Multilabel assays

The principle of time-resolved fluorometry has been described in detail elsewhere in this book (refer to the chapter by Biinzli [50]) this chapter oidy concerns clinical applications of time-resolved fluorometric assay (TRFIA) and comparison of this method with other assay techniques. The sensitivity of TRFIA is based on the use of chelates of rare earth metals (lanthanides), e.g., emopium (Eu ), terbium (Tb" ), samarium (Sm" ), and dysprosium (Dy ). Of these, Eu usually provides the highest sensitivity and has therefore been most widely used [6]. The emissimi spectra and decay times of the lanthanides differ from each other, and it has therefore been possible to develop multilabel assays for simultaneous detectiOTi of up to four different antigens [7]. Simultaneous determination of several antigens is especially... 

Lanthanides are particularly well-suited for multilabel assays due to their narrow line type emissions and varying decay times. Combination of different lanthanide donors where each of them may apply a set of different acceptors and create ET signal at different wavelength and decay times is also applied in bioanalytical assays, particularly in DNA hybridization assays in measuring single nucleotide polymorphic (SNP) alleles related to disease risk factors. 

Confocal microscopy Time-resolved microscopy Double photon scanning microscopy Multilabel assay... 

Quantify the luciferase activity using a commercial kit Luciferase assay system (PROMEGA) Load 10 pL of the lysed cells into a 96-well plate and place it into the lecture plate reader. Load the luciferase substrate to the injector. 50 pL of the luciferase substrate is injected via an injector, and the absorbance is read immediately at 563 nm on a Wallac Victor2 1420 Multilabel Counter (Perkin Elmer). 

Cell density was assessed using absorbance at a 600 nm wavelength (Abs oo nm)- For the bioreactor experiments, samples were diluted to the linear range of the assay, transferred to a 1-ml cuvette, and the absorbance was measured using a Spectronic 20 Genesys spectrophotometer. In the multiwell plate experiments, the plate cover was removed, and absorbance was measured using a Perkin Elmer Victor V 1420 multilabel counter. A second-order polynomial was fit (I =0.97) to describe the relationship between absorbance and dry mass of cells per liter (Eq. 1), and all data are reported as cell density (g/1). 

Lanthanide labelled antibodies or antigens, can be applied to most assays based on solid-phase separation. Reagents immobilized to microtitration plates allow an easy and efficient separation of the unbound fraction by using a plate washer. The bound fraction of the labelled gagent is quantified with a specially designed time-resolved fluorometer (DELFIA Research Fluorometer, Victor Multilabel Counter, Wallac Oy, Turku, Finland). 

Lanthanide-labelled immunoreagents have been applied in different types of single and multilabel immunoassays e.g. non-competitive, competitive assays (7,10,16,18) and in non-separation or homogeneous formats (20,21,22,23). 

Zhou M, Roovers J, Robertson GP, Grover CP (2003) Multilabeling biomolecules at a single site. 1. Synthesis and characterization of a dendritic label for electrochemiluminescence assays. Anal Chem 75(23) 6708-6717... 

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