Category: Assay Development and Screening

1164-E - Improving Throughput of an Unbound Bilirubin Assay: Microplate Based Assay Development of a Near Infrared Fluorescent Detection Assay on the CLARIOstar® for High Throughput Using the PHERAstar

Wednesday, February 7, 2018
11:30 AM - 12:30 PM

Bilirubin is a byproduct of hemoglobin turnover which is usually associated with albumin in plasma. However, in the case of hyperbilirubinemia, free bilirubin (Bf) can be present in the plasma and this Bf can pass through the blood-brain barrier and elicit neurotoxic effects. Recognizing that a direct measurement of Bf is much more useful in determining the risk of encephalopathy; Fluoresprobe Sciences have worked to design fluorescent assays for this purpose using fatty acid binding proteins (FABP) that are labeled with a fluorescent markers. Recently Fluoresprobe Sciences reported the development of a point of care device that employed a Bf sensitive FABP labeled with LICOR 700 DX and a Bf insensitive FABP labeled with LICOR 800 CW. The use of near infrared dyes provides a system that is ideal for use with undiluted blood where the LICOR 700 DX signal is quenched in the presence of Bf while the LICOR 800 CW should be unchanged. In order to improve throughput we sought to develop an assay that takes advantage of the simultaneous dual emission detection that is available on the PHERAstar line of microplate readers. To assist in the development of a suitable optic module the spectral scanning capabilities of the CLARIOstar equipped with a red-shifted PMT were employed. Excitation / emission scanning was performed on both fluorescently labeled FABP’s. Comparing both of these scans allowed us to select optimal components for a dual emission optic module. Finally tests were run on both the CLARIOstar and PHERAstar FSX to show that the expected responses to changing levels of Bf were observed. These tests both confirmed the performance of the optimized optic module and exemplified the outstanding sensitivity of the CLARIOstar that make it comparable to the PHERAstar and thus the ideal reader for this assay development.  

Carl Peters

Senior Applications Scientist
BMG Labtech
Cary, NC

Dr. Carl Peters is a microplate reader Senior Application Scientist with BMG LABTECH. He obtained a PhD in Cell and Molecular Biology from Northwestern University while studying Protein Kinase C signaling. He also has a B.S. in Biology from Hastings College. Prior to BMG LABTECH, he was an adjunct or assistant professor of Biology, Biochemistry and Molecular Biology at several different universities.