Production and Purification of Anti-Rhombomys opimus Immunoglobulins

BACKGROUND Zoonotic cutaneous leishmaniasis (ZCL) is an increasing public health problem in some endemic regions. Horseradish peroxidase (HRP) conjugated rabbit anti-Rhombomys opimus (R. opimus) Ig is needed for immunoblotting and ELISA tests used to explore the immune response of the rodents against the sand fly saliva. In this study, the production of HRP conjugated rabbit anti-R. opimus Ig was conducted for the first time. METHODS Rhombomys opimus Ig was purified from serum by protein G affinity chromatography column and injected into rabbit to produce anti-R. opimus Ig antibody. The titration of antibody against R. opimus Ig in rabbit serum was checked using indirect ELISA. Rabbit anti-R. opimus Ig was purified by Sepharose-4B-R. opimus Ig column. Reactivity of this antibody was assessed by indirect ELISA and was conjugated to HRP by periodate method. RESULTS Approximately 3.5 mg Ig was purified from 1 ml R. opimus serum using protein G affinity chromatography column. The molecular weight of purified R. opimus Ig was estimated about 150 kDa by SDS-PAGE. Nearly 2.3 mg rabbit anti-R. opimus Ig was purified from 1 ml immunized rabbit serum. The purified antibody was conjugated to HRP and the optimum titer of HRP conjugated rabbit anti-R. opimus Ig was determined as 1:8000 using direct ELISA. CONCLUSION HRP conjugated rabbit anti-Gerbil IgG has been produced by a few companies, but to our knowledge HRP conjugated rabbit anti-R. opimus Ig is not commercially available. Production of HRP conjugated rabbit anti-R. opimus Ig is considerably helpful for immunological studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries.