Immunological tagging

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

There are several immunological techniques in use. Enzyme multiplied immunological technique (EMIT) employs an enzymatic reaction to determine concentration, whereas radioimmunoassay (RIAl uses radioactively tagged reagents such as I lu measure concentration. 

Flumphreys, J.M. and Chappie, C. (2004) Immunodetection and quantification of cytochromes P450 using epitope tagging immunological, spectroscopic, and kinetic analysis of cinnamate 4-hydroxylase.. Immunol. Methods, 292, 97-107. 

Immunological methods have become important tools used to purify a protein, locate it in the cell, or quantify how much of the protein is present. These methods are predicated on the exquisite specificity of antibodies for their target proteins. Labeled antibodies provide a means to tag a specific protein so that it can be isolated, quantified, or visualized. 

The cellular locations of the mammalian PLD isozymes remain controversial. This is because immunological detection of the endogenous isozymes in cells has largely been unsuccessful, and most studies have utilized overexpression of epitope- or GFP-tagged PLDs. Consequently, the possibility of mislocalization of the overexpressed enzymes is a key issue in these studies. Subcellular fractionation studies have indicated that both PLDl and PLD2 are membrane bound, except when the expression level is high [17, 24, 25]. Phosphorylation and palmitoylation of fhe enzymes can alter their membrane association [17, 24—26]. These post-translational modifications will be considered in fhe next section. 

B. Treanor, P.M.P. Lanigan, K. Suhling, T. Schreiber, I. Munro, M.A.A. Neil, D. Phillips, D.M. Davis, P.M.W. French, Imaging fluorescence lifetime heterogeneity applied to GFP-tagged MFIC protein at an immunological synapse, J. Microsc. 217, 36-43 (2005)... 

Antibodies must be purified from the tissue culture supernatant, ascites fluid, or serum before they can be tagged" or otherwise effectively utilized in an immunoassay. Purification of antibodies can be accomplished to varying degrees employing a number of physical, biochemical and immunological techniques (10). 

The ability of both prokaryotic and eukaryotic cells to uptake Cr(Vl), reduce it to Cr(lll) and retain the Cr(III) products also has important practical applications. In haematology and immunology, cells are often tagged with small nontoxic doses of Cr(VI), which is reduced intracellularly to Cr(lll). Release of Cr(lll) by the cell is used as an indicator of membrane damage in tagged cells (e.g., by action of natural killer cells or during blood transfusion) (204, 205). In another application, increasing concern about Cr(Vl) as an environmental pollutant (Section lll.A) has prompted extensive research on bioremediation, which is based on uptake of Cr(Vl) by bacterial or plant cells and its reduction to relatively harmless Cr(lll) [(108, 115, 120, 128, 206, 207) and references cited therein]. 

In this approach, the bacteria are captured and preconcentrated from food samples with magnetic beads by immunological reaction with the specific antibody against Salmonella. After the lysis of the captured bacteria, further amplification of the genetic material by PCR with a double-tagging set of primers is performed to confirm the identity of the bacteria. Both steps are rapid alternatives to the time-consuming classical selective enrichment and... 

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