A spectrophotometric procedure for quantitation of antibody directed to bacterial antigens

Abstract

A spectrophotometric procedure was developed to study the reaction of whole Streptococcus mutans 6715 cells with purified IgG antibody directed to the group-specific antigen of these organisms. The nature of the reaction was studied by combining different concentrations of antibody with 10 8 formalin-killed S. mutans 6715 cells and monitoring the optical density at 530 nm periodically for up to 24 hr. The curves obtained by plotting the observed optical density values against time were characterized by three phases: an initial rise, a plateau and a fall. The optical density changes were related to the number and size of cell aggregates present at various times during the procedure. Aggregated cells, primarily those in intermediate-size complexes (21–150 cells) appeared to contribute to initial rise in optical density, while further changes appeared to most closely correspond to the formation and subsequent sedimentation of the larger-size complexes (> 150 cells). Apparent rate constants calculated for the formation of intermediate size aggregates did not significantly vary with antibody concentration, while the rate constants for large aggregate formation increased dramatically with increases in antibody concentration. Spectrophotometric analyses performed with 14 different antibody concentrations ranging from 7·6 to 380 μg/100 μl showed that a linear relationship existed, after 1 min of reaction, between the log of the antibody concentration and the change in optical density. This relationship, coupled with the demonstration that serum components did not contribute to the effects of antibody on bacterial cells, indicated that the concentration of antibody in immune serums could be measured. Comparison of the spectrophotometric procedure with direct elution of antibody from cells, using 5 different serum samples, showed a mean per cent difference between the 2 methods of 6·5% (range 0·9–16·1%). Further comparison of the spectrophotometric procedure with the reversed radial immunodiffusion technique (4 serums tested) showed a mean per cent difference of 10·4% (range 0·3–30·3%). The spectrophotometric procedure was at least as accurate as the other procedures utilized and had the distinct advantage of permitting determinations to be made after 1 min of reaction.