Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.
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.
Brown
Jeremy K
JK
Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Midlothian EH25 9RG, UK. Jeremy.brown2@ed.ac.uk
Pemberton
Alan D
AD
Wright
Steven H
SH
Miller
Hugh R P
HR
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000276:immunology*; D002470:Cell Survival; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007150:Immunohistochemistry / Q000379:methods*; D007583:Jejunum / Q000166:cytology / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D015534:Trans-Activators / Q000378:metabolism; D051176:beta Catenin
D000906:Antibodies; D000936:Antigen-Antibody Complex; C495267:CTNNB1 protein, mouse; C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D007140:Immunoglobulin Fab Fragments; D015534:Trans-Activators; D051176:beta Catenin