Enzyme-linked immunosorbant assay 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.)... 

Fig. 15. Schematic representation of the open sandwich enzyme-linked immunosorbent assay of a hapten.

Figure 8.3. Schematic representation of a non-competitive enzyme-linked immunosorbent assay using the sandwich technique.

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. 

A standard double-sandwich enzyme-linked immunosorbent assay (ELISA) or Western blot analysis is used. As the concentration of factor is very low in normal plasma (approximately 0.1 mg/1 for factor VIII), it is necessary to subject plasma samples to cryoprecipitation in order to concentrate the sample, prior to Western blot analysis. The cryoprecipitation protocol described by Bi et al. is as follows (Bi et al., 1996 Sarkar et al., 2000 Mah et al., 2003). Plasma samples are collected as described above for the Coatest assay. Plasma is then frozen at -80 °C overnight. Frozen samples are then subject to centrifugation at 7000 x g for 20 min at 4°C. The precipitate is washed with... 

Hefle, S.L., Bush, R.K., Yunginger, J.W., and Chu, F.S. 1994. A sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of selected peanut proteins in foods. J Food Protect 57 419 -23. 

S. Arakawa, H. Ishihara, O. Nishio, and S. Isomura, A sandwich enzyme-linked immunosorbent assay for kappa-carrageenan determination,/. Sci. Food Agric., 57 (1991) 135-140. 

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. 

Jakubik, J., Wess, J., 1999. Use of a sandwich enzyme-linked immunosorbent assay strategy to study mechanism of G protein-coupled receptor assembly. J. Biol. Chem. 274, 1349-1358. 

---