Construction of a <i>B</i>. <i>pseudomallei</i> protein microarray.

<p>The protein array was spotted with 20 different <i>B</i>. <i>pseudomallei</i> proteins and further internal positive and negative controls, for a total of 445 protein spots. All proteins were applied in triplicate and at five dilutions (0.01 mg/ml to 0.45 mg/ml) on glass slides, including human IgG and IgM controls and further other internal controls, i.e., murine IgG and IgM, bovine IgG, porcine IgG, caprine IgG and ovine IgG controls. All protein arrays were incubated <b>A</b> only with anti-human-IgG antibodies (empty control), <b>B</b> with melioidosis-positive or <b>C</b> melioidosis-negative blood sera or blood plasmas at 1:1000 dilutions (<b>A, B</b> and <b>C</b> are representative images). IgG antibodies bound to <i>B</i>. <i>pseudomallei</i> antigens were detected using horseradish-peroxidase (HRP) linked secondary anti-human-IgG antibodies and 3,3’,5,5’-tetramethyl-benzidine (TMB), which caused a blue precipitate. Protein arrays were read out by the ArrayMate (Alere Technologies GmbH, Germany). Highlighted are human IgG controls (green rectangle) and horseradish-peroxidase controls (red rectangle); all other controls are not shown.