Slemmon J R, Salvaterra P M, Saito K
J Histochem Cytochem. 1980 Jan;28(1):10-5. doi: 10.1177/28.1.6766153.
The preparation and characterization of a horseradish peroxidase-rabbit antiperoxidase Fab immunocomplex (HRP-Fab2) useful for immunocytochemical localization of primary tissue-bound rabbit antibody are described. Antisera with titer to horseradish peroxidase (HRP) were raised in rabbits. Anti-HRP-Fab fragments were prepared by controlled mercuripapain digestion of the purified rabbit IgG. The complex was formed during incubation of Fab fragments with HRP, and fractions containing HRP activity that were precipitable by goat anti-rabbit IgG serum were isolated by gel filtration. The major isolated complex had a molecular weight of approximately 150,000 daltons and migrated as a single band on cellulose acetate electrophoresis. Polyacrylamide gel electrophoresis in SDS indicated the major polypeptide components of the complex were HRP and Fab. RZ (absorbance at 403 nm/275 nm) determination indicated a molar ratio of 2 Fab:1 HRP. The complex was stable for at least 1 year at -20 degrees C and was used successfully in a number of immunocytochemical procedures.
本文描述了一种辣根过氧化物酶-兔抗过氧化物酶Fab免疫复合物（HRP-Fab2）的制备和特性，该复合物可用于免疫细胞化学定位与组织结合的兔源性初级抗体。用辣根过氧化物酶（HRP）对家兔进行免疫以产生抗血清。通过用控制量的巯基木瓜蛋白酶消化纯化的兔IgG来制备抗HRP-Fab片段。Fab片段与HRP孵育时形成复合物，通过凝胶过滤分离出含有可被山羊抗兔IgG血清沉淀的HRP活性的组分。分离得到的主要复合物分子量约为150,000道尔顿，在醋酸纤维素电泳上呈单一泳带。SDS聚丙烯酰胺凝胶电泳表明复合物的主要多肽成分是HRP和Fab。RZ（403nm处吸光度/275nm处吸光度）测定表明Fab与HRP的摩尔比为2:1。该复合物在-20℃下至少稳定1年，并成功用于多种免疫细胞化学程序。
