title: Assessment of free dye in solutions of dual-labeled antibody conjugates for in vivo molecular imaging.
abstract: 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.
authors.affiliation: Division of Molecular Imaging, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Sciences Center-Houston, Houston, TX, USA.
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Aldrich
authors.forename: Xuejuan
authors.initials: X
authors.lastname: Wang
authors.forename: Amy
authors.initials: A
authors.lastname: Hart
authors.forename: Sunkuk
authors.initials: S
authors.lastname: Kwon
authors.forename: Lakshmi
authors.initials: L
authors.lastname: Sampath
authors.forename: Milton V
authors.initials: MV
authors.lastname: Marshall
authors.forename: Eva M
authors.initials: EM
authors.lastname: Sevick-Muraca
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004396:Coloring Agents; D007202:Indicators and Reagents; D007205:Indium Radioisotopes; D012996:Solutions; D004369:Pentetic Acid; D000068878:Trastuzumab
references.citation: J Nucl Med. 1987 Jan;28(1):42-6
references.pmid: 3794811
references.citation: J Biomed Opt. 2008 Jul-Aug;13(4):041312
references.pmid: 19021320
references.citation: Curr Opin Biotechnol. 2009 Feb;20(1):74-82
references.pmid: 19233639
references.citation: Ann Surg. 1986 Sep;204(3):223-35
references.pmid: 3753057
references.citation: J Nucl Med. 1987 Nov;28(11):1736-44
references.pmid: 3668665
references.citation: Breast Cancer Res Treat. 2008 May;109(2):209-29
references.pmid: 17636396
references.citation: Eur J Nucl Med. 1990;16(3):149-56
references.pmid: 2364957
references.citation: Invest New Drugs. 2005 Oct;23(5):391-409
references.pmid: 16133791
references.citation: J Natl Cancer Inst. 2001 Aug 1;93(15):1141-6
references.pmid: 11481385
references.citation: Rev Recent Clin Trials. 2007 May;2(2):111-6
references.pmid: 18473995
references.citation: Curr Protoc Nucleic Acid Chem. 2008 Sep;Appendix:3D
references.pmid: 18819080
references.citation: J Nucl Med. 1987 Jan;28(1):34-41
references.pmid: 3794810
references.citation: Cancer Res. 1987 Nov 15;47(22):6093-9
references.pmid: 3499222
references.citation: Cancer Res. 1990 Feb 1;50(3 Suppl):949s-954s
references.pmid: 2297747
references.citation: Cancer Res. 1987 Jul 1;47(13):3572-6
references.pmid: 3472655
references.citation: Ann N Y Acad Sci. 2008;1131:13-36
references.pmid: 18519956
references.citation: Int J Clin Oncol. 2006 Jun;11(3):199-208
references.pmid: 16850126
references.citation: Nucl Med Commun. 2005 May;26(5):427-32
references.pmid: 15838425
references.citation: Curr Treat Options Oncol. 2007 Feb;8(1):47-60
references.pmid: 17660958
references.citation: J Nucl Med. 2007 Sep;48(9):1501-10
references.pmid: 17785729
references.citation: Nucl Med Biol. 2005 Jan;32(1):51-8
references.pmid: 15691661
references.citation: Proc Natl Acad Sci U S A. 1992 May 15;89(10):4285-9
references.pmid: 1350088
references.citation: Cancer Res. 1989 Mar 15;49(6):1600-8
references.pmid: 2647290
references.citation: Semin Oncol. 2001 Dec;28(6 Suppl 18):43-7
references.pmid: 11774205
references.citation: Cancer Biother Radiopharm. 2003 Jun;18(3):355-63
references.pmid: 12954122
references.citation: Semin Oncol. 2001 Oct;28(5 Suppl 16):115-24
references.pmid: 11706403
references.citation: J Biomed Opt. 2008 Jul-Aug;13(4):041303
references.pmid: 19021311
references.citation: Cell Mol Life Sci. 2004 Dec;61(23):2965-78
references.pmid: 15583858
references.citation: Cancer. 1975 Jun;35(6):1487-92
references.pmid: 50121
references.citation: Radiology. 1986 Nov;161(2):419-22
references.pmid: 3763912
references.citation: Cancer Res. 1980 Aug;40(8 Pt 2):2997-3000
references.pmid: 7397695
references.citation: Radiology. 2008 Mar;246(3):734-41
references.pmid: 18223125