Preparation of antibodies against xanthine oxidase from human milk.
1. Human xanthine oxidase [XO; EC 1.2.3.2.] was isolated by a non-proteolytic method from fresh human milk. Final purification of the protein was achieved by hydroxyapatite chromatography. Most (less than 95%) of the enzyme was released in the 0.40 M phosphate fraction at pH 6.8. 2. The specific activity of this preparation was found to be 0.047 microM min-1 mg-1 with xanthine as substrate. 3. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two subunits, each with a mol. wt approximately 122 kDa. 4. On non-denaturing acrylamide gels both of these subunits exhibited oxidase-like activity with xanthine as substrate in the presence of nitroblue tetrazolium and molecular oxygen. 5. Immunoconjugates of XO were prepared by the keyhole limpet hemocyanin (KLH)- and glutaraldehyde-crosslinking techniques. 6. Polyclonal antibodies to XO were raised by i.m. injection of these conjugates into female New Zealand rabbits. 7. Western blot analysis using the semi-dry technique was employed to confirm the specificity of the antibody.
Department of Pathology, Stanford University Medical School, CA 94305.
K
K
Graham
J E
JE
Fleming
R
R
Young
K G
KG
Bensch
D000818:Animals; D000917:Antibody Formation; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008895:Milk, Human / Q000201:enzymology*; D011817:Rabbits; D014969:Xanthine Oxidase / Q000276:immunology* / Q000302:isolation & purification
D014969:Xanthine Oxidase