Principle:

ELISA format
	Cells are lysed for releasing survivin protein. Anti-survivin monoclonal antibody was coated on 96 well microplate for survivin protein detection in cells of urine samples. The tracer is rabbit anti-survivin polyclonal antibody labeled with horseradish peroxidase. After adding tetramethylbenzidine (TMB) substrate and stopping by 1N HCl, the absorbent was measured at 450 nm for each well.

Kit content:

1. Survivin Microtiter Plate, One Plate of 96 Wells
3. Assay Buffer 20, 120 mL
4. Total Survivin Conjugate: anti survivin antibody-HRP, 11 mL,
5. Wash Buffer Concentrate, 100 mL,
6. human Survivin Standard, 500 pg/ tube
7. TMB Substrate, 12 mL,
8. Stop Solution 2, 11 mL,
9. Cell Lysis Buffer 2, 100 mL,
10. Protein assay reagent: coomassie blue
11. Protein concentration standard (BSA)

Storage:

All components of this kit are stable at 4 °C until the kit's expiration date.

ELISA procedure:

1. Add 100 ul lysis buffer to each sample for releasing survivin protein in cells
2. Samples diluted 10X by sample diluent.
3. Take enough coated antibody strips and put on strip holder.
4. Pipet 100 μL of Standards #1 through #6 into the appropriate wells.
5. Pipet 100 μL of the Samples into the appropriate wells.
6. Incubate at room temperature (21-25° C ) for 60 min.
7. Aspirate or shake out liquid from all wells. Add 280-300 uL of diluted 1X washing solution. Aspirate or shake out and turn plate upside down and blot on paper toweling to remove all liquid. Repeat the wash procedure 2 times (for a total of 3 washes).
8. Add 100 ul of conjugate to each well.
9. Incubate at room temperature (21-25° C ) for 60 min.
10. Wash with diluted 1X washing solution 3 times.
11. Add 100 ul of Substrate (TMB) substrate to each well.
12. Incubate at room temperature (21-25° C ) for 10 ± 2 min.
13. Add 100 ul of stop solution (1N HCl) to each well to stop the reaction. Tap the plate gently along the outsides, to mix contents of the wells.
14. Determine absorbance by reading assay plate at 450nm using 650nm as reference. The plate may be held up to 1 hour after addition of the Stop Solution before reading.

Total protein concentration for normalizing:

1. Cell lysate dilute 50X in PBS
2. Take non-coated strip and pipet 10 ul BSA standard diluted cell lysate in wells
3. Add coomassie blue at RT 10 min
4. Determine absorbance by reading assay plate at 590nm
5. Calculate the total protein concentration