Immunodot Assay

  1. Grow cells to be tested overnight to a dense lawn on solid media. Pipette 3 ml PBS (or other suitable buffer) onto the plate, and suspend cells by scraping with a glass spreader.

  2. Collect suspension by transfer pipette and place in a centrifuge tube.

  3. Autoclave tubes for 30 minutes at 121°C.

  4. Agitate the cells by vortexing or vigorous shaking.

  5. Pellet the cells by centrifugation at 2,500 rpm for 15 minutes at 4°C. Decant and retain supernatant.

  6. Add four volumes of ice-cold 95% ethanol to the supernatant. Mix and precipitate overnight at -20°C.

  7. Collect the precipitate by centrifugation for 15 minutes at 2,500 rpm at 4°C. Dry the pellet with tubes inverted over paper towels.

  8. Dissolve the dried pellet in a minimal amount of PBS (i.e. 0.5 ml, or 1/20th the volume of the original overnight culture).

  9. Serially dilute the suspension in a 96-well microtiter plate with PBS by pipetting 10 µl of PBS to each of six wells in a row of the plate. To the first well, add 1µ ul of the suspension to be tested and mix by pipetting up and down several times. Using a new pipette tip, transfer 10 µl from this well (1:2 dilution) to the next well in the row, creating a 1:4 dilution. Continue in this manner until all desired dilutions are represented (six wells allows a 1:64 dilution). It is critical to use a new pipette tip for each transfer.

  10. Spot 5 µl of each dilution (include an undiluted sample) to a piece of nitrocellulose membrane of a size appropriate to the number of samples to be tested (a 2" x 3" membrane allows for the testing of approximately three samples at seven dilutions (undiluted through 1:64), with room for a positive and negative control).

  11. Dry the nitrocellulose in air at room temperature or fix the samples by baking the membrane at 60°C-70°C for 30 minutes.

  12. Block the membrane by incubating in 10-15 ml (for the size membrane mentioned above) 5% skim milk/PBS/0.005% thimerasol for 2 hours to overnight at room temperature with rocking.

  13. Wash the membrane three times with 10-15 ml PBS/0.05% Tween-20.

  14. Prepare a 1:400 dilution of rabbit antisera in 9 ml PBS plus 1 ml of the blocking solution used above (25 µl rabbit antisera to 10 ml PBS/blocking solution). Absorb the rabbit antisera by dissolving 1 mg/ml of lyophilized wild-type or mutant bacteria appropriate to the experiment in the diluted rabbit antisera. Incubate in the cold room on a roller for 2 hours to overnight. Pellet the bacteria by centrifugation at 2,500 rpm, 4°C, for 15 minutes. Decant supernatant and filter through a 0.45 µm disposable filter.

  15. Incubate the membrane in the pre-absorbed antisera solution for 2 hours to overnight at room temperature with rocking.

  16. Discard the antisera solution and wash the membrane three times with 10-15 ml PBS/Tween.

  17. Incubate the membrane in 10 ml of a 1:2000 dilution of preabsorbed goat antirabbit alkaline phosphatase conjugate in 5% skim milk/PBS/Tween (5 µl conjugate in 10 ml of the skim milk/PBS/Tween solution. Absorb this antibody as outlined in step 13, using 5% skim milk/PBS/Tween as the diluent) for one hour.

  18. Discard the conjugate solution and wash the membrane three times with 10-15 ml PBS/Tween.

  19. Develop the membrane by incubating in substrate solution (BCIP/NBT; 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium), using enough to cover the membrane. Watch the color development closely (it is usually very rapid, 2 to 3 minutes) and stop the reaction by rinsing thoroughly several times with distilled water when development is sufficient.

  20. Photograph the membrane to provide a permenant record of the results.

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Revised: Sunday, September 03, 1995 1:24:52 PM