4986
Ehab Ali Mohamed Mohamed Fouad
Immunological studies on camels (Camelus dromedarius) in Egypt
Camel, Camelus dromedarius, Immunoglobulin G, HRP
As the labeled anti-camel Igs with enzymes for ELISA are unavailable in commercial level, the present investigation was directed for developing labeled anti-camel IgG with horseradish peroxidase. For purification of camel IgG whole molecule, camel sera were preliminary precipitated with 50% saturated ammonium sulphate and dialyzed against diluted PBS then concentrated. This preparation was followed by Protein A Sepharose affinity column chromatograhy for purification of camel IgG. The purity of the eluted camel IgG was tested by SDS-PAGE. Four bands of molecular weight 63, 52, 40 and 33 kDa, represent camel IgG, were detected. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. Then anti-camel IgG was purified by loading on Protein A Sepharose affinity column chromatography after precipitation and concentration. Whole molecule anti-camel IgG was conjugated with horseradish peroxidase. The obtained results showed that in fact glutraldehyde was effective and suitable reagent for producing protein-enzyme complexes that retained a part of their enzymatic and immunological specificity. Sensitivity of prepared conjugated secondary antibodies was detected using positive camel serum samples reacted with different antigens in ELISA compared with Protein A horseradish peroxidase. The recorded sensitivity is 100%. The specificity of the prepared conjugates was determined using negative serum samples in ELISA. The specificity is also 100% compared to 58- 75% of protein A horseradish peroxidase. The conjugates are stable for one year at -20ºC as proved by ELISA. Collectively, the current study introduces goat and rabbit anti-camel IgG whole molecules with simple,inexpensive method, with 100% sensitivity, 100% specificity and stability up to one year at -20ºC. The important facet of the current study is saving hard currency. Future investigations are necessary for preparation of IgG subclasses.
2015
Ph.d
Cairo
Veterinary Medicine