This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cleary, J. D. Articles by Lobb, C. J. Search for Related Content PubMed PubMed Citation Articles by Cleary, J. D. Articles by Lobb, C. J.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, Mar 1996, 637-641, Vol 40, No. 3
Copyright © 1996 by the American Society for Microbiology. All rights reserved.

Amphotericin B enzyme-linked immunosorbent assay

JD Cleary, SW Chapman, J Deng and CJ Lobb
Department of Clinical Pharmacy, University of Mississippi Medical Center, Jackson, 39216-4505, USA.

Our purpose was to develop and characterize an enzyme-linked immunosorbent assay (ELISA) which could measure the concentration of amphotericin B in serum. Amphotericin B was assayed by competition ELISA. Multiwell ELISA plates coated with amphotericin B (1.0 micrograms/ml) conjugated to bovine serum albumin were used to test replicates of serum samples spiked with amphotericin B. Purified rabbit polyclonal antibody against amphotericin B (1.4 micrograms/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 accuracy of the assay. The ability to measure lipid-associated amphotericin B was also evaluated in preliminary studies. Analysis of reference samples containing amphotericin B yielded a traditional sigmoidal curve. The limits of detection were 0.15 to 156 micrograms/ml. The sensitivity of the assay was affected by light and temperature exposure. Assay specificity was altered only by the presence of nystatin, a polyene antifungal agent similar to amphotericin B. Intrarun (coefficient of variation = 3.0%) and interrun (coefficient of variation = 12.8%) coefficients of variation were calculated and were comparable to those in similar assays. The assay's correlation coefficient (r = 0.907) demonstrated a statistically significant correlation between the optical density of the sample and the concentration of drug in the sample. The amphotericin B ELISA's ease, precision, and overall accuracy suggest that this assay could be used for assessments of serum amphotericin B concentrations. Multiple research questions concerning the role of serum amphotericin B concentrations in toxicity and efficacy have gone unanswered because of the labor-intensive nature of the assays which have been available to date. The ability to easily and rapidly measure 40 duplicate samples containing amphotericin B should also prove to be a distinct advantage for clinical research or reference laboratories in addressing these questions.

This article has been cited by other articles:

• Machard, S., Theodoro, F., Benech, H., Grognet, J.-M., Ezan, E. (2000). A Sensitive Amphotericin B Immunoassay for Pharmacokinetic and Distribution Studies. Antimicrob. Agents Chemother. 44: 546-550 [Abstract] [Full Text]