To further investigate the localization of antigens on the Lactobacillus cell envelope, we developed an indirect live-cell enzyme-linked immunosorbent assay (lcELISA). Lactobacillus cultures were grown overnight at 30uC, harvested and resuspended to an OD 600 of 1.0 in TG buffer [100 mM Tris-HCl pH.8, 50 mM glucose]. For cell wall digestion, 1 ml aliquots were resuspended in TG buffer with or without Lyz (250 kU/ml) for 5 or 45 min at 37uC. Cells were washed twice with TG buffer, resuspended in the same buffer supplemented with 3% BSA (Bovine Serum Albumin, Sigma), and incubated with mAb LA2.2 (1:500). Samples were washed twice and incubated for 30 min with goat anti-mouse IgG (H+L) antibodies conjugated to alkaline phosphatase (1:1000). After an extensive wash, labeled cells were incubated with pNPP Microwell Substrate System (KPL). Microtiter plates were loaded with 100 ml of each cellular suspension, and optical densities were measured at 405 nm by a Spectra MAX plus ELISA reader (Molecular Devices, Sunnyvale, CA).