Human serum is a good complement source with one problem: it already contains antibodies. Every adult donor has met pneumococci, meningococci and most of the other organisms vaccine programs target, and the antibodies they left behind will kill those bacteria in a bactericidal assay whether or not the vaccine antibody under test does anything. Antibody-depleted serum gets around this by taking the immunoglobulin out and leaving the complement in.
Why the antibodies have to go
A serum bactericidal assay (SBA) mixes the test antibody, live bacteria and an exogenous complement source, then counts survivors. An opsonophagocytic assay (OPA) adds phagocytes and asks the same question. In both, the complement source has to be functionally blind to the target. If it carries its own antibodies they will coat the bacteria, fix complement and produce killing that has nothing to do with the vaccine. Rabbit complement avoids this because young rabbits haven't encountered human pathogens; human complement has to be depleted first. Which to use, and when human complement gives the more relevant answer, is covered in choosing the right antibody-depleted human serum.
How depletion is done
The immunoglobulins are removed by immunoaffinity chromatography. Pooled human serum is passed over a resin carrying immobilized ligands that bind IgG and IgM. The antibodies stay on the column and the rest of the serum, complement included, flows through. Everything is done cold and quickly, because the complement proteins are far more fragile than the antibodies being removed. The depleted serum is then tested for residual IgG and IgM, titered for hemolytic activity, and frozen.
Three depletion levels are in routine use. IgG-depleted removes the dominant class and is enough when the assay's readout is insensitive to IgM. IgG/IgM-depleted is the standard for SBA and OPA work; IgM is a potent complement activator, so removing it matters. IgG/IgM/IgA-depleted also removes the secretory class, for pathogens with a mucosal component such as Neisseria gonorrhoeae or respiratory viruses.
Other uses
Outside vaccine assays, antibody-depleted serum is the matrix for immunoassay calibrators and negative controls, wherever a serum background without human immunoglobulin is needed, and it goes into proficiency panels. It also serves as a complement source in cytotoxicity and complement-deposition studies where human rather than rabbit complement is the relevant species.
Handling
Treat depleted serum as complement and store it at −70 °C or colder, thaw once in a 37 °C bath just until liquid, keep it on ice and use it within the day. Check the lot certificate for the hemolytic titer and the residual immunoglobulin levels, and run a no-antibody control in every assay to confirm the background is as low as you expect.
We've supplied double-depleted human serum to vaccine developers since 2017 and added the triple-depleted product for mucosal programs. Both are made in multi-liter lots so a clinical assay can stay on one lot from validation through the trial. Contact us for sample aliquots or to reserve a lot.