An immunoassay to evaluate human/environmental exposure to the antimicrobial triclocarban

Abstract

A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclocarban (TCC) was developed. The haptens were synthesized by derivatizing the para position of a phenyl moiety of TCC. The rabbit antisera were screened and the combination of antiserum 1648 and a heterologous competitive hapten containing a piperidine was further characterized. The IC(50) and detection range for TCC in buffer were 0.70 and 0.13-3.60 ng/mL, respectively. The assay was selective for TCC, providing only low cross-reactivity to TCC-related compounds and its major metabolites except for the closely related antimicrobial 3-trifluoromethyl-4,4'-dichlorocarbanilide. A liquid-liquid extraction for sample preparation of human body fluids resulted in an assay that measured low part per billion levels of TCC in small volumes of the samples. The limits of quantification of TCC were 5 ng/mL in blood/serum and 10 ng/mL in urine, respectively. TCC in human urine was largely the N- or N'-glucuronide. TCC concentrations of biosolids measured by the ELISA were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid, inexpensive, and convenient tool to aid researchers monitoring human/environmental exposure to TCC to better understand the health effects.

FIGURE 1

Design of immunizing haptens and their synthetic outline. Arrows on TCC structure represent sites where linkers are introduced. The chlorines at these sites are replaced by a hydrocarbon linker containing a functional –COOH group. In the isocyanate reaction scheme, R₁ and R₂ represent a hydrocarbon linker position.

FIGURE 2

ELISA inhibition curves for TCC determined on five different plates. Each symbol respresents the mean absorbance from 4 wells. Error bars represent the error on the mean absorbance of 5 analyses at each concentration. Some errors are smaller than the symbols. Sigmoidal fit for quadruplicate × n, n=5), Chi² = 0.337, R² = 0.998. Linear detection range = 0.10 – 3.6 ng/mL; low detection limit (IC₁₀) = 0.03 ng/mL; high detection limit (IC₉₀) = 10 ng/mL.

FIGURE 3

Comparison of TCC levels in urine following hydrolysis measured by LC-MS/MS and immunoassay. Each point represents the data from a single individual at each time point. Error bars are the standard deviation on 3 replicate analyses. Participants showered for 15 min.