Assay optimization for the detection of LTA in human serum. (a) Membrane insertion assay using the 25 nM α-Gram+ antibody for the detection of S. aureus LTA (1 µg/mL) incubated in control human serum with 1 h incubation at room temperature (teal line), 24 h incubation overnight at 4 °C (dotted blue line), and 24 h incubation overnight at 4 °C followed by sample processing (blue line). The background and non-specific signals are measured before the addition of LTA. (b) Detection using lipoprotein capture with the 25 nM α-Gram+ antibody to detect 1 µg/mL of S. aureus LTA following overnight incubation in control human serum using capture antibodies targeting HDL and/or LDL. (c) Concentration dependent detection of S. aureus LTA using lipoprotein capture with 25 nM of the α-LTA antibody in ELISA format measured in absorbance at 450 nm. (d) Comparison of the sensitivity of lipoprotein capture (HDL and LDL together) and membrane insertion assays for the detection of 100 ng/mL S. aureus LTA using the cocktail of 25 nM α-Gram+ monoclonal and 25 nM of α-Sau polyclonal antibody. All values given in (b–d) are the mean ± standard deviation derived from at least two independent determinations (n = 2). ELISA assays were performed in triplicate (n = 3). Statistical significance was determined by one-way ANOVA with Fisher’s least significant difference test used for post hoc analysis (***P < 0.001 or **P < 0.01).