HTRF Histamine Dynamic Detection Kit, 96 Assay Points

Assay principle

The kit is based on a competitive format involving a specific antibody labelled with Cryptate (donor) and histamine coupled to d2 (acceptor). Histamine produced by cells competes with d2-labelled histamine for binding to monoclonal anti-histamine Eu Cryptate.

Assay protocol

The histamine assay features a streamlined protocol with only two incubation steps: - Sample with acylation buffer and acylation reagent - Histamine detection using HTRF reagents. This protocol requires a single, three-hour incubation period following the acylation step.

Key features

Histamine detection upon allergen stimulation

Four independent blood samples (Donor A to D) were used to measure the spontaneous release of histamine, as well as upon stimulation with 5 allergen mixes including 6 grass mix, house dust mite, cat epithelium, inhalant mix and common ragweed allergen. ​*The anti IgE antibody (Ref # MABETS07) used as a positive control in the experiment can be purchased separately

Histamine detection upon REBL-2H3 stimulation

Rat basophils (RBL-2H3) were cultured in the presence or absence of 2.5 µM ionomycin to stimulate histamine release. Total histamine content was determined by lysing cells and spontaneous release was evaluated by incubating the cells in medium only. Histamine detection was performed following 30 minute cell stimulation at 37 ºC using the standard HTRF histamine assay protocol.

Histamine detection upon PBMC stimulation

Similar studies were performed using human peripheral blood basophils from two individual donors. Non-specific release of histamine using Ionomycin was compared to specific degranulation induced by cross-linking a membrane-bound IgE receptor with anti-IgE. Histamine detection was performed using the standard HTRF histamine assay protocol following 30 minutes of stimulation of peripheral blood basophils at 37ºC. Error bars represent a standard deviation of 3 replicates.