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A novel assay for monitoring internalization of nanocarrier coupled antibodies.

Authors :
Nielsen, Ulrik B
Kirpotin, Dmitri B
Pickering, Edward M
Drummond, Daryl C
Marks, James D
Source :
BMC Immunology; 2006, Vol. 7, p24-15, 15p, 3 Diagrams, 1 Chart, 1 Graph
Publication Year :
2006

Abstract

Background: Discovery of tumor-selective antibodies or antibody fragments is a promising approach for delivering therapeutic agents to antigen over-expressing cancers. Therefore it is important to develop methods for the identification of target- and function specific antibodies for effective drug delivery. Here we describe a highly selective and sensitive method for characterizing the internalizing potential of multivalently displayed antibodies or ligands conjugated to liposomes into tumor cells. The assay requires minute amounts of histidine-tagged ligand and relies on the non-covalent coupling of these antibodies to fluorescent liposomes containing a metal ion-chelating lipid. Following incubation of cells with antibody-conjugated liposomes, surface bound liposomes are gently removed and the remaining internalized liposomes are quantitated based on fluorescence in a high throughput manner. We have termed this methodology "Chelated Ligand Internalization Assay", or CLIA. Results: The specificity of the assay was demonstrated with different antibodies to the ErbB-2 and EGF receptors. Antibody-uptake correlated with receptor expression levels in tumor cell lines with a range of receptor expression. Furthermore, Ni-NTA liposomes containing doxorubicin were used to screen for the ability of antibodies to confer target-specific cytotoxicity. Using an anti-ErbB2 single chain Fv (scFv) (F5) antibody, cytotoxicity could be conferred to ErbB2-overexpressing cells; however, a poly(ethylene glycol)-linked lipid (DSPE-PEG-NTA-Ni) was necessary to allow for efficient loading of the drug and to reduce nonspecific drug leakage during the course of the assay. Conclusion: The CLIA method we describe here represents a rapid, sensitive and robust assay for the identification and characterization of tumor-specific antibodies capable of high drug-delivery efficiency when conjugated to liposomal nanocarriers. [ABSTRACT FROM AUTHOR]

Details

Language :
English
ISSN :
14712172
Volume :
7
Database :
Complementary Index
Journal :
BMC Immunology
Publication Type :
Academic Journal
Accession number :
29362636
Full Text :
https://doi.org/10.1186/1471-2172-7-24