Test development (WP3)
Aims and Overview
We have identified two areas where new tests could
be developed in order to improve high throughput phenotyping. New tests will be developed to study cardiac
arrhythmia which is a major contributor to human heart failure. In addition Luminex Multiplex-Bead-Array
technology will be further developed and tested for analysis of serum proteins.
1. Development of Luminex Multiplex-Bead-Array
platform for serum analysis
HZI, ICS & Ani.Rhône-Alpes (ENS-L)
Bastian Pasche, Andreas
Lengeling, Werner Müller,
Braunschweig, HZI, Laurent Monassier, ICS
tools gain increasing importance for the identification of biomarkers
indicative for disease development and progression. One of the ultimate
challenges is to gain more insight in systemic organismal responses by
quantifying in parallel multiple proteins and peptides associated with abnormal
physiology. This information can be subsequently be used to define the health
status of an individual. In this workpackage, we will develop new, primary
first-line phenotyping SOPs for mouse mutant lines that will allow measuring of
multiple serum proteins and peptides in a suspension array system. To
accomplish this we will use the Luminex Bead-Array System, which permits the
multiplexing of up to 100 different assays within a single sample. This system
uses a liquid suspension array of 100 sets of fluorophore-colour coded beads
coupled with individual antibodies that can be used to bind specific analytes.
Captured analytes are then subsequently quantified using a flow cytometer bead
array reader. We will use the Luminex system to develop SOPs for the detection
and quantification of proteins indicative for immune responses such as
immunoglobulins, cytokines, chemokines and acute phase proteins but also for
the measurement of other serum proteins that are important for secondary
screens as suggested by EUMODIC partners. Particular attention will be
given to adapt a number of hormonal assays to the Luminex technology. In fact
endocrine parameters are at present rarely determined because a large amount of
blood is required for adequate measurements. Multiplexing measurements will
remedy this problem.
2. Development of an arrhythmia screen in
Mamas Mamas, Elizabeth J.
Cartwright, Ludwig Neyses, Manchester; Laurent Monassier, ICS
Sudden cardiac death (SCD) contributes to
significant mortality in both acute myocardial infarction and chronic cardiac
failure, most commonly as a consequence of ventricular tachyarrhythmias.
SCD accounts for approximately 60-70% of deaths in patients with NYHA class II
congestive cardiac failure with rhythm disturbances being a major
component. It is estimated that SCD accounts for 400,000-500,000 deaths in
Europe each year. Screening for mutations which enhance
the propensity to rhythm disorders would therefore add significant value to any
large-scale mutational programme focussed on disease mechanisms.
At present, no thoroughly validated procedures for
rhythm disorders are available for use in primary screening. Injection of
pro-arrhythmic substances in mice has been used for years, but their use has
largely been empirical and has not been standardised. We therefore suggest the
development and standardisation of such tests in EUMODIC in order to identify
mutations with high susceptibility to arrhythmias.
Screening techniques will be developed to measure
QT duration, a major determinant of ventricular arrhythmias, by surface ECG.
These will identify animals with 'long-QT syndrome', one of the hallmarks of
both monogenic and polygenic arrhythmogenic diseases. The screening test will be fully optimised
and a standard operating procedure (SOP) will be instituted. Analysis will be carried out in mice under
basal conditions and under beta-blockade, which often unmasks latent QT
prolongation, particularly in mice whose heart rate is mostly controlled by
Pharmacological screening methods will be developed; under ECG
monitoring a variety of pro-arrhythmic substances including adrenaline (under
parasympathetic blockade) and ouabain (a digitalis-like compound) will be
injected to promote intracellular calcium overload and predispose mice to
rhythm disturbances. Calcium overload is known to contribute to ventricular
arrhythmias in cardiac failure, long-QT syndrome and other conditions.
Adrenaline and ouabain have been used previously, however, careful
dose-response curves and variability of sex- and strain-specific responses must
be established prior to use in primary screening. An SOP will be established for the procedure.
Overall, this development will lead to a highly
standardised screening method for ventricular arrhythmias, which belong to the
most frequent and dangerous manifestations of cardiovascular disease.