ELISA FOR DETECTING SECRETED ICAM-1

互联网2013-09-06

1611

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 4^o 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

Diluate mAb R6.5 (or CL203) to 10 ug/ml in PBS . Add 50 ul diluted antibody to wells. Incubate 37^o C for 1 h, or O/N at 4^o C.
Wash 4X with PBS.
Block remaining protein-binding sites by adding 200 ul/well of 1%BSA in PBS. Incubate 30 min at 37^o 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 37^o 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 37^o 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 37^o 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.

We get our best curves when the maximum Abs. for the 512 ng/ml standard is < 1.500, but less than 1.800.