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Fig. 4 | Journal of Translational Medicine

Fig. 4

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

Fig. 4

Peptides containing the narrow OspA236-239 region were successfully utilized for antibody competition and immunodepletion. a 600 pg of Bb Lyme antigen Grade 2 were spiked in human urine. Samples were processed through the Nanotrap particles and analyzed by western blot. Lane 2, 4 and 6 were obtained staining the western blot membranes with the mAb clone 0551 alone, the mAb neutralized with OspA219-253 peptide, and the mAb neutralized with a combination of OspA219-235 and OspA240-253, respectively. The peptide containing the OspA236-239 region successfully competed the mAb, whereas peptide missing the OspA236-239 region failed to compete the mAb clone ID 0551. b Peptide OspA219-235 was utilized for solid phase affinity depletion of the mAb clone 0551. The peptide (30 μg) was deposited on ELISA plate wells. The wells were washed and the excess peptide removed. The wells were blocked with PBS supplemented with 0.2 % I-Block, 0.1 % Tween 20. The mAb clone 0551 (3 μg) was incubated with the solid phase adsorbed peptides overnight. After incubation, the supernatant was recovered and brought to a volume of 3 mL in PBS supplemented with 0.2 % I-Block and 0.1 % Tween 20. In parallel, 600 pg of Bb Lyme antigen Grade 2 were spiked in urine and processed through the Nanotrap particles (lane 2 and 4). Lane 2 and 4 were obtained staining the western blot membranes with the mAb alone (3 μg) and the mAb after immunodepletion, respectively. There is no immunoreactivity in the mAb preparation after immunodepletion (lane 4). This is a further confirmation of the absence of non-specific signal in the mAb clone 0551 preparation

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