The cleanest way to prove a complement protein does something is to take it out and see what stops working. Depleted human serum is normal serum with one component removed by immunoaffinity and everything else left active. Add the purified protein back and the function should come back with it. Much of what we know about the cascade was worked out this way, and it's still the standard tool for asking which pathway an effect runs through.
Three pathways, one set of reagents
The classical pathway starts when C1q binds antibody on a surface. The lectin pathway starts when MBL or ficolins bind microbial sugars. The alternative pathway ticks over on its own and amplifies on any surface that lacks regulators. All three converge on C3 and then on the terminal sequence, C5 through C9. Take out a protein that belongs to only one pathway and you switch that pathway off.
C1q-depleted serum has no classical-pathway initiation. Lysis or deposition that survives in it is lectin or alternative. Lysis that disappears, and returns when purified C1q is added back, is classical.
Factor B- and Factor D-depleted serum remove the alternative pathway and its amplification loop, leaving classical and lectin activation to study on their own.
C3-depleted serum blocks all three pathways at their common point. It's the negative control that shows an effect is complement-dependent at all.
Terminal-component depletions (C5 through C9) let opsonization and C3 deposition proceed while preventing membrane attack, so you can separate the two.
We prepare human serum depleted of C1q, C1s, C2 through C9, Factor B, Factor D, Factor H, Factor I and Factor P. The full set is in our complement-depleted human serum collection.
What the experiments look like
A typical pathway study runs the same assay three ways: in normal serum, in the depleted serum, and in the depleted serum reconstituted with the purified component. Bacterial killing that disappears in C1q-depleted serum and returns on reconstitution is classical-pathway dependent. Killing that persists points to the alternative or lectin route. In work on complement resistance in Escherichia coli, for instance, blocking the classical pathway abolished most of the serum killing of the sensitive strain, which identified it as the main route of attack (PMC). The same design is used for antibody-mediated cytotoxicity, for testing complement inhibitors, and for asking whether a biomaterial or a nanoparticle activates complement, and if so through which pathway.
Regulator depletions answer a different question. Serum depleted of Factor H or Factor I has an unrestrained alternative pathway. Comparing it with normal serum shows how much a given surface relies on those regulators for protection.
Getting results you can trust
Run the depleted, normal and reconstituted conditions in the same experiment, from the same thaw. Confirm the depletion in your own assay before you draw conclusions, because "depleted" means below the detection limit of the release test, not absolute zero. Keep buffers free of chelators, thaw each vial once on ice, and use the same normal serum as the reconstitution baseline for the whole study.
If your experiment needs a component we don't list, or a reserved lot for a longer program, contact us and we'll tell you what we can prepare.