SPDP

BACKGROUND: Recently we reported a nanocontainer based reduction triggered release system through an engineered transmembrane channel (FhuA Delta1-160; Onaca et al., 2008). Compound fluxes within the FhuA Delta1-160 channel protein are controlled sterically through labeled lysine residues (label: 3-(2-pyridyldithio)propionic-acid-N-hydroxysuccinimide-ester). Quantifying the sterical contribution of each labeled lysine would open up an opportunity for designing compound specific drug release systems.

Liposome antibody conjugates are potentially useful as a means of targeting drugs to specific tissues. A new protocol for the conjugation of IgG to maleimide-containing liposomes was developed using 3-(2-pyridyldithio)propionic acid hydrazide (PDPH) as a cross-linker. Periodate-oxidized antibody was treated with PDPH to yield a hydrazone derivative. Deprotection with DTT produced a thiolated antibody which was then conjugated to liposomes containing N-[4-(p-maleidophenyl)butyryl]-1,2-sn-distearoylphosphatidyleth anolamine. The liposome-antibody conjugates were found to have in vitro properties similar to those of conjugates formed by the traditional 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) protocol but were cleared less rapidly in circulation. The PDPH protocol presents a viable alternative to SPDP, particularly for antibodies sensitive to amine modification.

T-cell epitope containing peptides covalently attached to macromolecular carriers can be considered as synthetic immunogens for the development of skin-test diagnostics and of vaccines. As a carrier, an amphoteric branched chain polypeptide, poly[Lys-(Glui-DL-Alam)] (EAK) with poly(L-lysine) backbone has been used. This polypeptide with free alpha-amino and gamma-carboxyl groups at the end of the side chains was conjugated with peptides representing two immunodominant regions of the 16 and 38 kDa proteins of Mycobacterium tuberculosis, respectively. Peptide C91SEFAYGSFVRTVSLPVGADE110 was elongated by Cys at the N-terminal and attached to the carrier containing protected SH groups to form disulfide bridges. Peptide 65FNLWGPAFHERYPNVTITA83 was conjugated to the 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) modified and acetylated EAK by introducing amide bond between the free alpha-amino group of peptide and the free gamma-COOH group of Glu at the terminal position of the branches.

A new homogeneous liposome immunoassay system was developed in which analyte-phospholipase C conjugates are used instead of complement or melittin. This system was applied for the determination of insulin. Liposomes incorporated with calcein as a marker were prepared by the reverse-phase evaporation method. The lysis of liposomes was determined by measuring the fluorescence intensity of calcein released from liposomes and it was increased with increasing concentration of phospholipase C. Insulin was conjugated to phospholipase C by a three-step procedure with hetero-bifunctional crosslinking reagents, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester. The lytic activity of phospholipase C was not affected by the reaction for conjugation. Both p-nitrophenylphosphatidylcholine hydrolytic activity and liposome lytic activity of insulin-phospholipase C conjugate were inhibited in the presence of insulin antiserum.