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Diagnostic Procedure
Required Reagent Volumes
Plate Layout-Antigen
Detection ELISA
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SAMPLE ADDITION
1. The remainder of the plate can be loaded with the test sample(s). Add 50 µl of sample 1 to wells 7 and 8 of rows A to H; second and third samples are added to rows A to H in wells 9 and 10, and 11 and 12, respectively (Plate Layout-Antigen Detection ELISA).
If more than three samples are to be run in the same test, other microtitre plates should be used as follows. Antigen controls are not required on these plates, but 50 µl of diluent buffer A should be dispensed to the wells (rows A to H) in columns 5 and 6 as buffer control columns. Five test samples may be added in 50 µl volumes in rows A to H, columns 1,2; 3,4; 7,8; 9,10 and 11,12, respectively.
Note that reference control antigens are required on only one microtitre plate for any given test run, provided that the same batch of plates and working preparations of reagents and buffers are used.
Use a fresh tip for each test sample and the control antigens; tips should be discarded into either disinfectant solution, or a container for eventual boiling.
2. Cover plates with lids and incubate on the orbital shaker at 37°C for 1 hour. Remove sufficient aliquots (Required Reagent Volumes) of OPD chromogen from -20°C and leave to thaw.
3. While plates are incubating, prepare working dilutions of the 'blocked' guinea pig detection sera in the appropriate order in an eight-well reservoir; 1/100 dilutions of each antiserum are used.
4. After incubation, wash plates by flooding with ELISA wash fluid three times, and empty residual fluid. Invert the plates and tap onto absorbent paper towelling to remove residual liquid. Proceed quickly to the next step.
5. Transfer 50 µl volumes of each guinea pig detecting serum dilution to each plate well in the appropriate order, e.g., rows A to H receive antisera to serotypes O, A, C, SAT1, SAT2, SAT3, ASIA 1 and SVDV, respectively.
6. Cover plates with lids, replace on the orbital shaker, and incubate at 37°C for 1 hour.
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