Table 1.
MAb476 cross-reactivity with other fungia.
Figure 1.
MAb476 recognizes in vivo Galf antigen in a murine model of IA and localizes in the bladder of infected mice.
A,B Sandwich ELISA detection of Galf-containing antigen in BAL and lung homogenates (panel A; N = 5–6) and serum (panel B; N = 2–6) of neutropenic mice, 2 days after aerosol infection with A. fumigatus. C, Organ distribution of 99mTc-MAb476 2 days after infection; Top, control mouse: 99mTc-label localization in liver and spleen; Bottom, infected mouse: 99mTc-label localized in the bladder, representative of 2 independent experiments (N = 3 per group).
Figure 2.
Urine has an inhibitory effect on MAb476-based immunoassays, which is abrogated by desalting or dialysis.
A, Comparison amongst PBS/blocking buffer, urine and desalted/dialyzed urine as a diluent for the secondary antibody in indirect ELISA. Antibody concentration to reach half maximal signal (EC50) in urine is significantly different than in PBS/blocking buffer or in desalted/dialyzed urine (p<0.001, F-test; data combined 3–9 independent assays). B,C, Comparison of PBS or blocking buffer vs. urine or desalted/dialyzed urine as a diluent for the EPA293 in sELISA. Panel B: Antigen concentration to reach half maximal signal (EC50) in urine is significantly different than in PBS/blocking buffer or in desalted/dialyzed urine (p<0.001, F-test; data combined 9–15 independent assays). Panel C: Comparison of mean signals at 40 ng/ml of EPA293 (urine vs. PBS/Blocking buffer and desalted/dialyzed urine, p = 0.0538 and 0.0009, respectively, unpaired t-test; N = 7–11 independent assays). D,E, Inhibitory effect of urine from 6 healthy volunteers in sELISA; EC50 values of all urines were significantly different from that of PBS (p<0.0001, F-test).
Figure 3.
Desalting/dialysis and/or concentration improve antigen detection in simulated urine samples in sELISA.
A, Detection of EPA293 in simulated (spiked) samples after desalting or dialysis at different MWCOs (40 KDa, 7 KDa, 3.5 KDa and 2 KDa); signal curves in PBS or in all treated urine were significantly (comparison of EC50; p<0.0001, F-test) different than in untreated urine. B, Detection of low, but biologically plausible (50 and 100 ng/ml), EPA293 concentrations in simulated samples after desalting (7 KDa), concentration (5–10 folds; 3 KDa spin-column), or both, compared to no treatment. (*) represents comparisons between a treatment and untreated urine with significant difference (p<0.01); (&) represents a trend (p = 0.0613) in comparison between a treatment and untreated urine; (#) represents comparison between concentration/desalting and concentration-only treatments with significant difference (p = 0.0077); all comparisons by One-way ANOVA with a Tukey's post-test for pair-wise comparisons; N = 2–3.
Figure 4.
MAb476 detects a urinary excreted Galf antigen in a GP model of pulmonary IA.
Processed (10-fold concentration/7 KDa-desalting) urines samples were tested in sELISA; 6 out of 8 infected guinea pigs had detectable antigenuria. Cut off (CO) value equals the mean+3SD of the absorbances of negative controls.
Figure 5.
MAb476-based Lateral flow immunochromatographic assay device (LFD) detects Galf antigen in simulated and human urine samples.
A, appearance of the assembled LFD; subsequent panels show scanned images of the reaction window. B, Comparison of urine and normal saline (NS) as diluent for EPA293 in LFD. C, Detection of EPA293 in simulated samples after concentration (5–10 folds, 3 KDa MWCO) and desalting (7 KDa MWCO), compared to no treatment. D, Detection of Galf-containing antigen in processed clinical urine samples; 2 patients have intermediate (++) positive signal and 2 have weak (+) positive signal.