AbstractA sandwich immunoassay has been developed for the detection of lipoteichoic acid (LTA), a major cell wall constituent of gram‐positive bacteria, from whole blood and ISOLATOR supernate. Monoclonal antibodies were produced to purified LTA fromStreptococcus mutans, BHT and were further characterized for crossreactivity with gram‐positive and negative bacteria and for reactivity to substituted and unsubstituted LTA. Eight monoclonal antibodies were identified that reacted exclusively with gram‐positive bacteria. Those antibodies able to capture H‐LTA were chosen to develop a sandwich immunoassay. The assay has a sensitivity of 0.2 ng LTA/mL in PBS, 0.5 ng/mL in whole blood and 2.0 ng/mL in whole blood that has been processed through the ISOLATOR. Further development of this assay may lead to the rapid detection of LTA from body