High Sensitivity Detection of Anti-DFS70 Antibodies by Radioimmunoprecipitation Assay (RIPA).

BACKGROUND: Autoantibodies against the chromosome associated protein DFS70/LEDGF (dense fine speckled 70/ lens epithelial growth factor; anti-DSF70) are increasingly being regarded as biomarkers for the diagnostic exclusion of systemic autoimmune rheumatic diseases (SARD). In routine ANA screening by indirect immunofluores-cence (IIFT) tests the presence of anti-DFS70 may first be presumed because of their characteristic immunofluo-rescence pattern (AC-2 pattern) and then be confirmed by antigen specific assays, a sequential approach, which may underestimate the prevalence of anti-DFS70 because of the inherent shortcomings of the ANA-IIFT. We therefore, for the first time, determined the prevalence of anti-DFS70 in patient sera by means of a sensitive and specific radioimmunoprecipitation assay (RIPA) as compared to a commercial ELISA. METHODS: Blood specimens referred for routine ANA screening (n = 1.100, ANA-Series) or for basic clinical chemistry tests (n = 350, CC-Series) were assayed for the prevalence of anti-DFS70 by RIPA using 35S-methionine labelled full-length DFS70 (FL-DFS70) as well as a C-terminal DFS70 fragment (CT-DFS70) generated by in vitro transcription/translation (ivTT) of the respective cDNAs. ELISA was performed using an anti-DFS70 test-kit (Eu-roimmun) and ANA-IIFT by means of commercial HEp-2 cells (INOVA) and appropriately chessboard titrated conjugates (Dianova). Accessory SARD markers (anti-dsDNA, anti-ENA) were determined in sera positive for anti-DFS70. RESULTS: The detection of anti-DFS70 by RIPA was considerably more sensitive than by ELISA, resulting in an overall detection rate of 9.0% (ANA-Series) and 8.0% (CC-Series) compared to ELISA revealing 4.6% (ANA-Se-ries) and 2.6% (CC-Series) anti-DFS70 positive sera. Of 99 RIPA reactive sera (ANA-Series) 72% were reactive against anti-FL-DFS70, 93% against CT-DFS70, polyspecific antibodies coexisted in 65%, reacting with both antigen specificities, 28% showed monospecific reaction with CT-DFS70 and 7% monospecific with FL-DFS70, indicating also the possible existence of antibodies specific for N-terminal epitopes in DSF70. Similar frequencies were seen in sera of the CC-series. The RIPA measured antibody concentrations (Rratio) obtained with FL-DSF70 antigen and CT-DSF70 antigen showed a correlation. There was also a correlation between the IIFT-ANA titers and Rratio found by RIPA. The consensus of suspected AC-2 pattern in ANA-IIFT and anti-DFS70 measured by RIPA was about 80%. No significant correlation existed between the antibody concentrations measured by RIPA and ELISA. Additional SARD markers were present in 24% of anti-DFS70 positive sera referred for ANA screening. No additional markers were seen in sera of the CC-Series. CONCLUSIONS: RIPA constitutes a highly-sensitive assay for detection of anti-DFS70 in human sera. ANA-IIFT screening performed under consideration of the AC-2 pattern for verification of antibodies to DFS70 under routine conditions may incorrectly estimate a considerable number of not only low but also high titer anti-DSF70 positive sera. The significance of RIPA reactive antibodies, especially of low titer range, in the context of SARD and healthy individuals now has to be scrutinized in further clinical studies.

Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

A fluorescence sedimentation assay for dsDNA antibodies.

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other assays but does also present some disadvantages as it utilizes radioactive-labelled dsDNA and requires high levels of technical expertise for safe handling. Here, a new precipitation assay, 'Fluoro-Farr' assay, is described. This assay maintains a high sensitivity and specificity for SLE but is based on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic efficiency of the assay was evaluated by testing 57 sera from SLE patients and 60 healthy controls. The Fluoro-Farr assay revealed a diagnostic sensitivity of 68% at a diagnostic specificity of 95% (ROC AUC 0.91). Furthermore, the new Fluoro-Farr assay was compared to the RIA-Farr assay, and showed a correlation of the outcomes from the two assays, but the Fluoro-Farr assay did not outperform the RIA-Farr assay due to its outstanding clinical diagnostic efficiency (ROC AUC 0.99). In conclusion, the Fluoro-Farr assay presents a viable alternative to the traditional RIA-Farr assay; especially in laboratories without facilities to perform assays with radioactivity-based read-out. As the RIA-Farr assay, the Fluoro-Farr assay has the advantage of being a precipitation assay allowing antibody:dsDNA interaction in solution using native dsDNA.

A clinico-radiological phenotype of voltage-gated potassium channel complex antibody-mediated disorder presenting with seizures and basal ganglia changes.

In childhood, central nervous system (CNS) presentations associated with antibodies to voltage-gated potassium channel (VGKC) complex include limbic encephalitis, status epilepticus, epileptic encephalopathy, and autistic regression. We report the cases of two individuals (a 6-year-old male and an 11-year-old female) who presented with an acute-onset explosive seizure disorder with positive VGKC complex antibodies and bilateral basal ganglia changes on magnetic resonance imaging (MRI). Both patients made a complete clinical recovery, without immunotherapy, with resolution of the MRI changes and normalization of the antibody levels. Extended antibody testing, including testing for leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein 2, and contactin-2 was negative. This could suggest that the clinico-radiological phenotype in our patients may in fact be associated with a novel autoreactive target(s) within the VGKC complex, as may be the case in other children with VGKC complex-mediated CNS disorders.

Detection of anti-ESA antibodies in human samples from PRCA and non-PRCA patients: an immunoassay platform comparison.

BACKGROUND: The immunological methods for detecting antibodies to erythropoiesis-stimulating agents (ESAs) differ in assay sensitivity. However, this parameter, routinely determined in clinical assays using a high-affinity non-human polyclonal antibody, gives a one-dimensional assessment of antibody detection. We compare three widely used immunological methods and evaluate the ability of each to detect mature human antibodies and human antibodies characteristic of an early immune response. METHODS: The detection of anti-ESA antibodies was compared between a radioimmunoprecipitation (RIP) assay, an electrochemiluminescence (ECL) bridging enzyme-linked immunosorbent assay and a surface plasmon resonance (SPR)-based immunoassay. All three methods were validated for sensitivity, specificity and precision. Specimens from clinical studies or post market testing were categorized as pure red cell aplasia (PRCA) or non-PRCA and then analyzed in each method. RESULTS: Among the antibody-mediated PRCA samples, which contain high affinity neutralizing antibodies, there was strong correlation between all methods. The results from non-PRCA sample analysis show high correlation between RIP and ECL methods; however, differences between the SPR immunoassay and the ECL and RIP were demonstrated. The samples that scored positive in the SPR immunoassay and negative by RIP and ECL were characterized to be of low antibody concentration, contained a high percentage of rapidly dissociating antibodies, or were antibodies of the IgM isotype. CONCLUSIONS: All three immunological methods are appropriate for detection of antibodies associated with antibody-mediated PRCA. However, the SPR immunoassay platform detected an early, low affinity IgG and IgM antibody response as well as detected and characterized a pathogenic antibody response associated with antibody-mediated PRCA.

Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients' and blood donors' sera.

AIM: To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. METHODS: Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunofluorescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. RESULTS: FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA. CONCLUSION: CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity.

Development of a highly sensitive diagnostic assay for muscle-specific tyrosine kinase (MuSK) autoantibodies in myasthenia gravis.

Autoimmune myasthenia gravis is usually characterized by the presence of autoantibodies against the acetylcholine receptor (~80-90% of patients) or muscle-specific tyrosine kinase (MuSK) (~5% of patients). In the remaining patients, no such antibodies (Abs) are detectable, but this could be due either to the presence of auto-Abs to yet unidentified antigens or to concentrations of circulating Abs below the threshold of the available assays. The most popular and sensitive assay for anti-MuSK Abs is a radioimmunoprecipitation assay (RIPA), which uses (125)I-labeled MuSK as test antigen. A serious limiting factor of the sensitivity of such RIPAs is that small volumes of test serum are required (maximum 5-20 µl) in order to avoid excessive background values. We have overcome this obstacle by the development of a two-step RIPA. This involves non-stringent affinity purification of anti-MuSK Abs from a large volume of patient's serum, followed by a regular RIPA step, with the isolated Abs, to determine the antibody titer. This two-step assay was shown to be 10-50 times more sensitive than the regular RIPA. All tested sera previously found to be positive in the regular RIPA and normal sera were also positive or negative in the two-step RIPA, respectively. Importantly, of seven tested sera previously characterized by regular RIPA as negative with titers that were above zero, but statistically not significantly higher from the background, two were found to be positive, while the others were clearly shown to be negative. We conclude that our diagnostic test can detect very low concentrations of circulating anti-MuSK Abs. The general principle of this two-step RIPA approach may also be applied to the detection of currently undetectable concentrations of circulating auto-Abs involved in other diseases.

Specific anti-nuclear antibodies in systemic sclerosis patients with and without skin involvement: an extended methodological approach.

OBJECTIVES: To determine how sensitively SSc-associated ANAs are detected by a standard identification algorithm compared with an extensive panel of ANA identification assays, and to assess the distribution of SSc-associated ANAs and SSc organ system involvement in patients without skin involvement (limited SSc). METHODS: Serum samples from 145 consecutive monocentric SSc patients fulfilling LeRoy and Medgser's criteria for early SSc were studied. ANAs were detected by IIF on HEp-2000 cells and identified by western blotting, protein radio-immunoprecipitation, RNA immunoprecipitation and line immunoassay (LIA). SSc organ involvement was assessed according to a modification of Medsger's disease severity scale. RESULTS: At least one specific ANA reactivity was present in 88% of the patients. The standard algorithm (IIF and LIA) found at least one specific ANA in 74% of the patients. The main reactivities missed by this algorithm were anti-RNA polymerase III, anti-PM/Scl and anti-Th/To. Eighty-three percent of the patients with limited SSc had at least one ANA. ACAs and anti-Th/To antibodies were significantly associated with limited SSc, whereas anti-topoisomerase I and anti-RNA polymerase III were observed less frequently. SSc organ system involvement was present in 63% of the patients with limited SSc, most of whom had lung involvement. CONCLUSIONS: Standard algorithms for ANA identification lack sensitivity for the detection of SSc-associated ANA and should be supplemented with additional assays, especially in a clinical environment that has particular interest in SSc. The spectrum of SSc-associated ANA differs according to the presence or absence of skin involvement.

Eliminating false-positive results in serum tests for neuromuscular autoimmunity.

In this study we describe the false-positive frequency in radioimmunoprecipitation assays for muscle acetylcholine receptor (AChR) and neuronal voltage-gated potassium channel (VGKC) autoantibodies, attributable to 125I-ligand immunoprecipitation. Sera were evaluated for AChR autoantibody (n = 34,095) and VGKC autoantibody (n = 11,028). We retested sera that yielded apparently positive results with 125I-ligand with and without detergent-solubilized cation-channel protein, indentified clinically validated fals-positive rates of 0.05% and 1.7% for AchR and VGKC autoantibodies, respectively. Specificity assurance in radioimmunoprecipitation assays requires subtraction of values for 125I-ligand binding.

Comparison of the Farr radioimmunoassay, 3 commercial enzyme immunoassays and Crithidia luciliae immunofluorescence test for diagnosis and activity assessment of systemic lupus erythematosus.

BACKGROUNDS: Among anti-double-strand (ds)DNA antibody assays, Farr radioimmunoassay is decreasingly used because it requires radioactive material and is labor intensive. We evaluated the performance of Farr, three commercial enzyme immunoassays (EIAs) and the Crithidia luciliae immunofluorescence test (CLIFT) in systemic lupus erythematosus (SLE). METHODS: Anti-dsDNA antibodies were determined in 99 SLE patients, 101 healthy subjects, and 53 patients with autoimmune rheumatic diseases. RESULTS: Farr performed better than the 3 EIAs and CLIFT for the diagnosis of SLE at the manufacturer's cut off and at the cut off set to achieve a specificity of 95%. To achieve a similar level of specificity, some EIAs had a decrease in sensitivity which was dramatic for some tests. Farr was also the best at distinguishing patients with quiescent to mildly active disease from patients with more active disease at the cut off value of 93 IU/ml. Using manufacturer's cut off did not allow distinguishing between patients with quiescent and active SLE. CONCLUSIONS: Farr was the best global test to assess the level of anti-dsDNA antibodies for both diagnosis and disease activity evaluation in SLE with adequately determined cut off values. Some EIA had low performances limiting their use in decision-making regarding diagnosis and/or treatment.

Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test.

The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Comparison of radioimmunoprecipitation with luciferase immunoprecipitation for autoantibodies to GAD65 and IA-2beta.

OBJECTIVE: To compare the sensitivity and specificity of luciferase immunoprecipitation (LIPS) with radioimmunoprecipitation (RIP) for the measurement of autoantibodies to the type 1 diabetes autoantigens glutamic acid decarboxylase 65 (GAD65) and insulinoma-associated protein (IA)-2beta. RESEARCH DESIGN AND METHODS: Sera from 49 type 1 diabetic patients and 100 nondiabetic control subjects from Diabetes Antibody Standardization Program 2007 were used to screen for autoantibodies to GAD65. An additional 200 type 1 diabetic patients and 200 nondiabetic control subjects were used to validate the GAD65 results and screen for autoantibodies to IA-2beta. RESULTS: LIPS showed equal sensitivity and specificity to RIP for detecting autoantibodies to GAD65 and IA-2beta. Receiver-operating characteristic analysis revealed that the detection of autoantibodies to GAD65 and IA-2beta by LIPS and RIP were not statistically different. CONCLUSIONS: The LIPS assay does not require the use of radioisotopes or in vitro transcription/translation and is a practical alternative at the clinical level for the RIP assay.

Comparison of insulin autoantibody: polyethylene glycol and micro-IAA 1-day and 7-day assays.

BACKGROUND: Older studies of diabetes development typically utilized a 7-day incubation polyethylene glycol competitive insulin autoantibody assay (CIAA). Our standard micro-IAA assay (mIAA) utilizes precipitation with proteins A/G and 1-day incubation (1-day mIAA), but is less sensitive compared to the CIAA assay. METHODS: We performed CIAA and mIAA assays in various conditions. We analyzed serum samples from 446 type 1 diabetes patients, from another set of 247 type 1 diabetes patients within 2 weeks of initiation of insulin treatment, from 150 healthy control donors, from 22 healthy participants in the diabetes autoimmunity study in the young (DAISY), and also coded sera from 50 patients with newly diagnosed type 1 diabetes and 50 blood donor control samples. RESULTS: In the process of our study, we found that the key condition was the incubation time. Therefore, we extended the incubation time to 7 days (7-day mIAA assay). No CIAA-negative control was positive with either 1-day or 7-day mIAA. In a new onset type 1 diabetes and at risk cohorts (DAISY study), the 7-day mIAA identified an additional 18% as being positive along with 16% of those who were initially 1-day mIAA negative and CIAA positive. Most subjects detectable only with the 7-day mIAA assay had intermediate levels of CIAA (80-300 nU/mL) (p = 0.01). CONCLUSIONS: The 7-day mIAA assay identifies a small but significant additional subset of individuals positive on the CIAA assay, while preserving specificity.

Angelica injection improves functional recovery and motoneuron maintenance with increased expression of brain derived neurotrophic factor and nerve growth factor.

This study assessed the neuroprotective effects of angelica injection in the rat sciatic nerve crush injury (SCI). Forty eight male Sprague Dawley rats were randomly divided into 4 groups: one was the sham group (S), which received sham surgery and given saline injection and the others were received SCI surgery and given saline injection, high and low dose angelica injection for 4 weeks, respectively. The sciatic functional index (SFI) in walking-track analysis, conductive velocity (CV), the number of fluorogold labeled motoneurons, and the expression patterns of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the sciatic nerve and spine were examined. The results showed that SFI descended gradually on day 7, and dropped more quickly on day 28 in treatment groups (Low and High dose group). The CV in treatment groups was higher than control group (C). The numbers of motoneurons in treatment groups were larger than C group (P<0.05), but less than that in S group (P<0.01). The expressions of BDNF and NGF protein in the groups received SCI surgery were significantly lower than in S group, but the protein expressions in the groups received angelica injections were significantly higher than that in C group (P<0.01). These findings suggested that angelica injection can improve the sciatic nerve crush injury, and the mechanism might be through the increase of BDNF and NGF protein expression.

Lateral flow immunoassay for diagnosis of Trypanosoma cruzi infection with high correlation to the radioimmunoprecipitation assay.

The incidence of blood donors seropositive for Trypanosoma cruzi in North America has increased with population migration and more rigorous surveillance. The United States, considered nonendemic for T. cruzi, could therefore be at risk to exposure to parasite transmission through blood or organ donations. Current tests show variable reactivity, especially with Central American sera. Here we describe the development of a lateral flow immunoassay for the rapid detection of T. cruzi infection that has a strong correlation to the radioimmunoprecipitation assay (RIPA) "gold standard" in the United States. Such a test could have utility in small blood banks for prescreening donors, as well as in cardiac transplantation evaluation. T. cruzi consensus and/or RIPA-positive sera from Central and South America were evaluated in enzyme immunoassays (EIAs). These included commercial panels from Boston Biomedica, Inc. (BBI) (n = 14), and HemaBio (n = 21). Other sources included RIPA-positive sera from the American Red Cross (ARC) (n = 42), as well as from Chile. Sera were tested with the multiepitope recombinant TcF. All but one of the BBI samples were positive and 7 of 21 HemaBio samples and 6 of 42 ARC samples were low positive or negative. This observation indicated the need for additional antigens. To complement TcF reactivity, we tested the sera with peptides 30, 36, SAPA, and 1.1, 1.2, and 1.3 His fragments of 85-kDa trans-sialidase. We identified a promising combination of the tested antigens and constructed a single recombinant protein, ITC6, that enhanced the relative sensitivity in U.S. blood donor sera compared to that of TcF. The data on its evaluation using RIPA-confirmed positive sera in EIA and lateral flow immunoassay studies are presented, along with an additional recombinant protein, ITC8.2, with two additional sequences for peptide 1 and Kmp-11. The latter, when evaluated in a dipstick assay with consensus positive sera, had a sensitivity of 99.2% and a specificity of 99.1%.

Aquaporin-4 antibodies in neuromyelitis optica and longitudinally extensive transverse myelitis.

BACKGROUND: There is increasing recognition of antibody-mediated immunotherapy-responsive neurologic diseases and a need for appropriate immunoassays. OBJECTIVES: To develop a clinically applicable quantitative assay to detect the presence of aquaporin-4 (AQP4) antibodies in patients with neuromyelitis optica and to characterize the anti-AQP4 antibodies. DESIGN: We compared a simple new quantitative fluorescence immunoprecipitation assay (FIPA) with both indirect immunofluorescence and an AQP4-transfected cell-based assay, both previously described. We used the cell-based assay to characterize the antibodies for their immunoglobulin class, IgG subclass, and ability to induce complement C3b deposition in vitro. SETTING: United Kingdom and Germany. PARTICIPANTS: Serum samples from patients with neuromyelitis optica (n = 25) or longitudinally extensive transverse myelitis (n = 11) and from relevant controls (n = 78) were studied. MAIN OUTCOME MEASURES: Comparison of different assays for AQP4 antibodies and characterization of anti-AQP4 antibodies in patients with neuromyelitis optica. RESULTS: We found antibodies to AQP4 in 19 of 25 patients with neuromyelitis optica (76%) using FIPA, in 20 of 25 patients with neuromyelitis optica (80%) using the cell-based assay, and in 6 of 11 patients with longitudinally extensive transverse myelitis (55%) with both assays; these assays were more sensitive than indirect immunofluorescence and 100% specific. The antibodies bound to extracellular epitope(s) of AQP4, were predominantly IgG1, and strongly induced C3b deposition. CONCLUSIONS: Aquaporin-4 is a major antigen in neuromyelitis optica, and antibodies can be detected in more than 75% of patients. Further studies on larger samples will show whether this novel FIPA is suitable for clinical use. The IgG1 antibodies bind to AQP4 on the cell surface and can initiate complement deposition. These approaches will be useful for investigation of other antibody-mediated diseases.

Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia.

The aim of the present study was to analyze the relations between the serum anti-erythropoietin antibody (AEAb) levels and the antibodies' neutralizing activity in 20 patients with renal anemia and rhEPO-induced antibodies. AEAb levels were determined by the enzyme-linked immunosorbent assay (ELISA, double antigen-bridging) and by radioimmunoprecipitation assay (RIPA). The bone marrow neutralization test was used to determine the neutralizing activity of the antibodies. RIPA and ELISA data resulted in closely correlated measurements. The relations between AEAb levels and the neutralizing activity of the antibodies are variable as shown by follow-up and cross-sectional evaluations of the data. Serum samples with a high antibody level (>1000 ng/ml) are associated with 100% neutralizing activity, whereas serum samples with lower AEAb levels show partial neutralizing activities or have no effect. Determining the neutralizing activity might be helpful when it comes to deciding of whether or not rhEPO therapy should be continued, specifically in patients who have low antibody levels. The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC(50)) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activities.

Evaluation of a new Trypanosoma cruzi antibody assay for blood donor screening.

BACKGROUND: This multicenter prospective study was designed to evaluate the performance characteristics of a new commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Trypanosoma cruzi in blood donors, the ORTHO T. cruzi ELISA Test System (Ortho-Clinical Diagnostics). STUDY DESIGN AND METHODS: Assay specificity was evaluated among 40,665 serum and ethylenediaminetetraacetate (EDTA) plasma specimens from volunteer blood donors and 481 T. cruzi antibody-negative specimens from a high-risk population. Sensitivity was evaluated among 106 T. cruzi-infected subjects identified by parasite detection, among 93 radioimmunoprecipitation assay (RIPA)-positive specimens from high-risk subjects, and 662 specimens presumed positive for the presence of T. cruzi antibodies by serologic methods. Also assessed were the equivalence of serum and plasma as specimen sources, performance equivalence of automated and semiautomated processing methods, nonspecific reactivity in specimens from other disease states or clinical conditions, and assay precision. RESULTS: Assay specificity was 99.998 percent in volunteer blood donors and 99.4 percent among high-risk subjects. Sensitivity was 100 percent among specimens positive by parasite detection, or by serologic methods, and 98.9 percent among RIPA-positive specimens from high-risk subjects. No differences were demonstrated between serum and plasma or between semiautomated and automated processing methods. Cross-reactivity was observed with known positive leishmaniasis specimens. Total inter- and intraassay variability was less than 10 percent with both the automated and the semiautomated methods. CONCLUSION: The ORTHO T. cruzi ELISA Test System is an effective, qualitative assay for screening blood donors for immunoglobulin G antibodies to T. cruzi. The assay was licensed for donor screening by the FDA in December 2006.

Column-based method to simultaneously extract DNA, RNA, and proteins from the same sample.

We describe a procedure for the simultaneous extraction of proteins and nucleic acids from the same experimental sample allowing for direct correlations between genetic, genomic, and proteomic data. This approach, using commercially available column-based nucleic acid extraction kits, requires no hazardous chemicals and is a quick, reliable, and consistent method for concomitant protein extraction. Buffer choice is critical to completely solubilize all proteins in the sample. Proteins solubilized in radioimmunoprecipitation assay (RIPA) buffer did not represent the entire profile when compared with conventionally extracted proteins using the same buffer at the one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) level, however proteins extracted from the columns and solubilized in a two-dimensional (2-D) electrophoresis lysis buffer showed a similar profile to conventionally extracted proteins when analyzed at both the 1-D and the 2-D level. We further showed that proteins extracted using these methods were compatible with Western blot analysis. This technique provides a simple and effective way to analyze protein and nucleic acids simultaneously from the same sample without affecting yield and quality.

Quantitation of autoantibodies in systemic autoimmune diseases: clinically useful?

Serial assessment of levels of autoantibodies has been proposed as being clinically useful in certain systemic autoimmune diseases. In particular, attention has been given to anti-dsDNA antibodies in systemic lupus erythematosus (SLE) and ANCA in the ANCA-associated vasculitides (AAV). Much controversy exists, however, concerning the value of serial testing in these diseases. We here review the various tests available for quantitation of anti-dsDNA and ANCA, and their capacity to detect changes in autoantibody levels that are associated with changes in clinical disease activity of the respective diseases. It is concluded that changes in anti-dsDNA as measured by the Farr assay and changes in ANCA as assessed by ELISA have predictive value for the occurrence of disease relapses, although this relationship is far from absolute. Consequently, treatment based on changes in levels of the respective autoantibodies only seems at present not justified, in view of the toxicity of currently available immunosuppressive regimens.