Protocol:Affinity Purified Monospecific Antibodies to Gel-Purified Proteins

Tony Rook

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Please find the link for the following protocol for

Affinity Purified Monospecific Antibodies to Gel-Purified Proteins
By: Ed Rybicki

http://www.mcb.uct.ac.za/manual/Monospec%20Antibodies.htm

Introduction

Since the introduction of sensitive immune assays, it has become increasingly apparent that previous criteria of protein purity are inadequate for the production of truly specific antisera. For example, I found that rabbit antisera raised against highly-purified Brome mosaic virus (BMV) reacted sufficiently well with plant components as to obscure the virus protein reaction in western blotting tests with infected plant samples (RYBICKI and VON WECHMAR 1982), even though only traces of plant proteins were visible after silver staining of SDS-PAGE fractionated purified virus used for rabbit immunisation (RYBICKI, unpublished). Because this problem necessitated the thorough and relatively laborious absorption of all sera to be used in such tests, I decided to investigate the feasibility of using preparative electroblotting to purify virus capsid protein to a sufficiently rigorous extent as to ensure the absence of any of the common host protein contaminants, and then to apply the method of OLMSTED (1981) for the affinity purification of virus-specific antibodies from antisera that also reacted with plant components. The latter technique entails using the virus protein immobilised on a porous support as a specific immunoadsorbent, from which antibodies may be eluted after specific binding. This investigation was designed to test the applicability of the outlined procedures to the routine small-scale production of antibodies suitable for use in enzyme-linked immunosorbent assays (ELISA), for viruses that occur only at very low concentration in plant sap, or which are extremely hard to purify because of their lability. There are also obviously many other applications.

References:
Rybicki, E.P. 1986. Affinity purification of specific antibodies from plant virus capsid protein immobilised on nitrocellulose. Journal of Phytopathology 116:30-38.

Olmsted, J.B. 1981. Affinity purification of antibodies from diazotised paper blots of heterogeneous protein samples. Journal of Biological Chemistry 256:11955-11957.

Rybicki, E.P. and M.B. von Wechmar. 1982. Enzyme-assisted immune detection of plant virus proteins electroblotted onto nitrocellulose paper. Journal of Virological Methods 5:267-278.