Download (Affinity and SRM) assays for detection of potential biomarkers for

Developing AFFIRM (Affinity and SRM) assays for detection of potential
biomarkers for relapsed breast cancer in plasma
Breast cancer is the leading cancer in women worldwide. Every year, 1.6 million women are diagnosed
with it, out of which 58% die due to the cancer. The incidence is rising rapidly in developing countries.
Even though there are several treatments, inability to detect the cancer at an early stage limits the
possibilities of curing it in time. After a patient has been treated for cancer and has completed his/ her
treatment they are happy to have won the biggest battle of their life and survived it. But after a few
months or a year, or even several years, they are struck by the news from the doctor that the cancer is
back, which is called a relapse. In most breast cancer cases, death is caused from the relapse rather than
the initial cancer. Therefore, it is necessary to develop methods which detect the relapse early and ensure
appropriate treatment can be given or relapse can be prevented. The most easily available sample for
testing is plasma from the patient’s blood. Presence of cancer can be detected by testing for certain
protein biomarkers (target proteins) that would be present in the plasma in case the patient is suffering
from the cancer or if the cancer is relapsing.
Antibodies are proteins that possess the property to bind specifically to targets. Since they are highly
sensitive and bind only to their targets, they are used to detect presence of proteins. ELISA is a standard
methods used to confirm presence of biomarkers. ELISA used 2 antibodies- primary to bind the target
specifically and secondary antibody which a fluorescent tag to measure the amount of bound target
protein.
Such antibody- based methods are sensitive but are available only for a few proteins and
developing them is very demanding and expensive. We have therefore developed assays for another
technology. Mass spectrometry is a technology that identifies proteins based on their mass to charge
ratio. Digesting a protein with an enzyme like Trypsin results in fragments of the protein called peptides.
The type of Mass spectrometry that was used in this study is called Selected Reaction Monitoring
(SRM), where we specify to the instrument the peptides that are unique to the target protein and it would
detect only these peptides. It is a much easier and faster technique as SRM looks for the specific peptides
rather than scanning all the peptides and then identifying the target. AFFIRM is a platform that combines
antibody and mass spectrometry technologies to detect and quantify proteins. We produced antibodies
that would bind specifically to the target proteins and the antibodies were bound to magnetic beads.
When the beads carrying the antibodies were exposed to the plasma spiked with the artificial target
proteins, the targets bind to the antibody. This is followed by digestion by an enzyme like Trypsin to cut
the protein into several peptides. The sample is then analyzed using the SRM. Development of SRM
assays for each of the 30 target proteins was attempted and 19 of them were successfully developed.
SRM assays help to pin point if a protein is present in the sample or not, and does it even when the
protein content is quite low (sensitive). This way we can identify at a much faster rate that the person is
suffering from breast cancer relapse. The developed assays showed promising results in lab tests; it
would next be confirmed using labelled heavy peptides. We aim to apply the developed assays to clinical
samples from breast cancer patients collected at primary diagnosis and at later recurrence. If successful,
it has potential to speed-up the diagnosis process for breast cancer relapse.