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.

Development of immunodetection system for botulinum neurotoxin type B using synthetic gene based recombinant protein.

Background & objectives: Botulinum neurotoxins (A-G) are among most poisonous substances in the world, produced by obligate anaerobic bacteria Clostridum botulinum. Among the seven serotypes A, B, E and F are of human importance. In India, the prevalence of C. botulinum as well as botulism outbreaks have been reported. Due to its extreme toxicity it has been classified in the Category A of biological warfare agent. So far, there is no commercial detection system available in India to detect botulism. The present study aims to develop an immuno detection system for botulinum neurotoxin serotype B using synthetic gene approach. Methods: The truncated fragment of the botulinum neurotoxin type B from amino acid 1-450 was synthesized using PCR overlap primers; the constructed gene was cloned in the pQE30UA vector and transformed to Escherichia coli SG 13009. The recombinant protein expression was optimized using various concentration of isopropylthiogalactoside (IPTG) induction, further the expression was confirmed by Western blot analysis using anti-His antibody. Recombinant protein was purified under denatured condition using Ni-NTA affinity chromatography. Antibody was generated against the recombinant protein using alum adjuvant in BALB/c mice and tested for cross reactivity with other serotypes of C. botulinum as well as closely related clostridia. An ELISA test was developed for the detection of botulinum neurotoxin and the minimum detection limit was also estimated. Results: The recombinant protein was expressed at maximum yield at 4.3 h of post-induction with 0.5 mM IPTG concentration. The recombinant protein was purified using Ni-NTA affinity chromatography up to the homogeneity level. The polyclonal antibodies were raised in mice with a titre of 1:2048000. The developed antibody was highly specific with a sensitivity of detecting approximately 15 ng/ml of recombinant protein and not showing any cross-reactivity with other serotypes. Interpretation & conclusions: There is no commercial immunodetection system available in India to detect botulism. The developed detection system is highly specific. It will be useful for growing food industry to detect botulinum neurotoxin in food samples as well as in clinical samples.

Protective effect of ethanolic and water extracts of sea buckthorn (Hippophae rhamnoides L.) against the toxic effects of mustard gas.

Ethanolic extract of H. rhamnoides L. leaf (HL-EOH), water and ethanolic extract of H. rhamnoides fruit (HF-W and HF-EOH), and H. rhamnoides flavone from fruit (HR-flavone) were evaluated against percutaneously administered sulphur mustard (SM), a chemical warfare agent. The animals administered with SM (9.7, 19.3 and 38.7 mg/kg) died at various days depending upon the dose and there was a significant reduction in the body weight. The H. rhamnoides extracts (1 g/kg; 3 doses; po) significantly protected the lethality, with a protective index of 2.4, 1.7, 1.7 and 2.2 for HL-EOH, HF-W, HF-EOH and HR-flavone respectively. Reduced glutathione (GSH) and oxidized glutalthione (GSSG) levels were reduced, and malondialdehyde (MDA) was elevated after percutaneous administration of SM. Oral administration of HL-EOH and HR-flavone significantly protected the body weight loss. Recovery in the levels of GSH, GSSG and MDA were also observed following oral administration of HL-EOH and HR-flavone. All the extracts were non-toxic and the LD50 was more than 5 g/kg. The present study shows that percutaneous administration of SM induces oxidative stress and ethanolic extract of leaf of H. rhamnoides and H. rhamnoides flavone from fruit can significantly protect it.

Covert bacteriuria in nephrotic syndrome.

Prevalence of covert bacteriuria in patients of nephrotic syndrome admitted for kidney biopsy was studied in 205 patients. Age ranged from 10 years to 65 years. 148 patients were male and 57 were female. Prevalence of covert bacteriuria was found in 38 patients (18.53%). In bacteriuric patients 30 were male, 8 were female. Escherichia coli was the commonest organism grown in bacteriuric patients (30 or 78.9%). Serum albumin was low and 24-hour urinary protein excretion was high in bacteriuric patients in comparison to abacteriuric patients. In bacteriuric patients membranous nephropathy was the commonest histopathological finding present in 15 patients (39.47%).