Duchenne Muscular Dystrophy Newborn Screening: Evaluation of a New GSP® Neonatal Creatine Kinase-MM Kit in a US and Danish Population.

Duchenne muscular dystrophy (DMD/Duchenne) is a progressive X-linked disease and is the most common pediatric-onset form of muscular dystrophy, affecting approximately 1:5000 live male births. DNA testing for mutations in the dystrophin gene confirms the diagnosis of this disorder. This study involves assessment of screening newborns for DMD using an immunoassay for muscle-type (MM) creatine kinase (CK) isoform-the GSP Neonatal CK-MM kit. Comparisons were made with CK activity determination by fluorescence measurement. In addition, the study evaluated the effect of gestational age, age of infant at time of sampling and how stable the CK-MM was over time. This assay discriminates well between normal, unaffected and Duchenne affected populations and is suitable for Duchenne newborn screening.

Development and validation of a time-resolved fluorometric immunoassay for screening of antichlamydial activity using a genus-specific europium-conjugated antibody.

The lack of high-throughput assays has limited the screening of new antimicrobials against obligate intracellular bacteria, including chlamydia, which cause a variety of diseases. In this study, a novel technological approach was developed to detect intracellular bacteria using time-resolved fluorometric immunoassay (TR-FIA), and the method was validated for susceptibility testing of Chlamydia pneumoniae. In this cell-based, 96-well plate assay, chlamydial inclusions are labeled with europium-conjugated antibody and quantified as time-resolved fluorometric signals by means of a multilabel counter. To confirm the reliability of the TR-FIA, susceptibilities of C. pneumoniae reference strain Kajaani 7 to a set of antimicrobial agents were determined by the TR-FIA, conventional immunofluorescence staining, and real-time polymerase chain reaction. Minimum inhibitory concentrations measured using the different methods demonstrated good to excellent correlation. Data relating to reproducibility (day-to-day variation 9.0%), as well as to the signal-to-background, signal-to-noise, and Z' values (6.5, 6.9, and 0.4, respectively), showed the suitability of the TR-FIA for screening. By means of dual labeling with sulfornodamine B the cytotoxicity of test compounds could be detected simultaneously with the susceptibility testing. In summary, the TR-FIA is a convenient, reliable, and objective alternative for detecting chlamydia in vitro. By being considerably less labor intensive and offering significantly higher throughput, the TR-FIA is especially suitable for screening of new antichlamydial compounds.

Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney.

Mice lacking exon 3 of perlecan (Hspg2) gene were generated by gene targeting. Exon deletion does not alter the expression or the reading frame but causes loss of attachment sites for three heparan sulfate (HS) side chains. Hspg2(Delta 3 / Delta 3) mice are viable and fertile but have small eyes. Apoptosis and leakage of cellular material through the lens capsule are observed in neonatal lenses, and lenses degenerate within 3 weeks of birth. Electron microscopy revealed altered structure of the lens capsule through which cells had formed extensions. No kidney malfunction, such as protein uria, was detected in Hspg2(Delta 3 / Delta 3) mutant mice, nor were ultrastructural changes observed in the glomerular basement membranes (BMs). To achieve further depletion in the HS content of the BMs, Hspg2(Delta 3 / Delta 3) mice were bred with collagen XVIII null mice. Lens defects were more severe in the newborn Col18a1(-/-) x Hspg2(Delta 3 / Delta 3) mice and degeneration proceeded faster than in Hspg2(Delta 3 / Delta 3) mice. The results suggest that in the lens capsule, HS chains have a structural function and are essential in the insulation of the lens from its environment and in regulation of incoming signals.