Enzyme-linked-immunosorbent antibody sandwich

Valdivieso-Garcia, A. Riche, E. Abubakar, O. Waddell, T. E. Brooks, B. W. A double antibody sandwich enzyme-linked immunosorbent assay for the detection of Salmonella using biotinylated monoclonal antibodies. J. Food Prot. 2001, 64,1166-1171. 

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. 

In a direct immunoassay the immobilized antibody binds to the corresponding antigen. The competitive immunoassay relies upon the competition of the analyte with a labelled analyte for antibody binding. These formats are widely used for high throughput affinity arrays. A sandwich immunoassay is based on the trapping or capture of the analyte by another antibody. In ELISA (enzyme linked immunosorbent assays) the second antibody is conjugated with an enzyme. The bound enzyme labelled antibody is detected by its ability to break down its substrate to a colored product. 

Enzyme-linked immunosorbent assay (ELISA) is based on the specific reaction between an antibody and an antigen. One of the reagents in the reaction is labeled with an enzyme that generates a colorimetric product that can be measured with a spectrophotometric device. The color intensity correlates with the concentration of specific antibody and the respective antigen. The reaction can be formatted in various ways in a multiwell plate (microtiter plate) with the common formats being the sandwich assay, the competitive assay, and the direct assay. (See Figure 11.1.)... 

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... 

Test wells exhibiting growth for the presence of monoclonal antibodies when the hybridoma growth covers approx 25% of the base of the cell well. The assay system used should mimic the final test format required. Commonly, Triple Antibody Sandwich (TAS) (5) enzyme-linked immunosorbent assay (ELISA) is use for screening hybridomas for specific antibody. It is important when testing hybri-... 

Fig. 1. The dual-antibody sandwich enzyme-linked immunosorbent assay.

Dual-Antibody Sandwich Enzyme-Linked Immunosorbent Assay... 

Antibodies can be used to measures levels of molecules in solution using the dual-antibody sandwich enzyme-linked immunosorbent assay. Two antibodies are used that become bridged in the presence of the substance of interest, leading to the production of a colored product by a reporter enzyme. 

The dual-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA Fig. 1) is a sensitive and specific technique for quantifying molecules in solution. The technique is dependent upon the availability of two antibodies recognizing separate epitopes upon the antigen to be measured such that they are able to bind to the molecule simultaneously (1,2). The capture antibody specific to the substance to be measured is first coated onto a high-capacity... 

Competitive enzyme-linked immunosorbent assay provides an alternative to dual-antibody sandwich enzyme-linked immunosorbent assay and is widely used for the measurement of substances in biological liquids. 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

Enzyme-Linked Immunosorbent Assay Both competitive and sandwich ELlSAs are available. Although the competitive ELISA is faster because it uses only one incubation with an antibody, it is reported to be less sensitive and exhibits large imprecision. ELISA can be performed on a microplate reader, allowing semiautomation. In the sandwich assay, the primary antibody (antialbumin antiserum) is fixed on the plastic plate, which is then washed. Samples, controls, and calibrators are added, and the complexes detected and quantified by a second antibody conjugated to an enzyme label. 

On the basis of an enzyme thermistor, Mattiasson et al. (1977) developed one of the first immunosensors. Immobilized antibodies against albumin are placed in a column and set into an ET. After injection of an albumin-sample and a known amount of enzyme-labeled albumin, both are separated from the sample matrix by antibody-antigen-interaction. After injection of a substrate, the change in heat is a measure of analyte concentration. The less heat produced means that more albumin has been bound. An elution step regenerates the ELISA. Due to its thermal detection principle, the procedure is called TELISA (thermometric enzyme-linked immunosorbent assay). Figure 3 shows the principle of the TELISA procedure in its sandwich configuration. 

An enzyme-linked immunosorbent assay has been developed for human Mn-SOD using the monoclonal antibody PG-11 (K5). As described above, human Mn-SOD is a tetramer composed of identical subunits. Therefore, the same monoclonal antibody could be used in the sandwich immunoassay as both captor and detector in the case of monomeric enzymes, two different antibodies should be used. 

The most prominent of these assays are radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). In the case of RIA, the analyte sample is either extracted or used directly, and mixed with a constant amount of antibody and radiolabeled analyte [tracer mostly fi-emit-ters (e.g., H, " C) or y-emitters (e.g., I)]. After equilibration and separation of the free and bound antigen, radioactivity is measured for quantification of the analyte. In the case of sandwich-RIA, two antibody preparations are used the first serves as the binding partner of the analyte, whilst the second - which is radiolabeled - is directed either against the analyte or the first... 

Protein arrays with 10-500 antibodies printed onto an array use detection techniques, including fluorescence and multiple sandwich enzyme-linked immunosorbent assays, but the broad application of these assays is restricted by lack of suitable antibodies for laboratory animals and some potential cross-reactivities between antibodies with similar affinities. Calibration, reproducibility, and identification of proteins are common problems for all of these technologies. A number of databases are available to help investigators identify the numerous proteins found using these separation techniques, particularly for mass spectrometer data. 

The sandwich technique can be improved even further if the second antibody is attached to an enzyme, such as alkaline phosphatase. The enzyme rapidly converts an added colorless substrate into a colored product, or a nonfluorescent substrate into a highly fluorescent product. These changes can be quantitated if the degree of change in color or fluorescence is proportional to the amount of hormone present in the patient sample. Less than a nanogram (10 g) of a protein can be measured by such an enzyme-linked immunosorbent assay (ELISA). 

Kerschbaumer R J, Hirschl S, Kaufmann A, et al. (1997). Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay. Anal. Biochem. 249 219-227. 

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. 

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