Additional file 1.Figure S1. 81 % of the total protein present in Bb Lyme antigen Grade 2 (American Research Products) is constituted by OspA. A dilution of a recombinant purified OspA protein (Genecopoeia) was analyzed by SDS PAGE and silver stained (100 to 1,000 ng, Lanes 3–7). The band intensities were quantified using ImageJ software and a calibration curve was built (insert graph). A linear regression curve was fitted (y = 5.39x+12281, R2 = 0.9873). 500 ng of Bb Lyme antigen Grade 2 were

more »

... aded on the gel (Lane 2) and compared to the recombinant OspA calibration curve (Lanes 3–7). From this procedure it derived that 81% of Bb Lyme antigen Grade 2 total protein content is OspA. In gel protein digestion and mass spectrometry (MS) analysis verified the predominant presence of tryptic peptides belonging to OspA in the bands at ~30 kDa and ~60 kDa present both in the Bb Lyme antigen Grade 2 and in the recombinant OspA. In particular, tryptic peptides containing the epitope for mAb clone 0551 OspA236-239 were present in both bands (MS-MS spectra are reported in the insert). Figure S2. Narrow OspA236-239 region binds to mAb clone 0551 with high specificity. Dot blot analysis revealed that only 2 out of 7 synthetic peptides with sequence reported in Table S1 show reactivity with the anti OspA monoclonal antibody clone 0551 used in this study. Figure S3. Light scattering analysis was used to determine the hydrodynamic diameter of the Nanotrap particles. As reported in Table S2, the hydrodynamic diameter of the Nanotrap particles was 1054.7 +/- 31.11 nm. Figure S4. A V/v ratio (V = Nanotrap suspension volume, v = sample volume) of 1/10 was optimized in order to maximize Lyme antigen capturing. a 2 ng of Lyme antigen was spiked in 500 µL urine aliquots. Urine samples were incubated with increasing amount of Nanotrap particle suspension (10–160 µL of Nanotrap at 5 mg/mL concentration). Lane 1 ladder; lane 2 Positive control (OspA 1ng); lane 3 10 µL of Nanotrap particles; lane 4 20 µL Nanotrap particles; lane 5 40 µL [...]