Elder Pupo

High‐efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels

We recently described a method for recovering polyacrylamide‐gel‐separated bacterial lipopolysaccharides (LPS) based on the sensitive on‐gel LPS detection (1—10 ng/band) with zinc‐imidazole followed by passive elution from 32 μm average size gel microparticles into water. With this procedure, the recovery of rough‐ or semismooth‐type LPS after 3 h elution is about 70—80%, while that of smooth LPS is only about 10%. Here we evaluated whether a simple replacement of water with other eluents would enhance the passive diffusion of LPS. We found that solutions of the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and Triton X‐100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the recovery of a smooth LPS band from Vibrio cholerae O1 in a concentration‐dependent manner. Furthermore, a quantitative recovery of rough or smooth LPS from V. cholerae O1, Escherichia coli O111:B4, E. coli K‐235, or Serratia marcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine after only 2 min. A simple dilution of SDS or DOC or evaporation of triethylamine rendered the eluted LPS preparations compatible with biochemical activity determination, as tested by Limulus amebocyte lysate assay. Thus, this improved micropurification method may be a suitable interface between analytical gel electrophoresis and further characterization or use of LPS.

Mice immunization with gel electrophoresis-micropurified bacterial lipopolysaccharides

Some evidence on the possible use of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to elicit antibodies against smooth‐ or rough‐type bacterial lipopolysaccharides (LPS) is shown. Gel‐separated LPS were negatively stained with zinc‐imidazole to precisely localize the bands of interest under fully reversible conditions. Then the bands of interest were excised and the resulting gel slices washed in a solution of a zinc‐complexing agent (e.g., 100 mM EDTA), after which they were extruded through a metal sieve of 32 μm average size contained in a 1 mL syringe, to generate homogeneous gel microparticles. The LPS‐containing gel slurries were used directly to immunize female BALB/c mice. Using this procedure, positive mouse polyclonal antibody responses against gel‐purified smooth‐ or rough‐LPS forms from Escherichia coli K‐235 or Bordetella pertussis were elicited, as tested by a dot‐immunoblotting assay. Our results may encourage the use of SDS‐PAGE‐micropurified LPS to develop optimized immunization procedures for the generation of specific antibodies against LPS bands of defined sizes, and therefore they constitute an intermediate step toward that aim.