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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

Nanotrap particles are necessary to detect urinary OspA in the urine of acute Lyme patients. Urinary OspA bands reverts to undetectable after successful treatment. Band specificity is assessed through competition assay. a Conditions for an optimal competition assay were established. 1 ng (lanes 2, 5, and 8) and 0.05 ng (lane 3, 6, and 9) of Bb Lyme antigen Grade 2 were spiked in urine. Lanes 2 and 3 were obtained with mAb clone 0551 alone. Lanes 5 and 6 were obtained with mAb neutralized with recombinant OspA. Reduced signal demonstrates high specificity. Lanes 8 and 9 were obtained with the mAb neutralized with Bb Lyme antigen Grade 2. Absence of signal with the neutralized antibody demonstrates specificity to OspA. b Competition assay was performed on all patient samples. Positive test outcomes show minimal or no binding of neutralized mAb to specific bands. Example competition assay for a Lyme disease patient is shown. 1 ladder; 2 Bb Lyme antigen Grade 2 OspA 1 ng; 3 initial Solution positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 4 supernatant positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 5 eluate positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 6 initial solution negative control (40 mL of urine processed through the Nanotrap particles); 7 supernatant negative control (40 mL of urine processed through the Nanotrap particles); 8 eluate negative control (40 mL of urine processed through the Nanotrap particles); 9 initial solution patient 180; 10 supernatant patient 180; 11 Eluate patient 180; 12 initial solution patient 180; 13 supernatant patient 180; 14 eluate patient 180. Membrane containing Lanes 1–11 was probed with the mAb alone, whereas membrane containing lanes 12–14 was probed with the mAb clone 0551 neutralized with recombinant OspA. cLane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12: example of patient urine samples demonstrating presence of OspA. Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller-than-full-lenght OspA C-terminal domain containing protein fragments in urine. d Nanotrap antigen test results on a representative sub-group of the 117 healthy volunteers (Table 1). Lane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12 example of patient urine samples demonstrating absence of OspA. e The OspA band is not detectable in the urine of acute stage Lyme patients after successful treatment. Lane 1 ladder; lane 2 Bb Lyme antigen Grade 2 in urine 0.1 ng; lane 3 negative control urine with no OspA; lane 4 Initial solution (=urine without Nanotrap particle pre-processing) of patient 120 before treatment; lane 5 eluate of patient 120 before treatment; lane 6 Initial solution patient 120 after treatment; lane 7 Eluate patient 120 after treatment
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Fig9: Nanotrap particles are necessary to detect urinary OspA in the urine of acute Lyme patients. Urinary OspA bands reverts to undetectable after successful treatment. Band specificity is assessed through competition assay. a Conditions for an optimal competition assay were established. 1 ng (lanes 2, 5, and 8) and 0.05 ng (lane 3, 6, and 9) of Bb Lyme antigen Grade 2 were spiked in urine. Lanes 2 and 3 were obtained with mAb clone 0551 alone. Lanes 5 and 6 were obtained with mAb neutralized with recombinant OspA. Reduced signal demonstrates high specificity. Lanes 8 and 9 were obtained with the mAb neutralized with Bb Lyme antigen Grade 2. Absence of signal with the neutralized antibody demonstrates specificity to OspA. b Competition assay was performed on all patient samples. Positive test outcomes show minimal or no binding of neutralized mAb to specific bands. Example competition assay for a Lyme disease patient is shown. 1 ladder; 2 Bb Lyme antigen Grade 2 OspA 1 ng; 3 initial Solution positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 4 supernatant positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 5 eluate positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 6 initial solution negative control (40 mL of urine processed through the Nanotrap particles); 7 supernatant negative control (40 mL of urine processed through the Nanotrap particles); 8 eluate negative control (40 mL of urine processed through the Nanotrap particles); 9 initial solution patient 180; 10 supernatant patient 180; 11 Eluate patient 180; 12 initial solution patient 180; 13 supernatant patient 180; 14 eluate patient 180. Membrane containing Lanes 1–11 was probed with the mAb alone, whereas membrane containing lanes 12–14 was probed with the mAb clone 0551 neutralized with recombinant OspA. cLane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12: example of patient urine samples demonstrating presence of OspA. Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller-than-full-lenght OspA C-terminal domain containing protein fragments in urine. d Nanotrap antigen test results on a representative sub-group of the 117 healthy volunteers (Table 1). Lane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12 example of patient urine samples demonstrating absence of OspA. e The OspA band is not detectable in the urine of acute stage Lyme patients after successful treatment. Lane 1 ladder; lane 2 Bb Lyme antigen Grade 2 in urine 0.1 ng; lane 3 negative control urine with no OspA; lane 4 Initial solution (=urine without Nanotrap particle pre-processing) of patient 120 before treatment; lane 5 eluate of patient 120 before treatment; lane 6 Initial solution patient 120 after treatment; lane 7 Eluate patient 120 after treatment

Mentions: Protein competition using a recombinant protein containing the OspA C-terminal mAb binding domain OspA236-239 was employed for the evaluation of patient urine specimens to ensure complete immunoassay specificity and to evaluate the presence of C-terminal fragments of OspA (Fig. 9a). A direct Western immunoblot (Fig. 9b) and a competition assay were performed on the patient’s urine sample (Fig. 9b). An individual patient’s urinary OspA was scored as positive only if both assay methods were judged positive. Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller OspA C-terminal domain protein fragments in urine. In some patients a high molecular weight band at ~60 kDa was detected and this was determined to be a dimer of OspA by mass spectrometry sequencing (Additional file 1: Figure S1). The higher molecular weight band was fully competable by the recombinant OspA (Fig. 9b).Fig. 9


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)

Nanotrap particles are necessary to detect urinary OspA in the urine of acute Lyme patients. Urinary OspA bands reverts to undetectable after successful treatment. Band specificity is assessed through competition assay. a Conditions for an optimal competition assay were established. 1 ng (lanes 2, 5, and 8) and 0.05 ng (lane 3, 6, and 9) of Bb Lyme antigen Grade 2 were spiked in urine. Lanes 2 and 3 were obtained with mAb clone 0551 alone. Lanes 5 and 6 were obtained with mAb neutralized with recombinant OspA. Reduced signal demonstrates high specificity. Lanes 8 and 9 were obtained with the mAb neutralized with Bb Lyme antigen Grade 2. Absence of signal with the neutralized antibody demonstrates specificity to OspA. b Competition assay was performed on all patient samples. Positive test outcomes show minimal or no binding of neutralized mAb to specific bands. Example competition assay for a Lyme disease patient is shown. 1 ladder; 2 Bb Lyme antigen Grade 2 OspA 1 ng; 3 initial Solution positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 4 supernatant positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 5 eluate positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 6 initial solution negative control (40 mL of urine processed through the Nanotrap particles); 7 supernatant negative control (40 mL of urine processed through the Nanotrap particles); 8 eluate negative control (40 mL of urine processed through the Nanotrap particles); 9 initial solution patient 180; 10 supernatant patient 180; 11 Eluate patient 180; 12 initial solution patient 180; 13 supernatant patient 180; 14 eluate patient 180. Membrane containing Lanes 1–11 was probed with the mAb alone, whereas membrane containing lanes 12–14 was probed with the mAb clone 0551 neutralized with recombinant OspA. cLane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12: example of patient urine samples demonstrating presence of OspA. Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller-than-full-lenght OspA C-terminal domain containing protein fragments in urine. d Nanotrap antigen test results on a representative sub-group of the 117 healthy volunteers (Table 1). Lane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12 example of patient urine samples demonstrating absence of OspA. e The OspA band is not detectable in the urine of acute stage Lyme patients after successful treatment. Lane 1 ladder; lane 2 Bb Lyme antigen Grade 2 in urine 0.1 ng; lane 3 negative control urine with no OspA; lane 4 Initial solution (=urine without Nanotrap particle pre-processing) of patient 120 before treatment; lane 5 eluate of patient 120 before treatment; lane 6 Initial solution patient 120 after treatment; lane 7 Eluate patient 120 after treatment
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Fig9: Nanotrap particles are necessary to detect urinary OspA in the urine of acute Lyme patients. Urinary OspA bands reverts to undetectable after successful treatment. Band specificity is assessed through competition assay. a Conditions for an optimal competition assay were established. 1 ng (lanes 2, 5, and 8) and 0.05 ng (lane 3, 6, and 9) of Bb Lyme antigen Grade 2 were spiked in urine. Lanes 2 and 3 were obtained with mAb clone 0551 alone. Lanes 5 and 6 were obtained with mAb neutralized with recombinant OspA. Reduced signal demonstrates high specificity. Lanes 8 and 9 were obtained with the mAb neutralized with Bb Lyme antigen Grade 2. Absence of signal with the neutralized antibody demonstrates specificity to OspA. b Competition assay was performed on all patient samples. Positive test outcomes show minimal or no binding of neutralized mAb to specific bands. Example competition assay for a Lyme disease patient is shown. 1 ladder; 2 Bb Lyme antigen Grade 2 OspA 1 ng; 3 initial Solution positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 4 supernatant positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 5 eluate positive control (0.5 ng in 40 mL of urine processed through the Nanotrap particles); 6 initial solution negative control (40 mL of urine processed through the Nanotrap particles); 7 supernatant negative control (40 mL of urine processed through the Nanotrap particles); 8 eluate negative control (40 mL of urine processed through the Nanotrap particles); 9 initial solution patient 180; 10 supernatant patient 180; 11 Eluate patient 180; 12 initial solution patient 180; 13 supernatant patient 180; 14 eluate patient 180. Membrane containing Lanes 1–11 was probed with the mAb alone, whereas membrane containing lanes 12–14 was probed with the mAb clone 0551 neutralized with recombinant OspA. cLane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12: example of patient urine samples demonstrating presence of OspA. Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller-than-full-lenght OspA C-terminal domain containing protein fragments in urine. d Nanotrap antigen test results on a representative sub-group of the 117 healthy volunteers (Table 1). Lane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 3–12 example of patient urine samples demonstrating absence of OspA. e The OspA band is not detectable in the urine of acute stage Lyme patients after successful treatment. Lane 1 ladder; lane 2 Bb Lyme antigen Grade 2 in urine 0.1 ng; lane 3 negative control urine with no OspA; lane 4 Initial solution (=urine without Nanotrap particle pre-processing) of patient 120 before treatment; lane 5 eluate of patient 120 before treatment; lane 6 Initial solution patient 120 after treatment; lane 7 Eluate patient 120 after treatment
Mentions: Protein competition using a recombinant protein containing the OspA C-terminal mAb binding domain OspA236-239 was employed for the evaluation of patient urine specimens to ensure complete immunoassay specificity and to evaluate the presence of C-terminal fragments of OspA (Fig. 9a). A direct Western immunoblot (Fig. 9b) and a competition assay were performed on the patient’s urine sample (Fig. 9b). An individual patient’s urinary OspA was scored as positive only if both assay methods were judged positive. Positive OspA bands are normally visible in the 28–30 kDa range although lower molecular bands can be detected and successfully competed suggesting the presence of smaller OspA C-terminal domain protein fragments in urine. In some patients a high molecular weight band at ~60 kDa was detected and this was determined to be a dimer of OspA by mass spectrometry sequencing (Additional file 1: Figure S1). The higher molecular weight band was fully competable by the recombinant OspA (Fig. 9b).Fig. 9

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