Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis.
The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive...
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| Format: | Article |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5562322?pdf=render |
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| author | Fauzia Jamal Pushkar Shivam Sarita Kumari Manish Kumar Singh Abul Hasan Sardar Pushpanjali Selvasankar Murugesan Shyam Narayan Anil Kumar Gupta Krishna Pandey V N R Das Vahab Ali Sanjiva Bimal Pradeep Das Shubhankar K Singh |
| author_facet | Fauzia Jamal Pushkar Shivam Sarita Kumari Manish Kumar Singh Abul Hasan Sardar Pushpanjali Selvasankar Murugesan Shyam Narayan Anil Kumar Gupta Krishna Pandey V N R Das Vahab Ali Sanjiva Bimal Pradeep Das Shubhankar K Singh |
| author_sort | Fauzia Jamal |
| collection | DOAJ |
| description | The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit. |
| first_indexed | 2024-12-21T16:18:24Z |
| format | Article |
| id | doaj.art-dd599a09148f4744bcf8665095043388 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T16:18:24Z |
| publishDate | 2017-01-01 |
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| spelling | doaj.art-dd599a09148f4744bcf86650950433882022-12-21T18:57:39ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01128e018247410.1371/journal.pone.0182474Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis.Fauzia JamalPushkar ShivamSarita KumariManish Kumar SinghAbul Hasan SardarPushpanjaliSelvasankar MurugesanShyam NarayanAnil Kumar GuptaKrishna PandeyV N R DasVahab AliSanjiva BimalPradeep DasShubhankar K SinghThe unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.http://europepmc.org/articles/PMC5562322?pdf=render |
| spellingShingle | Fauzia Jamal Pushkar Shivam Sarita Kumari Manish Kumar Singh Abul Hasan Sardar Pushpanjali Selvasankar Murugesan Shyam Narayan Anil Kumar Gupta Krishna Pandey V N R Das Vahab Ali Sanjiva Bimal Pradeep Das Shubhankar K Singh Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis. PLoS ONE |
| title | Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis. |
| title_full | Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis. |
| title_fullStr | Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis. |
| title_full_unstemmed | Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis. |
| title_short | Identification of Leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis. |
| title_sort | identification of leishmania donovani antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis |
| url | http://europepmc.org/articles/PMC5562322?pdf=render |
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