Working with the RarePlex Developer Kit
Video Transcript
Hello, my name is Edward Lo. I'm the scientist leading assay development at RareCyte and today I'm going to talk to you about working with the RarePlex Developer Kit.
A key problem in circulating tumor cell research is that very few assays are available to measure biomarker expression on CTCs. With new and interesting markers for cancer that are found every day, available commercial assays just can't keep up with demand. Our solution is to allow for the creation of custom assays with up to two biomarkers of interest by combining the Developer Kit with either an Enumeration Panel Kit or a Biomarker Panel Kit.
This system enables the investigation of your own biomarkers of interest using the 405 or 488 or both Developer Kits with a compatible RarePlex Panel Kit. This is the only available technology that allows adding two custom biomarkers and is designed to provide maximum flexibility for your needs, including the use of two antibodies of the same species in the developer channels.
The general workflow of creating your own custom assay is first to establish control samples, second to evaluate new antibody clones, and third, to titrate the selected clone. A last optional step is to optimize antigen retrieval, but should generally be unnecessary. Included with the Developer Kit are all necessary reagents and comprehensive instructions to help users quickly and effectively establish a new custom assay.
The first step in creating your own assay is to determine appropriate control samples. For each biomarker, we recommend finding a high, a low, and a biomarker negative cell line. Ideally, the high expressor should have clear separation above the negative cell line and the low expressor should have some overlap with the negative.
This will allow for better discrimination in performance for the rest of the workflow.
The next step in developing a custom assay is to evaluate antibody clones to your target of interest on model circulating tumor cells. In this example of a known nuclear biomarker, clone 1 has clean nuclear localization in the positive cell line and no staining on the negative cell line.
In contrast, the second clone has membranous staining on the positive cell line and nonspecific staining on platelets and white blood cells. Similar nonspecific staining is also observed in the marker negative cell line making this clone a poor candidate.
The final recommended step in creating your own assay is to titrate the primary antibody. Here we are looking for a plateau in the signal to background, which is the MFI on positive cells divided by the MFI on negative cells. This will make for a more stable assay with the highest signal to background possible. In this example, the highlighted concentration would be selected.
Bringing all these development steps together, this is an example of what the output of an assay looks like. Here, the positive cell line, A549, shows clear EGFR expression, while the negative cell lines, MDA-MB-453, have no expression. This is also reflected in the violin plots on the right, which show EGFR expression across the entire cell populations with clear separation between the positive and negative cells.
Expanding this work to two markers of interest, here we have staining of A549 cells for both Ki-67 and EGFR. The merged image on the left shows the expected nuclear localization for Ki-67 and membraneous staining for EGFR. After development, the custom assay can be applied to clinical samples. Here we have a prostate cancer sample stained using the Rrv7 panel kit with AR added using the Developer Kit. As expected ARv7 has clear nuclear localization, while AR is expressed both in the cytoplasm and the nucleus.
The RarePlex Developer Kit empowers you to create your own assays to study one or two of your own biomarkers of interest with sensitive and specific detection.
If you have any questions, please contact us at info@rareycte.com and we'll be happy to work with you to build an assay that addresses your particular needs.
Thank you for your attention and we hope to hear from you soon.