AMPHOTERICIN-B ENZYME-LINKED IMMUNOASSAY FOR CLINICAL USE - COMPARISON WITH BIOASSAY AND HPLC

OBJECTIVE: TO evaluate a new enzyme-linked immunosorbent assay (ELISA) for amphotericin B in serum samples. Results are compared with those obtained by HPLC and bioassay. DESIGN: Comparison of results obtained by ELISA, HPLC, and bioassay. METHODS: We developed a new ELISA using a polyclonal rabbit antibody to measure serum amphotericin B concentra tions, Blinded samples of amphotericin B in concentrations of 0.15-78 mu g/mL were prepared in human serum and assayed simultaneously by the ELISA, HPLC, and bioassay. The results of each assay were derived fro m standard curves and evaluated by using the Table Curve 2D computer p rogram. These data were compared by using correlation analysis with ev aluation of Pearson's correlation coefficient by Student's t-test. RES ULTS: ELISA and bioassay compared favorably at amphotericin B concentr ations of 0.3-20 mu g/mL with a correlation coefficient of r = 0.993, while ELISA and HPLC compared with a correlation coefficient of r = 0. 944. The average coefficient of variation over the range 0.3-20.0 mu g /mL was 28% +/- 7% for HPLC, 26% +/- 9% for ELISA, and 13% +/- 4% for bioassay. Comparison of all three assays revealed the highest correlat ion with the ELISA assay (r = 0.998) for the range of concentrations ( 0.3-20 mu g/mL) routinely achieved. Samples containing concentrations in excess of 20 mu g/mL could be diluted. Desiccation for concentratio ns less than 0.3 mu g/mL was not tested. CONCLUSIONS: The determinatio n of serum amphotericin B concentrations by ELISA gave results similar to those obtained by a bioassay and HPLC technique. Although variabil ity appears greater with ELISA, the ease of performing this assay expe dites the evaluation of many samples, Finally, this assay may allow th e determination of amphotericin B concentrations from lipid formulatio ns without interference from coadministered antibacterials or azole an tifungals.