Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis
Background and Aim: Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production o...
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| Format: | Article |
| Language: | English |
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Veterinary World
2024-04-01
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| Series: | Veterinary World |
| Subjects: | |
| Online Access: | https://www.veterinaryworld.org/Vol.17/April-2024/16.pdf |
| _version_ | 1797200670753816576 |
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| author | Susanti Susanti Pratiwi Pudjilestari Sudarmono N. L. P. Indi Dharmayanti Prasandhya Astagiri Yusuf |
| author_facet | Susanti Susanti Pratiwi Pudjilestari Sudarmono N. L. P. Indi Dharmayanti Prasandhya Astagiri Yusuf |
| author_sort | Susanti Susanti |
| collection | DOAJ |
| description | Background and Aim: Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis.
Materials and Methods: Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting.
Results: PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus.
Conclusion: IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis. |
| first_indexed | 2024-04-24T07:35:21Z |
| format | Article |
| id | doaj.art-99d31c4ff1844b4a882ee20ae3d7ad4a |
| institution | Directory Open Access Journal |
| issn | 0972-8988 2231-0916 |
| language | English |
| last_indexed | 2024-04-24T07:35:21Z |
| publishDate | 2024-04-01 |
| publisher | Veterinary World |
| record_format | Article |
| series | Veterinary World |
| spelling | doaj.art-99d31c4ff1844b4a882ee20ae3d7ad4a2024-04-20T06:30:01ZengVeterinary WorldVeterinary World0972-89882231-09162024-04-0117487187910.14202/vetworld.2024.871-879Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosisSusanti Susanti0https://orcid.org/0000-0001-6908-8053Pratiwi Pudjilestari Sudarmono1https://orcid.org/0000-0001-7637-2634N. L. P. Indi Dharmayanti2https://orcid.org/0000-0002-9399-8193Prasandhya Astagiri Yusuf3https://orcid.org/0000-0002-4212-0794Doctoral Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia.Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia.Research Center for Veterinary Science, Research Organization for Health, National Research and Innovation Agency, Bogor, 16911, Indonesia.Medical Physiology Biophysics Department and Medical Technology IMERI, Faculty of Medicine, Universitas Indonesia, Jakarta, 10430, Indonesia.Background and Aim: Microscopic agglutination test (MAT) for the diagnosis of leptospirosis requires live cultures and is serovar-specific, while polymerase chain reaction (PCR) requires expensive equipment and sample preparation. The rLipL32 protein is conserved and can be used for the production of immunoglobulin G (IgG) anti-rLipL32 antibody, which can be used as a biomarker for leptospirosis diagnosis. This study aimed to produce and characterize an IgG anti-rLipL32 antibody as a biomarker for leptospirosis diagnosis. Materials and Methods: Escherichia coli rLipL32 was cultured and analyzed by PCR and sequencing. Cultures were used for rLipL32 protein expression and purification and the rLipL32 protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The rLipL32 protein was used to produce anti-rLipL32 serum and was analyzed by enzyme-linked immunosorbent assay (ELISA). Serum was purified to obtain IgG anti-rLipL32 antibody and characterized by SDS-PAGE and western blotting. Results: PCR was able to amplify the LipL32 gene from E. coli rLipL32, and sequencing analysis showed 99.19% similarity with pathogenic Leptospira. SDS-PAGE analysis showed a 32-kDa band. ELISA results showed an increase in OD in anti-rLipL32 serum compared to preimmune serum. Western blotting results showed that the IgG anti-rLipL32 antibody was able to bind and cross-reacts with pathogenic Leptospira serovar but not with E. coli or Staphylococcus aureus. Conclusion: IgG anti-rLipL32 antibody has high specificity and sensitivity against Leptospira pathogens. These findings suggest that IgG anti-rLipL32 antibody is a promising biomarker for the diagnosis of leptospirosis.https://www.veterinaryworld.org/Vol.17/April-2024/16.pdfanti-rlipl32 serumimmunoglobulin g anti-rlipl32 antibodyleptospirarlipl32 protein |
| spellingShingle | Susanti Susanti Pratiwi Pudjilestari Sudarmono N. L. P. Indi Dharmayanti Prasandhya Astagiri Yusuf Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis Veterinary World anti-rlipl32 serum immunoglobulin g anti-rlipl32 antibody leptospira rlipl32 protein |
| title | Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis |
| title_full | Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis |
| title_fullStr | Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis |
| title_full_unstemmed | Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis |
| title_short | Production and characterization of immunoglobulin G anti-rLipL32 antibody as a biomarker for the diagnosis of leptospirosis |
| title_sort | production and characterization of immunoglobulin g anti rlipl32 antibody as a biomarker for the diagnosis of leptospirosis |
| topic | anti-rlipl32 serum immunoglobulin g anti-rlipl32 antibody leptospira rlipl32 protein |
| url | https://www.veterinaryworld.org/Vol.17/April-2024/16.pdf |
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