Date of CM pool and thaw: ____________

Date of assay: _____________

CAF/RMF line tested: ________________

DAY 1

• Stock Solution Preparation
  • Use within 2 months of reconstitution
  • Store at 4 °C
  • USE ULTRAPURE WATER AS A SOLVENT UNLESS OTHERWISE STATED
  • For EIA Buffer Preparation:
    • Dilute the vial of EIA buffer concentrate with 90 mL of ultrapure water
  • For Wash Buffer Preparation:
    • Dilute the 5 mL vial of wash buffer concentrate with 2 L of ultrapure water and add 1 mL of tween 20 (i.e. dilute the wash buffer 1:400 and add tween 20 at 0.5 mL//L soln)
• Standard Solution Preparation
  • Use the standard stock soln within 4 weeks of reconstitution, stored at 4 °C
  • Use all further diluted standard solns within 24 hrs
  • For the 10 ng/mL PGE2 stock standard:
    • Add 1 mL EIA buffer to the PGE2 standard vial
  • For the remaining serial dilution PGE2 standards:
    • *Use EIA buffer to further dilute UNLESS the samples will be diluted 1:10 in their respective culture medium; if this is the case, dilute the remaining standards in this culture medium diluent instead of EIA buffer

      Standard	Formulation	Final Concentration
      1	900 µl diluent* + 100 µl stock standard	1 ng/mL
      2	500 µl diluent* + 500 µl standard 1
      3	500 µl diluent* + 500 µl standard 2
      4	500 µl diluent* + 500 µl standard 3
      5	500 µl diluent* + 500 µl standard 4
      6	500 µl diluent* + 500 µl standard 5
      7	500 µl diluent* + 500 µl standard 6
      8	500 µl diluent* + 500 µl standard 7

• Prostaglandin E2 AChE Tracer Solution Preparation
  • Use within 2 weeks of reconstitution and store 4 °C
  • Reconstitute the 100 dtn PGE2 tracer with 6 ml EIA buffer
    • Add 60 µl tracer dye soln to 6 ml soln
• Prostaglandin E2 Monoclonal Ab Solution Preparation
  • Use within 4 weeks of reconstitution and store 4 °C
  • Reconstitute the 100 dtn soln with 6 ml EIA buffer
    • Add 60 µl antiserum dye soln to 6 ml soln
• Strip Plate Set Up
  • Place unused strips at 4 °C after use; be sure the packet is sealed with the desiccant inside
  • Use the suggested plate format; label appropriately
  • The minimum for a decent analysis:
    • 2 blanks (Blks)
    • 2 non specific binding wells (NSBs)
    • 3 maximum binding wells (B0s)
    • an eight point standard curve done in duplicate
  • To start the assay, pipet the above reconstituted reagents according to the table (volumes are in ul), and incubate 18 hrs 4 °C with plastic film cover:

    Well	EIA Buffer	Standard or Sample	Tracer	Antibody
    Blk	0	0	0	0
    TA	0	0	5 µl, day 2	0
    NSB	*100	0	50	0
    B0	*50	0	50	50
    Standard or Sample	0	50	50	50

    * if culture medium was used as the diluent instead of EIA buffer, add 50 µl culture medium to NSB and B0 wells, and 50 µl EIA buffer to NSB wells.

DAY 2

• Developing the Plate
  • Reconstitute 1 dtn vial of Ellman’s Reagent with 20 ml of ultrapure water.
  • This reconstituted soln is unstable and should be used the same day as it is prepared; protect from light.
  • Empty the wells and rinse 5x with wash buffer
  • Add 200 µl Ellman’s Reagent to each well and 5 µl of tracer to the TA wells
  • Cover the plate and incubate in the dark, shaking, for 60-90 min.

PGE2 Assay Protocol ( Click to Download )