Adapted by JAR 10/4/09

ELISA PROTOCOL ( Click to Download )

ELISA kit: cat # _____________ from EBioscience

Reagents:

Buffers:
1. Coating Buffer
1 packet ELISA coating buffer (cat # 00-0044-59)
1L DI water
filter using a 0.22 µm filter

2. Wash Buffer
1X PBS + 0.05% Tween

3. Assay Diluent
10 ml 5X assay diluent bottle (cat # 00-4202-43)
40 ml DI water

4. Capture Ab
48 µl of Capture Ab soln (cat # 14-7069-67)
12 ml coating buffer

5. Standard- rhIL6 at 1 µg/ml
To prepare 200 pg/ml soln, add 5 µl standard soln (cat # 39-8069-60) to 25 ml assay diluent
To prepare subsequent serial dilutions, use 500 µl of preceding standard soln and 500 µl assay diluent
*these are single use vials. Dilute as suggested above.

6. Detection Ab- Biotin conjugated anti-IL6
48 µl Detection Ab soln (cat # 13-7068-67)
12 ml assay diluent

7. Enzyme- Avidin HRP
48 µl Avidin HRP soln (cat # 18-4100-93)
12 ml assay diluent

8. Substrate Soln
Working dilution as prepared (cat # 00-4201-52)

9. Stop Soln
1 M H3PO4

Before Beginning ELISA

1. Prepare Coating Buffer.
2. Coat ELISA plate with 100 µl/well of capture antibody diluted in Coating Buffer.
3. Seal the plate and incubate overnight 4^oC.

Experimental Procedure

1. Aspirate wells and wash 5X with 200 µl/well wash buffer. Allow time for soaking between washes (1-2 minutes).
2. Blot the plate on a paper towel to remove any residual buffer.
3. Block wells with 200 µl/well 1X Assay Diluent. Incubate 1 hr RT.
4. Aspirate and wash as in step 1.
5. Add 100 µl diluted standards to each well (standards set up in duplicate). Perform 2 fold serial dilutions of the standards to generate a standard curve. Add 100 µl diluted samples to the appropriate wells (samples set up in triplicate). Seal the plate and incubate O/N 4^oC.
6. Aspirate and wash as in step 1.
7. Add 100 µl/well Detection Ab to all wells. Seal the plate and incubate RT 1 hr.
8. Aspirate and wash as in step 1.
9. Add 100 µl/well Avidin-HRP soln to all wells. Seal the plate and incubate RT 30 min.
10. Aspirate and wash 7X with 200 µl/well wash buffer. Allow 2 min soaking time between washes.
11. Add 100 µl/well Substrate soln to all wells. Seal the plate and incubate RT 15 min.
12. Add 50 µl stop soln to all wells.
13. Read the plate abs at 450 nm.