Three groups of male C57BL/6J mice (n = 5) were administered the native uricase, PEG-uricase, and PTMAO-uricase conjugates, respectively, via intravenous injection (25 U/kg weight) for 3 weeks (one dose per week). The mice sera were collected at various time points (0.1, 6, 12, 24, 48, and 72 hours) after the first and the third injection of uricase samples for evaluating the level of uricase/uric acid. The uricase concentration in plasma was estimated on the basis of the enzyme activity measured by AmplexTM Red Uric Acid/Uricase Assay kit. The circulation time of each uricase sample was calculated using PKSolver software following the instructions. The urate concentration in the mice sera was also measured by AmplexTM Red Uric Acid/Uricase Assay kit. All the mice were sacrificed on day 21, and their sera were harvested for antibody detection via ELISA tests as described above. For ELISA tests, uricase was used as the coated antigen for the detection of anti-uricase Ab, while PEG-uricase or PTMAO-uricase conjugate was used as the coated antigen for the detection of anticonjugate Ab.

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