255 - Development of a specific and robust electrochemiluminescence assay to measure serum darbepoetin concentrations in extremely premature infants

Friday, April 28, 2023

5:15 PM – 7:15 PM ET

Poster Number: 255
Publication Number: 255.13

Jessie Maxwell, University of New Mexico, albuquerque, NM, United States; Lauren Jantzie, Johns Hopkins University School of Medicine, Baltimore, MD, United States; Guohua An, University of Iowa, Iowa City, IA, United States; Sandra Beauman, University of New Mexico School of Medicine, Bernalillo, NM, United States; Robin K. Ohls, University of Utah School of Medicine, Salt Lake City, UT, United States

Presenting Author(s)

Jessie Maxwell, MD

Associate Professor of Pediatrics ad Neurosciences
University of New Mexico
albuquerque, New Mexico, United States

Background:

The erythropoiesis stimulating agents (ESAs) erythropoietin (Epo) and darbepoetin (Darbe) are being administered to preterm infants to stimulate red cell growth and decrease red blood cell transfusions. Darbe is a longer acting ESA compared to Epo, allowing for weekly administration. ESAs are also being evaluated for neurorepair, as previous studies have reported improved neuroprotection with hematopoietic doses of Epo and Darbe.

Objective:

To develop an assay that reliably measures serum Darbe concentrations in samples from preterm infants participating in a multicenter randomized Darbe trial.

Design/Methods:

A multi-array electrochemiluminescence Meso Scale Discovery (MSD) platform was used to develop the Darbe assay. The capture antibodies, calibrator protein, primary detection antibodies and secondary detection antibodies were evaluated from different sources and various concentrations optimized. Capture antibodies tested included the MSD Epo mouse monoclonal antibody, R&D Duoset Epo mouse monoclonal antibody, R&D AB286 rabbit polyclonal antibody, and R&D MAB2872 mouse monoclonal antibody. The calibrator standards evaluated included Darbepoetin drug (Amgen) and Epo DuoSet (R&D). Primary detection antibodies tested included R&D chicken polyclonal, biotinylated, R&D AB286 rabbit polyclonal, and R&D MAB2872 mouse monoclonal. The sulfo-tags trialed included streptavidin, anti-rabbit, and anti-mouse. Various concentrations and combinations of the above were evaluated during assay development phase.

Results:

R&D Duoset antibodies with streptavidin sulfo-tag resulted in consistent measurements of known Darbe concentrations. Darbe at a concentration of 100,000 pg/mL was diluted 4-fold and used as the standard calibrator. Participant samples are diluted based on the type of level (peak, random, trough, pg/mL) to ensure measurements are on the standard curve. The calibration curve ranges from 24.4 to 100,000 pg/mL, with a median lower limit of detection of 51 pg/mL. Both accuracy and precision of the method met the predefined acceptance criteria. Samples from infants enrolled in the trial are currently being measured.

Conclusion(s):

A specific, reliable, and accurate assay to measure Darbe levels in infant serum was developed and validated. This method has been successfully applied to quantify Darbe serum concentrations in extremely premature infants; the concentration data obtained will be used for population pharmacokinetic analysis to gain valuable insight on Darbe disposition in this vulnerable population as well as selection of the appropriate dose regimen of Darbe in infants.