Standardization of an opsonophagocytic assay for the measurement of functional antibody activity against Streptococcus pneumoniae using differentiated HL-60 cells

Abstract

Host protection against pneumococcal disease is primarily mediated by phagocytosis. We developed and standardized an opsonophagocytic assay using HL-60 cells (human promyelocytic leukemia cells). Fifty-five serum samples were analyzed for the presence of functional antibody against seven pneumococcal serogroups or serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) by using differentiated HL-60 cells (granulocytes) and peripheral blood leukocytes (PBLs). Six of the 55 serum samples were from unvaccinated adult volunteers, 31 serum samples were from adults who received one dose of the 14-valent or the 23-valent polysaccharide vaccine, and 18 serum samples were from 16-month-old infants who received four doses of an investigational 7-valent polysaccharide-protein conjugate vaccine. The results of an opsonophagocytic assay with HL-60 cells correlated highly with those of an assay with PBLs as effector cells (median r for seven serotypes ‫؍‬ 0.87; P < 0.01). Opsonophagocytic titers were compared with the immunoglobulin G antibody concentrations determined by enzyme-linked immunosorbent assay (ELISA). The r values for serogroups or serotypes 4

Contribution of serotype-specific IgG concentration, IgG subclasses and relative antibody avidity to opsonophagocytic activity against Streptococcus pneumoniae

“…Opsonophagocytic activity of serum antibodies was determined by measuring the killing of live pneumococci by fresh human polymorphonuclear leucocytes (PMNL) in the presence of antibody and complement. A modi®cation of the method described by Romero-Steiner et al was used [18]. Instead of differentiated HL-60 cells, PMNL were used.…”

Low‐cost, high‐throughput, automated counting of bacterial colonies

“…The opsonophagocytic killing assay (OPA) is a widely accepted method for the quantitation of functional antibodies against S. pneumonia (22). Unlike the enzyme-linked immunosorbent assay (ELISA), which measures both functional and nonfunctional antibodies (23), the OPA directly measures antibodies that both coat the surface of the bacteria (opsonization) and induce the subsequent uptake and killing (phagocytosis) of pneumococcal strains.…”

Mucosal immunisation with novelStreptococcus pneumoniaeprotein antigens enhances bacterial clearance in an acute mouse lung infection model

“…IgG serotype-specific antibodies to capsular polysaccharides have been shown to be both opsonic and bactericidal [37][38][39][40]. Similar functional antibody studies have not been reported for pneumococcal protein antigens, however, specific IgG to the streptococcal protein enolase, an ubiquitous surface-expressed protein, has been shown to effectively opsonise and enhance phagocytosis of both serotype 6 and 14 of group A streptococci [41].…”