Opsonophagocytic killing assays (OPKA/OPA) and multiplex OPKA (MOPA) evaluate the ability of antibodies to opsonize bacteria, facilitating their destruction by immune cells. Unlike ELISAs, which quantify antibodies, opsonophagocytic assays measure their functionality — specifically, whether they trigger immune responses that kill bacteria.
For vaccines targeting encapsulated bacteria like S. pneumoniae, OPKA data are especially vital to assess functional antibody response, as noted by the World Health Organization, which recommends OPKA for pneumococcal vaccine efficacy studies.
Our team has decades of experience supporting vaccine developers with OPKA reagent sourcing and optimization. Below are some of our scientists’ most important considerations and tips for reliable, reproducible assays.
Use consistent, well-differentiated phagocytes
Inconsistent cell populations increase variability and compromise accuracy. We recommend the following practices to avoid potential issues:
- Source HL60 cells from reputable suppliers (e.g., ATCC), and prepare your primary cell bank stock into a number of individual aliquots
- Expand a single aliquot to make several working cell banks to freeze
- Thaw working cell banks and expand cells carefully by recording each passage number
- Monitor the passage number and assess expression levels of CD71, CD35, and CD11b antigens before and after differentiation
Choose the right complement source
Complement proteins are essential in OPKA for facilitating opsonization. However, inconsistent complement activity can lead to false positives and reduced sensitivity.
Select a complement source (human or other animal) that undergoes strict quality control and standardized manufacturing processes. Our baby rabbit complement is a popular choice due to the availability of large pools (10 liters) that can be used for clinical testing over a large period of time using the same complement lot.
Large lot volumes help you save time since it is essential to test individual batches for non-specific killing. If you already have a qualified complement lot and need to source additional complement, we recommend obtaining a sufficient volume from multiple new lots to test in a head-to-head fashion.
These tests allow you to determine whether any of the new batches match your qualified lot to your satisfaction before purchasing large volumes. In certain instances the complement concentration used in your OPKA may have to be adjusted based on the specific lot to maintain assay sensitivity.
Learn more about baby rabbit complement in our previous blog post.
Minimize non-specific killing with heat-inactivation and appropriate controls
Heat-inactivation is critical to remove endogenous complement activity that can cause intrinsic bacterial killing. We recommend that all antisera tested in OPKAs is heat-inactivated at 56°C for 30 minutes.
Additionally, always run controls of complement, HL60 cells, and bacteria without antiserum to assess non-specific killing.
If you are experiencing higher than expected non-specific killing, here are a few troubleshooting ideas to try:
- Run the assay with a new aliquot of bacteria to see if the original one was compromised in some way that made it easier to be killed
- Use a different bacterial strain to see if you can replicate your findings
- Test a new HL60 cell preparation, preferably at either a higher or lower passage number
Leverage multiplex OPKAs for efficiency
In vaccine research, especially for pneumococcal vaccines, it’s often necessary to test multiple serotypes. Multiplex OPKAs (like MOPA4) allow simultaneous testing of several serotypes, reducing serum volume requirements. Multiplex assays increase throughput and reduce reagent costs, making your research more efficient.
Validate, qualify, and monitor OPKA performance with human serum
OPKA validation requires objective evidence that the assay consistently delivers quality data, which should fall within a specified range that is specific, accurate, and precise.
A negative human serum panel matrix can be used with seropositive serum samples to create human serum panels. These panels are necessary to qualify bacterial banks, cell banks, and complement sources — essential reagents for validation testing.
Negative human serum matrix is also required for dilutional linearity studies, and can facilitate construction of human seropositive controls to use as quality control specimens for data acceptance.
In addition, human serum panels are necessary to create proficiency panels to monitor OPKA performance throughout all Phase 3 studies and for follow-up post-licensure vaccine testing.
Effective vaccine research requires high-quality reagents
OPKA is a critical tool in functional immunology for vaccine development. Following best practices and using top-tier reagents enables reliable, reproducible results that help accelerate vaccine research.
For researchers conducting OPKA, using high-quality reagents — such as complement serums and antibody-depleted human serums — is essential for generating high-quality data across the entire vaccine testing lifecycle.
Ready to optimize your assays?
Pel-Freez offers a range of complement and serum products specifically designed to meet the rigorous demands of biofunctional assays.
Explore our portfolio of complement and antibody-depleted serums, or contact us for more information.
