However, many studies have shown that CL detection rate of rK39 assay is usually low [11, 12]. PIK-III patterns in response to various antigens have been mainly described for visceral leishmaniasis [2, 3]. Diagnostic tools that detect this serological response are often used in the laboratory identification of VL.L. donovanicomplex specific rK39 dipstick assay is usually a frequently used serological tool for VL detection [4C6]. Apart from its traditionally known clinical outcome leading to VL,L. donovaniis occasionally known to cause CL [7C10]. However, many studies have shown that CL detection rate of rK39 assay is usually low [11, 12]. ELISAs have been developed for the detection of CL with varying results [50.0% to 98.0% positivity] [13, 14]. Literature on CL caused byL. donovaniis scarce. Human leishmaniasis caused byL. donovaniis considered as an established disease in Sri Lanka [15C17]. LocalL. donovaniparasite was found to be genetically different from knownL. donovanistrains in other endemic settings [17]. CL remains the main clinical entity in Sri Lanka [18C20] with only few reported cases of VL and MCL [21, 22]. Ability of the local CL parasites to raise an antibody response has not been studied. In a previous attempt to elicit such a PIK-III response using rK39 immunochromatographic test on 24 cases of CL, none of them switched positive [10]. This indicated a poor or absent humoral response or, on the contrary, possible undetected antigenic differences of the local parasite variant. This project was designed to examine the serological response using an IgG-based in-house ELISA. Current study describes the first attempt to develop an in-house ELISA and its use to determine the prevalence of anti-IgG antibodies in CL acquired in Sri Lanka. 2. Materials and Methods 2.1. Sample Collection A total of 90 patients who presented to the routine leishmaniasis clinic at the Department of Parasitology, Faculty of Medicine, University of Colombo, with single or multiple skin lesions suggestive of cutaneous leishmaniasis were recruited after PIK-III obtainment of informed written consent. They were clinically evaluated and data were gathered using a PIK-III standard pretested questionnaire. Lesion aspirations (LA), slit-skin scrapings (SSS), and/or punch biopsies (PB) were collected from clinically suggestive skin lesions. Diagnosis of CL was established by light microscopic examination of LAs and scrapings. PCR was carried out on DNA extracted from punch biopsies of all microscopy negative patients [23, 24]. 2.2. Preparation of Crude Protein Lysate parasites were produced in M 199 culture medium according to the previously established protocols [24]. Cell pellets were prepared by centrifugation of parasites in late log phase at 3000?rpm for 10 minutes. Cells of a single culture isolate were used throughout the study to minimize errors. Crude protein lysate was obtained from the cell pellet by freeze-thaw method. Cell pellet was dissolved in sterile 1x PBS. Rapid freeze-thaw cycles were performed using liquid nitrogen. Pellet was dipped in liquid nitrogen until the freezing heat was achieved. Rabbit Polyclonal to GPR37 The pellet was then taken out and kept at 37C for 1 minute approximately until being completely dissolved. Procedure was repeated thrice to lyse proteins. Lysate was sonicated for 20 seconds followed by centrifugation and the supernatant was used for subsequent procedures. Protein estimation was carried out according to the altered Lowry procedure [25]. 2.3. Sera for Positive and Negative Controls A volume of 2?ml of intravenous blood was collected to a plain tube from a local patient with locally acquired and parasitologically confirmed VL (bone marrow examination by microscopy on direct smear andin vitrocultures), 20 apparently healthy individuals (HC) residing in a nonleishmanial area (Colombo), and ten patients with other skin diseases which clinically mimic CL (NCL) in the local setting (psoriasis, 2, leprosy, 3, eczema, 3, and allergic dermatitis, 2). Blood was allowed to clot at room heat and centrifuged at 3000?rpm for 10 minutes. Serum was separated and stored at ?20C for further use. Sera from the VL patient and 20 HCs were examined by rK39 assay and confirmed positive in case of VL and unfavorable in all HCs. HC samples were used as normal human serum for ELISA optimization. Sera of patients having other skin diseases (NCL) were tested for determination of cross reactions of the optimized ELISA. 2.4. ELISA Protocol/Methodology Checker board titration method was used. Different quantities of antigen (i.e., 0.5?+ 2SD) based on the values obtained for 10 nonimmune serum samples. Positive control had a mean absorbance value.