ABSTRACT
BACKGROUND: Antibodies targeting the NR1 subunit of the N-methyl-d-aspartate-receptor (NMDAR) are considered diagnostic for a novel form of autoimmune encephalitis. We report the validation of a qualitative indirect immunofluorescence antibody (IFA) test for the detection of anti-NMDAR IgG and describe the attributes of antibody-positive patients. METHODS: The anti-NMDAR IgG assay (Euroimmun Diagnosika, Lübeck, Germany) was validated with serum and cerebrospinal fluid (CSF) specimens from 30 healthy and 50 disease controls as well as 5 anti-NMDAR IgG-positive individuals. Consecutive specimens (n=1671) for anti-NMDAR IgG antibodies were evaluated and positive specimens titrated to end-point [starting dilutions: CSF; 1:1 and serum; 1:10]. In a subset of antibody-positive patients, we sought clinical information for correlation with diagnostic and treatment outcomes. RESULTS: The assay demonstrated excellent performance characteristics in all groups evaluated. Of the 1671 specimens tested, 1389 were unique cases with a positivity rate of 9.0% (n=123). For the antibody-positive samples, the female to male ratio was 2:1 with a prevalence of 46% in the pediatric population (≤17 years). Antibody titers were titrated to end-point for 106/123 specimens [45 CSF, 41 sera, and 20 CSF and serum pairs] with more than 75% having titers greater than 1:10 (CSF) and 1:20 (serum). Overall, high levels of these antibodies showed correlation to disease severity with variable response to treatment in the subset of patients evaluated. CONCLUSION: Our data suggests a high prevalence for anti-NMDAR antibody encephalitis irrespective of age and gender in our unselected disease cohort with support for measuring antibody titers in the evaluation of these patients.
Subject(s)
Anti-N-Methyl-D-Aspartate Receptor Encephalitis/diagnosis , Autoantibodies/blood , Autoantibodies/cerebrospinal fluid , Fluorescent Antibody Technique, Indirect/standards , Immunoglobulin G/blood , Immunoglobulin G/cerebrospinal fluid , Adolescent , Adult , Aged , Aged, 80 and over , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/blood , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/cerebrospinal fluid , Anti-N-Methyl-D-Aspartate Receptor Encephalitis/pathology , Case-Control Studies , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Receptors, N-Methyl-D-Aspartate/immunology , Severity of Illness IndexABSTRACT
Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3'-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3'-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.
Subject(s)
Fluorescent Dyes/chemical synthesis , Fluorides/chemistry , Leukemia Virus, Murine/enzymology , Oligonucleotide Probes/chemical synthesis , RNA-Directed DNA Polymerase/metabolism , DNA Primers/metabolism , Fluorescent Dyes/chemistry , Molecular Structure , Oligonucleotide Probes/chemistryABSTRACT
The development of a fluoride cleavable linker 1 for reversibly labeling (oligo)nucleotides is described here. The linker allows different ways of chemical attachment of a reporter molecule, for example, click chemistry or amide formation. The versatile attachment modes of labels are demonstrated by derivatizations with pyrene and fluorescein. Besides the synthesis of the new linker, we also show the derivatization of iodobenzene as a model compound and a nucleoside to demonstrate the applicability. Further, cleavability studies in solution and on a solid-supported oligonucleotide are shown. The linker can be applied in the synthesis of reversible terminators, useful for new DNA sequencing technologies like cyclic reversibly terminating (CRT) sequencing.
Subject(s)
Fluorescent Dyes/chemistry , Fluorides/chemistry , Oligonucleotides/chemistry , Sequence Analysis, DNA/methods , Staining and Labeling , Chromatography, High Pressure Liquid , Fluorescein/chemistry , Fluorescent Dyes/chemical synthesis , Pyrenes/chemistryABSTRACT
Reversible terminators having a fluoride cleavable 3'-O-blocking group are presented. Each nucleotide triphosphate is labelled by a fluorescent dye cleavable by the same reagent. We present here their synthesis, cleavage experiments and polymerase incorporation tests for a possible use in a process of Sequencing-by-Synthesis.
