Abstract
It has been suggested that some factor present in human plasma binds to Shiga toxin 2 (Stx2) and neutralizes it in vitro (Bitzan, M., Klemt, M., Steffens, R., and Muller-Wiefel, D. E. (1993) Infection 21, 140-145). This factor does not exist in other species (Caprioli, A., Luzzi, I., Seganti, L., Marchetti, M., Karmali, M., Clarke, I., and Boyd, B. (1994) Recent Adv. VTEC Infect. 353-356). Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it. Purification was carried out by serially subjecting human plasma to Con A-Sepharose, DEAE-Sepharose, hydroxyapatite, and gel-filtration high performance liquid chromatography (HPLC), using Stx2-neutralizing activity as the indicator. The gel-filtration HPLC fraction yielded a single band on SDS-polyacrylamide gel electrophoresis. Twenty N-terminal amino acid residues of this fraction were analyzed and found to correspond perfectly to human serum amyloid P component (HuSAP). Because commercially available HuSAP also showed Stx2 binding and neutralizing activity, we identified this factor as HuSAP.
Highlights
Shiga toxin (Stx)1-producing Escherichia coli (STEC) has been recognized as a pathogen that causes bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) mostly in developed countries [3]
Because analysis of this factor is important to understanding the pathology induced by Shiga toxin-producing Escherichia coli, we purified this factor from human plasma and identified it
Because commercially available human serum amyloid P component (HuSAP) showed Shiga toxin 2 (Stx2) binding and neutralizing activity, we identified this factor as HuSAP
Summary
Con A-Sepharose, DEAE-Sepharose, PD-10, NAP-5, and gel-filtration HPLC (Superose 6 HR 10/30) were from Amersham Pharmacia Biotech. The wells were washed with 1% BSA-PBS and blocked with 3% BSA-PBS at 37 °C for 1 h. After removing the blocking buffer, HuSAP serially diluted with 3% BSA-PBS was added to the wells, and the plates were incubated at 37 °C for 1 h. After washing with 1% BSA-PBS, the wells were incubated at 37 °C for 1 h with rabbit anti-HuSAP serum (Biogenesis) diluted 4000-fold with 3% BSA-PBS. The wells were washed with 1% BSA-PBS and incubated at 37 °C for 1 h with peroxidase-conjugated goat anti-rabbit immunoglobulin (Kirkegaard & Perry Laboratories) diluted 1:2000 with 3% BSA-PBS. After washing with 1% BSA-PBS, peroxidase activity was detected colorimetrically by adding TMB (Kirkegaard & Perry Laboratories)
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