Immunoassays homogeneous

Homogeneous immunoassay is conducted entirely in solutions. In this format, separation of the labeled antibody (Ab) and the antibody-antigen (Ab-Ag) complex is required, and this is usually achieved using CE separations. For instance, separation of Cy5-BSA from unreacted Cy5 and the complex (formed from Cy5-BSA and anti-BSA) was achieved in a flow-through sampling chip [567]. 

In addition, affinity CE separation was carried out to resolve monoclonal anti-BSA and BSA in dilute mouse ascites fluid. Then the affinity constant of this antigen-antibody interaction was measured [1004]. 

Separation of antigen and antibody-antigen complex was also achieved by dialysis with a polymeric microfluidic chip containing a PVDF dialysis membrane [821], 

In competitive homogeneous immunoassay, separation and quantitation of free and bound labeled antigen (cortisol) were carried out in a fused silica chip. Since the antibody-antigen complex was not detected, an internal standard (fluorescein) was added to aid quantitation. In addition, since most of the total cortisol was bound in the serum, a releasing agent, 8-anilino-l-naphthalenesulfonic acid (ANS), should be added [1006]. In other reports, competitive immunoassay for BSA was demonstrated after performing a CE separation on-chip [105,1005]. 

Competitive immunoassay was also conducted for serum T4 (3,5,3, 5 -tet-raiodo-L-thyroxine) on a fused silica chip. Again a T4-releasing agent (TAPS) was used to release T4 from serum [152]. 

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The advantages of homogenous immunoassays are simple formats and rapid data output producing user-friendly and cost-effective products. Technical challenges to consider, however, are the necessity to remove or minimize background interference from the reagents and nonspecific binding reactions. 

Homogeneous immunoassays rely on a change in the intensity of the label signal that occurs when labeled antigen binds with antibody. When the label is an antibody, a reduction in the rate of enzyme catalysis forms the basis for the assay. This technique... 

Fig. 14. General reaction scheme for homogeneous immunoassay. (Reprinted with permission from W. R. Heineman and H. B. Halsall, Anal. Chem. 1985,57,1321A. Copyright 1985, American Chemical Society)...

The main advantage of a homogeneous immunoassay, compared to a heterogeneous immunoassay, is the absence of a separation step. This translates into a simpler procedure and easier automation. However, homogeneous assays are typically less sensitive and more susceptible to sample interferences which are removed in a separation step. 

Fig. 10. Comparison of a typical homogeneous immunoassay (left) with a heterogeneous format (right). Depending on choice of reagents, there are many possible variations on these basic schemes...

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. 

Yamakawa Y, Ueda H, Kitayama A et al (2002) Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase. J Biosci Bioeng 93 537-542... 

Figure 14.3. Distinction between homogeneous and heterogeneous immunoassay formats. Haptenic analytes are indicated as triangles, and conjugated fluorescent probes are indicated by the letter F. In this hypothetical depiction, the homogeneous immunoassay is quantitated in the original reaction mixture. The heterogeneous immunoassay requires removal of unreacted tracer, further addition of reagents such as an enzyme to release a fluorescent molecule F, followed by quantitation.

Since FPIAs are conducted as homogeneous immunoassays, they are susceptible to effects from endogenous fluorophores and from intersample variations. Such problems and others due to the sample matrix are largely avoided by sample dilutions of several hundredfold. Low-affinity, nonspecific binding of tracers to sample proteins, when present in sufficiently high concentrations, can result in a falsely elevated polarization signal. Interference from sample proteins can be eliminated when warranted, by proteolytic hydrolysis with pepsin.(46)... 

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. 

A disposable, patterned, planar waveguide with a number of individual wells has been reported for a one-step homogeneous immunoassay of IgG.<133) The device is fabricated by an ion-exchange process, etching, and covalent reagent immobilization. The sample fills the waveguide by capillary action. The sample well, as well as fluorescent and nonfluorescent control wells are excited by an evanescent field, and individually scanned. The IgG detection limit is in the 10range. 

F. V. Bright and L. B. McGown, Homogeneous immunoassay of phenobarbital by phase-resolved fluorescence spectroseopy, Talanta 32, 15-18 (1985). 

Bromberg, A. and R. A. Mathies. Homogeneous immunoassay for detection of TNT and its analogues on a microfabricated capillary electrophoresis chip. Anal. Chem. 75, 1188-1195 (2003). 

Some electrochemically active substances that can generate photons on an electrode surface are suitable labels for homogeneous immunoassays. A labelled antigen exhibits an electrochemical reactivity and produces luminescence, but when it is immunochemically complexed, the labelled antigen loses its electrochemiluminescent properties. One optical immunosensor for homogeneous immunoassays was assembled by spattering platinum on the end surface of an optical fibre. Spattered platinum maintains optical transparency and functions as an electrode. An optical electrode efficiently... 

Figure 19-7 Scatchard plot for binding of antigen (X) to antibody (P). The antibody binds the explosive trinitrotoluene (TNT). The antigen is a fluorescent analog of TNT. From the slope, the binding constant for the reaction P + X PX is K 4.0 > 109M 1. [Derived from Figure 4 of A. Bromberg and R. A. Mathies, "Homogeneous Immunoassay for Defection of TNT on a Capillary Electrophoresis Chip"Anal. Chem. 2003, 75, 1188]...

Of particular interest is the development of homogeneous immunoassays based on cryptate 87 as label. They make use of a second, long range energy transfer... 

Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (L. M. Smith et al., 19 8 5), as a label in homogeneous immunoassay systems (Nithipatikom and McGown, 1987), to investigate specific interactions of proteins with cells surfaces (Hochman et al., 1988), and as an important fluorescent tag of antibodies in immunohistochemical staining techniques (Davidson and Hilchenbach, 1990). 

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