Purification of anti-apolipoprotein CIII antibodies by exploiting their affinity for apolipoprotein CIII linked to polyacrylamide gel after isoelectric focusing.
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.
Alsayed
N
N
INSERM U. 90, Service de Biochimie A, Paris, France.
Drüeke
T B
TB
Lacour
B
B
D000180:Acrylic Resins; D000906:Antibodies / Q000302:isolation & purification*; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007106:Immune Sera / Q000032:analysis; D007118:Immunoassay; D007525:Isoelectric Focusing
D000180:Acrylic Resins; D000906:Antibodies; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D007106:Immune Sera; C016680:polyacrylamide gels