人乳中黄嘌呤氧化酶抗体的制备。

Preparation of antibodies against xanthine oxidase from human milk.

Graham K, Fleming J E, Young R, Bensch K G

Department of Pathology, Stanford University Medical School, CA 94305.

Int J Biochem. 1989;21(7):715-22. doi: 10.1016/0020-711x(89)90201-2.

Abstract

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.

摘要

采用非蛋白水解方法从新鲜人乳中分离出人类黄嘌呤氧化酶[XO；EC 1.2.3.2]。通过羟基磷灰石层析实现该蛋白质的最终纯化。在pH 6.8时，大部分（少于95%）的酶在0.40 M磷酸盐组分中被洗脱出来。
以黄嘌呤为底物时，该制剂的比活性为0.047微摩尔·分钟⁻¹·毫克⁻¹。
十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）分离出两个亚基，每个亚基的分子量约为122 kDa。
在非变性丙烯酰胺凝胶上，在硝基蓝四唑和分子氧存在的情况下，以黄嘌呤为底物时，这两个亚基均表现出氧化酶样活性。
通过钥孔戚血蓝蛋白（KLH）和戊二醛交联技术制备XO免疫缀合物。
通过将这些缀合物肌肉注射到雌性新西兰兔体内来制备针对XO的多克隆抗体。
使用半干技术进行蛋白质印迹分析以确认抗体的特异性。