Abstract
Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20μl does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods.
Keywords: Homogeneous assay, homogeneous time resolved fluorescence, europium cryptate, histamine
Combinatorial Chemistry & High Throughput Screening
Title: Homogeneous Time Resolved Fluorescence Assay to Measure Histamine Release
Volume: 6 Issue: 8
Author(s): Emmanuel J. Claret, Josy Ouled-Diaf and Patrick Seguin
Affiliation:
Keywords: Homogeneous assay, homogeneous time resolved fluorescence, europium cryptate, histamine
Abstract: Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20μl does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods.
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Cite this article as:
Claret J. Emmanuel, Ouled-Diaf Josy and Seguin Patrick, Homogeneous Time Resolved Fluorescence Assay to Measure Histamine Release, Combinatorial Chemistry & High Throughput Screening 2003; 6 (8) . https://dx.doi.org/10.2174/138620703771826892
DOI https://dx.doi.org/10.2174/138620703771826892 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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