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DOI: 10.1369/jhc.3A6200.2004
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Journal of Histochemistry and Cytochemistry
Volume 52 (9): 1219-1230, 2004
Copyright ©The Histochemical Society, Inc.

Primary Antibody–Fab Fragment Complexes : A Flexible Alternative to Traditional Direct and Indirect Immunolabeling Techniques

Jeremy K. Brown, Alan D. Pemberton, Steven H. Wright and Hugh R.P. Miller

Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Midlothian, United Kingdom

Correspondence to: Jeremy K. Brown, Dept. of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Midlothian EH25 9RG, UK. E-mail: Jeremy.brown2{at}ed.ac.uk

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA. (J Histochem Cytochem 52:1219–1230, 2004)

Key Words: endogenous immunoglobulin • Fab • fluorescence • multicolor • multilabeling • flow cytometry • ELISA


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