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.

您知悉并同意，丁香通平台展示产品并非为平台自身产品，您发起询价或试用申请后，平台将会把您已提供或按要求提供的相关信息同步提供至所咨询的具体产品商家，由商家为您提供具体产品的价格咨询或产品试用注意事项及相关试用产品或完成相应购买，因此您知悉并授权平台将您的信息进行同步共享。您有权拒绝，但您知悉将无法参加询价或试用活动。