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3.4.6.2 The test procedureThe plate was washed in PBS -Tween 20 (washing solution) and were tapped dryEach well was sensitized with lOO1l1 of appropriately diluted hyperimmune mouseascitic fluid specific for a particular antigen. The hyperimmune mouse ascitic fluidwas diluted in 0.1 M carbonate buffer. The plate was incubated at 4[)C overnightafter which it was washed 4 times with washing solution. 100111 of appropriatelydiluted antigen in PBS-0.05% Tween 20 plus 1% skimmed milk (diluent), wasadded to the appropriate wells. Again, lOUIlI of appropriately diluted negative(normal mouse brain) antigen was added to appropriate wells. The plate was coveredand incubated at 37°C for 1 hour after which it was washed four times with washingsolution. The residual liquid was tapped onto paper towel. About 100111 of each testserum, positive human control serum, and negative human control scrum, diluted1:100 was added to apprpriate wells. The plate was covered and incubated at 37°C.Care was taken to avoid cross-contamination of the reagents in the dilution trays orplate during addition of reagents. The plate was washed four times with PBS-Tweenwashing solution and was tapped onto paper towel. Then 100111 of the conjugate(Cappel peroxides-conjugated Goat F (AB ') 2 Fragment to Human IgG), diluted1:20000 was added to all the wells. The plate was covered and incubated at 37°C forI hour and was washed six times with washing solution and tapped onto papertowel. About IOOll1 of the substrate was added to all the wells. The plate wasincubated in the drawer (equivalent to a dark room) for 10 minutes. Adding lOOlkl of4N H2S04 to all the wells stopped the reation. The plate was read at 450nmwavelength.3.5 VECTORAL STUDIESFurthermore, crepuscular/scoop net catches of 3395 mosquitoes from the rainforest and Sahel savannah zones were made, identified and pooled by species.Themosquitoes caught represented the two genera (Aedes, and Culex) which are quitecommon in Nigeria were trapped. Vectoral study became necessary for the reasonsstated below:3.5.1 Reasons for the vectoral studyAvailable reports have consistently indicated that the presence of a competentvector results in an appreciable risk of Dengue Fever occurring in that68