Enzyme-linked immunosorbent assay sandwich method

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. 

Among the many immunological assay methods, the enzyme-linked immunosorbent assay methods (ELISA) are the most popular methods. ELISA can detect both antigen molecules and antibody molecules with only a slight modification of the procedure. The direct-binding and sandwich methods that are used for the... 

Another technique, the enzyme-linked immunosorbent assay (ELISA), combines the specificity of antibodies with the sensitivity of an enzyme assay. The ELISA can be performed in a variety of combinations that involve either a specific antibody or the total cellular protein immobilized on a solid support, such as the wells of a plastic microplate. In one version of the method, the sandwich ELISA, the primary antibody is bound to the wells. When a mixture of proteins is added, the protein of interest binds to the antibody, and other proteins are washed away. A second labeled antibody, specific to a different epitope on the protein, is added, and the amoruit of signal is proportional to the amoimt of the particular protein in the sample. The method can be modified to detect specific antibodies in a mixture by using their antigen as the immobilized bait. ELlSAs also have the advantage of being able to be performed in 96-weU plates so many samples can be analyzed in one experiment. 

There are two common forms of heterogeneous assays used to detect and quantify disease markers the direct-labeling method, in which the proteins in a sample are labeled with a detectable tag and isolated from a complex sample by means of a bed of immobilized antibodies, and the indirect-labeling method, in which two antibodies are used for each marker. Sandwich-type immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), require two antibodies an immobilized antibody used to capture a molecule of interest - a protein, a virus, or a small molecule (e.g., a hormone. 

Enzyme-linked immunosorbent assays (ELISAs) in kit form are most widely used giving relatively rapid and inexpensive methods for multispecies identification. A typical format for such an ELISA is to coat different strips of the normal 12 x 8-well plate with antisera formed against serum albumin of the various species of interest. An extract of the meat product is added to the antibody-coated wells, incubated to ensure antibody binding of the serum albumin, and, after washing, a second antibody coupled with enzyme is introduced. The sandwich is visualized by addition of a substrate to the enzyme. ELISAs have been developed, also, for meat species identification in cooked meat products. These ELISAs are quite specific and sensitive ( 1% of each species can be detected) but are qualitative, or at best, semiquanti-tative. 

The conjugates were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) format for the determination of different metal ions including lead, mercury, cadmium and gallium. The study showed that gallium ions could be conveniently detected with ICso value of lO M, a linear concentration range of 5 x 10" to 1 X 10" M and a correlation coefficient of 0.9980. The assay detection limit which is defined as three standard deviations above the zero standard, was recorded to be 5 X 10 M. Moreover, the method showed a remarkable selectivity for Gallium relative to several other metals investigated. The selectivity was modulated by three... 

For diagnostic detection of BChE adducts of common G- and V-type nerve agents, enhanced sample throughput was achieved by automated processes in 96-well plate format, extracting plasma by inunimomagnetic separation (Knaack et al., 2012). An alternative method, based on the principle of a sandwich enzyme-linked immunosorbent assay (ELISA), was presented by Wang et al. (2011) to determine OP adducts in complete BChE. 

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