Enzyme linked immunosorbent assays defined

In each of the assays of potency the amount of the immunoglobulin and the amount of a corresponding standard preparation that are required to neutralize the infectivity or other biological activity of a defined amount of virus or to neutralize a defined amount of a bacterial toxin are determined. The two determined amounts and the assigned unitage of the standard preparation are then used to calculate the potency of the immunoglobulin in International Units (lU). ELISA, enzyme-linked immunosorbent assay. 

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. 

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%.

Methods for Determining Biomarkers of Exposure and Effect. No biomarker has been identified that can be quantitatively related to dinitrophenol exposure (see Section 2.5.1) however, the presence and the amount of 2,4-DNP and 2-amino-4-nitrophenol, a metabolite in the urine, can be used as rough indicators of the intensity of exposure (see Section 2.5.1). The methods presently available for determining 2,4-DNP and 2-amino-4-nitrophenol (diazotization) in urine are outdated (Gisclard and Woodward 1946). An enzyme-linked immunosorbent assay (ELISA) is available for the quantitation of 2-amino-4-nitrophenol in water samples, but is not effective in urine (Li et al. 1991). It would be useful to develop an updated routine method for determining 2,4-DNP, 2-amino-4-nitrophenol, and 4-amino-2-nitrophenol in urine with well-defined detection limits, precision, and accuracy. 

Gregorius, K., Theisen, M. (2001). In situ deprotection a method for covalent immobilization of peptides with well-defined orientation for use in solid phase immunoassays such as enzyme-linked immunosorbent assay. Anal. Biochem., 299, 84-91. 

Even more popular than dot blots are microtiter plate assays, so-called ELlSAs (enzyme-linked immunosorbent assay), in which antibody or antigen is loaded into the depression of polyvinyl chloride or polystyrene plates (Kemeny 1994). The depressions are then further coated with antibody, antigen, and enzyme-conjugated antibody in a defined sequence. The antigen is detected via an enzymatic color reaction (Table 6.2). Many companies (Nunc, Flow, Costar, Falcon) offer a palette of products such as 8- or 12-channel pipettes, automatic washing devices, ELISA readers, and so on that make life easier for the friends of ELISA. 

FIGURE 16.5 Metabolites of testosterone in Japanese quail show the resolution of LC-AMS used in defining the optimal metabolite for developing a field enzyme-linked immunosorbent assay (ELISA) test. Fractional resolution is shown for 8 Gaussian-fitted peaks. Two unresolved peaks have dashed Gaussian fits that predict a 2.6-min separation for a resolution of 0.93. 

This is directly proportional to the quantity of analyte present. This technique advanced rapidly when monoclonal antibodies became available, since this made it possible to produce unlimited quantities of antibodies with clearly defined characteristics [20]. This second type of assay is also known as a sandwich immunoassay, and is best suited to the assay of proteins rather than haptens. The widespread ELISA (Enzyme Linked Immunosorbent Assay) test belongs to this category [21-24]. 

The conjugates were employed in a sandwich enzyme-linked immunosorbent assay (ELISA) format for the determination of different metal ions including lead, mercury, cadmium and gallium. The study showed that gallium ions could be conveniently detected with ICso value of lO M, a linear concentration range of 5 x 10" to 1 X 10" M and a correlation coefficient of 0.9980. The assay detection limit which is defined as three standard deviations above the zero standard, was recorded to be 5 X 10 M. Moreover, the method showed a remarkable selectivity for Gallium relative to several other metals investigated. The selectivity was modulated by three... 

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