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Chapter 108 : Assay Methods for Measurement of Mediators and Markers of Allergic Inflammation

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Abstract:

With the discovery of immunoglobulin E (IgE) as the source of reaginic activity in serum, the release of histamine from leukocyte suspensions challenged with a specific antigen has been used as a reliable in vitro correlate of immediate hypersensitivity. Although histamine remains the most commonly measured mediator released following this IgE-mediated reaction, it is now well established that other mediators are also released from basophils and are important markers of allergic inflammation. This chapter highlights the growing interest in the use of flow cytometry assays to monitor activation-linked markers (e.g., CD63, CD69, and CD203c) on basophils and the applicability of such assays as surrogate indicators of mediator release. The radioenzymatic assay (REA) has the advantage of detecting very low levels of histamine, with a sensitivity of approximately 10 pg/ml. The REA is not subject to interference from high protein levels, which makes it an excellent protocol for measuring plasma histamine levels. Measurement of the release of preformed histamine from peripheral blood basophils challenged with a specific antigen is among several tests available, and histamine release remains a valuable in vitro correlate of immediate hypersensitivity reactions. Mediators such as LTC4 and IL-4, are generated by basophils upon IgE-dependent activation, and in vitro assays have recently been developed to measure these products. These assays, combined with those available for the detection of several other mediators occurring in biological fluids, have produced data that have significantly added to our understanding of the parameters, mechanisms, and pharmacologic control of allergic inflammation.

Citation: Schroeder J, Saini S. 2006. Assay Methods for Measurement of Mediators and Markers of Allergic Inflammation, p 964-974. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch108

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Figures

Image of FIGURE 1
FIGURE 1

Diagrammatic representation of the time course for histamine, LTC4, and IL-4 and IL-13 protein release by human basophils following IgE-dependent activation. Both preformed histamine and newly generated LTC4 are released within minutes after stimulation. The generation of IL-4 and IL-13 is thought to occur de novo, with protein detectable only after 1 to 2 h for IL-4 and after 4 h for IL-13, following stimulation.

Citation: Schroeder J, Saini S. 2006. Assay Methods for Measurement of Mediators and Markers of Allergic Inflammation, p 964-974. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch108
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Image of FIGURE 2
FIGURE 2

Diagrammatic representation of preparation of peripheral blood basophils by discontinuous Percoll density centrifugation. Leukocyte interface cells (buffy coat) are prepared from whole blood by centrifugation. Diluted buffy-coat cell suspensions are layered onto buffered isotonic gradients consisting of two Percoll densities (d) of 1.072 and 1.082 g/ml. Following centrifugation (700 ×, 20 min, at 24°C), basophils band on the 1.082-g/ml Percoll interface and range from 5 to 50% purity. v/v, vol/vol.

Citation: Schroeder J, Saini S. 2006. Assay Methods for Measurement of Mediators and Markers of Allergic Inflammation, p 964-974. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch108
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