Bioassays cytokine assay

Principles of Cytokine Assays Bioassay and immunoassay are the analytical techniques of choice to measure cytokines. However, newer instrumental techniques are also beginning to be used to measure cytokines and CKs. These techniques are used to quantify their (1) concentration and activity in biological fluids, (2) production by whole blood cells,(3) concentration of receptors, and (4) intracellular levels. 

As with the measurement of other analytes, cytokine assay methods changed from the original bioassays to immunoassays, flow cytometric analysis, and mi-croarray technology. 

Incubation periods in excess of 2 h were required before this activity was detected in cell-free supernatants. More recently, the use of cDNA probing of Northern transfers (to detect specific mRNA levels), the use of ELISA techniques (to detect protein levels immunologically) and the development of more specific bioassays (culture techniques in which a biomolecule stimulates proliferation in a particular cell line) have resulted in a more thorough analysis of IL-1 production by neutrophils. IL-1 is only poorly expressed in blood neutrophils because mRNA for this cytokine is detectable only at very low levels (if at all), and protein production is usually below the level of detection of most assays. However, exposure of neutrophils to lipopolysaccharide (LPS), or to cytokines such as GM-CSF, TNF or IL-1 itself, results in a rapid but transient increase in IL-1 expression. 

Endotoxicity results from the interaction of a bacterial cell envelope component (e.g., LPS or PG with a cell surface receptor constituting part of the nonspecific immune system, (i.e., a toll-like receptor on white blood cells). This results in the production of cytokines [e.g., interleukin 1 (IL-1) or tumor necrosis factor (TNF)] as part of an intracellular enzyme cascade which can cause severe tissue injury. Bioassays or immunoassays can be used to detect such reactions respectively. As noted above the most widely used bioassay is the LAL assay. A lysate of amoebo-cytes of the horseshoe crab (Limulus) contains an enzymatic clotting cascade which is activated by extremely low levels of LPS (nanogram levels or lower). There are variants of this assay that can detect PG, but they are not as widely used. As noted above, other bioassays employ cultured cell lines that respond to LPS or PG, respectively. Unfortunately bioassays are highly amenable to false positives (from the presence of cross-reactive substances) or false negatives from inhibition (by contaminants present in the sample) [10]. A detailed discussion of these assays is beyond the scope of this chapter and has been reviewed elsewhere [1]. 

Cytokines, which are protein mediators produced by immune cells, are involved in the regulation of cell activation, growth and differentiation, inflammation, and immunity. Measurement of cytokine production, as determined by techniques such as bioassay, radioimmunoassay, and enzyme-linked immunosorbent assay, has been used to examine various immune functions. 

The most relevant bioassays measure specific activities on immune function, either as effector or regulatory, for example, assay of cytokine IL-12 by the augmentation of the cytolytic function of natural killer cells (B9), IL-10 by the inhibition of the production of TNF-o from stimulated monocytes (B13), and IL-8 and eotaxin by the chemotaxis of neutrophils and eosinophils, respectively (C2). 

Lansky D. Validation of bioassays for quality control. In Brown F, Mire-Sluis AR, eds. Biological Characterization and Assay of Cytokines and Growth Factors. Dev Biol Stand. Basel Karger, 1999 97 157-168. 

The multiple actions of individual cytokines mean that cell lines rarely respond only to one cytokine. This is well illustrated by the efforts to develop specific bioassays for lL-1. Although the original mouse thymocyte proliferation assay was thought to be IL-1 specific, it is now clear that many cytokines influence the assay. IL-6 and TNF can replace IL-1 (G7, U4), and IL-2 and IL-4 can sygerize with... 

Bioassays are generally more sensitive than immunoassays for the measurement of cytokines in cell culture supernatants. The characteristics of calibration curves vary among different assays (Fig. 9) and limits of sensitivity must be set to use only the part of the curve that provides adequate precision. 

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