Open Access
Research
(Published
online: 23-01-2017)
15.
Preparation of goat and rabbit anti-camel
immunoglobulin G whole molecule labeled with horseradish
peroxidase -
Eman Hussein Abdel-Rahman, Jakeen Kamal El-Jakee, Mahmoud Essam
Hatem, Nagwa Sayed Ata and Ehab Ali Fouad
Veterinary World, 10(1): 92-100
doi:
10.14202/vetworld.2017.92-100
Eman Hussein Abdel-Rahman:
Department of Parasitology and Animal Diseases, National
Research Centre, Egypt.
Jakeen Kamal El-Jakee:
Department of Microbiology, Faculty of Veterinary Medicine, Cairo
University, Egypt.
Mahmoud Essam Hatem:
Department of Microbiology, Faculty of Veterinary Medicine, 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.
Received: 26-10-2016, Accepted: 21-12-2016, Published online:
23-01-2017
Corresponding author:
Ehab Ali Fouad, e-mail: ehabfoaud@gmail.com
Citation:
Abdel-Rahman EH, El-Jakee JK, Hatem ME, Ata NS, Fouad EA (2017)
Preparation of goat and rabbit anti-camel immunoglobulin G whole
molecule labeled with horseradish peroxidase,
Veterinary World, 10(1): 92-100.
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.
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