Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography
The significance of immunoglobulin Y (IgY) as a particular antibody equal to mammalian IgG is well understood. However, due to a lack of reliable purification procedures, producing highly pure IgY remains problematic. In this study, we aimed to optimize the recovery of pure IgY anti-MPT64 using thi...
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| Format: | Article |
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HH Publisher
2024-03-01
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| Series: | Progress in Microbes and Molecular Biology |
| Online Access: | https://journals.hh-publisher.com/index.php/pmmb/article/view/892 |
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| author | Sri Agung Fitri Kusuma Muhammad Fadhlillah Intan Timur Maisyarah Shintani Ayunda Khairunnisa Muhammad Yusuf Yaya Rukayadi Toto Subroto |
| author_facet | Sri Agung Fitri Kusuma Muhammad Fadhlillah Intan Timur Maisyarah Shintani Ayunda Khairunnisa Muhammad Yusuf Yaya Rukayadi Toto Subroto |
| author_sort | Sri Agung Fitri Kusuma |
| collection | DOAJ |
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The significance of immunoglobulin Y (IgY) as a particular antibody equal to mammalian IgG is well understood. However, due to a lack of reliable purification procedures, producing highly pure IgY remains problematic. In this study, we aimed to optimize the recovery of pure IgY anti-MPT64 using thiophilic adsorption chromatography. The purification of IgY anti-MPT64 was achieved by initial PEG lipid precipitation, then an optimized purification by varying the gradient concentration of elution buffer into five steps gradient (0-20, 20-40, 40-60, 60-80, and 80-100%v/v) for three injection column volume (CV) each and two steps gradient (0-50 and 50-100%v/v) in eight CV for each concentration. The obtained IgY was characterized by SDS-PAGE and dot-blot then determined its content levels using the Lowry method. The results showed that the five steps gradient purification was found to provide a better purity level of IgY than the two steps gradient. However, the IgY content obtained in the two steps gradient purification (2.2632± 0.011 mg/mL) was higher than the five steps gradient purification (1.35482 ± 0.023 mg/mL). Nevertheless, both purified IgY results can recognize MPT64 protein through a dot blot test. Therefore, it can be summarized that thiophilic adsorption chromatography with five steps gradient of purification was an efficient process to obtain a higher purity of IgY anti-MPT64, especially to be targeted as a diagnostic kit component for MPT64 detection.
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| format | Article |
| id | doaj-art-1a29093cde0441df9916027c1eefd2f5 |
| institution | Kabale University |
| issn | 2637-1049 |
| language | English |
| publishDate | 2024-03-01 |
| publisher | HH Publisher |
| record_format | Article |
| series | Progress in Microbes and Molecular Biology |
| spelling | doaj-art-1a29093cde0441df9916027c1eefd2f52025-02-04T08:39:40ZengHH PublisherProgress in Microbes and Molecular Biology2637-10492024-03-017110.36877/pmmb.a0000400Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography Sri Agung Fitri KusumaMuhammad FadhlillahIntan Timur MaisyarahShintani Ayunda KhairunnisaMuhammad YusufYaya RukayadiToto Subroto The significance of immunoglobulin Y (IgY) as a particular antibody equal to mammalian IgG is well understood. However, due to a lack of reliable purification procedures, producing highly pure IgY remains problematic. In this study, we aimed to optimize the recovery of pure IgY anti-MPT64 using thiophilic adsorption chromatography. The purification of IgY anti-MPT64 was achieved by initial PEG lipid precipitation, then an optimized purification by varying the gradient concentration of elution buffer into five steps gradient (0-20, 20-40, 40-60, 60-80, and 80-100%v/v) for three injection column volume (CV) each and two steps gradient (0-50 and 50-100%v/v) in eight CV for each concentration. The obtained IgY was characterized by SDS-PAGE and dot-blot then determined its content levels using the Lowry method. The results showed that the five steps gradient purification was found to provide a better purity level of IgY than the two steps gradient. However, the IgY content obtained in the two steps gradient purification (2.2632± 0.011 mg/mL) was higher than the five steps gradient purification (1.35482 ± 0.023 mg/mL). Nevertheless, both purified IgY results can recognize MPT64 protein through a dot blot test. Therefore, it can be summarized that thiophilic adsorption chromatography with five steps gradient of purification was an efficient process to obtain a higher purity of IgY anti-MPT64, especially to be targeted as a diagnostic kit component for MPT64 detection. https://journals.hh-publisher.com/index.php/pmmb/article/view/892 |
| spellingShingle | Sri Agung Fitri Kusuma Muhammad Fadhlillah Intan Timur Maisyarah Shintani Ayunda Khairunnisa Muhammad Yusuf Yaya Rukayadi Toto Subroto Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography Progress in Microbes and Molecular Biology |
| title | Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography |
| title_full | Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography |
| title_fullStr | Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography |
| title_full_unstemmed | Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography |
| title_short | Purification of Anti-Mycobacterium tuberculosis MPT64 Immunoglobulin-Y from Egg-Yolk Supernatant Using Thiophilic Adsorption Chromatography |
| title_sort | purification of anti mycobacterium tuberculosis mpt64 immunoglobulin y from egg yolk supernatant using thiophilic adsorption chromatography |
| url | https://journals.hh-publisher.com/index.php/pmmb/article/view/892 |
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