Hapten Synthesis, Antibody Generation, and Immunoassay Development for Linezolid Therapeutic Monitoring.

Abstract

Linezolid (LNZ) is a broad-spectrum antibiotic used for the treatment of severe Gram+ infections. Significant pharmacokinetic variability in different groups of patients requires therapeutic drug monitoring (TDM) to ensure optimal therapy. This article presents the first description of the development of immunoanalytical TDM systems for LNZ. Four LNZ derivatives (H1-H4) with spacer arms of a different length (0-6.2-9.4-11.8 Å) and chemistry (aliphatic and heterocyclic) were synthesized to prepare H1-H3-immunogens and H1/H4-coating antigens or H1/H2-enzyme tracers. Three distinct antibody types against H1-H3 haptens were generated in rabbits and served as the basis for the development of ELISAs in heterologous coated antigen and coated antibody formats. The length of the spacer in the immunogen had an insignificant effect on the quality of the antibody generated or the performance of the designed coated antibody-ELISA formats (IC50 = 0.48-0.75 ng/mL) and coated antigen ELISAs (IC50 = 11.5-28.3 ng/mL). The coated antigen-based assays were considered more appropriate for monitoring therapeutic levels of LNZ due to their more convenient reduced sensitivity but wider dynamic range than the coated antibody-based assays (2.0-160 vs 0.16-2.1 ng/mL). The best recovery of LNZ (82.1-99.6%) from spiked human serum among the sample pretreatments examined was provided by the trichloroacetic acid deproteinization procedure. Serum samples derived from surgical patients with superimposed Gram-positive infections treated with LNZ were evaluated by parallel ELISAs using different immunoreagents. The results obtained in the various assays were found to be concordant, thereby confirming the reliability of the measurements and the developed assay systems suitable for TDM purposes.