Quantitative immunoblot assay for assessment of liposomal antibody conjugation efficiency.
Routine direct assessment of immunoglobulin (Ig)-liposome(lp) conjugation efficiency has been impeded by phospholipid interference with standard protein and immunoassay methods. Rabbit IgG conjugated to anionic liposomes was quantitated in immunoblots using computer image analysis techniques. Lp-coupled Ig was separated from free Ig by dialysis in disposable Spectra/Por units (MWCO 300 kDa). Differential Lowry protein assay (DLA) of the thiolated Ig reactant and the dialyzate provided an estimate of conjugation efficiency that was compared to the results of the immunoblot assay (IBA). The color response of Ig-lp in the IBA was about an order of magnitude greater than rabbit IgG alone, requiring the synthesis of an Ig-lp standard in which the Ig conjugation efficiency was assessed by radiotracer methodology. The use of the same standard in three colorimetric protein assays verified the accuracy of the IBA and demonstrated that the colorimetric assays could be employed to determine Ig-lp conjugation efficiency. In terms of sensitivity and specificity, however, the IBA is better suited for routine assessment of laboratory-scale Ig-lp conjugation efficiencies. The DLA was found to be an unsatisfactory measure of conjugation efficiencies because an interfering substance was apparently released by Ig-lp preparations.
Klegerman
Melvin E
ME
EchoDynamics, Inc., College Park, Maryland 20742, USA. mk199@umail.umd.edu
Hamilton
Andrew J
AJ
Huang
Shao-Ling
SL
Tiukinhoy
Susan D
SD
Khan
Amer A
AA
MacDonald
Robert C
RC
McPherson
David D
DD
D000818:Animals; D001666:Binding Sites, Antibody; D015151:Immunoblotting / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology*; D007553:Isotope Labeling / Q000379:methods; D008081:Liposomes / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D011817:Rabbits
D007074:Immunoglobulin G; D008081:Liposomes; D011506:Proteins