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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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(a) Immunoblot reaction of rLipL21 protein recognized by different Leptospira spp. in serovar specific hyperimmune sera. Lane 1: His Tag monoclonal antibody; Lane 2: Australis (strain Ballico); Lane 3: Bataviae (strain Swart); Lane 4: Cynopteri (strain 3522C); Lane 5: Canicola (strain Hond Utrecht IV); Lane 6: Grippotyphosa (strain Moskva V); Lane 7: Hebdomadis (strain Hebdomadis); Lane 8: Javanica (Valrat Batavia 46); Lane 9: Pomona (strain Pomona); Lane 10: Icterohaemorrhagiae (strain RGA); Lane 11: Tarasovi (strain Perepelistin); Lane 12: Cellodoni (strain Cellodoni); Lane 13: Pyrogenes (strain Salinum); Lane 14: Hardjobovis (strain Sponselee); Lane 15: Hardjo (strain Hardjoprajito). (b) Immunoblots examining the reactivity of leptospirosis positive human sera with rLipL21. Lane 1–8: 0.5 μg of purified recombinant rLipL21 was probed with sera from Malaysian leptospirosis patients; Lane 1–5: leptospirosis patient sera detected rLipL21; and Lane 6–9: nonleptospirosis sera (leptospiral like clinical symptoms) not detected rLipL21. Molecular weight standards are indicated in kilodaltons.
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fig3: (a) Immunoblot reaction of rLipL21 protein recognized by different Leptospira spp. in serovar specific hyperimmune sera. Lane 1: His Tag monoclonal antibody; Lane 2: Australis (strain Ballico); Lane 3: Bataviae (strain Swart); Lane 4: Cynopteri (strain 3522C); Lane 5: Canicola (strain Hond Utrecht IV); Lane 6: Grippotyphosa (strain Moskva V); Lane 7: Hebdomadis (strain Hebdomadis); Lane 8: Javanica (Valrat Batavia 46); Lane 9: Pomona (strain Pomona); Lane 10: Icterohaemorrhagiae (strain RGA); Lane 11: Tarasovi (strain Perepelistin); Lane 12: Cellodoni (strain Cellodoni); Lane 13: Pyrogenes (strain Salinum); Lane 14: Hardjobovis (strain Sponselee); Lane 15: Hardjo (strain Hardjoprajito). (b) Immunoblots examining the reactivity of leptospirosis positive human sera with rLipL21. Lane 1–8: 0.5 μg of purified recombinant rLipL21 was probed with sera from Malaysian leptospirosis patients; Lane 1–5: leptospirosis patient sera detected rLipL21; and Lane 6–9: nonleptospirosis sera (leptospiral like clinical symptoms) not detected rLipL21. Molecular weight standards are indicated in kilodaltons.

Mentions: The immunoreactivity of the rLipL21 protein against human sera confirmed leptospirosis infection was assessed by immunoblot analysis (Figure 3(b)) and five sera samples from clinically confirmed leptospirosis patients (Figure 3(b)). Figure 3(b) presents the typical reaction of serum samples obtained from patients with (lanes 1 to 5) and without (lanes 6 to 9) leptospiral infection to detect the rLipL21. The rLipL21 protein with rabbit hyperimmune sera was tested. Antibody against the whole Leptospira spp. was detected by immunoblotting in all hyperimmune sera tested, with strong signal intensity as observed (Figure 3(a)).


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)

(a) Immunoblot reaction of rLipL21 protein recognized by different Leptospira spp. in serovar specific hyperimmune sera. Lane 1: His Tag monoclonal antibody; Lane 2: Australis (strain Ballico); Lane 3: Bataviae (strain Swart); Lane 4: Cynopteri (strain 3522C); Lane 5: Canicola (strain Hond Utrecht IV); Lane 6: Grippotyphosa (strain Moskva V); Lane 7: Hebdomadis (strain Hebdomadis); Lane 8: Javanica (Valrat Batavia 46); Lane 9: Pomona (strain Pomona); Lane 10: Icterohaemorrhagiae (strain RGA); Lane 11: Tarasovi (strain Perepelistin); Lane 12: Cellodoni (strain Cellodoni); Lane 13: Pyrogenes (strain Salinum); Lane 14: Hardjobovis (strain Sponselee); Lane 15: Hardjo (strain Hardjoprajito). (b) Immunoblots examining the reactivity of leptospirosis positive human sera with rLipL21. Lane 1–8: 0.5 μg of purified recombinant rLipL21 was probed with sera from Malaysian leptospirosis patients; Lane 1–5: leptospirosis patient sera detected rLipL21; and Lane 6–9: nonleptospirosis sera (leptospiral like clinical symptoms) not detected rLipL21. Molecular weight standards are indicated in kilodaltons.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: (a) Immunoblot reaction of rLipL21 protein recognized by different Leptospira spp. in serovar specific hyperimmune sera. Lane 1: His Tag monoclonal antibody; Lane 2: Australis (strain Ballico); Lane 3: Bataviae (strain Swart); Lane 4: Cynopteri (strain 3522C); Lane 5: Canicola (strain Hond Utrecht IV); Lane 6: Grippotyphosa (strain Moskva V); Lane 7: Hebdomadis (strain Hebdomadis); Lane 8: Javanica (Valrat Batavia 46); Lane 9: Pomona (strain Pomona); Lane 10: Icterohaemorrhagiae (strain RGA); Lane 11: Tarasovi (strain Perepelistin); Lane 12: Cellodoni (strain Cellodoni); Lane 13: Pyrogenes (strain Salinum); Lane 14: Hardjobovis (strain Sponselee); Lane 15: Hardjo (strain Hardjoprajito). (b) Immunoblots examining the reactivity of leptospirosis positive human sera with rLipL21. Lane 1–8: 0.5 μg of purified recombinant rLipL21 was probed with sera from Malaysian leptospirosis patients; Lane 1–5: leptospirosis patient sera detected rLipL21; and Lane 6–9: nonleptospirosis sera (leptospiral like clinical symptoms) not detected rLipL21. Molecular weight standards are indicated in kilodaltons.
Mentions: The immunoreactivity of the rLipL21 protein against human sera confirmed leptospirosis infection was assessed by immunoblot analysis (Figure 3(b)) and five sera samples from clinically confirmed leptospirosis patients (Figure 3(b)). Figure 3(b) presents the typical reaction of serum samples obtained from patients with (lanes 1 to 5) and without (lanes 6 to 9) leptospiral infection to detect the rLipL21. The rLipL21 protein with rabbit hyperimmune sera was tested. Antibody against the whole Leptospira spp. was detected by immunoblotting in all hyperimmune sera tested, with strong signal intensity as observed (Figure 3(a)).

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