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Electrophoresis 1981 Feb;2(5-6):279-87

Toward an improved immunoglobulin analysis by gel electrophoresis and electrofocusing

Chrambach A, An Der Lan B, Mohrmann H, Felgenhauer K

Abstract

The analysis of immunoglobulin by gel electrophoretic methods is presently burdened with two problems: the incapacity of conventional buffer systems to spread the polyclonal immunoglobulin pattern across the entire separation path length in gel electrophoresis, and the appearance of distinct bands in the conventional electrofocusing patterns, instead of he continuum of zones ¿ which would be expected form a polyclonal mixture of ¿charge isomers¿ and which is indeed obtained in gel electrophoresis. Polyclonal serum immunoglobulin differ from one another predominantly in net charge (i.e., they are ¿charge isomers¿). The operative pH of gel electrophoresis was therefore optimized. The acidic side of he isoelectric operative pH of 5.32, a discontinuous (multiphase) buffer system was found which was capable of stacking all immunoglobulin in a lyophilized preparation. In order to provide a magnified view immunoglobulin heterogeneity, the immunoglobulin mixture was fractionated into 26 groups of charge isomers using preparative isotachophoresis. When subjected to polyacrylamide gel electrophoresis in resolving gels operative at pH 4.85, 0°C, the isotachophoretic fractions spread between relative mobility values of 0.4 to 0.9 in the electrophoresis pattern, i.e., their sum total covered the entire separation path. Under these conditions, isotachophoretic fractions also revealed immunoglobulin oligomers. The number of oligomeric forms increased from increased from the leading (most basic) to the trailing (most acidic) immunoglobulin components within the stack. In polyacrylamide gel electrofocusing of the isotachophoretic fractions of immunoglobulin, using pI-renge 6¿9 ampholine, addition of a 0.01 M mixture of amino acids to the carrier ampholytes, and 20h or more electrofocusing time at 20 V/cm of gel, 0°C, polyclonal immunoglobulin patterns appeared continuous. Electrofocusing patterns of those isotachophoretic fractions of immunoglobulin which contained oilgomeric forms (as revealed by gel electrophoresis ) exhibited one or two major bands against the continuous background of stainable components. Monoclonal immunoglobulin produced distinct major bands under the same comditions. Thus, either polyacrylamide gel electrofocusing in the pI-range 6¿9 or polyacrylamide gel electrophoresis in a buffer system operative at pH 4.85 appear promising for the development of a clinically practical and artifact-free electrophoresis analysis for specific oligoclonal immunoglobulin.


Category: Journal Article
DOI: 10.1002/elps.1150020505
Includes FDA Authors from Scientific Area(s): Biologics
Entry Created: 2012-12-08
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