High-throughput Human Autoantibody Profiling

Technical Details

The HuScan human proteome PhIP-Seq library contains all annotated human protein sequences from an NCBI RefSeq database, including all published and computationally predicted splice variants and coding regions. The dataset was obtained in November 2015; after removing duplicates and partial entries through clustering at 99% sequence identity. Each protein was further divided into 49-amino acid peptides through a 25-AA sliding window approach. To eliminate redundant sequences from identical regions of the different isoforms and homologs, the 49-AA peptides were clustered and combined at 95% identity, where peptides with two or fewer differences in amino acids were represented by a single, representative sequence. The final library contains 731,724 unique peptide sequences that represent a full human proteome of 48,921 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. A more detailed description of this process is available (Mohan, et. al., 2018).

Service Details and Data Deliverables

A HuScan service involves case and control serum or plasma samples. These undergo a protein A/G pulldown assay, PCR amplification, and next-generation sequencing. Raw sequence data are run through a normalization and quantitation pipeline as previously described. Raw pipeline counts outputs are then provided to customers alongside normalized hit calls data for all individual 49mer peptides.

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.