A vote for robustness: Monitoring serum enzyme activity by thin-layer chromatography of dabsylated bradykinin products

Neuropeptide reporter assay for serum, capillary blood and blood cards

“…This MethodX article supplements work were we show the use of dabsylated substance P (DSP) as a neuropeptide reporter substance (NRS) for the monitoring of protease activity (PA) in serum [1] . This article is based on the assay developed for the use of dabsylated bradykinin (DBK) for the same purpose [2] , which was already employed in a clinical study [3] . Both peptides are of importance in research on inflammation and pain [4] .…”

“…Both peptides are of importance in research on inflammation and pain [4] . We have, therefore, evaluated the application of the assay with respect to other types of sampling such as capillary blood and blood cards in order to extend the application range of the assay, which was only used with serum so far [ 1 , 2 ]. Sampling thus becomes much easier, because little blood is needed, and outpatient studies are feasible as no trained personnel is necessary to prepare the blood cards.…”

“…An early extensive protocol for DBK-PAA is available for the interested user [6] . It was however modified and simplified later [2] . DSP-PAA [1] differs slightly from DBK-PAA and more adaptations were required for fresh and dried capillary blood.…”

“…DSP-PAA [1] differs slightly from DBK-PAA and more adaptations were required for fresh and dried capillary blood. Following initial in-house synthesis of DBK and DSP [ 1 , 2 ] we now order the labeled peptides from Peptide Specialty Laboratories (Heidelberg, Germany). Also, different analysis software was tested [ 2 , 6 ]; currently we prefer JustTLC (Sweday, Sodra Sandby, Sweden, Fig.…”

“…In both cases, the solution equivalent of 1 µl blood was subsequently incubated with 1 nmol dabsylated bradykinin (DBK; 37 °C, 60 min). For further sample preparation, six times the volume of ice-cold aceton was added followed by storage at −20 °C for 1 h. The supernatant was removed and dried for separation by TLC as described [2] . The dry residue was dissolved in 1 µL ethanol and spotted onto a TLC-plate; the sample vial was rinsed with another 1 µl which was added to the same spot.…”