An improved haemolytic plaque assay for the detection of cells secreting antibody to bacterial antigens.
Recent advances in the development of conjugate polysaccharide vaccines for human use have stimulated interest in the use of assays detecting antibody-secreting cells (AbSC) with specificity for bacterial antigens. Here we present improved haemolytic plaque-forming cell (PFC) assays detecting AbSC with specificity for tetanus and diphtheria toxoid as well as for Haemophilus influenzae type b and pneumococcal capsular polysaccharides. These assays were found to be less time consuming, more economical and yielded 1.9-3.4-fold higher plaque numbers than traditional Jerne-type PFC assays. In the case of anti-polysaccharide AbSC of the IgG isotype, the increase was as high as 7.4-11.8 times. Evidence is presented that the pronounced improvement in the detection of the latter is due to the presence of aggregating anti-IgG antibody from the beginning of the assay. It is proposed that in the case of low affinity of anti-polysaccharide antibodies aggregation of secreted monomeric antibody (IgG) is critical for plaque formation and increases the avidity of binding to target cells.
Barington
T
T
Laboratory of Medical Immunology, Medical Department TTA, Rigshospitalet, Copenhagen, Denmark.
Heilmann
C
C
D000906:Antibodies / Q000276:immunology; D000917:Antibody Formation; D000921:Antibody-Producing Cells / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology*; D006192:Haemophilus Infections / Q000175:diagnosis; D006193:Haemophilus influenzae; D006462:Hemolytic Plaque Technique; D006801:Humans; D011008:Pneumococcal Infections / Q000175:diagnosis; D012680:Sensitivity and Specificity; D013742:Tetanus / Q000175:diagnosis; D013745:Tetanus Toxoid
D000906:Antibodies; D000942:Antigens, Bacterial; D013745:Tetanus Toxoid