Wizard2 gamma counter

IgG was labeled with ¹²⁵I by the standard chloramine-T oxidation method. Briefly, IgG, Na¹²⁵I, and chloramine-T were mixed at 37 °C for 10 min under slight shaking. The synthesized ¹²⁵I-IgG was washed by 10 kDa ultrafiltration three times. To determine radiolabeling stability, ¹²⁵I-IgG was mixed with serum for 24 h at 37 °C. Portions of the mixture were sampled at different time intervals and filtered by 10 kDa ultrafiltration. The radioactivities retained on the filters were detected by Wizard2 Gamma Counter (PerkinElmer) to calculate radiolabeling stability.
For in vivo biodistribution of ¹²⁵I-IgG assay, B16F10 melanoma tumor-bearing mice were topically administrated with free ¹²⁵I-IgG and FCS/¹²⁵I-IgG ointment for different time points. Then, the major organs, including the liver, spleen, kidney, heart, lung, and tumor, were collected and measured by Wizard2 Gamma Counter (PerkinElmer). The cumulation efficacy was calculated by the formulation $\%\mathrm{ID}/g=R_n/R_0/m_n\times 100\%$ Where R_n was the radio intensity of the organ at the exact time point, m_n was the mass of the organ, R₀ was the radio intensity of the applied ointment.

Purified proteins (C4BP, IgA, IgD, IgE, IgG, IgM, IgG1, IgG2, IgG3, and IgG4) were diluted to 5 μg/ml in PBS and immobilized onto microtiter plates (Maxisorp breakapart, Nunc) at 4°C overnight. The plates were washed 3 times with wash buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween 20) and non-specific binding sites were blocked with 3% fish gelatin (Norland Products) in wash buffer. ¹²⁵I- labeled Protein H (50 kcpm) was diluted in binding-PBS (PBS supplemented with 0.1% Tween-20 and 0.1% BSA) and added to the immobilized proteins; in case of immobilized C4BP, indicated amounts of IVIG were added as stated in the individual experiments. After overnight incubation at 4°C and subsequent washing, using a Wizard² gamma counter (Perkin Elmer) radioactivity in the wells was measured. ¹²⁵I- labeled C4BP (500 Kcpm) was added to 5^*10⁵ bacteria in a final volume of 100 μl in the presence of indicated amounts of different IgG preparations and incubated at 37°C for 1 h. Bacteria were washed thrice with PBS prior to counting bacteria-associated radioactivity using a Wizard² gamma counter (Perkin Elmer).