Table 1.

Summary of autoantibody detection methodologies

Methodology nameHigh-throughputCostTimeAdvantagesDisadvantages
  • May take several days

  • This is the most time consuming of all the methods due to the need to construct the cDNA library

  • Allows detection from in vivo material

  • Use of multiantigen-specific patient serum allows the identification of several tumor-specific antigens in one experiment

  • Both the tumor-specific antigen and its coding cDNA are present in the same plaque when immunoscreening is performed that allows the subsequent sequencing of the matching cDNA immediately

  • Slightly more sensitive than SERPA

  • High likelihood of false-positive results

  • Does not detect alternate tumor-associated PTMs of antigens

  • Use of tumor tissue from a single cancer followed by screening with autologous patient serum limits identification of TAAs to that of a single patient

  • Parallel analysis of tumor proteins with healthy donor sera as controls cannot be performed easily

Phage displayYes—higher throughput than SEREXMore cost-effective if phage proteins are fused to antigens on bacteriophage surface
  • May take several days

  • Constructed directly from tumor tissue or patient tumor material–derived cell line

  • Does not detect alternate tumor-associated PTMs of antigens

Protein microarrayYesProduction of thousands of recombinant proteins is very expensive
  • Time restriction due to short shelf-life of protein arrays

  • Large numbers of antigens can be tested against large numbers of sera in a single test

  • Purified, recombinant or synthetic proteins may be used

  • Array platform may be 2D or 3D

  • Yeast or insect cells may be used as alternative expression systems to produce libraries with correct PTMs

  • 3D structure is often intact optimizing antigen–antibody interaction for recombinant proteins produced in mammalian systems

  • Requires only minute amounts of sera

  • High-quality protein synthesis is required

  • Other than high-quality antibodies or antigens, only commercially available proteins can be studied

  • Time restriction due to short shelf-life of protein arrays

  • High reproducibility is difficult to achieve

  • Enormous data collection requires specialized software tools

  • Production of thousands of recombinant proteins is very labor-intensive

  • Recombinant proteins produced in non-mammalian systems may not have the correct PTMs and may therefore be misfolded

  • Yes

  • Liquid-based separations are amenable to automation and the ELISA format can be coupled to mass spectrometric analysis that increases the throughput

More cost-effective than SEREX
  • May be completed within hours

  • Effective separation of a complex mixture of proteins based on their isoelectric points and molecular weights

  • Allows detection from in vivo material

  • Allows for the identification of tumor-specific PTMs and isoforms

  • Avoids the time-consuming construction of cDNA libraries

  • Parallel analysis of tumors proteins with healthy donor sera as controls can be performed easily

  • 2D immunoblots provide a global view of the antibody–tumor-associated antigen interaction

  • Limited identification of low-abundance and transmembrane TAAs

  • Because of the use of Western blot analyses only linear epitopes can be detected

  • Separation of cell membrane proteins remains a challenge due to their insoluble nature in aqueous buffers

  • This method of autoantibody detection is very labor-intensive

MAPPingYesSimilar cost-efficiency to SERPA
  • May be completed within hours

  • Tumor antigens are maintained in solution that allows the identification of structural epitopes

  • Restricted tumor antigen identification to antibody interactions with a low dissociation rate constant

  • Limited detection of tumor antigens in more complex protein solutions due to the use of immunoprecipitation