Sample Requirements for Antibody Sequencing
Typical Sample Requirements: monoclonal antibody, quantity>0.2mg, purity>95%.
Deliverable: A comprehensive report including the full sequences of the heavy and light chains, the supporting data for the sequence correctness, and other observations such as irregular modifications on the antibody. (Click here for and example of a coverage report.)
Turnaround time: Typically 1-2 weeks after receiving the sample.
Other Antibody Sequencing Techniques
Hybridoma sequencing sequences the cDNA encoding of the variable heavy (VH) and variable light (VL) domains of the hybridoma cell line. This is a mature technology and the most economical method when the antibody secreting cell line is available.
Edman sequencing has been around for several decades. It directly sequences the protein, but usually only capable of sequencing peptides up to 50 amino acids long. It is a low throughput process.
Intact Mass Analysis and Peptide Mapping
Intact mass analysis and peptide mapping are very often mistakenly associated with the sequencing technology. In fact those are great confirmation tools when the sequences of the antibody proteins are known. Intact mass analysis can confirm at the protein level whether the molecular weight is the same as the experimental observation. Peptide mapping can confirm at the peptide level, that the precursor of the peptides are observed. Both technologies lack the insights at the amino acid level, which is absolutely required in sequencing.
Also known as de novo antibody sequencing, this is direct sequencing of the antibody’s amino acid sequence using tandem mass spectrometry without reference to a protein or antibody sequence database. For more information, see Peptide Mapping vs Antibody Sequencing