Development of ELISA formats for polymyxin B monitoring in serum of critically ill patients

Treating infections in critically ill patients often requires the use of last-line antibacterial drugs such as polymyxins with a narrow therapeutic window and high toxicity. In critically ill patients, the drug pharmacokinetics changes significantly, and as a result, the antibiotic concentrations in blood and infection foci become suboptimal, which leads to therapeutic failures or toxic manifestations. For timely dosage adjustments, a competitive ELISA-based method using antibodies to polymyxin В (PMB) was developed. Among the several considered assays, a direct antibody-coated format was selected for its short duration (1.5 h) and the best agreement with the LC–MS/MS data (R2 = 98 %). The assay dynamic measurement range (IC20-IC80) could be substantially shifted by changing the ratio of immunoreagents. To conveniently measure the therapeutic range of PMB concentrations, it was adjusted to 5.0–192 ng/mL, allowing the samples to be analyzed after a simple 100-fold dilution with the assay buffer. The ELISA sensitivity expressed in half-inhibition concentration (IC50) and the limit of detection were 30.6 and 1.8 ng/mL, respectively. The assay cross-reactivity towards the related analogue colistin (COL) was 95 %, and this compound could also be adequately quantified by the same assay. The PMB and COL recovery from the spiked serum samples was similar and constituted 98–109 %. The trial drug monitoring was carried out in 3 patients with Gram-negative sepsis, and the established pharmacokinetic profiles of PMB revealed the necessity for individual dosage adjustment.