Spectramax m2e scanner

SOD activity in the serum samples of HC, NSVM and SVM was measured using a commercially available kit (Cayman Chemical, USA) following the manufacturer’s instructions. In brief, 10 μL of standard solution and samples (in duplicates) were added in the designated wells on a 96-well plate along with 200 μL of the diluted radical detector. Reactions were initiated by addition of 20 μL of diluted xanthine oxidase to all the wells. The plate was carefully vortexed for a few seconds and incubated on a shaker for 30 minutes at room temperature. The absorbance was measured at 440–460 nm using a SpectraMax M2e scanner (Molecular Devices, USA).

Eleven differentially abundant proteins were validated further in bigger cohorts of vivax malaria patients and controls [HC (n = 103), NSVM (n = 118), SVM (n = 34), DF (n = 26), and LEP (n = 12)] by ELISA. ELISA was performed using commercially available kits following the manufacturers’ directions: SAA, HP, Apo E, Apo A-I, HPX, RBP4, CP, and PLS (AssayPro, USA), SOD (Abcam, Cambridge, UK), TTN (CUSABIO Life science, China), and VTN (RayBiotech, Georgia, USA). Selection of the proteins for immunoassay-based validation was carried out on the basis of their fold-change, correlation with disease severity, association with malaria pathogenesis and commercial availability of the required ELISA kits. Quantitative direct/ competitive enzyme assay was carried out where standard and serum samples (malaria and controls) at a specific dilution were subjected to a microplate pre-coated with a polyclonal antibody specific for the target proteins. Optical densities were measured by using a SpectraMax M2e scanner (Molecular Devices, USA). Statistical significance of the average ratio of abundance was analyzed by Mann-Whitney U test (p < 0.05). Efficiency of the classifier proteins for prediction of NSVM, SVM and other two infectious diseases (DF and LEP) was analyzed by plotting ROC curves using GraphPad Prism software package (version 6.02).

Serum levels of TBARS in HC, NSVM and SVM were measured using a commercially available kit (Cayman chemicals, USA). Reagents and colorimetric standards were prepared according to manufacturer’s instructions. 100 μL of samples and standards (in duplicates) were added to labelled 5 mL vials containing 100 μL of SDS solution. 4 mL of color reagent was added and the vials were placed in boiling water for an hour. The vials were then immediately incubated on ice for 10 mins to stop the reaction. 150 μL from each vial was loaded onto clear plates at room temperature. The absorbance was measured at 530–540 nm using a SpectraMax M2e scanner (Molecular Devices, USA).