Homogenous immunoassay methods, 68-60

Direct Methods. Selective precipitation and homogeneous immunoassay methods have been used to measure LDL cholesterol directly. ... 

Abstract A significant number of immunochemical methods have been described for the determination of the most important emerging pollutants. The present chapter is a compilation of the information available today regarding immunochemical determination of industrial residues with a high potential risk of causing negative effects in the environment, wildlife, and public health. Homogeneous immunoassays, ELISAs, FIIAs, immunosensors, and selective immunoaffinity sample treatment methods have been reported for the analysis of an important number of these substances. The bases of these methods are briefly presented. 

Homogeneous TR-FIAs have been reported in which proprietary lanthanide chelates are used. In a homogeneous immunoassay for T4, a fluorescent europium chelate coupled to thyroxine is quenched by antibody binding. 90 A similar approach is used for estrone-3-glucuronide. 91 TRFIAs based on homogeneous methods have not yet become widely used. 

Other Immunoassay Methods. Other immunoassay methods can be used to quantitate the hapten these include homogeneous enzyme... 

This method was developed by Collaborative Research Inc. (F3). The technique, based on the use of immunoreactive liposomes, may be classified as another type of homogeneous immunoassay. The liposomes are microscopic vesicles (200-1000 nm in diameter) consisting of a relatively impermeable lipid bilayer that delineates and separates an internal aqueous compartment from the external aqueous medium. The principle is as follows (Fig. 3 and Table 6). An enzyme, alkaline phosphatase, is encapsulated in the liposomes [E] and sequestered from the substrate, p-nitrophenyl phos-... 

Fan and Zhang determined acetylcholine and choline in rat brain tissue by a fluorescence immunoassay method, making use of immobilized enzymes and chemiluminescence detection [50]. Tissue was homogenized with a 10 fold volume of 0.6 M HC104, the homogenates were kept on ice for 30 min, and then centrifuged at 2000 G for 20 min. The pellets were... 

In the homogeneous immunoassays there is no separation step because they are based on a changing signal by formation of the immune complex. The first assay of this type was the EMIT method (enzyme-modulated immunoassay technology) described by Engvall et al. (1970). The main disadvantages of homogeneous assays are low sensitivity and a more pronounced susceptibility to interferences. 

Heterogeneous or homogeneous immunoassays Certain assays require an additional method to remove unbound label (also termed free label) leaving bound label, and for a given assay these labelled components are either antibody or antigen, but not both. These assays are called heterogeneous assays, and common methods of separation include adsorption, precipitation, or commonly the use of a solid phase. In contrast, homogeneous assays do not require the removal of... 

Separation-free, homogeneous immunoassay protocols offer several advantages in comparison to heterogeneous methods. Because no separation is involved, the number of procedural steps is decreased, which decreases the time required per assay. Additionally, because the physical transfer step is avoided, potential sample loss related to this step is eliminated. Drugs with low molecular weights (amphetamines, digoxin) are commonly measured by separation-free homogeneous immunoassay protocols. ... 

Recent developments show that dry chemistry has also been advancing in the sector of immunological detection methods. The homogeneous immunoassay technique facilitated access to the immunological detection method. The substrate-bound fluoro-immunoassay (SLFIA) serves as an example for the determination of theophylline concentration. However, the radial partition immunoassay paved the way for determining the concentration of numerous drugs and hormones. 

The classification of immunoassay methods is based on (a) whether they are homogeneous, with no separation step needed prior to measurement, or heterogeneous, where a separation step is required (b) which species, antibody or antigen, is labeled and (c) the type of label employed. 

Both competitive and noncompetitive methods have been incorporated into homogeneous enzyme-labeled immunoassay kits that ultimately relate enzyme activity to analyte concentration.22 The competitive-binding assays are called enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescein immunoassay (SLFIA), apoenzyme reactivation immunoassay (ARIS), and cloned enzyme donor immunoassay (CEDIA), while a noncompetitive method is called enzyme inhibitory homogeneous immunoassay (EIHIA). 

Techniques for molecular analysis often require multiple steps, including sample preparation, amplification, and analysis. As a result, successful automation is critical for their routine adoption in the clinical laboratory. However, to corruptly paraphrase Henry David Thoreau in Civil Disobedience— I heartily adhere to the motto The best automation is not needing to automate. The simplest techniques are often the best. As for immunoassays, methods that require separation and washing steps are being replaced with nonseparation methods that are rapid and homogeneous. These and other simple methods hold great promise for the clinical laboratory. 

Antibodies are the key element in an immunoassay method. Typically, there are two types of antibodies monoclonal and polyclonal. Monoclonal antibodies are completely homogeneous with respect to antigen, while polyclonal... 

ECL is often confined to the surface of the electrode or its close vicinity. Reactive intermediates generated by the electrode processes are often very short lived, and the action distance from the surface toward the bulk solution is small. This feature can be exploited, for example, in developing methods for homogeneous immunoassay. 

In homogeneous immunoassays, the analyte is biochemically coupled to two specific antibodies labeled one with a LLB and the other by an organic acceptor. Emission from the organic acceptor is detected in time-resolved mode because the population of its excited state by intramolecular transfer from the LLB shifts its lifetime in the millisecond range. In this way, it is easy to discriminate between the luminescence emitted by uncoupled and coupled antibody molecules labeled with A similarly, since the luminescence of A is spectrally different from that of the LLB, interference from Ln luminescence emitted by the uncoupled antibody labeled with the Ln chelate is also discriminated. There is, therefore, no need to wash out unused reactants. A method using FRET for the... 

A pTAS must miniaturize the steps listed above (and any we may have omitted). There are two types of methods to do this. First, try to miniaturize the existing components used to accomplish these steps. Second, find new methods that accomplish the same result in a novel way appropriate to a pTAS. In the first family would be efforts to make microvalves and pumps, that move solutions around, mix them, and thereby miniaturize conventional chemistry. Homogeneous immunoassays involve efforts to find non-solution equivalents, such as controlled release of encapsulated reagents [14], or virtual separation using an evanescent wave optical signal [15], and are thus in the second category. 

DL Brandon, JW Corse, JJ Windle, LL Layton. Two homogeneous immunoassays for pyridoxamine. J Immunol Methods 78 87-94, 1985. 

---