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.