AMPHOTERICIN-B ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Our purpose was to develop and characterize an enzyme-linked immunosor bent assay (ELISA) which could measure the concentration of amphoteric in B in serum, Amphotericin B was assayed by competition ELISA, Multiw ell ELISA plates coated with amphotericin B (1.0 mu g/ml) conjugated t o bovine serum albumin were used to test replicates of serum samples s piked with amphotericin B, Purified rabbit polyclonal antibody against amphotericin B (1.4 mu g/ml) was added subsequent to the instillation of samples spiked with unknown amounts of amphotericin B. Experiments were performed to test the sensitivity, specificity, precision, and a ccuracy of the assay, The ability to measure lipid-associated amphoter icin B was also evaluated in preliminary studies, Analysis of referenc e samples containing amphotericin B yielded a traditional sigmoidal cu rve. The limits of detection were 0.15 to 156 mu g/ml. The sensitivity of the assay was affected by light and temperature exposure. Assay sp ecificity was altered only by the presence of nystatin, a polyene anti fungal agent similar to amphotericin B. Intrarun (coefficient of varia tion = 3.0%) and interrun (coefficient of variation = 12.8%) coefficie nts of variation were calculated and were comparable to those in simil ar assays, The assay's correlation coefficient (r = 0.907) demonstrate d a statistically significant correlation between the optical density of the sample and the concentration of drug in the sample, The amphote ricin B ELISA's ease, precision, and overall accuracy suggest that thi s assay could be used for assessments of serum amphotericin B concentr ations, Multiple research questions concerning the role of serum ampho tericin B concentrations in toxicity and efficacy have gone unanswered because of the labor-intensive nature of the assays which have been a vailable to date, The ability to easily and rapidly measure 40 duplica te samples containing amphotericin B should also prove to be a distinc t advantage for clinical research or reference laboratories in address ing these questions.