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Preparation of Goat and Rabbit Anti-Camel Immunoglobulin G Whole Molecule Labeled with Horseradish Peroxidase


Affiliations
1 Department of Parasitology and Animal Diseases, National Research Centre, Egypt
2 Department of Microbiology, Cairo University, Egypt
3 Department of Microbiology and Immunology, National Research Centre, Egypt
 

Aim: As the labeled anti-camel immunoglobulins (Igs) with enzymes for enzyme-linked immunosorbent assay (ELISA) are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP) to save hard curacy.
Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA.
Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at -20°C as proved by ELISA.
Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at -20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for preparation of IgG subclasses.

Keywords

Anti-Camel Immunoglobulin G, Camelus dromedarius, Conjugation, Horseradish Peroxidase, Purification.
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  • Nawito, M.F., Shalash, M. R., Hoppe, R. and Rakha, A.M. (1967) Reproduction in female camel. Egypt. Nat. Res. Cent. Bull., 2: 82.

  • Azwai, S.M., Carter, S.D. and Woldehiwet, Z. (1996) Immunoglobulins of camel (Camelus dromedarius) colostrum. J. Comp. Pathol., 114: 273-282.

  • Agab, H. (1993) Epidemiology of camel disease in Eastern Sudan, with emphasis on brucellosis. M.V.Sc. Thesis. University of Khartoum. p172-175.

  • Al-Ruwaili, M.A., Khalil, O.M. and Selim, S.A. (2012) Viral and bacterial infections associated with camel (Camelus dromedarius) calf diarrhea in North Province, Saudi Arabia. Saudi J. Biol. Sci., 19(1): 35-41.

  • Mohammed, M.A., Shigidy, M.T. and Al Juboori, A.Y. (2013) Sero-prevalence and epidemiology of Brucellosis in camels, sheep and goats in Abu Dhabi Emirate. Int. J. Anim. Vet. Adv., 5(2): 82-86.

  • El-Hewairy, H.M., Galal, S.A. and Mousa, W.M. (2014) New approach for diagnosis of Trypanosomes evansi in camel (Camelus dromedaries) by ELISA. Life Sci. J., 11(10): 1258-1263.

  • Abd El Hafez, S.M., Anwar, A.M., Ibrahim, A.M., Mahmoud, M.B. and Hassan, H.M. (2010) Preparation of fluoresce isothiocyanate conjugated IgG (FITC) anti-camel and anti-buffalo. Nat. Sci. J., 8: 342-347.

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the folin phenol reagent. Biol. Chem. J., 193: 265-275.

  • Laemmli, U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nat. J., 227: 680-685.

  • Muro, A., Ramajo, V., Lopez, J., Simon, F. and Hillyer, G.V. (1997) Fasciola hepatica vaccination of rabbits with native and recombinant antigens related to fatty acid binding proteins. Vet. Parasitol. J., 69: 219-229.

  • Paul, K.N. and Akira, K. (1974) Peroxidase-labeled antibody. A new method of conjugation. Histochem. Cytochem. J., 22: 1084-1091.

  • Avrameas, S. (1969) Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochem. J., 5: 43-52.

  • Roghaye, A., Mahdian, R., Behdani, M., Khanahmad, H., Langari, J., Namvarasl, N., Hassanzadeh-Ghasabeh, R. and Zeinali, S. (2014) Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation. Saudi J. Biol. Sci., 21(1): 35-39.

  • Mariam, S.H.S., Ooi, C.W., Tan, W.S., Janna, O.A., Arbakariya, A. and Tey, B.T. (2015) Purification of rabbit polyclonal immunoglobulin G with ammonium sulphate precipitation and mixed-mode chromatography. Sep. Purif. Technol. J., 144: 133-138.

  • Khamehchian, S., Zolfagharian, H., Dounighi, N. M., Tebianian, M. and Madani, R. (2014) A new approach to prepare Naja Naja oxiana antivenom as passive immunization for therapy. Hum. Vaccin. Immunother. J., 10: 1633-1638.

  • Blanc, M.R., Anouassi, A., Abed, M.A., Canepa, S., Labas, V. and Bruneau, G. (2009) A new method to discriminate immunogen-specific heavy-chain homodimer from heterotetramer immunoglobulin G directly in immunized dromedary whole plasma proteins: Western ligand blotting. Vet. Immunol. Immunopathol. J., 127: 340-349.

  • Jungbauer, A., Tauer, C., Reiter, M., Purtscher, M., Wenisch, E., Steindi, F., Buchacher, A. and Katinger, H. (1989) Comparison of protein A, protein G and copolymerized hydroxyapatite for the purification of human monoclonal antibodies. Chromatogr. J., 476: 257-268.

  • El-Hewairy, H.M., Moussa, W.M., El-Abeidy, A.A. and Seim, S.A. (2004) Preparation of anti-camel immunoglobulin-G conjugated with fluorescin isothiocyanate and alkaline phosphatase. 1st Anniversary Conference, FVM., Moshtohor. p59-66.

  • Mohammadian, T., Doosti, M., Paknejad, M., Siavoshi, F. and Massarrat, S. (2010) Preparative SDS-PAGE electroelution for rapid purification of alkyl hydroperoxide reductase from Helicobacter pylori. Iran. Public Health J., 39: 85-91.

  • Shaker, G.H. and Melake, N.A. (2012) Use of the single cell gel electrophoresis (comet assay) for comparing apoptotic effect of conventional antibodies versus nanobodies. Saudi Pharm. J., 20: 221-227.

  • Daley, L.P., Gagliardo, L.F., Duffy, M.S., Smith, M.C. and Appleton, J.A. (2005) Application of monoclonal antibodies in functional and comparative investigations of heavy-chain immunoglobulins in new world camelids. Clin. Diagn. Lab. Immunol. J., 12: 380-386.

  • Kataria, A.K. and Sharma, K.N. (2000) Chromatographic purification of serum and colostral immunoglobulins of camel (Camelus dromedarius). Camel Pract. Res. J., 7: 91-95.

  • Toaleb, N.I., Shaapan, R.M., Hassan, S.E. and El Moghazy, F.M. (2013) High diagnostic efficiency of affinity isolated fraction in camel and cattle toxoplasmosis. World Med. Sci. J., 8: 61-66.

  • Abdel-Rahman, E.H., Bashtar, A.M., Hassanain, M.A., Hassanain, N.A. and Toaleb, N.I. (2014) Evaluation of a vaccine candidate isolated from fasciola gigantica excretory-secretory products in rabbits. Glob. Vet., 13(5): 720-727.

  • Agindotan, B.O., Thottappilly, G., Uwaifo, A. and Winter, S. (2003) Production of monoclonal and polyclonal antibodies against a Nigerian isolate of banana streak virus. Afr. Biotechnol. J., 2: 171-178.

  • Imigawa, M., Yoshitake, S., Hamaguchi, Y., Ishikawa, E., Niitsu, Y., Urushizaki, I., Kanazawa, R., Tachibana, S., Nakazwa, N. and Ogawa, H.H. (1982) Characteristics and evaluation of horseradish peroxidase conjugates prepaed by using a maleimide compound, gluteraldehyde and periodate. Immunoassay J., 4: 207-211.

  • El-Hewairy, H.M. and Syame, S.M. (2008) Preparation of polyvalent anti-camel immuonoglobulins labeled with alkaline phosphates. Egypt. Comp. Pathol. Clin. Pathol. J., 21: 93-101.

  • Rae, P.F., Thrusfield, M.V., Higgins, A., Aitken, C.G.G., Jones, T.W. and Luckins, A.G. (1989) Evaluation of enzyme immunoassay in the diagnosis of camel (Camelus dromedarius) trypanosomiasis: A preliminary investigation. Infect. Sci. J., 102: 297-307.


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  • Preparation of Goat and Rabbit Anti-Camel Immunoglobulin G Whole Molecule Labeled with Horseradish Peroxidase

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Authors

Eman Hussein Abdel-Rahman
Department of Parasitology and Animal Diseases, National Research Centre, Egypt
Jakeen Kamal El-Jakee
Department of Microbiology, Cairo University, Egypt
Mahmoud Essam Hatem
Department of Microbiology, Cairo University, Egypt
Nagwa Sayed Ata
Department of Microbiology and Immunology, National Research Centre, Egypt
Ehab Ali Fouad
Department of Microbiology and Immunology, National Research Centre, Egypt

Abstract


Aim: As the labeled anti-camel immunoglobulins (Igs) with enzymes for enzyme-linked immunosorbent assay (ELISA) are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP) to save hard curacy.
Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA.
Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at -20°C as proved by ELISA.
Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at -20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for preparation of IgG subclasses.

Keywords


Anti-Camel Immunoglobulin G, Camelus dromedarius, Conjugation, Horseradish Peroxidase, Purification.

References