Fluorescence Immunoprecipitation Assays Research Articles

Distinctive characteristics of early-onset and late-onset neuromyelitis optica spectrum disorders

ABSTRACTObjectives: Little is known about patients with neuromyelitis optica spectrum disorders (NMOSD) as defined by onset age. This study aimed to analyze the different demographic, clinical, laboratory, and magnetic resonance imaging (MRI) characteristics in early-onset (≤50 years) NMOSD (EONMOSD) and late-onset (>50 years) NMOSD (LONMOSD). Materials and Methods: We enrolled 142 patients with NMOSD from Tianjin Medical University General Hospital, Tianjin, China, and categorized them into two groups according to the age of onset: EONMOSD and LONMOSD. Demographic, clinical, laboratory, and MRI characteristics were collected and compared between the two groups. Serum aquaporin-4 (AQP4) antibody levels were determined by cell-based assay and fluorescence immunoprecipitation assays. Results: Among the patients studied, 83 had early onset (≤50 years) and 59 had late onset (>50 years) of NMOSD. As compared with LONMOSD, EONMOSD patients had more severe visual disability according to functional scores in clinical parameters, significantly lower C3 and C4 serum levels, more frequent cervical lesions, and more lesions around the fourth ventricle, but fewer lesions in hemispheric white matter. LONMOSD patients suffered more motor and sensory disability than EONMOSD patients. Conclusions: In NMOSD, the clinical, laboratory, and MRI features differ according to age of onset, suggesting that differences in pathogenesis and treatment should be further investigated.

Evaluation of aquaporin-4 antibody assays.

Aquaporin-4 (AQP4) is a water channel protein that is most highly, but not exclusively, expressed in the central nervous system. In 2005 AQP4 was shown to be the antigenic target of neuromyelitis optica-immunoglobulin G (NMO-IgG, or AQP4-IgG), an antibody found specifically in patients with NMO and in formes frustes of NMO, such as longitudinally extensive transverse myelitis (LETM) or optic neuritis (ON). This discovery facilitated the clinical, pathological, and radiological distinction of NMO and the spectrum of NMO-related disorders from classical multiple sclerosis. In addition to its use as a diagnostic tool, AQP4-IgG predicts a high risk of relapse in patients with a clinically isolated syndrome of either LETM or ON. As disability in NMO is attack-related, early diagnosis and treatment are predicted to have a major effect on long-term disability. Thus, the importance of sensitive and specific assays to detect AQP4-IgG cannot be overstated. Both academic institutions and commercial companies have developed assays to identify AQP4-IgG in patients' sera or cerebrospinal fluid. Both AQP4 isoforms from different species have been used as the antigenic target in the form of frozen tissue sections in indirect immunofluorescence assays, partially purified protein for fluorescence immunoprecipitation assay, radioimmunoprecipita-tion assay or enzyme-linked immunosorbent assay, or transfected into cells for cell based assays or flow cytometry. We carried out a systematic review of the literature reporting different methodologies used to identify AQP4-IgG, examine whether longitudinal AQP4-IgG titers predict relapses in seropositive patients, and attempt to establish a reasonable timeframe for retesting negative serum samples.

Clinical Usefulness of Cell-based Indirect Immunofluorescence Assay for the Detection of Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder

BackgroundThe presence of antibodies to aquaporin-4 (AQP4) has been identified as a key characteristic of neuromyelitis optica spectrum disorder (NMOSD), an autoimmune inflammatory demyelinating central nervous system (CNS) disorder. We evaluated the performance of a cell-based indirect immunofluorescence assay (CIIFA) for detecting AQP4 antibodies using antigen prepared with a recombinant AQP4 peptide transfection technique and assessed the usefulness of CIIFA for diagnosis of NMOSD in routine clinical practice.MethodsForty-six serum samples from 36 patients as a comparison set and another 101 patients enrolled consecutively from a neurology clinic were included. CIIFA and fluorescence immunoprecipitation assays (FIPA) were performed. CIIFA was performed at 2 different institutions for comparison purposes.ResultsCIIFA and FIPA sensitivity in the comparison set was 86% and 79% in neuromyelitis optica (NMO) patients and 55% and 36% in high-risk NMO patients, respectively. The semiquantitative titer measured by CIIFA correlated well with the arbitrary unit (fluorescence units [FU]) derived from FIPA (r=0.66). Titers measured by CIIFA and FIPA were elevated in NMO patients compared to high-risk NMO patients (1:240 vs. 1:180 and 8,390 vs. 4,059 FU, respectively). The frequency of AQP4 antibody detection by CIIFA in 101 consecutively enrolled patients was 100% in NMO and 23% in high-risk NMO patients, while only 4.6% in control patients, including those with multiple sclerosis.ConclusionsDetection of AQP4 antibodies by CIIFA provides sensitive and highly specific diagnostic information for NMO and high-risk NMO patients, which can be used to differentiate these conditions from other demyelinating CNS diseases.

Serologic diagnosis of NMO

Neuromyelitis optica (NMO) immunoglobulin G (IgG) (aquaporin-4 [AQP4] IgG) is highly specific for NMO and related disorders, and autoantibody detection has become an essential investigation in patients with demyelinating disease. However, although different techniques are now used, no multicenter comparisons have been performed. This study compares the sensitivity and specificity of different assays, including an in-house flow cytometric assay and 2 commercial assays (ELISA and transfected cell-based assay [CBA]). Six assay methods (in-house or commercial) were performed in 2 international centers using coded serum from patients with NMO (35 patients), NMO spectrum disorders (25 patients), relapsing-remitting multiple sclerosis (39 patients), miscellaneous autoimmune diseases (25 patients), and healthy subjects (22 subjects). The highest sensitivities were yielded by assays detecting IgG binding to cells expressing recombinant AQP4 with quantitative flow cytometry (77; 46 of 60) or visual observation (CBA, 73%; 44 of 60). The fluorescence immunoprecipitation assay and tissue-based immunofluorescence assay were least sensitive (48%-53%). The CBA and ELISA commercial assays (100% specific) yielded sensitivities of 68% (41 of 60) and 60% (36 of 60), respectively, and sensitivity of 72% (43 of 60) when used in combination. The greater sensitivity and excellent specificity of second-generation recombinant antigen-based assays for detection of NMO-IgG in a clinical setting should enable earlier diagnosis of NMO spectrum disorders and prompt initiation of disease-appropriate therapies.

Non-radioactive serological diagnosis of myasthenia gravis and clinical features of patients from Tianjin, China

Purpose To establish non-radioactive assays for detection of antibodies (Abs) to the acetylcholine receptor (AChR) and to muscle specific kinase (MuSK). To show that the assays can be used in Tianjin for testing patients with MG. Method AChR and MuSK Abs in 51 Chinese MG patients' sera were tested in Oxford, UK, using the conventional radioimmunoprecipitation assay (RIPA), a recently described cell-based assay (CBA), and a new non-radioactive fluorescence immunoprecipitation assay (FIPA) which can measure both AChR and MuSK antibodies in one step. 102 MG sera were subsequently tested by CBA and FIPA in Tianjin, China. Based on the different serological subgroups, the clinical features of these 153 MG patients were analyzed. Results We first confirmed the sensitivity and specificity of the assays in Oxford. There was good agreement between the FIPA and the RIPA for AChR Abs (r 2 = 0.6; p < 0.0001) with 80% positivity in the RIPA and 76% in the FIPA. Two patients were positive for MuSK Abs (4% of total) by both RIPA and FIPA assays. CBA was more sensitive for MuSK Abs, identifying an additional 3 patients. In Tianjin, using the FIPA and CBA, 84/102 (82%) AChR Ab positive patients and four MuSK Ab positive patients were identified. In the whole group of 153 MG patients, there were no differences in clinical features between different antibody subgroups. However, there were 30 patients with thymomas (20%), of whom one had MuSK antibodies. Moreover, two patients with purely ocular MG also had MuSK antibodies. Conclusions Both the FIPA and CBA were established in China and should prove useful for diagnostic testing. Including the CBA for MuSK antibodies increased the number of MuSK-MG patients to 6% of the total, and 33% of the patients without AChR antibodies. The clinical features were mainly relatively mild but thymic tumors were common.

Frequency and prognostic impact of antibodies to aquaporin-4 in patients with optic neuritis

Background Antibodies to aquaporin-4 (AQP4-Ab) are found in 60–80% of patients with neuromyelitis optica (NMO), a severely disabling inflammatory CNS disorder of putative autoimmune aetiology, which predominantly affects the optic nerves and spinal cord. Objective To assess the frequency of AQP4-Ab in patients with optic neuritis (ON), and to investigate the prognostic implications of AQP4-Ab seropositivity in such patients. Patients and methods AQP4-Ab serum levels were determined in 224 individuals from Austria, Denmark, France, Germany, Italy, and Turkey using a newly developed fluorescence immunoprecipitation assay employing recombinant human AQP4. Results AQP4-Ab were detectable in 8/139 (5.8%) patients with acute monosymptomatic optic neuritis (AMON) and in 10/17 (58.8%) patients with established NMO and a last relapse of acute ON (NMO/ON), but not in 32 patients with multiple sclerosis or in 36 healthy controls. At last examination, 4/8 (50%) seropositive AMON patients had met the criteria for NMO but 0/128 seronegative AMON patients. Disease severity differed significantly between seropositive and seronegative AMON. Complete bilateral or unilateral blindness occurred in six AQP4-Ab positive patients, but only in one AQP4-Ab negative patient. AQP4-Ab levels did not vary between seropositive AMON and NMO/ON and did not correlate with disease severity. Female gender, a relapsing course, and concomitant autoimmunity were associated with AQP4-Ab seropositive status and risk of developing NMO. Conclusion AQP4-Ab is relatively rare among patients with AMON, but if present it predicts a high rate of conversion to NMO within one year.

Aquaporin-4 Antibodies in Neuromyelitis Optica and Longitudinally Extensive Transverse Myelitis

There is increasing recognition of antibody-mediated immunotherapy-responsive neurologic diseases and a need for appropriate immunoassays. 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. 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. United Kingdom and Germany. Serum samples from patients with neuromyelitis optica (n = 25) or longitudinally extensive transverse myelitis (n = 11) and from relevant controls (n = 78) were studied. Comparison of different assays for AQP4 antibodies and characterization of anti-AQP4 antibodies in patients with neuromyelitis optica. 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. 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.

Profiling of the CD4 receptor complex proteins

Intermolecular complexes produced by the CD4 molecule were studied. To preserve the integrity of weak protein-protein interactions of the CD4 antigen, cells were lysed in a mild nonionic detergent Brij97. Protein constituents of the complex were identified by our previously proposed fluorescence immunoprecipitation assay with subsequent mass spectrometry. In total, 26 proteins associated with CD4 were identified on CEM cells. The CD4 complex included the following major components: tyrosine phosphatase CD45, transferrin receptor CD71, tyrosine kinase Lck, and a lymphocyte phosphatase-associated phosphoprotein LPAP. The CD4 complex also contained some components of cytoskeleton and heat shock proteins. The association between CD4, CD71, and CD45 molecules was confirmed by immunoblotting. The CD4 complexes were not detected on the U937 myeloid cells lacking Lck and LPAP. We attempted to quantitatively characterize the CD4 complex composition.