Development and validation of an enzyme-linked immunosorbent assay for the quantification of gelonin in mouse plasma

ABSTRACT

This article details the development and validation of an enzyme-linked immunosorbent assay (ELISA) for the quantification of gelonin in mouse plasma. The ELISA was validated for intra- and inter-day variability and for accuracy over a standard curve range of 7.5–100 ng/mL. The assay was then applied to assess gelonin pharmacokinetics in mice. Results from the ELISA were compared to data obtained from a parallel study conducted with ¹²⁵Iodine-labeled gelonin, with quantification via gamma counting. The ELISA demonstrated good precision, as the percent coefficient of variation of quality control samples in intra-day and inter-day validation ranged from 5.4–9.3% and 2.9–7.3%, respectively. Sample recoveries ranged from 98.3–105% of nominal values. The ELISA method yielded lower plasma concentrations of gelonin than found from the less-specific gamma counting method. Consequently, pharmacokinetic analyses yielded significantly higher estimates for volume of distribution (106 ± 31 vs. 55.8 ± 13 mL/kg) and plasma clearance (34.7 ± 6.6 vs. 10.9 ± 2.1 mL/min/kg) for data determined by ELISA vs. by gamma counting.

KEYWORDS:

• ELISA
• gelonin
• protein toxin
• assay
• ribosome-inactivating protein
• pharmacokinetics

Funding

Funding for this research was provided by the University at Buffalo Center for Protein Therapeutics.

Additional information

Funding

Funding for this research was provided by the University at Buffalo Center for Protein Therapeutics.