Immunogenicity testing tells a vaccine developer whether a candidate produces a protective immune response. For serum bactericidal assays (SBA) and opsonophagocytic killing assays (OPKA) in particular, the complement source is a weak point: human serum comes with the donors' own antibodies, and those antibodies can kill the target organism whether or not the vaccine antibody does.
We developed a scalable, validated method for removing IgG, IgM and IgA from native human complement without losing its functional activity. This post is about the problem it solves and the data behind it.
The problem with native human complement
Any human serum used as a complement source contains IgG, IgM and IgA from the donors' past infections and vaccinations. Those antibodies activate the complement cascade on their own, so an SBA or OPKA shows killing that has nothing to do with the vaccine-induced antibody being tested. The background is high, it varies from lot to lot, and it makes results hard to interpret and hard to reproduce.
Labs used to work around this by hunting for rare seronegative donors or by depleting antibodies themselves, small-scale and slow. Neither is practical for a clinical assay program.
What the depletion process keeps
Our process selectively removes IgG, IgM and IgA from pooled human serum at liter scale while preserving complement function, the other major serum fractions and the matrix proteins, so what is left behaves like human complement without the background. Antibody-depleted human complement (ADHC) is made in large batches from that process.
Figure 1. Total complement hemolytic titer is retained following antibody depletion, enabling its use as a source of exogenous complement in vaccine efficacy and immunogenicity testing.
Figure 2. A) The Pel-Freez immunoglobulin removal process does not remove other major serum fractions, such as serum albumin, alpha1 globulins, alpha2 globulins, and beta globulins from pooled human serum, as demonstrated by serum protein electrophoresis. B) Comparison of pre- and post-depletion preparations by SDS-PAGE demonstrates similar protein profiles with the exception of IgG and IgM, which have been removed.
Figure 3. Analysis of pre- and post-depletion human serum by mass spectrometry indicates minimal differences in levels of critical matrix proteins (n = 3, *p > 0.05, †p < 0.05 due to increased serum concentration following depletion and diafiltration).
Where it has been used
Researchers in academia and the pharmaceutical industry have used ADHC and antibody-depleted human serum (ADHS) in immunogenicity assays against a wide range of bacterial and viral targets, including Neisseria gonorrhoeae, N. meningitidis, Haemophilus influenzae, respiratory syncytial virus, Staphylococcus aureus, SARS-CoV-2, Rickettsia, Klebsiella pneumoniae, Acinetobacter spp. and Mycoplasma genitalium. One complement source across that many targets means less assay-specific complement sourcing.
Figure 4. Distribution of literature citations by application since launch of ADHC and ADHS, n = 24.
Adoption has grown steadily, with more than 60 labs in 21 countries that used ADHC/ADHS in the first five years after launch, among them Sanofi, UCSF and UMass Chan Medical School, for routine assays and for new vaccine programs.
Figure 5. Example customer application: Comparison of Pel-Freez ADHC and a qualified complement source prepared by a vaccine developer in hSBA using sera from 12 vaccinated individuals.
Removing IgG and IgM takes the background out of the assay, so the result reflects the antibody you added. Lot-to-lot reproducibility is controlled by QC on every batch. Complement activity can be tuned by adjusting the ADHC concentration to suit the sensitivity the assay needs. And because it is made at volume, the same reagent carries from preclinical work into clinical trials.
If background antibody is a source of uncertainty in your immunogenicity assays, contact us for a sample, or look through the antibody-depleted human products.