Enzyme-linked-immunosorbent secondary antibody

An ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 5-28b). Proteins in a sample are adsorbed to an inert surface, usually a 96-well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins introduced in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody—an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibody-linked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. 

Sanchez, F.G., A.N. Diaz, A.F.G. Diaz, et al. 1999. Quantification of 2,4,5-trichlorophenoxyacetic acid by fluorescence enzyme-linked immunosorbent assay with secondary antibody. Anal. Chim. Acta 378 219-224. 

One form of heterogeneous immunoassay is called enzyme-linked immunosorbent immunoassay (ELISA). In one instance, electrochemical immunoassay was performed for anti-ferritin (antibody) in a PDMS/PMMA chip. First, DTSSP was self-assembled on the gold electrode deposited on the PMMA plate. Then horse spleen ferritin (antigen) was attached to the DTSSP layer. A 100-p.g/mL solution of anti-horse ferritin (rabbit serum) was added. Then a secondary anti-rabbit antibody (HRP-linked) was introduced. A substrate (4-CN) was finally added which was converted to a precipitate product. The precipitate caused a reduction... 

Figure 1. Dose-response antigen inhibition curves derived from ELISA (enzyme-linked immunosorbant assay), utilizing an antiserum raised against synthetic pigment-dispersing factor (PDF) of Romalea, Assay conditions antigen coat, 200 fmol Romalea PDF/well primary antibody dilution 1 140,000 secondary antibody (peroxidase-labelled antirabbit IgG), 1 1000 other details as described earlier (63). When no Romalea PDF is present in the assay, the obtained value is defined as 100%.

Competitive enzyme-linked immunosorbent assays (ELISA) Two types of ELISA have been used for the analysis of mycotoxins and both types are heterogenous competitive assays. One type, i.e. direct ELISA, involves the use of a mycotoxin-enzyme conjugate and the other system, i.e. indirect ELISA, involves the use of a protein-mycotoxin conjugate and a secondary antibody to which an enzyme has been conjugated. Although horseradish peroxidase (HRP) is most commonly used as the enzyme for conjugation, other enzymes such as alkaline phosphatase and beta-galactosidase, also have been used (5, 9, 13). 

IMSs have been employed as detectors for well-established methods for the determination of bacteria and enzyme-linked immunosorbent assays (ELISA). In ELISA methods, primary antibodies attach to epitopes on the bacterial wall. Each antibody has a structure containing numerous epitopes that can be associated with a secondary antibody. The secondary antibody also has a region with enzymatic activity. This enzymatic region is able to react with a substrate to cleave a product that either is colored and can be determined by a spectrophotometer or is volatile and can be determined by headspace analysis. In the method developed by Snyder et al. and quantitatively explored by Smith et al.," the final product exhibited a distinctive negative product ion peak, as observed with a mobility spectrometer. This was accomplished with the widely deployed military-grade CAM, which was used without modification and suggested that it could serve as a potential bacteria analyzer, provided reagent kits and an inlet adaptor were also distributed. 

At the same time that individual plants may be studied, various plant organs and tissues can be examined. Immunological methods and radioactive labeling techniques can be used to detect compounds at the cellular level. ELISA (enzyme-linked immunosorbent assay) serves to localize the site of accumulation of secondary metabolites by binding specific antibodies to a solid surface (Wilkinson et al., 1992). 

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