Vielen Dank für die Zusammenfassung in Englisch. Ich habe nun eine Antwort von unseren Mitochondrien -Spezialisten aus dem Labor erhalten.
Ich habe die Antwort nicht übersetzt um Übersetzungsfehler zu vermeiden:
"Let me start by saying these are very good questions and extremely difficult to answer as well. Here are the answers to your questions
Buffy coat samples to extract PBMC are available that have been stored at -196°C since the blood draw 20 years age. Are these samples appropriate for your assays?
It depends on the buffer in which they have been stored. I’m assuming from the temperature specification that the samples have been frozen in the vapor phase of liquid nitrogen. If they were frozen in freezing media containing 30 – 50% FBS + 10% DMSO + Standard Culture media, then they will be viable and in good condition to determine protein based end point biomarkers. If this is not the case, it will be difficult to get good proteomic data out of the samples because the freeze/thaw process may break the granulocytes and release proteases that will completely degrade the samples before you have a chance to work with them. These proteases are extremely potent and are difficult to inhibit with regular protease inhibitors available in the market.
Do you have any idea which markers would make sense in the context of Alzheimer’s disease (e.g. OXPHOS complexes)?
We have a fair amount of experience with Mitochondria, however our experience with the use of PBCSs as surrogate markers of disease states is more limited. We only have experience of this in the field of HIV and retroviral therapy, where OXPHOS proteins (CI and CIV) have been shown to be potential good surrogate markers of therapy toxicity in this subgroup of patients. Do you want to use PBMCs as a surrogate tissue for the discovery or for the validation of biomarkers of brain injury (Alzheimer’s Disease)?. If you are planning a DISCOVERY type of experiment, my suggestion is to look more broadly in the mitochondria proteome and even in the whole cellular proteome. Antibodies and Immunoassays are not very suited for this type of discovery research. There is a paper in the literature where they used a combined methodology (genomic/transcriptomic/proteomic profiling) to discover Alzheimer’s disease markers in the blood. This was published by a group at the University of Louisville Email: mailto:email@example.com. My suggestion is to email the senior author to discuss their results and potentially collaborate with them to select true candidate targets for validation.
Antibodies and Immunoassays are particularly useful in the validation stage, where you have selected a number of targets in the discovery phase and want to validate the results (remember that discovery phases have a large number of false positives even when this is controlled statistically with the use of “false discovery rate”). If you find in your talks with Professor Wang that the OXPHOS complexes or other mitochondria targets are in the list of candidate biomarkers, then the questions is how to use the best product/platform that measures that target, which is your next question.
Which technique would you suggest to use for a large-scale study? We are working on a tight budget
The answer to this question depends first on your accessibility to instrumentation. Do you have access to plate readers, are they colorimetric or fluorescent?. Do you have access to a flow cytometer? How many people will perform these experiments (data on 6000 samples with the use of immunoassays is not trivial), are they located in a single centralized large lab or in multiple labs around the world?
We have a platform called “Dipsticks” or lateral flow immunoassay which have been used by previous researchers in the past with PBMCs on other types of diseases. I can provide references with the use of Dipsticks in PBMCs in mitochondrial disease, frataxin, HIV/antiretroviral therapy and traumatic brain injury (which used PDH as a marker). These studies were a lot smaller than yours though. The main disadvantage of Dipsticks is that they are not amenable for high throughput. However researchers have used them in these studies, because they are extremely easy to use and require minimal training, for quantitative data they require a specialized reader “dipstick reader” which costs a fraction of what a regular microplate reader costs and could be easier to use in the future as potential “point of care research tests”. Bear in mind that all our products are RUO (research use only). Dipsticks are inexpensive but do require a large amount of “man power” and many of them have a higher sensitivity than the microplate counterpart. Our dipsticks measure the target proteins either in a sandwich ELISA or with an activity assay. Your other option could be microplates, which will allow you to run 96 tests at a time. I discourage you from using western blot in so many samples. Western blot is not quantitative when it comes to comparing results from one blot to the next, whereas the microplates or dipsticks are easier to compare from run to run. I encourage you to visit our metabolism page and look at different targets in these two platforms. On the main page there is a video about the dipsticks which will allow you to learn more about them. Once you decide on a particular target/platform we will happily help you with further scientific support.
The other page you should visit is our ELISA and activity assay kits page (below) for dipsticks and microplates
Ich hoffe diese Information ist hilfreich und wünsche Ihnen viel Erfolg mit Ihrer Studie.