تهيه و خالص سازي آنتي بادي با دي پلي كلونال بر عليه انسولين انساني

Production and Purification of Polyclonal Antibody to Human Indio

Insulin is a two-chain polypeptide hormone with molecular weight of 5.7 KD , which has 51 AA . By some disulfide bounds , these two chains are connected to each other . One of the most important diseases associating insulin is diabetes . In these patients , in the absence of insulin , cells cannot absorb glucose and glucose level of blood will increase and finally body tissues encounter a great energy shortage causing different acute and chronic implications . Nowadays for measuring this hormone , RIA methods are used . The aim of this research is designing of ELISA kit for measuring of insulin and the first step in designing such kits consists of providing and purifying antibody against human insulin . In order to provide and purify polyclonal antibody to insulin some practical fulfilled as follows . For produce antibody , we immunized Guinea pigs and after immunization stages , they were bleeded and their serum was separated . In order to assess and diagnose the produce anti-insulin by these pigs , RIA , DID and Dot blot methods were used . After making sure of the presence of insulin antibody in the serum , we purified antibody with saturated ammonium sulfate and then ion exchange chromatography . Also for the assessment of the antibody purification rate , the SDS-PAGE technique was used . Finally Western Blotting technique was used to make sure that there is not any unspecified bound .