Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma.
A sensitive assay was developed for human epidermal growth factors (hEGF) 1-48 (dosed), hEGF 1-53 (endogenous), without interference from potential metabolites hEGFs 1-47 or 1-46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 microgram ml-1, although a detection limit of 75 pg ml-1 appears feasible.
Kagel
J R
JR
Department of Pharmacokinetics and Drug Metabolism, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Co., Ann Arbor, MI 48105, USA.
Rossi
D T
DT
Nordblom
G D
GD
Dudeck
R C
RC
Barksdale
C M
CM
Kuo
B S
BS
Wright
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000097:blood*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits
D004815:Epidermal Growth Factor