**4.5. Serological analysis of recombinant cDNA expression libraries (SEREX)**

SEREX is one of the oldest methods for autoantigen discovery and utilises human cDNA expression libraries to profile autoantibody repertoires. The methodology for SEREX initially involves generation of a cDNA library from a cancer tissue or tumour cell line, followed by cloning of that library into a suitable expression vector, clonal separation of the library and expression of the encoded proteins in *Escherichia coli* cells grown on solid media. Colonies are then transferred to a nitrocellulose or PVDF membrane, lysed and the expressed recombinant proteins blotted with sera from patients and healthy controls. Sero-reactive proteins are then identified by sequencing the cDNA from positive colonies [63], which makes it more sensitive than the SERPA method since the latter relies on direct protein identification and is therefore limited by absolute protein abundance. Furthermore, the clonal separation of the members of the cDNA expression library provides greater resolving power than the gel-based SERPA method. However, as with all library-based screening methods, over-sampling is required to ensure that all members of the library are examined in SEREX: thus, if for example the cDNA library contains 10<sup>4</sup> unique clones, then at least 10<sup>5</sup> colonies would need to be screened for complete coverage, so even with the advent of colony picking and arraying robots, SEREX remains a relatively low throughput method.

The first cancer testis antigen, NY-ESO-1, was identified by SEREX by analysing tumour associated antigens (TAAs) that elicited a high titre IgG antibody in sera from patients with different types of cancer [64]. SEREX has also been successfully used to identify several TAAs that generate a humoral immune response in cancers such as those from the kidney, lung, breast and colon [63]. However, a fundamental limitation of SEREX is that the method lacks the ability to differentiate or detect post-translational modifications (PTMs) that are likely to play a significant role in autoimmune diseases [62] and cancers [63]. This approach also restricts the types of TAAs identified to those that can be expressed in a prokaryotic system and also effectively excludes TAAs that require folding mechanisms unique to eukaryotes to achieve the correct conformational epitope for recognition [63]. SEREX may also miss TAAs that are represented by truncated cDNAs in the library, since the encoded protein may lack specific epitopes or even whole domains. Furthermore, identification of TAAs is inherently limited to those that were expressed by the specific patient tumour or cell line from which the cDNA library was derived, which means that more than one cDNA library may be required to identify comprehensive set of TAAs for different cancers [63]. In addition, the presence of the crude prokaryotic cell lysate in every spot can give rise to high background binding in SEREX assays. Thus, for many of these reasons, SEREX has largely been superseded now by protein microarray technologies that are based on purified recombinant proteins.
