Eighty overlapping alpha-N biotinylated 15 residue synthetic peptides were obtained spanning the entire p43 sequence. Each peptide was bound 7.5 mcg/ml to duplicate streptavidin-coated wells of microtiter plates with streptavidin-only and conjugate-only controls. 100 mcl of human serum diluted 1:40 with PBS containing protease inhibitors was added to the wells and incubated for 1h. After washing, plates were developed using rabbit anti-human Ig-HRP conjugate followed by ABTS substrate. Plates were read at 405nm on an Anthos automated plate reader connected to a computer. A program was written which expressed the duplicate mean optical density (OD) for immunorecognition of each peptide by CD sera, against mean OD + 2SD for that peptide using normal sera. The resulting profile immediately demonstrates significant antibody binding by CD sera to specific sequence determinants within p43. Sera from 8 of 12 (75%) normal subjects showed weak antibody binding to one or more of 4 domains, peptides 22-24, 34-37, 40-43 and 64-67. Sera from all 9 (100%) CD patients so far tested demonstrated significant antibody binding (>mean normal + 2SD) to one or more of 4 different domains, peptides 28-32, 54-58, 71-73 and 74-80. Mean recognition of the major carboxyterminal epitope on p43 by CD sera was particularly strong exceeding mean normal +2 SD >twofold. IS900 has proved uniquely specific for M. paratuberculosis (M.ptb). The present findings are consistent with the concept that most of the human population has been exposed to M.ptb, and further implicate this organism as a chronic enteric pathogen in humans. A change in specific immune recognition of M.ptb may accompany the switch from benign intestinal cohabitation to disease state.