Assessment of influenza A neuraminidase (subtype N1) potency by ELISA.

Antibodies that inhibit neuraminidase (NA) activity of influenza virus provide resistance against disease and have been associated with milder epidemics. Although studies have demonstrated a correlation between NA inhibition antibody titers and vaccine efficacy, neither the quantity nor form of NA is measured in seasonal and pandemic influenza vaccines. In this report, we describe development of enzyme-linked immunosorbent assays (ELISAs) that are suitable for quantitation of the native form of NA of subtype N1. The assays use mouse monoclonal antibodies (mAbs) 1H5 and CD6 to capture NAs of viruses, and a different mAb 4E9 to detect bound antigen. The 1H5-capture ELISA detects NAs of seasonal and pandemic H1N1 viruses as well as H5N1 viruses and has a limit of quantitation (LOQ) of 5.5ng/mL for seasonal H1N1A/Brisbane/59/2007 NA. The CD6-capture ELISA is specific for NA of the 2009 pandemic viruses with a LOQ of 67ng/mL for A/California/07/2009 NA. The ELISA signals in both assays are proportional to NA enzymatic activity and correlate with NA immunogenicity. The ELISAs we describe may expedite the development of NA-based influenza vaccines by providing a practical assay to measure NA potency.

Economic high-throughput-identification of influenza A subtypes from clinical specimens with a DNA-oligonucleotide microarray in an outbreak situation.

Influenza A surface proteins H (haemagglutinin) and N (neuraminidase) occur in sixteen and nine distinct genotypes, respectively. The need for a timely production of vaccinations in case of pandemics or seasonal epidemics requires rapid typing methods for the determination of these alleles. The aim of the present study was to develop and improve a rapid and economic assay for determining H and N subtypes of influenza A from patient samples. The assay is based on the hybridisation of labelled amplicons from H and N reverse transcriptase-PCRs using consensus primer pairs to subtype-specific probes on microtiterstripe-mounted DNA-microarrays. An algorithm for semi-automatic data interpretation of raw data and assignment to H and N subtypes was proposed. Altogether, 191 samples were genotyped. This included 134 patient and 44 reference samples as well as controls. Under routine conditions sensitivity and specificity proved to be comparable to conventional nested or real-time PCRs. At least 130 out of 147 array-positive samples were unambiguously assignable. This included all sixteen variants of H as well as all nine variants of N. Furthermore, eighty-two samples from the 2009/2010 "novel H1N1/swine flu" (SF)-outbreak were correctly identified.

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression.

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation-sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease.