Development of ELISA based detection system for lethal toxin of Clostridium sordellii.

Background & objectives: Clostridium sordellii and its toxins are associated with diseases in animals as well as human. C. sordellii produces two protein toxins (lethal toxin and haemorrhagic toxin). Lethal toxin has gained more importance due its high toxicity. The present study was carried out to develop a sandwich ELISA for detection of lethal toxin of C. sordellii. Methods: The catalytic domain (1.6kb) of lethal toxin of C. sordellii was PCR amplified, cloned into pQE30 UA vector and transformed into Escherichia coli SG 13009. Expression conditions were optimized and the recombinant protein was purified under native condition using Ni-NTA affinity chromatography, confirmed by SDS-PAGE and Western blot. Antibody was generated against the purified recombinant protein using Freund’s complete and incomplete adjuvants (FCA and FIA) in BALB/c mice and rabbit. A sandwich ELISA was optimized for the detection of lethal toxin. Results: The maximum recombinant protein expression was achieved at 0.5 mM IPTG (isopropylthiogalactoside) induction 4.0 h of post-induction. The polyclonal antibody raised in mice and rabbit showed a titre up to 1:512000. The produced antibody was highly sensitive with the detection limit of 0.3 ng/ml of lethal toxin at 1:4000 dilutions of mice (capturing) and rabbit (revealing) antibody. Interpretation & conclusions: An ELISA based detection system was developed for the detection of lethal toxin of C. sordellii. The developed detection system was found to be specific as there was no cross-reactivity with any other clostridial toxins. It will be useful for the detection of lethal toxin of C. sordellii in clinical and environmental samples.

Standardization of anti-lethal toxin potency test of antivenoms prepared from two different Agkistrodon halys venoms

In Korea, antivenoms for the treatment of patients bitten by venomous snakes have been imported from Japan or China. Although there is cross-reactivity between these antibodies and venoms from snakes indigenous to Korea (e.g. Agkistrodon genus), protection is not optimal. Antivenoms specifically prepared to neutralize Korean snake venoms could be more effective, with fewer side effects. To this end, we established an infrastructure to develop national standards and created a standardized method to evaluate the efficacy of two horse-derived antivenoms using mouse lethal toxin test. Additionally, we determined the antivenoms neutralizing activity against lethal doses (LD50) of Agkistrodon halys (from Japan) and Jiangzhe Agkistrodon halys (from China) venoms. We also performed cross-neutralization tests using probit analysis on each pairing of venom and antivenom in order to check the possibility of using Jiangzhe A. halys venom as a substitute for A. halys venom, the current standard. Slope of A. halys venom with A. halys antivenom was 10.2 and that of A. halys venom with Jiangzhe A. halys antivenom was 9.6. However, Slope of Jiangzhe A. halys venom with A. halys antivenom was 4.7 while that of Jiangzhe A. halys venom with Jiangzhe A. halys antivenom was 11.5. Therefore, the significant difference in slope patterns suggests that Jiangzhe A. halys venom cannot be used as a substitute for the standard venom to test the anti-lethal toxin activity of antivenoms (p<0.05).(AU)

Inhibition of Lethal Toxin Expression in Bacillus anthracis Using Antisense Technology

Bacillus anthracis is the causative agent of anthrax primarily in animals and rarely in humans. B. anthracis producing 'anthrax toxin', however, could be a major agent of biological warfare. Anthrax toxin is produced from the pXO1 plasmid encoding the lethal toxin (LeTx) consisted of the protective antigen (PA) and the lethal factor (LF). In this study, we tested whether specific antisense oligonucleotide could inhibit the gene expression in B. anthracis. The antisense oligonucleotide was forced into bacterial cells either by lipofection or heat shock method. The expression of LeTx in B. anthracis was analyzed by the Western blot analysis and the MTT assay using to Raw 264.7 cells. The LeTx protein was purified and used for the production of specific antibodies. The expression of LeTx could be confirmed only in B. anthracis strains haboring pXO1 plasmid. B. anthracis treated with the antisense oligonucleotide through heat shock method markedly inhibited the production of PA. In the Western blot analysis, the expression of PA was inhibited from 25 micrometer and was completely inhibited at 50 micrometer of the antisense oligonucleotide. In the MTT assay, the cytotoxicity was reduced to 20% at 20 micrometer of the antisense oligonucleotide. Above results suggest that the antisense technology would be applied for the research on gene function in B. anthracis.