The overall goal of the following experiment is to use high throughput sequential Eliza in order to efficiently analyze large numbers of novel biomarkers that were discovered by proteomics. This is achieved by first adding samples to a source plate, which is used to create dilution for each biomarker and to store recovered plasma between tests. Next, Eliza's are run for each protein in order to determine their concentrations in patient samples.
Ultimately, the elevation of the protein levels of these biomarkers at the onset of clinical manifestations of acute graft versus host disease can be assessed. The main advantage of this technique over existing methods is that sequential analyzers can analyze multiple novel proteins that are not always available with over multiplexing platforms. The implications of this technique extend toward the diagnosis of acute graft versus host disease.
As biomarkers validated through this method are now being investigated for clinical care. Though this method can provide insight into complications in post-traumatic poetic stem cell transplantation. It can also be applied to biomarker discovery and validation for other diseases such as lung transplantation, complications, lung cancer, or potentially any disease for which a diagnostic blood test will be useful.
Visual demonstration of this method is critical, as it is important that the multiple steps are done consistently to ensure reproducibility and low batch variability On the day before the experiment, spin down thought plasma aliquot samples for 10 minutes at 15, 300 RCF and four degrees Celsius to separate the clots at the bottom and the lipids on the top from the plasma. Then plate 150 microliters of undiluted plasma from each sample onto a 96 well V bottom plate by manual pipetting according to predefined maps. Wrap the plate in Paraform and place it in a human chamber at four degrees Celsius for up to 72 hours.
Next, after reconstituting and diluting IL two receptor alpha and human growth factor, or HGF capture antibodies per the manufacturer's specifications plate 50 microliter aliquots of each antibody into each well of respective 96 well high binding half well plates. Then seal these plates with a plate sealer and incubate them overnight at four degrees Celsius. Now dilute reg three alpha with the manufacturer's coating buffer according to the manufacturer's protocol and plate 25 microliters of the solution into each well of a 3 84 well no maxor plate.
Then store this plate sealed at four degrees Celsius as well on the first day of the experiment. Begin by washing the IL two receptor alpha test plate and blocking it with blotto in TBS. After reconstituting the standard, set up an eight point standard curve as per the manufacturer's protocol.
Next plate, 50 microliters of undiluted plasma and 50 microliters of each standard in duplicate from the source plate to the ELIZA test plate. Then incubate the sealed plate at room temperature on a plate rotator set to 300 RPM. After two hours, reclaim the plasma from the IL two receptor alpha ELIZA test plate and place it back into the undiluted plasma source plate.
Then after completing the EIA per the manufacturer's protocol, read the optical density of each well using a plate reader set to 450 to 570 nanometers for the rich three alpha Eliza first transfer 10 microliters of undiluted plasma to a separate view bottom source plate, and then add 90 microliters of the manufacturer provided dilution buffer to each well to create a one to 10 dilution source plate. After completing the Reg three alpha Eliza, as per the manufacturer's protocol, add the stop solution to the 384 well plate and read the optical density of each well using a plate reader set to 450 to 620 nanometers. After reconstituting and diluting the elephant and tn FFR one capture antibodies per the manufacturer specifications code each well of the respective 96 well high binding half well plates with 50 microliters of the capture antibodies.
Then incubate the sealed test plates overnight at room temperature for the elephant plate and at four degrees Celsius for the TN FFR one plate. After completing the IL two receptor alpha Eliza transfer 60 microliters of undiluted plasma to a new source plate. Then at 60 microliters of 1%BSA in PBS to each well to make a one to two diluted plasma source plate.
Next wash and block the HGF test plate with BLOTTO in TBS and then set up the eight point standard curve dilution. After washing the plate, following the blocking step plate 50 microliter, duplicate aliquots of one to two diluted plasma and of each standard on the EISA test plate. Then incubate the sealed plate overnight at room temperature on a plate rotator set a 300 RPM.
The next morning, reclaim the one to two diluted plasma from the HGF Eliza test plate and place it back in the one to two diluted plasma source plate. After completing the EIA, according to the manufacturer's protocol, read the optical density of each well using a plate reader set to 450 to 570 nanometers on the second day of the experiment. First transfer 10 microliters of undiluted plasma to a new source plate, and then 190 microliters of 1%BSA in PBS to make 200 microliters of one to 20 diluted plasma.
Then after completing the elephant, Eliza, according to the manufacturer's protocol, read the optical density using a plate reader set to 450 to 570 nanometers. For the TN FFR one, Eliza first add 25 microliters of 1%BSA and PBS to the one to 20 source plate resulting in 125 microliters of one to 25 diluted plasma per well. Then after completing the TN FFR one EIA per the manufacturer's protocol, read the optical density of each well using a plate reader set to 450 to 570 nanometers for the I-L-H-E-I-A first transfer, 60 microliters of one to two diluted plasma to a new source plate, and then add 180 microliters of IL eight diluent to each well to make a one to six diluted plasma source plate.
Then after completing the ILH Eliza per the manufacturer's protocol, read the optical density of each well using a plate reader set to 450 to 570 nanometers. Finally, once all the Eliza have been completed, replace the unused talk plasma into the thought Eloqua and freeze for future use. In this graph, the optical densities for the standard curve of seven different ELIZA plates measuring the reg three alpha concentrations corresponding to the 1084 patients tested for the initial reg three alpha gastrointestinal graft versus host disease biomarker report are shown consistent optical densities between the plates assures consistent protein concentration measurements between experiments.
The concentrations of the proteins in the plasma samples were calculated by comparing the sample optical densities to the standard curve optical densities. A robotically clean handling platform can be used to further increase reproducibility while also decreasing the volume of plasma used by running Eliza and creative form well plates. While planning this procedure, it's important to run EISs that is undiluted plasma first so that it can be recovered and used in later experiments After its development.
This technique paved the way for researchers in the field of proteomics to explore candidate biomarkers in any disease of interest through the universal use of sequential Eliza s. After watching this video, you should have a good understanding of how to validate multiple biomarkers using sequential E while using small volumes of plasma and minimizing free cycles.
