Addressable Laser Bead Immunoassay Research Articles

Clinical significance of antibodies to Ro52/TRIM21 in systemic sclerosis

IntroductionAutoantibodies to Ro52 recently identified as TRIM21 are among the most common autoantibodies in systemic autoimmune rheumatic diseases, but their clinical association remains poorly understood. We undertook this study to determine the clinical and serologic associations of anti-Ro52/TRIM21 antibodies in patients with systemic sclerosis (SSc).MethodsDetailed clinical data and sera from 963 patients with SSc enrolled in a multicenter cohort study were collected and entered into a central database. Antibodies to Ro52/TRIM21 and other autoantibodies were detected with an addressable laser-bead immunoassay and different enzyme-linked immunosorbent assay (ELISA) systems. Associations between anti-Ro52/TRIM21 antibodies and clinical and other serologic manifestations of SSc were investigated.ResultsAnti-Ro52/TRIM21 antibodies were present in 20% of SSc patients and overlapped with other main SSc-related antibodies, including anti-centromere (by immunofluorescence and centromere protein (CENP)-A and CENP-B ELISA), anti-topoisomerase I, anti-RNA polymerase III, and anti-Pm/Scl antibodies. Anti-Ro52/TRIM21 antibodies were strongly associated with interstitial lung disease (odds ratio (OR), 1.53; 95% confidence interval (CI), 1.11 to 2.12; P = 0.0091) and overlap syndrome (OR, 2.06; 95% CI, 1.01 to 4.19; P = 0.0059).ConclusionsAnti-Ro52/TRIM21 antibodies were the second most common autoantibodies in this SSc cohort. In SSc, anti-Ro52/TRIM21 antibodies may be a marker of interstitial lung disease and overlap syndrome.

Correlation of anti-signal recognition particle autoantibody levels with creatine kinase activity in patients with necrotizing myopathy

Anti-signal recognition particle (anti-SRP) autoantibodies are associated with severe acquired necrotizing myopathies. The role of these autoantibodies remains elusive, and the evolution of anti-SRP levels over time is unknown. In this study, we developed an addressable laser bead immunoassay (ALBIA) technique to investigate a correlation between anti-SRP levels, serum creatine kinase (CK) levels, and muscle strength in patients with necrotizing myopathy. The diagnostic value of the ALBIA assay was determined by comparing serum levels of anti-SRP autoantibodies in 31 anti-SRP immunodot-positive patients to those in 190 healthy blood donors and 199 control patients with different inflammatory/autoimmune conditions or polyclonal hypergammaglobulinemia. Among the 31 anti-SRP-positive patients, serum samples from 8 patients were monitored over time for levels of anti-SRP autoantibodies and levels of CK (determined at least 3 times, consecutively, over a mean followup period of 783 days). The relationship between levels of anti-SRP autoantibodies and levels of CK was tested using a linear mixed model. The assay yielded positive results for anti-SRP in all anti-SRP immunodot-positive serum samples tested, while all control sera tested negative. The 8 anti-SRP-positive patients who were followed up longitudinally were found to have normalized CK levels and improved muscle strength. There was a striking correlation between the degree of myolysis, as measured by CK levels, in patients receiving therapy and the anti-SRP54 autoantibody levels in these same patients (P = 0.002). Anti-SRP-positive myositis appears to be one of the few autoimmune diseases in which specific autoantibody levels are correlated with surrogate disease activity markers. These results reveal the usefulness of monitoring anti-SRP autoantibody levels in patients receiving therapy, and may also suggest a possible pathogenic role for anti-SRP autoantibodies in the necrotizing myopathies.

Performance of a multiplex assay compared to enzyme and precipitation methods for anti-ENA testing in systemic lupus and systemic sclerosis

IntroductionTesting for autoantibodies to extractable nuclear antigens (ENA) is essential in the investigation of connective tissue disease. Counterimmunoelectrophoresis is an early described testing methodology for antibodies to ENAs, but is labour-intensive, only moderately sensitive, and reliant on high-quality reference sera. Enzyme-linked immunosorbent assay (ELISA) is automatable for relatively high sample throughput, but has issues with false positives. The addressable laser bead immunoassay (ALBIA) is a multiplex technology which can assess several antibody specificities simultaneously on a small serum sample. We report performance of an ALBIA system compared with CIE and ELISA. MethodsSamples from 100 systemic sclerosis patients attending Royal Free Hospital in 2007 and 99 SLE patients attending St Thomas's Hospital in 2007–2008 were studied. All samples were tested for antibodies to RNP, Sm, Ro, La, Scl-70, Jo-1 by in-house CIE, FIDIS™ ALBIA (BMD, France), and ELISAs (Phadia, Germany). Cohen's kappa coefficient was used to examine agreement of the different assay methods for the same antibody. McNemar's test was used to detect differences between methodologies. ResultsOne sample was positive for anti-Jo-1 by CIE, & confirmed by ALBIA & ELISA. All 198 remaining samples were anti-Jo-1 negative by all 3 methods. With respect to RNP, Ro, La, Scl-70 antibodies, there was good agreement in assay performance between CIE, ALBIA, and ELISA. For Sm, agreement was less good between CIE and ELISA (kappa 0.491), and ALBIA and ELISA (kappa 0.403). Using McNemar's test performance was no different between the 3 assays, with the following exceptions: between CIE and ELISA for Ro-60 (p<0.01) and RNP (p<0.05), and between ALBIA and ELISA for RNP (p<0.01). ConclusionsThe FIDIS™ ALBIA produced similar level of performance as CIE, but with advantages of automation, and less dependence on highly skilled operators. ALBIA represents a potential advancement applicable to routine Immunology diagnostics.

Autoantibodies to GW bodies and other autoantigens in primary biliary cirrhosis

Autoantibodies to intracellular targets in mitochondria and nuclei are serological hallmarks of primary biliary cirrhosis (PBC). One of the most recently identified cellular targets of PBC autoantibodies is a novel cytoplasmic structure referred to as GW bodies [GWB, G (glycine) W (tryptophan)-containing bodies (GWB)]. GWB are indentified as discrete cytoplasmic domains that are involved in mRNA processing via the RNA interference (RNAi) pathway. Key components of GWB include the proteins GW182, Ago2, RNA-associated protein 55 (RAP55) and Ge-1/Hedls. The primary objective was to study the frequency and clinical association of antibodies directed to GWB components, in 109 PBC patients. Autoantibodies to mitochondrial antigen-pyruvate dehydrogenase complex (M2), branched-chain 2-oxo-acid dehydrogenase complex and 2-oxo glutarate dehydrogenase complex (3E-BPO), gp210, sp100, promyelocytic leukaemia cell antigen (PML) and liver kidney microsomal-1 antigen (LKM-1) were detected by a line immunoassay and antibodies to GWB (GW182, RAP55, Ge-1, GW2, GW3) and glutamate receptor interacting protein (GRIP)-associated protein-1 (GRASP-1), by an addressable laser bead immunoassay (ALBIA). The most common GWB autoantigen targets were: RAP55-28%, GW182-12%, GW2-2% and antibodies to GRASP-1-17%. By comparison, the frequency of reactivity to established PBC autoantigens was: gp210, 27%; sp100, 27% and PML, 17%. None of the autoantibodies were associated with differences in Mayo risk score or liver decompensation. This study is the first study to show that antibodies to RAP55, GW182 and GRASP-1 are the most common GWB targets in PBC.

Measurement of autoantibodies using multiplex methodology in patients with systemic lupus erythematosus

Autoantibodies are central to the diagnosis and assessment of systemic lupus erythematosus (SLE). A recent technique for the measurement of autoantibodies utilizes addressable laser bead immunoassay technology (BioPlex 2200) which permits the simultaneous detection of multiple autoantibodies and improved efficiency due to the shorter time to perform the assay and low volume of test samples and reagents. In the current study we have compared this technique to more traditional measures of autoantibody detection. The clinical and laboratory data and stored serum samples from the enrollment visit into a long-term lupus registry at a single academic medical center were used. Sera were examined for a panel of autoantibodies using the BioPlex ANA screen. The results were compared to the historical data on autoantibody profiles using indirect immunofluorescence (IIF) and ELISA. The association with global and organ specific SLE disease activity (nephritis) was also examined. The study consisted of 192 patients who were predominantly female (87%) and Caucasian (91%) with mean disease duration of 8.8 years. The frequency of ANA and anti-dsDNA by IIF and ELISA was 81.3% and 46.6% respectively and was higher than that found with BioPlex (75.5% and 31.8%). The latter detected a higher proportion of patients with autoantibodies to Sm (7.5% vs 16.7%), RNP (21.8% vs 24.0%), Ro (37.4% vs 41.7) and La (13.9% vs 23.4%). Overall agreement between assays varied between 71.4% and 92.5%. Additional autoantibodies identified by BioPlex were anti-chromatin antibodies which were similar in frequency to anti-dsDNA antibodies (33.9% and 31.8% respectively). There was a low frequency of anti-ribosomal P (6.8%), anti-Scl-70 (5.2%), anti-centromere B (3.7%) and anti-Jo-1 (0.5%). Several autoantibodies revealed significant associations with SLEDAI scores but in a multivariate analysis the only autoantibodies that approached statistical significance were anti-Sm ( p = 0.094) measured by ELISA and anti-dsDNA ( p = 0.082) measured by BioPlex. There was no association between any of the autoantibodies regardless of the method of detection and cumulative organ damage scores. Fifty-three patients (27.6%) had lupus nephritis of which 17 (32%) had active nephritis at the time of autoantibody determination. There was no significant association between a positive ANA (IIF) and any autoantibodies detected by ELISA with either the cumulative occurrence of lupus nephritis or active nephritis. In contrast, there was an association between BioPlex detected anti-dsDNA with the cumulative occurrence of nephritis ( p = 0.074) which reached statistical significance with active nephritis at the time of antibody testing ( p = 0.012). This was confirmed by multivariate analysis ( p = 0.047). These results suggest reasonable agreement between the detection of lupus autoantibodies by ELISA and BioPlex. The latter demonstrated a better correlation with lupus nephritis.

Prevalence of Non–Organ-Specific Autoantibodies in Patients with Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a potentially life-threatening, organ-specific, autoimmune, blistering disease of the skin and mucous membranes. Although several reports suggest an association between pemphigus and other autoimmune connective tissue disorders, studies that measure non-organ-specific autoantibodies are lacking. To evaluate the prevalence of antinuclear antibodies (ANA), anti-double-stranded DNA (anti-dsDNA) antibodies, and antibodies against extractable nuclear antigens (ENAs) in PV patients. Serum samples were obtained from 59 PV patients and 50 healthy controls. Indirect immunofluorescence assays containing human epithelial cell substrates (HEp-2) and Crithidia luciliae were used to detect ANA and anti-dsDNA antibodies, respectively. A multiplexed addressable laser bead immunoassay was employed to measure autoantibodies to: Smith (Sm), ribonucleoprotein (RNP), Sjögren syndrome B (SSB/La), Sjögren syndrome A (SSA/Ro), histidyl transfer ribonucleic acid synthetase (Jo-1), topoisomerase I (Scl-70), and ribosome-P (Ribo-P) antigens. Positive ANAs were obtained in 22 of 59 (37.3%) PV patients compared with 4 of 50 (8.0%) healthy controls (p <or= .01). In both the patient and control populations, anti-dsDNA antibodies were absent. Antibodies against the ENAs were present in 1 of 59 (1.7%) PV patients compared with 0 of 50 (0%) controls. In the single ENA-positive PV patient, antibodies to SSB/La (1.7%) and SSA/Ro (1.7%) were detected. Non-organ-specific autoantibodies are prevalent in the PV population. As ANAs are detected in over one-third of PV patients, clinicians should screen for signs and symptoms of other connective tissue disease. Correlative clinical studies are warranted to determine the diagnostic, prognostic, and therapeutic significance of these findings.

AIT test has no problem in the detection of anti-ribosomal P – authors' response

In a recent article, Jearn and Kim assessed the ability of indirect immunofluorescence on a novel human macrophage cell line (IT-1) in an autoimmune target (AIT) assay to predict the presence of anti-ribosomal P (anti-Rib-P) antibodies [1], based on a previously described cytoplasmic and/or nucleolar staining pattern [2]. Despite the clearance of the AIT test by the South Korea Food and Drug Administration, to the best of our knowledge this test is not widely used as a screening method – neither is it approved by the Food and Drug Administration (FDA) USA or is it a Communaute Europeenne (CE) certified method for the detection of antinuclear antibodies. By contrast, many laboratories rely on HEp-2 cell substrates that are widely available as FDA and CE approved diagnostic kits from a variety of manufacturers. In their study, Jearn and Kim identified sera with anti-Rib-P reactivity based on a double immunodiffusion (ID) assay, and then used these sera to determine the positivity and staining patterns in the AIT test. This method raises concerns because it is generally known that immunodiffusion is a relatively insensitive technique for the detection of human autoantibodies when compared with other techniques such as ELISA and western immunoblotting (WB). In an example relevant to the anti-Rib-P system, Bonfa and colleagues clearly demonstrated that immunodiffusion has very low sensitivity for the detection of anti-Rib-P antibodies [3]. In their study, only 14% of anti-Rib-P-positive samples identified by WB were detected by immunodiffusion. Another study showed that ID exhibits significantly lower sensitivity compared with a multiplex system: 63/130 (48.5%) samples with at least one positive extractable nuclear antibody result were negative by ID, and all 11 sera with anti-Rib-P reactivity did not show a typical cytoplasmic staining pattern by indirect immunofluorescence [4]. While the aforementioned studies used HEp-2 cells as the substrate, there would be no reason to believe that the AIT assay would perform remarkably better. In the context of contemporary diagnostic assays such as WB, ELISA or multiplexed addressable laser bead immuno-assays, therefore, the data presented by Jearn and Kim do not support the claim that the AIT test represents a sensitive and reliable method for the detection of anti-Rib-P antibodies. A systematic study is mandatory to compare the sensitivity and specificity of the AIT test for the detection of anti-Rib-P antibodies and other antibody specificities using a variety of contemporary diagnostic platforms, not just ID. In addition to anti-Rib-P, such a study could also include the evaluation of anti-Jo-1, anti-SS-A (Ro60) and anti-Ro52 antibodies, which have been shown to produce false negative results in indirect immunofluorescence on HEp-2 cells [2,5].

Microbead-based technologies in diagnostic autoantibody detection

There is a rapid proliferation of new technologies to identify a spectrum of autoantibodies in medical conditions that range from organ-specific autoimmune diseases to systemic rheumatic diseases. Although many laboratories have adopted high-throughput diagnostic platforms such as enzyme linked immunoassays (ELISA), other technologies such as microbead-based assays are emerging as an alternative diagnostic platform. To understand the performance and importance of bead based immunoassays in clinical diagnostics and therapeutics. Current literature was reviewed using the PubMed search engine combining keywords of immunoassay and Luminex, as well as a personal literature database. Included in the evaluation and commentary are bead-based assays such as addressable laser bead immunoassays and related magnetic bead assays. Comparison with other conventional technologies has indicated that laser microbead immunoassays are reliable, accurate, cost-effective, highly sensitive and have rapid turn around time for results. While there are advantages to this diagnostic platform, there are challenges that must be addressed before wider acceptance or long-term use of this technology platform in the routine clinical diagnostic laboratory.

Limited reliability of the indirect immunofluorescence technique for the detection of anti-Rib-P antibodies

IntroductionAutoantibodies to the ribosomal P proteins represent a highly specific marker for the diagnosis of systemic lupus erythematosus, where they have been associated with certain clinical manifestations. Historically, autoantibodies against ribosomal P proteins have been detected by indirect immunofluorescence, immunodiffusion, immunoblot, and other immunoassays. More recently, enzyme-linked immunosorbent assays and line and addressable laser bead immunoassays have become more widely used. The primary goal of this study was to determine the sensitivity of indirect immunofluorescence using conventional HEp-2 substrates in the detection of sera with ribosomal P antibodies as detected by other immunoassays.MethodsAnti-ribosomal P-positive sera (n = 345) as detected by an addressable laser bead immunoassay were collected between 2003 and 2007 and analysed by indirect immunofluorescence. Furthermore, 51 anti-ribosomal P-positive samples from an unselected systemic lupus erythematosus cohort (n = 100) and the Centers for Disease Control and Prevention (CDC) anti-nuclear antibody (ANA) reference sera were tested for anti-ribosomal P reactivity.ResultsIn the cohort of 345 anti-ribosomal P-positive samples identified by addressable laser bead immunoassay, a low sensitivity (<30%) of indirect immunofluorescence on HEp-2 cell substrates was observed. Although the degree of sensitivity varied among different manufacturers, all immunofluorescence substrates exhibited limited sensitivity and false-negative results were not restricted to samples with low anti-ribosomal P titers. Even the anti-ribosomal P reactivity of CDC ANA reference serum number 12 was not clearly predictable by indirect immunofluorescence. Comparison of five different methods for the detection of anti-ribosomal P found moderate qualitative agreements.ConclusionsBased on our data, we conclude that indirect immunofluorescence on HEp-2 cells is not a reliable screening test for the prediction of ribosomal P antibodies. As this method is widely used as a first-line screening test for anti-nuclear and other autoantibodies, special considerations for the detection of ribosomal P antibodies are needed. As with many other autoantibodies, further effort is required for the standardisation of ribosomal P immunoassays.

Clinical and serological features of patients with autoantibodies to GW/P bodies

GW bodies (GWBs) are unique cytoplasmic structures involved in messenger RNA (mRNA) processing and RNA interference (RNAi). GWBs contain mRNA, components of the RNA-induced silencing complex (RISC), microRNA (miRNA), Argonaute proteins, the Ge-1/ Hedls protein and other enzymes involving mRNA degradation. The objective of this study was to identify the target GWB autoantigens reactive with 55 sera from patients with anti-GWB autoantibodies and to identify clinical features associated with these antibodies. Analysis by addressable laser bead immunoassay (ALBIA) and immunoprecipitation of recombinant proteins indicated that autoantibodies in this cohort of anti-GWB sera were directed against Ge-1/ Hedls (58%), GW182 (40%) and Ago2 (16%). GWB autoantibodies targeted epitopes that included the N-terminus of Ago2 and the nuclear localization signal (NLS) containing region of Ge-1/ Hedls. Clinical data were available on 42 patients of which 39 were female and the mean age was 61 years. The most common clinical presentations were neurological symptoms (i.e. ataxia, motor and sensory neuropathy) (33%), Sjögren's syndrome (SjS) (31%) and the remainder had a variety of other diagnoses that included systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary biliary cirrhosis (PBC). Moreover, 44% of patients with anti-GWB antibodies had reactivity to Ro52. These studies indicate that Ge-1 is a common target of anti-GWB sera and the majority of patients in a GWB cohort had SjS and neurological disease.

Autoantibodies in lupus nephritis patients requiring renal transplantation

The goal of this nested case-control study was to compare autoantibody profiles in systemic lupus erythematosus (SLE) patients with lupus nephritis (LN), lupus nephritis patients requiring renal transplantation (LNTP) and a SLE control group without nephritis (CON). Sera were assayed for a variety of autoantibodies by addressable laser bead immunoassay (ALBIA) and enzyme-linked immunoassay (ELISA) and to dsDNA by Crithidia luciliae assay. The frequency of nucleosome autoantibodies was significantly greater in the LNTP group (79%) compared to the LN (18%) and CON (9%) groups (P < 0.0005). The frequency of other autoantibodies, including anti-dsDNA, did not differ significantly between groups. Among patients with LN, the odds of progressing to renal transplantation was 16-fold higher (OR 16.5 [95% CI 2.5, 125.7], P = 0.0005) in patients testing positive for anti-nucleosome antibodies compared to those who tested negative. Furthermore, the level of anti-nucleosome antibodies was significantly ( P < 0.00005) higher in the LNTP group (3.69 +/- 2.79) than the LN (0.51 +/- 0.51) and CON (0.34 +/- 0.44) groups. Review of 48 renal biopsies from 29 patients indicated that there was no difference in renal histological classification among patients with anti-nucleosome antibodies compared to those who tested negative. Our observations suggest that nucleosome autoantibodies are a biomarker for more severe SLE renal disease requiring transplantation.

Heterogeneity of autoantibodies in 100 patients with autoimmune myositis: insights into clinical features and outcomes

The objective of this study was to determine the prevalence, mutual associations, clinical manifestations, and diagnoses associated with serum autoantibodies, as detected using recently available immunoassays, in patients with autoimmune myositis (AIM). Sera and clinical data were collected from 100 patients with AIM followed longitudinally. Sera were screened cross-sectionally for 21 autoantibodies by multiplex addressable laser bead immunoassay, line blot immunoassay, immunoprecipitation of in vitro translated recombinant protein, protein A assisted immunoprecipitation, and enzyme-linked immunosorbent assay. Diagnoses were determined using the Bohan and Peter classification as well as recently proposed classifications. Relationships between autoantibodies and clinical manifestations were analyzed by multiple logistic regression. One or more autoantibodies encompassing 19 specificities were present in 80% of the patients. The most common autoantibodies were anti-Ro52 (30% of patients), anti-Ku (23%), anti-synthetases (22%), anti-U1RNP (15%), and anti-fibrillarin (14%). In the presence of autoantibodies to Ku, synthetases, U1RNP, fibrillarin, PM-Scl, or scleroderma autoantigens, at least one more autoantibody was detected in the majority of sera and at least two more autoantibodies in over one-third of sera. The largest number of concurrent autoantibodies was six autoantibodies. Overall, 44 distinct combinations of autoantibodies were counted. Most autoantibodies were unrestricted to any AIM diagnostic category. Distinct clinical syndromes and therapeutic responses were associated with anti-Jo-1, anti-fibrillarin, anti-U1RNP, anti-Ro, anti-Ro52, and autoantibodies to scleroderma autoantigens. We conclude that a significant proportion of AIM patients are characterized by complex associations of autoantibodies. Certain myositis autoantibodies are markers for distinct overlap syndromes and predict therapeutic outcomes. The ultimate clinical features, disease course, and response to therapy in a given AIM patient may be linked to the particular set of associated autoantibodies. These results provide a rationale for patient profiling and its application to therapeutics, because it cannot be assumed that the B-cell response is the same even in the majority of patients in a given diagnostic category.

The Emergence of Multiplexed Technologies as Diagnostic Platforms in Systemic Autoimmune Diseases

In the last decade, there has been a rapid proliferation of new technologies that are capable of identifying an increasing spectrum of autoantibodies and other biomarkers in autoimmune diseases. These newer diagnostic technologies include line immunoassays, addressable laser bead immunoassays, microarrays in microfluidics platforms and nanobarcode particles. Multiplexed bead assays are a particularly robust platform because they are adaptable to the detection of a variety of disease specific biomarkers, such as autoantibodies, cytokines, adipokines, drugs, oligonucleotides and single nucleotide polymorphisms, Although many laboratories have adopted a variety of these diagnostic platforms to improve turn around times and meet budget constraints, there is an urgent need to ensure that the rapid adoption of new technologies is attended by an appropriate balance of assay sensitivity and specificity.

Advances and Applications of Multiplexed Diagnostic Technologies in Autoimmune Diseases

There is a rapid proliferation of new technologies to identify an ever increasing spectrum of autoantibodies in diverse medical conditions that range from organ-specific autoimmune diseases to systemic rheumatic diseases. Although many laboratories have adopted diagnostic platforms, such as enzyme linked immunoassays (ELISAs), to improve turn around times and meet budget constraints, the prevailing evidence is that the rapid adoption of new technologies is not attended by an appropriate balance of assay sensitivity and specificity. Emerging diagnostic technologies include addressable laser bead immunoassays, microarrays in microfluidics platforms and nanobarcode particles. Although these technologies provide advantages of high-throughput, multiplexed autoantibody assays that can be coupled to other disease specific biomarkers (ie, cytokines, single nucleotide polymorphisms) there is a clear need for standardization and internal validation before they are adopted into the clinical diagnostic laboratory.

Analysis of human sera that are polyreactive in an addressable laser bead immunoassay

Background: Following the introduction of addressable laser bead immunoassays (ALBIA) into our clinical laboratory, it was noted that certain sera would exhibit reactivity to numerous antigens in the array. To further understand the nature of this reactivity, we analyzed the reactivity of sequential sera that were identified over a 1 year period. Methods: Sera that demonstrated reactivity to 6 or more of the 8 antigens in an ALBIA kit (QuantaPlex 8: chromatin, Sm, RNP, Scl-70, ribosomal P protein, SS-A/Ro, SS-B/La, Jo-1) were tested for autoantibodies by indirect immunofluorescence (IIF) on HEp-2 cell substrates, for IgG, IgM and IgA rheumatoid factor, chromatin and ribosomal P protein by ELISA and by LINE immunoassay (LIA) and immunoblotting (IB). Results: In one calendar year, 40/4096 (0.8%) sera analyzed in a routine clinical laboratory setting demonstrated reactivity to 6 or more antigens in the QuantaPlex 8 kits. There was no common IIF pattern that could be attributed to the polyreactive sera. There was no apparent correlation of polyreactivity with IIF titers, indeed, 4/40 (10%) sera had a negative ANA at the screening dilution of 1/160. When subjected to IB, LIA and ELISA, polyreactivity to three or more antigens was confirmed for 12/40 (30%) of sera while 8/40 (20%) had reactivity to 1–2 antigens and 20 (50%) did not react with any antigens in these assays. Overall agreement of positive or negative tests between the ALBIA and IB, LIA and ELISA was 75% for chromatin, 50% for SS-A, 27.5% for Sm, 25% for Rib-P, 22.5% for RNP, 20% for Scl-70, 15% for Jo-1 and 7.5% for SS-B. 17/40 (42.5%) had a positive IgM, IgG or IgA rheumatoid factor, and 12/40 (30%) had all three isotype rheumatoid factors. Conclusions: On average, the agreement between ALBIA and other assays in this study of polyreactive sera was 30%. Approximately, one-half of sera that demonstrate reactivity to multiple autoantigens in a commercial ALBIA were confirmed to have reactivity to at least one autoantigen in another diagnostic assay and 30% could be regarded as polyreactive. Other sera, some of which had rheumatoid factor, appeared to have high background binding without demonstrating specific binding to any of the cognate antigens.

Anti‐p97/VCP Antibodies: An Autoantibody Marker for a Subset of Primary Biliary Cirrhosis Patients with Milder Disease?

We previously reported that 12.5% of primary biliary cirrhosis (PBC) sera reacted with a 95 kDa cytosol protein (p95c) that was subsequently identified as a p97/valosin-containing protein (VCP). The clinical features and course of the six anti-p97/VCP-positive PBC patients with Scheuer's stage 1 and 2 liver biopsies were monitored for an average of 15 years. This group was compared with 50 PBC patients that did not have detectable anti-VCP. Autoantibodies to a full-length recombinant p97/VCP were assayed by immunoprecipitation. All six PBC patients with anti-VCP had antibodies to the mitochondrial pyruvate dehydrogenase complex-E2 antigen as measured by an addressable laser bead immunoassay. The first was a male with no evidence of liver failure that died of cerebral infarction at the age of 85. The second was a 73-year-old female with Hashimoto's thyroiditis who has remained clinically stable without ursodeoxycolic acid (UDCA) treatment. Although the third had no HCV antibodies, he developed hepatocellular carcinoma at the age of 76 and died of renal failure at 78. The fourth was a 50-year-old female who remained clinically stable during follow-up and the fifth with Hashimoto's thyroiditis and stable liver function following UCDA treatment. The sixth was a male patient presenting a mild clinical course. The clinical course of these patients was in contrast to the 50 comparison group PBC patients who did not have anti-p97/VCP. As the six PBC patients with anti-p97/VCP antibodies had slowly progressive liver disease and no mortality related to autoimmune liver disease, our observations suggest that this autoantibody might be an indicator of a favourable prognosis.

Autoantibody Responses of Individuals in an Oil Sands Development Community

The authors studied autoantibodies in sera from 214 individuals in an oil sands community by indirect immunofluorescence, an addressable laser bead immunoassay, and the Crithidia luciliae assay. They compared results with exposure data that included urinary metabolites, health questionnaires, and overt disease as recorded by visits to a physician or hospitalization. The prevalence of autoantibodies was 13%, compared with 10% in 30 controls from a distant community (p > 0.05). The study group had higher autoantibody titers and some disease-specific autoantibodies, but these were not associated with reported autoimmune diagnoses. There was no evidence of increased autoimmune disease or response in this petroleum development population with low levels of exposure.