What is involved in a SISCAPA assay development project?

Before deciding to pursue a custom SISCAPA assay development project you may want to see if there is already an existing SISCAPA assay available to measure your protein of interest.  The best way to do this is to have a look at our current list of catalogue assays.  Each of the antibodies on this list is available in 1 mg quantities, enough to run at least 1000 samples, and usually ships within 2 weeks.  SISCAPA Assay Technologies (SAT) will also provide 4 nmol high purity stable isotope labeled and unlabeled peptides for each of our assays through our partnership with New England Peptide.

If you don't find a SISCAPA assay for your protein of interest in our catalogue, then we suggest SAT's custom assay development service.  This entails development of two target-specific reagents that determine which protein is measured: 1) a high affinity (low off-rate) anti-peptide antibody that is used to enrich the specific proteotypic, surrogate peptide to be measured and 2) a stable isotope labeled version of the peptide that serves as an internal standard for mass spectrometric quantitation.  Our monoclonal antibody (mAb) based assays are very stringently developed and provide significant increases in assay sensitivity compared to direct MRM measurements on sample digests.  Currently our standard assay development projects take about 6 months, but we also have an accelerated assay development protocol which takes only 3 months.   All of our assay development programs involve several key steps:

Selection of proteotypic peptide target
Selecting the right peptide target for a SISCAPA assay is a critical decision.  Our peptide targets are often tryptic, but we have also developed SISCAPA assays with LysC and GluC peptide targets.  SISCAPA peptides must be proteotypic (sequences found only in the protein of interest) in order to function as a true surrogate of the target protein, and they also must perform well in the mass spectrometer to allow facile and sensitive detection and quantification.  The peptides must also be able to serve as good antigens for antibody derivation. These and several additional criteria are built into proprietary algorithms developed by SAT to select a panel of candidate peptides for each custom SISCAPA assay development project that is undertaken.  Prior to immunization, peptides are synthesized and their LC-MS performance is determined empirically. 

Synthesis of the peptide target and preparation for use as an immunogen 
Once the peptides have been chosen, SAT develops an immunization strategy that will maximize the chances of raising an antibody that targets the desired peptide.  Post translational modifications are considered and immunogens designed accordingly.  SAT routinely employs specific conjugation strategies and variants of the target peptide in order to derive antibodies that will produce a robust and functional SISCAPA assay.  This critical phase of a custom assay project is based on experience drawn from developing hundreds of SISCAPA assays over the past fifteen years.

Generation of anti-peptide monoclonal antibodies and SISCAPA high-affinity clones screen
Between 1000 and 4000 hybridoma clones are produced during a typical custom SISCAPA assay project of which only a tiny fraction will end up being suitable for use in a SISCAPA assay.   To be a qualified capture reagent for a SISCAPA assay antibodies need to be of high affinity (specifically low off-rate) and high activity.  SAT screens all antibody candidates using methods that measure both their binding characteristics and their selectivity.  By using techniques that measure true antibody affinity, as opposed to most standard methods which measure avidity, SAT is able to predict which hybridoma clone will provide the best sensitivity in an SISCAPA assay.  

Full characterization of the new SISCAPA assay and delivery of the assay reagents
Once the selected monoclonal antibody is purified, the new SISCAPA assay undergoes a rigorous characterization that includes determination of the lower limit of detection (LLOD), the lower limit of quantitation (LLOQ) for the analyte, and mass spectrometric screening for interferences.  The results of the characterization are included in a comprehensive report delivered to the client along with the assay reagents.  For users engaged in high-throughput projects, SAT can also provide pre-developed robotic protocols on several laboratory liquid handling systems (currently Agilent Bravo and Beckman Biomek NXp systems).

We look forward to assisting you with developing a SISCAPA assay to measure your protein of interest!  Please don't hesitate to contact us if you'd like to request a quote or if you have any questions.  

 

Dried blood spots: Do it yourself!

Dried blood spots (DBS) have been around a long, long time - mainly for collecting and storing small heel-prick blood samples from newborn babies to test for inborn errors of metabolism.  Today that testing is typically done using mass spectrometry to look at a series of metabolites whose levels would be affected by absence of key enzymes (i.e., the inborn errors).

It turns out, happily, that drying a blood sample on filter paper (the original DBS "Guthrie" card format) is also an excellent way of collecting and preserving blood proteins.  In fact cards collected from newborns in Denmark have been saved for decades and the proteins shown to be analyzable, with little to no degradation.  This is in contrast to the normal clinical methods of blood sample collection that involve a phlebotomy nurse, needles and blood tubes, and long term sample storage in which aliquots of liquid serum, plasma or whole blood must be stored at -80C or below to preserve protein integrity.  The differences in cost and user convenience between these approaches is huge.

The conventional methods of blood sample collection tend to limit biomarker research to one or at most a few samples per subject, and thus focus on comparing study groups (e.g., healthy and sick) that are composed of different people.  Looking through the scientific literature it is very striking how few studies there are that track biomarker levels in the same person using an extended set of samples collected over time (so-called "longitudinal "studies).  The reason given is usually that collecting many conventional samples from a person involves too much cost (for the collection, for subsequent storage, and for the biomarker testing - so the grant doesn't get funded) and too much inconvenience (patients don't like getting jabbed repeatedly, so compliance is low). 

DBS sample collection opens up a new paradigm for research and clinical use of protein biomarkers: by allowing us to collect and preserve a blood sample wherever and whenever we want, they give us access to the domain of time.  In the first place, an extended history of samples collected and analyzed (a "biomarker history") would allow us to interpret changes in each biomarker against a personal baseline derived from that person's previous results - i.e., truly personalized diagnostics.  Because most biomarker proteins in blood vary less in a normal person from day to day than they do between people, a small change in the amount of a biomarker protein can be quite significant for the individual and still not large enough to fall outside the population's "normal range".

When I started thinking about this some years ago I decided to try practicing what I preach, and collect my own DBS samples.  This turned out to be easy to do using little disposable lancets (Medlance plus Extra by Strefa are my favorites) to prick my finger and Whatman 903 DBS cards to collect 5 drops of blood.  I'm a good bleeder and always get 5 nice drops out of the end of my little finger.  I keep the DBS samples in a Ziploc bag in the refrigerator with some desiccant to keep them dry.  All these supplies are freely available for purchase on the web, and it's easy to do the whole thing at home.

So I started DBS collection in August of 2008 and have just collected my 300th sample.  As a result, I think I may have the richest history of blood samples available anywhere, which allows me (using SISCAPA assay panels) to look very carefully at normal variation over time, and at specific things that have happened - including pneumonia, a series of "colds", onset and successful treatment of mild hypertension, and a gradual rise in hemoglobin A1c suggestive of the beginnings of diabetes.  Not everything we're interested in can be measured in DBS because of the limited sample volume, but a surprisingly rich set of proteins with known clinical significance can be measured at very modest cost (the SISCAPA story).

Whenever I get lazy I just remind myself that I'm never going to be able to go back and create these later.  How often do I collect samples?  During healthy periods maybe once a month or once a week - however there are times when I've taken samples every day to look at something that I think may cause interesting changes.  And when "interesting" things have happened over these 9 years, I have a set of samples to pin down biomarker changes that are directly correlated in time with the disease process involved.  N of 1 may make statisticians wince, but when that 1 is you, and you have been paying attention to other signs and symptoms (i.e.,  contextual data) needed to make sense of the biomarker history, you can help discover diagnostic (and ultimately treatment) possibilities that may never emerge from the conventional comparisons of disease vs healthy group.

Sometimes we need to bleed for progress...