Isolation of Polyclonal Antibodies to Collagen by Ion Exchange Chromatography on Column with DEAE-Sepharose

Olena Kravchenko, Larisa Oriabinska, Natalia Raksha, Larisa Ivashko, Mariia Abramova


Background. Development of new methods for isolation and purification of polyclonal antibodies.

Objective. Analysis of the efficiency of the ion exchange column with DEAE-Sepharose at isolation of polyclonal antibodies to collagen.

Methods. IgG were isolated from precipitated γ-globulin fraction of serum of immunized rabbit by ion exchange chromatography on column with DEAE-Sepharose.

Results. It was determined that the IgG output at volume ratio of 4:3 of starting material and the carrier applied to the column is 1.67 times higher than the ratio of 2:3. It is shown that IgG goes out of the column together with the protein that has a molecular weight of about 57–60 kDa. We found that 5.123 mg of IgG with minor impurities of serum proteins is obtained from 30 cm3 of rabbit’s serum.

Conclusions. The method of ion exchange chromatography on column with DEAE-Sepharose provides a high yield of IgG with few impurities and can be used as a way of pre-treatment of polyclonal antibodies.


Polyclonal antibodies; Collagen; Immunization; Ion exchange chromatography; DEAE-Sepharose


D. Low et al., “Future of antibody purification”, J. Chromatogr. B, vol. 848, no. 1, pp. 48–63, 2007. doi: 10.1016/j.jchromb. 2006.10.033

C. Newcombe and A.R. Newcombe, “Antibody production: Polyclonal-derived biotherapeutics”, J. Chromatogr. B, vol. 848, no. 1, pp. 2–7, 2007. doi: 10.1016/j.jchromb.2006.07.004

D. Koolivand et al., “Production of polyclonal antibody against grapevine fanleaf virus movement protein expressed in Esche­richia coli”, Plant Pathol. J., vol. 32, no. 5, pp. 452–459, 2016. doi: 10.5423/PPJ.OA.01.2016.0031

A.R. Parker et al., “Purification and characterization of anti-pneumococcal capsular polysaccharide IgG immunoglobulins”, Clin. Biochem., vol. 50, no. 1-2, pp. 80–83, 2017. doi: 10.1016/j.clinbiochem.2016.08.009

A.K. Lynn et al., “Antigenicity and immunogenicity of collagen”, J. Biomed. Mater. Res. Part B Appl. Biomater., vol. 71, no. 2, pp. 343–354, 2004. doi: 10.1002/jbm.b.30096

M. Yan et al., “Characterization of acid-soluble collagen from the skin of walleye pollack (Theragra chalcogramma)”, Food Chem., vol. 107, no. 4, pp. 1581–1586, 2008. doi: 10.1016/j.foodchem.2007.10.027

[7] K. Wangerin and H.-U. Wottge, “Immunological reactions against collagen”, Eur. J. Plast. Surg., vol. 17, pp. 287–291, 1994. doi: 10.1007/BF00181093

H. Bak and O.R.T. Thomas, “Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum”, J. Chromatogr. B, vol. 848, no. 1, pp. 116–130, 2007. doi: 10.1016/j.jchromb.2006.07.003

A.C. Grodzki and E. Berenstein, “Antibody purification: Ion-exchange chromatography”, Methods Mol. Biol., vol. 588, pp. 27–32, 2010. doi: 10.1007/978-1-59745-324-0_4

A. Ljunglof, “Ion exchange chromatography of antibody fragments”, Biotechnol. Bioeng., vol. 96, no. 3, pp. 515–525, 2007. doi: 10.1002/bit.21124

K.Y. Ko and K.Y. Ahn, “Preparation of immunoglobulin Y from egg yolk using ammonium sulfate precipitation and ion exchange chromatography”, Poultry Sci., vol. 86, no. 2, pp. 400–407, 2007. doi: 10.1093/ps/86.2.400

Z. Qin et al., “Purification of immunoglobulin and serum albumin from serum via strong anion exchange chromatography coupled with molecular exclusion chromatography”, Se Pu, vol. 30, no. 8, pp. 851–855, 2012. doi: 10.3724/SP.J.1123.2012.04002

U.K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4”, Nature, vol. 227, pp. 680–685, 1970. doi: 10.1038/227680a0

O.H. Lowry et al., “Protein measurement with the folin phenol reagent”, J. Biol. Chem., vol. 193, pp. 265–275, 1952.

M.M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding”, Anal. Biochem., vol. 72, no. 1-2, pp. 248–254, 1976. doi: 10.1016/0003-2697(76)90527-3

A.G. Gornall et al., “Determination of serum proteins by means of the biuret reaction”, J. Biol. Chem., vol. 177, pp. 751–756, 1949.

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