A homogeneous fluorescence-based method to measure antibody internalization in tumor cellsAnalytical Biochemistry


Haibiao Gong, Teresa Urlacher
Molecular Biology / Biochemistry / Biophysics / Cell Biology


oFluorescence le fl th e fl with by

Dy was ing based ns, lip nly on t also lso co molecu ernaliz s. Vario decrease of membrane-associated primary antibody, thereby procomplex is often trafficked to lysosomes for degradation by proteases. By exploring this phenomenon, we have developed a simple, but robust, fluorescence-based assay for antibody internalization. In this method, the antibody is sequentially dual-labeled with a fluorophore (IRDye 800CW, LI-COR Biosciences, Lincoln, signal increase was observed, indicating that IRDye 800CW fluores(data not shown). internalize incubate with phosphate-buffered saline (PBS) and lysed in RIPA buffer.

The cell lysates were then resolved by gel electrophoresis and scanned on an Odyssey CLx Infrared Imaging System to detect the fluorescent signal. The unquenched probe Pan800 (panitumumab labeled with only IRDye 800CW at the same dye/protein ratio ⇑ Corresponding author. Fax: +1 402 467 0825.

E-mail address: herbert.gong@licor.com (H. Gong). 1 Abbreviations used: EGFR, epidermal growth factor receptor; PBS, phosphatebuffered saline; SBR, signal-to-background ratio; ICG, indocyanine green.

Analytical Biochemistry 469 (2015) 1–3

Contents lists available at ScienceDirect io .eantibody internalization [6,7].

On antigen binding and internalization, the antibody/antigen either EGFR-expressing cells (F98-EGFR) or non-EGFR-expressing cells (F98-p). After 24 h of incubation at 37 C, cells were rinsedviding an indirect estimate of the internalized fraction of primary antibody [5]. Antibodies bearing an immunotoxin or intracellularly activated cytotoxic drug have also been employed as reporters of cence could be recovered on enzymatic proteolysis

To investigate whether Pan800QC can be undergo intracellular digestion, the probe washttp://dx.doi.org/10.1016/j.ab.2014.09.008 0003-2697/ 2014 Elsevier Inc. All rights reserved.d and d withdeveloped to detect antibody internalization. The traditional method relies on an acidic buffer to dissociate the surface-bound radioisotope-labeled antibody [2]. A variation of this method uses a fluorophore for signal detection and an anti-fluorophore antibody to quench the surface-associated fluorescence [3,4]. A fluorophorelabeled secondary antibody can also be employed to measure the

To test this idea, an anti-epidermal growth factor receptor (EGFR)1 therapeutic antibody, panitumumab, was dual-labeled as described above. By adjusting the ratio of IRDye QC-1 to IRDye 800CW, a quenching efficiency of 94% was achieved. This biomolecular probe was designated as Pan800QC. When Pan800QC was digested with proteinase K (Sigma, St. Louis, MO, USA), a 10.3-foldHomogeneous assay

The efficacies of many antibodybody–drug conjugates, immunotoxi geted gene delivery, depend not o and specificities of the antibodies bu properties. Internalizing antibodies a over noninternalizing antibodies in fore, characterization of antibody int ing is essential for these applicationtherapies, such as antiosomal drugs, and tarthe binding affinities on their internalization nfer certain advantages lar imaging [1]. Thereation after target bindus methods have been

NE, USA) and a quencher (IRDye QC-1, LI-COR Biosciences) using

NHS (N-hydroxysuccinimide) ester chemistry [8]. Due to the existence of multiple amine groups on the antibody molecule, the labeling product is heterogeneous. In the intact antibody, the fluorescence of the IRDye 800CW is decreased by the presence of the

IRDye QC-1 to variable degrees due to this heterogeneity. On antibody internalization and degradation, the fluorescence of IRDye 800CW is restored following separation from IRDye QC-1.Internalization

Antibody  2014 Elsevier Inc. All rights reserved.Notes & Tips

A homogeneous fluorescence-based meth internalization in tumor cells

Haibiao Gong ⇑, Teresa Urlacher

LI-COR Biosciences, Lincoln, NE 68504, USA a r t i c l e i n f o

Article history:

Received 6 May 2014

Received in revised form 11 September 2014

Accepted 13 September 2014

Available online 22 September 2014

Keywords: a b s t r a c t

We have developed a simp mab was dual-labeled with lecular probe Pan800QC. Th antibody. After incubation of Pan800QC was detected body and separation of IR to-background ratio of 8.5 and screening of internaliz

Analytical B journal homepage: wwwd to measure antibody uorescence-based method to monitor antibody internalization. Panitumue fluorophore IRDye 800CW and quencher IRDye QC-1 to yield the biomouorescence of IRDye 800CW is quenched by IRDye QC-1 on the same intact epidermal growth factor receptor (EGFR)-expressing cells, internalization an increase in fluorescence signal due to enzymatic digestion of the antie 800CW and IRDye QC-1. By optimizing reaction conditions, a signalobtained. This homogeneous assay can be applied in the characterization antibodies. chemistry lsevier .com/locate /yabio as Pan800QC) was included for comparison (Fig. 1). The upper bands (150 kDa) in lanes 3 and 6 of Fig. 1 represent intact probes because the molecular weights were equivalent to the control probes (Pan800QC and Pan800 diluted in PBS, loaded in lanes 1 and 4, respectively). The lower bands in lanes 3 and 6 (<10 kDa) presumably arose from intracellular digestion of the probes. These bands have a higher molecular weight compared with unconjugated IRDye 800CW dye (lane 7), indicating that IRDye 800CW is still attached to amino acids or polypeptide fragments after probe degradation. It is noteworthy that the lower bands in lanes 3 and 6 had similar fluorescence intensities, although the upper band of lane 3 was much weaker than that of lane 6 (the ratio of lane 3 upper band intensity to lane 6 upper band intensity is 1:6). These results provide additional evidence that the fluorescence signal of

Pan800QC can be restored following intracellular degradation of the probe.

Because minimal fluorescence signal is detected before cell treatment, a quenched probe allows the internalization assay to be conducted in a homogeneous manner, thereby simplifying the procedure. To this end, F98-EGFR and F98-p cells were cultured in 96-well plates and then incubated with Pan800QC. At different incubation time points, the plates were scanned on the Odyssey