Assessment of free dye in solutions of dual-labeled antibody conjugates for in vivo molecular imaging.
OBJECTIVE
Recent preclinical and clinical studies show that dyes that excite and fluoresce in the near-infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic.


METHODS
Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800 CW in (DTPA)(n)-trastuzumab-(IRDye 800)(m) conjugate sample preparations in which high-performance liquid chromatography (HPLC) assessment of free dye was not possible.


RESULTS
The SDS-PAGE assay was accurate and valid for free IRDye 800 CW amounts between 38 and 4 mol% of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined.


CONCLUSIONS
This method may be applicable to other near-infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging.
Aldrich
Melissa B
MB
Division of Molecular Imaging, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Sciences Center-Houston, Houston, TX, USA.
Wang
Xuejuan
X
Hart
Amy
A
Kwon
Sunkuk
S
Sampath
Lakshmi
L
Marshall
Milton V
MV
Sevick-Muraca
Eva M
EM
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D061067:Antibodies, Monoclonal, Humanized; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007205:Indium Radioisotopes / Q000737:chemistry; D057230:Limit of Detection; D004369:Pentetic Acid / Q000737:chemistry; D012996:Solutions; D019265:Spectroscopy, Near-Infrared; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004396:Coloring Agents; D007202:Indicators and Reagents; D007205:Indium Radioisotopes; D012996:Solutions; D004369:Pentetic Acid; D000068878:Trastuzumab
J Nucl Med. 1987 Jan;28(1):42-6
3794811
J Biomed Opt. 2008 Jul-Aug;13(4):041312
19021320
Curr Opin Biotechnol. 2009 Feb;20(1):74-82
19233639
Ann Surg. 1986 Sep;204(3):223-35
3753057
J Nucl Med. 1987 Nov;28(11):1736-44
3668665
Breast Cancer Res Treat. 2008 May;109(2):209-29
17636396
Eur J Nucl Med. 1990;16(3):149-56
2364957
Invest New Drugs. 2005 Oct;23(5):391-409
16133791
J Natl Cancer Inst. 2001 Aug 1;93(15):1141-6
11481385
Rev Recent Clin Trials. 2007 May;2(2):111-6
18473995
Curr Protoc Nucleic Acid Chem. 2008 Sep;Appendix:3D
18819080
J Nucl Med. 1987 Jan;28(1):34-41
3794810
Cancer Res. 1987 Nov 15;47(22):6093-9
3499222
Cancer Res. 1990 Feb 1;50(3 Suppl):949s-954s
2297747
Cancer Res. 1987 Jul 1;47(13):3572-6
3472655
Ann N Y Acad Sci. 2008;1131:13-36
18519956
Int J Clin Oncol. 2006 Jun;11(3):199-208
16850126
Nucl Med Commun. 2005 May;26(5):427-32
15838425
Curr Treat Options Oncol. 2007 Feb;8(1):47-60
17660958
J Nucl Med. 2007 Sep;48(9):1501-10
17785729
Nucl Med Biol. 2005 Jan;32(1):51-8
15691661
Proc Natl Acad Sci U S A. 1992 May 15;89(10):4285-9
1350088
Cancer Res. 1989 Mar 15;49(6):1600-8
2647290
Semin Oncol. 2001 Dec;28(6 Suppl 18):43-7
11774205
Cancer Biother Radiopharm. 2003 Jun;18(3):355-63
12954122
Semin Oncol. 2001 Oct;28(5 Suppl 16):115-24
11706403
J Biomed Opt. 2008 Jul-Aug;13(4):041303
19021311
Cell Mol Life Sci. 2004 Dec;61(23):2965-78
15583858
Cancer. 1975 Jun;35(6):1487-92
50121
Radiology. 1986 Nov;161(2):419-22
3763912
Cancer Res. 1980 Aug;40(8 Pt 2):2997-3000
7397695
Radiology. 2008 Mar;246(3):734-41
18223125