Multiplexed Flow Cytometry
for Opsonophagocytic Assays

Abstract

Investigators have proven the effectiveness of using fluorescent microbeads in opsonophagocytic assays instead of live infectious agents. FlowApps enhances those methods with a multiplexed cytometric approach by:

• Detecting functional antibodies with differentially labeled fluorescent targets
• Detecting antibody panels for multiple serotypes in a single assay
• Increasing throughput using automation

Introduction

Host protection against various bacterial diseases is mediated by opsonophagocytosis.  A variety of techniques have been used to determine opsonophagocytic activity of antibodies produced in response to vaccines or natural infections. Viability and fluorescent tag assays are the most common. The presence of functional antibodies in the host leads to complement activation, effective opsonization, phagocytosis, and recovery from infection.

Phagocytic assays make it possible to reproducibly determine the phagocytic titer of serum pre- or post-vaccination. Analytical methods with flow cytometry are flexible, fast, and precise.  Assay development is antigen specific, highly adaptable, and quickly customized.

The greatest barrier to the measurement of functional antibodies is the time required for testing individual samples.  To overcome this problem, FlowApps offers fluorescent bacterial and microsphere target assays suitable for Flow cytometric automation which increases relative sample throughput, reduces assay variability.

Methodology

There are currently two commonly used phagocytic assays for functional antibody measurement. The first is referred to as a "phagocytic killing assay" and measures the elimination or reduction in CFUs encapsulated organisms by phagocytic effector cells incubated with a fresh complement source, and heat-inactivated serum from individuals containing putative anti-capsular antibodies.

The second aproach is flow cytometric uptake assays that measure phagocytic titers of functional antibodies  An advanced development of this assay involves the use of polysaccharide coated fluorescent microsphere targets to replace bacterial targets.

Results

Correlations with the reference killing OPA for S. pneumoniae demonstrated acceptable correlations and the utility of multiplexed measurement of functional antibody mediated opsonophagocytosis against four different pneumococcal serotypes in a single assay.  Compared to the reference killing OPA, multiplexed cytometric assays offer great utility in being able to measure:

• Various antibodies including IgG, IgA, and IgM
• Innate immunity
• Monoclonal therapeutics
• Anti-protein antibodies, (e.g., rPA, PspA and pneumolysin)

Other advantages of cytometric methods include:  

• Automated High throughput analysis
• Multiplexed data collection
• Smal sample size
• Improved accuracy and precision
• Use of non-infectious standardized reagents with a long shelf-life
• Eliminates antibiotic interference
• Suitable for GMP compliance
  

FlowApps’ approach yields reproducible results similar to those obtained by 1st generation reference methods, but at a much higher serotype answer throughput. With autoloaders, batch analysis, and curve-fitting software, multiplexing technologies deliver even faster results.

Find out more:

Application of Biomarkers, Surrogate Endpoints, and Correlates of Protection to Vaccine Development

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