Autoantibodies In Human Sera Research Articles

Biosensor approaches for the detection of autoantibodies in human serum

The analysis of autoantibodies in human serum plays a crucial role in the diagnosis and follow-up of autoimmune diseases. The analytical tools available for the determination of these analytes, however, are still far from mature, lack standardization, and give low negative predictive values. Approaches using biosensor technology for analysis are an attractive alternative to classical techniques. In special applications, biosensors already have been proven to be effective for clinical diagnostics. This is due to the fact that real-time monitoring of the antigen/antibody interaction gives valuable information on autoantibody affinity to the respective antigenic structure. The potentials of biosensors for the serological analysis of autoantibodies are evident from the increasing number of publications on the subject. Thus, this review focuses on underlying biosensor techniques and published clinical trials. The advantages of multiplexed analyses of autoantibodies by use of microarrays are also emphasized. This promising bioanalytical technique is also particularly important for the structural identification of novel antigens.

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.

Rapid detection of anti-chromatin autoantibodies in human serum using a portable electrochemical biosensor

The current system employed electrochemical sensor technology for the measurement of autoantibodies in human sera. Anti-chromatin antibodies are an early and sensitive indicator of systemic lupus erythematosus (SLE) and are typically quantified using enzyme-linked immunosorbent assays (ELISA). Electrochemical detection compared favorably with ELISA using a sample of 30 SLE sera (r=0.9), and non-specific binding by normal serum immunoglobulin was undetectable. The electrochemical sensor assay required <20min processing time and utilized a hand-held apparatus with a disposable electrode. These results demonstrate the applicability of this technology to the rapid measurement of a clinically relevant analyte with an apparatus of potential simplicity and low cost.

A recombinant topoisomerase I ELISA: screening for IgG, IgM and IgA anti-topo I autoantibodies in human sera.

An ELISA for the detection of anti-topoisomerase I autoantibodies in sera from patients with suspected or manifest rheumatic diseases is described. The antigen source used in this assay consists of a recombinant protein containing the last 695 C-terminal amino acid residues of human topoisomerase I (topo I). The sensitivity of the assay was 61%, while the specificity was more than 98%. Using this ELISA, 47 sera from scleroderma patients and immunopositive for anti-topo I antibodies, were screened for the presence of the isotypes IgG, IgA and IgM to topo I. Our finding that relatively high levels of IgA antibodies to topo I are present in most of the sera tested is consistent with the results of Hildebrandt et al. [1]. In addition, it is demonstrated that the IgG and IgA antibodies in a serum may recognize different epitope regions on the topo I polypeptide.

Peptide-coated nanotube-based biosensor for the detection of disease-specific autoantibodies in human serum

We demonstrate a label-free peptide-coated carbon nanotube-based immunosensor for the direct assay of human serum. A rheumatoid arthritis (RA)-specific (cyclic citrulline-containing) peptide, was immobilized to functionalized single-walled carbon nanotubes deposited on a quartz crystal microbalance (QCM) sensing crystal. Serum from RA patients was used to probe these nanotube-based sensors, and antibody binding was detected by QCM sensing. Specific antibody binding was also determined by comparing the assay of two serum control groups (normal and diseased sera), and the native unmodified peptide. The sensitivity of the nanotube-based sensor (detection in the femtomol range) was higher than that of the established ELISA and recently described microarray assay systems, detecting 34.4 and 37.5% more RA patients with anti-citrullinated peptide antibodies than those found by ELISA and microarray, respectively. There was also an 18.4 and 19.6% greater chance of a negative test being a true indicator of a person not having RA than by either ELISA or microarray, respectively. The performance of our label-free biosensor enables its application in the direct assay of sera in research and diagnostics.

Antibody–antigenic peptide interactions monitored by SPR and QCM-D: A model for SPR detection of IA-2 autoantibodies in human serum

This work reports on a complementary use of surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D) technologies to study interactions between a peptide antigen and polyclonal antibodies, in an experimental format suitable for diagnostic assays of autoimmune diseases. In the chosen model, a synthetic peptide from the juxtamembrane region of IA-2 (a type 1 diabetes associated antigen) was immobilized by an optimized chemical protocol applicable to both BIACORE and QCM-D sensors. A thorough study of the peptide immobilization was performed to optimize the signal-to-noise ratio using mixed self-assembled monolayers (SAM) on a gold surface. Introduction of polyethylene glycol (EG6) chains into mixed SAM layers and addition of an anionic surfactant to the human serum reduced non-specific binding without modifying the viscoelasticity properties of the layer. Under our conditions, the antibody SPR detection limit was determined to be 0.2nM in diluted human serum. This value is in agreement with the reported rank distribution of IA-2 antibodies in diabetic patient sera. Label-free and real-time technologies such as SPR and/or QCM-D could be precious tools in future diagnostic assays.

Unraveling the Complex Proteome for Biomarker Discovery in Gastrointestinal and Liver Diseases

Unraveling the Complex Proteome for Biomarker Discovery in Gastrointestinal and Liver Diseases

Comparison of a multiplex, bead-based fluorescent assay and immunofluorescence methods for the detection of ANA and ANCA autoantibodies in human serum

Detection of antinuclear (ANA) and antineutrophil cytoplasmic (ANCA) antibodies is extensively used for establishing a diagnosis in patients with clinical features suggestive of autoimmune disorders. The most common methods for the identification of positive patients' sera for ANA or ANCA are indirect immunofluorescence (IIF) and ELISA-based procedures. Considerable effort has been made in developing simpler automated assays for routine laboratory use. Recently a commercially available microsphere-based fluorescent assay has been introduced for the detection of ANA and ANCA. The aim of this study was to compare this technology with routinely used IIF and ELISA procedures, in patients with a suggested autoimmune disorder. A highly significant correlation between ELISA procedures for specific antibodies and the microsphere-based assays were obtained for both ANA and ANCA as well as for extractable nuclear antigens ELISA screening, indicating that multiplex technology could replace individual ELISA tests for the measurement of specific autoantibodies. However, a low sensitivity for identifying IIF-positive cases was obtained for both ANA (58.0%) and ANCA (59.1%), although there was a significant correlation between the assays. In conclusion, our data show that a microsphere-based fluorescent assay may be a valid platform for the simultaneous determination of circulating individual ANA and ANCA autoantibodies. Furthermore, multiplexing technology offers several advantages that will probably make it an attractive tool in the future. Nevertheless, until further studies are conducted that determine the clinical performance of the multiplex technology, the initial screening of patients for autoantibodies with IIF is still considered necessary.

Protein microarrays.

With the introduction of DNA microarrays as novel analytical tools, the determination of thousands of binding events in one reaction became possible. The developed technology platforms are not limited to nucleic acids, and, in principle, every ligand-binding assay that works on solid phase can be miniaturized and brought into an array format. This unit explains how protein microarrays can be generated using equipment originally designed for DNA microarrays and how multiplexed assays for the quantification of proteins are set up. A protocol that describes a parallelized system for detecting autoantibodies in human serum is included as an example, and it is shown how existing sandwich immunoassays can be miniaturized and performed in array format. The unit also provides some theoretical background and commentary on the problems associated with this still-novel technology.

Lipid Analyses for the Management of Vascular Diseases

Despite a long history of studies on lipid abnormalities, numerous problems in laboratory technologies and techniques remain unresolved. One of the most commonly tested molecules is low-density lipoprotein (LDL) cholesterol, and a homogenous assay technique for measurement of this molecule has recently been introduced. Although the method represents remarkable technological breakthroughs with great potential for improving lipoprotein analysis, some discrepancies exist among assay protocols. Even for direct measurement of high-density lipoprotein (HDL) cholesterol, which has widely been accepted, there are still large discrepancies among data obtained by different protocols. Oxidatively modified LDL is an independent factor that is considered to directly contribute to the pathogenesis of early atherosclerosis. Lipid peroxidation products, surface charge, and spectrophotometric patterns are all applicable to the evaluation of in vitro oxidation. Only enzyme-linked immunosorbent assays using monoclonal antibodies have a potential for clinical use, but such methods are not yet standardized. There is also increasing evidence for the presence of anti-oxidized LDL autoantibodies in human sera, but the diagnostic utility remains controversial. In addition, small dense LDL has recently attracted much attention as an independent risk factor. Although this is a potential target of oxidization, a robust and simple analytical method does not yet exist. This review presents the current state of laboratory technologies for testing lipid abnormalities.

The use and abuse of commercial kits used to detect autoantibodies

The detection of autoantibodies in human sera is an important approach to the diagnosis and management of patients with autoimmune conditions. To meet market demands, manufacturers have developed a wide variety of easy to use and cost-effective diagnostic kits that are designed to detect a variety of human serum autoantibodies. A number of studies over the past two decades have suggested that there are limitations and concerns in the use and clinical application of test results derived from commercial kits. It is important to appreciate that there is a complex chain of users and circumstances that contributes to variations in the apparent reliability of commercial kits. The goal of this review is to identify the principal links in this chain, to identify the factors that weaken the chain and to propose a plan of resolution. It is suggested that a higher level of commitment and partnership between all of the participants is required to achieve the goal of improving the quality of patient care through the use of autoantibody testing and analysis.

Specificity of autoantibodies to SS-A/Ro on a transfected and overexpressed human 60 kDa Ro autoantigen substrate.

The objective of this study was to analyze apparently discrepant results that arose during the use of an indirect immunofluorescence (IIF) assay using transfected HEp-2 cells to detect anti-SS-A/Ro autoantibodies in human sera. Fourteen sera that had SS-A/Ro antibodies as detected on this commercial substrate, but did not have antibodies to SS-A/Ro as determined by double immunodiffusion (ID) or enzyme-linked immunosorbent assay (ELISA), were studied by immunoprecipitation (IP) of radiolabeled cell extracts and full-length recombinant SS-A/Ro. A multi-antigen strip immunoblotting (IB) assay containing both the 52- and 60-kDa antigens was included in the analysis. We confirmed that 12 of 14 of the sera under study had antibodies to SS-A/Ro protein antigens as determined by at least one other immunoassay. One serum had antibodies to hyRNA but no detectable reactivity with the 52- or 60-kDa antigens. One serum remained negative in all assays for SS-A/Ro autoantibodies.

Autoantibodies against oxidized low-density lipoprotein correlate with Achilles tendon xanthomas.

Although there is increasing evidence for anti-oxidized low-densitiy lipoprotein (LDL) autoantibodies in human sera, their diagnostic utility remains controversial. We examined the difference in autoantibody titers between patients with Achilles tendon xanthoma and control subjects. Fifteen hyperlipidemic patients with Achilles tendon xanthoma (group A+) and 94 hyperlipidemic patients without Achilles tendon xanthoma (group A-) were studied. Quantification of anti-oxidized LDL and anti-native LDL autoantibodies was performed using an ELISA method. To calculate antibody titers, we used the ratio between the spectrophotometric reading of anti-oxidized LDL and anti-native LDL wells. Using oxidized LDL that was purified by gel-permeation chromatography as antigen, immunoglobulin G level differed significantly between groups A+ and A- (p < 0.01). In contrast, using native and oxidized LDL as antigens without chromatographical purification revealed no significant difference between the two groups. Furthermore, immunoglobulin autoantibody titer did not correlate with age, body mass index, total cholesterol, high-density lipoprotein cholesterol, LDL cholesterol, or triglyceride in the entire group of subjects. Thus, immunoglobulin G autoantibody values appear to correlate with Achilles tendon xanthoma.

Method and manufacturer comparison for the detection of autoantibodies in human sera

Method and manufacturer comparison for the detection of autoantibodies in human sera

Anti-fucosyl-GM1 ganglioside IgG and IgM autoantibodies in human serum: no link to pathology

Anti-fucosyl-GM1 ganglioside antibodies were detected in sera of five persons: four patients with autoimmune neuropathies and, more recently, IgG antibodies in one with Graves' disease (Adler et al., Autoimmunity 18, 149–152, 1994) [1]. In the latter case, we were unable to find any relation between the occurrence of antibodies and thyroid disease. Now we report a detailed study on the anti-glycolipid antibodies in this patient. We found that her serum contained not only IgG but also a high level of anti-FucGM1 IgM antibodies, with a titer stable over a period of 5 years of treatment and follow-up. The carbohydrate structure of the epitope recognized by IgG and some of IgM antibodies seems to consist of Fuc-Gal-GalNAc-Gal- or a part of this sequence. Moreover, this patient's serum contained other IgM antibodies active against FucGM1 and also asialo GM1 glycolipids. Our results indicate that anti-FucGM1 ganglioside antibodies of G and M classes occur in serum of this patient with no apparent adverse health effects.

Radioimmunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands

Autoantibodies are an important marker of human autoimmune diseases and the development of simple, precise and reproducible immunoassays to detect autoantibodies is important to our understanding of human autoimmunity. GAD65 autoantibodies occur frequently in insulin-dependent diabetic patients and is a useful marker for IDDM. A RIA to detect immunoreactive GAD65 has not been described. In the present study we describe a semi-automated fluid-phase immunoassay for the rapid detection of GAD65 autoantibodies in human serum. We also developed a sensitive RIA to determine immunoreactive human GAD65 in biological fluids and in vitro cell systems. Using in vitro translated recombinant human GAD65 in a multiwell-adapted procedure, our GAD65Ab RIA combines high specificity and sensitivity with a high capacity to analyze a large number of samples. In this report the three critical steps in the GAD65Ab RIA, DNA preparation, in vitro translation and immunoprecipitation, have been optimized. In our RIA, GAD65Ab were detected in 116 155 (75%) new onset Swedish IDDM children and in 1 85 (1.2%) healthy controls. In an immunoassay to detect autoantibodies against the proinsulin converting enzyme 2 (PC-2) no such antibodies were detected in IDDM patients. In the GAD65 RIA the lower detection limit was 2 ng/ml (31 fmol/ml). Our data demonstrate that autoantigen radioligands produced by in vitro translation are useful in RIA for autoantibodies and autoantigens in studies of human autoimmunity.

Detection of autoantibodies to SS-A/Ro by indirect immunofluorescence using a transfected and overexpressed human 60 kD Ro autoantigen in HEp-2 cells.

The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp-2 cells to detect anti-SS-A/Ro autoantibodies in human sera. Seventy-three sera having SS-A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp-2 cell substrate that had been transfected with a full-length cDNA encoding a human 60 kD SS-A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS-A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty-nine of 73 (95%) SS-A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti-SS-A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS-A/Ro antibodies by IIF on standard HEp-2 substrates. We conclude that SS-A/Ro autoantibodies can be detected by an IIF assay using a HEp-2 cell substrate transfected with a SS-A/Ro cDNA. This new substrate detects SS-A/Ro antibodies that were not identified on standard HEp-2 substrates and by other immunoassays.

A radioimmunoassay for pancreatic elastase 1: use of [formula omitted] 1 conjugate as both standard and tracer to eliminate the influence of anti-elastase 1 autoantibodies

We have recently reported the occurrence of anti-elastase 1 autoantibodies in human sera (Asada et al., Biochim Biophys Acta, 1991;1080:34–39). The usual radioimmunoassay of elastase 1 in autoantibody-positive sera gave abnormally high levels of elastase 1 and low recoveries of elastase 1 added to the sera. Now we have established a new radioimmunoassay system for determining pancreatic elastase 1 in serum, in which α 1- antitrypsin elastase 1 conjugate, the major conjugation form of elastase 1 in serum, is used as a standard and α 1- antitrypsin-[ 125 I] elastase 1 as tracer, because we found that the conjugate could bind to the elastase 1 specific antiserum but not to the autoantibodies. Thus the new assay system completely eliminate the unfavorable effects of the autoantibodies. The elastase 1 levels determined by the new assay method exhibited a good correlation with those obtained by a commercial assay kit in the autoantibody-negative sera, while no correlation was observed in the autoantibody-positive sera.

Substrate Specificity for the Detection of Autoantibodies to Anterior Pituitary Cells in Human Sera

Using the indirect immunofluorescence test for the detection of pituitary autoantibodies in human serum, the results obtained with human fetal and non-human pituitary antigens were compared. Of the sera that were positive on human fetal substrate, 4% were recovered with adult baboon, 0% with fetal cymologous, 20% with porcine, 11% with bovine, 11% with ovine, and 7% with rat tissue. The rate of heterophilic antibodies to the above animal substrates was 5% to 14%. In contrast to human adult pituitaries, normal human sera did not bind to Fc receptors on ACTH-cells of human fetal pituitaries. This allowed the demonstration of ACTH-cell antibodies. The specificity of the reaction was proven by absorption studies with purified Fc fragments. These results suggest that human fetal tissue is the best source of antigen for the detection of pituitary autoantibodies. The use of animal tissue including non-human primate pituitary yields results that bear no clinical significance.

Autoantigens in human neuroblastoma cells

Neuroblastoma cells are frequently used as targets in studies of autoimmune diseases of the nervous system. We examined the human neuroblastoma cell line, LAN-5, for the presence of autoantigens that react with naturally occurring autoantibodies in human sera. Antibodies to the HNK-1 and Gal(ß1–3)GalNAc epitopes, which have been implicated in human autoimmune neuropathy and motor neuron disease, respectively, immunostained the surface of the neuroblastoma cells, and antibodies to the 200 kDa high molecular weight neurofilament protein (NFH) immunostained the cytoplasm and cell processes. The NHK-1 and Gal(ß1–3)GalNAc epitopes were associated with several glycoprotein bands in Western blots of the neuroblastoma cells, and the HNK-1 epitope was also shared by a glycolipid which co-migrated with 3-sulfoglucuronyl paragloboside (SGPG) from peripheral nerve, indicating that SGPG is synthesized in neuronal cells. Northern blot analysis revealed a single RNA band of 4800 bp for NFH in normal brain but two RNA species of 4800 and 3800 bp in both neuroblastoma and adrenal cells, confirming their common origin. The neuroblastoma cells appear to contain antigens that bind to naturally occurring autoantibodies in human serum and might therefore be useful for detecting and investigating the effects of anti-neuronal antibodies. The antibody populations being investigated, however, should be distinguished from other autoantibodies which might be present in the patients' serum.