Development of monoclonal antibody specific to yellow head virus (YHV) from P monodon

1. Development of monoclonal antibody specific to yellow head virus (YHV) from P monodon Introduction Shrimp diseases (viral infection) – economic losses PCR tech hi sensitive for virus detec YHV &WSSV the most hi virulent Al methods histo exam & symptodiagno Immuno tech – diagno many viral dis (human med, agriaplicat Prod antiserum YHV,WSSV – reported DevMab to YHV,WSSV – no reported

2. Material and methods • Experimental viral infection and immunogen preparation • Immunization • Hybridoma production • Screening methods • Immunocytochemestry • Viral antigen detection by monoclonal antibodies • Protein isolation by amoniumsulfat precipitation • SDS-PAGE and western blot analysis • Mab class and subclass determinaton

3. Experimental viral infection and immunogen preparation .JuvP mon (25-30 g) – tank 300 l 20 ppt .Prepargill with nat infections of WSSV – homogenized & .centrifuged 15min – membran filter .Suspenwas diluted (1:10) – injecarthrodialmembr .5 g gills(infected shrimp), homogenised .Haemolymp& gill prepar (non infected shrimp) – prepar same manne .Gill extracts & haemolymp extracts – test WSSV by PCR, RT-PCR for YHV

4. Immunization • Three swiss injected intraperitoneally with gill extract • Subsequently injected with gill extract • Adjusvant 3x 2wk intervals • Mouse antiserum was colected and tested against non and infected gill extract • Was boosted 3d before hybridoma production

5. Hybridoma production • A cell fusion procedure (adapted Kohler&Milstein,1976) • Fusion product from 1 mouse were plated on 30 micro culturplated • Identification – positive clones by screening methods, re-cloned by limited dilution methods

6. Screening methods

7. Immunocytochemestry

8. Viral antigen detection by monoclonal antibodies

9. Protein isolation by ammonium sulfate prcipitation

10. SDS-PAGE and western blot analysis

11. MAb class and subclass determination

12. Results and discussion • Histological analysis – fig.1 • Impure antigen – low yield hybridoma specific antibodies • Fusion trial – 600 hybridoma cell (30 micro culture plate) • First screening non infection &infection 2 positive hybridoma clones • Using immunocytochemestrycreening – cytoplasmaimmuno staining • Anti bodies produce (many other clones) – reactivity against gill extract • Trial produce low fusion yields (>3000 clones fusion) – improved hybridoma yield should greater specific antibodies • Immunocytochemestry studies using V3 2B – viral immuno reactivity/cytoplasma of epythel cell haemocytes (fig.1A)

13. Immunocytochemestry studies (infected) – YHV reacted positively with the antibodies (fig.3A) WSSV did not (fig.3B) • Control shrimp – no histological (immunocytochemestry) • Serially diluted haemolymph (infected shrimp) – immunoperoxidase methods V3 2B antibodies detected antigen (strong reactivity 1 : 50 dilution up) • Infected haemolymph was diluted non infected haemolymph (amoniumsulfat) – viral associated antigen detected 1 : 1000 / 1 : 1500 dilution • Modified methods (ELISA) combined – increase sensitivity