ELISA assays for purified representative peptides against three antisera Five peptides newly designed to encompass the regions R1-R5 were expressed as fusions to the C-terminus of thioredoxin (Trx) and purified from E. coli, using a modification of the self-cleaving elastin-like polypeptide tag method (ELP) (with purity over 80% as judged by SDS-PAGE) [29, 30] . For comparison, the Trx tag was used as the negative control. When mouse and goat antisera were employed as the first antibody, the wells were coated with 100 ng of peptides or Trx. But for human antisera, 10 ng of peptides or Trx control was used to decrease the background signal caused by unspecific interactions between the human antisera and the trace impurities from E. coli cells. After blocking with 10% Fetal Bovine Serum (FBS) diluted in 16PBS with 0.25% Tween-20 (PBST), serial dilutions were added to each well and incubated for 1 h at 37uC, followed by addition of 1:5,000 diluted HRP-conjugated secondary antibody. Assays were developed by adding 100 mL of 3,39,5,59-tetramethylbenzidine (TMB) substrate solution and the reactions were stopped with 50 mL of H 2 SO 4 (1 M). The assays were carried out in duplicates and each well was washed 4 times with PBST between steps. The absorbance at 450 nm was recorded on a SpectroMAX 190 Microtiter reader (Molecular Devices, CA).