Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis
Human leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the...
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Wiley
2020-01-01
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| Series: | International Journal of Analytical Chemistry |
| Online Access: | http://dx.doi.org/10.1155/2020/9289651 |
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| author | Bhagya Deepachandi Sudath Weerasinghe Himali Gunathilake Thisira P. Andrahennadi Mahendra N. Wickramanayake Shantha Siri Vishvanath Chandrasekharan Preethi Soysa Yamuna Siriwardana |
| author_facet | Bhagya Deepachandi Sudath Weerasinghe Himali Gunathilake Thisira P. Andrahennadi Mahendra N. Wickramanayake Shantha Siri Vishvanath Chandrasekharan Preethi Soysa Yamuna Siriwardana |
| author_sort | Bhagya Deepachandi |
| collection | DOAJ |
| description | Human leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the most virulent and visceralizing parasite, is increasingly reported to cause CL in many countries in the world. Although CL is generally not considered to evoke a humoral immune response except for a nonrobust and a variable response in minority of cases, VL is associated with a clear strong humoral response. However, humoral response in L. donovani-induced CL has not been well evaluated before. A suitable serology-based assay is an essential primary step in such a study. An indirect enzyme-linked immunosorbent assay (ELISA) based on Leishmania promastigote crude antigen (Ag) was designed and optimized in order to utilize in further serological studies on this new clinical entity. Optimization included quantification of crude Ag, checkerboard titration method for determination of optimal concentrations for coating Ag, human sera and secondary antibody (Ab) with suitable coating buffer, blocking buffer, and incubating temperatures. The selected coating buffer was 0.02 M phosphate buffer, pH 6.8, and the blocking buffer was 2% fetal bovine serum with 0.01 M phosphate-buffered saline. At least 1 μg of crude Ag was required for coating the ELISA plate, while 1 : 1000 serum was used as primary Ab. The optimized concentration of secondary Ab was 1 : 64000 which might be altered according to manufacturer recommendations. The assay specificity was pre-evaluated using sera (n = 20 from each category) from confirmed CL patients and controls (other skin diseases which mimic CL, other systemic diseases that mimic VL, nonendemic healthy controls, and endemic healthy controls). This procedure described an optimization procedure of an ELISA technique for detection of anti-Leishmania antibodies in patients with L. donovani caused CL. |
| format | Article |
| id | doaj-art-77fff1cd38914f1883ac8dc3e2a67a4c |
| institution | Kabale University |
| issn | 1687-8760 1687-8779 |
| language | English |
| publishDate | 2020-01-01 |
| publisher | Wiley |
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| series | International Journal of Analytical Chemistry |
| spelling | doaj-art-77fff1cd38914f1883ac8dc3e2a67a4c2025-02-03T06:46:37ZengWileyInternational Journal of Analytical Chemistry1687-87601687-87792020-01-01202010.1155/2020/92896519289651Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous LeishmaniasisBhagya Deepachandi0Sudath Weerasinghe1Himali Gunathilake2Thisira P. Andrahennadi3Mahendra N. Wickramanayake4Shantha Siri5Vishvanath Chandrasekharan6Preethi Soysa7Yamuna Siriwardana8Department of Parasitology, Faculty of Medicine, University of Colombo, Colombo 00800, Sri LankaDepartment of Parasitology, Faculty of Medicine, University of Colombo, Colombo 00800, Sri LankaDepartment of Parasitology, Faculty of Medicine, University of Colombo, Colombo 00800, Sri LankaDepartment of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Colombo 00800, Sri LankaDepartment of Chemistry, Faculty of Science, University of Colombo, Colombo 00300, Sri LankaNational Science Foundation, 47/5 Maitland Place, Colombo 00700, Sri LankaDepartment of Chemistry, Faculty of Science, University of Colombo, Colombo 00300, Sri LankaDepartment of Biochemistry and Molecular Biology, Faculty of Medicine, University of Colombo, Colombo 00800, Sri LankaDepartment of Parasitology, Faculty of Medicine, University of Colombo, Colombo 00800, Sri LankaHuman leishmaniasis which is considered a neglected tropical parasitic disease presents in three main clinical forms (i.e., cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL)) that are mainly determined by its causative species. Leishmania donovani, the most virulent and visceralizing parasite, is increasingly reported to cause CL in many countries in the world. Although CL is generally not considered to evoke a humoral immune response except for a nonrobust and a variable response in minority of cases, VL is associated with a clear strong humoral response. However, humoral response in L. donovani-induced CL has not been well evaluated before. A suitable serology-based assay is an essential primary step in such a study. An indirect enzyme-linked immunosorbent assay (ELISA) based on Leishmania promastigote crude antigen (Ag) was designed and optimized in order to utilize in further serological studies on this new clinical entity. Optimization included quantification of crude Ag, checkerboard titration method for determination of optimal concentrations for coating Ag, human sera and secondary antibody (Ab) with suitable coating buffer, blocking buffer, and incubating temperatures. The selected coating buffer was 0.02 M phosphate buffer, pH 6.8, and the blocking buffer was 2% fetal bovine serum with 0.01 M phosphate-buffered saline. At least 1 μg of crude Ag was required for coating the ELISA plate, while 1 : 1000 serum was used as primary Ab. The optimized concentration of secondary Ab was 1 : 64000 which might be altered according to manufacturer recommendations. The assay specificity was pre-evaluated using sera (n = 20 from each category) from confirmed CL patients and controls (other skin diseases which mimic CL, other systemic diseases that mimic VL, nonendemic healthy controls, and endemic healthy controls). This procedure described an optimization procedure of an ELISA technique for detection of anti-Leishmania antibodies in patients with L. donovani caused CL.http://dx.doi.org/10.1155/2020/9289651 |
| spellingShingle | Bhagya Deepachandi Sudath Weerasinghe Himali Gunathilake Thisira P. Andrahennadi Mahendra N. Wickramanayake Shantha Siri Vishvanath Chandrasekharan Preethi Soysa Yamuna Siriwardana Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis International Journal of Analytical Chemistry |
| title | Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis |
| title_full | Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis |
| title_fullStr | Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis |
| title_full_unstemmed | Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis |
| title_short | Prevalidation of an ELISA for Detection of a New Clinical Entity: Leishmania donovani-Induced Cutaneous Leishmaniasis |
| title_sort | prevalidation of an elisa for detection of a new clinical entity leishmania donovani induced cutaneous leishmaniasis |
| url | http://dx.doi.org/10.1155/2020/9289651 |
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