BLT Stages

The purpose of this research is to have a more purified sample with Avastin® at the end of the workflow, so that a better sensitivity with the LC-MS is created. To achieve this aim, the standard workflow is adjusted.

The standard workflow exists out of protein A immuno-enrichment, Trypsin/Lys-C digestion, peptide clean-up with C18 tips and suspension step. Three different ways to reach this aim will be tried. The first way is modifying the standard protein A immuno- enrichment workflow. The second is modifying the protein A immuno-enrichment by enzymatic elution by the protease IdeZ. The last is the use of nSMOL kit from Shimadzu, which is protein A capture with nanobeads.

A lot of parameters are optimized to improve the workflow in terms of sensibility, costs and runtime. The efficiency of the IdeZ is first proven, followed by optimizing the protocol to reduce the noise and overall cost. Many parameters are evaluated such as the LC gradient and the calibration points to make the comparison of the nSMOL kit and the standard workflow possible.

An example of improvement is removing the steps “Peptide clean-up” and “Resuspension step”. This improvement leads to a small loss of intensity but a larger sample volume.

This allows creation of replicates. Shortening the run time also gives a small loss of intensity but a higher throughput. The protein A immuno-enrichment with IdeZ shows good results. So it is optimized to be more efficient. After that a problem came up and a search started for the solution. The nSMOL kit and standard workflow comparison was made possible after changing many different parameters.

Because of the adjustments, the standard workflow takes up less time. The modified protein A immuno-enrichment by adding IdeZ protocol shows promise but it is more time consuming. The last modification is the nSMOL kit. The kit is compared with the standard protein A enrichment. This gives a 10- till 30-fold more area, is far more linear but less

accurate than the standard workflow. It’s also less time consuming and easy to perform.

A disadvantage is that the nSMOL kit is very expensive.

Alzheimer’s disease (AD) is the most common form of dementia worldwide. With an estimated 47 million cases of dementia, and an increasingly aging population, this number will only rise. Since today’s diagnosis, using CSF puncture, is highly invasive, the use of saliva thanks to its non-invasive sampling should be of interest.

The main objectives of the project were to develop sensitive ELISA assays for the quantification of beta-amyloid peptides in saliva of Alzheimer’s patients. For this purpose three different experiments were developed. The object of the first experiment was to detect beta-amyloid 42 in saliva. The second was to detect salivary oligomers of beta-amyloid. The focus of the third experiment was on detection of salivary autoantibodies against beta-amyloid 42.

For assay development, CSF samples were used from the collection of CHU Biological Resource Centre (CCBHM) in collaboration with CHU Nîmes and CHU Montpellier. Saliva samples were collected from healthy volunteers of the Institute for Regenerative Medicine & Biotherapy in Montpellier. For all three experiments, homebrew ELISA assays were developed. Obtained signals were measured using Meso Scale Discovery technology.

The developed ELISA assay based on beta-amyloid 42 makes it possible to measure levels of beta-amyloid in saliva with a LLOD of 105.29 pg/ml. However, for samples close to the LLOD, both high and low signals were obtained between different tests, causing high inter-assay variability. Signals were obtained using the homebrew ELISA developed for the oligomers experiment, but no significant results were obtained so far. During assay development of the autoantibody experiment, a significant increase was found of levels of CSF autoantibodies in AD patients with cerebral amyloid angiopathy-related inflammation (CAA-ri), in comparison with controls and patients with AD but without CAA-ri.

Contradictory results were obtained for levels of beta-amyloid peptides in saliva. Further development of the assay will be needed to be able to quantify beta-amyloid 42. Since no significant results were obtained for the oligomers experiment so far, the experiment needs to be continued using stable oligomeric beta-amyloid peptides for further development. The findings suggest an increase in CSF-levels of autoantibodies in patients with CAA-ri. In order to confirm these findings for salivary levels of autoantibodies, further assay development will be needed.