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aliquoted in lml amount in cryovials. Each cryovials was labeled with the virus,passage, lot number, source; date, 10% 5MB in borate I Tris O.lM, pH 9. The vialswere stored at _70°C til\ used.3.4.3 PREPARATION OF NORMAL MOUSE-BRAIN (NEGATIVECONTROL ANTIGEN)Suckling mice of the same age as those used for the antigens were harvested.With 18 gauge needle and syringe, the brain of the mouse was carefully sucked out.All the harvested mouse brains for a particular virus were. harvested and ground untoa smooth suspension. Appropriate amount of Borate buffer was added to the mousebrain suspension (l mouse brain to Iml of buffer). Appropriate amount of Trisbufferwas added to the suspension (For I mouse brain, O.1ml of Tris-buffer wasadded). The suspension was centrifuged at 10,000 rpm for 20 minutes. Thesupematant was aliquoted in lrnl amount into appropriately labeled cryovials. Allthe vials were stored at -70°C until tested3.4.3.1 TITMTION OF ANTIGEN AND MOUSE ASCITIC FLUIDThe technique developed by WHO CRORA, Institute Pasteur De Dakar, (IPD),Senegal, was adapted.1. Dilution of antigen.Each antigen was diluted serially starting with 1: 10 using carbonate buffer asdiluent in test tubes. Each dilution was added to the microtiter plate vertically (forexample antigen diluted W- 1 was added to wells Al to HI), covered and incubated at+4 oc overnight. The plate was washed 4 times with 0.5% PBS-Tween 20 washingsolution and tapped on filter paper to remove excess fluid. Apendix 22. Addition of mouse ascitic fluid (MAF)The mouse ascitic fluid is commercially available at WHO CRORA, InstitutePasteur De Dakar,(IPD), Senegal. The mouse ascitic fluid for each antigen used wasdiluted serially starting with 1:250 using the diluent (0.5% PBS-Tween plus 1%skimmed milk) in the test tubes. The dilution was added horizontally, for example1:250 was added to Al to AIO. The plate was covered and incubated at 37°C for 1hour.63