Search for diagnostic and prognostic biomarkers in Stroke.

The search for diagnostic and prognostic biomarkers in Stroke includes the study of:

-         molecules playing a role in ischemic stroke such as endogenous fibrinolysis, apoptosis or neogenesis

-         the mechanisms of hemorrhagic transformation after stroke mediated by matrix metalloproteinases

-         the molecular mechanisms of activation of the MMP9 by t-PA

-         the identification of markers that permit an “analytic” diagnostic of stroke

-         the biomarkers for growth and development of edema in hemorrhagic stroke and the predictors of recurrence

-         protein arrays in cerebral microdialyse and studies in the human cerebral parenquima (infartoteca).

Some of these findings might have therapeutic implications since biological markers as MMP-9 are well associated with Blood Brain Barrier disruption. In this direction, we have described MMP-9 predicting haemorraghic complications among patients receiving thrombolytic treatment. This fact might contribute to increase safety of reperfusion treatments. The impact of this research line is clear, since articles like this (Circulation 2003) have citacion index > 40 since its recent publication.

 The study of these molecules will also have diagnostic implications because we have proposed the biochemical diagnostic of stroke by means of the identification of a biomarkers panel that distinguish between a stroke and other stroke-mimicking conditions. All these results have been possible since the development of a “blood library” including more than 2000 stroke samples. We have also studied polymorphisms in many of these markers.

Also regarding MMPs and from a technical point of view, we have incorporated new study protocols. That is the case of “in situ zymography”, that will allow measuring gelatinases activities in cerebral parenchyma samples. We have optimized the method by working with stroke brain samples since we have created the first “brain infarct library” in our country as commented later in detail. The first studies with these samples confirm a different contribution of MMP-9 in the core and the penumbra infarcted area and by complementary techniques we demonstrated a different production by neurons, microglia and endothelial cells; and their contribution in the destruction of the Blood Brain Barrier barrier. We are the only group in our country with an available platform (SearchLight tech) to conduct MMPs protein arrays determinations. Moreover, among patients with malignant Middle Cerebral Artery infarctions, we have demonstrated the pass of MMP-2, MMP9 and their dimers (>200 kda) through microdialysis membranes and therefore the possibility of monitoring MMPs using these techniques.

SEARCHLIGHT

For the quantitative and qualitative analysis of different biomarkers studied in our laboratory,  such as the metalloproteinases, we use different techniques like ELISAs, immuno-analysis techniques used since the 70s and which permit the quantification of a multitude of proteins, in a more or less fast manner. In the last years appeared new technologies, based on the basically principles of ELISAs, that allow us to determine various protein at the same time, called “multiple ELISAs” or “protein arrays”. In our laboratory, we have been using these new technologies and we actually dispose of the SEARCHLIGHT system (ENDOGEN, Pierce Biotechnology).

1. Principle of the technique.

The SearchLight multiplex assay is a protein array based on the same principle as the ELISA sandwich, where we can analyse up to 16 different antibodies, bound to a 96-wells ELISA plate.

2. Technique.

This technique is simple and very similar to the ELISAs. The samples and standards are added to the wells and the proteins present in the samples will bind to the appropriate antibodies. We add a conjugate of streptavidin (streptavidin-horserardish peroxidase, SA-HRP) and a substrate (SuperSignal ELISA Femto Chemiluminescent Substrate). An image can be read through spots emitting chemiluminescence. The intensity of each spot ca be integrated when comparing with the references and the standard curve, in order to quantify the proteins of the samples.

3. Advantages

-5 to 50  ul  of sample is enough to analyse

  

 

-the researches obtain trustworthy results since it is not necessary to freeze and unfreeze the samples various times

 

-The SearchLight uses a substrate (SuperSignal ELISA Femto Chemiluminescent Substrate) that augments 2 to 10 times the sensibility compared to standard ELISAs

 

      -The specific software ArrayVision^TM permits to obtain better results.

 

 

 

Computer Software

 

    ArrayVision^TM is the software used to quantify the samples. It offers more that 8 curves for each molecule and automatically adjusts the samples results. The mean and standar deviations and automatically calculated and the data can be saved as a Microsoft Excel file, permitting the validation of results obtained.

 

Services

 

For the moment, we can analyse 125 human proteins and also various animal species. Different arrays are commercially available (angiogénesis, metalloproteinases, inflammation, TH1-TH2, etc), but it is also possible to develop new arrays. See protein arrays availables.

 

      We offer access to our technology to other groups. For more information, please refer to the “contact” section of this website.

       

It is equivalent to perform 16 ELISAs in 2 hours and in a unique assay.