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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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Immunoblot of panel of Leptospira spp. obtained by using rabbit anti-rLipL21-IgG antibody to detect single band in leptospiral LipL21 antigen in detergent phase. Lane 1: Canicola (strain Hond Utrecht IV); Lane 2: Hardjobovis (Sponselee); Lane 3: Autumnalis (strain Akiyami A); Lane 4: Icterohaemorrhagiae (strain RGA); Lane 5: Australis (strain Ballico); Lane 6: Pomona (strain Pomona); Lane 7: Grippotyphosa (strain Moskva V); Lane 8: L. kmetyi serovar Malaysia strain Bejo-iso 9T; Lane 9: Balum (strain Mus 127); Lane 10: Grippotyphosa (strain Moskva V); Lane 11: Hebdomadis (strain Hebdomadis); Lane 12: nonpathogenic species L. biflexa (strain Patoc 1). Molecular weight standards are indicated in kilodaltons.
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fig5: Immunoblot of panel of Leptospira spp. obtained by using rabbit anti-rLipL21-IgG antibody to detect single band in leptospiral LipL21 antigen in detergent phase. Lane 1: Canicola (strain Hond Utrecht IV); Lane 2: Hardjobovis (Sponselee); Lane 3: Autumnalis (strain Akiyami A); Lane 4: Icterohaemorrhagiae (strain RGA); Lane 5: Australis (strain Ballico); Lane 6: Pomona (strain Pomona); Lane 7: Grippotyphosa (strain Moskva V); Lane 8: L. kmetyi serovar Malaysia strain Bejo-iso 9T; Lane 9: Balum (strain Mus 127); Lane 10: Grippotyphosa (strain Moskva V); Lane 11: Hebdomadis (strain Hebdomadis); Lane 12: nonpathogenic species L. biflexa (strain Patoc 1). Molecular weight standards are indicated in kilodaltons.

Mentions: IgG-enriched fractions (anti-rLipL21-IgG) were able to recognize leptospiral native LipL21 antigen by immunoblot, which demonstrated that the antibody was specific against LipL21 from pathogenic Leptospira spp. (Figure 5). Preimmune serum did not react with the rLipL21 proteins (data not shown). When these antibodies were tested against triton X-114 fraction of pathogenic serovars of Leptospira spp., they bound to proteins that corresponded to the molecular weight of the native protein. From this result, it can be assumed that the recombinant forms of the candidate antigens effectively mimic the properties of the native form.


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)

Immunoblot of panel of Leptospira spp. obtained by using rabbit anti-rLipL21-IgG antibody to detect single band in leptospiral LipL21 antigen in detergent phase. Lane 1: Canicola (strain Hond Utrecht IV); Lane 2: Hardjobovis (Sponselee); Lane 3: Autumnalis (strain Akiyami A); Lane 4: Icterohaemorrhagiae (strain RGA); Lane 5: Australis (strain Ballico); Lane 6: Pomona (strain Pomona); Lane 7: Grippotyphosa (strain Moskva V); Lane 8: L. kmetyi serovar Malaysia strain Bejo-iso 9T; Lane 9: Balum (strain Mus 127); Lane 10: Grippotyphosa (strain Moskva V); Lane 11: Hebdomadis (strain Hebdomadis); Lane 12: nonpathogenic species L. biflexa (strain Patoc 1). Molecular weight standards are indicated in kilodaltons.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Immunoblot of panel of Leptospira spp. obtained by using rabbit anti-rLipL21-IgG antibody to detect single band in leptospiral LipL21 antigen in detergent phase. Lane 1: Canicola (strain Hond Utrecht IV); Lane 2: Hardjobovis (Sponselee); Lane 3: Autumnalis (strain Akiyami A); Lane 4: Icterohaemorrhagiae (strain RGA); Lane 5: Australis (strain Ballico); Lane 6: Pomona (strain Pomona); Lane 7: Grippotyphosa (strain Moskva V); Lane 8: L. kmetyi serovar Malaysia strain Bejo-iso 9T; Lane 9: Balum (strain Mus 127); Lane 10: Grippotyphosa (strain Moskva V); Lane 11: Hebdomadis (strain Hebdomadis); Lane 12: nonpathogenic species L. biflexa (strain Patoc 1). Molecular weight standards are indicated in kilodaltons.
Mentions: IgG-enriched fractions (anti-rLipL21-IgG) were able to recognize leptospiral native LipL21 antigen by immunoblot, which demonstrated that the antibody was specific against LipL21 from pathogenic Leptospira spp. (Figure 5). Preimmune serum did not react with the rLipL21 proteins (data not shown). When these antibodies were tested against triton X-114 fraction of pathogenic serovars of Leptospira spp., they bound to proteins that corresponded to the molecular weight of the native protein. From this result, it can be assumed that the recombinant forms of the candidate antigens effectively mimic the properties of the native form.

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