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.
The synthesis of novel Tb"' labels suitable for protein labelling are reported. Their luminescence properties as antibody conjugates were measured and compared to the results of corresponding Tb"' chelates of the parent ligand structures. When the lowest triplet-state energy level of the parent donor ligand was over 23 000 cm-I, i. e., the energy gap between the 5D, level of Tb"' and the lowest triplet-state energy level of the ligand exceeded 2600cm-', the label derivative with a long decay time (~=1.35-2.93 ms) and a high luminescence yield (E . 0 = 3 770-4 560) was found to be suitable for bioaffinity assays.Introduction. -Long-lifetime emitting lanthanide chelates as sensitive labels or probes, and time-resolved fluorometry in detection have already been used in a number of applications in bioaffinity assays [l].In commercial systems, due to the many requirements of optimal lanthanide chelate labels, compromises have been made, e.g., the detection comprises two separate steps, as a direct measurement of the analyte without any enhancement step is impossible [2]. In this respect, these technologies are not suitable for all applications, such as in situ hybridization, immunohistochemistry, homogeneous assays, and fluorescence imaging. In addition to sensitive bioaffinity assays, new highly luminescent and stable lanthanide labels permit the development of multilabel, miniaturized assay devices with simplified protocols and thus make it possible to perform multiparametric assays on sub-microliter volumes of samples. A lot of research effort has been directed to the design and synthesis of optimal labels also suitable for the applications mentioned above. The chelating agents commonly used for the sensitization of Eu"' and Tb"' ion luminescence include P-diketones
CK-MM can be reliably quantified in blood spots. The development of this CK-MM assay on a commercial immunoassay analyzer would enable standardized and high-throughput newborn blood spot screening of DMD.
The availability of an intrinsically fluorescent, inert, and stable Eu chelate label made it feasible to design one-step all-in-one immunoassays with time-resolved fluorometry for detection. Both competitive and noncompetitive immunoassays are performed in microtitration wells containing all assay-specific components in a stable dry form. Only the sample and one assay buffer common for all analytes need to be added. Model assays for human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), and progesterone all reached equilibrium in 15 min or less without compromising the performance characteristics of the measurements, all of which perform at least equivalent to state-of-the-art assays. The detection limits for hCG, AFP, and progesterone were 0.3 IU/L, 0.1 microgram/L, and 0.5 nmol/L, respectively. The assay ranges for hCG and AFP were linear to 5000 IU/L and 1200 micrograms/L, respectively. The immunoassay format can be readily implemented in a fully automated random-access immunoassay system with optimal performance characteristics and no handling of analyte-specific assay components.
Background: Type 1 diabetes mellitus results from destruction of the pancreatic insulin-producing beta cells by a chronic autoimmune process. Methods are needed for the detection of circulating autoantibodies to glutamic acid decarboxylase (GAD65), a major marker of this process. Methods: Streptavidin-coated microtiter plates were incubated with biotinylated GAD65, and after incubation with serum samples from patients with type 1 diabetes mellitus and control individuals, europium-labeled GAD65 was added. After washing steps, the delayed fluorescence was measured in duplicate in a fluorometer. Samples collected from 100 patients with newly diagnosed type 1 diabetes mellitus and 100 healthy controls were measured by the new assay and by a radiobinding assay. Results: The detection limit of the new assay was 1.49 WHO units/mL, the calibration curve was linear to 4 140 WHO units/mL, and no hook effect was observed up to 41 400 WHO units/mL. The intraassay CV was 2.1-6.3% over the calibration range. For patient serum samples, the intraassay, interassay, and total CVs were 5.4 -7.0%, 9.8 -13%, and 12-14%, respectively. Compared with conventional radioimmunologic methods, the analytical range was broader and the analysis time required to
We have performed a one-step all-in-one dual-label dry reagent time-resolved fluorometric assay measuring free and total prostate-specific antigen (PSA). All assay-specific components were predried in an ordinary microtitration well, including the Eu and Tb chelate-labeled monoclonal antibodies for the recognition of free and total PSA, respectively. Only the sample and assay buffer needed to be added. Optimally, equilibrium was reached in <15 min. New intrinsically fluorescent Eu and Tb chelates were used, which made it possible to read the fluorescence directly from the solid phase. The developed dual-label assay with the simple protocol is ideal for easy automation, as the reaction wells can be of alternative design. The performance characteristics are equivalent to those of the present state-of-the-art single-label assays. The detection limits for free and total PSA were 0.03 and 0.02 microg/L, respectively, and the assays were linear over the range tested (up to 300 microg/L).
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