MOESM1 of Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis
Creators: name: Magni, Ruben
name: Espina, Benjamin
name: Ketul Shah
name: Lepene, Benjamin
name: Mayuga, Christine
name: Temple Douglas
name: Espina, Virginia
name: Rucker, Sally
name: Dunlap, Ross
name: Petricoin, Emanuel
name: Kilavos, Mary
name: Poretz, Donald
name: Irwin, Gilbert
name: Shor, Samuel
name: Liotta, Lance
name: Luchini, Alessandra
Publisher: name: Figshare
Year: 2015
Subjects: subject: Biochemistry
subject: Space Science
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Marine Biology
subject: Cancer
subject: Inorganic Chemistry
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: 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 loaded 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 Nanotrap particles; lane 6 80 µL Nanotrap particles; lane 7 160 µL Nanotrap particles; b Band intensity was measured with ImageJ, plateau is reached with &gt;40 µL of Nanotrap particles. Figure S5. Interfering substances: the presence of a high amount of protein and blood in the urine does not interfere with Lyme antigen capture and detection. a 320 pg of Lyme antigen was spiked in samples of 40 mL of human urine. We tested the interference of albumin present in excess up to 10^8 fold. Increasing amounts of bovine serum albumin ranging from 0.31 mg/mL to 20 mg/mL were added to 40 mL of OspA containing human urine; urine samples were processed with Nanotrap particles. Ability of the Nanotrap particles to sequester OspA is not affected by increasing concentration of competing proteins in urine. Lane 1 ladder; lane 2 volunteer human urine in absence of OspA antigen (negative control); lane 3 OspA + BSA 20 mg/mL; lane 4 OspA + BSA 10 mg/mL; lane 5 OspA + BSA 5 mg/mL; lane 6 OspA + BSA 2.5 mg/mL; lane 7 OspA + BSA 1.25 mg/mL; lane 8 OspA + BSA 0.625 mg/mL; lane 9 OspA + BSA 0.31 mg/mL; lane 10 OspA + BSA 0.15 mg/mL. b Lyme antigen 320 pg was spiked in urine samples (40 mL). Increasing amounts of whole blood from 0.015 µL to 1 µL was added to the urine samples; urine samples were processed with Nanotrap particles and analyzed using western blot. Lane 1 ladder; lane 2 volunteer human urine in absence of OspA antigen (negative control); lane 3 OspA + 1 µL whole blood; lane 4 OspA + 0.5 µL whole blood; lane 5 OspA + 0.25 µL whole blood; lane 6 OspA + 0.125 µL whole blood; lane 7 OspA + 0.062 µL whole blood; lane 8 OspA + 0.031 µL whole blood; lane 9 OspA + 0.015 µL whole blood; lane 10 OspA + 0.007 µL whole blood. Figure S6. a Nanotrap particle preprocessing step is necessary to detect an OspA specific band in the urine of patient PD113, clinically positive for Lyme disease. Initial solution (IS) before Nanotrap particle processing. Eluate (E) after Nanotrap particle processing. b Positive and negative controls run with all Lyme patient samples. Lane 2 contains borrelia protein lysate (2 ng) in human urine. Lane 5 is a positive control 4 ng spiked in 40 mL of urine sample. Lane 6 and 7 are negative controls of 40 mL of volunteer urine processed through the Nanotrap particles. C=borrelia lysate control, IS=initial solution, S=supernatant, E=eluate. Table S1. Sequences of peptides tested for antibody binding. Table S2. The mAb epitope used herein (red rectangle) is conserved in common pathogenic species of Borrelia. BAA22342.1 in Borrelia garinii Taxonomy ID 29519, ADD14639.1 in Borrelia burgdorferi taxonomy ID 139, WP_012665647.1 in Borrelia valaisiana taxonomy ID 62088, WP_012665647.1 in Borrelia sp. SV1 taxonomy ID 498741, YP_003110622.1 in Borrelia burgdorferi 297 taxonomy ID 521009, NP_045688.1 in Borrelia burgdorferi B31 taxonomy ID 224326, WP_014023199.1 in Borrelia bissettii taxonomy ID 64897, ADG02035.1 in Borrelia afzelii taxonomy ID 29518, AAN65460.1 in Borrelia spielmanii taxonomy ID 88916. BLAST analysis was performed on the sequence KTSTLTISVNSKTTQLVFTKQDTITVQKYDSAGT (combination of peptide 5 and 6) with the following organisms: Treponema pertenue (taxonomy ID 168), Leptospiraceae (taxonomy ID 170), Treponema (taxonomy ID 157), Spirochaetes (taxonomy ID 203691) excluding Borrelia (taxonomy ID 138), Homo sapiens (taxonomy ID 9606), Epstein-Barr virus EBV (taxid:10376), Human cytomegalovirus (taxid:10359), herpes simplex virus 1 HSV-1 (taxid:10298), hepatitis C virus HCV (taxid:11103), Babesia txid5864, Anaplasma txid768, Ehrlichieae txid942, Bartonella  txid773, Rickettsias txid766.  No significant similarity found.  No homology was identified. Table S3. Light scattering analysis of Nanotrap particles. Table S4. Quantification of the amount of Remazol brilliant blue (RBB) dye covalently bound to the Nanotrap particles and percentage of reacted acrylic acid (AAc) moles. This information was obtained and recorded for every batch of produced Nanotrap particles (example batch # RM37B4 is reported here). Table S5. Clinical and diagnostic information of patients suspected of having early stage Lyme disease N=51 (N= 117 healthy volunteers were recruited under informed consent and included in the study). Treatment: Dx = doxycycline, Pd = prednisone, RC = rocepherin, sv = synovectomy, st = steroids, Am = amoxil, Zm = Zithromax. Urine collection timing and presence of symptoms: B = before treatment, PT = during or after treatment, symptoms present at the time urine was collected. Table S6. Post treatment patients being evaluated for recurrent or persistent disseminated Lyme disease derived from a Lyme endemic geographic region.