Rare earth cryptates and homogeneous fluoroimmunoassays with human sera

Abstract

The rare earth cryptates are used as long-lived fluorescent labels. They are formed by inclusion of a europium ion in the intramolecular cavity of a macropolycyclic ligand containing bipyridine groups as light absorbers. The use of fluoride ions in the measuring medium allows a total shielding of the label fluorescence. Here, I have established the conditions under which the signal of these cryptates can be amplified through the use of nonradiative energy transfer, and applied these conditions to homogeneous immunoassays by using allophycocyanin as the acceptor. Because only a low proportion of the cryptate label is involved in the transfer amplification, and because allophycocyanin emission occurs between the europium emission lines, time-resolved measurement of europium and allophycocyanin emission allows real-time correction of the optical properties of the assay medium. These features have allowed the development of a rapid homogeneous immunoassay of prolactin that can detect as little as 0.3 microgram/L (3rd International Standard 84/500).

First trimester maternal serum alpha-fetoprotein is not raised in pregnancies with open spina bifida

“…Maternal serum AFP was measured on the identified affected cohort and the control group using the Kryptor analyser using the heterogeneous immunoassay TRACE technology. 20 The performance characteristics of this analyser have been published previously. 21 A previously established, gestational median curve was used to convert AFP concentration units into MoMs corrected for maternal weight.…”

Comparison of LUMIPULSE® G1200 With Kryptor and Modular E170 for the Measurement of Seven Tumor Markers

“…Kryptor R (Thermo Fisher Scientific B.R.A.H.M.S, Asnières, France) is an automated analyzer with a patented detection system, Time Resolved Amplified Cryptate Emission (TRACE). TRACE technology is based on a nonradiative energy transfer between a donor (cage-like structure with an europium ion (cryptate)) and an acceptor (XL665, an algual alophycocianin) (29), both coupled to a mAb specific to each assay. Modular R Elecsys E170 (Roche Diagnostics, Meylan, France) is an electrochemiluminescence-based immunoanalyzer.…”

Photon Upconversion in Homogeneous Fluorescence‐based Bioanalytical Assays

“…However, the nonseparation nature and the modulation of the fluorescence signal also render homogeneous assay technologies very prone to interference from biological sample material, and traditionally, this has lead to inadequate assay sensitivities and a limited range of applications. 66,67 Major problems when using homogeneous fluorescence techniques in serum, plasma, or whole blood have been autofluorescence, scattered excitation light, and strong absorption of both ultraviolet (UV) and visible light. The light absorption can be avoided by using fluorescent probes with both excitation and emission wavelengths in the near-infrared window (650-1100 nm), where biological materials are most transparent to light, but autofluorescence and scattered excitation light will still limit the assay sensitivity.…”

“…68,69 Efficient solution for the autofluorescence and scattered excitation light is provided by the time-resolved fluorometry 70,71 and especially the application of longlifetime fluorescent lanthanide labels as donors in timeresolved FRET (TR-FRET) assays. 66,72,73 The need for UV excitation, however, has limited the use of TR-FRET assays with strongly colored samples, such as whole blood, which efficiently absorbs both UV and visible light. 74 Moreover, the time-resolved fluorometer requiring high-power pulsed UV excitation is rather expensive and inevitably less compact than fluorometers based on light emitting diode or laser diode excitation.…”