Immuno-Assay (Immuno-Assay.smt)

Before automation run -

• Priming ELx405 Wash Station
          Press [STOP] to end Loop program. 
          Press [NO] for home all axis. 
          Press [Main Menu] 
          Move tubing to TBST bottle. 
          Run 'Prime 1' program.
• Reagent prep.
  • 1% BSA in TBS (60ml for 2 plates). 
  • 0.05% TBST (for 1L) 
            Xml         H2O to final vol of 1 liter. 
            50ml        20x TBS 
            2.5ml       20% Tween 20 
  • His-Probe (1:3500 dil --- ~15ml for 2 plates) 
  • 1-step Turbo TMB (~25ml for 2 plates) 
  • 1M H2SO4 (~15ml for 2 plates)

After automation run -

• Prime and maintain ELx405 Wash Station.

Overview of Immuno-Assay

Preparation:

1. 1%BSA in TBS is kept in the refrigerator of the robot room. If it is running low or out, there is a powder mix in the cabinet of the second lab. Make according to the direction on the packet. 
2. 0.05% TBST is used as a wash for the plates. The following is the recipe for 1L: 
               947.5mL          Water 
               50mL               20X TBS 
               2.5mL              20% Tween 20 
   Mix thoroughly and dump into wash bucket for the EL405 machine. 
3. Check to be sure there is enough HIS tag for your experiment. If you open a new bottle it is reconstituted in 550µl of DI water. 
4. Prime the EL405 machine by: 
   press [STOP] on the face of the machine 
   press [NO] for home all axis 
   press [Main Menu] 
   Move the tube on the back side of the unit from the waste bottle to the TBST bottle 
   On the computer, select the SAMI program and choose the EL405 
   Run "Prime 1" program at least once

Procedure:

1. Select a plate from the refrigerator ("S" or "P") to place in the plate holder of the EL405 and run the program Asp1. This will remove all the liquid from the plate. 
2. If you choose you can aspirate two plates before running the next program on the Multimek. 
3. You will add the blocking buffer (1% BSA in TBS) to the plates with the method Block1_3, for one plate, or Block 2P, for two plates. 
4. These plates are then incubated at 37ºC for one hour. 
5. The plates then need to be washed of all the buffers. This is done in the EL405 with the program Immunowash1. 
6. The HIS probe is made by combining 37mL of TBST and 10.6ul of India tag. Be sure to make this immediately before use! 
7. The His probe can be added to one plate at a time with method HIS1_3 or to two plates at a time with HIS-2P. 
8. These plates are incubated for 30 minutes at room temperature. 
9. The plates must be handled one at a time from here on. It is important to allow the plates the proper amount of incubation time. Spacing between plates will be important so samples aren?t sitting in the light before going in the FluorStar for absorbance readings. 
10. After the 30 minute room temperature incubation the plate will need to be washed again using smWash.
11. Turbo TMB will be added to the plate with the TURBO3 method. 
12. Allow the plate to incubate at room temperature for 15-30 minutes until you see the development of blue color in most of the wells. It may be helpful to put the plate on the shaker to the left of the Multimek. 
13. If you are processing two plates at a time, this is a good time to perform steps 10-12 on the second plate. 
14. Sulfuric Acid is added to the plate to stop the reaction of the His Probe with Stop Reaction. Allow this plate to sit at room temperature about 30 seconds until the color changes to yellow. 
15. Select the FluoroStar program on the Robot computer. Highlight "User" and click "ok". At the bottom left of the screen is a box that reads either "Fluorescence" or "Absorbance". By clicking on that box you can change the function. Be sure it reads "Absorbance" for the Immuno-Assay procedure. 
16. Open the top of the FluoroStar machine and rotate the cords so the "Absorbance" cords are at the top. These are the gray cords. 
17. Back at the computer, click on "Immuno Assay". Label your experiment run with your plate id and the date. Click "Run Test Sample". 
18. Your results are put into an Excel spreadsheet. You can access this spreadsheet by opening FluoroStar and selecting the "X" -with a spreadsheet behind it- at the top of the screen in the middle of the tool bar. 
19. Repeat steps 10 through 16 for each plate you have.