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Fig. 9 | Journal of Translational Medicine

Fig. 9

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

Fig. 9

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. c Lane 1 ladder, lane 2 Bb antigen 1 ng; Lanes 312: 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 312 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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