"Sterile" and "low endotoxin" sound like the same promise, but they aren't: one says no living organisms; the other says no bacterial debris. A lot can pass sterility testing and fail an endotoxin limit, or the other way round, because they're different tests. Below, what each release test on a biological reagent measures, how it's done, and what the result does and doesn't tell you.
Sterility
Sterile serum, plasma and complement are made by passing the product through a 0.2 µm membrane into sterile containers under aseptic conditions, then confirmed by culturing samples of the filled lot in media that support bacterial and fungal growth for a defined period. The method for biological products is set out in the US regulations on sterility testing (21 CFR 610.12), and the ICH Q6B guideline on specifications for biological products lists sterility among the routine release tests (ICH).
Sterility is a sampling test, so it proves the sampled units were free of growth, not that every unit is, which is why the process (filtration validated for the filter and the product) matters as much as the result. It's also why we list sterile and non-sterile versions of many sera and plasmas, because filtration costs money. A coagulation assay that runs in an hour doesn't need it; a cell culture that runs for a week does.
Endotoxin
Endotoxin is the lipopolysaccharide of Gram-negative cell walls. It's released when bacteria die, it isn't removed by sterile filtration, and it activates immune cells at concentrations far below anything a protein assay would pick up. The test is the Limulus amebocyte lysate (LAL) assay, which uses a clotting cascade from horseshoe-crab blood that lipopolysaccharide triggers. Gel-clot, turbidimetric and chromogenic formats exist, and recombinant Factor C assays are replacing the animal-derived lysate. Results are reported in endotoxin units per milliliter or, for a purified protein, per milligram.
Since the debris can't be filtered out afterwards, endotoxin control means keeping bacteria out of the process from the start. Where an endotoxin specification applies, the result is on the lot's Certificate of Analysis. Where none is stated, assume the product wasn't tested for it and choose a low-endotoxin grade for cell or animal work.
The other release tests
- Hemolytic titer for complement, the functional assay that measures what the product is for.
- Appearance and hemolysis grade for serum and plasma: color, clarity and the presence of hemoglobin, which interferes with optical assays.
- Protein concentration and purity for albumins, gamma globulins and purified immunoglobulins, by electrophoresis or chromatography.
- Identity, confirming the species by immunological or electrophoretic methods, where the product's species is the specification.
- Mycoplasma and viral testing where the product is a complement or serum destined for cell-based assays. Human material also carries donor testing for HBsAg, HCV and HIV.
- pH and osmolality for liquid products where formulation matters to the assay.
Not every test applies to every product. The lot certificate lists the ones that were run and the specification each result was compared against.
Read the numbers, not the checkmarks
A result within specification is a floor, not a description. Two lots can both be "sterile, non-hemolyzed, titer ≥ 1:200" and behave differently in a sensitive assay. Record the actual values with your data and compare them when a result shifts. If your application is more demanding than the catalog specification, tell us. We can often test a lot against a tighter limit before it ships. Contact us with the specification your assay needs.