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The significantly acclaimed laboratory normal, Methods in Enzymology, is likely one of the so much hugely revered courses within the box of biochemistry. seeing that 1955, every one quantity has been eagerly awaited, usually consulted, and praised through researchers and reviewers alike. The sequence includes a lot fabric nonetheless appropriate this present day - really an important booklet for researchers in all fields of existence sciences
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Exp. Biol. Med. 47, 2 0 0 (1941). METHODS IN ENZYMOLOGY, VOL. 74 Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181974-4  FLUORESCENCE EXCITATION TRANSFER IMMUNOASSAY 29 cence" procedures require incubation of a tissue section with antibody toward a specific tissue antigen. The antibody may itself be labeled with a fluorescent tag or more frequently is indirectly labeled by washing away the unlabeled material and incubating the tissue a second time with labeled antibody to the first antibody.
Sci. A. 46, 1470 (1960). 32 IMMUNOASSAY METHODS  Second, with currently available dye pairs energy transfer from a fluorescent donor bound randomly to antibodies would be inefficient. Not only would some of the donors be too remote from the binding site to be efficiently quenched, but donors bound to nonspecific IgG would further add to background fluorescence. For these reasons the most practical labeling configuration requires fluorescent donors ("fluorescers") bound to a relatively pure antigen and antibody-bound acceptors ("quenchers") that are not fluorescent at the wavelengths of the donor emission.
Although powerful homogeneous enzyme immunoassay methods for proteins have recently been reported, r the fluorescence excitation transfer immunoassay 8 (FETI) that is described in this chapter offers significant advantages in terms of simplicity and generality of its application to both small molecules and proteins. Assay Principle One of the simplest concepts for detection of antibody binding is to measure the increase in proximity of the antibody and antigen occasioned by the binding event. Dipole-dipole-coupled electronic energy transfer between two chromophores is ideally suited to this type of measurement.