Homogenous immunoassay antigen

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... 

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]...

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 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]. 

In the on-chip theophylline homogeneous immunoassay, the antibody-antigen complex can be separated from the antigen. Why can such a separation be successful, but not in the case of the cortisol assay [330,1006] (2 marks)... 

Homogeneous immunoassay using FRET is an attractive assay format, since the antigen-antibody binding reaction is much accelerated compared to that of solid phase sandwich im-... 

A. Homogeneous immunoassay based on marker-labeled antigen Ag Ag Ab... 

The term homogeneous immunoassay may be defined as an immunoassay in which the extent of the antigen-antibody reaction can be determined without physical separation of the free and bound forms. This term is usually used for immunoassays such as enzyme and fluorescence immunoassays in which labeled substances (markers) are used. It does not include immunoassay systems such as nephelometry in which no labeled substances are used. Homogeneous immunoassays are widely used as routine tests because the procedures involved are simple. The principle of homogeneous immunoassay is based on changes in signals of the indicators by the antigen-antibody reaction (N5, U2). 

Homogeneous Immunoassay Based on Marker-Labeled Antigen... 

Several homogeneous immunoassays have been developed in which all of the reactions of markers occur when the reactants are closely linked by the antigen-antibody reaction. 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

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... 

Homogeneous immunoassay (HOIA) does not require physical separation of the free and antibody-bound antigen because the measured physical signal derived form the antibody-boimd, labeled material may be significantly different from that of the unbound entity. There may be an enhancement or an inhibition of enzyme activity upon binding of the antibody to the antigen. HOIA are simple to perform and automation can be carried out easily. Elimination of the separation step avoids a major source of imprecision in the assay. However selectivity may be compromised since interfering substances are not eliminated in the separation step. 

Competitive homogeneous immunoassays using enzyme-labeled hapten or antigen... 

The fluorescence of a tagged antigen is often quenched upon immunochemical reaction, and the decrease in fluorescence can be measured without going through a physical separation. This serves as the basis for homogeneous immunoassays, in contrast to heterogeneous immunoassays, which require a separation. 

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