ELISA sandwich

Fig. 5. Scheme of the last steps for the sandwich ELISA. Where Ah 1 represents the the surface-bound antibody, Ag, the antigen, and Ah 2, the... 

Shibahara, Y., Oka, M., Tominaga, K., li, T., Umeda, M., Uneo, N., Abe, A., Ohashi, E., Ushio, H., and Shiomi, K. (2007). Determination of crustacean allergen in food products by sandwich ELISA. Nippon Shokuhin Kagaku Kogaku Kaishi 54, 280-286. 

Sandwich ELISAs (Eigure 4) are the most common type of immunoassay used for the detection of proteins. A capture antibody is immobilized on the wells of a microplate. The solution containing the analyte is introduced and antibody-analyte... 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

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. 

Suter, M., and Butler, J.E. (1986) The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies. Immunol. Lett. 13, 313-316. 

FIGURE 11.25 Schematic illustration of sandwich ELISA mode. Adapted from http //pl.wikipedia. org/wiki/ELISA... 

The most common use of protein microarrays is in immunoassays. In particular, antibody-based immunoassays are the main stream of diagnostic assays due to their specificity. The assay usually runs in a multiplexed mode where the antibodies or other capture agents are immobilized and then exposed to a biological sample. There are four immunoassay formats direct binding, sandwich (ELISA), competitive, and displacement. Direct-binding and sandwich assays are the most common. There are some reports on the use of competitive assays and displacement assays, which are usually associated with high surface area/volume systems [72-76],... 

Fig. 7. Frequency distribution of plasma Lp(a) concentrations (a) and apo(a) alleles (b) in three populations. Plasma Lp(a) levels were measured in 381 subjects by the sandwich ELISA technique apo(a) allele size (number of K-4 repeats) was estimated using pulsed-field gel electrophoresis and genomic blotting. [With permission of Gaw et at. (G11).]...

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

Given their high degree of sensitivity, which is 2-5 times more than assays in which the antigen is directly bound to the solid phase, antibody sandwich ELISA is arguably the most useful of the immunosorbent... 

In general, four factors help to determine the sensitivity of the sandwich ELISA. These factors are (1) the number of molecules of the first antibody that are bound to tbe solid phase (2) the avidity of the first antibody for the antigen (3) the avidity of the second antibody for the antigen (4) the specific activity of the second antibody. By diluting or concentrating the antibody solution, the amount of capture antibody that is bound to the solid phase can be adjusted. In contrast, tbe avidity of the antibodies for the antigen can be altered only by substituting other antibodies. The specific activity of the second antibody is determined by the number and type of labeled moieties it contains. 

Immunological methods Methods such as gel-agglutination tests were introduced many years ago (according to Outcherlony) however they are not sufficiently sensitive. Reversed passive latex agglutination kits (SET-RPLA and TST-RPLA) are presently applied for enterotoxin detection and are more sensitive than gel diffusion (Wieneke, 1988). The ELISA technique is also very popular - toxins are detected via VIDAS STAPH ENTEROTOXIN SET and indirect double sandwich ELISA (Meyrand et al., 1998). 

Table 6.2 Assay Sensitivity for Antibody-Antigen Microarrays and Standard Sandwich ELISA... 

In reality, these forays represent miniaturization of the standard sandwich ELISA to attain higher throughput assays by multiplexing a limited number (<50) of analytes, e.g., cytokine panels. Even at these low densities, quantification problems arise in part due to a lack of robustness in the printing process and also in the selection and stability of monoclonal antibodies that must be highly specific and of high binding affinity to be useful for microarrays. 

The ELISA of cellular DNA fragmentation is a sandwich ELISA that measures apoptosis by quantitating the fragmentation and/or release of BrdU-labeled DNA. The commercial kit (Roche) can detect DNA fragments in the cell-free supernatants from cultured cells or cytoplasmic lysate of apoptotic cells prelabeled with BrdU... 

Very recently, a new noncompetitive assay principle called an open sandwich ELISA has been reported (S7) (Fig. 15). The assay mechanism could be regarded... 

Fig. 21. Principles of ELISA. A In a direct ELISA the unlabeled antigen is attached to the solid phase. Enzyme-conjugated antibody is then added, followed by the enzyme substrate solution and color is allowed to develop. B ELISA with unlabeled antibody attached to the solid support. A variable amount of antigen is then added. A secondary antibody labeled with enzyme, followed by substrate solution, is added to all wells. The amount of color produced is proportional to the amount of antigen present. C Sandwich ELISA assay with the antigen sandwiched between an immobilized, antigen-specific primary antibody and an antigen- or species-specific secondary antibody. An enzyme-labeled tertiary antibody increases the assay specificity and sensitivity...

Itoh, K., Suzuki, T. (2002). Antibody-guided selection using capture-sandwich ELISA. Methods Mol. Biol., 178, 195-199. 

Figure 11.13 Calibration curves of direct and sandwich ELISA.

Why does the sandwich ELISA method generally show a higher degree of sensitivity compared with the direct-binding ELISA method ... 

The extent of CD8+ T cell activation can alternatively be assessed by quantitative evaluation of cytokine (e.g., 7-interferon) production and release measured by standard sandwich ELISA. The assay to be used for evaluating the extent of T cell activation needs to be selected and... 

Fig. 2. Biochemical classification of the nia mutants, mutated domains and intragenic complementation. A collection of Nicotiana plumbaginifolia nia mutants (defective for NR apoenzyme) was tested for NR protein and activities (Cherel et al., 1990) and checked for in vivo and in vitro intragenic complementation (Pelsy Gonneau, 1991). NR protein amounts were measured by a sandwich ELISA test using as first antibody the anti-corn NR monoclonal antibody 96.9.25 (Ch6rel eta/., 1985) shown to be directed against the haem domain (M. Kavanagh, personal communication Meyer etal., 1991). The result is shown here as + or when a positive or negative ELISA test, respectively, was observed. Many class 4 mutants, most probably frameshift and deletion mutants, complement class 3 but not class 2 mutants.

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