To compare the immunogenicity of PEG and PTMAO, two groups of male C57BL/6J mice (n = 5) were administered the PEG-KLH and PTMAO-KLH conjugates, respectively, via subcutaneous injection (2 mg protein/kg weight) for 5 weeks (one dose per week). Mice sera were collected on day 7, 14, 21, 28, and 35 during the immunization study, and the anti-PEG or anti-PTMAO antibodies (IgM and IgG) in mice sera were detected using ELISA tests.

For ELISA tests, 100 μl of antigen solution (10 μg/ml of protein concentration), prepared in 0.1 M sodium carbonate buffer (pH 10.5), was used to coat each well of the 96-well plates. Antigens used in direct ELISAs for plate coating consisted of PEG–bovine serum albumin (BSA) (for the mice sera immunized with PEG-KLH) or PTMAO-BSA conjugates (for mice sera immunized with PTMAO-KLH). PEG-BSA and PTMA-BSAO conjugates were prepared, following the same preparation protocol as the PEG-KLH and PTMAO-KLH conjugates as described above. During coating procedure, plates were incubated at 4°C overnight. After removing antigen solutions, the plates were washed five times using PBS (pH 7.4) and then filled with blocking buffer [1% nonfat milk solution in 0.1 M tris buffer (pH 8.0)]. It is important to avoid using any buffer that contains PEG-like detergents, e.g., Tween 20 and Tween 80. After incubation at room temperature for 1 hour, blocking buffer was removed, and all wells were washed by PBS for another five times. Serial dilutions of monoclonal anti-PEG Abs in PBS containing 1% nonfat milk were added to the plates (100 μl per well), which were incubated for 1 hour at 37°C. The plates were then washed five times with PBS, followed by adding secondary antibody HRP conjugates (100 μl per well, 1:50,000 dilution; Bethyl Laboratories). After adding the secondary antibody, plates were incubated at room temperature for 1 hour and then washed five times using PBS before the addition of HRP substrate TMB (100 μl per well). The plates were shaken for 15 min, and 100 μl of stop solution (0.2 M H2SO4) was added to each well. Absorbance at 450 (signal) and 570 nm (background) was recorded by a microplate reader.

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