Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

The overall goal of this procedure is to measure human leukocyte antigen or HLA associated tumor antigens in patient fluids for stratification of patients to relevant immunotherapy. This is accomplished by first immobilizing T-cell receptor mimicking antibodies, known as TCRM onto the MICROPLATE surface. The second step is to add the patient sample along with serial dilution of target peptide HLA monomers to generate a standard curve, which allows for quantification of the target antigen in patient samples.

Next, binding of the target antigen to the antibody on the free label bioassay system is monitored until equilibrium is reached. The final step is to analyze the resulting data by using the standard curve to quantify the amount of target antigen in patient samples. Ultimately, label-free biosensor technology is used to detect cancer biomarkers in patient samples.

The main advantage of this technique over existing techniques such as lc MSS, is the rapid high throughput detection of HLA associated antigens. This allows for the rapid stratification of cancer patients to treatment modalities because it does serve as a biomarker detection platform. While this method is related to the screening of cancer biomarkers, the system actually is applicable to many applications including cell-based assays, immunophenotyping hybrid screening, or the screening of small molecule libraries.

We first had the idea for this method when we were developing a therapeutic T-cell receptor mimic antibody. We asked ourselves, how would we identify patients who would benefit most from this type of therapy? Demonstrating this procedure will be Kristen Dahl, a biotechnology graduate student working in my laboratory.

Begin by pre incubating the phosphate buffered saline or PBS at 28 degrees Celsius to minimize temperature related resonance shifts. Add 50 microliters of phosphate buffered saline or PBS pH 7.2 to the wells of avadon derivative coated plates. Next, open the bioassay scanner software.

Follow the setup wizard to define the experimental procedure, which includes selection of blanks, standards and samples in the plate layout, temperature setting, number of scans per minute and length of the experiment. Then insert the prepared assay plate into the label free bioassay scanner and start the first scan. The baseline is the initial set of scans collected at the start of the experiment.

If this is the first read performed on this specific plate, allow the scanner to run long enough to equilibrate the temperature and eliminate drift in the baseline. After the baseline has stabilized or after at least five scans, pause the read and eject the plate. Dump out the PBS and add 50 microliters of RL 21 a biotinylated antibody to all but control wells on the plate.

Then add 50 microliters of PBS to the control wells. Next, insert the plate back into the bioassay scanner and resume reading until saturation is reached. After approximately 1.5 hours, pause the read and eject the plate.

Then wash the plate three times with 200 microliters per well of PBS plus 0.05%Tween 20 or PBST. Follow the wash with three rinses of 200 microliters per well of PBS pH 7.2 without detergent. Tap excess PBS from the plate on paper towels prior to moving to the next step.

Next, add 50 microliters of PBS to all active wells. Then insert the plate back into the bioassay scanner and resume reading to monitor Post wash resonance. After reading, stop the read and eject the plate.

To add the analyte. Remove the PBS and add 50 microliters per well of a appropriate assay buffer for this assay. Commercially available normal human serum was diluted one to 20 in PBS.

Next, open the bioassay scanner software and follow the setup wizard. To define the experimental procedure, insert the plate into the bioassay scanner and begin a baseline read After the baseline has stabilized or after at least five scans, pause the read and eject the plate. Then remove the assay buffer from the wells.

Then spike the controls with HLAA oh 2 0 1 monomer bearing relevant or irrelevant peptides at concentrations ranging from 0.625 to 10 micrograms per milliliter in assay buffer. Then add 50 microliters of the standards to the control wells of the plate. Next, add 50 microliters of the analyte in assay buffer to the sample wells of the plate.

For this assay, spiked commercially available human serum was used. Insert the plate back into the bioassay scanner and resume reading until saturation is reached approximately 30 to 60 minutes following saturation, pause the read and eject the plate. Proceed to wash the plate three times with 200 microliters per well of PBST, followed by three rinses with 200 microliters per well of PBS as before then add 50 microliters of assay buffer to all active wells.

Now insert the plate back into the bioassay scanner and resume reading to monitor post wash resonance before stopping the read and ejecting the plate. Finally, analyze the data using the bioassay scanner software or export raw data files to preferred statistical analysis software. The bioassay scanner software automatically generates the binding curve based on the plate layout provided during experimental setup.

Biotinylated RL 21 A-T-C-R-M or IgG two A was immobilized on the AVID AVIDAN assay plate representative results show detection of the target tumor antigen F-L-S-L-H-L-A in a patient plasma sample at equilibrium and at post wash. IgG two A is used as a control for non-specific binding tissue sections were stained with a positive control and negative controls. And for RL 21 a staining of tumor tissue by RL 21 A confirms presentation of F-L-S-L-H-L-A complexes.

After watching this video, you should have a good understanding of how to measure HLA associated biomarkers in patient fluids using T-cell receptor mimicking antibodies, and monitoring the binding of the target antigen to the antibody on the label-free bioassay system. While attempting this procedure, it's important to remember to incubate all sample buffers prior to use to avoid temperature spikes and to use the correct sample buffer for each step.