Fluorescence polarization immunoassay based on fragmentary hapten for rapid and sensitive screening of polymyxins in human serum

Abstract

Polymyxin B (PMB) and polymyxin E (PME, or named colistin) are old polypeptide antibiotics and recently reintroduced as the last-line therapy for serious infections caused by multidrug-resistant Gram-negative pathogens. A rapid and reliable screening method for PMB and PME in human serum is urgently required due to their narrow therapeutic windows and individual differences. To develop fluorescence polarization immunoassay (FPIA) with high sensitivity and uniform specificity for PMB and PME, one novel fragmentary polymyxins hapten, named H1, was designed only containing the fatty acid acylated tripeptides and intendedly introducing sulfhydryl group for selective conjugation purpose. The H1 induced significantly higher titer and affinity antibody response than the PMB as hapten and produced five monoclonal antibodies (mAbs) with IC50 of 1.8–6.6 ng/mL for PMB and cross-reactions of 75.0–105.0 % for PME. To investigate the influence of tracer structure on the FPIA sensitivity, 12 tracers differing in spacer arm lengths, fluorophore types and hapten structures were fully paired with five mAbs, showing a safety length of spacer arm ranged from 4 to 17 Å. Under optimal conditions, the IC50 of FPIA was 3.7 ng/mL in buffer with a limit of detection of 1.4 μg/L in human serum for PMB and PME. The recoveries in spiked human serum were 73.8–91.8 % with CVs less than 12.8 % and whole assay time less than 25 min including sample preparation. These results showed that the FPIA was an efficient, accurate, and sensitive method for rapid screening of polymyxins and suitable for therapeutic drug monitoring.