Production and Purification of Anti-Rhombomys opimus Immunoglobulins

Background: Zoonotic cutaneous leishmaniasis (ZCL) is an increasing public health problem in some endemic re- gions. Horseradish peroxidase (HRP) conjugated rabbit anti-Rhombomys opimus (R. opimus) Ig is needed for im- munoblotting 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 in- jected into rabbit to produce anti-R. opimus Ig antibody. The titration of antibody against R. opimus Ig in rabbit se- rum was checked using indirect ELISA. Rabbit anti-R. opimus Ig was purified by Sepharose-4B-R. opimus Ig column. Re- activity 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.