Prostaglandin E2 EIA Kit Protocol

Prostaglandin E2 EIA Kit Protocol Adapted by Jenny 9/5/07

Date of CM pool and thaw: ______Date of assay: ______CAF/RMF line tested: ______

DAY 1  Stock Solution Preparation o Use within 2 months of reconstitution o Store at 4 C o USE ULTRAPURE WATER AS A SOLVENT UNLESS OTHERWISE STATED o For EIA Buffer Preparation: . Dilute the vial of EIA buffer concentrate with 90 mL of ultrapure water o 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 o Use the standard stock soln within 4 weeks of reconstitution, stored at 4 C o Use all further diluted standard solns within 24 hrs o For the 10 ng/mL PGE2 stock standard: . Add 1 mL EIA buffer to the PGE2 standard vial o 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 ul diluent* + 100 ul stock standard 1 ng/mL 2 500 ul diluent* + 500 ul standard 1 3 500 ul diluent* + 500 ul standard 2 4 500 ul diluent* + 500 ul standard 3 5 500 ul diluent* + 500 ul standard 4 6 500 ul diluent* + 500 ul standard 5 7 500 ul diluent* + 500 ul standard 6 8 500 ul diluent* + 500 ul standard 7  Prostaglandin E2 AChE Tracer Solution Preparation o Use within 2 weeks of reconstitution and store 4 C o Reconstitute the 100 dtn PGE2 tracer with 6 ml EIA buffer . Add 60 ul tracer dye soln to 6 ml soln

 Prostaglandin E2 Monoclonal Ab Solution Preparation o Use within 4 weeks of reconstitution and store 4 C o Reconstitute the 100 dtn soln with 6 ml EIA buffer . Add 60 ul antiserum dye soln to 6 ml soln

 Strip Plate Set Up o Place unused strips at 4 C after use; be sure the packet is sealed with the desiccant inside o Use the suggested plate format on the attached page; label appropriately o 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 o 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:

Standard or Well EIA Buffer Tracer Antibody Sample Blk 0 0 0 0 TA 0 0 5 ul, day 2 0 NSB *100 0 50 0 B0 *50 0 50 50 Standard or 0 50 50 50 Sample * if culture medium was used as the diluent instead of EIA buffer, add 50 ul culture medium to NSB and B0 wells, and 50 ul EIA buffer to NSB wells.

DAY 2  Developing the Plate o Reconstitute 1 dtn vial of Ellman’s Reagent with 20 ml of ultrapure water. o This reconstituted soln is unstable and should be used the same day as it is prepared; protect from light. o Empty the wells and rinse 5x with wash buffer o Add 200 ul Ellman’s Reagent to each well and 5 ul of tracer to the TA wells o Cover the plate and incubate in the dark, shaking, for 60-90 min. 