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

Abstract Views: 84  |  PDF Views: 1

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