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Production and characterization of a polyclonal antibody of anti-rLipL21-IgG against leptospira for early detection of acute leptospirosis.

Seenichamy A, Bahaman AR, Mutalib AR, Khairani-Bejo S - Biomed Res Int (2014)

Bottom Line: LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen.We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting.We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

ABSTRACT
Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.

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Expression analysis of rLipL21 on SDS-PAGE and Western blot. Lane U: uninduced lysates containing the pEL21 plasmid only; Lane I: auto induced lysates containing the pEL21 plasmid; Lane M: protein ladder.
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fig2: Expression analysis of rLipL21 on SDS-PAGE and Western blot. Lane U: uninduced lysates containing the pEL21 plasmid only; Lane I: auto induced lysates containing the pEL21 plasmid; Lane M: protein ladder.

Mentions: The rLip21 gene was cloned in an expression plasmid with an N-terminal His tag and the construct was then transformed into E. coli BL21. His tag was used for the purification of recombinant LipL21 (rLipL21) by Ni-NTA affinity column. Aliquots of E. coli-induced cultures were analyzed on 12% SDS-PAGE and expression of rLipL21 protein was confirmed by Western blot with anti-His MAb (Figure 2). The blotted membrane indicated the presence of corresponding band in the expressed rLipL21 protein.


Production and characterization of a polyclonal antibody of anti-rLipL21-IgG against leptospira for early detection of acute leptospirosis.

Seenichamy A, Bahaman AR, Mutalib AR, Khairani-Bejo S - Biomed Res Int (2014)

Expression analysis of rLipL21 on SDS-PAGE and Western blot. Lane U: uninduced lysates containing the pEL21 plasmid only; Lane I: auto induced lysates containing the pEL21 plasmid; Lane M: protein ladder.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016889&req=5

fig2: Expression analysis of rLipL21 on SDS-PAGE and Western blot. Lane U: uninduced lysates containing the pEL21 plasmid only; Lane I: auto induced lysates containing the pEL21 plasmid; Lane M: protein ladder.
Mentions: The rLip21 gene was cloned in an expression plasmid with an N-terminal His tag and the construct was then transformed into E. coli BL21. His tag was used for the purification of recombinant LipL21 (rLipL21) by Ni-NTA affinity column. Aliquots of E. coli-induced cultures were analyzed on 12% SDS-PAGE and expression of rLipL21 protein was confirmed by Western blot with anti-His MAb (Figure 2). The blotted membrane indicated the presence of corresponding band in the expressed rLipL21 protein.

Bottom Line: LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen.We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting.We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.

ABSTRACT
Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.

Show MeSH
Related in: MedlinePlus