ELISA principle

Kessler, C., Holtke, H.-J., Seibl, R., Burg, J., and Muhlegger, K. (1990) Nonradioactive labeling and detection of nucleic acids I. A novel DNA labeling and detection system based on digoxigenimantidigoxigenin ELISA principle (digoxigenin system). Mol. Gen. Hoppe-Seyler 371, 917-927. 

If the major aim of the ELISA is to obtain quantification of substances present in extremely low concentrations there are a number of adaptations to the technique that can be used. Such techniques often use alkaline phosphatase enzyme systems, which can be used, for example, to lock into a circular redox cycle producing an end product such as red formazan which, is hugely amplified in comparison to standard amplification methods (4). Chemiluminescent amplified ELISA principles have also been shown to give very high... 

Fig. 5. An assay cycle using the flow-ELISA principle. The sample supplied with a fixed amount of enzyme-labeled antigen is introduced at the arrow sample into the continuous flow stream. Buffer is fed for a shon period before substrate for the marker enzyme is given as a pulse. The system is rinsed by a pulse of glycine buffer, pH 2.2. After reconditioning, the system is ready for a new assay. (After Mattiasson et al. (135).)...

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

New detection methods of phenolic compounds are being developed. Based on the principle of the enzyme-linked immunosorbent assay (ELISA), a method has been developed to quantify phenolic compounds such as isoflavones (Vergne and others 2007). 

There exists a wide variety in the setup of ELISA assays (direct binding or competition setups) and the enzymatic reaction utilized [148]. A similar principle to enhance sensitivity by enzymatic coupling is realized after gel electrophoretic separation of proteins. Here proteins are transferred to nitrocellulose ( western blot ) and detected by antibody-coupled enzymes. 

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...

Noncompetitive ELISA. The usual principle here is the sandwich technique, which requires the antigen to have at least two antibody binding sites (epitopes). Unlabelled antibody is first fixed to microtitre plates a food sample containing antigen (analyte) is then added and allowed to react with the fixed unlabelled antibody (Figure 8.3). Unadsorbed material is washed out and enzyme-labelled antibody then added which reacts with a second site on the bound antigen. Unadsorbed Ab-E is washed off and enzyme activity assayed activity is directly related to the concentration of antigen. 

It is necessary to give some background information to understand the principle underlying immunochemical tests such as ELISA (Enzyme Linked Immuno Sorbent Assay). Such information is not always available to chemists, since chemistry and immunology have traditionally been separate disciplines. 

A radioisotope can be used as a tracer instead of the enzyme used in immunochemical tests. This method, introduced in 1960 for the measurement of insulin in serum, is called radio-immunology. It is the transposition in immuno-chemistry of the same principle as that used to determine the sulphate ion (cf. 17.5). Radioimmunoassays are similar to ELISA assays in the way in which the analysis is... 

Figure 19-13 illustrates the principle of an enzyme-linked immunosorbent assay, abbreviated ELISA in biochemical literature. Antibody 1, which is specific for the analyte of interest (the antigen), is bound to a polymeric support. In steps 1 and 2, analyte is incubated with the polymer-bound antibody to form a complex. The fraction of antibody sites that bind analyte is proportional to the concentration of analyte in the unknown. The surface is then... 

Principles of microfluidic ELISAs with electrochemical detection... 

The principle methods used for the determination of triazine-type herbicides are gas chromatography (Table 4.6) and high-performance liquid chromatography (Table 4.7). Other methods that have been used include isota-choelectrophoresis [369], ELISA [370-375], spectrophotometry [376,377] and thin-layer chromatography [378] (Table 4.8). 

Vascular endothelial growth factor 2000 C Factor 5 (IPCR 0.2 pg/ml fluorescence-ELISA 1 pg/ml) Sims et al. [49], real-time IPCR (see Section 2.2.3) detection of IPCR-amplificate was carried out online during PCR according to the TaqMan-principle, AbiPrism7700 sequence detector (Applied Biosystems)... 

In summary, the simple and flexible Universal-IPCR, with or without nonfunctionalized primary antibody, is well suited for a proof of principle of the general feasibility of the IPCR for a given problem in clinical laboratory applications, as successfully shown in several examples. It allows for an easy access to ultrasensitive research studies—if the necessary time and expertise for some fine-tuning of assay conditions is available. Under these conditions, a typical 1000- to 10,000-fold improvement of ELISA sensitivity is accessible in the research laboratory (see Table 1). 

The working principle of the ELISA described here involves the use of LHRH peptide-coated wells to capture any anti-LHRH antibodies present in the sera of vaccinated mice. Captured antibodies are detected through the use of a horseradish peroxidase-conjugated antibody that is specific for mouse immunoglobulins and a substrate is then added to induce a color change. In this case, the substrate utilized is 2,2 -azino-bis 3-ethylbenzthiazoline-sulfonic acid (ABTS Sigma-Aldrich, USA), which is converted to a green soluble end product by horseradish peroxidase (see Note 7). The level of substrate-induced... 

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