Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Abstract

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

Broadly Reactive Human Monoclonal Antibodies Targeting the Pneumococcal Histidine Triad Protein Protect against Fatal Pneumococcal Infection

“…The correlate of protection for current pneumococcal vaccines is based on the elicitation of anti-capsule antibodies that opsonize bacteria, leading to their phagocytosis by host immune cells and subsequent bacterial killing ( 73 , 74 ). Mouse MAbs isolated by vaccination with PhtD were previously shown to induce bacterial opsonophagocytosis, which was dependent on complement and macrophages ( 45 ).…”

“…An opsonophagocytic killing assay was performed as described previously ( 74 , 87 ), as adapted from an earlier protocol with modifications ( 88 ). TIGR4 stocks were incubated in triplicate wells in a 96-well round-bottom plate for 1 h at 37°C with the indicated antibodies (10 μg of antibody per well in a final volume of 100 μl per well) in opsonization buffer B (OBB; sterile 1× PBS with Ca 2+ /Mg 2+ , 0.1% gelatin, and 5% heat-inactivated FetalClone [HyClone]), with heat-inactivated FetalClone-only-treated TGR4 cells serving as a control.…”

Development and Immunogenicity of a Prototype Multivalent Group B Streptococcus Bioconjugate Vaccine

“…An opsonophagocytic killing assay was performed as described previously, , as adapted from an earlier protocol with modifications . GBS type Ia, Ib, and III stocks were incubated in triplicate wells in a 96-well round-bottom plate for 1 h at 37 °C with the indicated sera samples (5 μL serum/50 μL total reaction volume/well) in opsonization buffer B (OBB: sterile 1× PBS with Ca2 + /Mg2 +, 0.1% gelatin, and 5% heat-inactivated Hyclone FetalClone I Serum).…”

Evaluation of PLGA nanoparticles containing outer membrane proteins of Acinetobacter baumannii bacterium in stimulating the immune system in mice

“…The opsonophagocytic killing assay was performed using the method developed by Paschall et al . though with some modifications ( 25 ). This method is an experimental procedure in which phagocytic cells are co-cultured with bacterial units.…”