Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1.

Background/Objective: Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. Methods/Results: 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). Conclusions: The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥.

Interkit Reproducibility of the Indirect Immunofluorescence Assay on HEp-2 Cells Depends on the Immunofluorescence Reactivity Intensity and Pattern.

Introduction: The indirect immunofluorescence assay on HEp-2 cells (HEp-2/IFA) is used worldwide for screening for autoantibodies to cellular antigens. Cell culture and fixation methods influence the cell distribution of autoantigens and the preservation of epitopes. Therefore, discrepancy of results obtained using different HEp-2/IFA kits (interkit nonreproducibility) is a common phenomenon in the clinical laboratory routine. Objective: This study evaluated the interkit nonreproducibility of HEp-2/IFA results using samples from patients with systemic autoimmune disease (SAD), nonautoimmune diseases (NAD), and healthy blood donors (HBD). Methods: Serum from 275 SAD patients, 293 NAD patients, and 300 HBD were processed at 1:80 dilution using four HEp-2 kits according to the manufacturers' instructions. Interkit reproducibility was determined for positive/negative results and patterns. The agreement of positive/negative results among kits for each sample was determined as the reactivity agreement score (RAS). The pattern reproducibility score (PRS) in each sample was calculated as a function of the number of kits showing equivalent patterns. Qualitative variables and ordinal variables were analyzed by the Chi-square and Mann-Whitney U tests, respectively. Results: A total of 402 samples were nonreactive in all kits and were considered devoid of autoantibodies. Further analysis included the 466 reactive samples (238 SAD, 119 NAD, 109 HBD). Reactivity to the nucleus had the highest interkit reproducibility (RAS = 83.6), followed by the metaphase plate (RAS = 78.9), cytoplasm (RAS = 77.4), and nucleolus (RAS = 72.4). Interkit reproducibility was higher in SAD (RAS = 78.0) than in NAD (RAS = 70.6) and HBD (RAS = 71.3) groups. Samples with strong reactivity (++++/4 and +++/4) had higher interkit reproducibility than those with weak reactivity (+/4). In the SAD group, RAS for nuclear reactivity was 87.5% for strongly reactive samples as opposed to 4.4% for weakly reactive samples, and the same was observed for NAD and HBD samples. The most robust patterns were the centromere AC-3 (PRS = 78.4), multiple nuclear dots AC-6 (PRS = 73.6), nuclear coarse speckled AC-5 (PRS = 71.3), nuclear homogeneous AC-1 (PRS = 67.9), and the reticular cytoplasmic AC-21 (PRS = 68.6). Conclusion: Interkit nonreproducibility in HEp-2/IFA is prevalent and occurs with the highest frequency with weakly reactive samples. International initiatives with the engagement of in vitro diagnostic industry are encouraged to promote the harmonization of the properties and performance of HEp-2/IFA commercial kits.

Biological anti-TNF drugs: immunogenicity underlying treatment failure and adverse events.

INTRODUCTION: Genetically engineered monoclonal antibodies and fusion proteins directed against cytokines or their receptors represent a breakthrough in the treatment of various chronic immune-inflammatory diseases. Areas covered: Studies show high remission rates in several diseases, but clinical practice shows a significant percentage of individuals who do not exhibit the desired response. Loss of therapeutic benefit after initial successful response is designated secondary failure. Immune-biological agents are not self-antigens and are therefore potentially immunogenic. Secondary failure is frequently caused by antibodies against immune-biologicals, known as anti-drug antibodies (ADA). ADA that neutralize circulating immune-biologicals and/or promote their clearance can reduce treatment efficacy. Furthermore, ADA can induce adverse events by diverse immunological mechanisms. This review provides a comprehensive overview of ADA in rheumatoid arthritis patients treated with anti-TNF immune-biologicals, and explores the concept of therapeutic drug monitoring (TDM) as an effective strategy to improve therapeutic management. Expert opinion: Monitoring circulating ADA and therapeutic immune-biological drugs is helpful when evaluating patients with secondary failure. However, immunological tests for ADA vary considerably regarding their ability to detect different types of ADA. Several assays are not designed to determine ADA-induced drug neutralizing capacity, and they may report clinically non-relevant data, especially if drug is present in test samples.

Differential capacity of therapeutic drugs to induce Rods/Rings structures in vitro and in vivo and generation of anti-Rods/Rings autoantibodies.

Some HCV patients using ribavirin and interferon alpha (IFN-α) develop anti-rods and rings (RR) autoantibodies, the main target of which is inosine monophosphate dehydrogenase (IMPDH), the rate-determining enzyme in de novo GTP biosynthesis. In vitro inhibition of IMPDH by ribavirin induces RR formation. Here we investigate whether other commonly used drugs that interfere with GTP biosynthesis can induce RR structures in vitro and vivo and elicit generation of autoantibodies. HEp-2 cells treated for 24h with ribavirin, mycophenolic acid (MPA), azathioprine, methotrexate or acyclovir were positive for RR structures. However, adefovir, entecavir, tenofovir and lamivudine did not induce RR structures in these cells. Structures induced by ribavirin in HEp-2 cells are stable after 24h drug-washout, while structures induced by other drugs are relatively labile, disappearing within 2h. Looking at patients treated with these drugs, HCV patients treated with ribavirin (n=17) showed higher average percentage of RR-positive peripheral mononuclear cells than autoimmune patients treated with RR-inducing immunosuppressant drugs (n=21). Serum from 173 autoimmune patients who had been treated with MPA, azathioprine or methotrexate was tested for presence of anti-RR autoantibodies, and only one sample was found to be positive. Conversely, of 48 anti-RR autoantibody positive samples identified at Fleury Laboratories over 30months, 94% were from HCV patients treated with ribavirin plus IFN-α. These data indicate that RR structures can be induced by a variety of drugs in vitro and in vivo, but anti-RR autoantibody production is mostly restricted to HCV patients under ribavirin+IFN-α treatment.