Assay fluoroimmunoassays

Several other techniques for have evolved for biochemical assays. In chapter 2 of this book, Omann and Sklar report on a method of fluoroimmunoassay where the bound and unbound antigen are separated by the quenching of fluorescence that accompanies antibody binding. Then, in chapter 3, Holl and Webb show how they achieved a sensitive measurement of nucleic acids by the enhancement in fluorescence that accompanies the binding of fluorescent dyes to nucleic acids. Chandler et al, also used fluorescence enhancement to monitor calcium mobility in neutrophil cells. 

Fig.7. A demonstration of fluoroimmunoassay. Green circles (Tb-cored dendrimer complex) and red circles (conventional Eu complex) corresponds to different assays...

Enzyme-Linked Immunoadsorbent Assay (NRL) Multiplexed Sandwich Fluoroimmunoassay (LLNL) Capillary Electrophoresis (LLNL)... 

Nowadays, antibodies are utilized in numerous immunoanalytical methods. Those widely used in practice, such as radioimmunoassays, fluoroimmunoassays and enzyme-linked immunosorbent assays (ELISA), require labelled reagents. Millions of ELISA tests for diagnostics of various diseases are daily performed in clinical laboratories. The detection of analytes by two-antibody "sandwich" ELISA, is schematically outlined in Figure 3. 

Hemmila, I. (1985) Fluoroimmunoassays and immunofluorometric assays [Review]. Clin. Chem. 31, 359-370. 

D. J. Bucher, A. Mikhail, S. Popple, P. Graves, G. Meiklejohn, D. S. Hodes, K. Johansson, andP. E. Halonen, Rapid detection of Type A Influenza viruses with monoclonal antibodies to the M protein (Ml) by enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay, J. Clin. Microbiol. 29, 2484-2488 (1991). 

Enzymes can be linked to immunoassay reagents to amplify detection by the use of fluorogenic substrates. Enzyme-linked fluoroimmunoassays (ELFIAs) are very similar to photometric EIAs in format and workflow. EIAs are widely used, and many commercial ELFIA assays and systems are available/15 The most commonly used enzymes in ELFIAs are horseradish peroxidase, alkaline phosphatase, and fi-D-... 

Fluoroimmunoassays comprise a subclass of extrinsic labehng methods where various selective antigen (Ag)- antibody (Ab) immunoassay fluorescent labeling schemes yield a emission signal. One common scheme involves an enzyme-linked immunosorbent assay (ELISA) depicted in Figure 11.2 where the free Ab is tagged with a fluorophore. Numerous analytes can be detected via these types of selective lock-and-key methods. ... 

Fluoroimmunoassays. This assay requires the drug being assayed to be labelled with umbelliferyl-3 D-galactoside. The enzyme 3-galactosidase is added and the fluorescent products are released from the labelled antibiotic. The antibody in the bound fraction... 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Haupt K, Mayes AG, Mosbach K. Herbicide assay using an imprinted pol3mier-based system analogous to competitive fluoroimmunoassays. Anal Chem 1998 70 3936-3939. 

Fluorometry is a superior optical technique in terms of sensitivity and specificity. Merits of fluoroimmunoassays (FIAs) and fluoroimmuno-like assays (FILAs) include the stability and freedom from hazards of fluorescent labels compared to radioactive tracers, the moderate cost of analysis, the wide availability of the equipment needed, and the potential high sensitivity. In general, the sensitivity of fluorescence measurements is 10- to 1,000-fold higher than the absorption counterparts. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Fluoroimmunoassay These assays rely on the use of fluorescence-emitting labels. Typically, antibodies labelled with afluorophore are used, for example, fluorescein isothiocyanate. After washing, measured fluorescence relates to the amount of antigen. Another useful variant is called the homogeneous fluorescent polarization immunoassay that is commonly used to measure dmgs and analytes. 

All of the homogeneous fluoroimmunoassay systems suffer from the possibility of background interference from other drugs and metabolites. Enzyme labels may be inhibited by endogenous molecules, and there may be fluorescence enhancement or quenching from the same sources. This may be overcome by careful choice of assay conditions and the use of high dilutions of the biological sample. 

Immunoassays which employ drugs labelled with a substance which emits light when activated have been developed. The basic principles of these assays are similar to those of fluoroimmunoassays. The use of luminescent labels, however, has the advantage that light is not introduced into the system, so giving an increase in sensitivity. 

R F Schall and H J Tenoso, Alternatives to Radioimmunoassay Labels and Methods, C// Chem, 1981,27,1157-1164 D S Smith et al, A Review of Fluoroimmunoassay using Immunofluorometnc Assay, nn dm Biochem, 1981,28, 253-274... 

Luminescent RE + chelates have been successfully developed as labels and probes for highly sensitive and selective bioassays in the past two decades. Time-resolved Inmines-cence detection has been widely applied in fluoroimmunoassay, DNA hybridization assay, enzyme assay, cell activity assay and fluorescence imaging microscopy. ... 

Diamandis E, Bhayana V, Conway K, Reichstein E, Papanastasiou-Diamandis, A. A time-resolved fluoroimmunoassay of cortisol in serum A rapid non-isotopic assay. Chn Chem 1988 34 1216. 

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