Begin by labeling 96-well microtiter plates with the appropriate experimental information. Then, turn the plate in the vertical orientation and use a multichannel pipette to add 25 microliters of PBS to every well except the first well of the bottom back titration row. Add 50 microliters of the freshly prepared antigen solution of interest to the first well of the back titration row, followed by the addition of 25 microliters of RDE-treated serum samples to the first wells of the top 10 rows.
Add 25 microliters of the appropriate anti-serum to the first well of the 11th row as a positive control. Then, transfer 25 microliters from the first well of each row to their successive wells to perform serial two-fold dilutions. Pipette up and down 10 to 15 times for each dilution step, discarding the final 25 microliters from the last wells.
Next, add 25 microliters of the antigen solution to each well of rows 1 through 11, and 25 microliters of PBS only to each well of the back titration row. Tap the plate carefully 10 times on all four sides to mix, and cover the plate for 30 minutes at room temperature. At the end of the incubation, add 50 microliters of red blood cell solution to each well, and mix the plate with more tapping. Then, cover the plate again and incubate the red blood cells according to the species used, as outlined in the table.
After adding the red blood cells to the plate, adhere to the appropriate incubation time for the type of blood used in the assay. A too-short or too-long incubation will lead to an incorrect interpretation of the results.
At the end of the incubation, assess the hemagglutination by tilting the plate 90 degrees for 25 seconds, and mark the results for each well while the plate is still tilted on a printed scheme of the 96-well plate.
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