Immunosorbent systems

Figure 1. Capacity of model immunosorbent systems as a function of cycle number. (A) Goat anti-human IgC Fab fragment immobilized at 7 g/L. Column volume = 6 ml. (B) Same as above except that the initial antibody loading was 1 gL- and the immunosorbent volume was 4.2 ml.

Figure 2. Capacity of a model immunosorbent system as a function of cycle number.

ELISA Enzyme-linked immunosorbent assay EMS Eosinophilia-myalgia syndrome ENS Enteric nervous system EO Eosinophil... 

A very versatile piece of equipment that is affordable for individual laboratories is the microplate reader. This allows multiple samples to be analyzed at once, commonly in a 96-well format, although 384- and 1536-well formats are available. Typical measurements that can be performed include UV-Vis absorbance, fluorescence, or luminescence, allowing a range of assays to be performed, such as cell growth, enzyme kinetics, enzyme stability, or enzyme-linked immunosorbent assay [60-62]. Functionality can be increased by the use of liquid dispensing systems or automatic plate handling. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

In order to compare the specific activity of plant-derived C5-1 to that of the hybridoma-derived antibody, the antigen-binding capacity of antibodies produced in each system was assayed by enzyme-linked immunosorbent assay (ELISA). As shown in Table 1.2, antibodies from both sources demonstrated similar binding characteristics against human IgGs [8]. Furthermore, the stability of alfalfa-derived C5-1 in the blood stream of Balb/c mice was comparable to that of the hybridoma-derived IgG [8]. 

A continuous semiautomated FIIA system has been reported [208,235]. In this device the analyte-containing medium is allowed to flow through a column containing the antibodies immobilized on a support. First, the antibodies are saturated with a fluorescent dye-labeled analog of the analyte. As the analyte passes through the immunosorbent, some dye-labeled antigen is displaced and is then detected in a fluorometer located downstream from the column. The LOD achieved for the developed system is 1 pg L 1. 

McConnell RJ, Fitzgerald SP, Lamont JV (1992) Trenbolone and 19-nortestosterone residue analysis by immunoaffinity chromatography and high-performance liquid chromatography and/or an enzyme-linked immunosorbent assay. In Morgan MRA, Smith CJ, Williams PA (eds) Food safety and quality assurance applications of immunoassay systems. Elsevier, Barking, p 245... 

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the two-site format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing... 

K. Sato, M. Tokeshi, T. Odake, H. Kimura, T. Ooi, N. M. and T. Kitamori Integration of an Immunosorbent Assay System Analysis of Secretory Human Immunoglobulin a on Polystyrene Beads in a Microchip. Anal. Chem. 72, 1144 (2000). 

Kendall C, lonescu-Matiu I, Dreesman GR. (1983) Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA). J Immunol Methods 56, 329-39. 

Enzyme-linked immunosorbent assays. An indirect application of enzymes in analysis is as a marker or label in enzyme-linked immunosorbent assays (ELISA). In ELISA, the enzyme does not react with the analyte instead, an antibody is raised against the analyte (antigen or hapten) and labelled with easily assayed enzyme, usually a phosphatase or a peroxidase. The enzyme activity is proportional to the amount of antibody in the system, which in turn is proportional, directly or indirectly depending on the arrangement used, to the amount of antigen present (Morris and Clifford, 1984). 

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