Validation of a few differentially expressed proteins was performed using different immunoassay-based methods including immunoturbidimetric assay, ELISA and western blotting to confirm the results of proteomic analysis. Haptoglobin and apolipoprotein A-I (Apo A-I) concentrations were directly quantified turbidimetrically in the serum samples of malaria patients (n = 37), healthy subjects (n = 20) and febrile controls (n = 6). Compared to the healthy controls, both FM and VM patients found to have lower serum level of haptoglobin and Apo A-I (p,0.0001 in a Mann-Whitney test) ( Figure 3A and B). The mean haptoglobin concentration was found to be 0.20860.048 and 0.33360.06 g/L in FM and VM patients respectively, while the healthy and leptospirosis (FC) populations exhibited a mean values of 0.91860.1 g/L and 0.88860.056 g/L (mean 6 SE). Likewise, Apo A-I exhibited more than three times lower mean value in both the malaria patients compared to the healthy subjects (39.3965.43, 43.1964 .96 and 137.0565.33 mg/dL in FM, VM and HC respectively). While the febrile controls shown a mean value of 76.5264.12 mg/dL, which is around 2-fold higher than the malaria patients. Serum retinol-binding protein (RBP) concentration was measured by sandwich ELISA. The serum levels of RBP was found to be significantly (p,0.01) lower in malaria patients ( Figure 3C ). Western blot analyses of four differentially expressed targets proteins; haptoglobin, serum amyloid A, clusterin and retinol-binding protein were performed on a subset of control [HC (n = 12) and FC (n = 6)] and diseased samples [FM and VM (n = 12 each)] ( Figure 3D ). CBB staining of the SDS-PAGE gels and Ponceau staining of the transferred blots containing the resolved proteins indicated equal loading of the samples in each lane ( Figure S5 ). Western blot analysis showed up-regulation of serum amyloid A and downregulation of haptoglobin, clusterin and retinol-binding protein in FM and VM patients (p,0.01) compared to the healthy and febrile controls. These results confirmed our findings from the proteomic analysis, and are illustrated graphically in Figure 3E .