Immunodot Assay Research Articles

Clinicopathological features and treatment outcome of juvenile idiopathic inflammatory myopathies with anti-melanoma differentiation associated gene 5 antibodies: A case series study.

To characterize the clinicopathological features and treatment outcomes of juvenile idiopathic inflammatory myopathies (JIIM) with anti-melanoma differentiation associated gene 5 (MDA5) antibodies in a Chinese cohort. Anti-MDA5 antibody was detected by immunodot assay and indirect immunofluorescence assay on HEK293 cells in a series of Chinese JIIM cohort between 2005 and 2022. The clinical features, histological findings, and treatment outcomes of these anti-MDA5-antibody-positive patients were summarized. Of 59 JIIM patients, 3 (5.08%) were found to be anti-MDA5-antibody-positive. The frequency of anti-MDA5 antibody did not show significant difference between adult idiopathic inflammatory myopathies and JIIM cohorts (P = .720). The disease duration in patients with anti-MDA5 antibody was 2.83 ± 1.04 months. All 3 patients had typical skin lesions including Gottron sign and heliotrope rash, while interstitial lung disease and arthritis was only found in 1 patient. All 3 patients showed normal creatine kinase levels. On muscle biopsy, diffuse major histocompatibility complex class-I expression was seen in 3 patients and myxovirus-resistance protein A expression was found in 2 patients. All patients received long-term follow-up (6.42 ± 4.01 years). They were all drug-free and showed favorable treatment outcome with prednisone and additional immunosuppressant. Our study indicates that anti-MDA5 antibodies may not be common in Chinese JIIM. Anti-MDA5-positive JIIMs are characterized by typical skin lesions of dermatomyositis, normal CK levels, and increased major histocompatibility complex class-I expression. JIIMs with anti-MDA5 generally have good response to immunotherapies.

An immuno-DOT diagnostic assay for autoimmune nodopathy.

Autoimmune nodopathy (AN) is a life-threatening peripheral neuropathy mediated by four autoantibodies targeting axoglial cell adhesion molecules at the nodes ofRanvier: Neurofascin-155 (Nfasc155), PanNeurofascin (PanNfasc), Contactin-1 (CNTN1), and Contactin-associated protein 1 (CASPR1). Antibody detection is a strong biomarker for AN diagnosis and treatment monitoring. The aim of this study was to develop an immuno-dot assay (immuno-DOT) compatible with routine implementation in medical laboratories. This new approach was compared to standard techniques: indirect immunofluorescence assay, cell-based assay, and ELISA. Sensitivities (Se) and specificities (Sp) were calculated on a cohort composed of 58 patients diagnosed with AN, 50 seronegative patients with chronic inflammatory demyelinating polyradiculoneuropathy, 20 healthy controls, 30 patients with Guillain-Barré syndrome, 20 with monoclonal gammopathy and 20 with Charcot-Marie-Tooth disease. The patients were diagnosed with AN based on compatible electro-clinical arguments and at least two positive standard techniques. Immuno-DOT sensitivities and specificities were Se=91 %, Sp=97 % for anti-Nfasc155; Se=80 %, Sp=94 % for anti-PanNfasc; Se=93 %, Sp=98 % for anti-CNTN1; and Se=87 %, Sp=94 % for anti-CASPR1. Immuno-DOT allowed the diagnosis within 3 h and the accurate follow-up of the immune reactivity and isotype, and dot intensity correlated with antibody titers following treatments. A longitudinal study indicated that immuno-DOT yielded reliable results even after six months of storage at-20 °C. The diagnostic performance of immuno-DOT was satisfactory and compatible with routine implementation in medical laboratories.

ANCA and ASCA Profiles in a Moroccan Population With Inflammatory Bowel Diseases.

Diagnosing inflammatory bowel disease (IBD) is hindered by the invasive procedures required for accurate classification as Crohn's disease (CD) or ulcerative colitis (UC). As alternatives, non-invasive tests using anti-Saccharomyces cerevisiae antibodies (ASCA) and anti-neutrophil cytoplasmic antibodies (ANCA) have gained significance. This study evaluated ANCA and ASCA antibody frequencies in IBD and their role in disease characterization in a Moroccan population. Conducted at Marrakech's Mohammed VI University Hospital from 2014 to 2018, this cross-sectional study included patients with suggestive symptoms or confirmed IBD diagnosis based on clinical, endoscopic, and histological criteria. Immunological investigations detected p-ANCA, c-ANCA, and ASCA using immunofluorescence and immunodot assays. Among 60 participants (mean age: 33.1 ± 11.75 years), the 20-30-year age group was most affected (31.67%). CD, UC, and indeterminate colitis (IC) were diagnosed in 46.67%, 45%, and 8.33% patients, respectively. Gastrointestinal symptoms were prevalent (98.3%), with ANCA+/ASCA-profile in 41% of UC patients versus 11% in CD, and ANCA-/ASCA + profile exclusive to CD (50%). ANCA positivity was significantly associated with UC, rectal syndrome, and inflammatory syndrome, whereas ASCA positivity was significantly associated with CD and König's syndrome (p < 0.05). This study highlighted demographic and phenotypic particularities of IBD in a Moroccan population. Non-invasive tests using ASCA and ANCA antibodies offer valuable alternatives to invasive procedures, facilitating personalized management strategies. Variations in ANCA and ASCA profiles provide insights into disease characterization and inform tailored treatment approaches.

Immunoglobulin E-Mediated Food Sensitization in a Moroccan Pediatric Population with Celiac Disease

Introduction: Celiac disease is a chronic autoimmune disorder that occurs following the ingestion of gluten, in genetically predisposed individuals. Patients with celiac disease, especially children, are likely prone to develop allergic reactions to different food allergens. However, the relationship between food allergy and celiac disease remains not elucidated. The aim of this pioneering study was to evaluate the prevalence of allergic food sensitization in children with celiac disease in Morocco. Methods: A total of 57 children with confirmed celiac disease, including 25 males and 32 females with a mean age of 8.6 ± 4.4 years, underwent a food allergen-specific immunoglobulin E (IgE) screening. This screening was conducted using a multiparametric immunodot assay (Euroline Food “Maghreb,” Euroimmun). Statistical analysis was performed using R software. Results: Among the 57 cases tested, the overall rate of IgE-mediated sensitization to food allergens was found to be 48% (27/57), dominated by chicken, with 51.9% (14/27), followed by almond, 40.7% (11/27), sesame, 40.7% (11/27), potato 33.3% (9/27), and apple 18.5% (5/27). Of the s-IgE positive cases, 74% were sensitized at least to one allergen, 37% (10/27) were sensitized to both chicken and almond allergens. A significant correlation was observed between almond, sesame, chicken, and potato. Conclusion: The current study highlighted a high prevalence of food allergen sensitization in children with celiac disease. This underlines the potential benefit in screening for food allergy in celiac patients.

Rapid Immunodot AQP4 Assay for Neuromyelitis Optica Spectrum Disorder

Immunoglobulin G autoantibodies for aquaporin 4 (AQP4-IgG) serve as diagnostic biomarkers for neuromyelitis optica spectrum disorder (NMOSD), and the most sensitive and specific laboratory tests for their detection are cell-based assays (CBAs). Nevertheless, the limited availability of special instruments limits the widespread use of CBAs in routine laboratories. To validate an enzyme immunodot assay for simple and rapid detection of AQP4-IgG. This multicenter case-control study, conducted from May 2020 to February 2023, involved 4 medical centers (3 in China and 1 in Korea). The study included patients with AQP4-IgG-positive NMOSD, patients with other immune-related diseases, and healthy control individuals. Participants were excluded if they did not agree to participate or if their serum sample had turbidity. Serum AQP4 antibodies measured with immunodot assay. The main outcome was performance of the immunodot assay compared with the gold standard CBA for detecting AQP4-IgG. To examine generalizability, cross-validation in Korea and at a second site in China, validation of patients with other immune-related diseases, and follow-up validation of the original cohort were performed. A total of 836 serum samples were collected; 400 were included in the diagnostic study and 436 in the validation sets. In a head-to-head diagnostic study involving 200 patients with NMOSD with AQP4-IgG (mean [SD] age, 43.1 [13.5] years; 188 [94%] female) and 200 healthy controls, use of an immunodot assay demonstrated antibody detection performance comparable to that of the gold standard (κ = 98.0%). The validation sets included 47 patients with NMOSD and 26 patients with other autoimmune diseases from Korea, 31 patients with NMOSD at a second site in China, 275 patients with other diseases, and 57 patients with NMOSD at follow-up. In the validation study, of 436 cases, 2 (<1%) were false positive and none were false negative. The CBA identified 332 AQP4-IgG-positive samples and 504 negative samples (200 [40%] in controls and 304 [60%] in patients with other diseases); 2 of the positive cases (<1%) were false negative and 4 of the negative cases (<1%) were false positive. The overall sensitivity of the immunodot assay was 99.4% (95% CI, 97.8%-99.9%), and the specificity was 99.2% (95% CI, 98.0%-99.8%). This case-control study found that the immunodot assay was comparable to CBA for detecting AQP4-IgG. With its time- and cost-efficient characteristics, the immunodot assay may be a practical option for AQP4-IgG detection.

Detection of anti-ganglioside antibodies in Guillain-Barré syndrome.

Gangliosides are a class of glycosphingolipid molecules that are highly enriched in cellular membranes of the nervous system. The gangliosides associated with autoimmune diseases of the nervous system are mainly GM1, GD1a, GalNAc-GD1a, GM1b, GD3, CD1b, GT1a, and GQ1b. Multiple antibodies recognizing gangliosides are associated with some acute or chronic peripheral neuropathies, especially Guillain-Barré syndrome (GBS) and its clinical variants. Antibodies binding to gangliosides can activate complement system and recruit macrophages on the axolemma at the nodes of Ranvier of motor fibers, which are found in the course of GBS, causing axonal degeneration and reversible conduction block or conduction failure. Testing of anti-gangliosides autoantibodies is helpful for diagnosis of autoimmune peripheral neuropathies or support the diagnosis of the subtypes. These anti-gangliosides antibodies are usually detected by several qualitative or quantitative methods, particularly enzyme-linked immunosorbent assay (ELISA) and immunodot assays, which have been commercialized or established in-house worldwide. Herein, we introduce the methods and clinical applications of these assays in the diagnosis of autoimmune peripheral neuropathies. Anti-gangliosides antibodies are diagnostic markers of GBS subtypes. We use GBS as an example to explain the role of anti-gangliosides antibodies in the pathogenesis and diagnostic classification of neuropathies.

Optimization of Laboratory Diagnostics of Primary Biliary Cholangitis: When Solid-Phase Assays and Immunofluorescence Combine.

The laboratory diagnostics of primary biliary cholangitis (PBC) have substantially improved, thanks to innovative analytical opportunities, such as enzyme-linked immunosorbent assays (ELISA) and multiple immunodot liver profile tests, based on recombinant or purified antigens. This study aimed to identify the best diagnostic test combination to optimize PBC diagnosis. Between January 2014 and March 2017, 164 PBC patients were recruited at the hospitals of Parma, Modena, Reggio-Emilia, and Piacenza. Antinuclear antibodies (ANA) and anti-mitochondrial antibodies (AMA) were assayed by indirect immunofluorescence (IIF), ELISA, and immunodot assays (PBC Screen, MIT3, M2, gp210, and sp100). AMA-IIF resulted in 89.6% positive cases. Using multiple immunodot liver profiles, AMA-M2 sensitivity was 94.5%, while anti-gp210 and anti-sp100 antibodies were positive in 16.5% and 17.7% of patients, respectively. PBC screening yielded positive results in 94.5% of cases; MIT3, sp100, and gp210 were detected by individual ELISA test in 89.0%, 17.1%, and 18.9% of patients, respectively. The association of PBC screening with IIF-AMA improved the diagnostic sensitivity from 89.6% to 98.2% (p < 0.01). When multiple immunodot liver profile testing was integrated with AMA-IIF, the diagnostic sensitivity increased from 89.1% to 98.8% (p < 0.01). The combination of IIF with solid-phase methods significantly improved diagnostic efficacy in PBC patients.

Myositis-specific autoantibodies in clinical practice: Improving the performance of the immunodot

Background/objectivesIdiopathic inflammatory myopathies (IIM) diagnosis and sub-classification can be improved by detection of myositis specific antibodies (MSA) as a first step in diagnosis. However, when using semi-quantitative immunodots for MSA detection, clinical assay performance needs to be improved. MethodsA retrospective study was done for the “myositis” and “synthetase” immunodots (SRP, NXP2, TIF1gamma, SAE1/2, Mi2, MDA5, Jo1, PL7, PL12, EJ, OJ, KS, ZO and HA) from D-Tek used for 270 patients who had tested positive for MSA in a tertiary laboratory hospital. ResultsResults from this analysis revealed: (i) none of the 60 healthy controls presented MSA; (ii) a low assay specificity among patients who tested positive for MSA, 128/270 (47%) were labeled IIM based on the manufacturer's recommended threshold; (iii) in non-IIM patients (53%), the MSA spectrum overlaps predominantly with other autoimmune diseases or idiopathic interstitial lung disease; and (iv) use of a clinical cut-off improves assay specificity for anti-SRP, anti-NXP2, anti-MDA5, anti-Jo1 and anti-PL7 Abs. ConclusionDetermining the clinical threshold of the semi-quantitative immunodot assay for MSA is effective for improving its capacity to discriminate IIM from non-IIM and, when IIM diagnosis is excluded, another autoimmune disease or an idiopathic interstitial lung disease should be considered in front of a positive MSA.

Anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase antibodies: incidence using different testing criteria, and case series.

To estimate the incidence of anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies utilising different testing criteria, and review the clinical details of a series of patients with associated autoimmune myopathy. The incidence of anti-HMGCR antibodies in 2019 from 3 groups, South West London, Berkshire/Surrey and Southampton, were compared in the adult population. Anti-HMGCR antibodies were measured by commercial chemiluminescent and immunodot assays. The case notes of patients with anti-HMGCR antibodies were reviewed for the case series. The estimated incidence of anti-HMGCR antibodies in the first 2 groups was 1.94 per million adults per year, and in the third group 10.3 per million adults per year. In the first 2 groups the test criteria restricted analysis to specific clinician request for anti-HMGCR. In the third group test criteria included cases with less specific clinical features or a cytoplasmic indirect immunofluorescence anti-nuclear antibody pattern. The latter strategy had a positive predictive value of 66.1% for anti-HMGCR associated myopathy. A case series of 27 patients with anti-HMGCR antibodies revealed 19 with myopathy, oesophageal involvement in 26% and median peak CK 8000 IU/L. Response to treatment, including intravenous immunoglobulin, was good with CK normalising after median 5.5 months. In 8 cases there was no evidence of autoimmune muscle disease, 7 not statin exposed. Varying criteria result in a 5-fold difference in estimated incidence of anti-HMGCR antibodies, revealing positive cases without evidence of myopathy. Patients with anti-HMGCR myopathy respond well to immune suppression, supporting wider testing for these antibodies amongst patients with myopathy.

The graft framework: Quantitative changes in cell wall matrix polysaccharides throughout the tomato graft union formation

Plant cell walls provide essential functions in cell recognition, differentiation, adhesion and wound responses. Therefore, it is tempting to hypothesize that cell walls play a key role in grafting, but to date there are no quantitative studies targeting on cell wall changes during grafting. The aim of this work was to investigate the dynamics of pectic and hemicellulosic polysaccharides at the graft junctions in tomato homografts throughout the first 12 days after grafting. Cell wall fractionation, combined with ATR-FTIR spectroscopy and gas-chromatography, evidenced a marked increase in pectin content and a decrease in the degree of methyl-esterification of homogalacturonan in scion and rootstock throughout grafting. Also, recovery of tightly-bound hemicelluloses decreased at late times after grafting suggesting an increase of cross-linked hemicelluloses along grafting. In addition, immuno-dot assays revealed an increase in xyloglucan and arabinogalactan proteins in the first days after grafting, pointing to a presumed role in tissue adhesion-cohesion.

POLYCLONAL POLYCLONAL ANTIBODIES TO THE RECOMBINANT LST8/GΒL HOMOLOGUE OF TRITICUM AESTIVUM, A COMPONENT OF THE TORC1 SIGNALING

LST8 is a key component of the TOR signaling system, a central regulator of cell proliferation and growth in eukaryotic cells. In this study, polyclonal antibodies against LST8 of Triticum aestivum (TaLST8) were produced and characterized. cDNA of the wheat TaLST8 was isolated by reverse transcription-PCR. The amino acid sequence alignment of TaLST8 showed high homology with yeast LST8 (55% identity), human LST8 (51% identity), and Arabidopsis AtLST8 (85% identity). To obtain antibodies against TaLST8, a highly conserved part of the TaLST8 gene encoding a 150 amino acid protein (talst8/150) was cloned into pET-28c with a histidine tag (6xHis) and expressed in Escherichia coli. Rabbits were immunized with purified TaLST8/150 recombinant protein. The obtained antisera were purified to increase the specificity of recognition. The sensitivity and specificity of the produced antibody was analyzed by enzyme-linked immunosorbent assay, Western blotting and immunodot assay. Enzyme-linked immunosorbent assay showed that immunization with purified 6xHis-TaLST8/150 produced the high titer (1:64000) polyclonal antibodies with high specificity. Antibodies against TaLST8 allowed the sensitive detection of native and denatured protein in Western blot and immunodot assays. The purified polyclonal antibody raised against the recombinant TaLST8/150 protein is sufficiently specific and sensitive and could be a useful tool for future insights of the functioning of the TOR signaling system in wheat plants.

IgE-binding residues analysis of the house dust mite allergen Der p 23

House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P < 0.001). Atopic individuals who were only sensitized to HDM have a significantly higher number of IgE-binding residues than the individuals who were polysensitized to HDM and other crude allergens (P < 0.05). Individuals with allergic multimorbidity and moderate-to-severe allergic rhinitis also have a higher number of IgE-binding residues compared to those with single allergic disease and mild allergic rhinitis. The results prompt us to hypothesize that the individuals who have a higher number of IgE-binding residues may face a bigger challenge to be treated through immunotherapy due to the complexity in designing an effective hypoallergen with a high number of IgE-binding residues. We propose that the development of a refined molecular diagnostic assay, which includes alanine substitution of surface-exposed residues could be a more precise diagnostic strategy to identify all the IgE-binding residues of a major allergen for an atopic individual and the development could be another new dimension in allergy diagnosis and allergen immunotherapy treatment.

An immuno-dot blot assay for screening histidine kinase inhibitors

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.

Polyclonal antibody‐based farmer‐friendly flow‐through test for the detection of acute hepatopancreatic necrosis disease in shrimp

Early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) is currently the most significant disease of shrimp in farms of Vietnam, Thailand, Malaysia, China and Mexico, and there is a great risk that it may spread to other shrimp farming countries. Although, an array of sophisticated detection tools for AHPND available, there is a need for a sensitive, simple and rapid detection method. In this study, a simple, sensitive, rapid and polyclonal antibody-based farmer-friendly flow-through assay (FTA) test has been developed for the detection of AHPND pathogen. The recombinant Photorhabdus insect-related (Pir) A toxin-like protein of AHPND pathogen was used to immunize rabbits at 21-day interval observed for highest antibody titre after third booster by ELISA. The raised rabbit antiserum was purified by affinity chromatography and characterized by Western blot. The antiserum showed no cross-reactivity with AHPND-free Vibrio parahaemolyticus, V. anguillarum, White Spot Virus (WSV), Aeromonashydrophila and Aphanomycesinvadans. This polyclonal rabbit antiserum was used to develop a farmer-friendly FTA test for the detection of AHPND pathogen. This simple FTA testis is more sensitive and could detect PirAVP toxin up to 0.121 µg/ml, compared with 0.242 µg/ml by immunodot assay. Furthermore, FTA test requires only 8–10 min for completion, compared with 3 hr by immunodot thus found to be more sensitive, specific and cost-effective. Collectively, sensitive FTA test would help shrimp farmers to take real-time management decisions, especially emergency harvest and finally be a better hope for the prevention of AHPND.

Diagnostic yield of commercial immunodots to diagnose paraneoplastic neurologic syndromes.

ObjectiveTo investigate the diagnostic yield of commercial immunodots to detect onconeural antibodies associated with paraneoplastic neurologic syndromes (PNSs), we analyzed the proportion of confirmed positive results using alternative techniques.MethodsSera (n = 5,300) of patients with suspected PNS were tested by PNS+2 blot (Ravo Diagnostika; January 2016–May 2017) or EUROLINE PNS 12 Ag (Euroimmun; July 2017–November 2018). Positive samples were further explored by in-house indirect immunofluorescence and a third in-house technique (Western blot or cell-based assay) using recombinant protein. Those found negative by these 2 techniques were considered as nonconfirmed. We analyzed the relationship between band intensity and final confirmation. Clinical data were collected for all confirmed results and nonconfirmed EUROLINE immunodots.ResultsPNS+2 blot was positive in 128/1,658 (7.7%) sera and confirmed in 47/128 (36.7%). EUROLINE was positive in 186/3,626 (5.1%) and confirmed in 56/186 (30.1%). Confirmation was highly variable among the antibodies tested, from 7.2% (PNS+2 blot) and 5.8% (EUROLINE) for anti-Yo to 88.2% (PNS+2 blot) and 65.0% (EUROLINE) for anti-Hu. None of the 27 weak positive sera by EUROLINE was confirmed. Band intensity in confirmed cases was variable among the antibodies from strong positive for all anti-Yo (n = 3) and anti-Hu (n = 11) to positive (n = 19) or strong positive (n = 9) for anti-SOX1. Among patients with a nonconfirmed EUROLINE result and available clinical information, all had an alternative diagnosis, and only 6.7% had cancer.ConclusionsImmunodots may be useful for PNS screening, but a threshold should be established for each antibody, and clinical information and confirmation by other techniques are essential.Classification of evidenceThe study provides Class IV evidence that immunodot assays for onconeural antibodies accurately identify patients with paraneoplastic neurologic syndromes.

Characteristics of Systemic Sclerosis patients with positive anti-Th/To antibodies: About 6 patients and literature review

Among the antibodies described in Systemic Sclerosis (SSc), anti-Th/To antibodies (anti-Th/To) are rare and have been poorly studied. Thus, little is known about the profile of anti-Th/To positive patients. From our local Biobank (Marseille, France), we retrospectively selected data for 6 patients positive for anti-Th/To with an Immunodot assay. All of them suffered from SSc, sharing clinical and biological common features such as a limited cutaneous form of SSc, a decreased lung diffusing capacity and a speckled nuclear nucleolar immunofluorescence pattern of antinuclear antibodies screening on HEp-2 cells. In order to further characterize patients positive for anti-Th/To, we performed a thorough literature review. From 402 studied patients positive for anti-Th/To, we confirmed that these antibodies are associated with the limited cutaneous form of the disease (88% of the patients), and with an SSc related-pulmonary involvement (50%). The review analysis pointed out the rarity of the anti-Th/To with an estimated mean frequency of 3.4% of all SSc patients worldwide, their usual exclusivity with respect to the specific antibodies of scleroderma, and their high specificity (around 98%) for the diagnosis of SSc.

Citrullinated Inhibitor of DNA Binding 1 Is a Novel Autoantigen in Rheumatoid Arthritis.

ObjectiveTo explore the intrinsic role of inhibitor of DNA binding 1 (ID‐1) in rheumatoid arthritis (RA) fibroblast‐like synoviocytes (FLS) and to investigate whether ID‐1 is citrullinated and autoantigenic in RA.Methods RA patient serum ID‐1 levels were measured before and after infliximab treatment. RA FLS were transfected with a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 construct targeting ID‐1 to examine the effects of ID‐1 deletion. RA synovial fluid (SF) and homogenized synovial tissue (ST) were immunoprecipitated for ID‐1 and measured for citrullinated residues using an enzyme‐linked immunosorbent assay and Western blotting. Liquid chromatography tandem mass spectrometry (LC‐MS/MS) was performed on in vitro–citrullinated recombinant human ID‐1 (cit–ID‐1) to localize the sites of citrullination. Normal and RA sera and SF were analyzed by immunodot blotting for anti–citrullinated protein antibodies (ACPAs) to cit–ID‐1.Results RA patient serum ID‐1 levels positively correlated with several disease parameters and were reduced after infliximab treatment. RA FLS displayed reduced growth and a robust increase in interleukin‐6 (IL‐6) and IL‐8 production upon deletion of ID‐1. ID‐1 immunodepletion significantly reduced the levels of citrullinated residues in RA SF, and citrullinated ID‐1 was detected in homogenized RA ST (n = 5 samples; P < 0.05). Immunodot blot analyses revealed ACPAs to cit–ID‐1 but not to native ID‐1, in RA peripheral blood (PB) sera (n = 30 samples; P < 0.001) and SF (n = 18 samples; P < 0.05) but not in normal PB sera. Following analyses of LC‐MS/MS results for citrullination sites and corresponding reactivity in immunodot assays, we determined the critical arginines in ID‐1 for autoantigenicity: R33, R52, and R121.ConclusionNovel roles of ID‐1 in RA include regulation of FLS proliferation and cytokine secretion as well as autoantigenicity following citrullination.

The incidence of immunofluorescence patterns and specific autoantibodies observed in autoimmune patients in a tertiary care centre.

Objectives: Testing for antinuclear antibodies (ANA) facilitates the diagnosis of autoimmune diseases (ADs). Here, we report an incidence of ANA positivity and its patterns by indirect immunofluorescence (IIF) and specific autoantibodies through immunodot assay. Methods: Sera from 993 patients presenting with various ADs were tested by IIF and immunodot assay. Results: ANAs were detected in 39.7%, of which speckled pattern was predominantly observed (50.8%). 56.8% of samples were positive on the immunodot assay with SSA Ro 60 antibody being the most prevalent (30.7%). Discussion: A significant correlation (p minor 0.0001) was observed between patterns and auto-antibodies. Coarse speckled (CS) and homogeneous were overly represented by antibodies SSA Ro 60 (13%) and nucleosomes (5.8%) respectively. Mi-2, PL-7, PL-12, and SP-100 were the rarest autoantibodies specificities found. Conclusions: The presence of a particular IIF pattern is predictive of a specific autoantibody in the sample. Association of IIF patterns and specific autoantibody are relevant for a more accurate diagnosis of disease.

RANGING OF ANTIPHOSPOLIPID ANTIBODIES IN THE PATIENTS WITH THROMBOPHILIA AND RECURRENT MISCARRIAGE

Laboratory diagnosis of antiphospholipid syndrome (APS) is based on detection of antiphospholipid antibodies (aPLs). E.g., aPLs are directed against conformational epitopes of the so-called “co-factor” proteins: β2-gycoprotein 1 (β2-GP1), annexin V (An V) and prothrombin (Pt) that are formed during interaction with phospholipids – cardiolipin (CL), phosphatic acid (Pha), phosphatidylcholine (Pch), phosphatidylethanolamine (Pe), phosphatidylglycerol (Pg), phosphatidylinositol (Pi), phosphatidylserine (Ps). A routine methodology of detection based on ELISA testing is challenged by new tests when the antigen is absorbed on another kind of support like microbeads or membranes that can influence density of conformational epitopes for aPL’s binding. The aim of our study was to compare the results of aPLs detection by ELISA and multi-line immunodot assay (MLD). We collected blood serum samples from 45 patients with noncardioembolic ischemic strokes, 19 patients with recurrent deep vein thrombosis of lower limbs, 44 females with recurrent miscarriages, and 50 clinically healthy donors. To compare the results of aPL detection by ELISA and MLD kits, the test systems from different manufacturers were evaluated. We used an ELISA kits for detection of antibodies to CL IgG, aCL IgM, β2-GP1 produced by Euroimmun AG (Mr1) and Orgentec Diagnostica GmbH (Mr2) and MLD – for detection of antibodies to CL, β2-GP1, Pch, Pe, Pg, Pi, Ps, AnV and Pt (Medipan GmbH, Mr3). When a cut-off titer was used as the main index, 30.5% of patients were aPLs-positive with ELISA method by Mr1 and 38%, wiht Mr2. By MLD aPls were detected in 30% of patients. In the same cohort, medium and high aPLs titers (&gt; 40 U/mL) were determined in 12% of patients using ELISA kits. Positive and highly positive aPLs titers were determined in 16% when using a new method by Mr3. Medium and high titer were detected only for antibodies to β2-GP1, CL, An V, Pha and Phs. The use of ELISA approach for detection of aPLs in patients with thrombosis and obstetric pathology is associated with relatively high number of low-positive ELISA results. Due to higher sensitivity for medium and high aPLs titers, MLD testing may be used as a confirming method for APS diagnosis.

Nanogold – IgY antibodies. An immunoconjugated for the detection of house dust mite (Dermatophagoides) allergens

Conjugation of avian IgY antibodies to nanosensors has been extensively explored for the diagnostics of virus and parasite infection, as well as for the detection of pharmaceutically and toxicologically relevant molecules. However, to date this strategy has only been minimally applied the detection of allergens. In this study, gold nanoparticles (GNPs) were conjugated to a polyvalent IgY antibodies raised against Dermatophagoides group I allergens. GNPs were synthesized by HAuCL4 reduction using 1% trisodium citrate, and characterized them by absorption spectroscopy and transmission electron microscopy (TEM). The most stable immunoconjugates were obtained with 18-nm monodisperse GNPs and a minimal concentration of 12.5 μg/mL of IgY at pH 7.5. The immunoconjugate was capable of detecting up to 1.5 μg of a total Dermatophagoides farinae protein extract in an immuno-dot blot assay. This immunoreactant conjugate represents a new tool for the detection and control of indoor dust mite allergens.