Recommended ELISA procedure for human IFN alpha 2b determination:
1. Use 96-well Nunc Maxisorp plates for the assay. Coat microwells with 100 µl/well of antibody 9D3 (ab9386) diluted to 10 µg/ml in coating buffer (0.05 M Na-carbonate, pH 9.5). Incubate overnight at +4°C.
2. Aspirate wells and block with 150 µl/well of blocking buffer (2% BSA in PBS). Incubate at RT for 1 h.
3. Aspirate wells and wash 3 times with PBST (0.1% Tween-20 in PBS).
4. Prepare standard and sample dilutions in PBST. Recommended IFN alpha 2b concentrations: from 100 pg/ml to 100 ng/ml. Add 100 µl of each standard and sample into appropriate wells. Incubate at RT for 2 h.
5. Aspirate and wash 5 times with PBST.
6. Dilute detection antibody 4E10-HRP (ab5258) 1:1000 in PBST. Add 100 µl of diluted detection antibody to each well. Incubate at RT for 1 h.
7. Aspirate and wash 10 times with PBST.
8. Add 100 µl of substrate solution (TMB) to each well. Incubate at RT for 10-20 min.
9. Add 50 µl of stop solution (2 N H2SO4) to each well. Read absorbance at 450 nm.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Sandwich ELISA: 1/1000.
Not tested in other applications.
Optimal dilutions/concentrations should be determined by the end user.
This antibody can be used in two-site ELISA for the determination of human IFN-a2b.
It recognizes a different epitope than monoclonal antibody 9D3 (ab9386).
We recommend using this antibody (ab5258) as a detection antibody.
Produced by macrophages, IFN-alpha have antiviral activities.