Abstract
Staphylococcus aureus Cowan I containing protein A were rapidly fixed in hot 5% (w/v) trichloroacetic acid (TCA) for 6 min in order to remove the negatively charged cell wall polymer teichoic acid. This resulted in a stable IgG adsorbent able to bind 1.4 mg of human IgG/ml 10% (v/v) suspension for at least 5 months. This IgG binding ability is 25% less than for formalin-fixed bacteria, which were stable for at least 1 year. Preincubation of the bacterial adsorbents in 0.5% (v/v) Tween 20 after fixation prolonged stability and rendered prewashes prior to use unnecessary. IgG could be quantitatively eluted from TCA-fixed bacteria at pH 3.0, but not from formalin-fixed bacteria unless 80 mM MgCl 2 was included in the acid buffer. This is explained by ionic interaction between IgG and teichoic acid at the bacterial surface at low pH. Non-specific binding of different proteins to the bacterial adsorbents was also studied and buffers for reducing this effect are suggested.
Published Version
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