纤维结合蛋白的提纯、定量、活性检测及抗体制备

采用重复两次亲和层析法从血浆中提取纤维结合蛋白(简称FN)可达到电泳纯;免疫家兔,抗FN血清平均效价可达16~x;经吸收得单特异抗血清;再经(NH_4)_1SO_4沉淀、DE-AE-HW离子交换可制出高纯度抗FN IgG;建立了两种FN的定量测定方法:1.火箭电泳法,检查了89份健康人血浆,平均含量为293±45μg/ml;2.免疫浊度法,检测了20份血浆,平均值为344±52μg/ml。最后建立了促进BHK细胞伸展活性的检测法,敏感度可达5μg/ml,并初步应用于稳定性和热变性试验,为开展FN的应用提供了质控指标。对比了我们精制的FN与SIGMA试剂公司的FN,二者成分相同,活性也很相似。

Purification, Quantitation, Activity Determination and Antibody Preparation of Fibronectin

We have been carring out the study of fibronectin (FN) since J986. FN was purified by affinity chromatography from human plasma or cryoprecipitate and we proved that PAGE pure FN could be prepared only by two cycles of chromatography. By immunization of rabbit with FN, anti-FN titre reached 16. After absorption, monospecific antiserum was obtained. Further purification of the anti-serum was performed by (NH_4)_2 SO_4 precipitation and DEAE-HW ionexchange. Anti-FN IgG of high purity was obtained. Two quantitative methods have been established. We have determined 89 samples of normal plasma using rocket electrophoresis and the average FN level was 293±45μg/ml. 20 samples of normal plasma have been determined by immunoturbidity assay, the average level was 344±52μg/ml. Biological activity was determined by promoting BHK cell spreading ability. It's sensitivity reached 5μg/ml FN. The preliminary tests of stability and thermal denaturation have been performed, and these data provided the possibility of producing FN preparations in batch amount for therapeutic use.