Clinical Chemistry
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Clinical Chemistry 34: 49-52, 1988;
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Clinical Chemistry, Vol 34, 49-52, Copyright © 1988 by American Association for Clinical Chemistry

Purification of anti-apolipoprotein CIII antibodies by exploiting their affinity for apolipoprotein CIII linked to polyacrylamide gel after isoelectric focusing

N Alsayed, TB Drueke and B Lacour
INSERM U. 90, Service de Biochimie A, Paris, France.

A new immunoaffinity technique for purifying anti-apolipoprotein CIII antibodies has been developed by isolating bands of apo CIII2 from isoelectric focusing (IEF) gel. Total apo C was obtained from delipidated very-low-density lipoprotein by chromatography on Sephacryl S 200. Apo CIII2 was separated from the apo CII band by IEF on polyacrylamide gel with use of pH 4-6 ampholytes. After sonication of the band in distilled water and various different washes, we directly mixed with antiserum a suspension of apo CIII2 bound to IEF- polyacrylamide gel. After their elution, we tested the specificity of the antibodies by an enzyme immunoassay technique, using plates coated with apolipoprotein, 100 ng per well. No cross-contamination of eluted anti-apo CIII antibodies by anti-apo CII or anti-apo E antibodies was observed. This affinity technique is easy to use, rapid to perform, and no sophisticated apparatus is needed. The gels can be used repeatedly and yield reproducible results with a very good analytical recovery (94%). We anticipate that this technique will prove useful for purification of other antibodies, particularly antibodies to apolipoproteins such as anti-apo CII and anti-apo E.




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Copyright © 1988 by the American Association for Clinical Chemistry.