Amine-coated slides were prepared as described previously (50, 51). Serum samples from 112 patients infected with P. vivax malaria and 80 healthy individuals were tested against rPvMSP1-19 and rPvMSP8+1 using protein arrays. A series of double dilutions was developed to optimize the coating concentration (0.1 to 200 µg/ml) of recombinant proteins. The purified rPvMSP1-19, rPvMSP8+1, or PBS (100 µg/ml) was spotted in duplicate onto array slides and incubated for 2 h at 37°C. Each slide was blocked with 1 µl blocking buffer (5% bovine serum albumin in PBS with 0.1% Tween 20 [PBST]) and incubated for 1 h at 37°C. The chips were preabsorbed against wheat germ lysate (1:100 dilution) to block anti-wheat germ antibodies and then probed with serum samples of human malaria patients or healthy individuals (1:200 dilution). Alexa Fluor 546 goat anti-human IgG (10 µg/ml, Sigma) in PBST was used to detect antibodies, and the antibodies were scanned in a fluorescence scanner (ScanArray Express, Boston, USA). Fluorescence intensities of array spots were quantified by the fixed-circle method using ScanArray Express software (version 4.0; PerkinElmer). The cutoff value was equal to the mean plus 2 standard deviations of the mean intensity of 80 negative samples.

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