ELISA FOR DETECTING SECRETED ICAM-1
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1621
Purpose
Materials
- NUNC-Immuno Plate IIF.
- Horseradish Peroxidase-Streptavidin (Zymed; 43-4323)
- PBS or Balanced Salt Solution (BSS). Do not use RPMI or any medium that contains biotin.
- BSA (1% in PBS).
-
ABTS substrate and substrate buffer (Zymed; #00-2011)
- ABTS chromogen solution
- Hydrogen Peroxidase solution
- Citrate buffer
- Anti-ICAM-1 mAb R6.5 or CL203. Use as 10 ug/ml in PBS to coat plates.
-
Biotinylated mAb: Biotin RR1/1, R6.1 and Biotin R6.5 supplied.
- Use at a final concentration of 2 ug/ml (1:1000 dilution).
- Do not refreeze. Stable at 4o C for weeks.
-
Dilutions of sICAM-1 standards, 512 ng/ml -> 2 ng/ml, serial 2 fold dilutions in 1% BSA/PBS.
- 512 ng/ml
- 256 ng/ml
- 128 ng/ml
- 64 ng/ml
- 32 ng/ml
- 16 ng/ml
- 8 ng/ml
- 4 ng/ml
- 2 ng/ml
Procedure
- Diluate mAb R6.5 (or CL203) to 10 ug/ml in PBS . Add 50 ul diluted antibody to wells. Incubate 37o C for 1 h, or O/N at 4o C.
- Wash 4X with PBS.
- Block remaining protein-binding sites by adding 200 ul/well of 1%BSA in PBS. Incubate 30 min at 37o C.
- Wash 4X with PBS.
- Add 50 ul of culture supernatant containing secreted ICAM-1. Include positive control wells of purified ICAM-1 ranging from 512 ng/ml to 2 ng/ml. Incubate 30 min at 37o C.
- Wash 4X with PBS.
- Add 50 ul/well Biotinylated R6.5 diluted in 1% BSA/PBS to a final concentration of 2 ug/ml (1:1000 dilution of stock). Incubate 30 min at 37o C.
- Wash 4X with PBS.
- Add 50 ul/well HP-streptavidin diluted accoridng to manufacturers instructions. Current dilution is 1:2000 in 1% BSA/PBS. Incubate 30 min at 37o C.
- Wash 4X with PBS.
- Wash 1X with Substrate buffer without substrate.
- Add 200 ul/well Substrate buffer + ABTS Substrate .
-
Incubate at room temperature until sufficient color develops.
May be as rapid as 2 min or as long as 20 min. - Read absorbance at 414 nm, blanking against negative control well.
Expected Results
We get our best curves when the maximum Abs. for the 512 ng/ml standard is < 1.500, but less than 1.800.
