A one-step exclusion-binding procedure for the purification of functional heavy-chain and mammalian-type gamma-globulins from camelid sera.
A new approach has recently been proposed for the purification of 'mammalian-type' IgG, consisting of exclusion binding. The technique uses a gel ('Melon gel'; Pierce) that binds to all plasma proteins, but not to IgGs, thus allowing IgGs to be recovered in the FT (flow-through) fraction. Here, the technique was applied to camelid IgGs, which are known to be composed of not only classic mammalian-type IgGs (IgG1) but also HC-IgGs (heavy chain IgGs). Both mammalian type and HC-IgGs can be purified in the FT fraction of dromedary (Camelus dromedarius) plasma samples with less than 8.5% contamination, by making minor improvements to the conditions recommended by the manufacturer. The contaminant proteins, as determined by LC-MS/MS (liquid chromatography-tandem MS), are mainly transferrin and albumin. The recovery rate is elevated for both types of IgGs (95+/-14% and 88+/-25% for IgG1 and HC-IgGs respectively). IgGs thus purified maintain their ability to bind to their antigen, as measured by surface plasmon resonance and Western ligand blotting. Furthermore, IgGs can be purified from plasma samples of all camelid species in a similar manner, although the ratio of HC-IgGs to total IgGs was lower for Lama (llama) and Vicugna (vicuña) than for Camelus species. The 'Melon gel' technique can thus be used to satisfactorily purify IgG1 and HC-IgGs from all camelid species.
Blanc
Michel R
MR
INRA, UMR85 PRC (Physiologie de la Reproduction et des Comportements), Nouzilly, France.
Anouassi
Abdelhaq
A
Ahmed Abed
Mohanad
M
Tsikis
Guillaume
G
Canepa
Sylvie
S
Labas
Valérie
V
Belghazi
Maya
M
Bruneau
Gilles
G
D000818:Animals; D015153:Blotting, Western; D002162:Camelus / Q000097:blood*; D002853:Chromatography, Liquid; D007143:Immunoglobulin Heavy Chains / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D044967:Serum / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D005719:gamma-Globulins / Q000302:isolation & purification* / Q000378:metabolism
D007143:Immunoglobulin Heavy Chains; D005719:gamma-Globulins