9- Dr Mahmoudi Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 69 Original Article Production and Purification of Anti-Rhombomys opimus Immunoglobulins AA Akhavan1, R Ghods2, M Jeddi-Tehrani3, MR Yaghoobi-Ershadi1, A Khamesipour4, *AR Mahmoudi2 1Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 2Department of Immunochemistry, Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 3Department of Hybridoma, Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran 4Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran (Received 29 Aug 2011; accepted 6 Nov 2011) Abstract 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. Keywords: Rhombomys opimus, Polyclonal antibody, Zoonotic cutaneous leishmaniasis, Immunoglobulin, Iran Introduction Zoonotic cutaneous leishmaniasis (ZCL) is still a public health problem in some of the regions of the endemic areas. Leishmania major, the causative agent of the ZCL of the old world, is widely distributed in different populations of rodents in arid and savannah areas. The disease is transmitted to rodents and other vertebrate hosts by phelebotomi- nae sand flies (Gramiccia and Gradoni 2005). Rodents of the subfamily of Gerbillinae are the main reservoirs of ZCL in Iran and other countries where the disease is endemic (Du- brovsky 1979, Strelkoova 1996, Yaghoobi- Ershadi et al. 1996). Rhombomys opimus AA Akhavan and R Ghods contributed equally to this work. *Corresponding author: Ahmad Reza Mahmoudi, E-mail: mahmoudi@avicenna.ac.ir Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 70 (Cricetidae: Gerbillinae) is the main reser- voir host of the agent over the vast areas of the Turan lowland (west and south Kazakhstan and central Asia with adjacent parts of Af- ghanistan and Iran), Mongolia, and seemingly, in some provinces of China. Naturally in- fected R. opimus were found in more than 200 places of Turan lowland. This gerbil is also found to be naturally infected with L. turanica and L. gerbilli (Strelkova 1996, Akhavan et al. 2010a, 2010b, 2010c). Well- described stable ZCL system in central Asia, Afghanistan and Iran (central and north-east) are associated with R. opimus, the main res- ervoir, and Phlebotomus papatasi, the main vector (Javadian 1988, Yaghoobi-Ershadi and Javadian 1996, Javadian et al. 1998, Yag- hoobi-Ershadi et al. 1996, 2003, Gramiccia and Gradoni 2005, Yaghoobi-Ershadi 2008). 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 spe- cially producing anti Leishmania vaccine and/or transmission blocking vaccine (Akhavan 2011). 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 (Barral et al. 2000). Study on the immune re- sponse of R. opimus, the main reservoir host of ZCL in central and northeast of Iran, to infection of L. major, the causative agent of the disease, seems to be necessary. Horse- radish peroxidase (HRP) conjugated rabbit anti-R. opimus Ig is needed for immunoblot- ting and ELISA tests, used to find the im- mune response of the rodents against the sand fly saliva. As this material is not produced commer- cially in the world, its production was essen- tial and inescapable. To our knowledge, in the present study production of HRP conju- gated rabbit anti-R. opimus Ig has been pro- duced for the first time. Materials and Methods Rhombomys opimus serum collection To purify R. opimus Ig, sera were ob- tained from wild great gerbils collected from natural habitat in Sejzi rural district, Esfahan Province, central Iran. Great gerbils were an- aesthetized (ketamin hydrochloride 60 mg/kg and xylazine 5 mg/kg, intramuscularly) and then the blood sample was collected. The individual sera was isolated and kept at -20 °C until use. Purification of R. opimus Ig and polyclonal rabbit anti-R. opimus Ig Rhombomys opimus Ig was purified using HiTrap protein G HP affinity chromatogra- phy column (GE Healthcare, Uppsala, Swe- den). The 1:5 diluted serum in PBS (0.15M, pH= 7.2) was centrifuged, filtered by 0.2 µm filter and passed through the HiTrap protein G HP affinity chromatography column. Then the column was washed with PBS. The at- tached R. opimus Ig to the column was iso- lated from the column using Gly-HCl (0.2 M, pH= 2.5). Isolated Ig was dialyzed against PBS and finally the purified R. opimus Ig was stored at -20 ºC. For purification of rabbit anti-R. opimus Ig from rabbit serum, a Sepharose-4B-R. opimus Ig affinity chromatography column was prepared according to the Amersham Biosci- ences company instructions (71-7086-00 Edition AC). Using this column, the rabbit anti-R. opimus Ig was purified from rabbit serum as described for R. opimus Ig purifica- tion (see above). Concentration of purified R. opimus Ig and rabbit anti-R. opimus Ig was determined by reading the optical density (OD) of sam- ples at 280 nm and calculation of the con- centrations regarding to extinction coefficient of IgG molecule. Purity assessment of purified R. opimus Ig Molecular weight and purity of the R. opimus Ig were determined using SDS-PAGE Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 71 in non-reducing conditions, 8% separating gel and 4% stacking gel (according to Bio-Rad guideline). Immunization of New Zealand white rab- bit with R. opimus Ig A female New Zealand white rabbit aged 6–7 months was immunized with R. opimus Ig using Hudson and Hay’s method (Hudson and Hay 1991) with minor modifications. Rabbit was injected intramuscularly into the thigh muscle with 250 µg R. opimus Ig mixed with Complete Freund's Adjuvant, and 4 booster injections of 125 µ g of R. opimus Ig mixed with Incomplete Freund's Adjuvant at 4 weeks intervals between the first and sec- ond injection and 2 weeks intervals between subsequent injections. Before each immunization, blood sample was taken from the marginal vein of the rab- bit ear, centrifuged and the sera were checked for anti-R. opimus Ig antibody using ELISA method. Animal care and the procedures were con- ducted according to the guidelines of the ani- mal care and Ethics Committee of Avicenna Research Institute. Antiserum titration and reactivity assess- ment of purified polyclonal rabbit anti-R. opimus Ig by ELISA Rabbit anti-R. opimus Ig titer was evalu- ated using ELISA. Briefly, 10 µ g/ml of puri- fied R. opimus Ig in 100 µ l was added to each well of microtiter polystyrene strips (Max- isorp, Nunc, Roskilde, Denmark) and incu- bated at 37º C for 1.5 hours, 2 wells received only PBS as negative control, then the plate was washed 3 times with PBS-Tween (0.05%) (PBS-T), the wells were then blocked using 3% skim milk for 1.5 hours at 37º C. The plate was washed 3 times and serial dilutions of rabbit anti-R. opimus Ig serum from 1:1000 to 1:64000 dilutions were added and incubated for 1.5 hours at 37º C. Two other wells received PBS as primary antibody step negative control. After three times washing, HRP conjugated sheep anti-rabbit Ig (Avicenna Research Institute, Tehran, Iran) was added as secondary antibody and incubated for 1.5 hours at 37 ºC. After the last wash, the reaction was visualized using TMB substrate (USB, Cleveland, USA) and the optical density meas- ured by ELISA reader (Anthos 2020, Cam- bridge, England) at 450 nm. Reactivity of purified polyclonal rabbit anti- R. opimus Ig with related antigen was as- sessed by indirect ELISA test. ELISA proce- dures including coating antigen and secon- dary antibody were performed as described above. However, the serial dilutions of puri- fied rabbit anti-R. opimus Ig (by two fold dilutions from 10000 to 1.2 ng/ml) were added as primary antibody. Conjugation of polyclonal rabbit anti-R. opimus Ig with HRP Conjugation of polyclonal rabbit anti-R. opimus Ig with HRP was performed by pe- riodate method (Wilson and Nakane 1978). Briefly, HRP was oxidized by sodium perio- date. Oxidized HRP was mixed by rabbit anti- R. opimus Ig. Then, sodium borohydride was added to this mixture and finally the conjugate was purified from excess reagents by dialysis. Titration of HRP conjugated rabbit anti- R. opimus Ig by ELISA The optimum titer of HRP conjugated rabbit anti-R. opimus Ig was assessed by di- rect ELISA according to the procedure de- scribed for the antiserum titration ELISA. The wells were coated by R. opimus Ig (10 µ g/ ml) as coating antigen and two wells were coated with PBS only as negative control. Titration of HRP conjugated rabbit anti-R. opimus Ig was performed by two fold dilu- tions from 1:250 to 1:128000. Results From 1 ml R. opimus serum 3.5 mg R. opimus Ig was purified. The purified R. opimus Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 72 Ig related SDS-PAGE shows high purity of the purified Ig. Regarding to the molecular weight of mouse IgG as a molecular weight marker, the molecular weight of purified R. opimus Ig is about 150 kDa (Fig.1). The results showed that the specific antibodies were detectable following the first injection in rabbit. After the second injection, the antibody titer reached near the maximum level and there was no remarkable increase following subsequent injections. However, booster injections resulted in gradual and stepwise increase in the specific antibody titer against R. opimus Ig (Fig. 2). From 5 ml immunized rabbit serum 11.5 mg rabbit anti-R. opimus Ig was purified. It means that there was 2.3 mg rabbit anti-R. opimus Ig in each ml of the immunized rabbit serum. The results showed strong reactivity of this antibody with related antigen and this antibody can detect its antigen in very low concentrations of about 1.2 ng/ml (Fig. 3). After the HRP conjugation, it was neces- sary to find the optimum dilution of HRP conjugated rabbit anti-R. opimus Ig by direct ELISA. Optimum dilution for a specific con- jugate is the highest dilution of conjugate which can produces the highest signal. According to the HRP conjugated rabbit anti-R. opimus Ig titration curve, the optimum dilution of this conjugate is 1:8000 and it shows that the efficiency of conjugation is high (Fig. 4). Fig. 1. The SDS-PAGE analysis of purified R. opimus Ig by HiTrap Protein G HP affinity chromatography column Purified R. opimus Ig (≈150 KDa) Mouse IgG= 150 KDa BSA= 66 KDa Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 73 0 0.5 1 1.5 2 2.5 3 3.5 4 1/1000 1/2000 1/4000 1/8000 1/16000 1/32000 1/64000 Sera dilutions O D ( 4 5 0 n m ) Pre-immune 1st injection 2nd injection 3rd injection 4th injection 5th injection Fig. 2. Titration curve of rabbit anti-R. opimus Ig by indirect ELISA 0 0.5 1 1.5 2 2.5 3 3.5 10 00 0 50 00 25 00 12 50 62 5 31 2. 5 15 6. 2 78 .1 39 19 .5 9. 7 4. 8 2. 4 1. 2 Rabbit anti R. opimus Ig concentration(ng/ml) O D (4 5 0 n m ) Fig. 3. Reactivity test of purified rabbit anti-R. opimus Ig by indirect ELISA 0 0.5 1 1.5 2 2.5 3 3.5 4 1/ 25 0 1/ 50 0 1/ 10 00 1/ 20 00 1/ 40 00 1/ 80 00 1/ 16 00 0 1/ 32 00 0 1/ 64 00 0 1/ 12 80 00 HRP conjugated Rabbit anti R. opimus Ig dilutions O D (4 5 0 n m ) Fig. 4. Titration of HRP conjugated rabbit anti-R. opimus Ig by direct ELISA Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 74 Discussion HRP conjugated rabbit anti-Gerbil IgG is produced by a few companies; however, to our knowledge there is no record of produc- tion of HRP conjugated rabbit anti-R. opimus Ig. Production of HRP Conjugated Rabbit anti-R. opimus Ig is needed for serological and immunological studies of R. opimus, the main reservoir host of ZCL in Iran as well as some other countries, which may lead to plan immunological control of the disease (Akhavan 2011). Phlebotomus papatasi, vector of ZCL, co-injects Leishmania parasites along with saliva into the skin of R. opimus while feed- ing. Saliva from the sand fly is known to have pharmacological as well as immuno- modulatory activities such as vasodilatory and anticlotting effects which exacerbate the in- fectivity of the parasite (Kamhawi 2000). In- vestigation on the immune response of the host against salivary gland antigens of the vector, also interaction among the parasite, sand fly and host are necessary to find possi- ble tools to control the disease, especially in developing anti Leishmania vaccine and/or transmission blocking vaccine. Detecting the immune response of the host to saliva of the sand fly may also be used as a marker of transmission risk of the disease (Barral et al. 2000). The HRP conjugated rabbit anti-R. opimus Ig produced in this study helps the investigators to study on the immune response of R. opimus, to L. major exposure and in- fection. The HRP conjugated antibody is needed for immunoblotting and ELISA tests, which are used to detect the immune response of the rodents against the sand fly saliva. Purification of R. opimus Ig is a necessary step for production of polyclonal antibody. No established protocol was recorded for purification of R. opimus Ig. In this regard, considering the fact that R. opimus is a mam- mal belonging to the order Rodentia and ac- cording to previous studies (Kemp 2005), protein G could adsorb Ig molecules from many mammals and rodents such as human, mouse and rabbit. Thus it seems reasonable that protein G is a suitable candidate for pu- rification of IgG molecules from R. opimus serum. Using this method, 3.5 mg R. opimus Ig was obtained from 1 ml serum. Moreover, due to the lack of knowledge about the molecular size of R. opimus Ig, an- other rodent IgG (mouse) was used as a stan- dard for molecular weight determination in SDS-PAGE. The very similar migration of R. opimus Ig in the polyacrylamide gel to that of murine IgG allowed us to estimate it as around 150 kDa. A high titer of rabbit anti-R. opimus Ig production after the first injection of R. opimus Ig showed that R. opimus Ig mixed with adjuvant was immunogenic in rabbit, so that after the second injection, the antiserum titer in the rabbit hardly increased. However, we continued the immunizations to increase the avidity or affinity of the produced antibodies. In conclusion, this study describes the successful purification of R. opimus Ig and production of polyclonal antibodies against it in rabbit. Acknowledgements The authors are grateful to Ms L Balaie Goli, Ms M Babaei, Ms M Darzi, Mr J Mah- moudian, Ms L Eini and Mr A Moradzadeh, Monoclonal Antibody Research Center, Avi- cenna Research Institute, ACECR and Mr Arandian, Ms Shareghi, Mr Jafari and Mr Abdoli, Isfahan Health Research Center, Na- tional Institute of Health Research, TUMS for their kind collaboration. This research was financially supported by a grant of Avicenna Research Institute (ARI), Project No.1689- 33. The authors declare that they have no conflicts of interest. Iran J Arthropod-Borne Dis, 2011, 5(2): 69–76 AA Akhavan et al.: Production and Purification of ... 75 References Akhavan AA, Mirhendi H, Khamesipour A, Alimohammadian MH, Rassi Y, Bates P, Kamhawi S, Valenzuela JG, Aran- dian MH, Abdoli H, Jalali-Zand N, Jafari R, Shareghi N, Ghanei M, Yag- hoobi-Ershadi MR (2010a) Leishma- nia species: Detection and identification by nested PCR assay from skin sam- ples of rodent reservoirs. 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