Fig 1.
Diagram of data analysis plan.
Table 1.
Leishmania peptides previously identified by mass spectroscopy in individual urine samples of patients with VL from Brazil, Kenya and India.
Fig 2.
SDS-PAGE (A) and Western blot (B) analysis of purified recombinant Ld-mad1. Recombinant protein containing His-tag amino terminal residues were expressed in E. coli BL21(DE3)pLysS followed by purification by affinity chromatography using Ni-NTA agarose matrix. Purity was evaluated by SDS-PAGE (4–20% gradient polyacrylamide gel) Coomassie blue staining (A) and by Western blot using anti-His-tag mAb (B). Arrows point to a protein band with MW that matches the deduced MW of Ld-mad1. Several other bands of higher MW are seen in the Western blot; since these bands are revealed with an anti-His tag mAb they are possibly aggregates or polymers of Ld-mad1.
Fig 3.
Immunochemical detection of Ld-mad1 in L. donovani amastigotes.
Validation of Ld-mad1 as a genuine protein produced by L. donovani was performed by Western blot using amastigote and promastigote cell lysates from the parasite. L. donovani-infected macrophage DH82 was used as the source of the amastigote forms of the parasite. The antibody was a pool of three mAbs specific for Ld-mad1. rLd-mad1, purified recombinant protein; MWM, Markers and respective MWs; Lane 1, crude extract of macrophage DH82 infected with L. donovani; Lane 2, crude extract of non-infected macrophage DH82; Lane 3, crude extract of L. donovani promastigotes. The black arrow points to Ld-mad1 recombinant protein band recognized by the mAbs. A protein band of the same MW as Ld-mad1 that is present only in the cell lysate of L. donovani infected macrophages. No bands that correspond to the MW of Ld-mad1 were seen in membranes blotted with the same amastigote and promastigote cell lysates and probed with an irrelevant mAb.
Fig 4.
Mapping of epitope recognition by IgG mAbs specific to the leishmanial biomarkers Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1.
IgG mAbs were purified from supernatants of cloned hybridoma cells that recognized different peptides on the leishmanial biomarkers and were subsequently biotin labeled. Mapping of epitope recognition was performed by direct ELISA using plates coated with synthetic purified 20mer peptides covering the entire full length of each biomarker and overlapping by 10 amino acids. Letters in red and blue represent a sequence of 10–15 amino acids or the epitopes that were recognized by the indicated mAbs. Blue brackets illustrate the pair of antibodies that were selected as the capture and detecting reagents of the assembled capture ELISAs.
Table 2.
Number of generated hybridoma clones producing mAbs specific for different epitopes in each leishmanial biomarker.
Fig 5.
Determination of the limit of detection (LOD) of capture ELISAs assembled to individually detect the protein biomarkers Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1 spiked in urine samples of normal healthy subjects.
Capture mAbs at previously determined concentration of 200 ng/well were used to coat the ELISA plates that were prepare to detect each biomarker individually. Wells were then incubated with various concentrations of the antigen diluted in urine from normal healthy subjects followed by incubation with biotin labeled developer mAb (2 μg/ml). Reactions were developed after addition of streptavidin labeled peroxidase, substrate (H2O2) and the chromophore TMB. Results are expressed as OD read at 450nm. Note that for all six assays the LOD was ≤45 pg/ml of biomarker.
Fig 6.
Capture ELISA for detection of Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1 in urine of VL patients and local healthy controls from Brazil.
ELISA was assembled as indicated in Fig 5. Urine samples were VL patients (n = 24) and healthy control subjects (n = 10). (A) Results represent the sensitivity of the assays performed to detect each individual biomarker. (B) Is a representation of the combined best results obtained for each of the urine samples that was positive (or not) with at least one of the individual biomarker assays. Red solid lines represent the cutoff values, which were calculated using the average of the OD obtained from the urine of normal healthy control subjects + 3 SD. These are representative results of at least three experiments performed at different times with the same urine samples and same capture ELISA.
Fig 7.
Capture ELISA for detection of Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1 in urine of VL patients and local healthy controls from Kenya.
ELISA was assembled as indicated in Fig 5. Urine samples were from VL patients (n = 45) and healthy control subjects (n = 24). (A) Results represent the sensitivity of the assays performed to detect each individual biomarker. (B) Is a representation of the combined best results obtained for each of the urine samples that was positive (or not) with at least one of the individual biomarker assays. Solid red lines represent the cutoff values, which were calculated using the average of the OD obtained from the urine of normal healthy control subjects + 3 SD. These are representative results of at least three experiments performed at different times with the same urine samples and same capture ELISA.
Table 3.
Frequency of individual leishmanial biomarkers detected by capture ELISA in 24 urine samples obtained from VL patients from Brazil.
Table 4.
Frequency of individual leishmanial biomarkers detected by capture ELISA in 45 urine samples obtained from VL patients from Kenya.
Fig 8.
Initial clinical validation of a multiplexed assay for the diagnosis of VL from Brazil and from Kenya.
ELISA plates were coated with a pool of affinity purified antibodies specific for all six biomarkers (Li-isd1, Li-txn1, Li-ntf2, Ld-mao1, Ld-ppi1 and Ld-mad1) followed by blocking and overnight incubation with urine samples from VL patients from Brazil (n = 24), VL patients from Kenya (n = 45) and urine samples from non-VL patients with the following diseases: CL (cutaneous leishmaniasis), n = 6; CD (Chagas disease), n = 6; Sch (schistosomiasis), n = 6; and TB (tuberculosis), n = 12. In addition, urine from 35 healthy control subjects was also included. Plates were washed and wells were incubated with a second pool containing biotinylated mAbs specific for the six leishmanial antigens. Wells were then incubated with streptavidin labeled peroxidase, the substrate H2O2 and the chromophore TMB. OD was then read at 450nm. The dashed red line represents the cutoff value (0.26) calculated as described in the legend of Fig 6. This is a representative result of at least three experiments performed at different times with the same urine samples.