Enzyme-linked immunosorbent assays examples

The traditional microbiological methods are very time consuming and sometimes limited concerning their interpretation. For that reason fast analysis methods as well as automated methods have been developed the latter are often used in specialised microbiological laboratories. During the last few years more and more modern biotechnological methods have been implemented into quality control, for example the enzyme-linked immunosorbent assay or more recently the polymerase chain reaction, which allows the detection of very specific microorganisms. 

The products of gene expression (mRNA and proteins) can be measured by techniques such as the following. Northern blots are very similar to Southern blots except that the original sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radioactive probe. Microarrays are used to determine the differing patterns of gene expression in two different types of cells—for example, normal and cancer cells. Enzyme-linked immunosorbent assays (ELISAs) and western blots (immunoblots) are used to detect specific proteins. 

An example of an immunoassay is the enzyme-linked immunosorbent assay (ELISA), which uses 96-well plates with antibodies bound on the sides of the wells. The characteristic of interest is the binding constant for the immunoreaction, which is defined as the association constant between antibody and antigen. It is important to generate a good antibody with strong binding. 

An application example of Immuchip for the analysis of Interleukin IB by enzyme-linked immunosorbent assay is described in Procedure 50 (see in CD accompanying this book). 

As an example of microchip-based electrochemical immunoassays, we describe here the protocol established for the analysis of interleukin IB by enzyme linked immunosorbent assay (ELISA) with amperometric detection at the sub-pM level in DiagnoSwiss microfluidic chip called Immuchip . 

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa has proven its worth as an enzyme label for many years. It is stable, has a moderate size (140 kDa), a high turnover number, and can be assayed using a variety of different substrates. Its activity is easily detected by eye in, for example, immunoblots, and it can be quantified by changes in absorbance, fluorescence, or luminescence for use in enzyme-linked immunosorbent assays. 

There is an increasing trend to replace radioactive detection methods by approaches which exploit spectrophotometry, particularly UV/VIS absorption, fluorescence and chemiluminescence (Levy and Herrington 1995 Howard 1993). So, for example, radioimmunoassays (RIA) have been effectively replaced by enzyme-linked immunosorbent assays (ELISA) (Sect. 6.2.4) in which the enzyme-catalysed liberation of a... 

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