Assay enzymatic immunoassays

A significant amount of work on microdevices has recently focused on proteins and peptides. Some of this was initiated as part of the larger proteomics effort, but currently the work is centered largely on the transfer of current analytical methods to microchips. This includes enzymatic assays and immunoassays, both of which are routinely utilized in clinical laboratories. We have included these... 

Apart from immunoassays, enzyme assays can also be used to detect certain substrates in a clinical diagnostic setting. The benefits of performing enzymatic assays on microchips are the analytical power and minimal reagent use in microfluidic systems combined with the selectivity and amplification factors that come with biocatalysis. 

In order to avoid the described problems, antibody-based assays have been developed. As mentioned above, usually a primary antibody recognizes the modification, which in turn is then quantitated. One of these approaches is based on the technology of AlphaScreen (Amplified luminescent Proximity Homogeneous Assay) [51, 52], also known as luminescent oxygen channeling immunoassay (LOCI) [53], a method that can be used to study protein-protein interactions in general. In this case the enzymatic transfer of acetyl groups to a histone peptide is determined. 

Selected entries from Methods in Enzymology [vol, page(s)] Design, 178, 551 immunoassay, 178, 542 production, 178, 531 purification, 178, 543 substrates and enzymatic assay, 178, 544 derivatization with spectroscopic probe, 178, 567 ester cleavage assays, 178, 565 fluorescence quenching binding assay, 178,... 

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]. 

The use of ECL processes for the detection of biological compounds is a rapidly growing area of interest, both for quantitation of analytes and to measure biomolecular interactions. By using ECL active chromophores as labels for biological compounds, a variety of applications is possible, including assays for enzymatic activity, binding assays, immunoassays, and nucleic acid probe assays. 

Research on microchip protein analysis has been very active for cellular protein functional assay, clinical diagnostics, and proteomics studies. Once again, the microfluidic technology plays an important role in protein assays. Immunoassay, protein separation, and enzymatic assay will be described in detail in subsequent sections. 

The histamine content of the supernatants can be determined using the automated fluorometric analyzer (12). Other methods used include a radioisotopic-enzymatic assay (13) or an enzyme immunoassay (14). 

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