Biotechnol. Appl. Biochem. (2009) 54, 207–212 (Printed in Great Britain) doi:10.1042/BA20090208 207 A one-step exclusion-binding procedure for the purification of functional heavy-chain and mammalian-type γ -globulins from camelid sera Michel R. Blanc*†‡§, Abdelhaq Anouassi‖¶, Mohanad Ahmed Abed**, Guillaume Tsikis*†‡§, Sylvie Canepa*†‡§, Val´ erie Labas*†‡§, Maya Belghazi*†‡§ and Gilles Bruneau*†‡§ 1 *INRA, UMR85 PRC (Physiologie de la Reproduction et des Comportements), F-37380 Nouzilly, France, †CNRS, UMR6175 PRC, F-37380 Nouzilly, France, ‡Universit´ e Franc ¸ois Rabelais de Tours, F-37041 Tours, France, §Haras Nationaux, F-37380 Nouzilly, France, ‖Institut Agronomique et V´ et´ erinaire Hassan II, Rabat, Morocco, ¶Veterinary Research Center, Abu Dhabi, United Arab Emirates, and **Department of Medical Microbiology, College of Medicine, Kerbala University, P.O. Box 11252, Holly Kerbala, Iraq 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 determ- ined 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 (vicu ˜ na) than for Camelus species. The ‘Melon gel’ technique can thus be used to satisfactorily purify IgG1 and HC-IgGs from all camelid species. Introduction Camelid serum contains IgGs that are either of the mammalian type (heterotetramer: IgG1) or homodimers consisting of HC-IgGs (heavy chain IgGs), IgG2 and IgG3 as referred to by Hamers-Casterman et al. [1]. The absence of light chains and CH1 (heavy-chain gamma constant domain 1) in HC-IgGs confers on them a lower molecular mass (90 kDa versus 180 kDa) but a high binding affinity [2]. Diverse approaches have been proposed for the purification of IgG from serum, biological fluids or culture medium: ammonium sulfate precipitation, octanoic acid (‘caprylic acid’) treatment [3], thiophilic interaction [4] and affinity on immobilized Protein A, Protein G or Protein L (reviewed in [5]). A new approach (‘Melon gel’) has recently been proposed (manufacturer: Pierce, Perbio Science France, Brebieres, France) for the purification of various IgG isotypes from mammalian serum proteins based on a principle that has not been unveiled. In contrast with the immobilized Proteins A, G and L, which bind γ -globulins, in this approach, all non-γ -globulin proteins bind to Melon gel, whereas γ -globulins do not. Such a technique would present various potential advantages such as rapidity, the direct availability of IgGs (i.e. without the need for dialysis) and perhaps increased stability due to the absence of low pH treatment or precipitation at least for fragile isotypes. However, to the best of our knowledge and according to Pierce, it is not known whether camelid IgGs (especially the heavy-chain type, the binding capacity of which could be different from the mammal one) bind to Melon gel or not. Given these structural peculiarities of camelid IgG homodimers, we sought to determine if the Melon-gel technique could be applied to camelid IgGs. We showed that total IgGs, including HC-IgGs, can be purified from dromedary (Camelus dromedarius) plasma samples using this Key words: exclusion binding, flow-through (FT), heavy-chain IgG (HC-IgG), liquid chromatography–tandem MS (LC-MS/MS), Melon gel, surface plasmon resonance (SPR). Abbreviations used: ECL, enhanced chemiluminescence; FT, flow-through; FT1, the first FT fraction; HC-IgG, heavy-chain IgG; LC-MS/MS, liquid chromatography–tandem MS; ND, not detectable; RU, resonance unit; RY, recovery yield; SPR, surface plasmon resonance; WB, Western blotting; WLB, Western ligand blotting. 1 To whom correspondence should be addressed (email Gilles.Bruneau@tours.inra.fr). C  2009 Portland Press Ltd