Evaluation of T7 and lambda phage display systems for survey of autoantibody profiles in cancer patients.
In the current study we attempted to evaluate the suitability of T7 Select 10-3b and lambdaKM8 phage display systems for the identification of antigens eliciting B cell responses in cancer patients and the production of phage-displayed antigen microarrays that could be exploited for the monitoring of autoantibody profiles. Members of 15 tumour-associated antigen (TAA) families were cloned into both phage display vectors and the TAA mini-libraries were immunoscreened with 22 melanoma patients' sera resulting in the detection of reactivity against members of 5 antigen families in both systems, yet with variable sensitivity. T7 phage display system showed greater sensitivity for the detection of antibodies against members of CTAG, MAGEA and GAGE families, both systems showed equal performance in detecting the reactivity against MAGEC and SSX2 while only lambdaKM8 allowed the detection of anti-CTAGE5 antibodies. The biological properties of both phages turned out to be equally suitable for the production of antigen microarrays however in line with the plaque assay the sensitivity for the detection of various autoantibodies differed between the vectors. However, presumably due to the higher variability of the background signals in the microarray assay, it turned out to have comparable, in some cases even slightly lower sensitivity than the plaque assay. Next, we explored the repertoire of antigens that could be identified by screening T7 phage-displayed testis cDNA library with sera from melanoma patients. From the 243 antigens identified, only 24 represented known genes translated in their natural reading frame and included known TAAs like Annexin XI-A and a novel potential CT antigen SPAG8. Another 12 were uncharacterised genes but the remaining clones contained DNA fragments in non-natural reading frames that most likely represent mimotopes, nevertheless, they may turn out to be valid biomarkers.
Kalniņa
Zane
Z
Biomedical Research and Study Centre of Latvia, Riga, Latvia.
Siliņa
Karīna
K
Meistere
Irēna
I
Zayakin
Pawel
P
Rivosh
Alexander
A
Abols
Artūrs
A
Leja
Mārcis
M
Minenkova
Olga
O
Schadendorf
Dirk
D
Linē
Aija
A
D000912:Antibodies, Neoplasm / Q000097:blood* / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000302:isolation & purification; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D017123:Bacteriophage T7; D010582:Bacteriophage lambda; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D008545:Melanoma / Q000097:blood / Q000276:immunology*; D019151:Peptide Library; D040081:Protein Array Analysis / Q000379:methods*
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D001323:Autoantibodies; D019151:Peptide Library