PubMed · 2143801
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M Berger. 1990. Third component of human complement: C3.. https://doi.org/10.1016/0076-6879(90)84328-e
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Biotinylated small unilamellar liposomes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using avidin coupled to dextran as affinity ligand. In the absence of affinity ligand more than 90% of the liposomes partitioned in the poly(ethylene glycol)-rich top phase, whereas in its presence more than 95% partitioned in the dextran-rich bottom phase. For this redistribution to occur 10 mM and above of lithium sulphate, or other appropriate salts, had to be added to the two-phase system. Without added salt the liposomes with complexed avidin-dextran instead partitioned in the top phase. An extended mixing time for the system was required for maximum redistribution. Less than two biotin residues per liposome, coupled via a C6 spacer arm, was required to redistribute the liposomes to the bottom phase.
We have evaluated by chromatography two strategies of oligonucleotide binding to vitamin B12 (cobalamin). The first one was based on a covalent linkage of aminooligonucleotide to carboxycobalamin in presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Carboxycobalamin and EDC-cobalamin were eluted with a retention time of 16.5 and 21.6 min, respectively, in RP-HPLC, while aminooligonucleotide and oligonucleotide-cobalamin were coeluted at 19.4 and 19.8 min. In the second strategy, avidin was coupled to both biotinylated oligonucleotide and vitamin B12. Aminocobalamin and biotinylated cobalamin had respective retention times of 13 and 15.7 min in RP-HPLC and respective Rf values of 0.3 and 0.8 in thin-layer chromatography. Incubation of avidin with biotinylated cobalamin produced, in Superose 12 gel permeation, a peak with a retention time of 28 min, which corresponded to avidin-biotinylated cobalamin as it disappeared with an excess of either biotin or biotinylated oligonucleotide. In conclusion, we have prepared and purified by RP-HPLC and gel permeation chromatography an oligonucleotide-avidin-cobalamin complex which will be used as a vector complex of antisense oligonucleotides.
Miniaturization of ligand binding assays may reduce costs by decreasing reagent consumption, but it is less apparent that miniaturized assays can simultaneously exceed the sensitivity of macroscopic techniques by analyte "harvesting" to exploit the total analyte mass available in a sample. Capture reagents (avidin or antibodies) immobilized in 200-microm diameter zones are shown to substantially deplete analyte from a liquid sample during a 1-3-h incubation, and the assays that result sense the total analyte mass in a sample rather than its concentration. Detection of as few as 10(5) molecules of analyte per zone is possible by fluorescence imaging in situ on the solid phase using a near-infrared dye label. Single and multianalyte mass-sensing sandwich array assays of the IgG subclasses show the sensitivity and specificity of ELISA methods but use less than 1/100 the capture antibody required by the 96-well plate format.