Immunoassay separation-free homogeneous

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. ... 

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]. 

N5. Ngo, T. T., and Lenhoff, H. M., Recent advances in homogeneous and separation-free enzyme immunoassays. Appl. Biochem. Biotechnol. 6, 53-64 (1981). 

Accommodation of a homogeneous separation-free immunoassay on a dry matrix. This is possible for SLFIA and for ARIS. Other possibilities are labeling with gold particles or liposomes. The art lies in contact-free accommodation of all the reagents on the test strip, in such a way that no immune reaction takes place during storage. 

Like in RILAs, an advantage of fluorescence detection is the possibility of developing homogeneous FILAs using direct or indirect (competitive or displacement) approaches. The fluorescence polarization immunoassay (PFIA) and their homolog fluorescence polarization immuno-like assay (PFILA) are two of the most widely used procedures in homogeneous fluoroassays. Both are based on the principle that fluorescence polarization gives a direct measure of the bound/free ratio of the labeled analyte (tracer) without the need for their separation [23, 28]. 

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). 

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 immunoassays rely on a change in the intensity of a label signal that occurs when labeled antigen binds with antibody. When the label is an enzyme, a reduction in the rate of enzyme catalysis forms the basis for the assay. This technique is commonly known as an EMIT (enzyme multiplied immunoassay technique) assay. This enables free, labeled antigen to be distinguished from bound antigen with no separation step necessary. 

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. 

Homogeneous immunoassay Type of immunoassay that does not require separation of bound and free fractions... 

For a competitive immunoassay, a fixed quantity of tracer is incubated in the presence of a fixed (lesser) quantity of antibody and variable quantities of the analyte. After incubation, the free and bound fractions of the tracer are separated. Quantification of one or other of the fractions allows calculation of the amount of analyte present. This technique is partictdarly suited to hapten assay. This assay method, requiring a separation step, is known as a heterogeneous assay. In a few particular cases, the free and bound fractions of the tracer give different signals, eliminating the need for the separation step. This is known as a homogeneous-phase assay. 

In all cases development of a new non-isotopic immunoassay proceeds via the following steps (1) acquisition of specific polyclonal or monoclonal antibodies for the substance to be assayed, (2) preparation of a tracer, which may be either the analyte or the antibody modified by addition of label that can be detected at low levels, (3) selection of a separation method for the free and bound fractions of the tracer, except in the particular case of a homogeneous assay (discussed below),... 

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