尿激酶单克隆抗体制备,性质和应用

用高分子量尿激酶(HMW UK)免疫BALB/C小鼠,获得21株UK单抗(MoAb)分泌细胞株,并用部分株的培养上清液、腹水或纯化的抗体研究UK MoAb的性质,包括免疫球蛋白分类和亚类、抗体滴度及抗原表位等。有三株MoAb能抑制UK的纤溶活性。通过研究不同MoAb同时与UK抗原作用时其反应强度变化的特点。并确定MoAb在UK的重链或轻链的表位以后,选用部分MoAb建立UK ELISA和UK蛋白质印迹与酶联免疫染色分析法,用以分析比较两种不同UK注射剂和UK尿抽提物的UK抗原含量、比活性,以及UK分子量分布的状况。

Preparation,Characterization and Application of Monoclonal Antibodies against Urokinase

Twenty-one cloning hybridomas secreting monoclonal antibodies (MoAbs) to urokinase (UK) were obtained after immunization to Balb/C mouse with high molecular weihgt (HMW) UK and fusion of the cells immunized animal spleen and cultured FO myeloma cells.The properies of the MoAbs were studies with cloning culture supernatants, ascific fluid and purified anibodies. Results shoved that 6 out of the 18 MoAbs obtained were of the same subclass lgGl, all the rests were of IgG2a. Ten of the MoAbs assayed for concentration and titer in the culture supernatants exhibited high affinity of different degrees to UK antigen. Three of the 19 MoAbs studied were capable of neutralizing the UK enzyme activity. For the inhibition of 50% activity of 0.61 pmol UK, the amount of antibody needed was 0.85, 1.04 and less than 0.61 pmol respectively. These 3 MoAbs were prove (found) to bind specifically to the heavy (H) chain of UK protein, and the other 9, to the light (L) chain.Using the ELISA "saturation-pyramiding" method, 3 of the 8 MoAbs analyzed showed enhanced reactivi ty when any 2 of them were combined to react to UK antigen and that caused by any one singly. In order to develop a sensitive ELISA method for UK quantitative analysis, these cloning hybridomas were selected for expansion, ascites production and MoAb preparation. Two kinds of UK injections for clinical use and a urine UK extract for pharmaceutical production of UK agents were taken to analyze the UK antigen about their contents, specific activities and the dectrophorsis patterns by means of UK-ELISA as well as Western blot combined immunoenzyme-staining method using an anti-UK H chain-MoAb.