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A one-step exclusion-binding procedure for purification (...)

Biotechnol Appl Biochem. 2009 Dec ;54(4):207-12
A one-step exclusion-binding procedure for purification of functional heavy-chain and mammalian-type gamma-globulins from camelid sera.
Blanc MR, Anouassi A, Ahmed Abed M, Tsikis G, Canepa S, Labas V, Belghazi M, Bruneau G.

A new approach has recently been proposed for the purification of "mammalian-type" immunoglobulin G (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 flow-through fraction. Here, the technique was applied to camelid IgGs, which are known to be constituted not only of classic mammalian-type IgGs (IgG1) but also of IgGs containing only heavy chains (HC-IgGs). Both mammalian type and HC-IgGs can be purified in the flow-through fraction of dromedary 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 liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/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 could 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 and Vicugna than for Camelus species. The "Melon gel" technique can thus be used to satisfactorily purify IgG1 and HC-IgGs from all camelid species.


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