Abstract By means of an ELISA, we have found the pan-cancer biomarker HAAH to be closely associated with serum exosomes primarily in cancers of the breast, lung, colon, and prostate. This new understanding of the HAAH target has led to a dramatically improved serological detection assay, and in turn diagnostic reagent kit. In keeping with the multivalent particulate nature of exosomes, appropriately reformatting the ELISA assay has yielded a simplified so-called simultaneous-homologous format. Currently in this format, a single monoclonal anti-HAAH antibody (FB50) serves both as microplate capture and as a biotinylated detector. All reagents, including peroxidase-streptavidin, are co-incubated in the FB50- coated microplate simultaneously with serum samples, in the absence of intervening sequential steps. This considerably shorter exosome-enabled ELISA format requires half the time and yields generally on average a two-fold or higher increase in detection of HAAH. This format was evaluated with over 100 banked serum samples from several early stage cancers. While increasing the sensitivity for HAAH in cancer samples, the improved detection and performance has not caused any undesirable increases in false positives among more than 30 normal serum samples from healthy subjects. The reagent kit components include recombinant HAAH calibrators, positive /negative controls, vialed detection reagents and a Mylar packaged FB50 pre-coated microplate. The simplified reagent kit format has yielded close inter-operator and day to day trending consistency with a limit of detection (LOD) of 3 ng/mL. Recognizing the exosomal nature of the HAAH target has resulted in some changes in blood sample procurement, sample processing, and sample shipping. Such field testing of the HAAH reagent kit was recently done with serum samples from 48 high risk or mildly symptomatic volunteers with general concerns about cancer. Upon testing these samples 9 had an HAAH level greater than 3.0 ng/ mL (range 4.2 to 116.7) and 39 had less than 3.0 ng/ mL. Based upon the post-analysis diagnostic determination, there were two false negatives and one false positive, hence an overall accuracy of 93.8%. Citation Format: Mark A. Semenuk, Anokhi S. Cifuentes, Eleanor R. Ghanbari, Michael S. Lebowitz, Hossein A. Ghanbari. Improved detection of cancer specific serum exosomal aspartyl (asparaginyl) beta hydroxylase (HAAH) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 723. doi:10.1158/1538-7445.AM2017-723