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Biochim Biophys Acta. 1995 Nov 1;1239(2):157-67.

A novel strategy affords high-yield coupling of antibody Fab' fragments to liposomes.

Author information

1
Department of Biochemistry, McGill University, Montréal, Québec, Canada.

Abstract

A new assay for the production of reactive sulfhydryl-bearing antibody Fab' fragments has been utilized to develop conditions affording high efficiencies of coupling of mouse and rabbit IgG-derived Fab' fragments to lipid vesicles containing maleimidyl-functionalized phospholipids. Cysteine and mercaptoethylamine, but not dithiothreitol, reduce antibody F(ab')2 to Fab' fragments in very good yields under conditions where overreduction to heavy and light chains is minimized. Surprisingly, however, a large fraction of the Fab' fragments generated under these conditions can lack maleimide-reactive sulfhydryl groups, as demonstrated using a maleimidyl-poly(ethylene glycol) conjugate to shift selectively the electrophoretic mobility of the reactive sulfhydryl-bearing Fab' fragments. After modification of F(ab')2 reduction conditions specifically to maximize the yield of the latter fraction, it is possible to achieve high and very reproducible coupling of functional Fab' fragments to liposomes (equivalent to coupling of ca. 70% of total input protein and almost 100% of the reactive sulfhydryl-bearing Fab' fraction). A novel phospholipid-poly(ethylene glycol)-maleimide 'anchor' allows particularly efficient coupling of Fab' fragments to liposomes, even using relatively low liposome concentrations and molar percentages of the liposome-incorporated 'anchor' species. These results demonstrate that with appropriate optimization of the conditions for Fab' production and liposome coupling, Fab' fragments can be coupled to liposomes with efficiencies comparable to or exceeding those reported for coupling of intact antibodies. These results should facilitate the wider use of Fab' fragments as a potentially advantageous alternative to intact antibodies for liposomal targeting in various applications.

PMID:
7488620
DOI:
10.1016/0005-2736(95)00145-s
[Indexed for MEDLINE]
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