodies (AAbs) against tumor antigens present in the sera of patients. We have selected SPARC and Fetuin-A (also known as Alpha 2-HS Glycoprotein [AHSG]) for analysis as they are shown to be highly expressed at late stages of prostate cancer. The objectives of this study are: 1) To measure AAbs against SPARC and Fetuin-A in the sera of prostate cancer patients; 2) To determine whether there is a correlation between levels of SPARC and Fetuin-A AAbs in serum, disease and race status. Methods: Sera from prostate cancer patients and healthy controls were evaluated for AAbs against SPARC and Fetuin-A by using recombinant full-length proteins as substrates in an enzyme-linked immunosorbent assay (ELISA) assay. Sera from 117 Caucasian American (CA) n= and 111 African American (AA) prostate cancer patients with Gleason grades 6-10, and healthy controls (CA, n=52; AA, n=45) were analyzed in addition to sera from a biopsy cohort (n=99). The specificity of AAbs against the respective target proteins was confirmed by immunoblot analysis. Results: SPARC AAbs were detected in the sera, with significantly lower levels in both CA (p<0.0001; AUC=0.80), and AA prostate cancer patients (p<0.0001; AUC=0.82), compared to healthy controls. AAbs against Fetuin-A were significantly lower in prostate cancer patients in comparison to controls (p<0.0001; AUC=0.96). The range of AAb reactivity to SPARC and Fetuin-A was similar in both CA and AA prostate cancer patients. The results from biopsy cohort showed lower SPARC AAbs in cancer positive (n = 49) in comparison to cancer negative (n = 42) cases, and healthy controls. Conclusions: In this study, we established the presence of AAbs against SPARC in prostate cancer patient serum for the first time. More importantly, we observed highly significant differences between prostate cancer patient (low) and controls (high) sera, across different ethnic groups, similar to AAbs noted against Fetuin-A. These data support the further evaluation of SPARC and Fetuin-A AAbs as promising serum biomarkers for prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Shyh-Han Tan, Andy Martinez, Anshu Rastogi, Wei Huang, Sreedatta Banerjee, Lakshmi Ravindranath, Denise Young, Amina Ali, Indu Kohaar, Yongmei Chen, Jennifer Cullen, Gyorgy Petrovics, Albert Dobi, David G. McLeod, Jacob Kagan, Sudhir Srivastava, Isabell A. Sesterhenn, Inger L. Rosner, Shiv Srivastava, Alagarsamy Srinivasan. Tumor antigens Fetuin-A and Secreted Protein Acidic and Rich in Cysteine (SPARC) Autoantibodies as diagnostic and prognostic biomarkers in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3296.

Qualitative and Quantitative Assay for Detection of Circulating Autoantibodies against Human Aortic Antigen.

Increased amount of autoantibodies in human sera are the hallmark of autoimmune diseases ( Wang et al., 2015 ). In case of known antigen, detection of autoantibodies is done using laboratory based methods. However, in most autoimmune diseases, knowledge of self-antigen is still vague. We have developed an ELISA-based quantitative assay to detect the presence of autoantibodies as well as to measure the circulating autoantibodies in the sera of patients suffering from large vessel vasculitis (LVV), an autoimmune disease ( Chakravarti et al., 2015 ). Using this assay, we detected the increase in anti-aortic antibodies in LVV patient's sera. We have further verified the results by independent biochemical techniques and found the specificity to be > 94% ( Chakravarti et al., 2015 ). This method can be uniquely modified to suit any autoimmune, in particular organ specific, disease and thus has wider applications in the detection and quantification of autoantibodies.

Autoantibodies in the grocery shop: does quantity matter?

Testing for autoantibodies is an integrated part in the diagnostic process of many autoimmune diseases. As a consequence, the detection of autoantibodies is increasingly recognized in classification criteria and diagnostic guidelines. In this review, the question is addressed whether quantification of autoantibodies is clinically useful, not only in the diagnostic stage, but also during follow-up of patients with autoimmune diseases. It can be concluded that for a multitude of autoantibodies, there is sufficient evidence that the higher the amount of autoantibodies, the more likely that the patient has the respective autoimmune disease. Quantification is also of added value for monitoring therapeutic efficacy, although this depends on the type of therapy that is installed. The relation between rises in autoantibody levels and the occurrence of subsequent disease relapses, however, is far from absolute. Obviously, if quantification of autoantibodies is performed in the laboratory, results should also be reported quantitatively together with an appropriate interpretation for the clinician.

Pre-analytical effects of blood sampling and handling in quantitative immunoassays for rheumatoid arthritis

Variability in pre-analytical blood sampling and handling can significantly impact results obtained in quantitative immunoassays. Understanding the impact of these variables is critical for accurate quantification and validation of biomarker measurements. Particularly, in the design and execution of large clinical trials, even small differences in sample processing and handling can have dramatic effects in analytical reliability, results interpretation, trial management and outcome. The effects of two common blood sampling methods (serum vs. plasma) and two widely-used serum handling methods (on the clot with ambient temperature shipping, “traditional”, vs. centrifuged with cold chain shipping, “protocol”) on protein and autoantibody concentrations were examined. Matched serum and plasma samples were collected from 32 rheumatoid arthritis (RA) patients representing a wide range of disease activity status. Additionally, a set of matched serum samples with two sample handling methods was collected. One tube was processed per manufacturer's instructions and shipped overnight on cold packs (protocol). The matched tube, without prior centrifugation, was simultaneously shipped overnight at ambient temperatures (traditional). Upon delivery, the traditional tube was centrifuged. All samples were subsequently aliquoted and frozen prior to analysis of protein and autoantibody biomarkers. Median correlation between paired serum and plasma across all autoantibody assays was 0.99 (0.98–1.00) with a median % difference of −3.3 (−7.5 to 6.0). In contrast, observed protein biomarker concentrations were significantly affected by sample types, with median correlation of 0.99 (0.33–1.00) and a median % difference of −10 (−55 to 23). When the two serum collection/handling methods were compared, the median correlation between paired samples for autoantibodies was 0.99 (0.91–1.00) with a median difference of 4%. In contrast, significant increases were observed in protein biomarker concentrations among certain biomarkers in samples processed with the ‘traditional’ method. Autoantibody quantification appears robust to both sample type (plasma vs. serum) and pre-analytical sample collection/handling methods (protocol vs. traditional). In contrast, for non-antibody protein biomarker concentrations, sample type had a significant impact; plasma samples generally exhibit decreased protein biomarker concentrations relative to serum. Similarly, sample handling significantly impacted the variability of protein biomarker concentrations. When biomarker concentrations are combined algorithmically into a single test score such as a multi-biomarker disease activity test for rheumatoid arthritis (MBDA), changes in protein biomarker concentrations may result in a bias of the score. These results illustrate the importance of characterizing pre-analytical methodology, sample type, sample processing and handling procedures for clinical testing in order to ensure test accuracy.

Antiribosomal P protein IgG autoantibodies are highly specific for SLE and might be associated with disease activity

Background and objectivesSystemic lupus erythematosus (SLE) is characterised by the production of autoantibodies directed against a variety of nuclear and cytoplasmic antigens. One of these antigens is ribosomal P protein...

Prediction of Clinical and Mucosal Severity of Coeliac Disease and Dermatitis Herpetiformis by Quantification of IgA/IgG Serum Antibodies to Tissue Transglutaminase

We analysed whether the quantification of autoantibodies against tissue transglutaminase could be used to predict mucosal destruction and disease severity in patients with gluten sensitivity. One hundred seventy patients with coeliac disease (CD), comprising 52 children with severe malabsorption (group I), 59 children with mild symptoms (group II), 59 adults (group III), 134 patients with dermatitis herpetiformis (DH), and 131 disease controls, were studied. Serial serum samples of patients in groups I and II on a gluten-free diet were also included. Serum levels of antibodies against recombinant tissue transglutaminase were determined with ELISA using standard curves for quantification of antibodies. Immunoglobulin (Ig)A antibodies against tissue transglutaminase (IgA-TGA) were detected in all of the patients with CD and in 95% of the DH patients. The IgA-TGA and IgG-TGA levels were higher in group I (P < 0.001). The IgG-TGA levels and positivity rate in group I (100%) were higher than in group II (81%), group III (73%), and the DH group (67%). Elevated IgA-TGA and IgG-TGA levels in combination predicted a more severe small intestinal atrophy (P < 0.0001) with a specificity of 99% for Marsh IIIb-IIIc (flat) lesions. The kinetics of the IgA-TGA decrease during diet differed between groups I and II. High levels of IgA-TGA and IgG-TGA antibodies were associated with the grade of mucosal villous atrophy and a more severe clinical presentation. The combined measurement of IgA-TGA and IgG-TGA enables a noninvasive prediction of small intestinal villous atrophy with high accuracy, and may reduce the need for a biopsy in patients with suspected CD.

Abstract B57: UBE3A (E6AP) auto antibodies in hepatocellular carcinoma

Abstract B57 UBE3A (E6AP) auto antibodies in hepatocellular carcinoma 1Gebreselassie, D., 1Lan, R., 1Loffredo, C.A., 2Abdel-Hamid, M., 1Goldman, R. 1Lombardi Comprehensive Cancer Center Georgetown University Washington, D.C. &amp; 2National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt Patients with cancer produce auto antibodies that could serve as biomarkers for detection and classification of the disease. To identify auto antibodies associated with hepatocellular carcinoma (HCC), we used a protein array of 5,000 human proteins (Invitrogen) to compare sera of ten cancer free controls and ten patients with HCC matched on age, gender, and viral infection. Thirty six proteins significantly associated with HCC (p&amp;lt;0.05) were spotted on a custom array. The array experiments confirmed increased auto antibodies in HCC for 20 of the 36 candidates. The frequency of increase was as high as 45% for some of the auto antibodies in a comparison of 20 HCC cases and 10 matched controls. To further validate the observed auto antibody response to Ubiquitin protein ligase E3A (UBE3A), we fused cDNA for the protein, with an N-terminal FLAG tag and an enzyme reporter gene, humanized Renilla luciferase (hRluc). The construct was expressed in the Cos-1 mammalian cell line and crude cell lysates were used for quantification of auto antibodies in serum of the same individuals used in the protein array experiment. The assay is based on immunoprecipitation (IP) of the antigenic protein-construct on protein G beads followed by the quantification of hRluc chemiluminescence. Antibodies to the FLAG tag and the antigen of interest were used to optimize the assay. We found that HCC patient sera contain significantly elevated auto antibodies to UBE3A (t-test P&amp;lt;0.01). The autoantibody response to UBE3A was observed in approximately 20% of the HCC patients and correlates with the results of the protein array experiment. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B57.

Scleroderma Renal Crisis: A Rare Complication of Systemic Sclerosis with Poor prognosis-experience in a Medical Center in Central Taiwan

Objective: Scleroderma renal crisis (SRC) is the most life-threatening complication of systemic sclerosis (SSc), which is characterized by malignant hypertension and rapidly progressive renal failure. In this study, we investigate the incidence, clinical features, treatment and outcome of our SRC patients. Patients and Methods: We reviewed the records of 161 patients with SSc, seen from January 1991 to December 2005 in the Department of Rheumatology, Taichung Veterans General Hospital, Taiwan. Tests for anti-nuclear antibodies (ANA) in sera were screened against Hep-2 cells by indirect immunofluorescence assay. Quantification of autoantibodies to extractable nuclear antigen, including anti-Scl-70 antibody (Scl-70) and anti-centromere antibody, were tested with the AtheNa Multi-Lyte ANA test system. Data collected from the records included gender, age at onset, age at diagnosis, clinical manifestations, laboratory data, treatment and outcome. Fisher's exact test was used to compare differences in categorical variables. Results: Seventy-eight (48.4%) patients were limited type SSc and 83 (51.6%) patients were diffuse type SSc. Four (2.5%) patients of a total of 161 patients had SRC. All of our SRC patients were diffuse type with positive Scl-70. SRC occurred at 37.7±55.72 months (range from 1-120 months) after the diagnosis of SSc. The duration between Raynaud's phenomenon to disease onset was 3.75 ± 2.06 months. There were significant difference at myocardial involvement (p＜0.05) and pericardial effusion (p＜0.05) between the SRC and Non-SRC diffuse SSc patients. Two patients died within four months after the SRC, one patient required permanent dialysis and one patient partly regained renal function. Conclusion: SRC is a rare complication in Taiwanese SSc patients and has a poor prognosis. Whether early administration of angiotensin converting enzyme inhibitor might prevent or ameliorate the onset of SRC needs further investigation.

Laboratory testing in autoimmune rheumatic diseases

There are a number of pathological conditions in which tissue damage occurs in association with immune activation directed against components of normal tissue. The initial damaging events usually involve cells of the immune system, the T-cells, but the cell damage releases antigens that become targets for an antibody response. The detection and quantification of autoantibodies has become an important component in the diagnosis and management of autoimmune rheumatic diseases such as rheumatoid arthritis, systemic lupus erythematosus, the systemic vasculitides and systemic sclerosis. Each of these diseases is associated with a particular autoantibody or group of autoantibodies. They are usually detected by their reaction against tissue components using subjective methods such as indirect immunofluorescence. Any positive samples are further analysed using more specific and quantitative methods for the 'quantification' of the specific autoantibody concentration. It is important that these autoantibodies are not considered to be 'gold standard' tests: they are no more than markers of the disease with significant limitations. They are best used as part of a diagnostic panel rather than as a marker indicating one particular disease. Techniques are gradually improving, giving numerical results rather than titres, but a lack of standardization makes these results extremely variable. Many of the markers show no correlation with disease activity. Their use should be restricted to the initial investigation and not repeated every time the patient is followed up. Other markers do, however, correlate with disease activity and can be used to monitor disease. When investigating patients who have symptoms associated with autoimmune rheumatic diseases, analytes such as immunoglobulins, complement components and C-reactive protein may all be measured.

Use of synthetic peptides for the detection and quantification of autoantibodies.

The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins). Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B' synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity. Synthetic peptide antigens have been used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications.(ABSTRACT TRUNCATED AT 250 WORDS)