Time-Resolved Immunofluorometric Dual-Label Assay for Simultaneous Detection of Autoantibodies to GAD65 and IA-2 in Children with Type 1 Diabetes

Abstract

Background: Autoantibodies to glutamic acid decarboxylase (GADAs), specifically the 65-kDa isoform GAD65, and autoantibodies to the protein tyrosine phosphataselike molecule IA-2 (IA-2As) predict development of diabetes. Our aim was to develop a time-resolved immunofluorometric (TR-IFMA) dual-label assay method for the simultaneous detection of these autoantibodies and to evaluate the diagnostic sensitivity of the method compared with single-label TR-IFMA and fluid-phase radiobinding assay (RBA) in screening children with type 1 diabetes. Methods: We incubated combined biotinylated GAD65 and IA-2 proteins, glutathione S-transferase (GST)-IA-2, europium-labeled GAD65, terbium-labeled anti-GST antibody, and serum sample or calibrator and transferred aliquots to a streptavidin-coated 96-well microtiter plate for a second incubation. After washing, we added Delfia Enhancement solution to each well and measured the fluorescence of Eu. We developed the Tb fluorescence signal by use of the Delfia Enhancer solution and measured it. We analyzed serum samples from

Fluorescence Lifetime Measurements and Biological Imaging

“…A variety of homogeneous and heterogeneous time-gated assays facilitated by the commercial availability of lanthanides complexes suitable for bioconjugation and appropriate instrumentation such as time-gated plate readers have been described451,452 and reviewed 447. Stable lifetimes of the lanthanides have been applied for constructing FRET probes with the lanthanides as donors.…”

Simultaneous detection of Human Immunodeficiency Virus 1 and Hepatitis B virus infections using a dual-label time-resolved fluorometric assay

“…TRF technology using lanthanide chelates with high fluorescence intensity coupled to very low background signals, made possible by the temporal separation of long-lived emission signals, has the potential for achieving very high levels of sensitivity [2-5]. Consequently, lanthanide chelate-based TRF assays are available commercially for the detection of hormones, tumor markers, celiac disease markers and for neonatal screening.…”

“…The TRF assay described here differs from those reported earlier. It utilizes labels that provide optimal fluorescence without the need for a separate dissociation-based fluorescence enhancement of the DELFIA assays [2,4,5] or a non-dissociative signal development step of the LANFIA procedure [3] and permits measurement of the fluorescence directly from the dry surface of the microtiter wells.…”

“…Therefore, TRF cannot be employed to measure their signals and their detection is limited by autofluorecence. Further, the Eu 3+ and Tb 3+ tracers used in this study are inherently fluorescent, obviating the need for additional signal development steps as in the DELFIA and LANFIA methods [2-5], and can be measured directly from the dry surface of the microtiter wells. The simultaneous detection of two analytes combined with a relatively simpler assay format eliminating the extra signal development step, will contribute to both cost and time saving.…”

Ανάπτυξη Μεθόδου Ταυτόχρονου Ανοσοχημικού Προσδιορισμού Καρδιακών Δεικτών Με Τη Χρήση Τριχοειδούς Οπτικού Ανοσοαισθητήρα

“…Ιχνηθέτες όπως ραδιοϊσότοπα [12,[15][16][17][18][19], ένζυμα [20][21][22][23], φθορίζουσες ουσίες [24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] αλλά και ηλεκτρενεργά μόρια [39][40][41] έχουν υιοθετηθεί για την ανάπτυξη ανοσοπροσδιορισμών πολλαπλών αναλυτών. Εφόσον οι διάφοροι ιχνηθέτες έχουν διαφορετικές βέλτιστες συνθήκες λειτουργίας, η ταυτόχρονη χρήση τους απαιτεί συμβιβασμούς που αποβαίνουν σε βάρος της ευαισθησίας των αναπτυσσόμενων ανοσοπροσδιορισμών [14,42,43].…”