There are two ways to get IgG out of serum, and they sit at opposite ends of the purity-versus-cost scale. Ammonium sulfate precipitation is a century old, costs almost nothing, and gives a fraction that is mostly immunoglobulin. Protein A or G affinity chromatography costs real money per liter and gives IgG that is essentially pure. We use both methods: salt fractionation makes our animal gamma globulin fractions; affinity purification makes our human IgG and IgM. The two products are for different jobs.
Ammonium sulfate precipitation
Proteins stay in solution because water molecules surround them. Add enough salt and the water is drawn away, so the proteins aggregate and fall out, each at its own characteristic salt concentration. Immunoglobulins precipitate at around 40 to 45% saturation, while albumin, the other major serum protein, stays dissolved until roughly 50 to 55%. A cut at 40 to 45%, redissolved and repeated, gives a fraction enriched in IgG with some IgM, IgA and other globulins carried along.
It scales to any volume, needs nothing beyond a centrifuge and a cold room, and is gentle on the antibody. Purity is typically 70 to 90% immunoglobulin, and the product has to be dialyzed or desalted to get the ammonium sulfate out. Where the fraction will be a blocking protein, a calibrator for total immunoglobulin or an immunogen, that purity is exactly what's needed, and the price reflects it.
Protein A and Protein G affinity chromatography
Protein A, from Staphylococcus aureus, and Protein G, from streptococci, bind the Fc region of IgG with high affinity at neutral pH and let go at pH 3 or below. Serum is passed over a column of immobilized ligand, everything else washes through, and the IgG is eluted with acid and neutralized. One step gives IgG of 95% purity or better.
The two ligands differ in species and subclass coverage. Protein A binds human, rabbit, pig and guinea pig IgG well, but human IgG3 and mouse IgG1 poorly, and it doesn't bind goat, sheep, rat or chicken IgG usefully. Protein G binds all human and mouse subclasses and most ruminant IgG, which makes it the more general choice. Protein A has higher capacity and is preferred for the species it covers. The costs are the resin, its limited lifetime, and the low-pH elution, which some antibodies don't survive without aggregating.
Side by side
| Consideration | Ammonium sulfate | Protein A/G |
|---|---|---|
| Purity | 70–90% Ig | 95%+ IgG |
| Cost per gram | Low | High |
| Scale | Any volume | Limited by column size |
| Species coverage | All | Ligand dependent |
| Other isotypes | Retained (IgM, IgA) | Removed |
| Stress on antibody | Minimal | Acid elution |
Use salt fractionation when you need immunoglobulin in bulk and the other isotypes don't hurt. Use affinity chromatography when the product has to be IgG and nothing else, as for calibrators, conjugation, and therapeutic and in vivo use. A lot of processes use both, a salt cut to concentrate and remove albumin and then an affinity step to polish.
Our lyophilized gamma globulin fractions from rabbit, bovine, goat, sheep, pig, mouse and rat are salt-fractionated, which keeps the price suited to bulk diagnostic and blocking use. Our human IgG and human IgM are affinity purified for use as calibrators and controls. If you need an affinity-purified fraction from an animal species, or a different formulation, ask; both are routine custom requests.