# Diagnosis of the Multiepitope Protein rMLEEISH3 for Canine Visceral Leishmaniasis
## 3 Results and Discussion
### Diagnostic Performance of rMELEISH3 Antigen in Detecting CVL  
The evaluation of the antigenic potential of rMELEISH3 in the serodiagnosis of CVL was performed using a proprietary ELISA protocol and serum samples from previously characterized dogs. The results of the preliminary ELISA experiments (Figure 3) show that the rMELEISH3 antigen was efficient in discriminating between serum samples positive and negative _for L. infantum_ at all concentrations (100 to 400 ng per well). The accuracy of antibody detection _against L. infantum_ was determined from ROC curves generated for each antigen concentration (Figure 4), all showing sensitivities greater than 87.5% and specificities of 100%. However, assays performed with 100 ng of rMELEISH3/well showed slightly superior performance (AUC = 0.969) in terms of sensitivity and specificity compared to the other concentrations, and this antigen concentration was selected for subsequent ELISA experiments. Figure 2: SDS-PAGE analysis (12%) of the recombinant rMELEISH3 protein after affinity chromatography purification. The eluted fractions (E1–E5) show a main band migrating slightly below 45 kDa, which corresponds to the recombinant protein. Although this migration differs slightly from the expression profile in Figure 1A, the protein identity was confirmed by anti-His Western blot (Figure 1B). Abbreviations: MM, molecular marker; FT, continuous flow; L, wash fractions; E, elution fractions. Figure 4: Receiver Operating Characteristic (ROC) curves for preliminary enzyme-linked immunosorbent assays (ELISA) performed according to an in-house protocol with four rMELEISH3 protein concentrations (100, 200, 300, and 400 ng/well) and serum samples from _L. infantantum-infected_ (n = 16) and uninfected (n = 8) dogs. Cutoff values, area under the curve (AUC), sensitivity (SEN), and specificity (SPC) were determined for each antigen concentration. Analyses were performed using GraphPad Prism 5.0 software. Figure 3: Results of preliminary enzyme-linked immunosorbent assays (ELISA) performed according to an in-house protocol with four concentrations of rMELEISH3 protein (100, 200, 300, and 400 ng/well) and serum samples from _Leishmanian infantum-infected_ (n = 16) and uninfected (n = 8) dogs. Absorbances were recorded at 450 nm using a Multiskan(tm) FC microplate photometer, and cutoffs (shown by dashed lines) were determined for each antigen concentration. Abbreviations: AUC, area under the curve; SEN, sensitivity; SPC, specificity. Multiepitope recombinant proteins are promising alternatives for the serological diagnosis of CVL due to their high sensitivity even in asymptomatic dogs, as previously observed by [PERSON] et al. [33]. The results obtained with rMELEISH3 showed that the performance of this antigen is comparable to that of other recombinant proteins described in the literature for the diagnosis of CVL. For example, the chimeric protein ChimB, composed of B-cell epitopes originating from four _L. infantum proteins_, achieved 100% sensitivity and specificity in ELISA tests performed with human and canine samples [34]. Similarly, the recombinant _L. infantum protein rLc36_ used at a concentration of 1.0 \(\mathrm{\SIUnitSymbolMicro\SIUnitSymbolMicro mL}\) showed 85% sensitivity and 71% specificity in an ELISA test, and was able to differentiate positive and negative serum samples with 76% accuracy [35]. Furthermore, the rMELEISH protein characterized by [PERSON] et al. [17] achieved 100% sensitivity and specificity, while the LiHyQ protein _from L. infantum_ was reported as a promising diagnostic tool for CVL, even in asymptomatic dogs [36]. These examples highlight the potential of multiepitope antigen constructs in the development of highly sensitive and specific diagnostic tests for CVL.