Novel enzyme-linked immunoassay to determine nanogram levels of pneumocandins in human plasma.
A binding enzyme-linked immunosorbent assay (ELISA) has been developed for measuring nanogram concentrations of semisynthetic pneumocandin antifungal agents in human plasma. Semisynthetic pneumocandin L-733,560 was conjugated to succinylated hemocyanin by water-soluble carbodiimide and was used as an immunogen to produce polyclonal antibodies in rabbits. Pneumocandins were used to directly coat the wells of a microtiter plate, and quantitation was achieved by using rabbit polyclonal antibodies to pneumocandin L-733,560 and goat anti-rabbit immunoglobulin G conjugated to either alkaline phosphatase or horseradish peroxidase. Maximum binding of L-733,560 and most related analogs to the wells of the microtiter plate was found to occur in the first 5 min of incubation at 4 degrees C. Once bound to the plate, these pneumocandins could not be removed from the plate, either by treatment with 4.0 to 6.0 M urea or by treatment with 4.0 to 6.0 M guanidine hydrochloride for 24 h at 4 degrees C. The binding ELISA is linear with drug concentration and can detect levels of L-733,560 as low as 5 ng/ml in human plasma. The assay is also useful for quantitating plasma levels of related semisynthetic pneumocandins including clinical candidate MK-0991.
Karkhanis
Y D
YD
Department of Parasite Biochemistry & Cell Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA. yashkarkhanis@merck.com
Schmatz
D M
DM
D000818:Animals; D000900:Anti-Bacterial Agents / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D000935:Antifungal Agents / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D004306:Dose-Response Relationship, Immunologic; D054714:Echinocandins; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D010455:Peptides; D010456:Peptides, Cyclic / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D011817:Rabbits
D000900:Anti-Bacterial Agents; D000906:Antibodies; D000935:Antifungal Agents; D054714:Echinocandins; C089367:L 733560; D010455:Peptides; D010456:Peptides, Cyclic; C079465:pneumocandin B(0)