Detection Of Angiostatin In Human Urine And Its Clinic Application

Human Angiostatin, an endogenous protein of 38KD, is one of the endogenous angiogenesis inhibitors containing the first three or four disulfide-linked structures known as kringle from plasminogen. Each kringle has around 80 amino acid residues. Angiostatin may be produced from the degradation of plasminogen either by some enzymes secreted by tumor cells or by metalloelastases such as those derived from macrophages. Normal human blood concentration of Angiostatin is 1.6 + 0.2i mol.A large body of studies has demonstrated that angiostatin specifically suppresses proliferation of endothelial cells of tumor vessels and consequently inhibits the growth and metastasis of tumor. Now, clinic trial of angiostatin is under way. A lot of studies have been reported on the molecular biological characteristics and the manufacture and purification procedures of angiostatin.This study aimsa establishing an Angiostatin assay method todetect the angiostatin levels in fluid of tumor patients and to study the relationship between angiostatin levels and carcinogenesis, development and prognosis of some cancers.It has been proved that in the structure of angiostatin, there are two lysine-binding sites located in Kl and K4, respectively. According to the basic principles of ELISA, minipore boards were coated with lysine. After incubation sample (angiostatin), angiostatin would be bound to the fixed lysine residue and combine with the specific anti-angiostatin monoclonal antibody. The second antibody was labeled with enzymes.According to the principle and technical requirements of ELISA, in this study the titration method was used to define the optimal concentration of the coated, monoclonal antibody and the enzyme-labeled antibody. A purified angiostatin was used to control the quality of angiostatin ELISA and to draw the standard curve.Materials and methods:1. Isolation and purification of human angiostatinHuman angiostatin was isolated and purified through enzyme digestion,affinity chromatography and gel filtration chroma-tography using materials such as human plasma, elastase, sepharose 4B-lysine and aminohexylate.2. Conjugation of horseradish peroxidase (HRP) and rabbit anti- mouse IgGCrude antiserum from the salting-out of rabbit antiserum against mouse IgG by saturated ammonium sulphate was passedthrough Sephadex G-200 column for purifying. Purified rabbit anti- mouse IgG was conjugated to HRP with the modified sodium periodate method3. Determination and optimization of ELISA conditionsIn the 96-well plates coated with lysine, the purified angiostatin was added as standard, and the monoclonal antibody against human Kl-3 was used as the first antibody with the HRP conjugated rabbit anti-mouse IgG as the second antibody. According to the principle of ELISA technique, an array titration assay was used to determine the best concentration of the coating lysine, monoclonal antibody against human Kl-3, and the second antibody.4. Assay of angiostatin with standard curveStandard curve was drawn with the purified angiostatin. The sensitivity of this standard curve was detected. Human angiostatin was detected according to the standard curve. Statistics:The data were analyzed by chi-square test, F test, the least significant difference-t test or analysis of variance. Summary statistics were expressed as mean values. Intergroup differences were considered statistically significant at a =0.05 Results:1 .The electrophoreses of extracted and purified angiostatin in this study were made and showed a clear band at 38kD with no other protein band. Western blotting demonstrated that the purifiedprotein was the angiostatin and its purity was>95%, satisfying the requirment of ELISA.2.The purified and labeled HRP-rabbit anti mouse IgG had a labeling rate of 0.41 and could be used as the second antibody.3.A procedure of ELISA and linear arrangement of the procedure was determined. The lowest detection limit was Ing/ml, and upper detection value was 200ng/ml.4.Detection results...