Purification of human alpha 2-antiplasmin with chicken IgY specific to its carboxy-terminal peptide.
alpha 2-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to alpha 2-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and alpha 2-antiplasmin, pure alpha 2-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of alpha 2-antiplasmin activity; and (b) the alpha 2-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the alpha 2-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.
Lee
S C
SC
Warren Medical Research Institute, University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Lee
K N
KN
Schwartzott
D G
DG
Jackson
K W
KW
Tae
W C
WC
McKee
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007136:Immunoglobulins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011092:Polyethylene Glycols; D000979:alpha-2-Antiplasmin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
C016512:IgY; D007136:Immunoglobulins; D010446:Peptide Fragments; D000979:alpha-2-Antiplasmin; D011092:Polyethylene Glycols