Every lot of complement we ship carries one number that matters more than the rest: its hemolytic titer. It tells you how much lytic activity is in the vial, and it's the figure to compare when a bactericidal or cytotoxicity assay stops behaving.
What CH50 measures
The classical complement pathway ends in a membrane attack complex that punches holes in a target cell. The standard way to measure that activity is to give the pathway an easy target, sheep red blood cells coated with anti-sheep antibody, and see how much serum it takes to lyse half of them. The CH50 is the reciprocal of the serum dilution that produces 50% hemolysis, expressed per milliliter, so a higher CH50 means more functional complement. The 50% point is used because the dose-response curve is steepest there, which makes the titer reproducible (Modern Complement Analysis, PMC).
The assay needs every component from C1 to C9 to work in sequence, so a normal CH50 says the whole classical pathway is intact and a low one says something along the chain is missing or has been used up. There are variants for the alternative pathway (AH50, using rabbit erythrocytes without antibody) and for individual components, but CH50 is the one that appears on a reagent Certificate of Analysis.
How it's run
Serum is serially diluted in a buffer containing calcium and magnesium, mixed with a fixed number of sensitized sheep cells, and incubated at 37 °C for 30 to 60 minutes. The tubes are spun and the hemoglobin released into the supernatant is read at 541 nm against a fully lysed control. Plot percent lysis against dilution, read off the 50% point, and that's the titer. Every part of the assay is sensitive to handling. The cells have to be fresh and evenly sensitized, the buffer's divalent cations exact, and the serum kept cold until the moment it is diluted.
Using the titer
The titer on the certificate is a lot-release value measured on the day of filling, so treat it as a ceiling. Complement loses activity with time at −20 °C, quickly at 4 °C, and with every freeze-thaw. That's why we recommend storing it at −70 °C or colder and thawing a vial once, right before use. When you compare lots, compare titers. When an assay drifts, re-titer the working stock before you change anything else. Products sold as complement sources, baby rabbit complement and pooled human complement among them, are titered before release, and the lot certificate is searchable on our Certificates of Analysis page.
The clinical version
The same assay is run on patient serum. A low CH50 with a normal alternative-pathway titer points to a classical-pathway deficiency or to consumption by immune complexes, as in active lupus. A CH50 near zero suggests a complete deficiency of one component. Reagent titers and patient titers are read the same way, which is part of why the assay has stayed standard for so long.
Assays differ in how much complement they need. An opsonophagocytic assay tolerates a lower titer than a serum bactericidal assay against a resistant strain. Tell us the assay and the target organism and we can suggest a complement source and titer range that other laboratories have found to work.