High-throughput Murine Autoantibody Profiling

Technical Details

The first whole proteome murine autoantibody discovery platform reported; the first available commercially. Each protein in the GRCm38.p5 mouse proteome was downloaded and divided into 62-amino acid peptides with 19-AA overlap. Redundant sequences from identical regions of the different isoforms and homologs were eliminated if they had >95% identity overlap. The final library contains 482,672 unique peptide sequences that represent a full murine proteome of 50,135 proteins and protein isoforms.

This was ordered as an oligonucleotide library, PCR-amplified with adaptors for cloning, and packaged into a T7 phage display vector that was expanded in E. Coli. An aliquot from this library is then reacted with diluted patient serum or other antibody-containing fluid. Bound antibodies are immunoprecipitated with protein A/G beads, the precipitate amplified by PCR, and the sequences quantified by a next-generation sequencing and analysis pipeline that compares patient-sample IP read counts to negative controls with no antibody input (mock IPs) in the context of overall clonal frequency of individual peptides in the parent library. Output data are then created at both the peptide level.

Service Details and Data Deliverables

Sample Requirements

Serum or plasma20 μL aliquot per sample

Cerebrospinal fluid (CSF)250 μL aliquot per sample

Other antibodies (IgG monoclonals, B cell supernatants, etc)250 μL aliquot per sample at 0.01 mg/mL concentration

12 sample minimumSold in multiples of x12 samples (i.e., x12, x24, x36, x48 – full rows of a standard 96-well plate), and any number after 48 samples

Cohort balancing (studies bigger > 336 samples)

Individual PhIP-Seq studies are prepared in x96 well plates using aliquots of our phage library; each study requires x48 controls pooled plasma, known polyclonal, and protein A/G beads-only internal control samples per sequencer run (provided free-of-charge). Data are most reproducible within a single sequencer run. Sequencer runs are currently limited to 4x 96-well plates (336 experimental samples + x48 controls per run).

Sample Shipping and Sample Return

We will include shipping details in your quote – you must cover the cost of shipping samples to CDI Labs. Typically, after you receive your report, CDI Labs keeps the remaining samples for two months and then disposes of them. When shipping to us, please let us know if you want the remaining samples returned after the study is complete. Return shipping will be charged.