Haitham Amer

Citation:

Amer, H. M. M., Production and characterization of monoclonal antibodies to avian reovirus, , Giza, Cairo University, 2001.

Thesis Type:

Abstract:

In the present study, we produced and characterized a panel of monoclonal antibodies against the reference strain of avian reoviruses (S1133) which is widely used as a vaccine against such virus worldwide. The following steps were conducted to meet the objective of the current work:
1- Avian reovirus strain S1133 was propagated for six successive passages in the Vero cell line.
2- The propagated virus was titrated and characterized by Serum neutralization test and used for preparation of the virus antigen.
3- The avian reovirus was subsequently concentrated using ammonium sulfate precipitation method and prepared for mice immunization.
4- SDS-PAGE was used to confirm the presence of the avian reovirus structural proteins in the concentrated preparations.
5- Six- to eight- weeks-old Balb/C mice were injected with the concentrated avian reovirus antigen mixed with complete Freund`s adjuvant. Two weeks after, mice were boostered by intraperitonieal injection of the concentrated antigen mixed with incomplete Freund`s adjuvant. Ten days later, a final booster with the concentrated antigen was given in three following days.
6- The mice spleen cells were harvested for fusion with Myeloma cells using polyethylene glycol aided method.
7- Screening of the culture supernatant for hybridomas producing antibodies specific to the avian reovirus was performed using Enzyme linked immunosorbent assay (ELISA).
8- Hybridomas that revealed high positive ELISA titers (n=10) in the first screening were cloned 1-6 times by limiting dilution method. Of the cloned ten hybridomas, seven clones that producing monoclonal antibodies against the avian reovirus S1133 strain were selected for characterization.
9- Characterization of the seven produced monoclonal antibodies in solid phase ELISA, dot ELISA and immunofluorscent test revealed that all the monoclonal antibodies were specific to the avian reovirus (strain S1133), non of them had any neutralizing activity against the avian reovirus infection in cell culture. The polypeptide specificity of the produced monoclonal antibodies was tested using Western immunoblotting assay. Three of which were specific to the outer capsid proteins, μB/ μBC, while only one monoclonal antibody reacted specifically with the σB protein. The rest of monoclonal antibodies failed to recognize any of the viral proteins in Western blot.
10- Trials for application of such monoclonal antibodies in developing an improved diagnostic techniques (Antigen capture ELISA and antibody capture ELISA) revealed promising results.
The results obtained in current study should reveal an added information on the circulating reoviruses among poultry population in Egypt. Further studies concerning with the application of the proposed monoclonal antibodies needed to be addressed.

Notes:

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