Protein A-containing fixed bacteria as solid phases

Protein A-containing fixed bacteria as solid phases 

SpA-containing Staphylococci (Section 3.3), fixed with trichloroacetic acid (TCA) or formalin (Section 3.3.1), may also serve as immunosorbent for many mammalian antibodies (Table 7.1). Both fixation procedures are satisfactory but yield products with different properties. Fixation of Staphylococci with hot TCA (Lindmark, 1982) removes the negatively charged cell-wall polymer teichoic acid, producing an IgG-sorbent which can bind 1.4 mg human IgG per ml of a 10% (v/v) suspension of bacteria and is stable for about 5 months. Formalin-fixed bacteria (Kessler, 1976) bind 35% more IgG and are stable for at least 1 year. However, IgG can be eluted quantitatively from TCA-fixed bacteria but not from formalin-fixed bacteria, probably due to the interaction between IgG and teichoic acid, unless 80 mM MgCh is included in the acid buffer. 

The production of Staphylococci and their fixation with formalin has been described in Section 3.3.1. For the fixation of the bacteria with TCA, the bacterial suspension is heated in a boiling water bath for 2 min and an equal volume of hot 10% TCA is then added while swirling. The mixture is heated for 6 min at 90°C 

Teichoic acid may also be removed from formalin-fixed bacteria by alkaline hydrolysis (Roger and Garret, 1963). 

---