Immunoassays, cytokines

Antibodies have been used extensively as diagnostic tools in many different formats, especially for measurement of protein and cytokine abundances. Applications of antigen arrays can be found in reverse immunoassays for detection of allergens and autoimmune antibodies. 

Hybridoma technology (Chapter 13) facilitated development of immunoassays capable of detecting and quantifying cytokines. 

This author and coworkers at Beckman Coulter first described the use of a low form 96-well plastic microplate for automated micro-ELISA immunoassays (Matson et al., 2001). The polypropylene plate was first modified by a radiofrequency plasma amination process (Matson et al., 1995) followed by conversion to an acyl fluoride surface chemistry for rapid covalent attachment of biomolecules. Proteins (1 to 2 mg/mL) were prepared in 50 mM carbonate buffer, pH 9, containing 4% sodium sulfate (to improve spot uniformity) and printed using a conventional arrayer system. Approximately 200-pL droplets of monoclonal antibodies (anti-cytokine) were deposited into the bottom of the microwells using a Cartesian PS7200 system equipped... 

While in most instances the RCA microarray immunoassay results were comparable to published performance reports on commercial ELISA (Quan-tikine, R D Systems), there were notable exceptions in which ELISA appeared to out-perform the microarray by 10- to 50-fold sensitivity. Such differences may be due to variations in binding affinities exhibited by the particular capture antibodies employed in the assay. In studies involving lipopolysaccharide (LPS)-induced secretion of cytokines from human... 

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]. 

Wadhwa M, Thorpe R. Standardization and calibration of cytokine immunoassays meeting report and recommendations. Cytokine (1997) 9 791-793. 

Ray CA, Bowsher RR, Smith WC, et al. Development, validation and implementation of a multiplex immunoassay for the simultaneous determination of five cytokines inhuman semm./. Pharm. Biomed. Anal. (2005) 36 1037-1044. 

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

The enzyme immunoassay (EIA) applies a single antibody to measure small molecules. The assay works on the principle that two antigens, enzyme-labeled and unlabeled analytes, compete for binding to the limited number of binding sites on the primary antibody, which is subsequently bound to the immobilized anti-IgG. The amount of labeled antigen bound is inversely proportional to the amount of unlabeled antigen (e.g., a cytokine) present in the sample (S7). 

Sensitive and specific immunoassays to detect rabbit IL-8 and MCP-1 cytokines that mediate leukocyte recruitment to the lungs. J. Immunol. Methods 197,19-29. 

Pro-inflammatory cytokines are important mediators of inflammation and tissue destruction. This section describes two cell-based assays that were used to screen for inhibitors of cytokine production and some of the compounds discovered using these screens. The two screens were important elements of a collaboration between Xenova Ltd and the Suntory Institute of Biomedical Research to find microbial metabolites with potential utility for the treatment of rheumatoid arthritis. Both screens were cell stimulatory assays with similar formats, the principle of which is illustrated in Figure 3. Treatment of cells with a particular stimulus activates a signal transduction pathway, one of the end results of which is production of a cytokine, which is secreted into the assay medium. After a separation step, the cytokine of interest is measured quantitatively in the supernatant by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA) using a europium-labeled tertiary antibody. At the same time, cytotoxic properties of test substances are determined by assessing their effect on proliferation of the separated cells. 

Cytokines are peptides used by cells for interceUular communication and for controlling the inner environment of the cells in which they operate2 They are produced by cell types that have important roles in the immune response, inflammation, hemopoiesis, healing, and systemic response to injury. Many cytokines are measured by bioassay and immunoassay,. ... 

In practice, all analyses for cytokines must be performed within a narrow interval of time (not more than 5 hours) after sample collection to prevent cellular interactions and possible cytokine release. Blood samples must be collected with endotoxin-free heparin. The same is true for immunoassays deafing with cytokine production by blood cells. 

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. 

Historically the functions of cytokines have been elucidated first with bioassays preceding immunoassays for cytokine quantification. The bioassay of a given cytokine is based on its bioactivity in a defined biological model, normally based on a certain cell line. Various approaches have been reported (1) proliferation tests— induction of cell growth (e.g., B9 cell line for IL-6) (2) tests for cytotoxicity— TNFa on WEHI164... 

Currently, immunoassay is the practical method of choice for measuring cytokines and their receptors. As cytolanes are proteins, specific antibodies can be raised against recombinant cytokines and have also been measured as an indicator of cytokine presence, The general characteristics of cytokine immunoassays are comparable with the classical immunoassays, with monoclonal, oligoclonal, or polyclonal antibodies all being used (see Chapter 9). The most popular formats are immunoradiometric assay (IRMA) and ELISA, which use a first monoclonal antibody for the capture and a second antibody labeled with a radioisotope or an enzyme. 

The main disadvantage of immunoassays is that they measure total cytokine, which contains both functional and nonfunctional levels. Cross-reactivity with the precursors or degradation products of the cytokine is frequently observed. The detection limit (about 1 to lOpg/mL) of immunoassays is higher than bioassays. A 1995 study reported a lack of comparability between the different kits for IL-2, IL-6, IL-8, and TNFa. The major advantages of immunoassays are their excellent analytical performance and their ability to be automated. 

The comparative characteristics of bioassays and immunoassays when applied to cytokine measurements in biological fluids are listed in Table 22-9. ... 

Malone D, Napolitano LM, Genuit T, Bochicchio GV, Kole K, Scalea TM. Total cytokine immunoassay a more accurate method of cytokine measurement J Trauma 2001 50 821-5. 

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