C3NeF detection in serum samples was performed as described previously by Paixão- Cavalcante and collaborators [24 (link)] with several modifications. Briefly, residual Bb was detected using a monoclonal anti-Bb antibody (A227, Quidel) (1:500; 1 h, 37 °C), followed by peroxidase-conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) (1:5000; 1 h). Color was developed using o-phenylenediamine dihydrochloride (Sigma-Aldrich, Madrid, Spain) and absorbance was measured at 492 nm. Samples were considered positive when optical density was higher than 0.3 units of absorbance.
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