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评价用鼠巨噬细胞集落刺激因子(CSF-1)免疫的兔抗血清与人尿CSF-1的交叉反应性。一种交叉反应的抗血清用来提纯人尿中的CSF。将来自正常兔血清的IgG组分和抗CSF血清分别结合到溴化氰活化的琼脂糖凝胶上。取10升人尿用超滤法浓缩后,按顺序通过正常IgG柱和抗血清IgG柱。交叉反应蛋白质通过IgG柱去除,而CSF被抗CSF柱吸附。彻底冲洗抗体柱后,以4 mol硫氰酸钠洗脱CSF。根据在十二烷基硫酸钠(SDS)-丙烯酰胺凝胶上的迁移来判断,这一组分地含有4～5种污染的蛋白质。在进一步的提纯步骤中,CSF被刀豆素A(ConA)琼脂糖选择性地保留,然后用α-甲基葡萄糖苷洗脱。经这种方法提纯的CSF比活性为0.8～2.4×10~7U/mg蛋白质。一种单一的主要污染物质通过反相高效液相色谱(HPLC)法去除。最终提纯的CSF比活性为2.5～4.4×10~7U/mg蛋白质。每10升尿液可产生18～20μg的纯物质,收率为15%～25%。这一技术与以往报道的长时间、多步骤的方法相比,具有提纯速度快,CSF-1纯品产量高的优点。
To evaluate the cross-reactivity of rabbit antiserum immunized with murine macrophage colony-stimulating factor (CSF-1) to human urinary CSF-1. A cross-reacting antiserum is used to purify CSF in human urine. IgG fractions from normal rabbit serum and anti-CSF serum were separately bound to cyanogen bromide activated Sepharose. Ten liters of human urine were concentrated by ultrafiltration and passed sequentially through a normal IgG column and an antiserum IgG column. The cross-reacting protein is removed by the IgG column, whereas the CSF is adsorbed by the anti-CSF column. After washing the antibody column thoroughly, the CSF was eluted with 4 mol of sodium thiocyanate. Judging from the migration on the sodium dodecyl sulfate (SDS) -acrylamide gel, this fraction contains 4 to 5 contaminating proteins. In a further purification step, CSF is selectively retained by concanavalin A (ConA) agarose and then eluted with alpha-methylglucoside. The CSF specific activity purified by this method is 0.8 to 2.4 × 10 -7 U / mg protein. A single major contaminant is removed by reversed-phase high-performance liquid chromatography (HPLC). The final purified CSF specific activity was 2.5-4.4 × 10-7 U / mg protein. Each 10 liters of urine can produce 18 ~ 20μg of pure substance, the yield was 15% ~ 25%. Compared with the long-time and multi-step methods reported in the past, this technology has the advantages of high purification speed and high yield of pure CSF-1.