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Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis.

Magni R, Espina BH, Shah K, Lepene B, Mayuga C, Douglas TA, Espina V, Rucker S, Dunlap R, Petricoin EF, Kilavos MF, Poretz DM, Irwin GR, Shor SM, Liotta LA, Luchini A - J Transl Med (2015)

Bottom Line: Specificity of the urinary OspA test for the clinical symptoms was 40/40.Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

View Article: PubMed Central - PubMed

Affiliation: George Mason University, Manassas, VA, USA. rmagni@gmu.edu.

ABSTRACT

Objectives: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB.

Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation.

Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10(-6)). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.

Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

No MeSH data available.


Related in: MedlinePlus

Narrow OspA236-239 region is conserved across different Borrelia species and binds to mAb clone 0551. a Crystallography structure of OspA (Protein Data Bank PDB ID# 1FJ1): the epitope of the mAb is highlighted in red. b BLAST search against different Borrelia strains and species shows that the mAb clone 0551 epitope is highly conserved whereas the flanking regions are variable. c Synthetic peptides mimicking the OspA236-239 region interact with the mAb in a dose dependent manner (dot blot analysis, 1, 2, 3 = Bb Lyme antigen 0.5, 5, and 10 ng, respectively; 4, 5, and 6 = OspA219-253 0.5, 1, and 2 μg, respectively; 7, 8, and 9 = OspA219-235 0.5, 1, and 2 μg, respectively; 10, 11, and 12 = OspA240-253 0.5, 1, and 2 μg, respectively). Negative control peptides (OspA219-235 and OspA240-253) containing flanking regions but lacking the OspA236-239 sequence were devoid of immunoreactivity with the mAb clone 0551
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Fig3: Narrow OspA236-239 region is conserved across different Borrelia species and binds to mAb clone 0551. a Crystallography structure of OspA (Protein Data Bank PDB ID# 1FJ1): the epitope of the mAb is highlighted in red. b BLAST search against different Borrelia strains and species shows that the mAb clone 0551 epitope is highly conserved whereas the flanking regions are variable. c Synthetic peptides mimicking the OspA236-239 region interact with the mAb in a dose dependent manner (dot blot analysis, 1, 2, 3 = Bb Lyme antigen 0.5, 5, and 10 ng, respectively; 4, 5, and 6 = OspA219-253 0.5, 1, and 2 μg, respectively; 7, 8, and 9 = OspA219-235 0.5, 1, and 2 μg, respectively; 10, 11, and 12 = OspA240-253 0.5, 1, and 2 μg, respectively). Negative control peptides (OspA219-235 and OspA240-253) containing flanking regions but lacking the OspA236-239 sequence were devoid of immunoreactivity with the mAb clone 0551

Mentions: The epitope of OspA recognized by the mAb clone 0551 was sequenced and verified by peptide dot blot and peptide competition. First, the Bb Lyme antigen Grade 2 (81 % OspA content, Additional file 1: Figure S1) was partially digested with pepsin and the protease derived fragments were split into two aliquots. One aliquot was analyzed by western blotting with the anti-OspA mAb clone 0551. In parallel, the second aliquot of pepsin fragments was analyzed by SDS-PAGE and silver stained. The band in the SDS-PAGE mirroring the smallest peptide fragment recognized by the mAb was cut out and processed for mass spectrometry analysis (Fig. 2). The fragment KTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGT (OspA219-253, Fig. 2, Additional file 1: Table S1) was the smallest sequence that reacted with the mAb clone 0551. This sequence is located on the C-terminal region of the protein (Fig. 3a, b). Two overlapping peptides, Peptide 5 (OspA219-239) = KTSTLTISVNSKKTTQLVFTK and peptide 6 (OspA234-253) = QLVFTKQDTITVQKYDSAGT (Additional file 1: Table S1), were synthesized and evaluated against control peptides from other regions of the molecule for their reactivity with the mAb (Fig. 3c, Additional file 1: Figure S2). The overlapping region of these peptides (OspA236-239 = VFTK) was found to be necessary and sufficient for antibody recognition via dot blot (Additional file 1: Table S1, Fig. 3c), solution phase competition and immunoaffinity solid phase competition (Fig. 4). The flanking regions, highly variable in the Borrelia burgdorferi sensu strictu and across different Borrelia species were devoid of immunoreactivity with the mAb clone 0551 (Figs. 3, 4, Additional file 1: Table S1).Fig. 2


Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis.

Magni R, Espina BH, Shah K, Lepene B, Mayuga C, Douglas TA, Espina V, Rucker S, Dunlap R, Petricoin EF, Kilavos MF, Poretz DM, Irwin GR, Shor SM, Liotta LA, Luchini A - J Transl Med (2015)

Narrow OspA236-239 region is conserved across different Borrelia species and binds to mAb clone 0551. a Crystallography structure of OspA (Protein Data Bank PDB ID# 1FJ1): the epitope of the mAb is highlighted in red. b BLAST search against different Borrelia strains and species shows that the mAb clone 0551 epitope is highly conserved whereas the flanking regions are variable. c Synthetic peptides mimicking the OspA236-239 region interact with the mAb in a dose dependent manner (dot blot analysis, 1, 2, 3 = Bb Lyme antigen 0.5, 5, and 10 ng, respectively; 4, 5, and 6 = OspA219-253 0.5, 1, and 2 μg, respectively; 7, 8, and 9 = OspA219-235 0.5, 1, and 2 μg, respectively; 10, 11, and 12 = OspA240-253 0.5, 1, and 2 μg, respectively). Negative control peptides (OspA219-235 and OspA240-253) containing flanking regions but lacking the OspA236-239 sequence were devoid of immunoreactivity with the mAb clone 0551
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4634744&req=5

Fig3: Narrow OspA236-239 region is conserved across different Borrelia species and binds to mAb clone 0551. a Crystallography structure of OspA (Protein Data Bank PDB ID# 1FJ1): the epitope of the mAb is highlighted in red. b BLAST search against different Borrelia strains and species shows that the mAb clone 0551 epitope is highly conserved whereas the flanking regions are variable. c Synthetic peptides mimicking the OspA236-239 region interact with the mAb in a dose dependent manner (dot blot analysis, 1, 2, 3 = Bb Lyme antigen 0.5, 5, and 10 ng, respectively; 4, 5, and 6 = OspA219-253 0.5, 1, and 2 μg, respectively; 7, 8, and 9 = OspA219-235 0.5, 1, and 2 μg, respectively; 10, 11, and 12 = OspA240-253 0.5, 1, and 2 μg, respectively). Negative control peptides (OspA219-235 and OspA240-253) containing flanking regions but lacking the OspA236-239 sequence were devoid of immunoreactivity with the mAb clone 0551
Mentions: The epitope of OspA recognized by the mAb clone 0551 was sequenced and verified by peptide dot blot and peptide competition. First, the Bb Lyme antigen Grade 2 (81 % OspA content, Additional file 1: Figure S1) was partially digested with pepsin and the protease derived fragments were split into two aliquots. One aliquot was analyzed by western blotting with the anti-OspA mAb clone 0551. In parallel, the second aliquot of pepsin fragments was analyzed by SDS-PAGE and silver stained. The band in the SDS-PAGE mirroring the smallest peptide fragment recognized by the mAb was cut out and processed for mass spectrometry analysis (Fig. 2). The fragment KTSTLTISVNSKKTTQLVFTKQDTITVQKYDSAGT (OspA219-253, Fig. 2, Additional file 1: Table S1) was the smallest sequence that reacted with the mAb clone 0551. This sequence is located on the C-terminal region of the protein (Fig. 3a, b). Two overlapping peptides, Peptide 5 (OspA219-239) = KTSTLTISVNSKKTTQLVFTK and peptide 6 (OspA234-253) = QLVFTKQDTITVQKYDSAGT (Additional file 1: Table S1), were synthesized and evaluated against control peptides from other regions of the molecule for their reactivity with the mAb (Fig. 3c, Additional file 1: Figure S2). The overlapping region of these peptides (OspA236-239 = VFTK) was found to be necessary and sufficient for antibody recognition via dot blot (Additional file 1: Table S1, Fig. 3c), solution phase competition and immunoaffinity solid phase competition (Fig. 4). The flanking regions, highly variable in the Borrelia burgdorferi sensu strictu and across different Borrelia species were devoid of immunoreactivity with the mAb clone 0551 (Figs. 3, 4, Additional file 1: Table S1).Fig. 2

Bottom Line: Specificity of the urinary OspA test for the clinical symptoms was 40/40.Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

View Article: PubMed Central - PubMed

Affiliation: George Mason University, Manassas, VA, USA. rmagni@gmu.edu.

ABSTRACT

Objectives: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB.

Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation.

Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10(-6)). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e(-15)). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein.

Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative.

No MeSH data available.


Related in: MedlinePlus