Introduction: Phelebotomine sand flies, vectors of leishmaniasis, co-inject Leishmania parasites along with saliva into the skin of the host while feeding. Saliva from phlebotominae sand flies is known to have pharmacological as well as immunomodulatory activities such as vasodilatory and anticlotting effects which exacerbate the infectivity of the parasite. Investigation on immunogenic components of sand fly saliva and the immune response of the host against it, also interaction among the parasite, sand fly and host is necessary to find possible tools to control the disease specially producing anti Leishmania vaccine and/or transmission blocking vaccine. Detecting the immune response of the host to saliva of sand flies can also be used as a marker of transmission risk of the disease. Study on the immune response of Rhombomys opimus, the main reservoir host of zoonotic cutaneouse leishmaniasis(ZCL) in Central and northeast of Iran, to infection of L. major, the causative agent of the disease, seems to be necessary. HRP conjugated Rabbit anti- R. opimus Ig is needed for immunoblotting and ELISA tests, which are used to find the immune response of the rodents against the sand fly saliva. As this material is not produced commercially in the world, its production in Iran was essential and inescapable. To our knowledge, production of HRP conjugated Rabbit anti- R. opimus Ig has been produced for the first time in Iran and the world as well.
Materials and methods: R. opimus Ig were purified by protein G affinity chromatography column and were injected to rabbit for production of polyclonal antibody. The raising of antibody level against R. opimus Ig in rabbit serum were assessed by ELISA test. Then Seph-4B- R. opimus Ig were prepared and Rabbit anti R. opimus Ig were purified from rabbit ascitis by this column. Reactivity of purified Rabbit anti R. opimus Ig was assessed by ELISA test. This antibody were conjugated to Horseradish peroxidase (HRP) and the optimum titer of HRP-Rabbit anti R. opimus were also determined by ELISA test.
Results: Approximately 4.88 mg Ig were purified from R. opimus serum by Protein G column. Following injections of purified R. opimus Ig to Rabbit, increasing of rabbit anti R. opimus titer were examined using ELISA test. Bleeding from the rabbit was done and from 5 cc rabbit serum about 11.5 mg rabbit anti R. opimus Ig were purified using Seph-4B-R. opimus Ig column. This polyclonal antibody has been conjugated to HRP and the optimum titer was determined as 1/1000 by ELISA test.
Discussion: HRP Conjugated Rabbit anti-Gerbil IgG (h+l) is being produced by a few companies such as Immunology Consultants Laboratory Inc (ICL), but to our knowledge HRP Conjugated Rabbit anti-R. opimus Ig is not produced by any company.
Production of HRP Conjugated Rabbit anti-R. opimus Ig considerably helps serologic and immunologic studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries, which can be led to immunologic control of the disease.