Normal Serum Cytotoxicity for P32-Labeled SmoothEnterobacteriaceaeIII. Isolation of a γG Normal Antibody and Characterization of Other Serum Factors Causing P32Loss

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Abstract
Spitznagel, JohnK. (University of North Carolina School of Medicine, Chapel Hill). Normal serum cytotoxicity for P32-labeled smoothEnterobacteriaceae. III. Isolation of a γG normal antibody and characterization of other serum factors causing P32loss. J. Bacteriol.91:401–408. 1966.—Gram-negative bacteria lost metabolically incorporated P32when suspended in serum only if the serum contained heat-labile in addition to heat-stable factors. Gram-positive bacteria labeled with P32and included for comparison lost P32in heat-inactivated as well as in fresh normal serum. Further investigation of gram-negative bacteria showed that a smoothEscherichia coli(O117:H27) lost P32only if suspended in serum containing complement fractions C′1, C′2, C′3, and C′4 “normal” antibody and lysozyme. The normal antibody was recovered by absorption on and subsequent elution fromE. coliO117:H27 cell walls. Immunoelectrophoresis showed that it was a γG-globulin. Its P32-releasing activity was destroyed by 2-mercaptoethanol. Lysozyme was found to potentiate the P32-releasing action of normal antibody plus complement. Evidence was obtained suggesting that β1Cglobulin was the component absorbed to zymosan during serum absorption at 15 C. Reduction of the β1Clevel evidently upsets an important balance that exists in normal serum among complement, antibody, and lysozyme. This balance is essential for maximal P32release from labeled bacteria, or possibly for a maximal antibacterial effect from normal serum. The possible relationships of bactericidal, bacteriolytic, and opsonic action of normal serum are discussed.