Adsorption of pooled serum samples was carried out to fully remove ID-8 recognizing antigens expressed in vitro. Pooled serum samples from chickens infected with M. gallisepticum were first adsorbed using A-MG. Native cell pellets were resuspended in pooled sera with addition of 10 mM EDTA and a complete cocktail of protease inhibitors (Roche, Germany). The serum–cell suspension was mixed for 1.5 h at 4 °C. The cells were repelleted and the absorbed serum was recovered by aspiration. This adsorption procedure was repeated three times. Pooled sera were subsequently serially adsorbed against B-MG and C-MG and then serially adsorbed against A-EC, B-EC and C-EC preparations. Briefly, in total, 16 adsorption steps were performed. A 10 μl aliquot of pooled sera was saved after each adsorption step to evaluate the efficacy of the process by ELISA and Western blot analyses. Using Western blot analysis, we saw that most of the antibodies was eliminated immediately after the first adsorption step and then a progressive decrease in reactivity of sera was detected until the seventh step (data not shown). Using ELISA, which is a more sensitive method, a progressive decrease in reactivity of the absorbed pooled sera was detected till the fifteenth step, where the decrease of the antibodies level reached a plateau (data not shown).
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