High-performance capillary electrophoresis for characterization of hapten-protein conjugates used for production of antibodies against soyasaponin I.

Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten-protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-alpha-D-galactoside; PNPG) coupled to KSTI. The PNPG-KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten-protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.

Does complement kill E. coli by producing transmural pores?

Three lines of evidence are presented to indicate that C5b-9 kills serum-sensitive E. coli K 12 cells by generating functional pores across the outer and inner bacterial membrane. First, viable cells carrying C5b-8 complexes are impermeable to o-nitrophenyl-beta-D-galactoside (ONPG), but lose viability and become permeable to this marker upon post-treatment with purified C9 in the absence of lysozyme. Cells killed with colicin E1 or gentamicin are also impermeable to ONPG but take up the marker if they are post-treated with lysozyme-free serum. Second, killing by C5b-9 is highly effective, deposition of only a small number of complexes being lethal. This has been demonstrated in experiments where viable cells carrying 2000-4000 C5b-7 complexes per CFU were permitted to multiply in broth culture, and the daughter generations subsequently treated with purified C8 and C9. Fifty percent killing was observed in the fifth to sixth generation, corresponding to a dilution of C5b-7 complexes to 50-100 molecules/CFU. In the presence of 2 mM EDTA, further dilution of C5b-7 down to 8-30 complexes/CFU still caused 50% killing of daughter cells. Third, treatment of C5b-7 cells with purified CC8 and C9 results in the release of intracellular K+, which commences immediately after addition of C8/C9. This was shown in experiments where C5b-7 cells were packed to high density in saline, post-treated with C8 + C9, and K+ directly measured in the cell supernatants. Based on these results, we propose that C5b-9 pores deposited in the outer bacterial membrane periodically fuse with the inner membrane, the transmural pores thus generated permitting rapid K+ efflux, with cell death ensuing through the collapse of membrane potential.