Production and characterization of anti-Her 2 monoclonal antibodies

Breast cancer is the most common cancer among women in the world. Early diagnosis of this cancer is a key element for its treatment. One of the approaches for diagnosis of breast cancer is detection of its tumour-associated markers. Hence, Her2 has been the main focus of the researches in the field. For diagnosis of Her2 overexpression, monoclonal antibodies [mAb] reacting against Her2 were produced in this study. For this purpose, two peptides from extracellular domain of Her2 were selected and the mAbs reacting against them were produced by hybrodoma technology. Reactivity of these antibodies were then evaluated in different immunological assays including ELISA, Immunoflurescence [IF], western blot [WB] and immunoprecipitation [IP]. Total of 5 clones were produced from two separate fusions, and antibody isotyping revealed that all clones were IgM. These mAbs showed appropriate reactivities in the following assays: ELISA, immunofluresence by staining of breast cancer cell line [SKBR3], WB and IP by detecting the 185 KD band of Her2. In conclusion, it seems that the mAbs are useful diagnostic tools for detection of Her2 expression in patients with breast cancer

[Streptokinase: extraction, cloning and overexpression of recombinant protein with simplified purification technique]

Streptokinase has been widely prescribed as a fibrinolytic agent for myocardial infarction. Simple structural characteristics of this protein have provided techniques for production of different recombinant types of this protein. The present study was designed to prepare equisimilisH46A subtype of streptokinase in our country. Having extracted the DNA, the streptokinase gene was replicated and cloned in pGEX-4T-2. The recombinant product was transformed in BL21 [DE3] plysS'. Then the recombinant streptokinase expression and performance was assessed by lasic densitometry and special test for S2251 substrate. Use of a restriction enzyme for both sides of a gene may facilitate its cloning in different expressive carriers. Expression rate of recombinant protein [45%] confirmed successful cloning. Use of pGEX-4T-2 carrier was not only associated with active recombinant streptokinase production, added GST to its amine ending that could facilitate purification process