Purification method directly influences effectiveness of an epidermal growth factor-coupled targeting agent for noninvasive tumor detection in mice

Joy L. Kovar, William M. Volcheck, Jiyan Chen, Melanie A. Simpson

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled EGF was determined by binding and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation. Both assays indicated a reduction of up to 60% of the fluorescence intensity with damaged agents. Using a research prototype imaging system optimized for IRDye 800CW detection, we compared the efficacy of intact and damaged targeting agents for imaging subcutaneous tumors in mice. In live animal images and in sections of the excised tumors, damaged targeting agents consistently yielded diminished fluorescence signals corresponding to the reduction observed in microplate assays. This is the first study to directly correlate targeting agent signal strength in whole cell binding, In-Cell Western, and in vivo near-infrared imaging.

Original languageEnglish (US)
Pages (from-to)47-54
Number of pages8
JournalAnalytical Biochemistry
Volume361
Issue number1
DOIs
StatePublished - Feb 1 2007

Keywords

  • Binding affinity
  • EGF receptor
  • IRDye 800CW EGF
  • In-Cell Western assay
  • Noninvasive
  • Whole animal imaging

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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