Immobilization of Proteinase K for urine pretreatment to improve diagnostic accuracy of active tuberculosis
Fig 4
A) Shows the concentration curve (0–1000 μg /mL) for IPK that would give optimal results when used for the pretreatment of urine spiked with LAM. The inset shows 400 μg /mL of IPK (red) to be the optimal concentration that gives the best results compared to the urine spiked with LAM that did not see any IPK (dark blue). B) Showing the standard Proteinase K (SPK) treatment of urine from a healthy volunteer spiked with LAM purified from M.tb CDC1551 cells. The blue line with circles shows the effect of SPK in sequestering LAM from the protein/s or other inhibitors present in urine compared to the red line with triangles where the urine spiked with LAM has not been pretreated with SPK. The yellow line shows the background signal obtained with the urine and the green line with diamonds shows that Proteinase K itself does not have any effect on blank urine. C) Shows the optimal time required for the pretreatment, with IPK at 400 μg /mL, of urine spiked with LAM. At 60 min (grey line) and 120 min (yellow line) (see inset) compared to the 0 min (pink line) pretreatment time, more LAM seems to be available for binding to the antibody. D) Shows the optimal temperature needed (55°C) for the IPK pretreatment of urine spiked with LAM for 60 min at 400 μg/mL of IPK.