Development of Improved Method of Obtaining of Fc-fragments of Human IgM

Автори

The aim of work was to develop the improved methods of obtaining and allocating Fc-fragments of human IgM. To achieve this goal IgH hydrolysis with subsequent purification (Fс)5μ-fragments was optimized. The improved method for obtaining Fc-fragments of IgM provides papain hydrolysis of immunoglobulin in the nitrogen environment for 30 minutes allowing to achieve maximum output of Fcμ-fragments without their further degradation: isolation and purification of Fcμ-fragments by two-stage gel filtration on sephacryl S-300; control the purity of the target product in electrophoresis in polyacrylamide gel with SDS and immunodiffusion for Ouchterlony. By employing the proposed scheme, we can obtain Fcμ-fragments of high purity. Outcome of Fsμ-fragments after all stages of purification was about 15 % of the initial IgM amount in the preparation. Molecular weight of Fcμ-fragments was approximately 95 kDa.

Publication year: 
2012
Issue: 
3
УДК: 
577.27:57.083.33:543.54
С. 7—11. Іл. 6. Бібліогр.: 17 назв.
References: 

1. Ройт А., Бростофф Дж., Мейл Д. Иммунология. — М.: Мир, 2002. — 592 с.
2. W. Bains, Biotechnology from A to Z. Oxford, GB: IRL Press, 1998, 422 pр.
3. Імунологія: Підручник / А.Ю. Вершигора, Є.У. Пастер, Д.В. Колибо та ін.; передм. С. Комісаренка; за заг. ред. Є.У. Пастер. — К.: Вища шк., 2005. — 598 с.
4. Якобисяк М. Імунологія / Пер. з польської за ред. проф. В.В. Чоп’як. — Вінниця: Нова книга, 2004. — 672 с.
5. Иммунология: Практикум / Е.У. Пастер, В.В. Овод, В.К. Позур, Н.Е. Вихоть. — К.: Вища шк., 1989. — 304 с.
6. Антитела. Методы: Кн. 1 / Пер. c англ.; под ред. Д. Кэтти. — М.: Мир, 1991. — 287 с.
7. S.-E. Svehag et al., “Ultrastructure of papain and pepsin digestion fragments of human IgM globulins”, J. Exp. Med., vol. 130, pp. 691—705, 1969.
8. G.M. Butchko and F.P. Inman, “Mercaptan-induced fragmentation of a subunit-like proteolytic fragment of lnmunoglobulin M”, Biochem. J. (Great Britain), vol. 127, pp. 801—807, 1972.
9. K. Onoue et al., “Structure of human immunoglobulin M. II. Isolation of a high molecular weight Fc fragment of IgM composed of several Fc subunits”, J. Immunol., vol. 100, pp. 238—244, 1968.
10. I.D. Wilson, “Studies on the products of peptic digestion of IgA”, Immunol., vol. 20, pp. 327—339, 1971.
11. I.V. Nikolayenko et al., “Preparation of highly purified human IgG, IgM, and IgA for immunization and immunoanalysis”, Ukrainica Bioorganica Acta, vol. 2, no. 2, pp. 3—11, 2005.
12. Hsiao Shu-his and F.W. Putman, “The cleavage of human g-globulin by papain”, J. Biol. Chem., vol. 236, no 1, pp. 122—135, 1961.
13. J. Goding, Monoсlonal antibodies. Principles and practice. San Diego: Acedemic press, 1996, 492 рp.
14. U.R. Laemmli, “Cleavage of structural proteins during the assembly of the of bacteriophage T4”, Nature, vol. 227, pp. 680—685, 1970.
15. Михайлов А.Т., Симирский В.Н. Методы иммунохимического анализа в биологии развития (практическое руководство). — М.: Наука, 1991. — 288 с.
16. E. Harlow and D. Lane, Antibodies. A laboratory manual. New York: Cold Spring Harbor, 1988, 726 pp.
17. Иммунологические методы / Под ред. Х. Фримеля. — М.: Мир, 1979. — 520 с.

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